Two peptide toxins with antimicrobial activity, lycotoxins I and II, were identified from venom of the wolf spider Lycosa carolinensis (Araneae: Lycosidae) by virtue of their abilities to reduce ion and voltage gradients across membranes. Both peptides were purified to homogeneity by reversed-phase liquid chromatography and determined to have the following primary structures by Edman microsequencing: IWLTALKFLGKHAAKHLAKQQLSKL-NH2 for lycotoxin I and KIKWFKTMKSIAKFIAKEQMKKHLGGE-OH for lycotoxin II. The predicted secondary structures of the lycotoxins display amphipathic alpha-helix character typical of antimicrobial pore-forming peptides. Antimicrobial assays showed that both lycotoxins potently inhibit the growth of bacteria (Escherichia coli) and yeast (Candida glabrata) at micromolar concentrations. To verify its hypothesized pore-forming activity, lycotoxin I was synthesized and shown to promote efflux of Ca2+ from synaptosomes, to cause hemolysis of erythrocytes, and to dissipate voltage gradients across muscle membrane. The lycotoxins may play a dual role in spider-prey interaction, functioning both in the prey capture strategy as well as to protect the spider from potentially infectious organisms arising from prey ingestion. Spider venoms may represent a potentially new source of novel antimicrobial agents with important medical implications.

Voltage-gated potassium channels (Kv channels) are involved in repolarization of excitable cells. In pancreatic ␤-cells, prolongation of the action potential by block of delayed rectifier potassium channels would be expected to increase intracellular free calcium and to promote insulin release in a glucose-dependent manner. However, the specific Kv channel subtypes responsible for repolarization in ␤-cells, most importantly in humans, are not completely resolved. In this study, we have investigated the expression of 26 subtypes from Kv subfamilies in human islet mRNA. The results of the RT-PCR analysis were extended by in situ hybridization and/or immunohistochemical analysis on sections from human or Rhesus pancreas. Cell-specific markers were used to show that Kv2.1, Kv3.2, Kv6.2, and Kv9.3 are expressed in ␤-cells, that Kv3.1 and Kv6.1 are expressed in ␣-cells , and that Kv2.2 is expressed in ␦-cells. This study suggests that more than one Kv channel subtype might contribute to the ␤-cell delayed rectifier current and that this current could be formed by heterotetramers of active and silent subunits. Diabetes 53: 597-607, 2004

beta-Adrenergic receptor (betaAR) activation and/or increases in cAMP regulate growth and proliferation of a variety of cells and, in some cells, promote cell death. In the current studies we addressed the mechanism of this growth reduction by examining betaAR-mediated effects in the murine T-lymphoma cell line S49. Wild-type S49 cells, derived from immature thymocytes (CD4(+)/CD8(+)) undergo growth arrest and subsequent death when treated with agents that increase cAMP levels (e.g., betaAR agonists, 8-bromo-cAMP, cholera toxin, forskolin). Morphological and biochemical criteria indicate that this cell death is a result of apoptosis. In cyc(-) and kin(-) S49 cells, which lack G(s)alpha and functional protein kinase A (PKA), respectively, betaAR activation of G(s)alpha and cAMP action via PKA are critical steps in this apoptotic pathway. S49 cells that overexpress Bcl-2 are resistant to cAMP-induced apoptosis. We conclude that betaAR activation induces apoptosis in immature T lymphocytes via G(s)alpha and PKA, while overexpression of Bcl-2 prevents cell death. betaAR/cAMP/PKA-mediated apoptosis may provide a means to control proliferation of immature T cells in vivo.

The discovery of novel therapeutic agents that act on voltage-gated sodium channels requires the establishment of high-capacity screening assays that can reliably measure the activity of these proteins. Fluorescence resonance energy transfer (FRET) technology using membrane potential-sensitive dyes has been shown to provide a readout of voltage-gated sodium channel activity in stably transfected cell lines. Due to the inherent rapid inactivation of sodium channels, these assays require the presence of a channel activator to prolong channel opening. Because sodium channel activators and test compounds may share related binding sites on the protein, the assay protocol is critical for the proper identification of channel inhibitors. In this study, high throughput, functional assays for the voltage-gated sodium channels, hNa(V)1.5 and hNa(V)1.7, are described. In these assays, channels stably expressed in HEK cells are preincubated with test compound in physiological medium and then exposed to a sodium channel activator that slows channel inactivation. Sodium ion movement through open channels causes membrane depolarization that can be measured with a FRET dye membrane potential-sensing system, providing a large and reproducible signal. Unlike previous assays, the signal obtained in the agonist initiation assay is sensitive to all sodium channel modulators that were tested and can be used in high throughput mode, as well as in support of Medicinal Chemistry efforts for lead optimization.

. Hanatoxin (100 nM) inhibited total Kv current by 65% at +20 mV. Taken together, these data provide evidence of at least two distinct types of Kv channels in human pancreatic β-cells and suggest that more than one type of Kv channel may be involved in the regulation of glucose-dependent insulin secretion.