Pomegranate juice and extract extended lifespan and reduced intestinal fat deposition in Caenorhabditis elegans.

Pomegranate juice with a high content of polyphenols, pomegranate extract, ellagic acid, and urolithin A, have anti-oxidant and anti-obesity effects in humans. Pomegranate juice extends lifespan of Drosophila melanogaster. Caenorhabditis elegans (C. elegans) (n = 6) compared to the control group in each treatment, lifespan was increased by pomegranate juice in wild type (N2, 56 %, P < 0.001) and daf-16 mutant (daf-16(mgDf50)I) (18 %, P = 0.00012), by pomegranate extract in N2 (28 %, P = 0.00004) and in daf-16(mgDf50)I (10 %, P < 0.05), or by ellagic acid (11 %, P < 0.05). Pomegranate juice reduced intestinal fat deposition (IFD) in C. elegans (n = 10) N2 (-68 %, P = 0.0003) or in the daf-16(mgDf50)I (-33 %, P = 0.0034). The intestinal fat deposition was increased by pomegranate extract in N2 (137 %, P < 0.0138) and in daf-16(mgDf50)I (26 %, P = 0.0225), by ellagic acid in N2 (66 %, P < 0.0001) and in daf-16(mgDf50)I (74 %, P < 0.0001), or by urolithin A in N2 (57 %, P = 0.0039) and in daf-16(mgDf50)I (43 %, P = 0.0001). These effects were partially mediated by the daf-16 pathway. The data may offer insights to human aging and obesity due to homology with C. elegans.

Spiroplasma found in the eyes of scrapie affected sheep.

OBJECTIVE: Scrapie, a transmissible spongiform encephalopathy (TSE) occurring naturally in sheep, characteristically shows a severe retinopathy that is well developed in the terminal phases of the disease. In this study, we set out to demonstrate similar retinal changes in our ruminant spiroplasmosis TSE model. PROCEDURE: The eyes from deer, sheep, and goats that were inoculated intracranially with the laboratory strain of spiroplasma (suckling mouse cataract [SMCA] strain of Spiroplasma mirum) or with Spiroplasma sp. isolated from the brains affected with scrapie or with chronic wasting disease were examined by light microscopy for pathologic changes and by immunocytochemistry for distribution of spiroplasma antigen. The eyes were also obtained from a research flock of sheep with terminal scrapie, from which the intraocular tissues were submitted aseptically for culture assay in M1D broth or as explants on bovine corneal endothelia (BCE). RESULTS: The eyes from the spiroplasmosis ruminant models showed retinopathy remarkably similar to eye lesions seen in sheep with scrapie. The spiroplasma antigen accrued in the ruminant model eye tissues, particularly in the retina, the vitreous humor, and the corneal endothelia. A Spiroplasma sp. grew out of the scrapie-affected eyes both in the M1D broth and in the BCE cultures but did not expand. These new spiroplasma isolates differed immunologically from SMCA. CONCLUSION: These data showed a clear association of spiroplasma with scrapie suggesting that these bacteria have a role in the pathogenesis of TSE and that the eye should be a research focus for future studies of TSE.

Spatio-temporal thermal kinetics of in situ MWCNT heating in biological tissues under NIR laser irradiation.

Carbon nanotubes have many potential applications in life sciences and engineering as they have very high absorbance in the near-infrared (NIR) spectrum, while biological tissues do not. The purpose of this study was to determine the effect of 1064 nm NIR laser power levels on the spatial temperature distribution and the temperature kinetics in mammalian tissue at both macroscopic and microscopic scales. The model tissue was the 'flat' of a chicken wing (the section containing the radius and ulna), which was injected under the skin in the subcutaneous layer of tissue. Specimens were exposed to laser radiation and an infrared thermography system was used to measure and record the temperature distributions in the specimens at both the macroscopic and microscopic scales. Experimental results concluded that power levels of 1536 mW easily achieved hyperthermic temperatures with localized values as high as 172.7 °C.

Effects of cecropin B transgene expression on Mannheimia haemolytica serotype 1 colonization of the nasal mucosa of calves.

OBJECTIVE: To express a cecropin B transgene on bovine nasal mucosa and determine the effect on Mannheimia haemolytica serotype 1 (S1) colonization. ANIMALS: 27 crossbred beef calves. PROCEDURE: The antibacterial efficacy of cecropin B against M. haemolytica S1 was first determined by measuring its minimum inhibitory concentration (MIC). The peptide was also diluted in pooled bovine nasal secretions, and its antibacterial activity was evaluated. The nasal passages of 16 calves were aerosolized with 25, 50, or 100 microg of plasmid DNA/nostril, whereas 11 control calves were aerosolized with only the transfection reagent. In 2 of the experiments, 12 treated and 8 control calves were exposed intranasally with an aerosol of M. haemolytica S1. Nasal swab specimens and secretions were collected and analyzed by use of polymerase chain reaction (PCR), real-time PCR, real-time reverse-transcriptase PCR, ELISA, and bacterial culture. RESULTS: In vitro, cecropin B inhibited M. haemolytica S1 at an MIC of 2 microg/mL and its antibacterial activity was not affected by proteolytic activity in nasal secretions. Cecropin B transgene expression was detected in calves transfected with 50 or 100 microg of DNA/nostril. Antibacterial activity against M. haemolytica S1 was observed in all calves transfected with 100 microg of DNA/nostril but in only 2 of the 4 calves transfected with 50 microg of DNA/nostril. CONCLUSIONS AND CLINICAL RELEVANCE: In vitro, cecropin B has an effective antibacterial activity against M. haemolytica S1 and can prevent colonization of the nasal mucosa after transfection of a vector expressing cecropin B in vivo.

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

In addition to their fermentable dietary fiber and the soluble ß-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 < .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 < .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 < .01). Additional glucose further increased expression 1.5-fold of the 4 genes in N2 (P < .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 < .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.

Prowashonupana barley dietary fibre reduces body fat and increases insulin sensitivity in Caenorhabditis elegans model.

Prowashonupana barley (PWB) is high in ß-glucan with moderate content of resistant starch. PWB reduced intestinal fat deposition (IFD) in wild type Caenorhabditis elegans (C. elegans, N2), and in sir-2.1 or daf-16 null mutants, and sustained a surrogate marker of lifespan, pharyngeal pumping rate (PPR), in N2, sir-2.1, daf-16, or daf-16/daf-2 mutants. Hyperglycaemia (2% glucose) reversed or reduced the PWB effect on IFD in N2 or daf-16/daf-2 mutants with a sustained PPR. mRNA expression of cpt-1, cpt-2, ckr-1, and gcy-8 were dose-dependently reduced in N2 or daf-16 mutants, elevated in daf-16/daf-2 mutants with reduction in cpt-1, and unchanged in sir-2.1 mutants. mRNA expressions were increased by hyperglycaemia in N2 or daf-16/daf-2 mutants, while reduced in sir-2.1 or daf-16 mutants. The effects of PWB in the C. elegans model appeared to be primarily mediated via sir-2.1, daf-16, and daf-16/daf-2. These data suggest that PWB and ß-glucans may benefit hyperglycaemia-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 alpha-hordothionin and wheat beta-purothionin, revealed that anions can trigger dynamic and structural changes in the thionin antiparallel double alpha-helix core. Analysis of the molecular dynamics simulations demonstrated that anions induced unfolding of the alpha2 and alpha1 helices at the carboxyl ends which are located on the opposite ends of the alpha-helix core. An internalized water molecule was observed inside the unfolded alpha2 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 alpha2 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 alpha2 C-end were necessary for unfolding of the alpha1 C-end. Anions interacted primarily with residues of the phospholipid-binding site and the R30 network while the alpha1/alpha2 hydrophobic region was void of anions. However, during strong interactions of anions with the R30 network and phospholipid-binding site, the alpha1/alpha2 hydrophobic region attracted anions which interacted with conserved residues of the alpha1 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 alpha-helix core by interactions of anions with the phospholipid-binding site may be relevant to a mechanism of membrane-permeabilizing activity.

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 beta-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. Hecate, hecate-betaCG 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 betaCG 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-betaCG via the lateral tail vein. The ability of hecate-betaCG to destroy xenografts of human breast cancer cells (MDA-MB-435S) in nude mice was demonstrated for the first time. We conclude that hecate-betaCG and LHRH-hecate conjugates could serve as useful drugs for the treatment of breast cancer.

Effects of a lytic peptide conjugated to beta 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 beta-chain of hCG (hecate-beta 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-beta 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-beta 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-beta 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-beta 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-beta hCG, and estradiol + hecate-beta 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 < 0.05). Immunocytochemical studies revealed the expression of LH/hCG receptor protein in the OVCAR-3 cells and tumor tissues. CONCLUSION: Hecate-beta hCG is a putative candidate for treating ovarian 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 beta-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 < 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.