The Morphological Effects of Two Antimicrobial Peptides, Hecate-1 and Melittin, on Escherichia Coli

The effects of the 26 amino acid, cationic, amphipathic, antibacterial peptide melittin and hecate-1, a 23 amino acid analog of it, on the gram negative bacterium Escherichia coli were investigated using scanning electron microscopy (SEM), transmission electron micros-copy (TEM), and freeze-fracture. Both peptides killed virtually all bacteria at the peptide concentration and cell density used. TEM and SEM revealed aggregates of bacteria entangled with material extruded from the bacterial surfaces. SEM revealed irregular bacterial surfaces with bleb-like projections. TEM and freeze-fracture indicate that the bacterial inner and outer membranes, as well as the peptidoglycan layer between, were extensively damaged. The cytoplasmic contents of the cells, however, did not appear radically disturbed, providing little evidence for osmotically induced cytolysis

Oat consumption reduced intestinal fat deposition and improved health span in Caenorhabditis elegans model

© 2015 The Authors. In addition to their fermentable dietary fiber and the soluble β-glucan fiber, oats have unique avenanthramides that have anti-inflammatory and antioxidant properties that reduce coronary heart disease in human clinical trials. We hypothesized that oat consumption will increase insulin sensitivity, reduce body fat, and improve health span in Caenorhabditis elegans through a mechanism involving the daf-2 gene, which codes for the insulin/insulin-like growth factor-1-like receptor, and that hyperglycemia will attenuate these changes. Caenorhabditis elegans wild type (N2) and the null strains sir-2.1, daf-16, and daf-16/daf-2 were fed Escherichia coli (OP50) and oat flakes (0.5%, 1.0%, or 3%) with and without 2% glucose. Oat feeding decreased intestinal fat deposition in N2, daf-16, or daf-16/daf-2 strains (P \u3c.05); and glucose did not affect intestinal fat deposition response. The N2, daf-16, or sir-2.1 mutant increased the pharyngeal pumping rate (P \u3c.05), a surrogate marker of life span, following oat consumption. Oat consumption increased ckr-1, gcy-8, cpt-1, and cpt-2 mRNA expression in both the N2 and the sir-2.1 mutant, with significantly higher expression in sir-2.1 than in N2 (P \u3c.01). Additional glucose further increased expression 1.5-fold of the 4 genes in N2 (P \u3c.01), decreased the expression of all except cpt-1 in the daf-16 mutant, and reduced mRNA expression of the 4 genes in the daf-16/daf-2 mutant (P \u3c.01). These data suggest that oat consumption reduced fat storage and increased ckr-1, gcy-8, cpt-1, or cpt-2 through the sir-2.1 genetic pathway. Oat consumption may be a beneficial dietary intervention for reducing fat accumulation, augmenting health span, and improving hyperglycemia-impaired lipid metabolism

Structural changes induced in thionins by chloride anions as determined by molecular dynamics simulations

Computational analysis of two membrane-permeabilizing peptides, barley α-hordothionin and wheat β-purothionin, revealed that anions can trigger dynamic and structural changes in the thionin antiparallel double α-helix core. Analysis of the molecular dynamics simulations demonstrated that anions induced unfolding of the α2 and α1 helices at the carboxyl ends which are located on the opposite ends of the α-helix core. An internalized water molecule was observed inside the unfolded α2 C-end. Strong interactions of anions with the R30 regulating network or simultaneous interactions of anions with the phospholipid-binding site and the R30 hydrogen bonding network triggered unfolding of the α2 C-end. An increase of anion density for two residues of the phospholipid-binding site (K1, R17, and Q22) or R17 and R19 and a preceding unfolding of the α2 C-end were necessary for unfolding of the α1 C-end. Anions interacted primarily with residues of the phospholipid-binding site and the R30 network while the α1/α2 hydrophobic region was void of anions. However, during strong interactions of anions with the R30 network and phospholipid-binding site, the α1/α2 hydrophobic region attracted anions which interacted with conserved residues of the α1 C-end. Analysis of anion-induced rearrangements pointed to auxiliary residues of the R30 network and the phospholipid-binding site. Induction of conformational changes on the opposite ends of the α-helix core by interactions of anions with the phospholipid-binding site may be relevant to a mechanism of membrane-permeabilizing activity. © 2009

Effects of a lytic peptide conjugated to β hCG on ovarian cancer: Studies in vitro and in vivo

Objective. The aim of this study was to determine the in vitro and in vivo effects of the lytic peptide, hecate, alone and conjugated to a 15-amino-acid fragment of the β-chain of hCG (hecate-β hCG) on the ovarian carcinoma cell line NIH: OVCAR-3 and determine the expression of luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptors in cell cultures and tumor tissues. Methods. For in vitro studies, hecate or hecate-β hCG was added to cultures of ovarian cancer cells in the presence or absence of estradiol or follicle stimulating hormone. The cytotoxicity of lytic peptides was measured by trypan blue exclusion and lactate dehydrogenase release. For in vivo studies, OVCAR-3 xenografts were established in female athymic nude mice which were then treated once per week for 3 weeks with hecate or hecate-β hCG via the lateral tail vein. An immunohistochemical method was used to analyze the expression of LH/hCG receptor in tumor and culture cells. Results. In in vitro studies, both hecate-β hCG and hecate destroyed ovarian cancer cells (NIH: OVCAR-3) in a dose-dependent manner. Removal of steroids from the culture medium reduced the sensitivity of the OVCAR-3 cell line to the hecate-β hCG in a reversible manner. In in vivo studies, the average tumor volume and tumor burden in lytic peptide treated animals were reduced. In the groups of animals treated by hecate, hecate-β hCG, and estradiol + hecate-β hCG, tumor volumes after treatment expressed as a percentage of increase (197.4 ± 21.72, 199.0 ± 18.57, and 193.8 ± 22.94%, respectively) were reduced, compared to control (263.0 ± 21.72%) animals (P \u3c 0.05). Immunocytochemical studies revealed the expression of LH/hCG receptor protein in the OVCAR-3 cells and tumor tissues. Conclusion. Hecate-β hCG is a putative candidate for treating ovarian cancer. © 2002 Elsevier Science (USA)

Membrane disrupting lytic peptide conjugates destroy hormone dependent and independent breast cancer cells in vitro and in vivo

We have prepared conjugates of a membrane disrupting lytic peptide (hecate) and a 15-amino acid segment of the β-chain of CG and hecate and the decapeptide, luteinizing hormone releasing hormone (LHRH). We have tested the concept that these conjugates will target breast cancer cells expressing LH/CG or LHRH receptors. In previous studies, we were able to destroy prostate cancers in vitro and in vivo with lytic peptide conjugates [1]. Hecate, hecate-βCG and LHRH-hecate were added to cultures of the human breast cancer cell lines MCF-7 and MDA-MB-435S. Hecate and its conjugates showed concentration dependent toxicity to both cell lines. The lytic peptide alone showed similar EC50 values for both cell lines; however, there was a significant difference between the EC50 values when the conjugates were tested. The hormone dependent MCF-7 cell line was less sensitive to the βCG conjugate than to the LHRH conjugate; the reverse was found for the hormone independent MDA-MB-435S cells. Removal of steroids decreased the sensitivity of MCF-7 cells to both lytic peptide conjugates and this sensitivity could be restored by adding estradiol. Activation of protein kinase C further increased the sensitivity to the drug. MDA-MB-435S xenografts were established in intact female athymic nude mice, which were treated once a week for 3 weeks with hecate-βCG via the lateral tail vein. The ability of hecate-βCG to destroy xenografts of human breast cancer cells (MDA-MB-435S) in nude mice was demonstrated for the first time. We conclude that hecate-βCG and LHRH-hecate conjugates could serve as useful drugs for the treatment of breast cancer

Human prostate cancer cells and xenografts are targeted and destroyed through luteinizing hormone releasing hormone receptors

BACKGROUND. A conjugate of a lytic peptide, hecate, and a 15-amino acid segment of the β-chain of chorionic gonadotropin (CG) destroyed human prostate xenografts in nude mice by targeting LH receptors. Since these xenografts also express LHRH receptors, we prepared a LHRH-hecate conjugate and tested its ability to destroy PC-3 cells in vitro and in vivo. MATERIALS AND METHODS. LHRH-hecate was added to cultures of PC-3, BRF 41 T, DU145, and LNCaP cells in the presence and absence of steroids. PC-3 xenografts were established in nude male mice, which were treated with LHRH-hecate. RESULTS. Injections of LHRH-hecate resulted in tumor growth arrest and marked reduction of tumor burden (62.2 mg/g body weight in saline controls vs. 10.5 mg/g body weight in treated mice; P \u3c 0.0001); unconjugated LHRH and hecate had no effect on tumor burden and tumor viability (48.5 mg/g body weight in LHRH treated animals vs. 63.2 mg/g body weight in hecate treated mice). Marked tumor necrosis occurred in conjugate treated mice. Removal of steroids from the culture media decreased the sensitivity of LNCaP and PC-3 cells to the LHRH-hecate; adding estrogen restored the sensitivity. CONCLUSIONS. LHRH-hecate may be effective in treating hormone dependent and independent prostate cancers. © 2003 Wiley-Liss, Inc

Targeted destruction of androgen-sensitive and -insensitive prostate cancer cells and xenografts through luteinizing hormone receptors

BACKGROUND. We have prepared a conjugate of a lytic peptide (hecate) and a 15-amino acid segment of the β-chain of LH to test the concept that this conjugate will target cancer cells expressing LH receptors. METHODS. Hecate-βLH was added in vitro to cultures of Chinese hamster ovary (CHO) cells with and without LH receptors and to prostate cancer cells in the presence or absence of steroids, follicle-stimulating hormone (FSH), epidermal growth factor (EGF), or βLH. PC-3 xenografts were established in male athymic nude mice and treated once a week for 3 weeks with hecate-βLH via the lateral tail vein. RESULTS. The conjugate showed concentration-dependent toxicity for the following prostate cancer cell lines: BRF 41 T\u3eDU145\u3ePC-3\u3eLNCaP, according to their LH receptor capacities. Steroid removal reduced sensitivity to the drug in a reversible manner. Hecate-βLH reduced the tumor burden in the nude mice from 60 to 12.5 mg/g body weight. CONCLUSION. We conclude that the hecate-βLH conjugate selectively kills androgen-dependent and-independent prostate cancer cells both in vivo and in vitro; its toxicity depends on the number of LH receptor sites present. © 2001 Wiley-Liss, Inc

Light-microscopic studies of 3T3 cell plasma membrane alterations mediated by melittin

Various light microscopic techniques were used to study the effect of melittin, a major toxic constituent of honey bee venom, on plasma membranes of 3T3 mouse fibroblasts. Bright-field light microscopy and Trypan Blue dye exclusion were used to demonstrate changes in membrane permeability after exposure to melittin. Differential interference contrast (DIC) microscopy showed that membrane vesiculation induced by melittin was dose dependent. Using both fluorescent lipid and glycoprotein markers, we found that membrane vesicles were primarily composed of lipids. A sequence of events associated with vesicle formation was depicted by DIC and fluorescence microscopy. Confocal laser scanning fluorescence microscopy demonstrated a translocation of membrane glycoproteins from the plasma membrane to the cytosol following melittin treatment. The significance of membrane vesiculation and translocation of membrane glycoproteins in damaged cells is discussed