Antimicrobial peptides from the skin of the Japanese mountain brown frog Rana ornativentris: evidence for polymorphism among preprotemporin mRNAs

A previous study led to the isolation of antimicrobial peptides belonging to the temporin and brevinin-2 families from a pooled extract of the skin of adult specimens of the Japanese mountain brown frog Rana ornativentris Werner 1903. In order to ascertain whether individual frogs expressed the full complement of temporin genes, we individually cloned cDNAs encoding the temporin precursors from total RNA extracted from the skins of 12 frogs by RT-PCR using a set of preprotemporin-specific primers. All the specimens examined contained mRNAs directing the synthesis of the novel, but inactive, temporin-1Oe (ILPLLGNLLNGLL x NH2). Nucleotide sequence analysis revealed marked polymorphism among individual frogs. Twenty-seven distinct preprotemporin-1Oe mRNAs were identified that contained synonymous substitutions in the antimicrobial peptide region and both synonymous and non-synonymous substitutions in the signal peptide and intervening sequence regions. Up to eight preprotemporin-1Oe mRNA variants were found within a single frog. In addition, several cDNAs encoding preprotemporin-1Oa and -1Ob and a single cDNA encoding preprotemporin-1Oc were characterized. Peptidomic analysis of norepinephrine-stimulated skin secretions revealed the presence of temporin-1Oe, temporin-1Of (SLILKGLASIAKLF x NH2), temporin-1Og (FLSSLLSKVVSLFT x NH2), four members of the ranatuerin-2 family and one member of the palustrin-2 family in addition to previously characterized temporin and brevinin-2 peptides.     

Discovery of a novel azepine series of potent and selective 5-HT2C agonists as potential treatments for urinary incontinence

A range of heterocycle fused azepines were synthesized in order to find a CNS penetrant, selective 5-HT_2C agonist for the treatment of incontinence. The pyridazo-azepines such as compound 11 were shown to be potent 5-HT_2C agonists and have potential for CNS penetration and good in vitro ADME properties but lacked selectivity against 5-HT_2B. Fusing a further heterocycle gave the selective triazolopyrimido-azepines. An example of this series, compound 36, was shown to be potent, selective, metabolically stable in vitro and efficacious in an in vivo model of stress urinary incontinence.     

Isolation and characterization of proSS1-32, a peptide derived from the N-terminal region of porcine preprosomatostatin

A peptide derived from the N-terminal region of porcine prosomatostatin, proSS1-32, has been purified to homogeneity from extracts of porcine upper intestine. Amino acid analysis revealed that the peptide consists of 32 residues. The complete primary structure was determined as: A P S D P R L R Q F L Q K S L A A A A G K Q E L A K Y F L A E L. This sequence obviously comprises residues 1-32 of porcine prosomatostatin since it is identical to the corresponding sequence in human preprosomatostatin. The postulated cleavage site in porcine prosomatostatin is a Leu-Leu bond between residues 32 and 33, thus confirming previous studies of the processing of the somatostatin precursor in the rat and transgenic mouse.     

Assessment of the potential of temporin peptides from the frog Rana temporaria (Ranidae) as anti‐diabetic agents

Temporin A (FLPLIGRVLSGIL‐NH₂), temporin F (FLPLIGKVLSGIL‐NH₂), and temporin G (FFPVIGRILNGIL‐NH₂), first identified in skin secretions of the frog Rana temporaria, produced concentration‐dependent stimulation of insulin release from BRIN‐BD11 rat clonal β‐cells at concentrations ≥1 nM, without cytotoxicity at concentrations up to 3 μM. Temporin A was the most effective. The mechanism of insulinotropic action did not involve an increase in intracellular Ca²⁺ concentrations. Temporins B, C, E, H, and K were either inactive or only weakly active. Temporins A, F, and G also produced a concentration‐dependent stimulation of insulin release from 1.1B4 human‐derived pancreatic β‐cells, with temporin G being the most potent and effective, and from isolated mouse islets. The data indicate that cationicity, hydrophobicity, and the angle subtended by the charged residues in the temporin molecule are important determinants for in vitro insulinotropic activity. Temporin A and F (1 μM), but not temporin G, protected BRIN‐BD11 cells against cytokine‐induced apoptosis (P < 0.001) and augmented (P < 0.001) proliferation of the cells to a similar extent as glucagon‐like peptide‐1. Intraperitoneal injection of temporin G (75 nmol/kg body weight) together with a glucose load (18 mmol/kg body weight) in C57BL6 mice improved glucose tolerance with a concomitant increase in insulin secretion whereas temporin A and F administration was without significant effect on plasma glucose levels. The study suggests that combination therapy involving agents developed from the temporin A and G sequences may find application in Type 2 diabetes treatment.     

Identification of the C-terminally alpha-amidated amino acid in peptides by high-performance liquid chromatography

A sensitive method for the rapid identification of the C-terminally amidated amino acid in peptides is described. Peptides containing the alpha-amide group at the C-terminus were cleaved with endopeptidases. The fragments released (oligopeptides, amino acids and the C-terminally amidated residue) are coupled to phenylisothiocyanate. The phenylthiocarbamoyl derivative of the amino acid alpha-amide is selectively extracted from the mixture by alkaline butyl acetate and identified by a high-performance liquid chromatography system that enables rapid and complete separation of the derivatives of 17 amino acid amides at a detection limit of 20-50 pmol. The C-terminal alpha-amides of neurokinin-A (Met-NH2), mammalian secretin (Val-NH2), pancreatic polypeptide (Tyr-NH2) and peptide HI (Ile-NH2) are unequivocally determined at a level of 0.5-2 nmol per peptide. This method was used to characterize a crude peptide fraction prepared from porcine brain. Cholecystokinin-58 was identified in this fraction by detection of phenylthiocarbamoyl-phenylalaninamide. The method is suitable for the identification of the C-terminal alpha-amidated residue of purified peptides, but can also be used as a screening strategy to isolate from complex biological extracts novel peptides containing an alpha-amidated amino acid at the C-terminus.     

IL 1 expression in a clone of human T cells

Three human T cell clones, all of which are T3+, T4+, T8-, and T11+, were examined for IL 1 production. Two clones were found to express readily detectable, membrane-bound IL 1 activity upon stimulation with OKT3 antibody, rIL 2, and PMA. Northern blot analysis of RNA from one of the clones shows that cells can be induced to express the genes for both IL 1 alpha and IL 1 beta. Furthermore, the pattern of expression in response to different stimuli suggests that the genes for IL 1 alpha and IL 1 beta are regulated independently.     

Scyliorhinin I and II: two novel tachykinins from dogfish gut

Two peptides with tachykinin-like ability to contract longitudinal muscle from the guinea pig ileum were isolated from the intestine of the common dogfish, Scyliorhinus caniculus. The amino acid sequence of scyliorhinin I was established as Ala-Lys-Phe-Asp-Lys-Phe-Tyr-Gly-Leu-Met-NH2 and this peptide cross-reacted with antisera directed against the C-terminal region fo substance P. The amino acid sequence of scyliorhinin II was established as Ser-Pro-Ser-Asn-Ser-Lys-Cys-Pro-Asp-Gly-Pro-Asp-Cys-Phe-Val-Gly-Leu-Met- NH2 and this peptide cross-reacted with antisera directed against the C-terminal region of neurokinin A. The mammalian peptides substance P and neurokinin A were absent from the dogfish intestinal tissue.     

Effect of streptozotocin administration upon the serotonin content of the pancreas and small intestine of the rat

The study has examined the effect of streptozotocin-induced diabetes of 3 days and 10 weeks duration upon the serotonin content of the rat pancreas and small intestine. Streptozotocin administration (65 mg/kg) resulted in a significant (p less than 0.001) decrease in pancreatic serotonin after 3 days (to 18% of the non-diabetic content). Diabetes of both short- and medium-term duration had no significant effect upon the serotonin content of the small intestine suggesting that changes in mucosal serotonin levels are not responsible for the diarrhea frequently observed in streptozotocin-treated animals. The diabetogenic effect of streptozotocin and the reduction in pancreatic serotonin were abolished by prior injection of nicotinamide thus providing further evidence for co-storage of insulin and serotonin in the B cell.     

The induction of tolerance to DNFB contact sensitivity by using hapten-modified lymphoid cells. III. Effects of hapten concentration on the ability of MLS-disparate cells to induce rapid unresponsiveness

We investigated genetic restrictions in the induction of immediate tolerance to DNFB contact sensitivity in mice. Using spleen cells from various donor strains haptenated at 500 micro M DNFB, we were unable to detect any restrictions in tolerance induction in recipients that were either syngeneic or allogeneic to the donor strain. However, if the concentration of hapten used in the in vitro labeling was decreased (from 500 micro M to 2.5 to 5 micro M DNFB), differences in tolerogenesis between the various donor strain haplotypes were found. Haptenated spleen cells labeled with 5 micro M DNFB produced a profound level of unresponsiveness in allogeneic recipients but produced minimal tolerance in syngeneic animals. This tolerant state was shown to be antigen-specific and was not produced by unmodified allogeneic cells alone. Further genetic analysis demonstrated that an efficient tolerant state was produced when the donor of the tolerogen and recipient differed at the MLS locus rather than at either the MHC or minor regions. This phenomenon required viable, Thy 1-bearing cells in the haptenated donor population for efficient tolerogenesis to DNFB contact sensitivity.