Comparative sequence analysis of citrate synthase and 18S ribosomal DNA from a wild and mutant strains of Aspergillus niger with various fungi

A mutation was induced in Aspergillus niger wild strain using ethidium bromide resulting in enhanced expression of citric acid by three folds and 112.42 mg/mL citric acid was produced under optimum conditions with 121.84 mg/mL of sugar utilization. Dendograms of 18S rDNA and citrate synthase from different fungi including sample strains were made to assess homology among different fungi and to study the correlation of citrate synthase gene with evolution of fungi. Subsequent comparative sequence analysis revealed strangeness between the citrate synthase and 18S rDNA phylogenetic trees. Furthermore, the citrate synthase movement suggests that the use of traditional marker molecule of 18S rDNA gives misleading information about the evolution of citrate synthase in different fungi as it has shown that citrate synthase gene transferred independently among different fungi having no evolutionary relationships. Random amplified polymorphic DNA (RAPD-PCR) analysis was also employed to study genetic variation between wild and mutant strains of A. niger and only 71.43% similarity was found between both the genomes. Keeping in view the importance of citric acid as a necessary constituent of various food preparations, synthetic biodegradable detergents and pharmaceuticals the enhanced production of citric acid by mutant derivative might provide significant boost in commercial scale viability of this useful product.

Abbreviations

CS - Citrate synthase, CA - Citric acid, RAPD - Random amplified polymorphic DNA, TAF - Total amplified fragments, PAF - Polymorphic amplified fragments, CAF - Common amplified fragments.     

Modelling the Effect of a Functional Endothelium on the Development of In-Stent Restenosis

Treatment of stenosed coronary arteries by balloon angioplasty and stenting results in arterial injury including severe damage to the endothelium at the site of treatment and initiates a complex cascade of inflammatory processes that may lead to the development of in-stent restenosis (ISR). Many clinical and biological factors involved in the progression of restenotic lesions have been studied in detail over the past few years but the mystery behind the pathophysiological mechanisms of this disease is still unresolved. In the present work, the effects of re-endothelialization and nitric oxide release on neointimal growth are investigated in-silico using a two dimensional multi-scale model of ISR. The effect of stent deployment depths on the development of ISR is studied as a function of time after stenting. Two dimensional domains were prepared by deploying bare metal stent struts at three different deployment depths into the tissue. Shear stress distribution on endothelial cells, obtained by blood flow simulations, was translated into nitric oxide production that keeps the smooth muscle cells in quiescent state. The cellular growth trends were plotted as a function of time and the data indicate a positive correlation between the neointimal growths and strut deployment depths in the presence of a functional endothelium, in qualitative agreement with in-vivo data. Additionally, no ISR is observed if a functional endothelium appears much earlier.     

Simple inhibition studies for distinction between homodimeric and heterodimeric isoenzymes of glutathione transferase

Simple inhibition studies in which fractional velocity is measured as a function of inhibitor concentration were used to distinguish heterodimeric from homodimeric isoenzymes of glutathione transferase. Homodimeric isoenzymes from rat, mouse, and human tissues were shown to give graphs of fractional velocity versus the logarithm of inhibitor concentration that were characterized by a sigmoid curve shape and a maximal slope of -0.58 at 50% inhibition, characteristic for linear inhibition of monomeric or non-cooperative oligomeric enzymes. In contrast, inhibition curves for heterodimeric isoenzymes from rat liver displayed significant deviations from these characteristics. The basis for the identification of heterodimers was the finding that the kinetic properties of subunits were additive such that the inhibition curve of a heterodimeric isoenzyme could be predicted from those of the corresponding homodimers. The method should be valuable in the differentiation between the multiple forms of glutathione transferase in tissues not previously characterized. It is suggested that the method should be applicable for discrimination also in other isoenzyme families consisting of oligomeric structures of identical and nonidentical subunits.     

Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota

Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+) ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA"). We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.     

Clinical presentation of depression among Malaysian women in Penang Island

Objectives To identify the aetiology and clinical presentation of depression among Malaysian women.Methods A cross-sectional study was conducted at the Psychiatry Clinic, Public Hospital, Pulau Penang, Malaysia. Retrospective evaluations of records were conducted between January 2002 and December 2007. The data were analysed using the statistical software, SPSS v. 131®.Results Ninety-six (56.8%) of the patients were Chinese, the mean (± SD) age of the patients was 45 ± 17.8 years, with a majority (72; 42.6%) aged over 50 years. The incidence of depression with comorbid hypertension and comorbid diabetes mellitus was significant among women aged over 50 (P < 0.001 (hypertension) P < 0.015 (diabetes mellitus)). Marital and relationship problems were found to significantly affect Chinese women aged 15–30 years (P = 0.019). In terms of the clinical presentation of depression among Malaysian women, suicidal ideation and somatic symptoms like reduced energy/being easily fatigued were more frequent among Chinese.Conclusion Symptoms of being short-tempered, crying, restless and doubtful/distracted should not be neglected in primary care because of the possibility of mental health disorders. The timely evaluation of diabetic and hypertensive patients is an ideal strategy to prevent mental health disorders.     

New bioactive natural products from Launaea nudicaulis

Ethyl acetate soluble fraction of methanolic extract of Launaea nudicaulis was subjected to chromatographic purification to get four new compounds including a quinic acid derivative (1), a pentahydroxy acetylene analog: trideca-12-ene-4,6-diyne-2,8,9,10,11-pentaol (2), a flavone glycoside (3) and a sesquiterpene lactone (4) together with 10 known compounds. The structures of the new isolates were established by using 1D, 2D NMR techniques and high-resolution mass spectrometry, whereas, the known isolates were identified based on 1D NMR and mass spectrometric information and in comparison with the reported data in the literature. The structure of 4 was also confirmed through single X-ray crystallographic analysis. Cholistaquinate (1) exhibited significant activity in DPPH free radical scavenging assay with an IC₅₀ value of 60.7 μM, whereas, nudicholoid (4) exhibited a moderate inhibitory activity against the enzyme butyrylcholinesterase with an IC₅₀ value of 88.3 μM.     

Gene expression profiles of vitrified in vitro- and in vivo-derived bovine blastocysts

Vitrification is becoming a preferred method for pre‐implantation embryo cryopreservation. The objective of this study was to determine the differentially expressed genes of in vivo‐ and in vitro‐produced bovine embryos after vitrification. In vitro‐ (IVF) and in vivo‐derived (IVV) bovine blastocysts were identified as follows: in vitro‐produced fresh (IVF‐F), in vitro‐produced vitrified (IVF‐V), in vivo‐derived fresh (IVV‐F), in vivo‐derived vitrified (IVV‐V). The microarray results showed that 53 genes were differentially regulated between IVF and IVV, and 121 genes were differentially regulated between fresh and vitrified blastocysts (P < 0.05). There were 6, 268, 962, and 17 differentially regulated genes between IVF‐F × IVV‐F, IVF‐V × IVV‐V, IVF‐F × IVF‐V, and IVV‐F × IVV‐V, respectively (P < 0.05). While gene expression was significantly different between fresh and vitrified IVF blastocysts (P < 0.05), it was similar between fresh and vitrified IVV blastocysts. Significantly up‐regulated KEGG pathways included ribosome, oxidative phosphorylation, spliceosome, and oocyte meiosis in the fresh IVF blastocyst samples, while sphingolipid and purine metabolisms were up‐regulated in the vitrified IVF blastocyst. The results showed that in vitro bovine blastocyst production protocols used in this study caused no major gene expression differences compared to those of in vivo‐produced blastocysts. After vitrification, however, in vitro‐produced blastocysts showed major gene expression differences compared to in vivo blastocysts. This study suggests that in vitro‐produced embryos are of comparable quality to their in vivo counterparts. Vitrification of in vitro blastocysts, on the other hand, causes significant up‐regulation of genes that are involved in stress responses. Mol. Reprod. Dev. 79: 613–625, 2012. © 2012 Wiley Periodicals, Inc.     

Complete Genome Sequencing of a Velogenic Viscerotropic Avian Paramyxovirus 1 Isolated from Pheasants (Pucrasia macrolopha) in Lahore,Pakistan

We report the complete genome sequence of avian paramyxovirus 1 (APMV-1) isolated from an acute and highly contagious outbreak in pheasants (Pucrasia macrolopha) in Lahore, Pakistan. Biological and serological characterization showed a velogenic strain of APMV-1, which was further confirmed by the sequence analysis of the cleavage site in the fusion protein. Complete genome sequencing and phylogenetic analysis indicated that this isolate belonged to genotype VII, specifically to subgenotype VIIa, and clustered closely with isolates characterized from Indonesia. Notably, the isolate showed significant differences from previously characterized APMV-1 from Pakistani commercial and rural chicken.