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81.
Multiple transporters associated with malaria parasite responses to chloroquine and quinine 总被引:1,自引:0,他引:1
Mu J Ferdig MT Feng X Joy DA Duan J Furuya T Subramanian G Aravind L Cooper RA Wootton JC Xiong M Su XZ 《Molecular microbiology》2003,49(4):977-989
Mutations and/or overexpression of various transporters are known to confer drug resistance in a variety of organisms. In the malaria parasite Plasmodium falciparum, a homologue of P-glycoprotein, PfMDR1, has been implicated in responses to chloroquine (CQ), quinine (QN) and other drugs, and a putative transporter, PfCRT, was recently demonstrated to be the key molecule in CQ resistance. However, other unknown molecules are probably involved, as different parasite clones carrying the same pfcrt and pfmdr1 alleles show a wide range of quantitative responses to CQ and QN. Such molecules may contribute to increasing incidences of QN treatment failure, the molecular basis of which is not understood. To identify additional genes involved in parasite CQ and QN responses, we assayed the in vitro susceptibilities of 97 culture-adapted cloned isolates to CQ and QN and searched for single nucleotide polymorphisms (SNPs) in DNA encoding 49 putative transporters (total 113 kb) and in 39 housekeeping genes that acted as negative controls. SNPs in 11 of the putative transporter genes, including pfcrt and pfmdr1, showed significant associations with decreased sensitivity to CQ and/or QN in P. falciparum. Significant linkage disequilibria within and between these genes were also detected, suggesting interactions among the transporter genes. This study provides specific leads for better understanding of complex drug resistances in malaria parasites. 相似文献
82.
Wagh S Ramaiah A Subramanian R Govindarajan R 《Pigment cell research / sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society》2000,13(6):442-448
In melanocytes, enzymes involved in the generation of melanin monomers are present and active in coated vesicles which are known to be acidic. Melanin polymerization however, occurs only in melanosomes. In vitro, it is not possible to generate melanin at the acidic pH of melanosomes using 3,4-dihydroxyphenylalanine (DOPA) and tyrosinase alone whereas melanin readily forms at higher pH with these reagents. Dimerization and elongation of the melanin polymer is known to require deprotonation. We have hypothesized that the amino acid side chains of melanosomal proteins act as proton acceptors to initiate polymerization and that the protonated basic groups serve to attract the negatively charged oligomers thus aiding polymerization and binding to proteins. We show that basic model proteins and basic premelanosomal proteins promote polymerization at an acidic pH and that positively charged surfaces allow binding of the growing melanin polymer. With progressive polymerization and exhaustion of the proton abstracting ability of melanosomal proteins, melanosomal pH drops further, which, we argue, is an additional controlling step that limits tyrosinase activity and melanin polymerization. 相似文献
83.
Liszewski MK Leung M Cui W Subramanian VB Parkinson J Barlow PN Manchester M Atkinson JP 《The Journal of biological chemistry》2000,275(48):37692-37701
Membrane cofactor protein (MCP; CD46), a widely distributed regulator of complement activation, is a cofactor for the factor I-mediated degradation of C3b and C4b deposited on host cells. MCP possesses four extracellular, contiguous complement control protein modules (CCPs) important for this inhibitory activity. The goal of the present study was to delineate functional sites within these modules. We employed multiple approaches including mutagenesis, epitope mapping, and comparisons to primate MCP to make the following observations. First, functional sites were located to each of the four CCPs. Second, some residues were important for both C3b and C4b interactions while others were specific for one or the other. Third, while a reduction in ligand binding was invariably accompanied by a parallel reduction in cofactor activity (CA), other mutants lost or had reduced CA but retained ligand binding. Fourth, two C4b-regulatory domains overlapped measles virus interactive regions, indicating that the hemagglutinin docks to a site important for complement inhibition. Fifth, several MCP regulatory areas corresponded to functionally critical, homologous positions in other CCP-bearing C3b/C4b-binding proteins. Based on these data and the recently derived crystal structure of repeats one and two, computer modeling was employed to predict MCP structure and examine active sites. 相似文献
84.
Recombinant production and purification of novel antisense antimicrobial peptide in Escherichia coli 总被引:9,自引:0,他引:9
Haught C Davis GD Subramanian R Jackson KW Harrison RG 《Biotechnology and bioengineering》1998,57(1):55-61
A fusion protein was genetically engineered that contains an antimicrobial peptide, designated P2, at its carboxy terminus and bovine prochymosin at its amino terminus. Bovine prochymosin was chosen as the fusion partner because of its complete insolubility in Escherichia coli, a property utilized to protect the cells from the toxic effects of the antimicrobial peptide. This fusion protein was purified by centrifugation as an insoluble inclusion body. A methionine linker between prochymosin and the P2 peptide enabled P2 to be released by digestion with cyanogen bromide. Cation exchange HPLC followed by reversed-phase HPLC were used to purify the P2 peptide. The recombinant P2 peptide's molecular mass was confirmed by mass spectrometry to within 0.1% of the theoretical value (2480.9 Da), and the antimicrobial activity of the purified recombinant P2 against E. coli D31 was determined to be identical to that of the chemically synthesized peptide (minimal inhibitory concentration of 5 mg/mL). Although the yield of the fusion protein after expression by the cells was high (16% of the total cell protein), the percentage recovery of the P2 peptide in the inclusion bodies was relatively low, which appears to be due to losses in the cyanogen bromide digestion step. 相似文献
85.
Livers with Constitutive mTORC1 Activity Resist Steatosis Independent of Feedback Suppression of Akt
Heidi L. Kenerson Savitha Subramanian Rebecca McIntyre Machiko Kazami Raymond S. Yeung 《PloS one》2015,10(2)
Insulin resistance is an important contributing factor in non-alcoholic fatty liver disease. AKT and mTORC1 are key components of the insulin pathway, and play a role in promoting de novo lipogenesis. However, mTORC1 hyperactivity per se does not induce steatosis in mouse livers, but instead, protects against high-fat diet induced steatosis. Here, we investigate the in vivo mechanism of steatosis-resistance secondary to mTORC1 activation, with emphasis on the role of S6K1-mediated feedback inhibition of AKT. Mice with single or double deletion of Tsc1 and/or S6k1 in a liver-specific or whole-body manner were generated to study glucose and hepatic lipid metabolism between the ages of 6–14 weeks. Following 8 weeks of high-fat diet, the Tsc1-/-;S6k1-/- mice had lower body weights but higher liver TG levels compared to that of the Tsc1-/- mice. However, the loss of S6k1 did not relieve feedback inhibition of Akt activity in the Tsc1-/- livers. To overcome Akt suppression, Pten was deleted in Tsc1-/- livers, and the resultant mice showed improved glucose tolerance compared with the Tsc1-/- mice. However, liver TG levels were significantly reduced in the Tsc1-/-;Pten-/- mice compared to the Pten-/- mice, which was restored with rapamycin. We found no correlation between liver TG and serum NEFA levels. Expression of lipogenic genes (Srebp1c, Fasn) were elevated in the Tsc1-/-;Pten-/- livers, but this was counter-balanced by an up-regulation of Cpt1a involved in fatty acid oxidation and the anti-oxidant protein, Nrf2. In summary, our in vivo models showed that mTORC1-induced resistance to steatosis was dependent on S6K1 activity, but not secondary to AKT suppression. These findings confirm that AKT and mTORC1 have opposing effects on hepatic lipid metabolism in vivo. 相似文献
86.
Lactic acid is an intermediate-volume specialty chemical for a wide range of food and industrial applications such as pharmaceuticals, cosmetics and chemical syntheses. Although lactic acid production has been well documented, improved production parameters that lead to reduced production costs are always of interest in industrial developments. In this study, we describe the production of lactic acid at high concentration, yield and volumetric productivity utilizing a novel homofermentative, facultative anaerobe Enterococcus faecalis CBRD01. The highest concentration of 182 g lactic acid l−1 was achieved after 38 h of fed-batch fermentation on glucose. The bacterial isolate utilized only 2–13% of carbon for its growth and energy metabolism, while 87–98% of carbon was converted to lactic acid at an overall volumetric productivity of 5 g l−1 h−1. At 13 h of fermentation, the volumetric productivity of lactate production reached 10.3 g l−1 h−1, which is the highest ever reported for microbial production of lactic acid. The lactic acid produced was of high purity as formation of other metabolites was less than 0.1%. The present investigation demonstrates a new opportunity for enhanced production of lactic acid with potential for reduced purification costs. 相似文献
87.
Manikandan Subramanian Lale Ozcan Devram Sampat Ghorpade Anthony W. Ferrante Jr. Ira Tabas 《PloS one》2015,10(8)
Obesity-induced inflammation in visceral adipose tissue (VAT) is a major contributor to insulin resistance and type 2 diabetes. Whereas innate immune cells, notably macrophages, contribute to visceral adipose tissue (VAT) inflammation and insulin resistance, the role of adaptive immunity is less well defined. To address this critical gap, we used a model in which endogenous activation of T cells was suppressed in obese mice by blocking MyD88-mediated maturation of CD11c+ antigen-presenting cells. VAT CD11c+ cells from Cd11cCre
+
Myd88
fl/fl vs. control Myd88
fl/fl mice were defective in activating T cells in vitro, and VAT T and B cell activation was markedly reduced in Cd11cCre
+
Myd88
fl/fl obese mice. However, neither macrophage-mediated VAT inflammation nor systemic inflammation were altered in Cd11cCre
+
Myd88
fl/fl mice, thereby enabling a focused analysis on adaptive immunity. Unexpectedly, fasting blood glucose, plasma insulin, and the glucose response to glucose and insulin were completely unaltered in Cd11cCre
+
Myd88
fl/fl
vs. control obese mice. Thus, CD11c+ cells activate VAT T and B cells in obese mice, but suppression of this process does not have a discernible effect on macrophage-mediated VAT inflammation or systemic glucose homeostasis. 相似文献
88.
Sachi Horibata Tommy V. Vo Venkataraman Subramanian Paul R. Thompson Scott A. Coonrod 《Journal of visualized experiments : JoVE》2015,(99)
Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells. 相似文献
89.
90.
Ramasamy Krishnamoorthy Chang-Gi Kim Parthiban Subramanian Ki-Yoon Kim Gopal Selvakumar Tong-Min Sa 《PloS one》2015,10(6)
Arbuscular Mycorrhizal Fungi (AMF) play major roles in ecosystem functioning such as carbon sequestration, nutrient cycling, and plant growth promotion. It is important to know how this ecologically important soil microbial player is affected by soil abiotic factors particularly heavy metal and metalloid (HMM). The objective of this study was to understand the impact of soil HMM concentration on AMF abundance and community structure in the contaminated sites of South Korea. Soil samples were collected from the vicinity of an abandoned smelter and the samples were subjected to three complementary methods such as spore morphology, terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) for diversity analysis. Spore density was found to be significantly higher in highly contaminated soil compared to less contaminated soil. Spore morphological study revealed that Glomeraceae family was more abundant followed by Acaulosporaceae and Gigasporaceae in the vicinity of the smelter. T-RFLP and DGGE analysis confirmed the dominance of Funneliformis mosseae and Rhizophagus intraradices in all the study sites. Claroideoglomus claroideum, Funneliformis caledonium, Rhizophagus clarus and Funneliformis constrictum were found to be sensitive to high concentration of soil HMM. Richness and diversity of Glomeraceae family increased with significant increase in soil arsenic, cadmium and zinc concentrations. Our results revealed that the soil HMM has a vital impact on AMF community structure, especially with Glomeraceae family abundance, richness and diversity. 相似文献