Maurotoxin is a toxin isolated from the venom of the Tunisian chactoid scorpion Scorpio maurus. It is a 34-amino-acid peptide cross-linked by four disulfide bridges. Maurotoxin competes with radiolabeled apamin and kaliotoxin for binding to rat-brain synaptosomes. Due to its very low concentration in venom (0.6% of the proteins), maurotoxin was chemically synthesized by means of an optimized solid-phase technique. The synthetic maurotoxin was characterized. It was lethal to mice following intracerebroventricular injection (LD,,, 80 ng/mouse). The synthetic maurotoxin competed with '*'I-apamin and 'Z51-kaliotoxin for binding to rat-brain synaptosomes with half-maximal effects at concentrations of 5 nM and 0.2 nM, respectively. Synthetic maurotoxin was tested on K' channels and was found to block the Kvl.1, Kv1.2, and Kv1.3 currents with half-maximal blockage (I&) at 37, 0.8 and 150 nM, respectively. Thus, maurotoxin is a scorpion toxin with four disulfide bridges that acts on K' channels. The half-cystine pairings of synthetic maurotoxin were identified by enzymatic cleavage. The pairings were Cys3 -Cys24, Cys9-Cys29, Cysl3-Cysl9 and Cys31 -Cys34. This disulfide organization is unique among known scorpion toxins. The physicochemical and pharmacological properties of synthetic maurotoxin were indistinguishable from those of natural maurotoxin, which suggests that natural maurotoxin adopts the same half-cystine pairing pattern. The conformation of synthetic maurotoxin was investigated by means of circular dichroism spectroscopy and molecular modeling. In spite of its unusual half-cystine pairings, the synthetic-maurotoxin conformation appears to be similar to that of other short scorpion toxins.Keywords: maurotoxin; scorpion toxin; half-cystine pairing ; apamin-sensitive K' channels ; voltage-gated K' channels.Because polypeptide animal toxins interact with ion channels and modulate their activities [ 1 -31, these toxins are useful pharmacological probes to investigate ion-specific channel proteins and their function. In recent years, toxins acting on various K+ channels have been isolated from diverse scorpion venoms [4]. Maurotoxin has recently been purified from the venom of the chactoid scorpion Scorpio maurus, and characterized (Kharrat, R., Mansuelle, P., Sampieri, F., Crest, M., Martin-Eauclaire, M. F., Rochat, H. and El Ayeb, M., unpublished results). Maurotoxin is a basic toxin of 34 amino acid residues cross-linked by four disulfide bridges. Maurotoxin was found to compete with radiolabeled apamin and kaliotoxin for binding to rat-brain synaptosomes (Kharrat, R., Mansuelle, P., Sampieri, F., Crest, M., Martin-Eauclaire, M. F., Rochat, H. and El Ayeb, M., unpublished results). Thus, it is a scorpion toxin with four disulfide bridges that acts on K' channels. Due to its sequence, maurotoxin does not belong to any of the four groups of K+-channel Maurotoxin is only 0.6% of the total proteins in a crude venom, which is not readily available. Thus, we performed chemical solid-phase synthesis of this toxin to e...

Recently, we demonstrated that U87 glioblastoma xenograft tumors treated with anti-adrenomedullin (AM) antibody were less vascularized than control tumors, suggesting that AM might be involved in neovascularization and/or vessel stabilization. Angiogenesis, the sprouting of new capillaries from preexisting blood vessels, is a multistep process that involves migration and proliferation of endothelial cells, remodeling of the extracellular matrix and functional maturation of the newly assembled vessels. In our study, we analyzed the role of AM on human umbilical vein endothelial cell (HUVEC) phenotype related to different stages of angiogenesis. Here we report evidence that AM promoted HUVEC migration and invasion in a dose-dependent manner. The action of AM is specific and is mediated by the calcitonin receptor-like receptor/receptor activity-modifying protein-2 and -3 (CRLR/RAMP2; CRLR/RAMP3) receptors. Furthermore, AM was able to induce HUVEC differentiation into cord-like structures on Matrigel. Suboptimal concentrations of vascular endothelial growth factor (VEGF) and AM acted synergistically to induce angiogenic-related effects on endothelial cells in vitro. Blocking antibodies to VEGF did not significantly inhibit AM-induced capillary tube formation by human endothelial cells, indicating that AM does not function indirectly through upregulation of VEGF. These findings suggest that the proangiogenic action of AM on cultured endothelial cells via CRLR/RAMP2 and CRLR/RAMP3 receptors may translate in vivo into enhanced neovascularization and therefore identify AM and its receptors acting as potential new targets for antiangiogenic therapies.

The human immunodeficiency virus (HIV) genome codes for a trans-activating regulatory protein, tat. Using chemically synthesized tat, it was found that 125I-tat and 12s51_ t384;6 specifically bound to rat brain synaptosomal membranes with moderate affinity (K0.5 = 3 ,uM). Interaction of tat with nerve cells was also revealed by flow cytometry, which showed its binding to rat glioma and murine neuroblastoma cells, using both direct fluorescence with fluorescein isothiocyanate-labeled tat and indirect immunofluorescence assays. This interaction was investigated with electrophysiology using isolated excitable frog muscle fibers and cockroach giant interneuron synapses. tat acted on the cell membrane and induced a large depolarization, accompanied by a decrease in membrane resistance, thereby modifying cell permeability. It is now established that infection with human immunodeficiency virus type 1 (HIV-1) is often complicated by neurological syndromes that include dementia, subacute encephalitis, and vacuolar degeneration of the spinal cord (9,19,28,32). The identification and isolation of HIV-1 from the brain suggests that the retrovirus itself is responsible for the neurological disorders observed in HIV-infected patients. Other lentiviruses, including visna virus (15) and simian immunodeficiency virus (22), are also associated with brain infections. Among central nervous system (CNS) cells, monocyte and macrophage lines are preferentially infected by HIV, but infection of other neural cell types has also been discussed (20,35).The pathogenic mechanism by which the virus causes encephalopathy remains unknown. It was reported recently that the HIV envelope glycoprotein manifests neurotoxic activity by increasing free Ca2+ in rat neurons, thus causing cellular damage (4, 7). This effect can be prevented by Ca2+ channel antagonists.As an approach to another possible cause of neurological dysfunction, we investigated whether other HIV proteins could be implicated in this pathology. For this study, numerous peptides were chemically synthesized on an Applied Biosystems peptide synthesizer (model 430A) with the stepwise solid-phase method (25,29).By testing the neurotoxicity of synthetic fragments of various lengths, derived from gp160, p25, nef, and tat proteins, we discovered that the intracerebroventricular injection of tat or some tat fragments caused toxic and lethal effects in mice. The 86-residue tat protein from HIV-1 has been previously reported to be critical for virus replication through its role in viral trans activation (1,11,12,14,18,33 We have further investigated tat neurotoxicity by structure-activity relationships, using binding experiments and electrophysiology. We first investigated the capacity of radiolabeled tat3886 from HIV-1, LAVBru isolate, to bind to rat brain synaptic nerve ending particles (synaptosomal membranes) prepared by the method of Gray and Whittaker (13). Protein was measured by a modified Lowry method (24). 125I-tat38 86 (>10-8 M), the most active peptide in vivo (Table 1), bound to...

Adrenomedullin (AM) is a multifunctional peptide vasodilator that transduces its effects through calcitonin receptor-like receptor/receptor activity modifying protein-2 and -3 (CLR/RAMP2 and CLR/RAMP3). Previously, we reported on the development of an anti-AM antibody that potently inhibits tumor cell proliferation in vitro and tumor growth in vivo. Here, we report the effect of anti-AM receptor antibodies (alphaAMRs) on angiogenesis and tumor growth. We demonstrate that alphaAMRs decrease in a dose-dependent manner the growth of U87 glioblastoma cells and HT-29 colorectal cancer cells, but not A549 lung cancer cells, in vitro. In vivo, AM in Matrigel plugs induces angiogenesis by promoting recruitment of endothelial cells, pericytes, myeloid precursor cells, and macrophages and by promoting channel formation. Remarkably, systemic administration of alphaAMRs every 3 d markedly reduced neovascularization of Matrigel plugs in a dose-dependent fashion, as demonstrated by reduced numbers of the recruited cells and vessel structures. Several human tumor xenografts in athymic mice were used to examine the effect of alphaAMR treatment on tumor angiogenesis and growth. AlphaAMR treatment significantly suppressed the growth of glioblastoma, lung, and colon tumors. Histological examination of alphaAMR-treated tumors showed evidence of disruption of tumor vascularity with decreased microvessel density, depletion of endothelial and pericyte cells, and increased tumor cell apoptosis. These findings support the conclusion that alphaAMR treatment inhibits tumor growth by suppression of angiogenesis and tumor growth and suggest that AMRs may be useful therapeutic targets.