Publications

Human serum albumin (HSA) is efficiently taken up by cancer cells as a source of carbon and energy. In this study, we prepared a monomodified derivative of HSA covalently linked to an EDTA derivative and investigated its efficacy to shuttle weakly anti-proliferative EDTA associating ligands such as vanadium, into a cancer cell line. HSA-S-MAL-(CH2)2-NH-CO-EDTA was found to associate both with the vanadium anion (+5) and the vanadium cation (+4) with more than thrice the associating affinity of those ligands toward EDTA. Both conjugates internalized into glioma tumor cell line via caveolae-mediated endocytosis pathway and showed potent anti-proliferative capacities. IC50 values were in the range of 0.2 to 0.3 µM, potentiating the anti-proliferative efficacies of vanadium (+4) and vanadium (+5) twenty to thirty fold, respectively. HSA-EDTA-VO++ in particular is a cancer permeable prodrug conjugate. The associated vanadium (+4) is not released, nor is it active anti-proliferatively prior to its engagement with the cancerous cells. The bound vanadium (+4) dissociates from the conjugate under acidic conditions with half maximal value at pH 5.8. In conclusion, the anti-proliferative activity feature of vanadium can be amplified and directed toward a cancer cell line. This is accomplished using a specially designed HSA-EDTA-shuttling vehicle, enabling vanadium to be anti-proliferatively active at the low micromolar range of concentration.

Tuftsin-PhosphorylCholine (TPC) is a novel bi-specific molecule which links tuftsin and phosphorylcholine. TPC has shown immunomodulatory activities in experimental mouse models of autoimmune diseases. We studied herein the effects of TPC ex vivo on both peripheral blood mononuclear cells (PBMCs) and temporal artery biopsies (TABs) obtained from patients with giant cell arteritis (GCA) and age-matched disease controls. GCA is an immune-mediated disease affecting large vessels. Levels of 18 cytokines in supernatants, PBMC viability, T helper (Th) cell differentiation of PBMCs and gene expression in TABs were analyzed. Treatment ex vivo with TPC decreased the production of IL-1 beta, IL-2, IL-5, IL-6, IL-9, IL-12(p70), IL-13, IL-17A, IL-18, IL-21, IL-22, IL-23, IFN gamma, TNF alpha, GM-CSF by CD3/CD28 activated PBMCs whereas it negligibly affected cell viability. It reduced Thl and Th17 differentiation while did not impact Th22 differentiation in PBMCs stimulated by phorbol 12-myristate 13-acetate plus ionomycin. In inflamed TABs, treatment with TPC down-regulated the production of IL-1 beta, IL-6, IL-13, IL-17A and CD68 gene expression. The effects of TPC were comparable to the effects of dexamethasone, included as the standard of care, with the exception of a greater reduction of IL-2, IL-18, IFN-gamma in CD3/CD28 activated PBMCs and CD68 gene in inflamed TABs. In conclusion our results warrant further investigations regarding TPC as an immunotherapeutic agent in GCA and potentially other autoimmune and inflammatory diseases.

Despite aggressive therapy, existing treatments offer poor prognosis for glioblastoma multiforme patients, in part due to poor penetration of most drugs across the blood-brain barrier (BBB). We propose a minimal-invasive combined treatment approach consisting of local BBB disruption in the tumor in parallel to systemic drug administration. Local BBB disruption is obtained by convection-enhanced delivery of a novel BBB disruption agent, enabling efficient/targeted delivery of the systemically administered drug by the tumors own vasculature. Various human serum albumin (HSA) analogs were synthesized and screened for BBB disruption efficacy in custom in vitro systems. The candidate analogs were then delivered into naïve rat brains by convection-enhanced delivery and screened for maximal BBB disruption and minimal brain toxicity. These studies found a noncationized/neutralized analog, ethylamine (EA)-HSA, to be the optimal BBB-opening agent. Immunocytochemical studies suggested that BBB disruption by EA-HSA may be explained by alterations in occludin expression. Finally, an efficacy study in rats bearing intracranial gliomas was performed. The rats were treated by convection-enhanced delivery of EA-HSA in parallel to systemic administration of Methotrexate, showing significant antineoplastic effects of the combined approached reflected in suppressed tumor growth and significantly (∼x3) prolonged survival.

We found that human serum albumin (HSA) contains a single binding domain for derivatives of long-chain fatty acid (LCFA)-like molecules in which the carboxylate is replaced by sulfonate. Accordingly, we have synthesized 16-sulfo-hexadecanoic acid-N-hydroxysuccinimide ester [HO₃S-(CH ₂)₁₅-CONHS], an agent that reacts selectively with the amino side chains of peptides and proteins. A macromolecule containing a single 16-sulfohexadecanoate moiety associating with albumin with a K_a value of 0.83 ± 0.08 × 10⁶ M^-1, a sufficient affinity to extend the actions in vivo of such short-lived peptides and proteins. Subcutaneous administration of insulin-NHCO-(CH₂) ₁₅-SO₃^- into mice facilitated a glucose-lowering effect 4.3 times in duration and 6.6 times in area under the curve (AUC) as compared to an in vitro equipotent amount of Zn²⁺-free insulin. Similarly, subcutaneous and intravenous administration of exendin-4-NHCO-(CH₂)₁₅-SO₃^- to mice yielded prolonged and stable reduction in glucose level, 5-9-fold longer than that of exendin-4. Also, a single subcutaneous administration of human interferon-α2-[NH-CO-(CH₂)₁₅-SO₃^-]₃ to mice yielded circulating antiviral activity over a period of 40 h. In conclusion, a simple, hydrophilic reagent has been engineered, synthesized, and studied. Its linkage to peptides and proteins in a monomodified fashion yielded hydrophilic, prolonged acting derivatives, due to their acquired ability to associate with serum albumin after administration.

chelators hold great promise as disease-modifying drugs for Alzheimer's therapy, and recent research efforts have focused on designing multi-target chelators with increased targeting and efficacy through rational drug design. In this review, we discuss our research studies on the rational design of new multi-target chelators with the potential not only to simultaneously modulate several disease-related targets, but also contain features designed to improve the BBB permeability, increase the brain targeting, and minimize potential side effects. These new chelators include neuroprotective chelators with brain selective monoamine oxidase (MAO) A/B inhibitory activity, acetylcholinesterase (AChE) inhibitors with site-activated chelating and neurogenesis activity, and AChE-MAO A/B inhibitors with site-activated chelating and neurogenesis activity.

The finding that acetylcholinesterase (AChE) colocalizes with β-amyloid (Aβ) and promotes and accelerates Aβ aggregation has renewed an intense interest in developing new multifunctional AChE inhibitors as potential disease-modifying drugs for Alzheimer's therapy. To this end, we have developed a new class of selective AChE inhibitors with site-activated chelating activity. The identified lead, HLA20A, exhibits little affinity for metal (Fe, Cu, and Zn) ions but can be activated following inhibition of AChE to liberate an active chelator, HLA20. HLA20 has been shown to possess neuroprotective and neurorescuing activities in vitro and in vivo with the ability to lower amyloid precursor holoprotein (APP) expression and Aβ generation and inhibit Aβ aggregation induced by metal (Fe, Cu, and Zn) ion. HLA20A inhibited AChE in a time and concentration dependent manner with an HLA20A-AChE complex constant (K_i) of 9.66 × 10^-6 M, a carbamylation rate (k₊₂) of 0.14 min^-1, and a second-order rate (k_i) of 1.45 × 10 ⁴ M^-1 min^-1, comparable to those of rivastigmine. HLA20A showed little iron-binding capacity and activity against iron-induced lipid peroxidation (LPO) at concentrations of 1-50 μM, while HLA20 exhibited high potency in iron-binding and in inhibiting iron-induced LPO. At a concentration of 10 μM, HLA20A showed some activity against monoamine oxidase (MAO)-A and -B when tested in rat brain homogenates. Defined restrictively by Lipinski's rules, both HLA20A and HLA20 satisfied drug-like criteria and possible oral and brain permeability, but HLA20A was more lipophilic and considerably less toxic in human SHSY5Y neuroblastoma cells at high concentrations (25 or 50 μM). Together our data suggest that HLA20A may represent a promising lead for further development for Alzheimer's disease therapy.

Many peptides with the potential of therapeutic action for brain disorders are not in clinical use because they are unable to cross the blood-brain barrier (BBB) following peripheral administration. We have developed two potential strategies for the delivery of peptides to the brain and demonstrated their feasibility with enkephalins. In the first approach, designated induced reversible lipophilization, Leu/Met Enkephalins were converted to 9-fluorenylmethoxycarbonyl (Fmoc) derived lipophilic prodrug analogues, which undergo slow, spontaneous hydrolysis under physiological conditions, generating the native agonists. In contrast to Enkephalin, Fmoc-Met-Enkephalin was found to facilitate an analgesic effect following intraperitoneal administration in mice. Fmoc-Leu-Enkephalin was not analgesic. In the second approach, Enkephalin was linked to BBB transport vectors through an Fmoc based linker spacer, forming conjugates that slowly release Enkephalin under physiological conditions. A pronounced antinociceptive response was thus obtained following intraperitoneal administration of either cationized-human serum albumin-Fmoc-Enkephalin or polyethylene glycol₅-Fmoc-Enkephalin. Derivatives of Enkephalin covalently linked to the same BBB-transport vectors through a stable (nonreversible) chemical bond were not analgesic. In summary, we have demonstrated that lipophilicity can be conferred to hydrophilic peptides to a degree permitting the permeation of the BBB by passive diffusion, without the drawback of agonist inactivation, which is often caused by irreversible derivatization. Similarly, in the second strategy, the conjugation to BBB-permeable vectors overcomes the obstacle of peptide inactivation by releasing the active form in the central nervous system.

Amyloids are pathological fibrillar aggregates of proteins related to over 20 diseases. Amyloid fibers are characterized by the cross-beta motif, which is minimally defined as a series of beta-strands extended perpendicular to the fiber axis, joined by hydrogen bonds parallel to the fiber direction. Several structures, all in agreement with the cross-beta definition, have been proposed for specific amyloids. We study the correlation among the suprastructural chirality, molecular structure, and molecular chirality of amyloids. Here we investigate the suprastructure chirality of different (all-S) serum amyloid A (SAA) truncated peptides. We found that the suprastructure chirality of amyloid fibers from segments SAA(2-6), SAA(1-11) and the majority of those from SAA(2-9) is left-handed, which is consistent with the beta-sheet protofilament model. In contrast, SAA(1-12) and SAA(2-12) as well as SAA(1-12), where the C-terminal aspartic acid was point mutated to either leucine or alanine, form right-handed helical amyloid fibers. Such a suprastructure switch indicates a molecular change in the protofilament structure. This is supported by the behavior observed in the FTIR spectra, where the amide I peak of all of the right-handed fibers is red shifted relative to the left-handed amyloid fibers. This work is a case study where isolated short fragments of SAA containing the same amyloidogenic core sequence fold into different amyloid structures. We show that core sequences, supposed to start the misfolding aggregation of the full-length amyloid peptides, may have structures different from those assumed by the isolated segments.

Estrogen has a key role in the regulation of skeletal growth and maintenance of bone mass. Recently, we developed peptides having estrogen-like activity as potential estrogen-based new drugs. The aim of the present study was to evaluate the influence of long-term administration of the most efficacious of these peptides, the hexapeptide EMP-1 (VSWFFE), on bone mass and development. EMP-1 was injected daily to ovariectomized (OVX) and intact young, sexually mature female mice for 10 weeks. Whole femora, including the cartilaginous growth plates were analyzed by micro-computed tomography (μCT). We found that peptide EMP-1 restrains bone growth in OVX mice: it inhibited dramatically bone longitudinal growth (40%), and decreased femoral diaphyseal diameter. Peptide EMP-1 had no effect on bone growth in normal mice, and did not influence the OVX-induced bone loss. We then developed a new μCT methodology to evaluate uncalcified and calcified growth plate parameters. In the OVX mice, peptide EMP-1 reduced volume and thickness of the uncalcified growth plate, a possible cause for the inhibition of bone longitudinal growth. Peptide EMP-1 may be used as a lead compound for the development of drugs to treat acromegalic patients.

We attempted to engineer a novel long-acting insulin based on the following properties: (i) action as a prodrug to preclude supraphysiological concentrations shortly after injection; (ii) maintenance of low-circulating level of biologically active insulin for prolonged period; and (iii) high solubility in aqueous solution. A spontaneously hydrolyzable prodrug was thus designed and prepared by conjugating insulin through its amino side chains to a 40 kDa polyethylene glycol containing sulfhydryl moiety (PEG₄₀-SH), employing recently developed hetero-bifunctional spacer 9-hydroxymethyl-7(amino-3-maleimidopropionate)-fluorene-N-hydroxysucinimide (MAL-Fmoc-0Su). A conjugate trapped in the circulatory system and capable of releasing insulin by spontaneous chemical hydrolysis has been created. PEG₄₀-Fmoc-insulin is a water-soluble, reactivatable prodrug with low biological activity. Upon incubation at physiological conditions, the covalently linked insulin undergoes spontaneous hydrolysis at a slow rate and in a linear fashion, releasing the nonmodified immunologically and biologically active insulin with a t_1/2 value of 30 h. A single subcutaneous administration of PEG₄₀-Fmoc-insulin to healthy and diabetic rodents facilitates prolonged glucose-lowering effects 4- to 7-fold greater than similar doses of the native hormone. The beneficial pharmacological features endowed by PEGylation are thus preserved. In contrast, nonreversible, "conventional" pegylation of insulin led to inactivation of the hormone.

Pegylation is a powerful technology to prolong the action of proteins in vivo, but it is impractical for low-molecular-weight (LMW) drugs, which are usually inactivated upon such modification. Here, we have applied a recently developed strategy of reversible pegylation to gentamicin, a LMW antibiotic. Variable length polyethyleneglycol (PEG-SH) chains were covalently linked to gentamicin using two heterobifunctional agents, each containing a spontaneously hydrolyzable bond. The inactive derivatives regained full antibacterial potency upon incubation under physiological conditions in vitro, and following systemic administration to rats, they released native active gentamicin with half-lives 7- to 15-fold greater than those of systemically administered nonderivatized gentamicin. In conclusion, reversibly pegylated prodrug derivatives of gentamicin were found to be capable of releasing gentamicin for prolonged periods in vivo. Most importantly, the major drawback of conventional pegylation, namely, the loss of pharmacological potency following irreversible derivatization, has been overcome.

Vasoactive intestinal peptide (VIP) is a prominent neuropeptide, exhibiting a wide spectrum of biological activities in mammals. However, the clinical applications of VIP are mainly hampered because of its rapid degradation in vivo. Peptide glycosylation, a procedure frequently used to increase peptide resistance to proteolytic degradation and consequently increase peptide metabolic stability, has not been performed yet on VIP. The presence of three N-glycosylation sites on VIP receptor type 1 (VFAC1) was previously demonstrated. Therefore, glycosylation of the VIP ligand could potentially increase its receptor affinity because of glyco-glyco interactions between the ligand and the receptor. In order to enhance VIP's metabolic stability and to increase its ligand-receptor binding/activation, eight glycosylated VIP derivatives were successfully synthesized by the solid-phase procedure. Each VIP analog was monoglycosylated by a monosaccharide addition to one amino-acid residue along the sequence. Glycosylation did not affect the α-helical structure shown by the native VIP in organic environment. Few glycosylated VIP analogs displayed highly potent VPACI receptor binding and cAMP-induced activation; only 4-6 fold lower in comparison to the native VIP. Furthermore, the peptide analog glycosylated on Thr¹¹ ([11Glyc]VIP) showed a significantly enhanced stability toward trypsin enzymatic degradation in comparison to VIP. Analysis of the degradation products of (11Glyc)VIP showed that differently from VIP, incubation of the peptide [11Glyc]VIP with trypsin resulted in no cleavage at the Arg¹²-Leu¹³ peptide bond, suggesting that VIP glycosylation may lead to enhanced metabolic stability.

Affecting an estimated 5% of adults over 65 years of age, Parkinson's disease and Alzheimer's disease are the most common neurodegenerative disorders. Accumulating evidence suggests that oxidative stress induced by the breakdown of iron homeostasis is a major contributor to the neuronal loss observed in neurodegeneration. Thus, brain-permeable iron chelators may present potential therapeutic benefits. In the present study, iron-chelating hydroxamate groups were introduced into the NAP (NAPVSIPQ) peptide, whose neuroprotective qualities have been widely demonstrated. Our experiments revealed that the novel dihydroxamate peptide 3 is capable of inhibiting iron-catalyzed hydroxyl radical formation and lipid peroxidation, abilities that are not part of the repertoire of its parent peptide. In addition, peptide 3 was superior to native NAP in protecting human neuroblastoma cell cultures against the toxicity of hydrogen peroxide. These results suggest that NAP-based iron chelators deserve further investigation in the search for drug candidates for neurodegeneration.

Dysfunction of the ubiquitin-proteasome system (UPS) and accumulation of iron in substantia nigra (SN) are implicated in the pathogenesis of Parkinson's disease (PD). UPS dysfunction and iron misregulation may reinforce each other's contribution to the degeneration of dopamine (DA) neurons. In the present study, we use a new brain-permeable iron chelator, VK-28 [5-(4-(2-hydroxyethyl) piperazin-1-yl (methyl)-8-hydroxyquinoline], and its derivative M30 [5-(N-methyl-N-propargyaminomethyl)-8-hydroxyquinoline] in vivo to test their neuroprotective and neurorestorative properties against proteasome inhibitor (lactacystin) -induced nigrostriatal degeneration. Bilateral microinjections of lactacystin (1.25 μg/side) into the mouse medial forebrain bundle were performed. Administration of VK-28 (5 mg/kg, once a day) or M30 (5 mg/kg, once a day) was applied intraperitoneally 7 days before or after the lactacystin microinjection until the mice were sacrificed 28 days after microinjection. We found that VK-28 and M30 both significantly improved behavioral performances and attenuated lactacystin-induced DA neuron loss, proteasomal inhibition, iron accumulation, and microglial activation in SN. In addition, M30 restored the Bcl-2 level, which was suppressed after lactacystin injection. These findings suggest that brain-permeable iron chelators can improve DA neuron survival under UPS impairment. Furthermore, M30, a derivative of VK-28 and neuroprotective agent rasagiline, may serve as a better neuroprotective therapy for PD.

Accumulation of iron at sites where neurons degenerate in Parkinson's disease (PD) and Alzheimer's disease (AD) is thought to have a major role in oxidative stress induced process of neurodegeneration. The novel non-toxic lipophilic brain- permeable iron chelators, VK-28 (5- [4- (2- hydroxyethyl) piperazine- 1- ylmethyl]- quinoline- 8- ol) and its multi-functional derivative, M-30 (5-[N-methyl-N-propargylaminomethyl]-8-hydroxyquinoline), as well as the main polyphenol constituent of green tea (-)-epigallocatechin-3-gallate (EGCG), which possesses iron metal chelating, radical scavenging and neuroprotective properties, offer potential therapeutic benefits for these diseases. M-30 and EGCG decreased apoptosis of human SH-SY5Y neuroblastoma cells in a neurorescue, serum deprivation model, via multiple protection mechanisms including: reduction of the pro-apoptotic proteins, Bad and Bax, reduction of apoptosis-associated Ser139 phosphorylated H2A.X and inhibition of the cleavage and activation of caspase-3. M-30 and EGCG also promoted morphological changes, resulting in axonal growth-associated protein-43 (GAP-43) implicating neuronal differentiation. Both compounds significantly reduced the levels of cellular holo-amyloid precursor protein (APP) in SH-SY5Y cells. The ability of theses novel iron chelators and EGCG to regulate APP are in line with the presence of an iron-responsive element (IRE) in the 5-untranslated region (5UTR) of APP. Also, EGCG reduced the levels of toxic amyloid-beta peptides in CHO cells over-expressing the APP "Swedish" mutation. The diverse molecular mechanisms and cell signaling pathways participating in the neuroprotective/neurorescue and APP regulation/processing actions of M-30 and EGCG, make these multifunctional compounds potential neuroprotective drugs for the treatment of neurodegenerative diseases, such as PD, AD, Huntington's disease and amyotrophic lateral sclerosis.

Objectives: Administration of intravenous Ig (IVIG) is a recognized, safe and efficient mode of immunomodulatory therapy for many autoimmune diseases. Anti-idiotypic antibody binding to pathogenic autoantibodies and hence inhibition of binding to the corresponding antigen is one postulated mechanism of the beneficial effect of IVIG. The aim of this study was to fractionate the anti-beta-2-glycoprotein-I (β₂GPI) anti-idiotypic antibodies from a commercial IVIG preparation and use it as a treatment in an experimental antiphospholipid syndrome (APS) mouse model. Methods: Anti-β₂GPI polyclonal antibodies were purified on a β₂GPI column. The purified antibodies were bound to CN-Br-activated sepharose and employed for purification of IVIG-anti-anti-β₂GPI (anti-idiotypic antibodies), defined as specific intravenous Ig (sIVIG). The idiotype specificities were confirmed by competition assays. The effect of sIVIG in vitro was tested in a trophoblast and choriocarcinoma matrigel/invasion assay (i.e. proliferation and metalloproteinase (MMP)2/MMP9 expression) and in vivo in a fetal loss model of APS. Results: Anti-β₂GPI antibodies inhibited human trophoblast cell invasion in vitro. The function was attributed to the Fab portion of the anti-β₂GPI Igs, since β₂GPI-related synthetic peptides specific for the Fab part of the anti-β₂GPI antibodies neutralized its activity. APS sIVIG fraction reduce human trophoblast invasion in vitro by 560 times more than the whole IVIG compound and improved the MMP2 and MMP9 production by trophoblast cells. sIVIG improved significantly (200 times more) the pregnancy outcome in BALB/c mice passively infused with anti-β₂GPI antibodies, in comparison to treatment with IVIG (P

Luliberin analogues modified at the N-terminus were synthesized to search for drugs exerting a cytotoxic effect on cells of hormone-dependent tumors. A synthetic scheme effective in the preparation of analogues containing fatty acid residues was proposed. The cytotoxic effect of the peptides was studied on a number of cell lines of human tumors in vitro. The dependence of the antitumor effect on the length of peptide chain, amino acid sequence, and structure of the N-terminal group was demonstrated. Modification with palmitic acid was found to result in highly active compounds in the case of analogues containing more than ten aa, whereas modifications with lauric, caproic, or trimethylacetic acid led to compounds with significantly lower activities. Analogues of luliberin containing a palmitic acid residue and effectively inhibiting the growth of tumor cells in vitro were synthesized.

Polymyxin B (PMXB) blocks the action of insulin on glucose uptake in vitro. In vivo, it reverses hypoglycemia induced by exogenous insulin. Here we have treated mature male rats daily with PMXB over a period of two weeks. This therapy has decreased body weight by 11%, adipose fat mass by 46% and triglyceride levels by 39%, with no indication of liver or kidney toxicity. Two suboptimal parameters, however, were a decrease in food intake in the first week of treatment and some increase in fasting glucose levels. We have screened for PMXB-analogs having less associating affinity with rat-muscle phospholipids, and revealed that the same therapy using PMXB-derived peptide (nona-PMXB) is most optimal. This PMXB-analog is devoid of antibacterial activity and is four times less toxic than PMXB. Nona-PMXB therapy lower by 10, 32, 35 and 6% body weight gain, fat mass, circulating triglycerides and fasting glucose levels, respectively, in spite of normal or even elevated food intake in nona-PMXB treated rats. In summary, we found that nona-PMXB therapy is capable if inducing leanness in mature rats, particularly at the expense of decreasing fat-mass in adipose tissue. By and large, we suggest that lowering the action of insulin, on fat build-up solely, may be a therapeutically feasible task to fight with human adiposity in the future.

Five linear analogs of GnRH containing a p-aminophenylalanine (Pap) residue in their sequence and their six corresponding azo-bridged cyclic derivatives were synthesized. The precyclic peptides were prepared on solid-support, while azocyclization was performed in solution by diazotization of the p-aminophenylalanine residue followed by intramolecular coupling of the formed diazo salt with either tyrosine or histidine side chains present in the sequence. All peptides were examined for their binding ability to the GnRH receptor expressed on rat pituitary membranes and for their LH-release activity from dispersed rat pituitary cells. Linear analogs 1 i.e [Pap⁵] GnRH and 3, i.e. [Tyr³, Pap⁵] GnRH, were found to bind to the GnRH receptors only slightly less avidly than native GnRH. Their cyclization, however, led to a marked reduction in the binding capacity, i.e. from IC₅₀ of 10^-9 M to the 10^-7 M range, and in biopotency, i.e. LH-release. All other linear and cyclic peptides were found to bind selectively to the GnRH receptor only in the low μM range. Only peptide 1 was found comparable to native GnRH in respect to LH-release activity and thus may potentially be a good agonist of the parent peptide. Peptides 1-4, the most potent GnRH receptor binders, were examined for their conformational properties using CD. Cyclic-azo peptides 2 and 4 were further evaluated by NMR spectroscopy in solution combined with molecular modeling. The structural information obtained explains in part the GnRH-like biological activity observed.

A large body of data indicates that a cascade of events contributes to the neurodegeneration in Alzheimer's disease (AD) and Parkinson's disease (PD). Metal (Fe, Cu, Zn) dyshomeostasis and oxidative stress are believed to play a pivotal role in the pathogenesis of these diseases. Accordingly, multifunctional compounds combining metal chelating and antioxidative activity hold a great promise as potential drugs for treating AD and PD. In this study, two novel NAPVSIPQ (NAP) analogs (M98 and M99) with potential antioxidant-metal chelating ability were designed and investigated, aiming to improve the poor metal chelating and antioxidative activity of NAP. Our studies showed that both M98 and M99 formed stable metal (Fe, Cu, Zn) complexes in water and demonstrated good metal (Fe, Cu, Zn) chelating properties as opposed to the poor metal (Fe, Cu, Zn) chelating properties of their parent peptide NAP. M98 and M99 exhibited significant inhibition of iron-induced lipid peroxidation in rat brain homogenates at concentrations of ≥30 μM, while NAP failed to show any inhibition even at 100 μM. In human neuroblastoma cell (SH-SY5Y) culture, M98 and M99 at 1 μM completely protected against 6-hydroxydopamine (60HDA) toxicity with potency similar to NAP and desferal (DFO), a strong iron chelator and a highly potent radical scavenger. In PC12 cell culture, M98 at the range of 0.001-1 μM displayed potent protection against 6-OHDA toxicity, comparable to NAP and DFO. These results suggest that M98 and M99 deserve further investigation as potential drug candidates for neuroprotection.

Iron and monoamine oxidase activity are increased in brain of Parkinson's disease (PD). They are associated with autoxidation and oxidative deamination of dopamine by MAO resulting in the generation of reactive oxygen species and the onset of oxidative stress to induce neurodegeneration. Iron chelators (desferal, Vk-28 and clioquinol) but not copper chelators have been shown to be neuroprotective in the 6-hydroxydoapmine and MPTP models of Parkinson's disease (PD), as are monoamine oxidase B inhibitors such as selegiline and rasagiline. These findings prompted the development of multifunctional anti PD drugs possessing iron chelating phamacophore of VK-28 and the propargylamine MAO inhibitory activity of rasagiline. M30 is a potent iron chelator, radical scavenger and brain selective irreversible MAO-A and B inhibitor, with little inhibition of peripheral MAO. It has neuroprotective activity in in vitro and in vivo models of PD and unlike selective MAO-B inhibitors it increases brain dopamine, serotonin and noradrenaline. These findings indicate beside its anti PD action, it may also possess antidepressant activity, similar to selective MAO-A and nonselective MAO inhibitors. These properties make it an ideal anti PD drug for which it is being developed.

Antioxidants and iron chelating molecules are known as neuroprotective agents in animal models of neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD). In this study, we designed and synthesized a novel bifunctional molecule (M10) with radical scavenging and iron chelating ability on an amino acid carrier likely to be a substrate for system L, thus targeting the compound to the central nervous system (CNS). M10 had a moderate iron affinity in HEPES buffer (pH 7.4) with log K₃ = 12.25 ± 0.55 but exhibited highly inhibitory action against iron-induced lipid peroxidation, with an IC₅₀ value (12 μM) comparable to that of desferal (DFO). EPR studies indicated that M10 was a highly potent ^.OH scavenger with an IC₅₀ of about 0.3 molar ratio of M10 to H₂O₂. In PC12 cell culture, M10 was at least as potent as the anti-Parkinson drug rasagiline in protecting against cell death induced by serum-deprivation and by 6-hydroxydopamine (6-OHDA). These results suggest that M10 deserves further investigation as a potential agent for the treatment of neurodegenerative disorders such as AD and PD.

A series of novel derivatives of neuropeptides with a metal-chelating moiety was synthesized and examined for various properties related to iron (Fe) chelation and neuroprotective action. All derivatives chelated Fe to form stable Fe complexes in water. Some strongly inhibited Fe-induced lipid peroxidation with an IC50 value of about 12 mu M. In PC12 cell culture, several compounds, at concentrations as low as 1 mu M, attenuated serum-free stimulated cell death and improved cell survival by 20-35%. At this concentration, these analogs also protected against 6-hydroxydopamine (6-OHDA)-induced cell death, increasing cell viability by 20-30%. Electron paramagnetic resonance (EPR) studies indicated that besides being good Fe chelators, these analogs act as radical scavengers to directly scavenge hydroxyl radicals. Together, the data indicate that some of the analogs could be further developed as possible neuroprotective agents for treatment of neurodegenerative diseases such as Parkinson's, Alzheimer's, and Huntington's diseases, Friedreich's atxia, amyotrophic, and lateral sclerosis where Fe misregulation has been reported.

We report that oral administration of vanadium (+5) combined with L-glutamic acid γ-monohydroxamate at 1:2 stoichiometry [L-Glu(γ)HXM·VO₃^-] is highly effective in reducing blood glucose levels (BGLs) in a wide variety of diabetic rodents. In streptozocin-treated rats, a single administration (0.28 mmol/kg body wt) decreased BGL from 490 to 360 mg/dl within 1 h of administration, keeping this reduced level for additional 22 h, and a daily dose of 0.14 mmol/kg was found optimal. In Zucker diabetic fatty (ZDF) rats, a single dose of 0.14 mmol/kg normalized BGL within 8 h of administration, and maintained normal value for additional two days. In db/db mice, a single L-Glu(γ)HXM·VO ^3- administration of 0.2 mmol/kg decreased BGL from 500 ± 50 to 240 ± 20 mg/dl at 2 h, but was less effective afterwards. In high-carbohydrate (CHO)-fed Psammomis obesus, a single oral dose (0.14 mmol/kg) normalized BGL over a period of two days, and a daily dose of 0.07 mmol/kg/d, at the time P. obesus was transferred from low- to high-CHO diet, fully arrested the development of hyperglycemia characterizing this diabetic rodent. Finally, we found that the index of toxicity of orally administered L-GLU(γ)HXM-vanadate in rodents is 5-7 times lower than that of free sodium vanadate.

Type I gonadotropin-releasing hormone (GnRH) receptor (GnRHR) is unique among mammalian G-protein-coupled receptors (GPCRs) in lacking a C-terminal tail, which is involved in desensitization in GPCRs. Therefore, we searched for inhibitory sites in the intracellular loops (ICLs) of the GnRHR. Synthetic peptides corresponding to the three ICLs were inserted into permeabilized αT3-1 gonadotrope cells, and GnRH-induced inositol phosphate (InsP) formation was determined. GnRH-induced InsP production was potentiated by ICL2 > ICL3 but not by the ICL1 peptides, suggesting they are acting as decoy peptides. We examined the effects of six peptides in which only one of the Ser or Thr residues was substituted with Ala or Glu. Only substitution of Ser ¹⁵³ with Ala or Glu ablated the potentiating effect upon GnRH-induced InsP elevation. ERK activation was enhanced, and the rate of GnRH-induced InsP formation was about 6.5-fold higher in the first 10 min in COS-1 cells that were transfected with mutants of the Gn-RHR in which the ICL2 Ser/Thr residues (Ser¹⁵¹, Ser¹⁵³, and Thr¹⁴²) or only Ser ¹⁵³ was mutated to Ala as compared with the wild type GnRHR. The data indicate that ICL2 harbors an inhibitory domain, such that exogenous ICL2 peptide serves as a decoy for the inhibitory site (Ser¹⁵³) of the Gn-RHR, thus enabling further activation. GnRH does not induce receptor phosphorylation in αT3-1 cells. Because the phosphomimetic ICL2-S153E peptide did not mimic the stimulatory effect of the ICL2 peptide, the inhibitory effect of Ser¹⁵³ operates through a phosphorylation-independent mechanism.

A combination of light, oxygen and a photosensitizer is used to induce death of cancer cells by photodynamic therapy. In this study, we have synthesized several new methyl helianthrone derivatives and compared their phototoxicity with that of hypericin. In contrast to hypericin, methyl helianthrones are soluble in aqueous solutions and have a broad range of light absorbance, which allows the use of polychromatic light Structural modifications of methyl helianthrone demonstrated that substitution of hydrogen atoms of methyl helianthrone at Positions 2 and 5 with Br atoms or methylation of its phenolic hydroxyls, significantly increases the corresponding singlet oxygen quantum yield and their phototoxicity toward αT3-1, M2R and LNCaP cells. The phototoxicity of some of these compounds was similar to that of hypericin. Methyl helian-thrones, like hypericin, accumulated mainly in the perinuclear region as evident by confocal microscopy. Irradiation of cells pretreated with methyl helianthrone derivatives generates intracellular reactive oxygen species and lipid free radicals, as shown by a fluorescentic probe and electron paramagnetic resonance methods, respectively. The phototoxicity of these methyl helianthrones as well as their ability to oxidize membrane lipids were significantly decreased on addition of specific Type-II inhibitors, suggesting the involvement of singlet oxygen as the main oxidant.

Keywords: Rheumatology

Keywords: POLYETHYLENE-GLYCOL

The IgG-derived immunomodulating peptide tuftsin, Thr-Lys-Pro-Arg, is recognized by specific receptors on phagocytic cells, notably macrophages, and is capable of targeting proteins and peptides to these sites. Aiming to target 3-azido-3-deoxythymidine (AZT) to HIV-infected macrophages, a conjugate of AZT with tuftsin was synthesized. The AZT-tuftsin chimera possesses the characteristic capacities of its two components. Thus, like AZT, it inhibits reverse transcriptase activity and HIV-antigen expression, and similarly to tuftsin, it stimulates IL-1 release from mouse macrophages and augments the immunogenic function of the cells. Importantly, the conjugate is not cytotoxic to T-cells. The results suggest that the AZT-tuftsin conjugate might have potential use in AIDS therapy.

Iron has been shown to accumulates at site where neurons degenerate in neurodegenerative diseases of Parkinson's disease, Alzheimer's disease, Huntington disease, amyotrophic lateral sclerosis and Friedreich ataxia. Iron is thought to participate or initiate oxidative stress via generation of reactive oxygen species (ROS), such as hydroxyl radical. Iron chelators are neuroprotective and prevent 6-hydroxydoapmine and MPTP dopaminergic neurotoxicity in rats and mice. However, their action on monoamine oxidase (MAO) A and B have not been determined previously since MAO-B inhibitors have been shown to be neuroprotective in cellular and animal models of Parkinson's disease. The chelators 8-hydroxyquinoline, O-phenanthroline, 2,2-dipyridyl, U74500A and U74600F showed a preference for inhibition of rat brain mitochondrial MAO-A over MAO-B. Their IC₅₀ ranged from 10^-3M to 101M, with 21-amino steroids (U74500A and U74006F) showing a greater selectivity and potency for MAO-A. Desferrioxamine (desferal), a prototype potent iron chelator, exhibited relatively poor MAO inhibitory. The inhibitions of MAO-A and B by 21-amino steroids (Lazaroids) were time dependent and irreversible. Those initiated by 8-hydroxyquinoline, 2,2-dipyridyl and O-phenanthroline were fully reversible by enzyme dilution experiments. Both Fe²⁺ and Fe³⁺ reverse the MAO-A and B inhibition induced by the latter chelators, but not those initiated by 21-amino steroids. The data infer that either the inhibition of MAO by 21-amino steroids is either the resultant of their conversion to an irreversible covalently bound ligand or that the iron chelation moiety and MAO inhibitory activity in these compounds are not mutually shared. The results suggest that bifunctional brain penetrable drugs with iron chelating property and MAO inhibitory activity in could be the most feasible approach for neuroprotection in neurodegenerative diseases. Such drug would prevent participation of elevated iron in oxidative stress and formation of reactive hydroxyl radical, via its interaction with H₂O2 (Fenton chemistry), generated as a consequence MAO and other oxidative enzyme reactions to generative cytotoxic reactive hydroxyl radical. We have now developed several of these compounds with neuroprotective, MAO inhibitory and iron chelating properties from our prototype iron chelators, VK-28 possessing propargylamine moiety of our anti-parkinson drug, rasagiline.

Many peptide and protein drugs have a short circulatory half-life in vivo. The covalent attachment of polyethylene glycol (PEG) chains (PEGylation) can overcome this deficiency, but pegylated peptides and proteins are often inactive. In this study, we present a novel PEG-IFNα2 conjugate, PEG ₄₀-FMS-IFNα2, capable of regenerating native interferon α2 (IFNα2) at a slow rate under physiological conditions. A 2-sulfo-9-fluorenylmethoxycarbonyl (FMS) containing bifunctional reagent, MAL-FMS-NHS, has been synthesized, enabling the linkage of a 40 kDa PEG-SH to IFNα2 through a slowly hydrolyzable bond. By use of a BIAcore binding assay, the in vitro rate of regeneration of native interferon was estimated to have a half-life of 65 h. Following subcutaneous administration to rats and monitoring circulating antiviral activity, active IFNα2 levels peaked at 50 h, with substantial levels still being detected 200 h after administration. This value contrasts with a half-life of about 1 h measured for unmodified interferon. The concentration of active IFNα2 scaled linearly with the quantity injected. Comparing subcutaneous to intravenous administration of PEG₄₀-FMS-IFNα2, we found that the long circulatory lifetime of IFNα2 was affected both by the slow rate of absorption of the PEGylated protein from the subcutaneous volume and by the slow rate of discharge from the PEG in circulation. A numerical simulation of the results was in good agreement with the results observed in vivo. The pharmacokinetic profile of this novel IFNα2 conjugate combines a prolonged maintenance in vivo with the regeneration of active-native IFNα2, ensuring ready access to peripheral tissues and thus an overall advantage over currently used formulations.

Currently used antiestrogenic drugs against hormone-dependent breast cancer, and estrogenic drugs used in treatment of osteoporosis, are associated with risk factors. Therefore, there is a strong need to develop selective estrogen receptor modulators with better tissue selectivity. In a recent study (Peptides, 2002, Vol. 3, 573-580), we used a monoclonal antibody to estradiol (mAb-E2) to screen a phage-display peptide library. We identified a 15-mer peptide (peptide H5) that recognizes mAb-E2 (IC₅₀ 1 μM) and estrogen receptor (ER)α (IC₅₀ 500 μM) but not ERβ, and displays estrogen-like activity in vitro and in vivo. In this study, we designed and prepared peptides based on peptide H5, which possess improved estrogenic activity, by evaluating their binding to mAb-E2 and to ERs. Initially, we determined the minimal binding sequence of peptide H5 capable of binding mAb-E2 and ER. Subsequently, systematic single-residue replacements of the minimal sequence, followed by multiple-residue replacements, yielded hexa- and heptapeptides with increased affinities to mAb-E2 and to ER. The most promising peptides, VSWFFE (EMP-1) and VSWFFED (EMP-2) (EMP: estrogen-mimetic peptide), bind mAb-E2 with high affinity (IC₅₀ of 6 and 30 nM, respectively), recognize ERs with increased affinity (IC₅₀ of 100 μM for ERα, and 100-250 μM for ERβ), and possess estrogenic activity in vivo. The short peptides described in this study may be used as potential lead compounds for developing new ER ligands.

Photodynamic therapy uses a combination of light, oxygen, and a photosensitizer to induce the death of malignant cells. To improve the selectivity of a photosensitizer toward cancerous cells that express gonadotropin-releasing hormone (GnRH) receptors, protoporphyrin IX (PpIX) was conjugated to a GnRH agonist, [D-Lys⁶]GnRH, or to a GnRH antagonist, [D-pGlu¹, D-Phe², D-Trp³, D-Lys ⁶]GnRH. The condensation of the peptide with PpIX was carried out in a homogeneous solution using benzotriazole-1-yloxytris(pyrrolidinophosphonium) hexafluorophosphate as a coupling reagent. Although these conjugates had lower binding affinity to rat pituitary GnRH receptors than their parent analogues, they fully preserved their agonistic or antagonistic activity in vitro and in vivo. The GnRH agonist conjugate proved to be long-acting in vivo. Thus, 24 h after its administration to rats (2 nmol/rat), serum LH concentrations were significantly higher than in rats treated with the same amount of the parent peptide. The conjugates, notably the agonist, were more phototoxic toward pituitary gonadotrope αT3-1 cell line than was unconjugated PpIX. In contrast to PpIX, the phototoxicity of the conjugates toward αT3-1 cells or to human breast cancer cells (MCF-7 cells that were transfected with human GnRH receptors) was alleviated by co-incubation with the parent peptide, indicating that phototoxicity is receptor-mediated. The selectivity of the GnRH antagonist conjugate to gonadotrope cells in a primary pituitary culture was ∼10 times higher than that of the unconjugated PpIX. Thus, GnRH-based conjugates may affect cancer cells not only by acting as classic GnRH analogues to reduce the plasma levels of steroids by desensitization of the pituitary gland but also by selective photodamage of cells that express GnRH receptors.

Exendin-4, a glucagon-like peptide-1 agonist, is a relatively short-lived species in the circulatory system in vivo. We have linked three moieties of 2-sulfo-9-fluorenylmethoxycarbonyl (FMS) to the three amino functions of exendin-4. FMS₃-exendin-4 thus obtained has about 0.1% the glucose-lowering potency of the native peptide. Upon in vitro incubation at physiological conditions, the FMS moieties undergo hydrolysis generating the parent fully active, exendin-4. A single subcutaneous administration of FMS₃-exendin-4 to healthy and type II diabetic mice has induced a glucose-lowering profile that exceeds in length several times that obtained by the native peptide. The reduction of blood glucose level began 1h after administration and was maximal 3-4h later. The blood glucose level returned to pre-administered values with t_1/2 of 12±0.7, 26±2, and 44±3h with doses of 1, 10, and 100μg/mouse, respectively. In sum, we have synthesized and characterized FMS₃-exendin-4, a prodrug derivative of the native insulinotropic peptide, and found it to facilitate a prolonged and stable, glucose-lowering action in healthy and diabetic mice.

Intensive studies have been carried out during the last two decades, on the insulinomimetic effects of vanadium. Vanadium compounds mimic most of the metabolic effects of insulin on the main tissues of the hormone in vitro. Vanadium therapy induces normoglycemia and improves glucose homeostasis in insulin deficient and insulin resistant diabetic rodents. Improved sensitivity to insulin in liver and muscle tissues of Type II diabetic patients following vanadium therapy was observed as well. The key mechanisms involved are inhibition of protein-phosphotyrosine phosphatases and activation of nonreceptor protein-tyrosine kinases, in an insulin-receptor tyrosine kinase independent fashion. Vanadate activates glucose-metabolism in vitro at a site preceding activation of phosphatidylinositol-3-kinase (PI3-kinase). Regarding inhibition of lipolysis, vanadate (but not insulin) acts at a site downstream to the activation of PI3 kinase. Additional vanadium-dependent mechanism, operating in vivo, is the restoration of glucose-6-phosphate levels in liver, muscle and adipose tissue of hyperglycemic diabetic rats. This is attributed to vanadate-dependent inhibition of liver glucose-6-phosphatase, and of nonspecific hexose-6-phosphatases of the diabetic muscle and adipose tissues. Initial clinical studies were already performed. Several beneficial effects were documented. The potential usage of vanadium in the future care of diabetes in human, however, depends on manipulations that would elevate the insulinomimetic efficacy of vanadium without increasing its toxicity. Organically chelated vanadium compounds, in particular, the L-isomer of Glu(γ) monohydroxamate (L-Glu(γ)HXM) are active in potentiating the capacity of free vanadium to activate glucose metabolism, in vitro and in diabetic rats in vivo. L-Glu(γ)HXM differs from other vanadium ligands in being an amino acid derivative that permeates into peripheral tissues through the amino acid transport system. In rat adipocytes, L-Glu(γ)HXM itself activates partially glucose metabolism, by permeating into cell interior, associating with the minute quantity of intracellular vanadium, and turning it into an insulinomimetic active species. L-Glu(γ)HXM, associates with the vanadyl (+4) cation, and the vanadate (+5) anion, at neutral pH with nearly the same binding affinity. Both these oxidation states of vanadium are insulinomimetic. The therapeutical potency of L-Glu(γ)HXM-vanadium complexes is actively studied. Preliminary results on this issue are to be presented.

Accelerated neuronal death brings about cognitive as well as motor and other dysfunctions. A major neuropeptide, vasoactive intestinal peptide (VIP), has been shown to be neuroprotective. However, VIP-based drug design is hampered by the instability of the peptide and its limited bioavailability. Two independent approaches were thus taken to exploit VIP as a lead drug candidate: (1) Potent neuroprotective lipophilic analogs of VIP were synthesized, e.g. [stearyl-norleucine-17] VIP (SNV); and (2) potent neuroprotective peptide derivatives were identified that mimic the activity of VIP-responsive neuroprotective glial proteins. VIP provides neuronal defense by inducing the synthesis and secretion of neuroprotective proteins from astrocytes; activity-dependent neuroprotective protein (ADNP) was discovered as such glial cell mediator of VIP- and SNV-induced neuroprotection. In subsequent studies, an eight-amino-acid peptide, NAP, was identified as the smallest active element of ADNP exhibiting potent neuroprotective activities. This paper summarizes the biological effects of SNV and NAP and further reports advances in NAP studies toward clinical development. An original finding described here shows that NAP, while protecting neurons, demonstrated no apparent effect on cell division in a multiplicity of cell lines, strengthening the notion that NAP is a specific neuroprotective drug candidate.

Polymyxin B nonapeptide (PMBN), a cationic cyclic peptide derived from the antibacterial peptide polymyxin B, is capable of specifically increasing the permeability of the outer membrane (OM) of Gram-negative bacteria toward hydrophobic antibiotics. In this study, we evaluated the contribution of the hydrophobic segment of PMBN (i.e., D-Phe⁵-Leu⁶) to this activity. Accordingly, we synthesized four analogs of PMBN by replacing D-Phe⁵ with either with D-Trp or D-Tyr and Leu⁶ with Phe or Ala and evaluated their ability to bind cell-free lipopolysaccharide (LPS) and increase bacterial OM permeability. Compared with PMBN, [D-Tyr⁵]PMBN and [Ala⁶]PMBN possessed reduced LPS affinity (IC₅₀ = 2.5, 25, and 12 μM, respectively) and significantly reduced OM permeability and LPS neutralization activity. [Phe⁶]PMBN exhibited rather similar affinity to cell-free LPS (IC₅₀ = 5 μM) and the same OM permeability capacity as PMBN. However, [D-Trp⁵]PMBN, despite its similar affinity to cell-free LPS (IC₅₀ = 4 μM), had moderately reduced OM permeability capacity. These results demonstrate the significant role of the PMBN hydrophobic segment in promoting biological activity.

Vasoactive intestinal peptide (VIP) stimulates the neuroblastoma cell line (NMB) to proliferate. Neuropeptide activity can be inhibited by neutral endopeptidases that function intracellularly and in the extracellular milieu. NMB cells express neutral endopeptidase (NEP) activity that can be specifically inhibited by phosphoramidon (PA). Our data now show that phosphoramidon treatment increases the efficacy of VIP-stimulated neuroblastoma proliferation. These results suggest that membrane endopeptidases modulate VIP-associated cell proliferation and enhancement of endopeptidase activity may serve as a target for cancer therapy.

Most low-molecular-weight drugs are short-lived species in the circulatory system, being rapidly eliminated by glomerular filtration in the kidney. However, binding to human serum albumin (HSA) can slow clearance and prolong lifetime profile in vivo. In this study, we have engineered a gentamicin derivative with affinity to albumin by linking three (2-sulfo)-9-fluorenylmethoxy-carbonyl (FMS) to three amino groups of gentamicin C₁. FMS₃-gentamicin associates with HSA with a K_a value of (1.31 ± 0.2) x 10⁵ M^-1. It has less than 1% the antibacterial potency of native gentamicin. Upon incubation at pH 8.5 and 37 °C, the FMS moieties from FMS₃gentamicin undergo slow hydrolysis (t_1/2 = 8.0 ± 0.2 h), leading to a linear regeneration of the antibacterial potency with a t_1/2 value of 11 ± 0.7 h. FMS₃-gentamicin is a long-lived species in the rat circulatory system. Following a single subcutaneous or intravenous administration, it maintains a prolonged pharmacokinetic profile with a peak and a "through" concentration of immuno/antibacterial active gentamicin exceeding 4-5 times the duration obtained by administered native gentamicin. To sum up, an approach aimed at elongating the lifetime of low-molecular-weight drugs in vivo has been examined here with gentamicin. Two to three FMS per mole of compound are to be introduced to obtain an albumin associating affinity of K_d = 7.6-9.2 μM and, hence, to significantly extend the drug's lifetime in situ following administration. By use of this technology, the loss of pharmacological potency with derivatization is of no consequence, since FMS moieties are hydrolyzed and activity is generated at physiological conditions.

The effects of a (N-stearyl, Norleucine(17)) vasoactive intestinal peptide hybrid ((SN)VIPhybrid) on cells stably transfected with VPAC(1), VPAC(2), or PAC(1) receptors were investigated. (SN)VIPhybrid inhibited specific I-125-VIP binding to membranes derived from CHO cells transfected with VPAC(1) or VPAC(2) receptors with high affinity (IC50 = 30 and 50 nM). (SN)VIPhyb inhibited specific I-125-PACAP-27 binding to membranes derived from NIH/3T3 cells transfected with PAC(1) receptors with high affinity (IC50 = 65 nM). PACAP-27 caused cAMP elevation in NIH/3T3 cells transfected with PAC(1) receptors and the increase cAMP caused by pituitary adenylated cyclase (PACAP) was inhibited by (SN)VIPhyb. Also, the increase in cAMP caused by VIP using CHO cells transfected with VPAC(1) or VPAC(2) receptors was antagonized by (SN)VIPhyb. These results indicate that (SN)VIPhyb is an antagonist for VPAC(1), VPAC(2), and PAC(1) receptors.

The antiphospholipid syndrome (APS) is characterized by the presence of pathogenic autoantibodies against β2-glycoprotein-I (β2GPI). The factors causing production of anti-β2GPI remain unidentified, but an association with infectious agents has been reported. Recently, we identified a hexapeptide (TLRVYK) that is recognized specifically by a pathogenic anti-β2GPI mAb. In the present study we evaluated the APS-related pathogenic potential of microbial pathogens carrying sequences related to this hexapeptide. Mice immunized with a panel of microbial preparations were studied for the development of anti-β2GPI autoantibodies. IgG specific to the TLRVYK peptide were affinity purified from the immunized mice and passively infused intravenously into naive mice at day 0 of pregnancy. APS parameters were evaluated in the infused mice on day 15 of pregnancy. Following immunization, high titers of antipeptide [TLRVYK] anti-β2GPI Ab's were observed in mice immunized with Haemophilus influenzae, Neisseria gonorrhoeae, or tetanus toxoid. The specificity of binding to the corresponding target molecules was confirmed by competition and immunoblot assays. Naive mice infused with the affinity-purified antipeptide Ab's had significant thrombocytopenia, prolonged activated partial thromboplastin time and elevated percentage of fetal loss, similar to a control group of mice immunized with a pathogenic anti-β2GPI mAb. Our study establishes a mechanism of molecular mimicry in experimental APS, demonstrating that bacterial peptides homologous with β2GPI induce pathogenic anti-β2GPI Ab's along with APS manifestations.

The effects of a vasoactive intestinal peptide (VIP) receptor antagonist (VIPhyb) on human glioblastoma cells were characterized. Pituitary adenylate cyclase activating polypeptide (I-125-PACAP-27) bound with high affinity to U87, U118, and U373 cells. Specific I-125-PACAP-27 binding to U87 cells was inhibited, with high affinity, by PACAP but not VIP or VIPhyb (IC50 = 10, 1500, and 500 nM, respectively). By reverse transcriptasepolymerase chain reaction (RT-PCR), a major 305bp band was observed indicative of PAC1 receptors. PACAP-27 caused cAMP elevation and the increase in cAMP caused by PACAP-27, was inhibited by the VIPhyb. Also, PACAP-27 caused cytosolic Ca2+ elevation in Fura-2AM loaded U87 cells and the VIPhyb inhibited this increase. Using the MTT growth assay, the VIPhyb was shown to inhibit glioblastoma growth in a concentration dependent manner. Using a clonogenic assay in vitro, 10 muM VIPhyb significantly inhibited proliferation of U87, U118, and U373 cells. In vivo, 0.4 mug/kg VIPhyb inhibited U87 xenograft proliferation in nude mice. These results suggest that the VIPhyb antagonizes PAC1 receptors on glioblastoma cells and inhibits their proliferation.

We have determined the crystal structure at 1.8 Å resolution of a complex of α-bungarotoxin with a high affinity 13-residue peptide that is homologous to the binding region of the α subunit of acetylcholine receptor. The peptide fits snugly to the toxin and adopts a β hairpin conformation. The structures of the bound peptide and the homologous loop of acetylcholine binding protein, a soluble analog of the extracellular domain of acetylcholine receptor, are remarkably similar. Their superposition indicates that the toxin wraps around the receptor binding site loop, and in addition, binds tightly at the interface of two of the receptor subunits where it inserts a finger into the ligand binding site, thus blocking access to the acetylcholine binding site and explaining its strong antagonistic activity.

In an attempt to develop an efficient chemotherapeutic agent targeted at malignant cells that express receptors to gonadotropin releasing hormone (GnRH) we coupled [D-Lys⁶]GnRH covalently to an emodin derivative, i.e. emodic acid (Emo) to yield [D-Lys⁶(Emo)]GnRH. Emodin is a naturally occurring anthraquinone which is widely used as a laxative and has other versatile biological activities. Physico-chemical studies employing electron paramagnetic resonance and electrochemistry of the conjugate as well as the (Emo) moiety showed that these compounds could be easily reduced either chemically, photochemically or enzymatically to their corresponding semiquinones. In the presence of oxygen the semiquinones generated reactive oxygen species (ROS), mainly superoxide and hydroxyl radicals, which were detected by the spin trapping method. Moreover, upon irradiation with visible light these compounds produced ROS and a highly reactive excited triplet state of Emo, which by itself may cause the oxidation of certain electron acceptors such as amino acids and bases of nucleic acids. Thus, [D-Lys⁶]GnRH-photosensitizer conjugates may be potentially used for targeted photodynamic chemotherapy aimed at treating cancer cells that carry GnRH receptors. These conjugates may also induce cytotoxicity in the dark similar to common conventional chemotherapeutic agents. The peptidic moiety, [D-Lys⁶]GnRH, was found to be stable toward highly reactive ROS generated either from enzymatic reduction or upon photoirradiation. The physico-chemical properties of Emo were only marginally influenced by the peptidic [D-Lys⁶]GnRH carrier.

Nanomolar concentrations of vasoactive intestinal peptide (VIP), picomolar concentrations of stearyl-norleucine 17-VIP (SNV) and femtomolar concentrations of NAPVSIPQ (NAP), an 8-amino-acid peptide derived from the VIP-responsive activity-dependent neuroprotective protein, provide broad neuroprotection. In rat cerebral cortical cultures, 10 ^-16-10 ^-7 M NAP increased intracellular cyclic guanosine monophosphate (cGMP) (2.5-4-fold) and 10 ^-10 M NAP increased extracellular nitric oxide (NO) by 60%. In the same culture system, VIP and SNV (at micromolar concentrations) increased extracellular NO by 45-55%. The NAP dose required for cGMP increases correlated with the dose providing neuroprotection. However, the concentrations of NAP, SNV and VIP affecting NO production did not match the neuro-protective doses. Thus, NO may mediate part of the cell-cell interaction and natural maintenance activity of VIP/SNV/NAP, while cGMP may mediate neuroprotection.

Mastoparan, an amphiphilic cationic tetradecapeptide was previously shown to block activation of the NADPH oxidase in the cell-free system presumably by association with a cytosolic component/s of the enzyme. Blockade of oxidase activation was now demonstrated in the semirecombinant NADPH oxidase system. The structural basis of the inhibitory effect of MP on oxidase assembly was explored employing a variety of truncated and specifically substituted synthetic peptide analogs. The data indicated that an α helical fold, positive net charge, hydrophobicity and amphiphilicity were essential for the inhibitory potency and that peptide analogs below eleven residues were inactive. To identify the MP-binding oxidase subunit three different binding assays were carried out utilizing free or immobilized recombinant p47-phox, p67-phox, p40-phox and Rac1 in conjunction with immobilized MP or soluble ¹²⁵I-tyr-MP, respectively. The data implicated p67-phox as the main MP-binding component. The binding site on the p67-phox was localized to the 1-238 aminoterminal fragment of the molecule. NADPH oxidase activation supported by this fragment was inhibitable by MP. In addition, SH3 domains of p47-phox and p40-phox and the carboxyterminal SH3 domain of p67-phox exhibited a low affinity towards MP.

The effects of vasoactive intestinal peptide (VIP) antagonists on breast cancer cells were investigated. (N-stearyl, norleucine¹⁷)VIP hybrid ((SN)VIPhyb) inhibited specific ¹²⁵I-VIP binding to MCF7, SKBR3, T47D ZR75-1 and MDA-MB231 cells with high affinity (IC₅₀ values of 0.03-0.06 μM). (SN)VIPhyb,1 μM, inhibited the ability of 10 nM VIP to cause elevation of cAMP and to increase c-fos mRNA. Micromolar concentrations of (SN)VIPhyb inhibited the proliferation of MDA-MB231 or MCF7 cells using a MTT and clonogenic assay. Using a MTT assay, (SN)VIPhyb enhanced the ability of taxol and doxorubicin to inhibit breast cancer growth. Using nude mice bearing MDA-MB231 xenografts, VIPhyb potentiated the ability of taxol to inhibit proliferation. The results indicate that VIP receptor antagonists increase the ability of chemotherapeutic drugs to kill breast cancer cells.

Background: α-Bungarotoxin (α-BTX) is a highly toxic snake venom α-neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. We describe the design and synthesis of peptides that bind α-BTX with high affinity, and inhibit its interaction with AChR with an IC₅₀ of 2 nM. The design of these peptides was based on a lead peptide with an IC₅₀ of 3 × 10^-7 M, previously identified by us [M. Balass et al., Proc. Natl. Acad. Sci. USA 94 (1997) 6054] using a phage-display peptide library. Results: Employing nuclear magnetic resonance-derived structural information [T. Scherf et al., Proc. Natl. Acad. Sci. USA 94 (1997) 6059] of the complex of α-BTX with the lead peptide, as well as structure-function analysis of the ligand-binding site of AChR, a systematic residue replacement of the lead peptide, one position at a time, yielded 45 different 13-mer peptides. Of these, two peptides exhibited a one order of magnitude increase in inhibitory potency in comparison to the lead peptide. The design of additional peptides, with two or three replacements, resulted in peptides that exhibited a further increase in inhibitory potency (IC₅₀ values of 2 nM), that is more than two orders of magnitude better than that of the original lead peptide, and better than that of any known peptide derived from AChR sequence. The high affinity peptides had a protective effect on mice against α-BTX lethality. Conclusions: Synthetic peptides with high affinity to α-BTX may be used as potential lead compounds for developing effective antidotes against α-BTX poisoning. Moreover, the procedure employed in this study may serve as a general approach for the design and synthesis of peptides that interact with high affinity with any desired biological target.

The Gram-negative bacterial endotoxin lipopolysaccharide (LPS) is a major inducer of sepsis. The natural cyclic peptide polymyxin B (PMB) is a potent antimicrobial agent, albeit highly toxic, by virtue of its capacity to neutralize the devastating effects of LPS. However, the exact mode of association between PMB and LPS is not clear. In this study, we have synthesized polymyxin B nonapeptide, the LPS-binding cyclic domain of PMB, and its enantiomeric analogue and studied several parameters related to their interaction with LPS and their capacity to sensitize Gram-negative bacteria toward hydrophobic antibiotics. The results suggest that whereas the binding of the two enantiomeric peptides to E. coli and to E. coli LPS is rather similar, functional association with the bacterial cell is stereospecific. Thus, the L-enantiomer is capable of synergism with the hydrophobic antimicrobial drugs novobiocin and erythromycin, whereas the D-enantiomer is devoid of such activity. The potential of understanding and consequently utilizing the PMB-LPS association for novel, nontoxic PMB-derived drugs is discussed.

Polymyxin B nonapeptide (PMBN), a cationic cyclic peptide derived by enzymatic processing from the naturally occurring peptide polymyxin B, is able to increase the permeability of the outer membrane of Gram-negative bacteria toward hydrophobic antibiotics probably by binding to the bacterial lipopolysaccharide (LPS). We have synthesized 11 cyclic analogues of PMBN and evaluated their activities compared to that of PMBN. The synthetic peptides were much less potent than PMBN in their capacity to sensitize Escherichia coli and Klebsiella pneumoniae toward novobiocin and to displace dansyl-PMBN from Escherichia coli LPS. Moreover, unlike PMBN, none of the analogues were able to inhibit the growth of Pseudomonas aeruginosa. The structural - functional features of PMBN were characterized and identified with regard to the ring size, the distance between positive charges and peptide backbone, the chirality of the DPhe-Leu domain, and the nature of the charged groups. Apparently, the structure of PMBN is highly specific for efficient perturbation of the outer membrane of Gram-negative bacteria as well as for LPS binding. The present study further increases our understanding of the complex PMBN-LPS and may, potentially, enable the design of compounds having enhanced permeabilization potency of the Gram-negative outer membrane.

We have previously determined that Ac-D-Trp-Leu-Asp-Ile-Ile-Trp (peptide I), an endothelin antagonist, binds specifically (K(i) = 1.9 μM) to the rat pituitary gonadotropin-releasing hormone (GnRH) receptor. Moreover, peptide I exhibits a GnRH agonistic activity, mediated directly by the GnRH receptor. We now report structure-activity studies of peptide I in respect to its interactions with the GnRH receptor. Our studies suggest that the bioactive conformation of peptide I, recognized by the GnRH receptor, is of a cyclic nature. Thus cyclic analogues of peptide I exhibit higher affinity to the GnRH receptor and increased agonistic potencies as compared to peptide I itself. A linear peptide, Ile-Ile-Trp-D-Trp-Leu-Asp, which presumably forms a similar cyclic conformation, was also shown to be a GnRH agonist. Intraperitoneal administration of Ac-Ile-Ile-Trp-D-Trp-Leu-Cys-OH (K(i) = 0.32 μM), one of the cyclic hexapeptides that we have synthesized, to rats induces secretion of luteinizing hormone (LH) with a potency which is only 1 order of magnitude less than that of GnRH itself. Moreover, plasma levels of LH remained elevated for a longer period of time following the administration of the cyclic hexapeptide. This novel class of GnRH agonists may prove useful in the development of new therapeutics.

Vasoactive intestinal peptide (VIP) is a recognized growth factor affecting many cell types. We have previously developed a series of lipophilic VIP analogues containing an N-terminal covalently attached stearyl moiety. The current studies identified stearyl-Nle¹⁷-VIP and stearyl-Nle¹⁷-neurotensin_6-11VIP_7-28, acting at μM concentrations, as cytotoxic to human keratinocytes. The core C-terminal active VIP-derived peptide, stearyl-Lys-Lys-Tyr-Leu-NH₂ (St-KKYL-NH₂), was identified as being responsible for the observed cytotoxicity. Cytotoxicity coincided with marked reduction in intracellular cyclic GMP and was abolished by co-treatment with the endonuclease inhibitor, aurine-tricarboxylic acid, suggesting apoptotic mechanisms. Stearyl-VIP derivatives thus offer lead compounds for future drug development against hyperproliferative skin conditions. Copyright (C) 2000 Federation of European Biochemical Societies.

Most mammalian cells contain vanadium at a concentration of about 20 nM, the bulk of which is probably in the reduced vanadyl (+4) form. Although this trace element is essential and should be present in the diet in minute quantities, no known physiological role for vanadium has been found thus far. In the late 1970s the vanadate ion was shown to act as an efficient inhibitor of Na⁺, K⁺-ATPase as well as of other related phosphohydrolases. In 1980 vanadium was reported to mimic the metabolic effects of insulin in rat adipocytes. During the last decade, vanadium has been found to act in an insulin-like manner in all three main target tissues of the hormone, namely skeletal muscles, adipose, and liver. Subsequent studies revealed that the action of vanadium salts is mediated through insulin-receptor independent alternative pathway(s). The investigation of the antidiabetic potency of vanadium soon ensued. Vanadium therapy was shown to normalize blood glucose levels in STZ-rats and to cure many hyperglycemia-related deficiencies. Therapeutic effects of vanadium were then demonstrated in type II diabetic rodents, which do not respond to exogenously administered insulin. Finally, clinical studies indicated encouraging beneficial effects. A major obstacle, however, is overcoming vanadium toxicity. Recently, several organically chelated vanadium compounds were found more potent and less toxic than vanadium salts in vivo. Such a newly discovered organic chelator of vanadium is described in this review. (C) 2000 Elsevier Science Inc.

A molecular homology has been demonstrated between sequences of the heavy chain variable regions of the anti-DNA, anti-cardiolipin monoclonal antibody, 2C4C2, isolated from C3H.SW mice with induced systemic lupus erythematosus, and sequences of the anti-DNA monoclonal antibody BW16 originating in the lupus-prone (NZBXNZW)F1 mice. It was of interest to determine whether these homologous sequences function also as immunodominant T-cell epitopes, in order to establish a connection between spontaneous and induced experimental models. Therefore, three peptides were designed and synthesized based on the complementarity determining region (CDR)1, CDR2 and CDR3 of the heavy chain of the monoclonal antibody 2C4C2. In the present study, we compare these peptides with the CDR1- and CDR3-based peptides of another murine anti-DNA antibody; namely, 5G12. The comparison was carried out by analyzing the ability of the peptides to induce T-cell activation in (NZBXNZW)F1 lupus-prone mice and in mouse strains susceptible to induction of experimental systemic lupus erythematosus. Immunization of (NZBXNZW)F1 mice with the 2C4C2 mAb or with its CDR-based peptides, as well as immunization with the 5G12-based CDR peptides, induced significant lymph node proliferation to the pCDR3 of the 5G12 mAb. Naive (NZBXNZW)F1 splenocytes exhibited activation to the same peptide. It is also shown that MHC class II molecules of (NZBXNZW)F1 macrophages bind preferentially the 5G12-based pCDR3. It is proposed that the CDR3-based peptide of 5G12 mAb of experimental lupus is also a dominant and relevant epitope in the (NZBXNZW)F1 lupus-prone mice. Copyright (C) 2000 Elsevier Science B.V.

Vanadate mimics the metabolic actions of insulin. In diabetic rodents, vanadate also sensitizes peripheral tissues to insulin. We have analyzed whether this latter effect is brought about by a mechanism other than the known insulinomimetic actions of vanadium in vitro. We report that the levels of glucose 6-phosphate (G-6-P) in adipose, liver, and muscle of strepto-zotocin-treated (STZ)-hyperglycemic rats are 77, 50, and 58% of those in healthy control rats, respectively. Normoglycemia was induced by vanadium or insulin therapy or by phlorizin. Vanadate fully restored G-6-P in all three insulin-responsive peripheral tissues. Insulin did not restore G-6-P in muscle, and phlorizin was ineffective in adipose and muscle. Incubation of diabetic adipose explants with glucose and vanadate in vitro increased lipogenic capacity three- to fourfold (half-maximally effective dose = 11 ± 1 μM vanadate). Lipogenic capacity was elevated when a threshold level of ~7.5 ± 0.3 nmol G-6-P/g tissue was reached. In summary, 1) chronic hyperglycemia largely reduces intracellular G-6-P in all three insulin-responsive tissues; 2) vanadate therapy restores this deficiency, but insulin therapy does not restore G-6-P in muscle tissue; 3) induction of normoglycemia per se (i.e., by phlorizin) restores G-6-P in liver only; and 4) glucose and vanadate together elevate G-6-P in adipose explants in vitro and significantly restore lipogenic capacity above the threshold of G-6-P level. We propose that hyperglycemia-associated decrease in peripheral G-6-P is a major factor responsible for peripheral resistance to insulin. The mechanism by which vanadate increases peripheral tissue capacity to metabolize glucose and to respond to the hormone involves elevation of this hexose phosphate metabolite and the cellular consequences of this elevated level of G-6-P.

Several ligands, when complexed with vanadium, potentiate its insulinomimetic activity both in vivo and in vitro. We have recently found that L-Glu-γ-monohydroxamate (HXM) and L-Asp(β)HXM were especially potent in this regard. In the present study, we used vanadium-enriched adipose cells and cell-free experimental systems to determine the features of L-Glu(γ)HXM and L-Asp(β)HXM that turn these ligands into optimal-synergizing vanadium chelators. We found that L-Glu(γ)HXM and L-Asp(β)(HXM) possess teristics: 1) They associate with vanadium(+5) at pH 7.2 within a narrow range of an apparent formation constant of 1.3 to 1.9 x 10²M^-1; 2) they have nearly the same binding affinity for the vanadyl(+4) cation and the vanadate(+5) anion at physiological pH values; and 3) they form intense ultraviolet absorbing complexes upon associating with vanadium(+4) at 1 and 3 M stoichiometry, respectively, at pH 3.0. Vanadium ligands lacking any of these three defined criteria synergize less effectively with vanadium to activate glucose metabolism.

In the course of our studies toward the development of novel analogs of the decapeptide gonadotropin releasing hormone (GnRH), we have examined a hexapeptide that is an antagonist of endothelin (ET). It was found that this peptide, AC-D-Trp-Leu-Asp-Ile-Ile-Trp (peptide 1), binds specifically to the pituitary GnRH receptor. Moreover, peptide 1 exhibits a GnRH agonistic activity (i.e., it induces luteinizing hormone release from rat pituitary). This activity is mediated directly by the GnRH receptor and is suppressed by a GnRH antagonist. Removal of the acetyl group of peptide I results in a hexapeptide (peptide 2) with binding properties similar to those of GnRH but with a diminished affinity toward the ET receptor. Several other ET antagonists were screened for a potential interaction with the GnRH receptor. Two of these, the hexapeptide PD145065 and the cyclic pentapeptide BQ-123, expressed GnRH agonistic activity at micromolar concentrations in vitro. BQ- 123, previously approved for trials on humans as an ET antagonist, is demonstrated to act in vivo as a GnRH agonist, in a dose that was demonstrated previously as the minimal required dose for significant ET antagonism. The GnRH agonistic activity of ET antagonists may therefore result in interference with the physiological control of the reproductive system. Such effects may be most severe when ET antagonists are used chronically. Thus, the major practical message of this study is the need to circumvent the potential side effects of ET antagonist-based drugs.

Oxidative stress is a common associative mechanism that is part of the pathogenesis of many neurodegenerative diseases. Vasoactive intestinal peptide (VIP) is a principal neuropeptide associated with normal development and aging. We have previously reported that VIP induced the secretion of proteins from glial cells, including the novel survival-promoter: activity-dependent neurotrophic factor (ADNF). ADNF-9, a nine amino acid peptide derived from ADNF, protects neurons from death caused by various toxins. In the present study, we examined the neuroprotective effect of VIP against oxidative stress in a pheochromocytoma cell line (PC12). In addition, a lipophilic derivative of VIP, Stearyl-Nle¹⁷-VIP (SNV), and two femtomolar-acting peptides: ADNF-9 and a 70% homologous peptide to ADNF-9, NAP were tested as well. PC12 cells were treated with 100 μM H₂O₂ for 24 h resulting in a reduction in cell survival to 35-50% as compared to controls. Addition of VIP or SNV prior and during the exposure to 100 μM H₂O₂ increased cell survival to 80-90% of control values. Culture treatment with ADNF-9 or NAP in the presence of 100 μM H₂O₂ increased cell survival to 75-80% of control values. Messenger RNA expression analysis revealed that incubation with VIP resulted in a twofold increase in VIP mRNA, whereas NAP treatment did not cause any change in VIP expression, implicating different mechanisms of action. Furthermore, addition of an ADNF-9 antibody prevented the ability of VIP to protect against oxidative stress, suggesting that VIP protection is partially mediated via an ADNF-like protein.

Keywords: LIPOPOLYSACCHARIDE; ANTIBIOTICS

The effects vasoactive intestinal peptide (VIP) antagonists were investigated on pancreatic cancer cell lines. (N-Stearyl, Norleucine¹⁷) VIP hybrid ((SN)VIPhyb) inhibited ¹²⁵I-VIP binding to human Capan-2 cells with an IC₅₀ value of 0.01 μM whereas VIPhybrid had an IC₅₀ value of 0.2 μM. By RT-PCR and Northern blot, VPAC₁ receptor mRNA was detected in CAPAN-2 cells. One μM (SN)VIPhyb and 10 μM VIPhyb inhibited the ability of 30 nM VIP to elevate cyclic AMP and increase c-fos mRNA. (SN)VIPhyb, 1 μM inhibited the clonal growth of CAPAN-2 cells in vitro. In vivo, (SN)VIPhyb (10 μg/day s.c.) inhibited CAPAN-2 xenograft growth in nude mice. These results indicate that (SN)VIPhyb is a pancreatic cancer VPAC receptor antagonist.

Previous reports indicate that VIP and the structurally related peptide PACAP, inhibit IL-2 and IL-10 production in antigen-stimulated T lymphocytes. Intracellular cAMP elevation appears to be the primary transduction pathway involved. However, in the lower concentration range, an additional, cAMP-independent transduction pathway appears to mediate the VIP inhibition of cytokine production. Here, we address this question by using VIP agonists and antagonists which act through cAMP-dependent and -independent pathways. The antagonists based on the neurotensin-VIP hybrid molecule did not affect the inhibitory effect of VIP/PACAP on IL-2 and IL-10 production, confirming that astrocytes and T lymphocytes express different receptors. A lipophilic antagonist with increased membrane permeability, partially reversed the inhibitory effect of VIP/PACAP, forskolin, prostaglandin E2, and 8-bromo-cAMP without significantly affecting cAMP levels, suggesting that it acts downstream of cAMP. Two VIP inhibit agonists IL-2 and IL-10 production. One of the agonists increases cAMP, whereas the second one does not induce cAMP/cGMP. Our results indicate that VIP inhibits cytokine production in stimulated CD4+ T cells through two separate mechanisms, which involve both cAMP-dependent and cAMP-independent transduction pathways. Copyright (C) 1999 Elsevier Science B.V.

In vitro autoradiography with [¹²⁵I]vasoactive intestinal peptide revealed that the vasoactive intestinal peptide analogue, stearyl-norleucine¹⁷ vasoactive intestinal peptide, reported to be inactive at adenylyl cyclase-linked receptors in astrocytes, displaced a subset of vasoactive intestinal peptide binding sites on rat brain sections. These sites were widespread in adult rat brains and enriched in the olfactory bulb and thalamus, and corresponded to previously demonstrated GTP-insensitive vasoactive intestinal peptide binding sites. Stearyl-norleucine¹⁷ vasoactive intestinal peptide also identified receptors in rat lung and liver. In the adult brain, the stearyl-norleucine analog displaced only GTP-insensitive vasoactive intestinal peptide binding sites. In contrast, stearyl-norleucine¹⁷ vasoactive intestinal peptide-displaceable sites in the embryonic day 9 mouse appeared to include both GTP-sensitive and GTP-insensitive binding sites. This observation suggested the presence of an embryonic vasoactive intestinal peptide receptor with distinct pharmacological properties. Treatment of whole cultured mouse embryos with stearyl-norleucine¹⁷ vasoactive intestinal peptide resulted in stimulation of embryonic growth, with the stearyl-norleucine analog equipotent to vasoactive intestinal peptide, but less efficacious at higher concentrations (10^-7M). Embryonic growth was inhibited by pituitary adenylyl cyclase-activating peptide and 8-bromoadenosine 3',5'-cyclic monophosphate. In addition, 8-bromoadenosine 3',5'-cyclic monophosphate inhibited stearyl-norleucine¹⁷ vasoactive intestinal peptide-stimulated growth.The results of the current study support the hypothesis that vasoactive intestinal peptide regulation of early postimplantation embryonic growth occurs, at least in part, independently of adenylyl cyclase stimulation. Copyright (C) 1999 IBRO.

Antiphospholipid syndrome (APS) is characterized by recurrent fetal loss, repeated thromboembolic phenomena, and thrombocytopenia. The syndrome is believed to be caused by antiphospholipid beta-2-glycoprotein-I (β2GPI)- dependent Abs or anti-β2GPI Abs by themselves. Using a hexapeptide phage display library, we identified three hexapeptides that react specifically with the anti-β2GPI mAbs ILA-1, ILA-3, and H-3, which cause endothelial cell activation and induce experimental APS. To enhance the binding of the peptides to the corresponding mAbs, the peptides were lengthened to correspond with the site of the β2GPI epitope being recognized by these mAbs. As a result, the following three peptides were prepared: A, NTLKTPRVGGC, which binds to ILA-1 mAb; B, KDKATFGCHDGC, which binds to ILA-3 mAb; and C, CATLRVYKGG, which binds to H-3 mAb. Peptides A, B, and C specifically inhibit both in vitro and in vivo the biological functions of the corresponding anti-β2GPI mAbs. Exposure of endothelial cells to anti- β2GPI mAbs and their corresponding peptides led to the inhibition of endothelial cell activation, as shown by decreased expression of adhesion molecules (E-selectin, ICAM-1, VCAM-1) and monocyte adhesion. In vivo infusion of each of the anti-β2GPI mAbs into BALB/c mice, followed by administration of the corresponding specific peptides, prevented the peptide- treated mice from developing experimental APS. The use of synthetic peptides that focus on neutralization of pathogenic anti-β2GPI Abs represents a possible new therapeutic approach to APS.

The understanding of the molecular mechanisms leading to peptide action entails the identification of a core active site. The major 28-aa neuropeptide, vasoactive intestinal peptide (VIP), provides neuroprotection. A lipophilic derivative with a stearyl moiety at the N-terminal and norleucine residue replacing the Met-17 was 100-fold more potent than VIP in promoting neuronal survival, acting at femtomolar-picomolar concentration. To identify the active site in VIP, over 50 related fragments containing an N- terminal stearic acid attachment and an amidated C terminus were designed, synthesized, and tested for neuroprotective properties. Stearyl-Lys-Lys-Tyr- Leu-NH₂ (derived from the C terminus of VIP and the related peptide, pituitary adenylate cyclase activating peptide) captured the neurotrophic effects offered by the entire 28-aa parent lipophilic derivative and protected against β-amyloid toxicity in vitro. Furthermore, the 4-aa lipophilic peptide recognized VIP-binding sites and enhanced choline acetyltransferase activity as well as cognitive functions in Alzheimer's disease-related in vivo models. Biodistribution studies following intranasal administration of radiolabeled peptide demonstrated intact peptide in the brain 30 min after administration. Thus, lipophilic peptide fragments offer bioavailability and stability, providing lead compounds for drug design against neurodegenerative diseases.

Keywords: HUMAN-LEUKOCYTE ELASTASE; CATHEPSIN-G; EXTRACELLULAR-MATRIX; PHYSIOLOGICAL-ROLE; HUMAN-NEUTROPHILS; PEPTIDES; ADHESION; GLYCOPROTEINS; INHIBITION; SEQUENCE

Recently, multiple receptors for vasoactive intestinal peptide (VIP) have been molecularly cloned and our understanding of VIP chemistry and mechanisms of action has been broadened. The following review outlines the physiological effects of the hormone from growth regulation, reproduction, bronchodilation, vasodilation and immune interactions to neurotrophism. VIP- based drug design and non-invasive innovative delivery modes are discussed with emphasis on tumor diagnosis and treatment, impotence treatment and neuroprotection.

Serum amyloid A (SAA), an acute-phase protein, exists normally in the serum while complexed with high-density lipoprotein 3 (SAA-HDL3). Its levels increase markedly during inflammatory diseases. The pentapeptide Tyr-Ile-Gly-Ser-Asp (YIGSR-like) and the tripeptide Arg-Gly-Asn (RGD-like), related to the cell adhesion domains of laminin and fibronectin, respectively, exist in SAA within close proximity (YIGSDKYFHARGNY; amino acid residues 29-42). A structure-function study of linear and head-to-tail cyclic peptides, related to the amino acid residues 29-42 and 70-76 (GRGAEDS) of human SAA, was performed in order to evaluate their ability to inhibit adhesion of human T-lymphocytes to surfaces coated with extracellular matrix purified from bovine corneal cells.

Tumor necrosis factor alpha (TNF alpha) an early cytokine produced by activated macrophages, plays an essential role in normal and pathological inflammatory reactions. The excessive production of TNF alpha is prevented by the so-called "macrophage-deactivating factors." This study examines the role of two structurally related neuropeptides, the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating peptide (PACAP), as inhibitors of TNF alpha. Both VIP and PACAP inhibit TNF alpha production from lipopolysaccharide-stimulated RAW 246.7 cells in a dose- and time-dependent manner. Although the activated cells express mRNA for all three VIP/PACAP receptors, agonist and antagonist studies indicate that the major receptor involved is VIP1R. VIP/PACAP inhibit TNF alpha gene expression by affecting both NF-kB binding and the composition of the cAMP responsive element binding complex (CREB/c-Jun). Two transduction pathways, a cAMP-dependent and a cAMP-independent pathway, are involved in the inhibition of TNF alpha gene expression and appear to differentially regulate the transcriptional factors involved. Because TNF alpha plays a central role in various inflammatory diseases such as endotoxic shock, multiple sclerosis, cerebral malaria, and various autoimmune conditions, the down-regulatory effect of VIP/PACAP may have a significant therapeutic potential.

Cytotoxic T-lymphocytes (CTLs) kill abnormal cells. CTLs recognize major histocompatibility complex class I molecules in complex with peptides derived from relevant antigens. The identification of tumor associated antigen peptides enabled the design of anti-tumor and anti-metastatic vaccines in a murine lung carcinoma.

Peptide p259-271 of the human acetylcholine receptor α-subunit, preferentially stimulates T cells of patients with myasthenia gravis (MG) and is an immunodominant epitope for T cells of BALB/c mice. A p259-271 specific T cell line of BALB/c origin was established and was shown to induce experimental MG in naive mice. Seven analogs of p259-271 were synthesized, and two of them were found to inhibit the p259-271 specific proliferative responses of the line and of p259-271 primed lymph node cells. Moreover, the most efficient inhibitor, analog 262Lys, prevented the MG related manifestations in mice inoculated with the line, and might be of potential value for the treatment of MG.

At the end of neuronal migration, the neopallial germinative zone produces glial cells destined to colonize the upper layers of neocortex. High densities of binding sites for vasoactive intestinal peptide (VIP) have been found in the rodent germinative zone just after completion of neuronal migration, suggesting a possible role of VIP in neocortical astrocytogenesis. In the present study, administration of a VIP antagonist at embryonic days 17 and 18 to pregnant mice was followed by a dramatic depletion of astrocytes in the upper cortical layer of the offspring. The depletion of astrocytes was dose-dependent, with a 42% reduction in the density of astrocytes observed with 50 μg of antagonist. The antagonist effect was reversed by cotreatment with VIP or pituitary adenylate cyclase-activating polypeptide (PACAP), suggesting the involvement of a receptor common to these two neuropeptides. VIP antagonist-induced inhibition of astrocytogenesis was also blocked by Ro 25-1553, a long-acting cyclic VIP analogue selective for the PACAP II VIP2 receptor subclass. Our results demonstrate that VIP and/or PACAP play a crucial physiological role in neocortical astrocytogenesis, possibly through interaction with PACAP II VIP2 receptors.

Activity-dependent neurotrophic factor (ADNF) is a glia-derived protein that is neuroprotective at femtomolar concentrations. A 14-amino acid peptide of ADNF (ADNF-14) has been reported that protects cultured neurons from multiple neurotoxins. Structure-activity relationships of peptides related to ADNF-14 now have been determined. A 9-amino acid core peptide (ADNF-9) has been identified that has greater potency and a broader effective concentration range (10^-16 to 10^-13 M) than ADNF or ADNF-14 in preventing cell death associated with tetrodotoxin treatment of cerebral cortical cultures. Deletions or conservative amino acid substitutions to ADNF-9 resulted in reduced potency, narrower effective concentration range and/or decreased efficacy. Removal of the N-terminal serine or the COOH- terminal isoleucine-proline-alanine from ADNF-9 produced a significant reduction in survival-promoting activity. Comparative studies of ADNF-9 action in mixed (glia plus neurons) vs. glia-depleted neuronal cultures indicated that ADNF-9 can act directly on neurons, although the potency of the peptide was 10,000-fold greater in mixed cultures. Kinetic studies showed that exposure to ADNF-9 for only 2 hr was sufficient to produce a 4-day protection against the cell-killing action of tetrodotoxin. Treatment with bafilomycin A1 (an inhibitor of receptor-mediated endocytosis) for 2 hr prevented the ADNF-and ADNF-9-mediated neuroprotection. ADNF-9, like ADNF- 14, was neuroprotective against N-methyl-D-aspartate and the β-amyloid peptide (amino acids 25-35), and had a much broader range of effective concentrations than ADNF-14. These studies identify ADNF-9 as an attractive lead compound for the development of therapeutic agents against neurodegenerative diseases.

In an attempt to produce efficient cytotoxic derivatives of luteinizing hormone-releasing hormone (LH-RH), two novel 1,4-naphthoquinone derivatives of [D-Lys⁶]-LH-RH were synthesized primarily by solid-phase peptide synthesis, in good yield and high purity. The ability of each analog to produce reactive oxygen species using enzymatic reduction, i.e. NADPH-cytochromxe P-450 reductase, was evaluated employing electron spin resonance (ESR) spectroscopy and spin-trapping techniques. The ESR results suggest that the novel cytotoxic analogs are extremely effective in generating oxygen radicals.

CTLs recognize antigenic peptides bound to MHC class I Ags on the cell surface of tumor cells. Tumor-associated Ag (TAA) peptides are 8 to 10 amino acids long and can be derived from normal, mutated, or viral proteins. The majority of T cell-defined Ags have been identified in human melanoma tells. These were shown to be commonly expressed by different allogeneic melanomas that share the same MHC molecule. We have recently isolated Kb-restricted TAA peptides, which are mutations of the gap junction protein connexin 37, from the spontaneous C57BL/6 Lewis lung carcinoma (3LL). These peptides, named MUT 1 and MUT 2, serve as CTL epitopes and can induce CTL activity in vivo. Using CTL cross-reaction assays, peptide extraction, HPLC fractionation, and reverse transcriptase-PCR amplification, we show that clones of another spontaneous C57BL/6 lung carcinoma, CMT 64, share TAA peptides with the 3LL carcinoma. Vaccination with synthetic MUT 1 or MUT 2 induces CTLs that efficiently lyse CMT 64-derived clones, protects mice from CMT 64 metastasis, and affords therapy of established CMT 64 metastases. Hence, shared CTL epitopes exist between two spontaneous murine lung carcinomas.

The effects of VIP receptor antagonists were investigated using non-small cell lung cancer (NSCLC) cells. By Northern blot and RT-PCR, VIP, receptors were detected on NSCLC cell line NCI-H1299. VIPhybrid,(N-Stearyl-Norleucine(17)) VIPhybrid ((SN)VIPhybrid) and PTC4495 inhibited I-125-VIP binding to NCI-H1299 cells with IC50 values of 500, 30 and 5000 nM respectively. (SN)VIPhybrid (1 mu M) had no effect on basal cAMP but strongly inhibited the increase in cAMP caused by 10 nM VIP. The order of peptide potency to inhibit cAMP was (SN)VIPhybrid > VIPhybrid > PTC4495. (SN)VIPhybrid was more potent than VIPhybrid at inhibiting NCI-H1299 colony formation. Also, (SN)VIPhybrid was more potent than VIPhybrid at inhibiting NCI-H1299 xenograft formation in nude mice. These data suggest that (SN)VIPhybrid antagonizes VIP, receptors on NSCLC cells.

The effects of pituitary adenylate cyclase activating polypeptide (PACAP) hybrid, a synthetic antagonist, was investigated on NIH/3T3 cells containing PACAP receptor (R) splice variants (SVs). PACAPhybrid inhibited ¹²⁵I-PACAP-27 binding to NIH/3T3 cells stably expressing PACAP-R basic, SV-1, SV-2 or SV-3 with an IC₅₀ of 1000 nM. PACAPhybrid antagonized the ability of PACAP-27 to elevate cAMP regardless of the PACAP-R SV used. PACAP was more efficacious at increasing cytosolic Ca²⁺ in NIH/3T3 cells containing PACAP-R SV-2 than PACAP-R basic, SV-1 or SV-3. PACAPhybrid antagonized the increase in cytosolic Ca²⁺ caused by PACAP-27 regardless of the PACAP-R SV used. PACAP was more potent at elevating c-fos mRNA using NIH/3T3 cells transfected with PACAP-R SV-2 than PACAP-R basic, SV-1 or SV-3. PACAPhybrid antagonized the increase in c-fos mRNA caused by PACAP-27. These data suggest that PACAPhybrid is a useful PACAP receptor antagonist for PACAP-R SVs.

Mast cells are known to accumulate in various inflammatory processes, some of which are known to be associated with increased local and systemic levels of acute-phase reactants such as serum amyloid A (SAA) or with amyloid deposition. The mechanism(s) by which mast cells are recruited to these sites, however, has not been fully elucidated. It has recently been shown that SAA interacts with extracellular matrix (ECM) components and thereby acts as a chemoattractant and regulator of immune cell migration. On the basis of these observations, we examined the effect of SAA on mast cell adhesion to ECM, an essential step in cellular transmigration. We could first demonstrate strong specific binding of recombinant human SAA (rSAA) to murine mast cells using flow cytometry. Moreover, radiolabeled rSAA was found to bind, in a saturable manner, to mast cells, reaching a binding affinity of 10^-8 M. When immobilized by preincubation with ECM, SAA or its proteolytically degraded amyloid A fragment (amino acid residues 2-82), which contains RGD-related adhesion motif but not the COOH-terminal portion of SAA (amino acid residues 77-104), induced the adhesion of resting mast cells to ECM or laminin. SAA and AA, in soluble or immobilized forms, did not activate mast cells to release mediators. Mast cell adhesion to the immobilized ECM- SAA complex appeared to occur through an integrin recognition, inasmuch as adhesion was calcium dependent and could be blocked by an RGD-containing peptide or by anti-CD29 monoclonal antibody. Genistein also inhibited adhesion, indicating that tyrosine kinase activity was involved. These data suggest that SAA bound to ECM may serve as an important inducer of mast cell adhesion, thus regulating mast cell recruitment and accumulation at these sites, which in turn could potentiate further pathology.

Extended peptides that derive from the primary sequence of the acute phase reactant C-reactive protein (CRP) are shown to inhibit in vitro the enzymatic activities of human leukocyte elastase (hLE) and human leukocyte cathepsin G (hCG), which are associated with the tissue damage that occurs during the course of several chronic inflammatory conditions. Major inhibitory activity was observed in the peptides CRP_70-98 and CRP_50-98 towards hLE (K_i = 4.0 μM) and hCG (K_i = 1.4 μM), respectively. In contrast to the inability of intact CRP pentamers to inhibit both enzymes, CRP subunits (monomers) inhibited hLE (3.0 μM) and hCG (3.6 μM) activity.

Four novel 2,4-methano amino acids (MAAs, 1-aminocyclobutane-1- carboxylic acids) were synthesized. These include the basic MAA analogs of lysine (16), ornithine (5), and arginine (6) and the neutral methanovaline (22), related to proline. The above MAAs, as well as the MAA analog of homothreonine (7), were incorporated into the peptide chain of the immunomodulatory peptide tuftsin, Thr-Lys-Pro-Arg, known to enhance several biological activities mediated by phagocytic cells. The synthetic methano tuftsin analogs were assayed for their ability to stimulate interleukin-6 (IL-6) secretion by mouse peritoneal macrophages and for their stability in human serum toward enzymatic degradation. It was found that, at 2 x 10^-7 M, [MThr¹]tuftsin (24) and an isomer of [MVal³]tuftsin (27a) were considerably more active than the parent peptide in augmentation of cytokine release. [MOrn²]Tuftsin (25) was equally potent. The analogs [MThr¹]tuftsin (24) and [MOrn²]tuftsin (25), both pertaining to the proteolytically sensitive Thr- Lys bond of tuftsin, exhibited high resistance to enzymatic hydrolysis as compared to tuftsin. Using specific rabbit anti-tuftsin antibodies in a competitive enzyme-linked immunosorbent assay (ELISA) revealed that none of the MAA analogs can cross-react with tuftsin. It may indicate that the peptides assume global structures different than that of tuftsin.

Breast cancer vasoactive intestinal peptide (VIP) receptors were characterized. Using in vitro autoradiographic techniques, ¹²⁵I-labeled VIP bound with high affinity to breast biopsy sections. ¹²⁵I-labeled VIP bound specifically to five breast cancer cell lines examined using receptor- binding techniques. Specific ¹²⁵I-labeled VIP binding to MDA-MB-231 cells was inhibited with high affinity by VIP and pituitary adenylate cyclase- activating polypeptide (IC₅₀ = 2 nM) and with moderate affinity by the VIP hybrid (IC₅₀ = 0.5 μM). VIP elevated the cAMP in a dose-dependent manner, and VIP hybrid (10 μM) inhibited the increase in cAMP caused by VIP. Using Northern blot analysis, VIP (10 nM) stimulated c-fos and c-myc mRNA, and the increase caused by VIP was reversed by the VIP hybrid. The VIP hybrid inhibited breast cancer growth in vitro and in vivo using nude mice bearing breast cancer xenografts. These data suggest that the VIP hybrid is a breast cancer VIP receptor antagonist.

Serum amyloid A (SAA), a prototypic acute phase protein reactant, exists naturally in the serum of healthy individuals. However, the levels of SAA in serum and its presence in sites of inflammation increase during certain chronic diseases associated with a local elevation of cytokine concentrations. Although the chemical structure of SAA is defined, its putative immunologic role(s) is still obscure. Nevertheless, it has been shown that 1) SAA acts as a chemoattractant and regulator of the migration of monocytes, polymorphonuclear cells, and T lymphocytes through endothelial cell monolayers; and 2) SAA and its proteolytically degraded N-terminal amyloid A fragment contain an extracellular matrix (ECM)-related cell adhesion epitopes. Herein, we examined whether SAA can associate with specific ECM moieties, and whether immobilized SAA-ECM complexes affect T lymphocyte adhesion. Radiolabeled human rSAA ([¹²⁵I]rSAA) interacted avidly (K(d) = 10^-9 M) and transiently with intact ECM, laminin, and vitronectin, but not with fibronectin or collagen type II. The binding of [¹²⁵I]rSAA to ECM and laminin was inhibited by unlabeled rSAA and by the AA fragment, but not by the C-terminal portion of SAA (amino acid residues 2- 82 and 77-104, respectively). Upon interactions with SAA or amyloid A, immobilized ECM, laminin, and vitronectin induced the adhesion of resting human CD4⁺ T cells in an apparently β₁-integrin-mediated manner. Thus, the ECM appears to serve as a temporary anchorage site for SAA and amyloid A, and these ECM-complexed molecules seem to be involved in regulating the recruitment and accumulation of immunocytes in extravascular inflammatory compartments.

Bis-[ortho-carboxyaryl]- and bis[ortho-amidoaryl]methylphosphine oxides, in acidic medium, spontaneously cyclodehydrate to give trigonal bipyramidal (TBP) spirodioxy- and azoxyphosphoranes, respectively. Regioselectivity with a few nucleophiles and electrophiles, unexpectedly, accompany some of their reactions. Their structural similarity to pentacoordinate intermediates, obtained on the hydrolysis of organophosphorus (OP) poisons, and their stability at the physiological pH, renders them, as transition state analogs (TSA), useful promoters of catalytic antibodies.

L-Histidine, N(α)-cbz-L-histidine, L-tryptophan and L-proline react with 1,4-naphthoquinone or 2,3-dichloro-1,4-naphthoquinone to afford modified N-quinonyl amino acids. With free α-amino acids, the quinone moiety is attached to the α-amino group. With blocked α-amino acids the quinone moiety is attached to the heterocyclic nitrogen atom.

Neurodegenerative diseases, in which neuronal cells disintegrate, bring about deteriorations in cognitive functions as is evidenced in millions of Alzheimer patients. A major neuropeptide, vasoactive intestinal peptide (VIP), has been shown to be neuroprotective and to play an important role in the acquisition of learning and memory. A potent lipophilic analogue to VIP now has been synthesized, [stearyl-norleucine¹⁷]VIP ([St-Nle¹⁷]VIP), that exhibited neuroprotection in model systems related to Alzheimer disease. The β-amyloid peptide is a major component of the cerebral amyloid plaque in Alzheimer disease and has been shown to be neurotoxic. We have found a 70% loss in the number of neurons in rat cerebral cortical cultures treated with the β-amyloid peptide (amino acids 25-35) in comparison to controls. This cell death was completely prevented by cotreatment with 0.1 pM [St- Nle¹⁷]VIP. Furthermore, characteristic deficiencies in Alzheimer disease result from death of cholinergic neurons. Rats treated with a cholinergic blocker (ethylcholine aziridium) have been used as a model for cholinergic deficits. St-Nle-VIP injected intracerebroventricularly or delivered intranasally prevented impairments in spatial learning and memory associated with cholinergic blockade. These studies suggest both an unusual therapeutic strategy for treatment of Alzheimer deficiencies and a means for noninvasive peptide administration to the brain.

The cure of micrometastases following surgery is the major goal of cancer immunotherapy. We have recently isolated tumour-associated antigen (TAA) peptides, MUT 1 and MUT 2, derived from a mutated connexin 37 gap-junction protein, from the malignant 3LL-D122 murine lung carcinoma. We now report that synthetic MUT 1 or MUT 2 induces effective antitumour cytoxic T lymphocytes. Peptide vaccines protect mice from spontaneous metastases of 3LL-D122 tumours. Moreover, peptide vaccines reduce metastatic loads in mice carrying pre-established micrometastases. Tumour-specific immunity was primarily mediated by CD8⁺ T cells. This is the first evidence that peptide therapy may be effective in treatment of residual tumours and provides a rationale for the development of peptide vaccines as a modality for cancer therapy.

Thermoplasma 20 S proteasomes are composed of only two different types of subunits (designated as α and β) but are nearly indistinguishable in their quaternary structure from eukaryotic 20 S proteasomes consisting of 14 distinct subunits. In this study, we compared both the nature and the rate of the proteolytic activities of Thermoplasma and of granulosa cell proteasomes on the neurohormone, gonadotropin-releasing hormone (GnRH), the degradation products of which can be unequivocally identified. Both Thermoplasma and granulosa proteasome degrade the decapeptide GnRH at the Trp³-Ser⁴, Ser⁴- Tyr⁵, Tyr⁵-Gly⁶, and Gly⁶-Leu⁷ bonds. While the main product of Thermoplasma proteasomes was a GnRH-(1-4) fragment, the main product of granulosa cell proteasome was a GnRH-(1-5) fragment, indicating that the principal degrading activity of Thermoplasma proteasome targets Ser⁴-Tyr⁵ bond, while the principal degrading activity of granulosa cell proteasome targets the Tyr⁵-Gly⁶ bond of GnRH. These differences in the degradation pattern of the neurohormone were observed when proteasome activities were compared both at 60 °C, the optimal temperature for Thermoplasma proteasomal activity, and at 37 °C, the optimal temperature of granulosa proteasome proteolytic activity. Although the catalytic mechanism is probably conserved from archaebacterial to eukaryotic proteasomes, our results suggest that there are striking differences in the preferred cleavage site of GnRH. This reflects the changes in the proteasomal subunit repertoire during evolution.

Healthy individuals manifest natural T cell reactivity to epitopes of the 60-kDa heat shock protein (hsp60) of both self and bacterial origin. The present studies were done to learn whether defined peptides of hsp60 could function as T cell carrier epitopes for a poorly immunogenic T-independent capsular polysaccharide, the Vi Ag of Salmonella typhi. Homologous peptides were synthesized from the mouse self-hsp60 molecule (CP1m), from the closely related human hsp60 molecule (CP1h), and from the more distant Escherichia coli (CP1ec) and mycobacterial (CP1mt) hsp60 molecules. The peptides were conjugated to Vi and tested for their immunogenicity in BALB/c (H-2(d)) and H-2 congenic mice (H-2(k) and H-2^b). We now report that the self-CP1m and cross-reactive CP1h peptides were as immunogenic as was the non-cross- reactive foreign CP1 ec peptide. Small amounts of the CP1 peptide, even in PBS, sufficed to induce anti-Vi Abs of the IgG1 (T-dependent) isotype in naive mice. The carrier effect was associated with the ability of the peptides to bind to APC and to induce T cell proliferation. H-2(d) and H- 2(k) mice, but not H-2^b mice responded to CP1m/h and CP1ec. None of the mice responded to CP1mt. No signs of inflammation or autoimmune disease were detected. Thus, natural T cell autoimmunity exists and can be harnessed to provide T cell help for Ab production to a foreign bacterial molecule in a synthetic vaccine.

Keywords: VASOACTIVE-INTESTINAL-PEPTIDE; ENVELOPE PROTEIN; DENDRITIC SPINES; SURVIVAL; RAT; CELLS; RETARDATION; ANTAGONIST; DYSGENESIS; MITOSIS

Synthetic peptides related to amino acid residues 2942 of human serum amyloid A (SAA), TyrIleGlySerAspLysTyrPheHisAlaArgGlyAsnTyr, were found to inhibit the adhesion of human Tlymphocytes and of mouse M4 melanoma cells to surfaces coated with the major cell adhesive glycoproteins of the extracellular matrix, laminin or fibronectin. Correspondingly inhibitory activity was manifested by the entire 14residue peptide, by its YIGSD lamininrelated domain, and by RGN, the fibronectinrelated domain. Intact recombinant SAA (rSAA) and its 176 fragment, an amyloid A (AA) protein, also inhibited cell adhesion. The peptides did not inhibit collagen and ADPinduced aggregation of human platelets. Proteolysis of SAA by lysosomal enzymes originating from human neutrophils led to generation of specific peptide segments some of which pertain to the 2942 domain. It is suggested that the acutephase protein SAA might be involved, either directly or via its peptide fragments, in inhibition of inflammatory reactions or metastatic processes which depend on integrin and possibly other extracellularmatrixspecific receptors mediated specific recognition and interactions with immobilized components of bloodvessel walls.

The in vitro antiviral activity of two amphiphilic synthetic peptides, modelin1 (mod1) and modelin5 (mod5), and of the natural antibacterial peptide magainin2 (mag2) against herpes simplex viruses type 1 (HSV1) and 2 (HSV2) were evaluated. The peptides were incubated with the virus, i.e. direct inactivation, and their effects examined by means of plaque reduction assay and/or reduction in virus yield. Only mod displayed a strong antiviral effect against HSV1 and HSV2, with 50% effective dose (ED₅₀) values of 4.6 and 4.1 μg/mL, respectively. Mag2, mod5 and a mixture of both had no significant inhibitory effect. Addition of mod1 up to a concentration of 100μg/mL to the culture medium had no significant cytotoxic effect on host vero cells, as measured by the trypan blueexclusion method. It showed, however, considerable hemolytic activity against human red blood cells. Experiments including acyclovir (ACV) as a reference viral inhibitor indicated that the mode of action of mod1 is different from that of ACV. In contrast to ACV, the peptide inactivates the virus following a very short incubation before vero cell infection, suggesting some kind of direct interaction of the peptide with the viral envelope, rather than inhibition of viral DNA replication or gene expression. Our results suggest that mod1 may be an effective topical antiviral agent against herpes viruses.

The high concentration of gonadotropin-releasing hormone (GnRH) in milk of several species implies that the mammary gland is either a site of synthesis for this neuropeptide or that it is efficiently concentrated from plasma by this organ. By PCR amplification of mammary gland cDNA, we have demonstrated expression of the mRNA for GnRH. The GnRH mRNA was present in the mammary gland of pregnant and lactating rats but not of virgin rats, implying that expression of the GnRH gene is activated during pregnancy, probably by prolactin. In contrast, actin mRNA was evident in all the preparations of mammary glands. Since GnRH is also known to be synthesized by the placenta, it is likely that the placenta and the mammary gland are complementary units by which the mother exercises control over the development and the metabolism of the infant during pregnancy as well as after parturition. In addition, GnRH synthesized by the mammary gland may also affect the mother by a paracrine and/or an endocrine mechanism.

Receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) have been subdivided into type I. receptors and II receptors. Using rat brain stem as a tissue containing type I. receptors and rat lung as a tissue containing type II receptors, we investigated the binding of ¹²⁵I-labelled PACAP, vasoactive intestinal peptide (VIP) and the related peptide, heloderrnin to these and, by chemical cross-linking, the relative molecular weight of the receptors. Type I. receptors showed an M_r of 58,000 and type II an M_r of 54,000. After deglycosylation type I. receptors showed an M_r of 49,000 and type II receptors an M_r of 41,000 demonstrating a high degree of glycosylation of both subtypes. This compares with the predicted (unglycosylated) M_rs of the recently cloned rat braintype I. receptor and rat lung type II receptor of 56,000 and49,000, respectively. We also studied a hybrid VIP receptor antagonist (VIP-neurotensin) and showed it was unable to displace ¹²⁵I-PACAP from type I. receptors (K_i>10, µM) but was effective at type II receptors and propose that this peptide could be used to differentiate between PACAP effects at the two receptor subtypes.

The most prevalent lung cancer, non-small cell lung cancer (NSCLC) has receptors for vasoactive intestinal peptide (VIP). Here the effects of a VIP antagonist (VIP-hyb) on NSCLC growth were investigated. In vivo, when VTPhyb (10 μg, s.c.) was daily injected into nude mice, xenograft formation was significantly inhibited by ≈80%. In vitro, VIP (100 nM) stimulated colony formation ≈2-fold, whereas 1 μM VIPhyb inhibited colony formation by ≈50% when adenocarcinoma cell line NCI-H838 was used. The attenuation of tumor proliferation is receptor mediated, as VIPhyb inhibited specific ¹²⁵I-labeled VIP binding to cell lines NCI-H157 and NCI-H838 with an IC₅₀ of 0.7 μM. VIP (10 nM) increased the cAMP levels 5-fold when cell line NCI-H838 was used, and 10 μM VIPhyb inhibited the increase in cAMP caused by VIP. Northern blot analysis and radioimmunoassays have shown VIP mRNA and VIP-like immunoreactivity in NSCLC cells. These data suggest that VIP may be a regulatory peptide in NSCLC and that VIPhyb is a VIP receptor antagonist that inhibits proliferation.

Keywords: Endocrinology & Metabolism

The synthesis of 11 peptides, ranging in composition from 9 to 17 amino acid residues, by solid-phase methodology was accomplished with the purpose of studying how the amphiphilic and hydrophobic character, the size of the molecule, and the charge distribution modulate the antibacterial activity. It was found that peptides composed of 16 and 17 amino acid residues, with high hydrophobic (mainly due to Trp or Phe) and hydrophilic (due to Lys) character distributed along opposite amphiphilic faces, showed considerable antibacterial activity against clinically isolated bacteria together with Gram positive and Gram negative ATCC bacterial strains. However, the hemolytic capacity of the peptides was also significant. Decreasing the hydrophobic character of the molecule by replacing Trp or Phe with Leu residues while maintaining the basic contribution of Lys drastically reduced the hemolytic activity and only slightly decreased the bioactivity. Peptides composed of 910 amino acid residues with high hydrophobic and basic nature possess antibacterial activity but, in general, are less active than the larger counterpart peptides. By replacing all Trp residues of a short peptide by Leu residues, the activity was considerably reduced. Circular dichroism studies and antibacterial assays showed that shorter peptides with very low helical content, and thus deprived of amphiphilic character, still have appreciable bioactivity. This observation, coupled with the fact that due to their small size they cannot span the bacterial outer lipid bilayer, may suggest different mechanisms of action for long-chain vis-a-vis short-chain peptides.

Human Creactive protein (CRP) is shown to mediate a release of enzymatic activity from neutrophils which promotes its own degradation in the extracellular medium. This egress of proteolytic activity, which was upregulated by phorbol 12myristate 13acetate (PMA), was found to occur from both the cytoskeleton and membrane fractions of neutrophils and was dependent on the time of incubation of CRP with the cells and the concentration of CRP. Neutrophil kinases activated by PM A are found to be involved in uprcgulating the activity of the CRPdegrading protease. The apparent molecular weight of the CRPdegrading protease associated with the conditioned medium from PMAstimulated neutrophils and neutrophil membrane and cytoskeleton preparations, was found by size exclusion chromatography to be 600 kD and migrated on 313% SDSPAGE as four discrete bands to positions corresponding to apparent molecular weights of 209 kD, 316 kD, 398 kD and 501 kD.

The external envelope glycoprotein (gp120) of the human immunodeficeincy virus (HIV) has been shown to be toxic to neurons in culture. To further investigate the neurological effects of gp120, the involvement of this protein with the acquisition of spatial discrimination was assessed. Both native and recombinant gp120 were administered into the cerebral ventricles of adult rats and performance was evaluated in the Morris swim maze. Gp120 treatment retarded acquisition after daily administration of 12 ng. The specificity of this impairment was demonstrated in that the performance of animals given the same amount of gp160 from recombinant baculovirus was not different from animals given saline. Vasoactive intestinal peptide (VIP) has been shown to block gp120-induced neurotoxicity in culture and a VIP receptor antagonist has displayed toxic properties to neurons in culture. We show here that this antagonist, which competitively inhibits VIP binding and blocks VIP-mediated functions in cell cultures from the CNS, also produced an impairment of performance. This retardation was attenuated by cotreatment with VIP, supporting the specificity of the observed impairment. Thus, gp120 and the VIP antagonist produced similar retardation of spatial discrimination, suggesting that both may impair memory for spatially related stimulus control.

Six Thr¹ (Oglyco)derivatives of the \u201cphagocytosis stimulating peptide\u201d tuftsin, HThrLysProArgOH and the Nglycosylated undecapeptide HThrLysProArgGluGlnGlnTyrAsn(βdGlcNAc)SerThrOH, which correspond to the \u201ctuftsinregion\u201d at the Fcdomain of immunoglobulin G (amino acid residues 289299), were evaluated in comparison with tuftsin and rigin, HGlyGlnProArgOH, for their capacity to evoke the release of interleukin1 and tumor necrosis factor from mouse peritoneal macrophages and from human monocytes. Several glycosylated tuftsin derivatives were found to modulate, in a rather dosedependent manner, the release of the two cytokines from both cell types.

A vasoactive intestinal peptide (VIP) antagonist was synthesized and used to investigate the interactions of VIP with its receptors present in the central nervous system (CNS). The VIP antagonist is a hybrid peptide consisting of a portion of VIP and a portion of neurotensin, designed to change the membrane permeability of the VIP portion. The hybrid antagonist displaced 80 to 90% of [¹²⁵I]VIP binding to cell cultures from cerebral cortex, hippo-campus or spinal cord. The displacement curve was biphasic, suggesting two binding sites. In the case of cortical astrocytes, the antagonist had a K(i) of 45 pM at one site and a K(i) of 74 nM at the other. At the lower affinity binding site, the antagonist was about 10-fold more potent than VIP in displacing radiolabeled VIP. The accumulation of cyclic AMP (cAMP) in VIP-stimulated cortical glia cultures was decreased by the new antagonist (EC₅₀, 59 nM). This decrease in cAMP was greater than that achieved in the presence of other putative VIP antagonists. Finally, the addition of 1 nM hybrid antagonist to dissociated spinal cord cultures resulted in a 42% reduction in neuronal cell counts as compared with controls, and the EC₅₀ of this effect was about 30 pM, which corresponded closely to the K(i) of antagonist displacement of [¹²⁵I]VIP binding at the high-affinity site. The antagonist appears to be a competitive blocker for both VIP-mediated increases in cAMP formation or VIP-associated maintenance of neuronal survival in spinal cord cultures. Thus, we describe a potent VIP antagonist which interacts with two functionally distinct VIP receptors in the CNS.

We have recently provided evidence that C-reactive protein (CRP) could act as an up-regulatable substrate for membrane-associated neutrophil serine protease(s). The resultant degradation of CRP yielded small soluble bioactive peptides that inhibit many of the proinflammatory functions of activated neutrophils and could oppose the tissue destructive potential of these cells. We report on the reverse phase HPLC separation of the small TCA-soluble peptides obtained when CRP is degraded with non-stimulated or PMA-stimulated neutrophils and purified neutrophil membranes. The amino acid sequence of seven peptides isolated from the CRP digest has been ascertained and synthetic peptides homologous to these sequences have been synthesized. Three of the synthetic peptides corresponding to residues 201-206 (CRP-III), 83-90 (CRP-IV), and 77-82 (CRP-V) of the intact protein were identified to significantly inhibit Superoxide production from activated neutrophils at 50 μM whereas CRP-III and CRP-V in addition inhibited neutrophil chemotaxis at this concentration. These peptides act additively and their action likely involves the signal transduction pathways for neutrophil activation.

Normal human monocytes and macrophages generate potent procoagulant activity (PCA) resembling tissue factor (TF) in response to various stimuli. In this study we show that tuftsin, a natural stimulator of many functions of monocytes and macrophages, also stimulates a potent PCA in mixed mononuclear cells and monocytes, and a mild PCA in lymphocytes and cell lines of monocytic origin (U937 and THP). No activity was generated by several lymphoid cell lines and HL-60 cells. The PCA resembled TF in that it accelerated clotting through the extrinsic coagulation pathway and was inhibited by concanavalin-A and by monoclonal anti-TF antibodies. The induction of TF-like activity by tuftsin was dose- and time-dependent. It was located in the cell membrane and did not require T cells for expression. Generation of TF-like activity was prevented by actinomycin D, while cytarabine had no effect on this process, suggesting that expression of the activity depends on protein synthesis. Studies with various tuftsin analogs suggest that tuftsin stimulates generation of TF-like activity, as well as other functions of monocytes via the same receptors. The results with the monocytic cell lines show that tuftsin affects mainly mature cells. The induction of TF-like activity in mononuclear cells by tuftsin constitutes an important link between mononuclear cells and the immune and coagulation systems. It may play a major role in the pathogenesis of thromboembolism and fibrin deposition in various inflammatory and immunologic disorders.

The hydroxylic sidechain functional groups of serine, threonine, hydroxproline and tyrosine, the α and εamino moieties of lysine and the thiol group of cysteine were masked by the 3nitro2pyridinesulfenyl (Npys) protecting group. Deprotection was mildly affected by thiolysis with either 2mercaptopyridine and 2mercaptomethyl imidazole (O and NNpys) or with 3mercaptoacetic acid and 2mercaptoethanol (SNpys). Thiolysis was monitored spectrophotometrically and was completed in a rather short time. Incorporation of the Npys group into a whole and single thiolyzable deprotection scheme is suggested.

Peritonitis caused by Candida albicans is a major complication of continuous ambulatory peritoneal dialysis (CAPD). Increasing the activity of the peritoneal macrophages- the predominant cell type found in the peritoneal cavity-may be of great importance in the prevention and therapy of peritonitis. Therefore, the activating effect of tuftsin was studied on human peritoneal macrophages from CAPD patients. Tuftsin induced a biphasic effect on macrophage activity within a range of 2×10^-9-2×10^-6M, with a maximal activity 2×10^-7M. At this concentration,tuftsin enhanced by twofold cell association with radiolabelled candida (from 2±0.2 to 4±0.2 candida per macrophage) and superoxide anion production in response to exposure to candida (from 150±20 to 300±20 nmoles/mg). These results suggest the potential use of tuftsin as a therapeutic drug.

The participation of gonadal steroid hormones in regulation of the vasoactive intestinal peptide (VIP) gene expression in the hypothalamus was studied using a quantitative densitometric hybridization assay. In the female rat the levels of VIP mRNA were found to be significantly decreased following ovariectomy (4.41 ± 0.7 arbitrary units of absorbance vs. 8.52 ± 0.18). This decrease was largely reversed after three days of treatment with estradiol dibenzoate. In contrast to the female rats, no significant change in VIP mRNA levels was observed in the male rats, following orchidectomy. These results suggest a sexual dimorphism with regard to the steroid regulation of hypothalamic VIP gene expression in the rat.

Treatment of human neutrophils with C-reactive protein (CRP) causes a concentration-dependent decrease in the extent of activation of superoxide production and of granule secretion, induced by phorbol-12-myristate-13-acetate (PMA) or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF). The same treatment also causes a significant reduction in the degree of PMA- and fMLF-stimulated phosphorylation of several cell proteins. These include the proteins of 43-47 kDa, whose extent of phosphorylation correlates with the activation of superoxide production and of secretion. Contrary to the effects exerted on protein phosphorylation, CRP does not affect the fMLF-elicited increase in neutrophil cytosolic Ca²⁺.

Synthesis of some modified rigins is described in which either Dgluconic acid or 2amino2deoxyβDglucopyranose have been linked to the parent molecule through amide bonds involving the αamino function, αcarboxyl function or the γamide function of glutamine in position 2. Glu²rigin and DgluconylGlu²rigin have also been synthesized. Binding and phagocytosis assays have been carried out on the rigin derivatives and on some glycosylated tuftsin derivatives as well. Of all the tested peptides only rigin enhanced the phagocytic capacity of mouse peritoneal macrophages to the same extent as tuftsin. The peptides HThrLysProArgNHGlc and NαgluconylGlyGluProArgOH slightly enhanced phagocytosis. HThr[(α + β)Oglucosyl]LysProArgOH was found to displace ³Htuftsin even better than tuftsin but lacked the ability to stimulate phagocytosis.

Three hexadecapeptides which correspond to the putative Ca²⁺ binding domains II and III of calmodulin were synthesized employing solid phase methodology. One of the peptides contained an internal cystine bridge which was formed while the corresponding linear peptide was still attached to the polymeric carrier. The interaction of the synthetic peptides with calcium ions was investigated using Tb³⁺mediated fluorescence. Binding was of the order Cal2 > Cal3 > Cal3C (Fig. 1) with binding constants K_Tb= 0.68 times 10⁵, 0.54 times 10⁵, and 0.21 times 10⁵ M¹ respectively. Biological activity of the compounds was assessed by measuring their stimulatory effect on erythrocyte membrane (Ca²⁺⁺ Mg²⁺)ATPase activity For 50% activity as compared with CaM, the concentration of peptides required was for Cal2, Cal3 and Cal3C, 50, 100 and 167 times higher than CaM, respectively. The results suggest that the three synthetic peptides possess certain calmodulinlike features.

We have synthesized a tetradecapeptide corresponding to residues 354367 of the deltasubunit of Torpedo acetylcholine receptor. This peptide contains the sequence ArgArgSerSer which has been proposed as the site for phosphorylation of the acetylcholine receptor (AChR) by an endogenous cAMPdependent protein kinase. We have shown that the synthetic peptide can be phosphorylated by the catalytic subunit of bovine heart cAMPdependent protein kinase. Antibodies elicited against peptide 354367 were shown to crossreact with native AChR and to bind specifically to the delta and gammasubunit as detected by immunoblotting. Furthermore, antipeptide antibodies were shown to inhibit specifically the cAMPdependent phosphorylation of both the delta and gammasubunits. This suggests that the phosphorylation sites in the delta and gammasubunits are highly crossreactive, and is in agreement with the demonstration that an endogenous cAMPdependent kinase phosphorylates these two subunits, probably on homologous sequences. Tryptic digestion of the deltasubunit isolated from phosphorylated AChR yields a single 25kd phosphorylated fragment. Immunoblotting experiments allowed us to map peptide 354367 within this phosphorylated fragment.

To identify the VIP biosynthetic pathways, we have isolated the human VIP gene, using synthetic oligodeoxynucleotides. These specific hybridization probes were constructed according to the neuroblastoma VIP-cDNA sequence and contained up to 39 bases. The gene structure was deduced by direct chemical nucleotide sequencing. Six exons were thus far discovered; among them two short exons, one encoding VIP and the second encoding PHM-27 (a peptide having a N-terminal histidine and C-terminal methionine amide, closely related in sequence and activity to VIP). As a model system for VIP gene expression, we used a human buccal tumor producing elevated amounts of VIP. In these cells, a major transcript of the VIP-gene was identified as a long RNA containing intron sequences. The occurrence of elevated quantities of a high molecular weight, intron containing, gene transcript which is not processed directly into mature RNA suggests that VIP gene expression may be regulated at the RNA processing level.

Analysis of the structure and function of a protein such as the epidermal growth factor receptor is facilitated by the use of antibodies directed against discrete portions of the protein. Here, we describe the characterization and use of antibodies directed against synthetic peptides corresponding to specific portions of the epidermal growth factor receptor and/or v-erbB protein. In particular, one useful antiserum has allowed us to compare the protein kinase activities of the epidermal growth factor receptor and the v-erbB proteins and to conclude that the v-erbB protein is a protein-tyrosine specific kinase as is its homologue the avian epidermal growth factor receptor.

Resistance to methotrexate was developed by continuous exposure of P388 murine leukemia cells in vitro to increasing concentrations of methotrexate up to 1×10^-7 M. Once established, the resistance to methotrexate was stable. This was also found in methotrexate-resistant cells that were maintained in methotrexate-free medium for more than 4 months. The sensitivity of the methotrexate-resistant P388 cells to doxorubicin was comparable to the sensitivity measured in the parental cell line. Another methotrexate-resistant cell line was developed, in a similar way, from doxorubicin-resistant P388 cells. This methotrexate-resistant cell line maintained its original resistance to doxorubicin. In methotrexate-sensitive cells, the dimethyl and dibutyl esters of methotrexate were 18.3- and 2.7-fold less active, respectively, than the free methotrexate in inhibiting cell growth. In methotrexate-resistant cells, the inhibitory effect of the methotrexate dimethyl ester was similar to its effect on the methotrexate-sensitive cell line. The activity of the methotrexate dibutyl ester was 3.3-fold lower than its activity in the parental cell line. However, both esters of methotrexate were much more active than free methotrexate in the methotrexate-resistant cell line. In the doxorubicin-resistant cell line, the activity of free methotrexate was comparable to its activity in the doxorubicin-sensitive parent cell line. However, this cell line was remarkably resistant to the ester analogs of methotrexate. The clinical implications of these findings are discussed.

The transforming protein v-erbB of avian erythroblastosis virus (AEV) displays extensive sequence homology with the presumptive protein-tyrosine kinase domain of the human EGF receptor and with the src protein-tyrosine kinase family of oncogenes. However, no kinase activity has previously been demonstrated for the v-erbB protein. Here antibodies generated against a synthetic peptide from the C terminus of human EGF receptor are shown to immunoprecipitate the EGF receptor from human and avian cells, as well as the v-erbB proteins from AEV-transformed cells that become phosphorylated on tyrosine residues upon the addition of γ-³²P-ATP. The immunoprecipitates are also able to phosphorylate exogenous tyrosine-containing substrates. Hence, it is likely that both avian EGF receptor and v-erbB proteins are protein tyrosine-specific protein kinases. Since the kinase activity of v-erbB protein cannot be regulated by EGF, it is proposed that the tyrosine protein kinase function of v-erbB may be constitutively activated.

A fluorescent analog of the phagocytosis stimulating peptide tuftsin was prepared by coupling tetramethyl rhodamine isothiocyanate to a C-terminal elongated derivative of tuftsin. This analog, Thr-Lys-Pro-Arg-Gly-Lys(N^ε-tetramethyl rhodamine)-OH, was used to visualize tuftsin receptors on mice macrophage cells by fluorescent image intensification. Fluorescent labelling was carried out at 37°C, using a concentration of 200 nM and 2 μM of the fluorescent tuftsin derivative. The formation of peptide-receptor clusters and their subsequent internalization, as discerned by image intensification, were rapid processes, 5 min and 5-30 min, respectively. Preincubation of macrophages with tuftsin for various time intervals, followed by quantification of the tuftsin receptor using radiolabelled tuftsin, suggest that tuftsin receptors are initially increased in amount (5-7 min) and subsequently reduced (after 10-15 min) as judged by sites available for tritiated tuftsin. The binding studies are rather complementary to the fluorescence observations and support the assumption that the tuftsin receptor on the membrane of the mice macrophage cell is rapidly mobilized.

Peptides corresponding to sequences of the Fc-portion of immunoglobulin G (IgG) surrounding and containing the tuftsin molecule were synthesized. The compounds were assayed for their ability to compete with [³H-Arg⁴]tuftsin in binding to mouse peritoneal macrophages and to stimulate the cell's capacity to phagocytize. Despite the sensitivity that tuftsin has demonstrated to various chemical modifications and structural alterations which usually cause reduction or total loss of biological activity, IgG-related analogs possess potent tuftsin-like activity. The activity is not caused by enzymatic breakdown and release of tuftsin. The fact that the elongated tuftsin analogs can specifically be attached to and activate macrophages may indicate a possible connection between Fc and tuftsin's receptors.

Six new analogs of the phagocytosisstimulating peptide tuftsin were synthesized with the eventual aim of characterizing and isolating the tuftsin receptor. These analogs can be classified as follows : (a) photoaffinity label ling analogs for the specific covalent attachment to the tuftsin receptor; (b) fluorescent analogs containing either rhodamine or dansyl fluorescent probes for microscopic visualization of the tuftsin receptor; (c) biotin analog for separation and purification of the receptor by affinity methods. In this paper we describe the various synthetic pathways employed to introduce sensitive prosthetic groups into the tuftsin molecule while preserving its biological activity. Activities of the various analogs synthesized as compared to tuftsin in biological and receptorbinding assays are described. All analogs are able to stimulate phagocytosis of the macrophage cell as well as compete specifically for tuftsin binding sites on these cells.

Degradation of luteinizing hormone releasing hormone (LH-RH) by purified plasma membranes from rat pituitaries was investigated. Synthetic LH-RH (0.5 mg/ml) was incubated (20 min, 37°C) with pituitary plasma membranes (750 μg protein/ml). The reaction was stopped by centrifugation at 4°C. The degradation products were isolated by high pressure liquid chromatography using a reversed-phase column. Amino acid analysis of the degradation products indicated that the N-terminal tripeptide (pGlu-His-Trp) and the N-terminal hexapeptide (pGlu-His-Trp-Ser-Tyr-Gly) sequence of LH-RH are the main degradation products. These results suggest that the main cleavage sites of LH-RH by the pituitary plasma membrane-bound enzymes are the Gly⁶-Leu⁷ and the Trp³-Ser⁴ bonds of the neurohormone.

Insoluble 2mercaptopyridine and 2mercaptonitrobenzene derivatives were prepared by modification of commercially available polystyrene. Applicability of these polymers as reagents for the thiolytic removal of the 2nitrophenylsulphenyl aminoprotecting group and as supports for preparation of polymeric active esters was evaluated. Polymeric 2mercaptopyridine (PMP) was found efficient for both purposes. It was used in the stepwise synthesis of Leuenkephalin via the polymeric reagent approach, serving as an Npscleaver. Polymeric esters derived from PMP and Bocamino acids proved to be excellent acylators. Their usefulness is exemplified in the preparation of two dipeptides, which were produced rapidly and in high yields and purity.

Sixteen new analogs of the phagocytosis-stimulating peptide tuftsin have been synthesized. The biological activities of these synthetic peptides, in which either the C-terminal or both C- and N-terminals are chemically altered, were evaluated by studying their effects on the phagocytosis of heat-killed yeasts and on the reduction of the dye nitroblue tetrazolium by normal human polymorphonuclear leukocytes. The results demonstrate that the integrity of the guanidine side chain of arginine at position four of tuftsin is crucial for maximal activity. Modification, even in side chain length, of the guanidine leads to decreasing activity. Preservation of the positive charge of position four of tuftsin yields analogs possessing considerable activity. Simultaneous alterations of both C- and N-terminal results in diminishing activites. The results of this study are discussed in relation to the structural features of tuftsin. It appears that interaction between the carboxyl of Arg⁴ and the amino group of Thr¹ which would indicate a specific conformation such as a 4 → 1 β-turn are not favored.

The serum concentrations of the phagocytosis-stimulating peptide, tuftsin, were determined by radioimmunoassay in 21 patients with sickle cell disease and in 12 healthy controls. The mean serum tuftsin concentration was significantly lower in patients with haemoglobin SS disease (154.3 ± 35.1 ng/ml; 308.6 ± 70.2 nmol/l, P

Phorbol myrystate acetate (PMA) activates nitroblue tetrazolium reduction in human polymorphs. The activation is inhibited by dibutyryl cyclic AMP, theophylline and phenylbutazone, but is not influenced by hydrocortisone in vitro, nor is it inhibited by leukocytes from patients treated with prednisone. Peptide analogues of Tuftsin also had no effect on this stimulatory activity. We conclude that the action of PMA on the nitroblue tetrazolium reduction is mediated through cyclic nucleotides.

The conformation and conformational transitions of poly(HisAlaGlu) have been investigated by ir, nmr, and CD measurements. The results obtainedas well as the results of our previous investigations by potentiometric titration and hydrodynamic techniques [Goren et al., Biopolymers (1977) 16, 15411555]indicate that when dissolved in water, the copolymer assumes a disordered conformation. On changing the pH of the solution, the states of ionization of the sidechain imidazole and carboxyl groups change in the same manner as in lowmolecularweight model compounds. Concomitantly, the overall shape of the macromolecule is altered, while the conformation of the polypeptide backbone changes from one disordered state to another but never assumes a regular form. In water/methanol and water/trifluoroethanol mixtures, transitions from a disordered state to the αhelix conformation were observed on increasing the alcohol content of the system. The conformational transitions followed pathways which differ from one another according to the experimental conditions employed. Conformational landmarks (intermediates) have been identified along these pathways.

The hydrolysis of pnitrophenyl acetate is catalyzed by imidazole, free in solution or as the side chain in poly(HisAlaGlu). This is based on the observations that the reaction is first order in ester and first order in nonprotonated imidazole. Catalysis of pnitrophenyl acetate hydrolysis is dependent on solvent conditions. The effect of low concentrations of ethanol, dioxane, and trifluoroethanol were investigated. As the concentration of organic solvent is increased, the secondorder rate constant for imidazole catalysis decreases. The decrease, however, is greater for imidazole than for poly(HisAlaGlu). In 2% trifluoroethanol/water solution, free imidazole has twice the catalytic activity of polymeric imidazole, while in 40% trifluoroethanol/water they have equal activity. Since under the latter solvent conditions poly(HisAlaGlu) is partially αhelical, the relative improvement in polymericimidazole catalysis may be attributed to imidazole hydrogenbonded to a carboxylate ion. With this assumption the carboxylateimidazole hydrogenbonded system has been calculated to have three times the base catalytic activity of imidazole.

The thiolysis of N^im2, 4dinitrophenylhistidine peptides with 2mercaptoethanol demonstrates an optimal rate between pH 8.5 and 9.0. Base lability of both reactants and product was investigated as a possible cause for the pH optimum. N2, 4Dinitrophenylimidazole, a model for N^im2, 4dinitrophenylhistidine peptides, 2mercaptoethanol and the product of thiolysis, 2, 4dinitrophenylSmercaptoethan2ol, were subjected to aqueous basic conditions (pH 8.5 to 12.0). The reactions were followed spectrophotometrically, and products were identified by comparison of their ultraviolet spectra with commercially available or synthetic compounds. Mechanisms and rate constants for base hydrolysis of 2, 4dinitrophenylthio ethers are presented. Although N2, 4dinitrophenylimidazole and 2, 4dinitrophenylSmercaptoethan2ol dohydrolyze, it is the oxidation of 2mercaptoethanol to 2mercaptoethanol disulfide which results in the decreasing rate of thiolysis above pH 9.0.

Poly(LhistidylLalanylαLglutamic acid) has been prepared in order to test the acidbase catalytic ability of a carboxylimidazole hydrogenbonded system. Two different blocked histidylalanylglutamic acid monomers were used in the polymerization step. The imidazole ring was blocked with either a dinitrophenyl or a tbutyloxycarbonyl group. The γcarboxyl of glutamic acid was protected as the benzyl ester. Both the coupling reactions and the polymerization step were via the Nhydroxysuccinimide active ester method. Thiolysis removed the dinitrophenyl group, while hydrogen bromide removed the tbutyloxycarbonyl and the benzyl groups. The watersoluble unblocked polymers obtained were fractionated on Sephadex G50 or BioGel P30. Fractions within a range of average molecular weights of 2,000 to 25,000 were isolated. Enzymatic oxidation of the acid hydrolyzate of the polymers revealed that no detectable racemization had occurred.

A crude preparation of Kallikrein inactivator, which inhibits the gonadotropin-releasing hormone (GnRH)-degrading enzyme(s) from rat hypothalamus and anterior pituitary, was fractionated by passage through an ion-exchange column. The enzyme-inhibiting fraction was coupled to Sepharose and the resin obtained was used for affinity-chromatography purification of the GnRH-degrading enzyme. The enzyme from crude tissue preparations was retained on this column and eluted by 0.05 M phosphate buffer. A 9-12 fold increase in the specific activity of the enzyme was achieved. Bacitracin, an effective peptide inhibitor of the degradation of GnRH, was also coupled to Sepharose. Three different such Se-pharose-bacitracin conjugates were synthesized, two of which inhibited the degradation of GnRH by hypothalamic and pituitary extracts. They all failed, however, to separate the active enzymic fraction from the bulk of accompanying proteins, using affinity chromatographic techniques.