Species-specific variations in the molecular heterogeneity of the platelet-activating factor

The molecular heterogeneity of platelet-activating factor (PAF) synthesized by unstimulated and Ca2+ ionophore (A23187)-stimulated PMN from rat, mouse, and guinea pig and by rat basophilic leukemia (RBL) cells was investigated by gas chromatography-negative ion chemical ionization mass spectrometry. Several molecular species of PAF ranging from C14:0 to C19:0 were detected in all of the cells studied. PAF produced by each cell type exhibited a unique pattern of molecular species distribution. Although C16:0 was the major PAF molecular species of rat PMN and RBL cells representing 96% and 85% of the total PAF, respectively, PAF from mice PMN contained 81% of C16:0, 10% of C18:1, and 6% of C18:0. Alternatively, A23187-stimulated guinea pig PMN yielded PAF molecular species 35% in C16:0, 35% in C17:0, 8% in C18:1, and 3% in C18:0. Small but significant differences in the PAF molecular species distribution of resting and ionophore stimulated cells were also observed. In contrast to the PAF molecular species composition, the precursor 1-O-alkyl-2-acyl-glycero-3-phosphocholine of all the cell types was predominantly hexadecyl (C16:0) alkyl chain in the sn-1 position, representing 60 to 80% of the total 1-O-alkyl-2-acyl-glycero-3-phosphocholine. Thus, these results not only indicate a high degree of selectivity for utilization of precursor substrates for PAF biosynthesis, but also demonstrate that the selectivity is species specific.     

Fatty acid composition of diacyl, alkylacyl, and alkenylacyl phospholipids of control and arachidonate-depleted rat polymorphonuclear leukocytes

Phospholipid fatty acid composition and phospholipid subclass distribution of control and arachidonate-depleted rat polymorphonuclear leukocytes (PMN) were compared. The 20:4-depleted PMN contained significantly higher amounts of 16:1, 18:1 and 20:3 (delta 5,8,11) and lower amounts of 18:2 and 20:4 than the phospholipids from control cells. Choline-containing glycerophospholipids (CGP) were the major phospholipids of both control and 20:4-depleted cells representing 34% and 37% of the total phospholipids, respectively. Significant amounts of ethanolamine-containing glycerophospholipids (EGP) (29% and 30%) and sphingolipids (20% and 18%) were also present in both cell types. Serine-containing glycerophospholipids (SGP) together with inositol-containing glycerophospholipids (IGP) constituted 16% and 13% of the phospholipids in control and 20:4-depleted cells, respectively. CGP from control cells had significantly higher amounts of 16:0 and 18:2 and lower amounts of 18:0 and 20:4 than EGP, whereas CGP from 20:4-depleted cells has higher amounts of 16:0 and 16:1 and lower amounts of 20:3 than EGP. Analysis of the subclass composition of CGP and EGP revealed that both control and 20:4-depleted cells contained significantly large amounts of alkylacyl-GPC and alkenylacyl-GPE. Small amounts of alkylacyl-GPE and alkenylacyl-GPC were also observed. The predominant fatty acyl residues found in the 1,2-diacyl-GPC, alkylacyl-GPC of control cells were 16:0, 18:0, 18:1, 18:2, and 20:4, while those of 20:4-depleted cells were 16:0, 16:1, 18:0, 18:1, and 20:3. More than 60% of CGP-bound 20:4 of control cells and about 70% of the CGP-bound 20:3 of 20:4-depleted cells were found in their alkylacyl species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cold stress induces in situ phospholipid molecular species changes in cell surface membranes

The structural organization of Tetrahymena pyriformis is such that its cilia are remote from the main centers of lipid metabolism. As a result, the ciliary membrane lipid composition of cells exposed to low-temperature stress is initially unaffected by the significant metabolic changes induced in microsomal membranes. Nevertheless, changes in the ciliary membrane lipid composition can be detected during the first 4 h of cold exposure. A combination of in vivo and in vitro experiments has provided strong evidence for a substantial retailoring of ciliary phospholipid molecular species in situ in the absence of any importation of lipids from the cell interior or change in overall ciliary fatty acid composition. The mechanism responsible for the ciliary lipid changes is independent of the one(s) triggering internal acclimation responses. Our observations establish for the first time that chilling stress can simultaneously induce separate and distinctive lipid modification responses in different parts of a cell. This finding could be important in identifying the molecular ‘sensor’ capable of actuating stress-induced lipid changes.     

Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Mulberry (Morus alba L.) under different abiotic stresses

Molecular Biology Reports - Mulberry (Morus alba L.) is the sole food source for the mulberry silkworm, Bombyx mori and therefore important for sericulture industry. Different abiotic stress...     

Utilization of 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide (EDCI)/1-hydroxybenzotriazole (HOBt) as a polymerizing agent

The commonly used coupling reagents in peptide synthesessuch as dicyclohexylcabodiimide, diisopropylcarbodiimide and3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide with or without1-hydroxybenzotriazole or N-hydroxysuccinimide have been used as polymerizing agents in the synthesis of elastic/plastic protein-based polymers. It was found that 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide with 1-hydroxybenzotriazole gave equally good polymers comparable toconventional p-nitrophenol approach. Further, we present here the polymerization and characterization of structural andfunctional properties of poly(Val-Pro-Gly-Val-Gly), which is themost striking repeating sequence in the bovine and porcine elastins. The polymers obtained by both p-nitrophenol and 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide approach werecharacterized by carbon-13 and proton nuclear magnetic resonancespectroscopy, differential scanning calorimetry, circular dichroism and fourier transform infrared spectroscopy. These results conclude that poly(Val-Pro-Gly-Val-Gly) obtained by bothmethods were identical in all respects of physical and chemicalproperties indicates that 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide with 1-hydroxybenzotriazole method can be conveniently employed to synthesize these polymers.     

Biochemical and molecular analyses of copper-zinc superoxide dismutase from a C4 plant Pennisetum glaucum reveals an adaptive role in response to oxidative stress

Cadherin 23 (CDH23) is an important constituent of the hair cell tip link in the organ of Corti. Mutations in cdh23 are associated with age-related hearing loss (AHL). In this study, we proposed that the Cdh23(nmf308/nmf308) mice with progressive hair cell loss had specific morphological changes and suffered a base to apex gradient and age-related hearing loss, and that mutations in cdh23 were linked to AHL. The Cdh23(nmf308/nmf308) mice produced by the N-nitrosourea (ENU) mutagenesis program were used as an animal model to study AHL and progressive hair cell loss. RT-PCR was performed to confirm the cdh23 mutation in Cdh23(nmf308/nmf308) mice and genetic analysis was used to map the specific mutation site. Distortion product otoacoustic emission (DPOAE) assay and acoustic brainstem evoked response (ABR) threshold analysis were carried out to evaluate the AHL. Cochlear histology was examined with scanning electron microscope (SEM) and transmission electron microscope (TEM), as well as the nuclear labeling by propidium iodide staining; terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and caspase-3 activities were examined to evaluate cell apoptosis. Genetic mapping identified the candidate gene linking AHL in Cdh23(nmf308/nmf308) mice as cdh23. A mutation in exon3 (63 T>C) was screened as compared with the sequence of the same position of the gene from B6 (+/+) mice. The cochleae outer hair cells were reduced from 5-10% at one month to 100% at three months in the basal region. DPOAE and ABR exhibited an increasing threshold at high frequencies (≥16kHz) from one month of age. Morphological and cellular analysis showed that Cdh23(nmf308/nmf308) mice exhibited a time course of histological alterations and cell apoptosis of outer hair cells. Our results suggest that the cdh23 mutation may be harmful to the stereociliary tip link and cause the hair cell apoptosis. Due to the same cdh23 mutations in human subjects with presbycusis (Petit et al., 2001; Zheng et al., 2005), the Cdh23(nmf308/nmf308) mouse is an excellent animal model for investigating the mechanisms involved in human AHL.     

Fusarium proliferatum, an endophytic fungus from Dysoxylum binectariferum Hook.f, produces rohitukine, a chromane alkaloid possessing anti-cancer activity

Rohitukine is a chromane alkaloid possessing anti-inflammatory, anti-cancer and immuno-modulatory properties. The compound was first reported from Amoora rohituka (Meliaceae) and later from Dysoxylum binectariferum (Meliaceae) and Schumanniophyton problematicum (Rubiaceae). Flavopiridol, a semi-synthetic derivative of rohitukine is a potent CDK inhibitor and is currently in Phase III clinical trials. In this study, the isolation of an endophytic fungus, Fusarium proliferatum (MTCC 9690) from the inner bark tissue of Dysoxylum binectariferum Hook.f (Meliaceae) is reported. The endophytic fungus produces rohitukine when cultured in shake flasks containing potato dextrose broth. The yield of rohitukine was 186 μg/100 g dry mycelial weight, substantially lower than that produced by the host tissue. The compound from the fungus was authenticated by comparing the LC–HRMS and LC–HRMS/MS spectra with those of the reference standard and that produced by the host plant. Methanolic extract of the fungus was cytotoxic against HCT-116 and MCF-7 human cancer cell lines (IC₅₀ = 10 μg/ml for both cancer cell lines).     

Virus isolation and molecular epidemiology of highly pathogenic avian influenza A(H5N8) from an outbreak in free-ranging wild birds,India 2016

We report the molecular epidemiology of highly pathogenic avian influenza (HPAI) virus involved in an outbreak causing death in free-ranging wild birds at Mysore, Karnataka state of India. The virus was typed as HPAI A(H5N8) by conventional and TaqMan probe based real-time PCR assays. Six isolates of HPAI virus were recovered in 9-day-old embryonated chicken eggs. Haemagglutinin gene-based phylogeny of virus isolates showed >?99.9% nucleotide sequence identity with HPAI A(H5N8) isolates from migratory birds and domestic poultry from China and Korea indicating either these wild birds have routed their migration through Korea and/or eastern China or these dead birds must have directly or indirectly contacted with wild birds migrating from Eastern China and/or Korean regions. The study emphasises the role of migratory wild birds in spread of HPAI across the globe.     

Synonymous codon usage pattern among the S,M, and L segments in Crimean-congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever (CCHF) virus is one among the major zoonosis viral diseases that use the Hyalomma ticks as their transmission vector to cause viral infection to the human and mammalian community. The fatality of infectious is high across the world especially in Africa, Asia, Middle East, and Europe. This study regarding codon usage bias of S, M, and L segments of the CCHF virus pertaining to the host Homo sapiens, reveals in-depth information about the evolutionary characteristics of CCHFV. Relative Synonymous Codon Usage (RSCU), Effective number of codons (ENC) were calculated, to determine the codon usage pattern in each segment. Correlation analysis between Codon adaptation index (CAI), GRAVY (Hydrophobicity), AROMO (Aromaticity), and nucleotide composition revealed bias in the codon usage pattern. There was no strong codon bias found among any segments of the CCHF virus, indicating both the factors i.e., natural selection and mutational pressure shapes the codon usage bias.