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Proinflammatory cytokine-induced tight junction remodeling through dynamic self-assembly of claudins
Christopher T. Capaldo Attila E. Farkas Roland S. Hilgarth Susanne M. Krug Mattie F. Wolf Jeremy K. Benedik Michael Fromm Michael Koval Charles Parkos Asma Nusrat 《Molecular biology of the cell》2014,25(18):2710-2719
Tight junctions (TJs) are dynamic, multiprotein intercellular adhesive contacts that provide a vital barrier function in epithelial tissues. TJs are remodeled during physiological development and pathological mucosal inflammation, and differential expression of the claudin family of TJ proteins determines epithelial barrier properties. However, the molecular mechanisms involved in TJ remodeling are incompletely understood. Using acGFP-claudin 4 as a biosensor of TJ remodeling, we observed increased claudin 4 fluorescence recovery after photobleaching (FRAP) dynamics in response to inflammatory cytokines. Interferon γ and tumor necrosis factor α increased the proportion of mobile claudin 4 in the TJ. Up-regulation of claudin 4 protein rescued these mobility defects and cytokine-induced barrier compromise. Furthermore, claudins 2 and 4 have reciprocal effects on epithelial barrier function, exhibit differential FRAP dynamics, and compete for residency within the TJ. These findings establish a model of TJs as self-assembling systems that undergo remodeling in response to proinflammatory cytokines through a mechanism of heterotypic claudin-binding incompatibility. 相似文献
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Characterization of the alanine racemases from two mycobacteria 总被引:2,自引:0,他引:2
D-Alanine is a necessary precursor in the biosynthesis of the bacterial peptidoglycan. The naturally occurring L-alanine isomer is racemized to its D-form through the action of a class of enzymes called alanine racemases. These enzymes are ubiquitous among prokaryotes, and with very few exceptions are absent in eukaryotes, making them a logical target for the development of novel antibiotics. The alanine racemase gene from both Mycobacterium tuberculosis and M. avium was amplified by PCR and cloned in Escherichia coli. Overexpression of the proteins in the E. coli BL21 system, both as native and as His-tagged recombinant products, has been achieved. The proteins have been purified to electrophoretic homogeneity and analyzed biochemically. A D-alanine requiring double knock-out mutant of E. coli (alr, dadX) was constructed and the cloned genes were able to complement its deficiencies. 相似文献
44.
Claudia A. Vilo Michael J. Benedik Daniel A. Kunz Qunfeng Dong 《Journal of bacteriology》2012,194(23):6618-6619
We report here the 6.97-Mb draft genome sequence of Pseudomonas fluorescens strain NCIMB 11764, which is capable of growth on cyanide as the sole nitrogen source. The draft genome sequence allowed the discovery of several genes implicated in enzymatic cyanide turnover and provided additional information contributing to a better understanding of this organism''s unique cyanotrophic ability. This is the first sequenced genome of a cyanide-assimilating bacterium. 相似文献
45.
The nitrilases are enzymes that convert nitriles to the corresponding acid and ammonia. They are members of a superfamily, which includes amidases and occur in both prokaryotes and eukaryotes. The superfamily is characterized by having a homodimeric building block with a αββα–αββα sandwich fold and an active site containing four positionally conserved residues: cys, glu, glu and lys. Their high chemical specificity and frequent enantioselectivity makes them attractive biocatalysts for the production of fine chemicals and pharmaceutical intermediates. Nitrilases are also used in the treatment of toxic industrial effluent and cyanide remediation. The superfamily enzymes have been visualized as dimers, tetramers, hexamers, octamers, tetradecamers, octadecamers and variable length helices, but all nitrilase oligomers have the same basic dimer interface. Moreover, in the case of the octamers, tetradecamers, octadecamers and the helices, common principles of subunit association apply. While the range of industrially interesting reactions catalysed by this enzyme class continues to increase, research efforts are still hampered by the lack of a high resolution microbial nitrilase structure which can provide insights into their specificity, enantioselectivity and the mechanism of catalysis. This review provides an overview of the current progress in elucidation of structure and function in this enzyme class and emphasizes insights that may lead to further biotechnological applications. 相似文献
46.
The dinI homolog of S. marcescens was cloned from a plasmid library by virtue of its ability to inhibit nuclease expression from the S. marcescens nucA gene integrated in the genome of E. coli. The S. marcescens DinI protein is 68% identical to DinI of E. coli. It has a similar effect on other SOS regulated genes and likely exerts it effect on nuclease expression, which is most pronounced
as the cells entered stationary phase, through inhibition of basal SOS expression.
Received: 12 April 2001 / Accepted: 14 May 2001 相似文献
47.
Filimonova MN Gubskaya VP Nuretdinov IA Benedik MJ Cherepanova NA Leshchinskaya IB 《Biochemistry. Biokhimii?a》2001,66(3):323-327
The mechanism of action of p-chloromercuribenzoate (PCMB) on Serratia marcescens nuclease was investigated. The analysis showed that PCMB forms complexes with DNA. Binding of C7H5O2Hg+ to DNA changes the secondary structure of the DNA. These changes alter the enzymatic activity of S. marcescens nuclease, which was previously found to be sensitive to the secondary structure of the substrates. The nuclease activity was either suppressed or stimulated in the presence of PCMB depending on the C7H5O2Hg+ to nucleotide equivalent ratio. Binding of C7H5O2Hg+ to DNA did not form an abortive enzyme–substrate complex. Binding of Mg2+ to the C7H5O2Hg–DNA complex caused appropriate changes in secondary structure of the substrate. Since Mg2+ and C7H5O2Hg+, though differing in the type of metal cation, are similar in their mechanisms of influence on enzymatic activity of S. marcescens nuclease, the identity of other metal-containing effectors in their mechanism of action on Serratia marcescens nuclease is assumed. 相似文献
48.
Jandhyala DM Willson RC Sewell BT Benedik MJ 《Applied microbiology and biotechnology》2005,68(3):327-335
Recombinant forms of three cyanide-degrading nitrilases, CynD from Bacillus pumilus C1, CynD from Pseudomonas stutzeri, and CHT from Gloeocercospora sorghi, were prepared after their genes were cloned with C-terminal hexahistidine purification tags and expressed in Escherichia coli, and the enzymes purified using nickel-chelate affinity chromatography. The enzymes were compared with respect to their pH stability, thermostability, metal tolerance, and kinetic constants. The two bacterial genes, both cyanide dihydratases, were similar with respect to pH range, retaining greater than 50% activity between pH 5.2 and pH 8 and kinetic properties, having similar Km (6–7 mM) and Vmax (0.1 mmol min–1 mg–1). They also exhibited similar metal tolerances. However, the fungal CHT enzyme had notably higher Km (90 mM) and Vmax (4 mmol min–1 mg–1) values. Its pH range was slightly more alkaline (retaining nearly full activity above 8.5), but exhibited a lower thermal tolerance. CHT was less sensitive to Hg2+ and more sensitive to Pb2+ than the CynD enzymes. These data describe, in part, the current limits that exist for using nitrilases as agents in the bioremediation of cyanide-containing waste effluent, and may help serve to determine where and under what conditions these nitrilases may be applied. 相似文献
49.
Mutant analysis shows that alanine racemases from Pseudomonas aeruginosa and Escherichia coli are dimeric 下载免费PDF全文
Alanine racemases are ubiquitous prokaryotic enzymes providing the essential peptidoglycan precursor D-alanine. We present evidence that the enzymes from Pseudomonas aeruginosa and Escherichia coli function exclusively as homodimers. Moreover, we demonstrate that expression of a K35A Y235A double mutation of dadX in E. coli suppresses bacterial growth in a dominant negative fashion. 相似文献
50.
Michael J. Benedik 《Molecular & general genetics : MGG》1989,218(2):353-354
Summary This report demonstrates high efficiency transduction of enteric bacteria using single strand plasmids packaged in M13 phage capsids. Transformation by plasmid DNA is usually a very inefficient process in many enteric bacteria other than Escherichia coli K12. Plasmids carrying an M13 origin of replication can be replicated and packaged when cells carrying such plasmids are infected with M13 or a derivative helper phage. By introducing an F plasmid into E. coli, Serratia marcescens, Citrobacter freundii, and Enterobacter aerogenes, these species can now be infected at high efficiency with M13 phage and with packaged single strand plasmids, yielding an efficient method to introduce cloned DNA fragments into these bacteria. The titer of colony forming units in a lysate was essentially equivalent in all the bacteria, demonstrating an equal efficiency of transduction of these other enteric bacteria compared to E. coli. 相似文献