Virulent Mycobacterium bovis Beijing Strain Activates the NLRP7 Inflammasome in THP-1 Macrophages

Mycobacterium bovis is the causative agent of tuberculosis in a wide range of mammals, including humans. Macrophages are the first line of host defense. They secrete proinflammatory cytokines, such as interleukin-1 beta (IL-1β), in response to mycobacterial infection, but the underlying mechanisms by which human macrophages are activated and release IL-1β following M. bovis infection are poorly understood. Here we show that the ‘nucleotide binding and oligomerization of domain-like receptor (NLR) family pyrin domain containing 7 protein’ (NLRP7) inflammasome is involved in IL-1β secretion and caspase-1 activation induced by M. bovis infection in THP-1 macrophages. NLRP7 inflammasome activation promotes the induction of pyroptosis as well as the expression of tumor necrosis factor alpha (TNF-α), Chemokine (C-C motif) ligand 3 (CCL3) and IL-1β mRNAs. Thus, the NLRP7 inflammasome contributes to IL-1β secretion and induction of pyroptosis in response to M. bovis infection in THP-1 macrophages.     

Genetic analysis of the pnp-deaD genetic region reveals membrane lipoprotein NlpI as an independent participant in cold acclimatization of Salmonella enterica serovar Typhimurium

Plasmid pAMI7 of the methylotrophic bacterium Paracoccus aminophilus JCM 7686 (Alphaproteobacteria) encodes a functional type II restriction-modification (R-M) system designated PamI. Homologous systems were identified in the genomes of distinct taxonomic groups of Bacteria and Archaea, which provides evidence that horizontal gene transfer has contributed to the wide dissemination of R-M modules - even between domains. Analysis of the cleavage specificity of the R.PamI endonuclease revealed that this protein is an isoschizomer of restriction enzyme NcoI. Interestingly, bioinformatic analyses suggest that R.PamI and NcoI are accompanied by methyltransferases of different methylation specificities (C5-methylcytosine and N4-methylcytosine methyltransferases, respectively), which possibly exemplifies recombinational shuffling of genes coding for individual components of R-M systems. The PamI system can stabilize plasmid pAMI7 in a bacterial population, most probably at the postsegregational level. Therefore, it functions in an analogous manner to plasmid-encoded toxin-antitoxin (TA) systems. Since the TA system of pAMI7 is nonfunctional, it is highly probable that this lack is compensated by the stabilizing activity of PamI. This indicates the crucial role of the analyzed R-M system in the stable maintenance of pAMI7, which is, to our knowledge, the first report of 'symbiosis' between a R-M system and a plasmid in the Alphaproteobacteria.     

Trypsin inhibition by a peptide hormone: crystal structure of trypsin-vasopressin complex

The large variety of serine protease inhibitors, available from various sources such as tissues, microorganisms, plants, etc., play an important role in regulating the proteolytic enzymes. The analysis of protease-inhibitor complexes helps in understanding the mechanism of action, as well as in designing inhibitors. Vasopressin, an anti-diuretic nonapeptide hormone, is found to be an effective inhibitor of trypsin, with a K(i) value of 5 nM. The crystal structure of the trypsin-vasopressin complex revealed that vasopressin fulfils all the important interactions for an inhibitor, without any break in the scissile peptide bond. The cyclic nature due to a disulfide bridge between Cys1 and Cys6 of vasopressin provides structural rigidity to the peptide hormone. The trypsin-binding site is located at the C terminus, while the neurophysin-binding site is at the N terminus of vasopressin. This study will assist in designing new peptide inhibitors. This study suggests that vasopressin inhibition of trypsin may have unexplored biological implications.     

Diversification of Ferredoxins across Living Organisms

Ferredoxins, iron-sulfur (Fe-S) cluster proteins, play a key role in oxidoreduction reactions. To date, evolutionary analysis of these proteins across the domains of life have been confined to observing the abundance of Fe-S cluster types (2Fe-2S, 3Fe-4S, 4Fe-4S, 7Fe-8S (3Fe-4s and 4Fe-4S) and 2[4Fe-4S]) and the diversity of ferredoxins within these cluster types was not studied. To address this research gap, here we propose a subtype classification and nomenclature for ferredoxins based on the characteristic spacing between the cysteine amino acids of the Fe-S binding motif as a subtype signature to assess the diversity of ferredoxins across the living organisms. To test this hypothesis, comparative analysis of ferredoxins between bacterial groups, Alphaproteobacteria and Firmicutes and ferredoxins collected from species of different domains of life that are reported in the literature has been carried out. Ferredoxins were found to be highly diverse within their types. Large numbers of alphaproteobacterial species ferredoxin subtypes were found in Firmicutes species and the same ferredoxin subtypes across the species of Bacteria, Archaea, and Eukarya, suggesting shared common ancestral origin of ferredoxins between Archaea and Bacteria and lateral gene transfer of ferredoxins from prokaryotes (Archaea/Bacteria) to eukaryotes. This study opened new vistas for further analysis of diversity of ferredoxins in living organisms.     

柑橘果糖激酶基因的克隆及表达

植物果糖激酶(FRK)在果糖磷酸化中起重要作用.通过PCR技术从温州蜜柑(Citrus unshiu Marc.)基因组中扩增得到编码果糖激酶基因的2个基因组DNA片段,分别命名为Cufrk1、Cufrk2,利用RT-PCR从果实中分离到了与Cufrk1外显子序列一致的cDNA序列,并通过RACE技术分离到这个基因的全长cDNA序列,命名为CuFRK1(GenBank号:AY561840).Cufrk1与Cufrk2编码氨基酸序列相似性为68%.CuFRK1 cDNA全长为1 459 bp,5'端和3'端的非翻译区分别为167 bp和239 bp,该序列含有一个完整的开放读码框,编码350个氨基酸,蛋白质分子量约为37.5 kD,等电点为5.03,含有2个果糖激酶糖特异结合域及3个ATP结合域,其氨基酸序列与其他植物中已分离的果糖激酶基因相似性在62%～78%.Northern分析显示,CuFRK1(Cufrk1)与Cufrk2在柑橘幼叶、发育初期果实中表达量较高,在果皮和茎中不表达,在花瓣及成熟果实中表达模式有一定差异.酶活性分析表明,果实中的果糖激酶活性随果实的发育而降低,同时,果实中的果糖不断积累,在果实整个发育过程中果糖含量与果糖激酶活性呈极显著负相关.     

Interactions of mouse glycophorin A with the dRTA-related mutant G719D of the mouse Cl-/HCO3- exchanger Ae1

The AE1 mutation G701D, associated with recessive distal renal tubular acidosis (dRTA), produces only minimal erythroid phenotype, reflecting erythroid-specific expression of stimulatory AE1 subunit glycophorin A (GPA). GPA transgene expression could theoretically treat recessive dRTA in patients and in mice expressing cognate Ae1 mutation G719D. However, human (h) GPA and mouse (m) Gpa amino acid sequences are widely divergent, and mGpa function in vitro has not been investigated. We therefore studied in Xenopus oocytes the effects of coexpressed mGpa and hGPA on anion transport by erythroid (e) and kidney (k) isoforms of wild-type mAe1 (meAe1, mkAe1) and of mAe1 mutant G719D. Coexpression of hGPA or mGpa enhanced the function of meAe1 and mkAe1 and rescued the nonfunctional meAe1 and mkAe1 G719D mutants through increased surface expression. Progressive N-terminal truncation studies revealed a role for meAe1 amino acids 22-28 in GPA-responsiveness of meAe1 G719D. MouseN-cyto/humanTMD and humanN-cyto/mouseTMD kAE1 chimeras were active and GPA-responsive. In contrast, whereas chimera mkAe1N-cyto/hkAE1 G701DTMD was GPA-responsive, chimera hkAE1N-cyto/mkAe1 G719DTMD was GPA-insensitive. Moreover, whereas the isolated transmembrane domain (TMD) of hAE1 G701D was GPA-responsive, that of mAe1 G719D was GPA-insensitive. Thus, mGpa increases surface expression and activity of meAe1 and mkAe1. However, the G719D mutation renders certain mAe1 mutant constructs GPA-unresponsive and highlights a role for erythroid-specific meAe1 amino acids 22-28 in GPA-responsiveness.     

Prey selection and food habits of three sympatric large carnivores in a tropical lowland forest of the Eastern Himalayan Biodiversity Hotspot

Prey selection and the feeding habits of tiger Panthera tigris, leopard Panthera pardus and Asiatic wild dog Cuon alpinus were investigated from June 2009 to December 2011 in Pakke Tiger Reserve, Arunachal Pradesh. A total of 422 scats were analyzed of which, 109 scats were of tigers, 150 were of leopard and 163 scats were of dholes. Multinomial Likelihood ratio test was used to estimate the prey selectivity of predators and Ivlev index, Pianka index were used to estimate prey preference and overlap respectively. Biomass consumption for three sympatric predators varied from 254.3 kg for dholes to 599.1 kg for tigers. Sambar, barking deer, wild pig were preyed more than their availability by all the predators. Ivlev index shows barking deer and sambar were preferred more than available prey for tiger where as leopard preferred sambar more than available and avoided barking deer. Dhole preferred more than available wild pig and barking deer. There was a high overlap between tiger–leopard (85.3%) and tiger–dhole (77.5%). To the best of our understanding, this study provides the first reliable information on prey selection and food habits of sympatric large carnivores in a protected area of Eastern Himalayan tropical rainforest.     

Densitometric Quantification and Optimization of Polyphenols in Phyllanthus maderaspatensis by HPTLC

Quantifying and optimizing the polyphenol content of Phyllanthus maderaspatensis was accomplished using a single-solvent HPTLC system. Analyzing hydroalcoholic extracts for kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid, we simultaneously quantified and optimized their concentration. In the experiment, the methanol to water ratio (%), temperature (°C), and time of extraction (min) were all optimized using a Box-Behnken statistical design. Kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid were among the dependent variables analyzed. In the HPTLC separation, silica gel 60F254 plates were used, and toluene, ethyl acetate, and formic acid (5:4:1) made up the mobile phase. For kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid, densitometric measurements were carried out using the absorbance mode at 254 nm. Hydroalcoholic extract of P. maderaspatensis contains rutin (0.344), catechin (2.62), gallic acid (0.93), ellagic acid (0.172), quercetin (0.0108) and kaempferol (0.06). Further, it may be affected by more than one factor at a time, resulting in a varying degree of reaction. A negative correlation was found between X1 (extraction time (min)) and X2 (temperature), as well as X1 and X3 (solvent ratios). Taking these characteristics into consideration, the method outlined here is a validated HPTLC method for measuring kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid.     

Visceral Adipose MicroRNA 223 Is Upregulated in Human and Murine Obesity and Modulates the Inflammatory Phenotype of Macrophages

Obesity in humans and mice is typified by an activated macrophage phenotype in the visceral adipose tissue (VAT) leading to increased macrophage-mediated inflammation. microRNAs (miRNAs) play an important role in regulating inflammatory pathways in macrophages, and in this study we compared miRNA expression in the VAT of insulin resistant morbidly obese humans to a non-obese cohort with normal glucose tolerance. miR-223-3p was found to be significantly upregulated in the whole omental tissue RNA of 12 human subjects, as were 8 additional miRNAs. We then confirmed that miR-223 upregulation was specific to the stromal vascular cells of human VAT, and found that miR-223 levels were unchanged in adipocytes and circulating monocytes of the non-obese and obese. miR-223 ablation increased basal / unstimulated TLR4 and STAT3 expression and LPS-stimulated TLR4, STAT3, and NOS2 expression in primary macrophages. Conversely, miR-223 mimics decreased TLR4 expression in primary macrophage, at the same time it negatively regulated FBXW7 expression, a well described suppressor of Toll-like receptor 4 (TLR4) signaling. We concluded that the abundance of miR-223 in macrophages significantly modulates macrophage phenotype / activation state and response to stimuli via effects on the TLR4/FBXW7 axis.