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91.
92.
Tsiang M Jones GS Niedziela-Majka A Kan E Lansdon EB Huang W Hung M Samuel D Novikov N Xu Y Mitchell M Guo H Babaoglu K Liu X Geleziunas R Sakowicz R 《The Journal of biological chemistry》2012,287(25):21189-21203
tert-Butoxy-(4-phenyl-quinolin-3-yl)-acetic acids (tBPQA) are a new class of HIV-1 integrase (IN) inhibitors that are structurally distinct from IN strand transfer inhibitors but analogous to LEDGINs. LEDGINs are a class of potent antiviral compounds that interacts with the lens epithelium-derived growth factor (LEDGF) binding pocket on IN and were identified through competition binding against LEDGF. LEDGF tethers IN to the host chromatin and enables targeted integration of viral DNA. The prevailing understanding of the antiviral mechanism of LEDGINs is that they inhibit LEDGF binding to IN, which prevents targeted integration of HIV-1. We showed that in addition to the properties already known for LEDGINs, the binding of tBPQAs to the IN dimer interface inhibits IN enzymatic activity in a LEDGF-independent manner. Using the analysis of two long terminal repeat junctions in HIV-infected cells, we showed that the inhibition by tBPQAs occurs at or prior to the viral DNA 3'-processing step. Biochemical studies revealed that this inhibition operates by compound-induced conformational changes in the IN dimer that prevent proper assembly of IN onto viral DNA. For the first time, tBPQAs were demonstrated to be allosteric inhibitors of HIV-1 IN displaying a dual mode of action: inhibition of IN-viral DNA assembly and inhibition of IN-LEDGF interaction. 相似文献
93.
Ana Gvozdenovic Matthias J. E. Arlt Carmen Campanile Patrick Brennecke Knut Husmann Walter Born Roman Muff Bruno Fuchs 《PloS one》2013,8(4)
Osteosarcoma (OS) is the most frequent primary malignant bone cancer in children and adolescents with a high propensity for lung metastasis. Therefore, it is of great importance to identify molecular markers leading to increased metastatic potential in order to devise more effective therapeutic strategies that suppress metastasis, the major cause of death in OS. CD44, the principal receptor for the extracellular matrix component hyaluronan (HA), is frequently found overexpressed in tumor cells and has been implicated in metastatic spread in various cancer types. Here, we investigated the effects of stable shRNA-mediated silencing of CD44 gene products on in vitro and in vivo metastatic properties of the highly metastatic human 143-B OS cell line. In vitro, CD44 knockdown resulted in a 73% decrease in the adhesion to HA, a 57% decrease in the migration rate in a trans-filter migration assay, and a 28% decrease in the cells'' capacity for anchorage-independent growth in soft agar compared to the control cells, implicating that CD44 expression contributes to the metastatic activity of 143-B cells. However, making use of an orthotopic xenograft OS mouse model, we demonstrated that reduced CD44 expression facilitated primary tumor growth and formation of pulmonary metastases. The enhanced malignant phenotype was associated with decreased adhesion to HA and reduced expression of the tumor suppressor merlin in vivo. In conclusion, our study identified CD44 as a metastasis suppressor in this particular experimental OS model. 相似文献
94.
95.
Marie Brázdová Lucie Navrátilová Vlastimil Tichy Kate?ina Němcová Matej Lexa Roman Hrstka Petr Pe?inka Matej Adámik Borivoj Vojtesek Emil Pale?ek Wolfgang Deppert Miroslav Fojta 《PloS one》2013,8(3)
Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition in vitro and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed. 相似文献
96.
Bactericidal peptidoglycan recognition protein induces oxidative stress in Escherichia coli through a block in respiratory chain and increase in central carbon catabolism 下载免费PDF全文
Des R. Kashyap Marcin Kuzma Dominik A. Kowalczyk Dipika Gupta Roman Dziarski 《Molecular microbiology》2017,105(5):755-776
Mammalian Peptidoglycan Recognition Proteins (PGRPs) kill both Gram‐positive and Gram‐negative bacteria through simultaneous induction of oxidative, thiol and metal stress responses in bacteria. However, metabolic pathways through which PGRPs induce these bactericidal stress responses are unknown. We screened Keio collection of Escherichia coli deletion mutants and revealed that deleting genes for respiratory chain flavoproteins or for tricarboxylic acid (TCA) cycle resulted in increased resistance of E. coli to PGRP killing. PGRP‐induced killing depended on the production of hydrogen peroxide, which required increased supply of NADH for respiratory chain oxidoreductases from central carbon catabolism (glycolysis and TCA cycle), and was controlled by cAMP‐Crp. Bactericidal PGRP induced a rapid decrease in respiration, which suggested that the main source of increased production of hydrogen peroxide was a block in respiratory chain and diversion of electrons from NADH oxidoreductases to oxygen. CpxRA two‐component system was a negative regulator of PGRP‐induced oxidative stress. By contrast, PGRP‐induced thiol stress (depletion of thiols) and metal stress (increase in intracellular free Zn2+ through influx of extracellular Zn2+) were mostly independent of oxidative stress. Thus, manipulating pathways that induce oxidative, thiol and metal stress in bacteria could be a useful strategy to design new approaches to antibacterial therapy. 相似文献
97.
Roman A. Sidorov Anatoly V. Zhukov Vasily P. Pchelkin Andrei G. Vereshchagin Vladimir D. Tsydendambaev 《化学与生物多样性》2013,10(6):976-988
Small amounts of a mixture of fatty acid short‐chain‐alkyl esters (FASCAEs) were obtained from the fruits of twelve plant species of Celastraceae family, and in five of them the FASCAEs were present not only in the arils but also in the seeds. These mixtures contained 32 individual FASCAE species, which formed four separate fractions, viz. FA methyl, ethyl, isopropyl, and butyl esters (FAMEs, FAEEs, FAIPEs, and FABEs, resp.). The FASCAE acyl components included the residues of 16 individual C14–C24 saturated, mono‐, di‐, and trienoic FAs. Linoleic, oleic, and palmitic acids, and, in some cases, also α‐linolenic acid predominated in FAMEs and FAEEs, while myristic acid was predominant in FAIPEs. It can be suggested that, in the fruit arils of some plant species, FAMEs and FAEEs were formed at the expense of a same FA pool characteristic of a given species and were strongly different from FAIPEs and FABEs esters regarding the mechanism of their biosynthesis. However, as a whole, the qualitative and quantitative composition of various FASCAE fractions, as well as their FA composition, varied considerably depending on various factors. Therefore, separate FASCAE fractions seem to be synthesized from different FA pools other than those used for triacylglycerol formation. 相似文献
98.
Birger Scholz Karl Sk?ld Kim Kultima Celine Fernandez Sofia Waldemarson Mikhail M. Savitski Marcus S?derquist Mats Borén Robert Stella Per Andrén Roman Zubarev Peter James 《Molecular & cellular proteomics : MCP》2011,10(3):M900229-MCP200-MCP200
Little is known about the nature of post mortem degradation of proteins and peptides on a global level, the so-called degradome. This is especially true for nonneural tissues. Degradome properties in relation to sampling procedures on different tissues are of great importance for the studies of, for instance, post translational modifications and/or the establishment of clinical biobanks. Here, snap freezing of fresh (<2 min post mortem time) mouse liver and pancreas tissue is compared with rapid heat stabilization with regard to effects on the proteome (using two-dimensional differential in-gel electrophoresis) and peptidome (using label free liquid chromatography). We report several proteins and peptides that exhibit heightened degradation sensitivity, for instance superoxide dismutase in liver, and peptidyl-prolyl cis-trans isomerase and insulin C-peptides in pancreas. Tissue sampling based on snap freezing produces a greater amount of degradation products and lower levels of endogenous peptides than rapid heat stabilization. We also demonstrate that solely snap freezing related degradation can be attenuated by subsequent heat stabilization. We conclude that tissue sampling involving a rapid heat stabilization step is preferable to freezing with regard to proteomic and peptidomic sample quality.The evolving maturation of the field of proteomics has, in the same way as in genomics, highlighted the need of better sampling procedures and sample preparation methodologies to minimize the effect of post mortem alterations. The aspect of sample quality is not new in any way and is relevant in most biomedical fields but has only lately started to receive adequate attention. The main factors influencing sample quality is storage temperature of the body until tissue removal (foremost a problem in clinical settings and extraction of less accessible tissue samples from model organisms) and post mortem interval (PMI)1 (1–3). Post mortem degradation in during PMI is a well known compromising problem when studying endogenous peptides (2, 3) and has also been proven to affect the results of polypeptide (here defined as proteins larger than 10 kDa) studies (3–8). PMI degradation has mainly been studied on human or mouse brain tissue, using two-dimensional electrophoresis (2-DE), SDS-PAGE, and immunoblotting (1, 3–12). There are also a few proteomic studies on muscle tissue degradation in livestock (13–16).We and others have previously explored the effect of focused microwave irradiation with regard to sample quality, demonstrating that this method is more reliable than snap freezing in liquid nitrogen, especially with regard to post-translational modification (PTM) stability (2, 3, 17–20). An alternative method based on cryostat dissection with subsequent heat treatment through boiling has also been reported to improve endogenous peptide sample quality (21). Besides focused microwave irradiation, which is specifically used for rodent brain tissue sampling, we have also demonstrated the efficiency of rapid heat stabilization through conductivity with regard to sample degradation (3, 22). Although somewhat constrained by its dependence on how quickly the tissue is harvested from the body, the latter procedure has the added advantage that it can be used on any type of tissue and species, fresh as well as frozen. This study will compare effects of sampling procedures on the liver and pancreas degradome following rapid heat stabilization, the more traditional snap freezing, or the combination of snap freezing with subsequent heat stabilization.To summarize, this study investigated the effects of post mortem degradation in pancreas and liver. Both tissues are well studied because of their multiple functions in the body and their involvement in different diseases such as diabetes or hepatocarcinoma. Pancreas is especially interesting in this context as it displays endocrine secretion of peptides, and exocrine secretion of digestive enzymes, the later making it a protease rich tissue. We used both two-dimensional difference in gel electrophoresis (2D-DIGE) and label free liquid chromatography mass spectrometry (LC-MS) based differential peptide display (2, 18), the later to better investigate changes in small molecular fragment that are not easily detectable by gel-based methods. 2D-DIGE is an unrivaled methodology to characterize alterations in isoform patterns, which is an important aspect considering that post-translational modifications (PTMs) such as phosphorylations are especially sensitive to post mortem influence within a few minutes PMI (3). The peptidomics approach has been used in several studies to point out early post mortem changes and protein degradation that tissue undergo following sampling and is therefore a well-suited method (3, 18, 22). 相似文献
99.
Leucine aminopeptidase (LAP) is an integral membrane glycoprotein localized to the apical membrane domain of intestinal and kidney epithelial cells. By indirect immunofluorescence, we have shown that antibodies raised against rat intestinal LAP recognized a similar protein concentrated in the bile canalicular (BC) domain of the hepatocyte in situ (Roman, L.M., and A.L. Hubbard, 1983, J. Cell Biol., 96:1548-1558). We have extended this localization to the ultrastructural level. When a saponin-permeabilized, agarose-embedded plasma membrane (PM) fraction was incubated with affinity-purified anti-LAP, 85% of the protein A-gold particles associated with the three recognizable PM domains were present in the BC. The levels of labeling on the other two domains (sinusoidal and lateral) did not exceed that observed with nonimmune controls. The concentration of LAP in the BC domain in isolated PM sheets prompted us to use this antigen for the affinity isolation of BC membrane (Roman, L.M., and A.L. Hubbard, 1984, J. Cell Biol., 98:1497-1504, companion paper). 相似文献
100.
Xu X Martin B Comstock JP Vision TJ Tauer CG Zhao B Pausch RC Knapp S 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2008,117(2):221-233
Carbon isotope composition (delta(13)C) and leaf water-use efficiency vary in concert in C3 plants, making delta(13)C useful as a proxy for plant water-use efficiency. A QTL for delta(13)C was detected in the Solanum pennellii chromosome fragment of IL5-4, an introgression line with S. lycopersicum cv. M82 background. M82 and IL 5-4 were crossed, and RFLP markers in the target region converted to PCR-based markers. Forty-one recombinants with an introgression fragment ranging in length from 1.1 to 11.4 cM were identified by marker assisted selection (MAS) among approximately 2000 F2 plants. A total of 29 markers were mapped within the introgression fragment unique to IL5-4. These markers divided the about 9 cM target region into nine intervals. A dominant QTL for delta(13)C, designated QWUE5.1 that explained 25.6% of the total phenotypic variance was mapped to an interval about 2.2 cM long. Twenty-one plants with a S. pennellii chromosome fragment shortened to a length of 2.0-9.1 cM by a second recombination event were generated by MAS of 1,125 F4 plants. Two near isogenic lines with high delta(13)C (small negative value) and carrying QWUE5.1 on the shortest introgression fragments (about 7.0 cM) were identified. The markers and genetic stocks developed are valuable for cloning the gene underlying QWUE5.1, MAS of QWUE5.1, and fine-mapping genes/QTL located in this region. 相似文献