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91.
Acharya A Chavan YG Mukhopadhyaya PN Nagee A Kunjadia P Misra RN 《Roumanian archives of microbiology and immunology》2012,71(1):29-38
Human MECP2 gene located at q28 arm of X chromosome was identified as target for thermal co-amplification with HIV-1 proviral DNA of infected individuals. The selected MECP2 gene-specific primers functioned at a wide range of annealing temperature, extension time and exhibited no significant interaction with pathogen specific primers. A 466 bp PCR amplicon originating from human MECP2 gene was found to be diagnostic for inhibition-free PCR reaction when co-amplified with the HIV-1 target gene in a multiplexed, nested PCR reaction. The 5' end of the MECP2 primers were engineered to position an EcoRI restriction endonuclease site to facilitate rapid cloning in various DNA vector molecules at the corresponding EcoRI sites. Cell mass of Escherichia coli (XL1Blue) harboring the recombinant plasmid when added to pleural fluid of HIV-1 infected individuals co-infected with Mycobacterium tuberculosis, generated the diagnostic 466 bp MECP2 PCR amplicon as well as the 194 bp PCR amplicon of target gene from M. tuberculosis. The experiment underlined potential of the region spanning nucleotide position 4118099 to 4118552 of human MECP2 gene (NCBI accession number NT_011726.13) as a reliable target for multiplex PCR to accommodate a wide range of thermal cycling and multiplex reaction conditions. In both cases of this study, electrophoresis-based separation of the 466 bp MECP2 fragment and the 232 bp and 194 bp HIV-1 and M. tuberculosis fragments respectively was distinct and unambiguous. The potential of this human MECP2 gene available from human genome or recombinant plasmid as a potent target to monitor PCR inhibition for a range of different PCR reactions is discussed. 相似文献
92.
JA Ochoa-Alvarez H Krishnan Y Shen NK Acharya M Han DE McNulty H Hasegawa T Hyodo T Senga JG Geng M Kosciuk SS Shin JS Goydos D Temiakov RG Nagele GS Goldberg 《PloS one》2012,7(7):e41845
Cancer is a leading cause of death of men and women worldwide. Tumor cell motility contributes to metastatic invasion that causes the vast majority of cancer deaths. Extracellular receptors modified by α2,3-sialic acids that promote this motility can serve as ideal chemotherapeutic targets. For example, the extracellular domain of the mucin receptor podoplanin (PDPN) is highly O-glycosylated with α2,3-sialic acid linked to galactose. PDPN is activated by endogenous ligands to induce tumor cell motility and metastasis. Dietary lectins that target proteins containing α2,3-sialic acid inhibit tumor cell growth. However, anti-cancer lectins that have been examined thus far target receptors that have not been identified. We report here that a lectin from the seeds of Maackia amurensis (MASL) with affinity for O-linked carbohydrate chains containing sialic acid targets PDPN to inhibit transformed cell growth and motility at nanomolar concentrations. Interestingly, the biological activity of this lectin survives gastrointestinal proteolysis and enters the cardiovascular system to inhibit melanoma cell growth, migration, and tumorigenesis. These studies demonstrate how lectins may be used to help develop dietary agents that target specific receptors to combat malignant cell growth. 相似文献
93.
Treatment of blood loss with plasma expanders lowers blood viscosity, increasing cardiac output. However, increased flow velocity by conventional plasma expanders does not compensate for decreased viscosity in maintaining vessel wall shear stress (WSS), decreasing endothelial nitric oxide (NO) production. A new type of plasma expander using polyethylene glycol conjugate albumin (PEG-Alb) causes supra-perfusion when used in extreme hemodilution and is effective in treating hemorrhagic shock, although it is minimally viscogenic. An acute 40% hemodilution/exchange-transfusion protocol was used to compare 4% PEG-Alb to Ringer's lactate, Dextran 70 kDa and 6% Hetastarch (670 kDa) in unanesthetized CD-1 mice. Serum cytokine analysis showed that PEG-Alb elevates monocyte chemotactic protein-1 (MCP-1), a member of a small inducible gene family, as well as expression of MIP-1α, and MIP-2. MCP-1 is specific to increased WSS. Given the direct link between increased WSS and production of NO, the beneficial resuscitation effects due to PEG-Alb plasma expansion appear to be due to increased WSS through increased perfusion and blood flow rather than blood viscosity. 相似文献
94.
TK Lai G Biswas S Chatterjee A Dutta C Pal J Banerji N Bhuvanesh JH Reibenspies K Acharya 《Chemistry & biodiversity》2012,9(8):1517-1524
Two new lanostane-type triterpenes, 1 and 2, were isolated from Astraeus hygrometricus. The structures were established by IR, (1) H- and (13) C-NMR, MS, and X-ray crystallographic experiments. The triterpenes exhibited excellent in vitro toxicities against Candida albicans, comparable to standard antifungal antibiotics. The triterpene 2 significantly inhibited the growth of Leishmania donovani promastigotes in vitro. The triterpene skeleton may be considered a template structure in search for new compounds with anticandidal and leishmanicidal activity. 相似文献
95.
Estradiol‐induced structural dimorphisms exist in the songbird brain. However, how they arise is not clear since there is a scarce distribution of ERα and lack of ERβ in song control nuclei. This suggests that other receptors are involved. The G‐protein coupled membrane‐bound estrogen receptor, GPR30, is a candidate but has never been investigated in songbirds. In this study, we characterized its gene and protein in the zebra finch brain. Analysis of the putative GPR30 protein sequence revealed a strong similarity to avian and mammalian homologues. Quantitative PCR indicated that the gene was elevated in the telencephalon of both sexes from posthatching day (P) 15 to P45, with a male‐biased sex difference at P21 and P30. In comparison, expression at younger posthatching ages and in adults was significantly less. At P21, GRP30 protein was widespread, nonuniform, and overlapped with song control nuclei. Of particular interest, the number of immunoreactive cells was greatest in HVC and RA, but less in LMAN and Area X. Labeling in HVC was also dimorphic; with more cells present in males than in females. In parallel with the gene, by adulthood, protein expression was reduced across most brain regions. Taken together these data suggest that GPR30 may contribute to differences in song system development by mediating dimorphic responses to estrogens. In addition, the extensive protein distribution indicates that it may also have a role in general brain development in both sexes. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012 相似文献
96.
Anders CK Acharya CR Hsu DS Broadwater G Garman K Foekens JA Zhang Y Wang Y Marcom K Marks JR Mukherjee S Nevins JR Blackwell KL Potti A 《PloS one》2008,3(1):e1373
Purpose
To define the biology driving the aggressive nature of breast cancer arising in young women.Experimental Design
Among 784 patients with early stage breast cancer, using prospectively-defined, age-specific cohorts (young ≤45 years; older ≥65 years), 411 eligible patients (n = 200≤45 years; n = 211≥65 years) with clinically-annotated Affymetrix microarray data were identified. GSEA, signatures of oncogenic pathway deregulation and predictors of chemotherapy sensitivity were evaluated within the two age-defined cohorts.Results
In comparing deregulation of oncogenic pathways between age groups, a higher probability of PI3K (p = 0.006) and Myc (p = 0.03) pathway deregulation was observed in breast tumors arising in younger women. When evaluating unique patterns of pathway deregulation, a low probability of Src and E2F deregulation in tumors of younger women, concurrent with a higher probability of PI3K, Myc, and β-catenin, conferred a worse prognosis (HR = 4.15). In contrast, a higher probability of Src and E2F pathway activation in tumors of older women, with concurrent low probability of PI3K, Myc and β-catenin deregulation, was associated with poorer outcome (HR = 2.7). In multivariate analyses, genomic clusters of pathway deregulation illustrate prognostic value.Conclusion
Results demonstrate that breast cancer arising in young women represents a distinct biologic entity characterized by unique patterns of deregulated signaling pathways that are prognostic, independent of currently available clinico-pathologic variables. These results should enable refinement of targeted treatment strategies in this clinically challenging situation. 相似文献97.
Post-translational tyrosine nitration of eosinophil granule toxins mediated by eosinophil peroxidase
Ulrich M Petre A Youhnovski N Prömm F Schirle M Schumm M Pero RS Doyle A Checkel J Kita H Thiyagarajan N Acharya KR Schmid-Grendelmeier P Simon HU Schwarz H Tsutsui M Shimokawa H Bellon G Lee JJ Przybylski M Döring G 《The Journal of biological chemistry》2008,283(42):28629-28640
Nitration of tyrosine residues has been observed during various acute and chronic inflammatory diseases. However, the mechanism of tyrosine nitration and the nature of the proteins that become tyrosine nitrated during inflammation remain unclear. Here we show that eosinophils but not other cell types including neutrophils contain nitrotyrosine-positive proteins in specific granules. Furthermore, we demonstrate that the human eosinophil toxins, eosinophil peroxidase (EPO), major basic protein, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), and the respective murine toxins, are post-translationally modified by nitration at tyrosine residues during cell maturation. High resolution affinity-mass spectrometry identified specific single nitration sites at Tyr349 in EPO and Tyr33 in both ECP and EDN. ECP and EDN crystal structures revealed and EPO structure modeling suggested that the nitrated tyrosine residues in the toxins are surface exposed. Studies in EPO(-/-), gp91phox(-/-), and NOS(-/-) mice revealed that tyrosine nitration of these toxins is mediated by EPO in the presence of hydrogen peroxide and minute amounts of NOx. Tyrosine nitration of eosinophil granule toxins occurs during maturation of eosinophils, independent of inflammation. These results provide evidence that post-translational tyrosine nitration is unique to eosinophils. 相似文献
98.
Requirement of Rad5 for DNA polymerase zeta-dependent translesion synthesis in Saccharomyces cerevisiae 下载免费PDF全文
In yeast, Rad6-Rad18-dependent lesion bypass involves translesion synthesis (TLS) by DNA polymerases eta or zeta or Rad5-dependent postreplication repair (PRR) in which error-free replication through the DNA lesion occurs by template switching. Rad5 functions in PRR via its two distinct activities-a ubiquitin ligase that promotes Mms2-Ubc13-mediated K63-linked polyubiquitination of PCNA at its lysine 164 residue and a DNA helicase that is specialized for replication fork regression. Both these activities are important for Rad5's ability to function in PRR. Here we provide evidence for the requirement of Rad5 in TLS mediated by Polzeta. Using duplex plasmids carrying different site-specific DNA lesions-an abasic site, a cis-syn TT dimer, a (6-4) TT photoproduct, or a G-AAF adduct-we show that Rad5 is needed for Polzeta-dependent TLS. Rad5 action in this role is likely to be structural, since neither the inactivation of its ubiquitin ligase activity nor the inactivation of its helicase activity impairs its role in TLS. 相似文献
99.
Mutations in the signature motif in MutS affect ATP-induced clamp formation and mismatch repair 总被引:1,自引:0,他引:1
Acharya S 《Molecular microbiology》2008,69(6):1544-1559
MutS protein dimer recognizes and co-ordinates repair of DNA mismatches. Mismatch recognition by the N-terminal mismatch recognition domain and subsequent downstream signalling by MutS appear coupled to the C-terminal ATP catalytic site, Walker box, through nucleotide-mediated conformational transitions. Details of this co-ordination are not understood. The focus of this study is a conserved loop in Escherichia coli MutS that is predicted to mediate cross-talk between the two ATP catalytic sites in MutS homodimer. Mutagenesis was employed to assess the role of this loop in regulating MutS function. All mutants displayed mismatch repair defects in vivo . Biochemical characterization further revealed defects in ATP binding, ATP hydrolysis as well as effective mismatch recognition. The kinetics of initial burst of ATP hydrolysis was similar to wild type but the magnitude of the burst was reduced for the mutants. Given its proximity to the ATP bound in the opposing monomer in the crystal and its potential analogy with signature motif of ABC transporters, the results strongly suggest that the loop co-ordinates ATP binding/hydrolysis in trans by the two catalytic sites. Importantly, our data reveal that the loop plays a direct role in co-ordinating conformational changes involved in long-range communication between Walker box and mismatch recognition domains. 相似文献
100.