Conformational analysis and in vitro immunomodulatory and insulinotropic properties of the frog skin host-defense peptide rhinophrynin-27 and selected analogs

The study investigates conformational analysis and the in vitro cytokine-mediated immunomodulatory and insulin-releasing activities of rhinophrynin-27 (ELRLPEIARPVPEVLPARLPLPALPRN; RP-27), a proline-arginine-rich peptide first isolated from skin secretions of the Mexican burrowing toad Rhinophrynus dorsalis (Rhinophrynidae). In both water and 50% trifluoroethanol-water, the peptide adopts a polyproline type II helical conformation with a high degree of deviation from the canonical collagen-like folding and a pronounced bend in the molecule at the Glu13 residue. Incubation of mouse peritoneal cells with RP-27 significantly (P < 0.05) inhibited production of the pro-inflammatory cytokines TNF-α and IL-1β and stimulated production of the anti-inflammatory cytokine IL-10. The peptide significantly (P < 0.01) stimulated release of insulin from BRIN-BD11 rat clonal β-cells at concentrations ≥ 1 nM while maintaining the integrity of the plasma membrane and also stimulated insulin release from isolated mouse islets at a concentration of 10−6 M. Increasing the cationicity of RP-27 by substituting glutamic acid residues in the peptide by arginine and increasing hydrophobicity by substituting alanine residues by tryptophan did not result in analogues with increased activity with respect to cytokine production and insulin release. The combination of immunosuppressive and insulinotropic activities together with very low cytotoxicity suggests that RP-27 may represent a template for the development of an agent for use in anti-inflammatory and Type 2 diabetes therapies

Isolation and characterization of cytotoxic and insulin-releasing components from the venom of the black-necked spitting cobra Naja nigricollis (Elapidae)

Four peptides with cytotoxic activity against BRIN-BD11 rat clonal β-cells were purified from the venom of the black-necked spitting cobra Naja nigricollis using reversed-phase HPLC. The peptides were identified as members of the three-finger superfamily of snake toxins by ESI-MS/MS sequencing of tryptic peptides. The most potent peptide (cytotoxin-1N) showed strong cytotoxic activity against three human tumour-derived cell lines (LC50 = 0.8 ± 0. 2 µM for A549 non-small cell lung adenocarcinoma cells; LC50 = 7 ± 1 µM for MDA-MB-231 breast adenocarcinoma cells; and LC50 = 9 ± 1 µM for HT-29 colorectal adenocarcinoma cells). However, all the peptides were to varying degrees cytotoxic against HUVEC human umbilical vein endothelial cells (LC50 in the range 2-22 µM) and cytotoxin-2N was moderately hemolytic (LC50 = 45 ± 3 µM against mouse erythrocytes). The lack of differential activity against cells derived from non-neoplastic tissue limits their potential for development into anti-cancer agents. In addition, two proteins in the venom, identified as isoforms of phospholipase A2, effectively stimulated insulin release from BRIN-BD11 cells (an approximately 6-fold increase in rate compared with 5.6 mM glucose alone) at a concentration (1 µM) that was not cytotoxic to the cells suggesting possible application in therapy for Type 2 diabetes

Peptidomic Analysis of Skin Secretions of the Caribbean Frogs Leptodactylus insularum and Leptodactylus nesiotus (Leptodactylidae) Identifies an Ocellatin with Broad Spectrum Antimicrobial Activity

International audienceOcellatins are peptides produced in the skins of frogs belonging to the genus Leptodactylus that generally display weak antimicrobial activity against Gram-negative bacteria only. Peptidomic analysis of norepinephrine-stimulated skin secretions from Leptodactylus insularum Barbour 1906 and Leptodactylus nesiotus Heyer 1994, collected in the Icacos Peninsula, Trinidad, led to the purification and structural characterization of five ocellatin-related peptides from L. insularum (ocellatin-1I together with its (1-16) fragment, ocellatin-2I and its (1-16) fragment, and ocellatin-3I) and four ocellatins from L. nesiotus (ocellatin-1N,-2N,-3N, and-4N). While ocellatins-1I,-2I, and-1N showed a typically low antimicrobial potency against Gram-negative bacteria, ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH 2) was active against an antibiotic-resistant strain of Klebsiella pneumoniae and reference strains of Escherichia coli, K. pneumoniae, Pseudomonas aeruginosa, and Salmonella typhimurium (minimum inhibitory concentrations (MICs) in the range 31.25-62.5 µM), and was the only peptide active against Gram-positive Staphylococcus aureus (MIC = 31.25 µM) and Enterococcus faecium (MIC = 62.5 µM). The therapeutic potential of ocellatin-3N is limited by its moderate hemolytic activity (LC 50 = 98 µM) against mouse erythrocytes. The peptide represents a template for the design of long-acting, non-toxic, and broad-spectrum antimicrobial agents for targeting multidrug-resistant pathogens

Direct observation of mammalian cell growth and size regulation

We introduce a microfluidic system for simultaneously measuring single cell mass and cell cycle progression over multiple generations. We use this system to obtain over 1,000 hours of growth data from mouse lymphoblast and pro-B-cell lymphoid cell lines. Cell lineage analysis revealed a decrease in the growth rate variability at the G1/S phase transition, which suggests the presence of a growth rate threshold for maintaining size homeostasis

Identification of an Antimicrobial Peptide from the Venom of the Trinidad Thick-Tailed Scorpion Tityus trinitatis with Potent Activity against ESKAPE Pathogens and Clostridioides difficile

Envenomation by the Trinidad thick-tailed scorpion Tityus trinitatis may result in fatal myocarditis and there is a high incidence of acute pancreatitis among survivors. Peptidomic analysis (reversed-phase HPLC followed by MALDI-TOF mass spectrometry and automated Edman degradation) of T. trinitatis venom led to the isolation and characterization of three peptides with antimicrobial activity. Their primary structures were established asTtAP-1 (FLGSLFSIGSKLLPGVFKLFSRKKQ.NH2), TtAP-2 (IFGMIPGLIGGLISAFK.NH2) and TtAP-3 (FFSLIPSLIGGLVSAIK.NH2). In addition, potassium channel and sodium channel toxins, present in the venom in high abundance, were identified by CID-MS/MS sequence analysis. TtAP-1 was the most potent against a range of clinically relevant Gram-positive and Gram-negative aerobes and against the anaerobe Clostridioides difficile (MIC = 3.1–12.5 µg/mL). At a concentration of 1× MIC, TtAP-1 produced rapid cell death (Acinetobacter baumannii and Staphylococcus aureus). The therapeutic potential of TtAP-1 as an anti-infective agent is limited by its high hemolytic activity (LC50 = 18 µg/mL against mouse erythrocytes) but the peptide constitutes a template for the design of analogs that maintain the high bactericidal activity against ESKAPE pathogens but are less toxic to human cells. It is suggested that the antimicrobial peptides in the scorpion venom facilitate the action of the neurotoxins by increasing the membrane permeability of cells from either prey or predator