Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Antiviral therapy of human immunodeficiency virus (HIV) infection is currently based on inhibition of reverse transcriptase by dideoxynucleosides, such as azidothymidine. Because of widespread toxicity it is reasonable to selectively target these drugs to infected cells. This may be accomplished utilizing drug-LDL conjugates, which are internalized via cell specific receptor pathways. With respect to HIV infection, scavenger receptors of the macrophage system seems to offer a hopeful perspective. This pathway requires chemical modification of surface polarity of the LDL. Cell experiments were conducted in HepG2 hepatocytes, which express apolipoprotein B receptors, and in P388 macrophages, which express scavenger receptors. LDL particles to be conjugated were isolated from blood donor plasma and from LDL-apheresis waste material. Non-covalent LDL conjugation with amphiphilic nucleoside derivatives produced only an unspecific nucleoside transfer to cell membranes, due to instability of the LDL conjugates. An experimental method (coincubation test) was developed to identify those conjugates that are stable in the presence of other lipophilic compartments. Covalent coupling of nucleosides to the apolipoprotein B moiety of LDL particles resulted in stable conjugates. As a consequence, the surface charge became negative, and the LDL displayed scavenger receptor affinity rather than apolipoprotein B receptor affinity. Selective targeting of nucleosides to macrophages can be accomplished by covalent coupling to LDL.
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PMID:Preparation of nucleoside-LDL-conjugates for the study of cell-selective internalization: stability characteristics and receptor affinity. 176 41

During the differentiation of human enterocytes, the secretion of apolipoprotein B is switched from the apoB-100 form seen almost exclusively in fetal cells to the apoB-48 form seen almost exclusively in adult cells. This switch is accomplished by the post-transcriptional editing of the messenger RNA for apoB-100. We report that a similar switch occurs during the differentiation of the human colonic carcinoma cell line, Caco-2, and that this is accomplished by the same mRNA editing mechanism. Caco-2 cells cultured on Millipore filters developed confluent electrically resistant monolayers, and on Western blot analysis, using a monoclonal antibody directed against the amino terminal region of human apoB-100 (Mab C1.4), secreted greater than 50% apoB-48 (of total apoB-100) into culture media, while Caco-2 cells grown on plastic secreted greater than 95% apoB-100. To assess whether mRNA editing was responsible for the switchover from apoB-100 to apoB-48, apoB cDNA fragments spanning nucleotides 6504-6784 of apoB mRNA were synthesized using RNA isolated from Caco-2 cells grown on filters and Caco-2 cells grown on plastic. The appropriate oligonucleotide primers and Moloney murine leukemia virus reverse transcriptase were used. The resulting cDNA fragments were amplified by the polymerase chain reaction (PCR), and PCR products were subcloned and sequenced. A single cytosine/thymine base change occurred in 8/20 clones of cDNA derived from Caco-2 cells grown on filters (corresponding to a cytosine/uridine change in mRNA) and in 1/25 clones of cDNA derived from Caco-2 cells grown on plastic. PCR products of genomic sequences from Caco-2 cells did not contain the stop codon.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Editing of apolipoprotein B messenger RNA in differentiated Caco-2 cells. 235 74

To investigate the coagulation system in crustacean decapoda, a homodimeric glycoprotein of 380 kDa was purified from the hemolymph of tiger shrimp (Penaeus monodon) by sequential DEAE anion exchange chromatography. The purified protein was coagulated by the shrimp hemocyte transglutaminase in the presence of Ca2+. The clottable protein contains 44% alpha helices and 26% beta sheets as determined by circular dichroism spectra. Its conformation is stable in buffer of pH 4-9. To solve its primary structure, partial sequences of the purified polypeptides from cyanogen bromide cleavage and endopeptidase digestion were also determined. A shrimp cDNA expression library was constructed. By combination with antibody screening, reverse transcriptase PCR using degenerate primers from determined amino acid sequences and cDNA library screening with digoxigenin-labeled DNA probes, the entire cDNA of 6124 bp was obtained. This cDNA encodes a protein of 1670 amino acids, including a 14-amino acid signal peptide. With four potential N-glycosylation sites, the clottable protein was found to contain 3.8% high-mannose glycan; and Man8GlcNAc and Man9GlcNAc were released upon endo-beta-N-acetylglucosaminidase hydrolysis. Upon conducting a protein sequence database survey, the shrimp clottable protein shows 36% identities to the crayfish clotting protein and lower similarities to members of insect vitellogenins, apolipoprotein B and mammalian von Willebrand factor. Notably, a region rich in Gln residues, a polyGln motif and five Ser-Lys-Thr-Ser repeats are present in the shrimp protein, suggesting this protein might be a transglutaminase substrate. Northern blot analysis revealed that the clottable protein is expressed in most of the shrimp tissues but not in the mature hemocytes.
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PMID:Molecular cloning and characterization of a hemolymph clottable protein from tiger shrimp (Penaeus monodon). 1056 6

Our objective was to determine whether HIV-infected children treated with protease inhibitors (PIs) have different blood lipid, insulin, and glucose levels and body composition than HIV-infected children not treated with PIs. A cross-sectional cohort study was performed; in which 23 children were treated with combination antiretroviral therapy including a PI for at least 6 months and 12 children were treated with nucleoside reverse transcriptase inhibitors only (no-PI group). Levels of lipids, apolipoprotein B (apoB), insulin, and glucose were determined in the fasting state. Body composition and fat distribution were determined by anthropometric measurements and dual energy X-ray absorptiometry (DEXA) scan. Total cholesterol levels were higher in the PI-treated children (5.33 +/- 0.87 mM) than in the no-PI children (3.69 +/- 0.59 mM) (p < 0.0001). Similarly, low-density lipoprotein (LDL) levels were also elevated in the PI-treated children (3.27 +/- 0.76 vs. 2.14 +/- 0.51 mM) (p < 0.0001). ApoB and high-density lipoprotein (HDL), and to a lesser degree triglyceride levels, were also increased in the PI-treated children. Apart from percent arm fat as measured by DEXA, there were no differences between the two groups in measures of body composition or in their fasting glucose and insulin levels. The results from this cross-sectional cohort study suggest that the predominant lipid abnormalities associated with treatment with combination antiretroviral therapy including a PI in HIV-1-infected children are elevated total and LDL cholesterol.
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PMID:Metabolic abnormalities in HIV type 1-infected children treated and not treated with protease inhibitors. 1152 81

C127, a murine mammary tumor-derived cell line, is capable of lipidating and secreting apolipoprotein B-41 (apoB-41) in the apparent absence of microsomal triglyceride transfer protein (MTP). Using a semiquantitative reverse transcriptase-coupled polymerase chain reaction, mouse MTP mRNA was detected in C127 cells at approximately 10-20% of the relative abundance of human MTP in HepG2 cells. Radiolabeling of C127 cells with [35S]methionine and [35S]- cysteine followed by immunoprecipitation with anti-MTP antibodies identified a band with an electrophoretic mobility identical to that of authentic mouse MTP. Cotransfection of apoB-41 and the MTP 97-kDa subunit in C127 cells enhanced apoB secretion by approximately 5-fold relative to apoB-41 transfection alone, suggesting that MTP is limiting in these cells. To establish that MTP expression is responsible for apoB-containing lipoprotein assembly in C127 cells, the effects of the MTP inhibitor BMS-200150 were examined. Secretion of apoB-41 by C127 cells was inhibited to the same extent observed in COS-1 cells cotransfected with apoB-41 and MTP. These results suggest that low MTP expression, and not the expression or overexpression of another known or novel factor(s), is responsible for apoB assembly and secretion in C127 cells and further supports the essential nature of MTP in the biogenesis of apoB-containing lipoproteins. .
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PMID:Lipoprotein assembly capacity of the mammary tumor-derived cell line C127 is due to the expression of functional microsomal triglyceride transfer protein. 1171 59

The aim of this study was to identify the first abnormalities of apolipoprotein B (apoB) metabolism in HIV-infected patients treated by antiretroviral therapy (ART) with protease inhibitors (PIs). The influence of ART on the metabolism of apoB in VLDL, IDL, and LDL was investigated in six patients receiving dual nucleoside reverse transcriptase inhibitors (NRTIs) and PI, and in five patients receiving NRTI and nevirapine. None of the patients had lipodystrophy. The study was performed in the fed state. Each subject received an intravenous injection of a 0.7 mg.kg-1 bolus of l-[1-13C]leucine, immediately followed by a 16 h constant infusion at 0.7 mg.kg-1.h-1. The VLDL- and IDL-apoB concentrations were significantly higher in PI-treated patients compared to non-PI-treated patients. The VLDL-apoB and IDL-apoB production rates were markedly higher in PI-treated patients compared to non-PI-treated patients (54.5 +/- 30.1 vs. 30.9 +/- 8.4 mg.kg-1.d-1, P = 0.04; and 43.5 +/- 20.0 vs. 18.7 +/- 7.8 mg.kg-1.d-1, P = 0.04, respectively). In conclusion, our study shows that patients receiving ART with PI present altered metabolism of the VLDL-IDL-LDL chain compared with patients treated without PI. These data confirm that PI therapy is associated with a physiopathological mechanism for dyslipidemia in addition to the effect of lipodystrophy on lipid metabolism.
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PMID:Increased VLDL-apoB and IDL-apoB production rates in nonlipodystrophic HIV-infected patients on a protease inhibitor-containing regimen: a stable isotope kinetic study. 1286 87

The orphan hepatic nuclear factor (HNF) HNF4alpha is of pivotal importance for liver development and hepatocellular differentiation and plays an essential role in a regulatory circuitry to control a wide range of metabolic processes. It also targets genes in other organs, including pancreas, kidney, intestine, and colon; promotes expression of an epithelial phenotype; triggers de novo formation of functional tight junctions; and contributes to epithelial cell polarity. In particular, HNF4alpha dysfunction leads to metabolic disorders, including diabetes. We used the chromatin immunoprecipitation (ChIP) cloning procedure and a bioinformatic approach to search for candidate genes associated with impaired liver, pancreas, and kidney function. We identified two novel targets regulated by HNF4alpha, which participate in the control, at least in part, in cell-cycle regulation and are members of the mitogen-activated kinase pathway. In multiple ChIP assays, ribosomal S6 kinase 4 (RSK4) and p21-activated kinase 5 (PAK5) were confirmed, and in vitro binding of HNF4alpha was evidenced by electrophoretic mobility shift assays (EMSA) using oligonucleotides, which harbor novel binding sites. We also used EMSA to probe for binding sites in promoters of HNF1alpha, apolipoprotein B, alpha1-antitrypsin, and angiotensinogen. We further studied RSK4 and PAK5 kinase expression in streptozotocin-induced diabetic rat kidney and brain and observed significant repression of HNF4alpha, RSK4, and PAK5 as determined by quantitative real-time reverse transcriptase-polymerase chain reaction. RSK4 and PAK5 may provide a molecular rationale for late-stage complications in disease, and further studies are warranted to explore these targets for the treatment of diabetic nephro- and neuropathy, frequently seen in patients with HNF4alpha dysfunction.
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PMID:RSK4 and PAK5 are novel candidate genes in diabetic rat kidney and brain. 1561 95

C-reactive protein (CRP) is frequently deposited in the lesions of the arterial intima; however, the origin and pathological significance of CRP in these lesions are not completely understood. In this study, we measured CRP levels in the plasma of hypercholesterolemic rabbits and investigated CRP expression at both the mRNA and protein levels using rabbit and human atherosclerotic specimens. CRP levels were significantly elevated in both cholesterol-fed and Watanabe heritable hyperlipidemic rabbits, and CRP levels were clearly correlated with aortic atherosclerotic lesion size. Immunohistochemical staining coupled with Western blotting analysis revealed that CRP-immunoreactive proteins were found at all stages of atherosclerosis from the early to advanced lesions. CRP was present extracellularly and co-localized with apolipoprotein B but was rarely associated with the cytoplasm of macrophages and foam cells. Real-time reverse transcriptase-polymerase chain reaction analysis revealed that CRP mRNA in atherosclerotic lesions was barely detectable, and isolated macrophages did not express CRP mRNA, suggesting that CRP proteins found in the lesions were essentially derived from the circulation rather than synthesized de novo by vascular cells. These results suggest that there is a link between plasma CRP and the degree of atherosclerosis and that inhibition of plasma CRP may represent a therapeutic modality for the treatment of cardiovascular disease.
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PMID:C-reactive protein in atherosclerotic lesions: its origin and pathophysiological significance. 1650 18

The introduction of highly active antiretroviral therapy (HAART) combining potent drugs that can inhibit reverse transcriptase, integrase and protease activities has changed the natural history of the human immunodeficiency virus (HIV) type 1 disease. Unfortunately, poor penetrability into different anatomic compartments, toxicity and drug resistance are some of the problems related to their prolonged use. The ability of HIV to mutate and become resistant, along with the ongoing viral replication during HAART, can lead to the emergence of independently evolving viral strains in different anatomic compartments (i.e., brain, testes, lymph nodes, etc.). In addition, HAART predominantly effects the viral replication in the activated or differentiating CD(+) T lymphocytes, but appears to have a very limited effect on HIV-1 preintegration complexes in the latently infected cells. Existing drug therapies do not eliminate these viral reservoirs, nor do they prevent their formation. New strategies are needed for eliminating protected areas of HIV-1 in vivo. Therefore, the persistence of latent HIV-1 reservoirs is the principal barrier in the complete eradication of HIV-1 infection in patients by antiretroviral therapy at present. African non-human primates (NHPs) naturally infected with various simian immunodeficiency viruses (SIVs) appear not to develop immunodeficiency or AIDS, whereas Asian NHPs, which are unnatural hosts, infected with SIVs, as well humans infected with HIV-1, will nearly always develop progressive loss of CD(+) T lymphocytes and a gradual destruction of immune functions. Understanding the difference in the host responses between natural and unnatural hosts, and deciphering which host factors are responsible for the non-pathogenic course of natural SIV infections, would be valuable in developing more-effective treatment or prevention strategies for HIV/AIDS. A number of factors encoded by host cells have been identified that appear to play critical roles in the SIV infection process. Two of these factors, TRIM5alpha (a member of a large family of proteins known as the TRIM proteins) and cellular apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like-3G (APOBEC3G) have been recently identified. APOBEC3G genes belong to a family of primate genes that produce enzymes (in this case, APOBEC3G) that 'edit' RNA by replacing cytosine with guanine into viral particles as the virus undergoes reverse transcription in the cytoplasm of the host cell. HIV-1, in turn, counters with a protein called viral infectivity factor (Vif), which binds to the APOBEC3G enzyme that degrades it. Several other blocking factors have been described, including lentiviral blocking factor (Lv)1 and 2. These factors appear to block the infection at a postentry step; after reverse transcription has occurred, but before proviral integration. Thus, it is crucial to understand the molecular mechanisms involved in the establishment, maintenance and reactivation of lentiviral latency. This review presents various models of HIV-1 latency and forward a new unified model of lentiviral latency.
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PMID:A unified concept of HIV latency. 1704 12

The aim of this study was to determine whether systemic elevation of tumor necrosis factor (TNF)-alpha induces intestinal-derived apolipoprotein B (apoB)48-containing very low-density lipoprotein (VLDL) production in hamsters after fat loading and whether TNF-alpha disturbs the related mRNA expression in inflammatory, insulin and lipoprotein signaling pathways in primary enterocytes. In vivo TNF-alpha and Triton-WR1339 infusion, Western blotting and reverse transcriptase-polymerase chain reaction were combined to explore the mechanisms underlying intestinal overproduction of apoB48-containing chylomicrons and VLDL(1) particles by TNF-alpha. TNF-alpha infusion increased intestinal production of chylomicron and VLDL(1)-apoB48 in postprandial (fat load) states. Following TNF-alpha-treatment in enterocytes, there was enhanced gene expression of Il1alpha and beta, Il6 and Tnf and decreased mRNA levels of components of the insulin signaling pathway including the insulin receptor (Ir), Ir substrate-1 and 2, PI3 k, and Akt, but increased phosphatase and tensin homolog deleted on chromosome ten (Pten) protein and mRNA expression. TNF-alpha also induced Cd36 and peroxisome proliferators-activated receptor (Ppar)gamma expression, as well as microsomal triglyceride transfer protein (Mtp) protein and mRNA, but suppressed the sterol regulatory element binding protein (Srebp)1c protein and mRNA level. Systemic elevation of TNF-alpha stimulates the postprandial overproduction of apoB48-containing chylomicrons and VLDL(1) particles by disturbing intestinal gene expression of the inflammatory, insulin and lipoprotein pathways. These findings provide mechanistic links among the inflammatory factor, TNF-alpha, intestinal inflammatory/insulin insensitivity and the overproduction of intestinal apoB48-containing lipoproteins.
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PMID:Elevation of tumor necrosis factor-alpha induces the overproduction of postprandial intestinal apolipoprotein B48-containing very low-density lipoprotein particles: evidence for related gene expression of inflammatory, insulin and lipoprotein signaling in enterocytes. 2040 35


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