基于iTRAQ技术对植物乳杆菌FS5-5的耐盐特性分析

【目的】对大酱中耐盐性较好的植物乳杆菌进行蛋白质组学研究,为植物乳杆菌盐胁迫应激机制的研究提供实验数据。【方法】本项研究以筛选自东北传统农家大酱的耐盐性较好的植物乳杆菌FS5-5为研究对象,绘制了其在0%、6.0%、7.0%和8.0%(W/V)Na Cl浓度下的生长曲线,并利用i TRAQ技术研究了其在0%、6.0%、7.0%和8.0%(W/V)Na Cl浓度下的蛋白质表达情况。【结果】植物乳杆菌FS5-5在0%、6.0%、7.0%和8.0%(W/V)Na Cl浓度下到达对数生长期中期的时间点分别为5、10、12和12 h;以差异倍数在1.2倍以上且P0.05为筛选条件对6.0%、7.0%和8.0%(W/V)Na Cl浓度下与0%进行差异蛋白质的筛选,共筛选出1271个差异蛋白质。这些差异蛋白质主要参与糖代谢、氨基酸代谢、脂肪酸代谢、核苷酸代谢、应激反应、转运、PTS系统和核糖体代谢等。【结论】植物乳杆菌在高盐浓度下生长与能量合成蛋白质、应激蛋白质以及相容性溶质转运蛋白质的表达上调有密切关系。     

In vitro examination of anti-parasitic,anti-Alzheimer,insecticidal and cytotoxic potential of Ajuga bracteosa Wallich leaves extracts

This research study is mainly focused to evaluate the anti-parasitic, insecticidal, cytotoxic and anti-alzheimer potential of various leaf extracts of Ajuga bracteosa Wallich ex Bentham. 04 different extracts were prepared using solvent of different polarity to determine the best candidate for potent bioactivity i.e. n-hexane (NH), Ethyl acetate (EA), Ethanol (EL) and Chloroform (CH). Concentrations of each extracts were made specified for all activities. All extracts were exploited for broad range of biomedical applications including leishmaniasis, in vitro anti-Alzheimer, insecticidal and cytotoxic studies. Our results showed that A. bracteosa n-hexane extract was highly active against Leishmania Tropica with significant inhibition of 58 ± 1.61 for promastigote and 63 ± 2.29 for amastigote at 1000 μg/mL. Furthermore, promising anti-alzheimer activity acetylcholinesterase (AChE) 46 ± 0.83 and butrylcholineterase (BChE) 49 ± 1.17 was noted for n-hexane. The insecticidal potential of these extracts were test against five different insects (Rhyzopertha dominica, Trogoderma granarium, Tribolium castaneum, Sitophilus oryze, and Callosobruchus analis). The higest mortality rate of insecticidal activity was recorded by n-hexane followed by Ethyl acetate whereas ethanol extract was found to be less effective against all the test species. Significant cytotoxic potential of each plant sample against Artemia salina thus aware us for further detailed research to find out novel drugs. Based on our results we believe that Ajuga bracteosa could be used to develop as a potential botanical insecticide against different insect and pests, such as aphids as well as an excellent source for the compound isolation as anti-tumor agent.     

Human leukocyte antigen alleles,genotypes and haplotypes frequencies in renal transplant donors and recipients from West Central India

BACKGROUND:

Human leukocyte antigen (HLA) is comprised of a highly polymorphic set of genes which determines the histocompatibility of organ transplantation. The present study was undertaken to identify HLA class I and class II allele, genotype and haplotype frequencies in renal transplant recipients and donors from West Central India.

MATERIALS AND METHODS:

HLA typing was carried out using Polymerase Chain Reaction-Sequence Specific Primer in 552 live related and unrelated renal transplant recipients and donors.

RESULTS:

The most frequent HLA class I and class II alleles and their frequencies in recipients were HLA-AFNx0101 (0.1685) and AFNx0102 (0.1649), HLA-BFNx0135 (0.1322), and HLA-DR beta 1 (DRB 1)FNx0115 (0.2192), whereas in donors, these were HLA-AFNx0102 (0.1848) and AFNx0101 (0.1667), HLA-BFNx0135 (0.1359), and HLA-DRB1FNx0115 (0.2409). The two-locus haplotype statistical analysis revealed HLA-AFNx0102-B61 as the most common haplotype with the frequency of 0.0487 and 0.0510 in recipients and donors, respectively. Further, among the three locus haplotypes HLA-AFNx0133-BFNx0144-DRB1FNx0107 and HLA-AFNx0102-BFNx0161-DRB1FNx0115 were the most common haplotypes with frequencies 0.0362 and 0.0326, respectively in recipients and 0.0236 and 0.0323, respectively in donors. Genotype frequency revealed a high prevalence of genotype HLA-AFNx0102/AFNx0124 in recipients (0.058) compared to donors (0.0109) whereas low prevalence of HLA-AFNx0101/AFNx0102 in recipients (0.0435) than in donors (0.0797). The phylogenetic and principal component analysis of HLA allele and haplotype frequency distribution revealed genetic similarities of various ethnic groups. Further, case control analysis provides preliminary evidence of association of HLA-A genotype (P < 0.05) with renal failure.

CONCLUSION:

This study will be helpful in suitable donor search besides providing valuable information for population genetics and HLA disease association analysis.     

Effect of sequential deletion of extra N-terminal residues on the structure and stability of yeast iso-1-cytochrome-c

A sequence alignment of yeast cytochrome-c (y-cyt-c) with mammalian cyts-c shows that the yeast protein has a five residue long N-terminal extension. A question arises: Does this N-terminal extension play any roles in the stability, structure, and folding of the yeast protein? To answer this question, in silico and in vitro studies were carried out on the wild type (WT) protein and its five deletants (Δ(?5/?5), Δ(?5/?4), Δ(?5/?3), Δ(?5/?2), and Δ(?5/?1) where Δ denotes the deletion and the numbers refer to the residues deleted, e.g. Δ(?5/?1) denotes the deletion of residues numbered from ?5 to ?1 (TEFKA), while Δ(?5/?2) denotes the deletion of resides numbered from ?5 to ?2 (TEFK) and so on). The main conclusion of the in silico study is that the order of stability of deletants and WT protein is Δ(?5/?4) > WT > Δ(?5/?3) > Δ(?5/?5) > Δ(?5/?1) ~ Δ(?5/?2). In vitro studies involved (i) measurements of thermodynamic stability of all proteins by differential scanning calorimetry and from sigmoidal curves of two different structural properties ([θ]₂₂₂, a probe for detecting change in secondary structure, and Δε₄₀₅, a probe for detecting alteration in the heme environment), and (ii) characterization of all proteins by various spectral properties. The main conclusions of the in vitro studies are as follows: (i) The order of thermodynamic stability of all proteins is in excellent agreement with that predicted by in silico studies, and (ii) A sequential deletion of the N-terminal extension has no effects on protein structure and folding.     

Genetic diversity,demographical history and conservation aspects of the endangered yew tree Taxus contorta (syn. Taxus fuana) in Pakistan

The western Himalayan yew (Taxus contorta Griffith, synonym Taxus fuana Nan Li & R.R. Mill), one of the highly prized tree species for its anticancer properties, is experiencing severe reduction in populations across its range in Pakistan. We examined a chloroplast DNA region (trnL-F) and ten nuclear microsatellite loci variations among seven populations to investigate the levels of genetic diversity and reconstruct the demographic history of T. contorta in Pakistan. A low haplotype diversity (H _T?=?0.182), moderate level of microsatellite diversity (H _E?=?0.541?±?0.034), significant population differentiation (F _ST?=?0.107) and high level of inbreeding (F _IS?=?0.219–0.418) characterized this species. Results of an approximate Bayesian computation (ABC), mismatch distribution analysis, neutrality tests and data on glacial chronologies of the western Himalaya indicated a historic demographic expansion of T. contorta populations from small ancestral populations. This expansion is estimated to have occurred at least 37.5 thousand years ago (kya) during the late Pleistocene. An unprecedented level of habitat disturbance over the last few decades, coupled with the highly variable climate may have played an important role in shaping the genetic structure of the extant T. contorta populations. We have discussed a range of sustainable management measures for the ecological restoration of remnant populations as well as for a sustainable exploitation of this plant of high economic potential.     

In Macrophages,Caspase-1 Activation by SopE and the Type III Secretion System-1 of S. Typhimurium Can Proceed in the Absence of Flagellin

The innate immune system is of vital importance for protection against infectious pathogens. Inflammasome mediated caspase-1 activation and subsequent release of pro-inflammatory cytokines like IL-1β and IL-18 is an important arm of the innate immune system. Salmonella enterica subspecies 1 serovar Typhimurium (S. Typhimurium, SL1344) is an enteropathogenic bacterium causing diarrheal diseases. Different reports have shown that in macrophages, S. Typhimurium may activate caspase-1 by at least three different types of stimuli: flagellin, the type III secretion system 1 (T1) and the T1 effector protein SopE. However, the relative importance and interdependence of the different factors in caspase-1 activation is still a matter of debate. Here, we have analyzed their relative contributions to caspase-1 activation in LPS-pretreated RAW264.7 macrophages. Using flagellar mutants (fliGHI, flgK) and centrifugation to mediate pathogen-host cell contact, we show that flagellins account for a small part of the caspase-1 activation in RAW264.7 cells. In addition, functional flagella are of key importance for motility and host cell attachment which is a prerequisite for mediating caspase-1 activation via these three stimuli. Using site directed mutants lacking several T1 effector proteins and flagellin expression, we found that SopE elicits caspase-1 activation even when flagellins are absent. In contrast, disruption of essential genes of the T1 protein injection system (invG, sipB) completely abolished caspase-1 activation. However, a robust level of caspase-1 activation is retained by the T1 system (or unidentified T1 effectors) in the absence of flagellin and SopE. T1-mediated inflammasome activation is in line with recent work by others and suggests that the T1 system itself may represent the basic caspase-1 activating stimulus in RAW264.7 macrophages which is further enhanced independently by SopE and/or flagellin.     

5S ribosomal ribonucleic acid sequences in Bacteroides and Fusobacterium: evolutionary relationships within these genera and among eubacteria in general

The 5S ribosomal ribonucleic acid (rRNA) sequences were determined for Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides capillosus, Bacteroides veroralis, Porphyromonas gingivalis, Anaerorhabdus furcosus, Fusobacterium nucleatum, Fusobacterium mortiferum, and Fusobacterium varium. A dendrogram constructed by a clustering algorithm from these sequences, which were aligned with all other hitherto known eubacterial 5S rRNA sequences, showed differences as well as similarities with respect to results derived from 16S rRNA analyses. In the 5S rRNA dendrogram, Bacteroides clustered together with Cytophaga and Fusobacterium, as in 16S rRNA analyses. Intraphylum relationships deduced from 5S rRNAs suggested that Bacteroides is specifically related to Cytophaga rather than to Fusobacterium, as was suggested by 16S rRNA analyses. Previous taxonomic considerations concerning the genus Bacteroides, based on biochemical and physiological data, were confirmed by the 5S rRNA sequence analysis.