Effects of C-terminal amidation and heptapeptide ring on the biological activities and advanced structure of amurin-9KY, a novel antimicrobial peptide identified from the brown frog, Rana kunyuensis

Rana kunyuensis is a species of brown frog that lives exclusively on Kunyu Mountain,Yantai,China.In the current study,a 279-bp cDNA sequence encoding a novel antimicrobial peptide (AMP),designated as amurin-9KY,was cloned from synthesized double-strand skin cDNA of R.kunyuensis.The amurin-9KY precursor was composed of 62 amino acid (aa) residues,whereas the mature peptide was composed of 14 aa and contained two cysteines forming a C-terminal heptapeptide ring (Rana box domain) and an amidated C-terminus.These structural characters represent a novel amphibian AMP family.Although amurin-9KY exhibited high similarity to the already identified amurin-9AM from R.amurensis,little is known about the structures and activities of amurin-9 family AMPs so far.Therefore,amurin-9KY and its three derivatives (amurin-9KY1-3) were designed and synthesized.The structures and activities were examined to evaluate the influence of C-terminal amidation and the heptapeptide ring on the activities and structure of amurin-9KY..Results indicated that C-terminal amidation was essential for antimicrobial activity,whereas both C-terminal amidation and the heptapeptide ring played roles in the low hemolytic activity.Circular dichroism (CD) spectra showed that the four peptides adopted an α-helical conformation in THF/H2O (v/v 1∶1) solution,but a random coil in aqueous solution.Elimination of the C-terminal heptapeptide ring generated two free cysteine residues with unpaired thiol groups,which greatly increased the concentration-dependent anti-oxidant activity.Scanning electron microscopy (SEM) was also performed to determine the possible bactericidal mechanisms.

Identification and characterization of two novel cathelicidins from the frog Odorrana livida

Antimicrobial peptides(AMPs) are a group of gene-encoded small peptides that play pivotal roles in the host immune system of multicellular organisms.Cathelicidins are an important family of AMPs that exclusively exist in vertebrates. Many cathelicidins have been identified from mammals, birds, reptiles and fish. To date, however, cathelicidins from amphibians are poorly understood. In the present study, two novel cathelicidins(OL-CATH1 and 2) were identified and studied from the odorous frog Odorrana livida.Firstly, the cDNAs encoding the OL-CATHs(780 and735 bp in length, respectively) were successfully cloned from a lung cDNA library constructed for the frog. Multi-sequence alignment was carried out to analyze differences between the precursors of the OL-CATHs and other representative cathelicidins.Mature peptide sequences of OL-CATH1 and 2 were predicted(33 amino acid residues) and their secondary structures were determined(OL-CATH1 showed a random-coil conformation and OL-CATH2 demonstrated α-helical conformation). Furthermore,OL-CATH1 and 2 were chemically synthesized and their in vitro functions were determined. Antimicrobial and bacterial killing kinetic analyses indicated that OL-CATH2 demonstrated relatively moderate and rapid antimicrobial potency and exhibited strong anti-inflammatory activity. At very low concentrations(10 μg/mL), OL-CATH2 significantly inhibited the lipopolysaccharide(LPS)-induced transcription and production of pro-inflammatory cytokines TNF-α, IL-1βand IL-6 in mouse peritoneal macrophages. In contrast, OL-CATH1 did not exhibit any detectableantimicrobial or anti-inflammatory activities. Overall,identification of these OL-CATHs from O. livida enriches our understanding of the functions of cathelicidins in the amphibian immune system. The potent antimicrobial and anti-inflammatory activities of OL-CATH2 highlight its potential as a novel candidate in anti-infective drug development.

Intestinal microflora in patients with liver disease

Intestinal microflora is closely related to the occurrence and development of liver diseases.The imbalance of intestinal microflora can cause the increase of endotoxin and change the metabolic pathways such as lipid metabolism,thus causing the occurrence of liver diseases.It is beneficial to explore the effects of intestinal microflora on metabolism and its relationship with liver disease,which maybe reveal the pathogenesis of liver disease and find new therapeutic targets for liver disease.This paper reviewed the effects of intestinal microflora on endotoxin,short-chain fatty acid metabolism,fatty acid metabolism,bile acid metabolism,choline metabolism and endogenous alcohol production,and analyzed the relationship between intestinal microflora and liver disease.

Dietary Guidelines for Chinese Residents(2016):comments and comparisons

A high quality diet is believed to play a functional role in promoting the healthy growth of mankind and preventing many kinds of chronic degenerative diseases, including cancer, cardiovascular disease, diabetes, and obesity. Adherence to a high quality diet has been strongly associated with a lower risk of mortality. To help promote healthy lifestyles and physical strength, the Chinese government has produced a new revised version of the Dietary Guidelines for Chinese Residents （2016） and the Chinese Food Pagoda, as guidance for dietary intake among its population. Similarly, the Japanese government has produced the Japanese Food Guide Spinning Top Model, and the US government has recently published revised dietary recommendations in its 2015-2020 eighth edition of Dietary Guidelines for Americans. The evidence from all respective cohort studies involved in producing these guidelines shows a reduced risk of many chronic diseases and mortality if the guidelines are followed. All scientific findings support encouraging the general population to consume a broad variety of food on the basis of nutrient and food intakes in order to prevent deficiency diseases and a surplus of energy and nutrients, and recommend daily physical activity for health promotion.

Colorectal cancer cell growth inhibition by linoleic acid is related to fatty acid composition changes

Polyunsaturated fatty acids （PUFAs） possess anti-cancer action both in vitro and in vivo. In the present study, we detected cell viability with methyl thiazolyl tetrazolium （MTT） assay and cell membrane permeability with propidium iodide （PI） fluorescence dyeing, and calculated cell membrane fluidity change as fluorescence anisotropy. Fatty acid content in cells was measured by gas chromatography/mass spectroscopy （GC/MS）, and the relationship between fatty acid composition and cell viability was studied. We observed that n-6 PUFA linoleic acid （LA） inhibited tumor cell growth at high concentrations （〉300 μmol/L）, while low concentrations （100-200 μmol/L） seemed to promote cell proliferation. Analyses of cell membrane permeability, cell membrane fluidity, and cell fatty acid composition suggested that the anti-cancer action of LA could be related to changes in the ratio of n-6 to n-3 PUFAs. We observed that pre-incubation of cancer cells with 100 μmol/L LA for 24 h enhanced cell sensitivity to the cytotoxic action of LA, whereas undifferentiated cell line LoVo seemed to have a distinct path in LA-induced death. These results showed that one of the mechanisms by which supplementation of LA induces cancer cell death could be altering the ratio of n-6/n-3 PUFAs, and this may be related to celt differentiation status.

Epigallocatechin-3-gallate affects the growth of LNCaP cells via membrane fluidity and distribution of cellular zinc

Objective:To evaluate effects of epigallocatechin-3-gallate (EGCG) on the viability, membrane properties, and zinc distribution, with and without the presence of Zn2+, in human prostate carcinoma LNCaP cells. Methods: We examined changes in cellular morphology and membrane fluidity of LNCaP cells, distribution of cellular zinc, and the incorporated portion of EGCG after treatments with EGCG, Zn2+, and EGCG+Zn2+. Results: We observed an alteration in cellular morphology and a decrease in membrane fluidity of LNCaP cells after treatment with EGCG or Zn2+. The proportion of EGCG incorporated into liposomes treated with the mixture of EGCG and Zn2+ at the ratio of 1:1 was 90.57%, which was significantly higher than that treated with EGCG alone (30.33%). Electron spin resonance (ESR) studies and determination of fatty acids showed that the effects of EGCG on the membrane fluidity of LNCaP were decreased by Zn2+. EGCG accelerated the accumulation of zinc in the mitochondria and cytosol as observed by atomic absorption spectrometer. Conclusion: These results show that EGCG interacted with cell membrane, decreased the membrane fluidity of LNCaP cells, and accelerated zinc accumulation in the mitochondria and cytosol, which could be the mechanism by which EGCG inhibits proliferation of LNCaP cells. In addition, high concentrations of Zn2+ could attenuate the actions elicited by EGCG.

Molecularly imprinted polymers as the extracted sorbents of clenbuterol ahead of liquid chromatographic determination

A pre-treatment methodology for clenbuterol hydrochloride （CLEN） isolation and enrichment in a complex matrix environment was developed through exploiting molecularly imprinted polymers （MIPs）. CLEN-imprinted pol- ymers were synthesized by the combined use of ally-β-cyclodextrin （ally-13-CD） and methacrylic acid （MAA）, allyl-β-CD and acrylonitrile （AN）, and allyl-β-CD and methyl methacrylate （MMA） as the binary functional monomers. MAA-linked allyl-β-CD MIPs （M-MAA） were characterized by Fourier transform-infrared （FT-IR） spectroscopy and a scanning electron microscope （SEM）. Based upon the results, M-MAA polymers generally proved to be an excellent selective extraction compared to its references： AN-linked allyl-β-CD MIPs （M-AN） and MMA-linked allyl-β-CD MIPs （M-MMA）. M-MAA polymers were eventually chosen to run through a molecularly imprinted solid-phase extraction （MISPE） micro-column to enrich CLEN residues spiked in pig livers. A high recovery was achieved, ranging from 91.03% to 96.76% with relative standard deviation （RSD） ≤4.45%.

Effects of astaxanthin on oxidative stress induced by Cu^(2+) in prostate cells

Astaxanthin（AST）, a carotenoid molecule extensively found in marine organisms and increasingly used as a dietary supplement, has been reported to have beneficial effects against oxidative stress. In the current paper, the effects of AST on viability of prostate cells were investigated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide（MTT） assay; cell apoptosis and intracellular reactive oxygen species（ROS） levels were determined by flow cytometry; the mitochondrial membrane potential（MMP） was measured by fluorospectrophotometer; and activities of malondialdehyde（MDA）, superoxide dismutase（SOD）, catalase（CAT）, and glutathione peroxidase（GSH-Px） were evaluated by a detection kit. The results show that copper ion（Cu^2＋） induced apoptosis, along with the accumulation of intracellular ROS and MDA, in both prostate cell lines（RWPE-1 and PC-3）. AST treatments could decrease the MDA levels, increase MMP, and keep ROS stable in RWPE-1 cell line. An addition of AST decreased the SOD, GSH-Px, and CAT activities in PC-3 cell line treated with Cu^2＋, but had a contrary reaction in RWPE-1 cell lines. In conclusion, AST could contribute to protecting RWPE-1 cells against Cu^2＋-induced injuries but could cause damage to the antioxidant enzyme system in PC-3 cells.