Proteome expression and carbonylation changes during Trypanosoma cruzi infection and Chagas disease in rats

Inflammation and oxidative stress, elicited by Trypanosoma cruzi infection, are important pathologic events during progressive Chagasic cardiomyopathy. In this study, we infected Sprague-Dawley rats with T. cruzi, and treated with phenyl-α-tert-butylnitrone (PBN-antioxidant) and/or benznidazole (BZ-anti-parasite). We employed two-dimensional gel electrophoresis/mass spectrometry to investigate (a) the plasma proteomic changes associated with infection and disease development, and (b) the beneficial effects of PBN and BZ in controlling the disease-associated plasma profile. Matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) tandem MS (MS/MS) analysis of differentially expressed (total 146) and oxidized (total 48) protein spots yielded 92 unique proteins. Our data showed that treatment with PBN and BZ restored the differential expression of 65% and 30% of the disease-associated proteins to normal level, respectively, and PBN prevented development of oxidative adducts on plasma proteins. Western blotting to detect dinitrophenyl-derivatized carbonyl-proteins revealed plasma proteins were maximally oxidized during acute infection. Functional and disease/disorder analyses allocated a majority of the differentially expressed and oxidized proteins into inflammation/immunity and lipid metabolism categories and to molecular pathways associated with heart disease (e.g. cardiac infarction, contractile dysfunction, hypertrophy, and hypertension) in chagasic rats, and to curative pathways (e.g. ROS scavenging capacity, immune regulation) in infected rats treated with PBN and/or BZ. We validated the two-dimensional gel electrophoresis results by Western blotting, and demonstrated that the disease-associated increased expression of gelsolin and vimentin and release of cardiac MYL2 in the plasma of chagasic rats was returned to control level by PBN/BZ treatment. Increased plasma levels of gelsolin, MYL2 and vimentin were directly correlated with the severity of cardiac disease in human chagasic patients. Together, these results demonstrate the plasma oxidative and inflammatory response profile, and plasma detection of cardiac proteins parallels the pathologic events contributing to Chagas disease development, and is of potential utility in diagnosing disease severity and designing suitable therapy for management of human chagasic patients.     

酶法催化降解2,4,6-三硝基甲苯的研究进展

2,4,6-三硝基甲苯(TNT)作为一种广泛使用的含能材料,发挥巨大作用的同时也给环境带来了严重的污染,对人类健康构成一定威胁。目前国内外的TNT处置主要有物理、化学、生物及酶法等方法,其中酶法作为一种新兴的方法,显示了良好的应用潜力,受到研究者的广泛关注,。比较了各类处理方法的优缺点,重点介绍近年来涉及TNT降解的酶学研究进展,并对酶法在TNT废水处理和土壤修复中的应用前景进行展望。     

Mutations in the hydrophobic core of ubiquitin differentially affect its recognition by receptor proteins

Ubiquitin (Ub) is one of the most highly conserved signaling proteins in eukaryotes. In carrying out its myriad functions, Ub conjugated to substrate proteins interacts with dozens of receptor proteins that link the Ub signal to various biological outcomes. Here we report mutations in conserved residues of Ub's hydrophobic core that have surprisingly potent and specific effects on molecular recognition. Mutant Ubs bind tightly to the Ub-associated domain of the receptor proteins Rad23 and hHR23A but fail to bind the Ub-interacting motif present in the receptors Rpn10 and S5a. Moreover, chains assembled on target substrates with mutant Ubs are unable to support substrate degradation by the proteasome in vitro or sustain viability of yeast cells. The mutations have relatively little effect on Ub's overall structure but reduce its rigidity and cause a slight displacement of the C-terminal β-sheet, thereby compromising association with Ub-interacting motif but not with Ub-associated domains. These studies emphasize an unexpected role for Ub's core in molecular recognition and suggest that the diversity of protein-protein interactions in which Ub engages placed enormous constraints on its evolvability.     

Klebsiella pneumoniae multiresistance plasmid pMET1: similarity with the Yersinia pestis plasmid pCRY and integrative conjugative elements

Background

Dissemination of antimicrobial resistance genes has become an important public health and biodefense threat. Plasmids are important contributors to the rapid acquisition of antibiotic resistance by pathogenic bacteria.

Principal Findings

The nucleotide sequence of the Klebsiella pneumoniae multiresistance plasmid pMET1 comprises 41,723 bp and includes Tn1331.2, a transposon that carries the bla _TEM-1 gene and a perfect duplication of a 3-kbp region including the aac(6′)-Ib, aadA1, and bla _OXA-9 genes. The replication region of pMET1 has been identified. Replication is independent of DNA polymerase I, and the replication region is highly related to that of the cryptic Yersinia pestis 91001 plasmid pCRY. The potential partition region has the general organization known as the parFG locus. The self-transmissible pMET1 plasmid includes a type IV secretion system consisting of proteins that make up the mating pair formation complex (Mpf) and the DNA transfer (Dtr) system. The Mpf is highly related to those in the plasmid pCRY, the mobilizable high-pathogenicity island from E. coli ECOR31 (HPI_ECOR31), which has been proposed to be an integrative conjugative element (ICE) progenitor of high-pathogenicity islands in other Enterobacteriaceae including Yersinia species, and ICE_Kp1, an ICE found in a K. pneumoniae strain causing primary liver abscess. The Dtr MobB and MobC proteins are highly related to those of pCRY, but the endonuclease is related to that of plasmid pK245 and has no significant homology with the protein of similar function in pCRY. The region upstream of mobB includes the putative oriT and shares 90% identity with the same region in the HPI_ECOR31.

Conclusions

The comparative analyses of pMET1 with pCRY, HPI_ECOR31, and ICE_Kp1 show a very active rate of genetic exchanges between Enterobacteriaceae including Yersinia species, which represents a high public health and biodefense threat due to transfer of multiple resistance genes to pathogenic Yersinia strains.     

A functional gene array for detection of bacterial virulence elements

Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known or novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples.     

Modification of sterculia gum with methacrylic acid to prepare a novel drug delivery system

The present paper deals with the modification of the sterculia gum with methacrylic acid (MAAc) to hydrogels for use in drug delivery. The hydrogels were characterized by SEMs, FTIR and swelling studies. The release dynamics of model anti-ulcer drug (ranitidine hydrochloride) from the hydrogels has been studied for the evaluation of the release mechanism. The values of the diffusion exponent 'n' (0.55, 0.54 and 0.59) and gel characteristic constant 'k' (2.109 x 10(-2), 3.698 x 10(-2) and 2.427 x 10(-2)) have been obtained, respectively, in distilled water, pH 2.2 buffer and pH 7.4 buffer. The release of the drug from the hydrogels occurred through non-Fickian diffusion mechanism.     

Biphenylsulfonyl-thiophene-carboxamidine inhibitors of the complement component C1s

Complement activation has been implicated in disease states such as hereditary angioedema, ischemia-reperfusion injury, acute respiratory distress syndrome, and acute transplant rejection. Even though the complement cascade provides several protein targets for potential therapeutic intervention only two complement inhibitors have been approved so far for clinical use including anti-C5 antibodies for the treatment of paroxysmal nocturnal hemoglobinuria and purified C1-esterase inhibitor replacement therapy for the control of hereditary angioedema flares. In the present study, optimization of potency and physicochemical properties of a series of thiophene amidine-based C1s inhibitors with potential utility as intravenous agents for the inhibition of the classical pathway of complement is described.     

Purification and Characterization of Lectin from Seeds of Delonix regia

A lectin from the crude extract of seeds of Delonix regia (DRL) has been purified by ammonium sulphate fractionation followed by specific adsorption on Sephadex G-50 column and subsequent displacement with 100 mM D-glucose. The purified lectin (yield 1.41 mg g^?1 dry seed) is a hetero-tetramer of 156 kD in size, consisting of four polypeptides (M_r of 32, 36, 42 and 46 kD) as detected on SDS-PAGE. It is a thermostable protein and remains active between pH 2.0–11.0. The lectin agglutinated erythrocytes of human and other primates. The hemagglutinating activity was not affected by cations and chelating agents. Of the 23 different sugars tested for specificity, maximum inhibition of the hemagglutination was shown by D-glucose. The immunological crossreactions of DRL with monospecific antibodies against SBA, Con A, PNA, DBA and PHA-E indicate that DRL is very closely related to Concanavalin A.     

GH10 XynF1 and Xyn11A: the predominant xylanase identified in the profiling of extracellular proteome of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> LC1

Advanced techniques of enzyme production and purification have become prerequisite due to their diverse industrial applications. There is an utmost requirement for screening of new strains capable of synthesising industrially useful enzymes. The present study reports the production and profiling of extracellular proteins expressed by the newly isolated strain of a filamentous fungus, Aspergillus oryzae LC1. The extracellular enzyme production was done by submerged fermentation using Mendel’s and Sternberg’s medium (MSM), and its optimisation was done using one factor at a time (OFAT). The presence of xylanase was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography. In addition, the profiling of extracellular proteome of Aspergillus oryzae LC1 was carried out by liquid chromatography coupled tandem mass spectrometry (LC-MS/MS). In this study, media optimisation showed 5.7-fold increase in xylanase activity. The multiple bands observed in zymography revealed the presence of various forms of xylanase. A total of 73 proteins were identified in LC-MS/MS analysis. Functional classification showed that the hydrolytic enzymes consisted of 48% glycoside hydrolase, 11% proteases, 1% polysaccharide lyase and esterase’s, 9% oxidoreductases and 30% other proteins. A total of 26 families of glycosidic hydrolase were detected with other protein families such as serine peptidase, S, LysM, G-D-S-L, M35, carboxyl esterase (CE1), pectate lyase (PL) and oxidoreductases. Among the huge diversity of synergistically acting biomass cleaving enzymes, endo-1, 4-β xylanase with isoforms: xyn F1, xyn B, β xylanase and xyn 11A belonging to GH10 family covered the major portion of the total percentage of identified proteins. As per our knowledge, this is the first report of extracellular proteome analysis of Aspergillus oryzae LC1 suggesting its capability for recombinant expression and evaluation in hemicellulose deconstruction applications.