Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

There is marked variability in the extent to which the three classes of antiretroviral (ARV) drugs bind to plasma proteins (<5 to >99%). Protease inhibitors (PIs), with the exception of indinavir, are more than 90% protein bound, mainly to alpha1-acid glycoprotein (AAG). Efavirenz, a nonnucleoside reverse transcriptase inhibitor (NNRTI), is more than 99% bound, mainly to albumin. Nucleoside reverse transcriptase inhibitors (NRTIs) are not highly protein bound. The pharmacological activity of ARV drugs is dependent on unbound drug entering cells that harbor the human immunodeficiency virus (HIV). There has been concern that changes in protein binding could impact on antiviral activity and management. However, for PIs and NNRTIs, and for many drugs given orally, altered plasma binding would not be expected to influence the average exposure to unbound (active) drug after chronic oral dosing. Nevertheless, there will be a change in the relationship between total and unbound concentrations that will be important if, as part of therapeutic drug monitoring, the total rather than the unbound drug is measured. Measuring drug concentrations that are needed to inhibit different HIV strains (wild type and drug resistant) in vitro could also cause confusion because most methods employ bovine serum in the assay medium, and unbound concentrations are not directly measured. Estimating unbound drug concentrations in human plasma and in incubation media can be highly method dependent and thus may affect the calculated IC50 (the concentration of drug that results in 50% inhibition of viral replication). Because inhibitory quotients (IQs = C(trough)/IC50) are becoming part of pharmacokinetic/pharmacodynamic (PK/PD) analyses of clinical trial data, the strengths and weaknesses of the methods used for the determination of unbound drug concentration in plasma and in vitro systems--ultracentrifugation, ultrafiltration, and equilibrium dialysis--need to be understood. Consensus on standard procedures must be reached. In June 2002, a panel of experts assembled by the Forum for Collaborative HIV Research met in Washington, DC, to review the basic principles of protein binding of ARV drugs, and to discuss the impact that changes in plasma protein binding may have on the PKs and activity of ARV drugs as well as on therapeutic drug monitoring. The purpose of the meeting was to discuss the following topics: (1) basic principles of protein binding and how changes in binding can impact on drug PKs and drug exposure in vivo, (2) variability in plasma protein binding among patients taking ARV drugs, (3) the impact of HIV infection and concomitant diseases on the extent of plasma protein binding, (4) the likelihood of clinically relevant drug interactions at the level of plasma protein binding, (5) the evidence that measuring unbound concentrations of ARV drugs in the plasma of patients gives more meaningful information than total drug concentration and, therefore, should be considered in routine therapeutic drug monitoring of ARV agents, (6) optimal method(s) for measuring the unbound concentration of drugs in vitro (for IC50 determination) and in vivo, and (7) future studies that need to be considered to fully understand the importance of plasma protein binding in therapeutic drug monitoring. This report summarizes the topics discussed at this meeting. It guides the reader through the discussions that allowed the panel to formulate a series of statements regarding the significance of plasma protein binding of ARV drugs when studied in vitro and in vivo. The roundtable participants also identified research priorities that are important for understanding the sources of inter- and intraindividual variability in protein binding in patients. These include obtaining data on unbound as well as on total concentrations in PK studies; looking at variants of AAG and whether they differ in binding affinity; and emphasizing the importance of developing a standard procedure for drug susceptibility assays used to determine IC50 values.
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PMID:Protein binding in antiretroviral therapies. 1458 13

Breakthroughs during lamivudine therapy were assessed according to hepatitis flares and mutational polymorphism of hepatitis B virus (HBV) infecting patients. Of 42 patients with chronic hepatitis B and positive for hepatitis B e antigen in serum, 13 (30%) harbored HBV mutants with lamivudine resistance after a mean duration of 29 months on lamivudine. The virological breakthrough occurred 14.5 months after the start of lamivudine treatment, and all the patients with it developed breakthrough hepatitis 3 months later. The clinical course of breakthrough hepatitis was self-limited except in one patient who had already developed cirrhosis at the baseline. One year after breakthrough hepatitis, serum ALT, albumin, prothrombin time and platelet counts were maintained well on conventional treatments without resorting to interferon. Major HBV mutants during breakthrough hepatitis were those with M552I in the YMDD motif of viral DNA polymerase/reverse transcriptase in 7 patients (54%), M552I/L528M in 4 patients (31%) and M552V/L528M in 2 patients (15%). There were no patients in whom mutations at nucleotide 529 occurred including the 2 who later developed hepatocellular carcinoma. There was no clear relationship between distinct mutational patterns and clinical courses. Further studies are needed for making out the effects of lamivudine-resistant mutants on clinical outcomes, taking into considerations genotypes of HBV.
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PMID:Subclones of drug-resistant hepatitis B virus mutants and the outcome of breakthrough hepatitis in patients treated with lamivudine. 1468 51

The aim of the study was to determine the correlation between the expression of tissue factor (TF) and the receptor for advanced glycation end products (RAGEs) and vascular complications in patients with longstanding uncontrolled type 2 diabetes (T2D). TF and RAGE mRNAs as well as TF antigen and activity were investigated in 21 T2D patients with and without vascular complications. mRNA expression was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) in nonstimulated and advanced glycation end product (AGE) albumin-stimulated peripheral blood mononuclear cells (PBMCs). TF antigen expression was determined by enzyme-linked immunosorbent assay (ELISA) and TF activity by a modified prothrombin time assay. Basal RAGE mRNA expression was 0.2 +/- 0.06 in patients with complications and 0.05 +/- 0.06 patients without complications (P =.004). Stimulation did not cause any further increase in either group. TF mRNA was 0.58 +/- 0.29 in patients with complications and 0.21 +/- 0.18 in patients without complications (P =.003). Stimulation resulted in a nonsignificant increase in both groups. Basal TF activity (U/10(6) PBMCs) was 18.4 +/- 13.2 in patients with complications and 6.96 +/- 5.2 in patients without complications (P =.003). It increased 3-fold in both groups after stimulation (P =.001). TF antigen (pg/10(6) PBMCs) was 33.7 +/- 28.6 in patients with complications, 10.4 +/- 7.8 in patients without complications (P =.02). Stimulation tripled TF antigen in both groups of patients (P =.001). The RAGE/TF axis is up-regulated in T2D patients with vascular complications as compared to patients without complications. This suggests a role for this axis in the pathogenesis of vascular complications in T2D.
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PMID:Increased expression of tissue factor and receptor for advanced glycation end products in peripheral blood mononuclear cells of patients with type 2 diabetes mellitus with vascular complications. 1520 87

In this study, xyloglucan (XG) was used as a new synthetic extracellular matrix (ECM) for primary mouse hepatocyte attachment in Ca-alginate (AL) capsules. The rates of hepatocytes adhesion onto collagen type I-, XG-coated and uncoated polystyrene (PS) surface were 89.1%, 91.1% and 25.5%, respectively, at 4 h after incubation at 37 degrees C. From the inhibition study in a cell adhesion assay, the adhesion rates of freshly isolated hepatocytes and preincubated hepatocytes with 20 mm galactose onto the XG-coated surface were 55.7 and 17.3%, respectively, after 30 min incubation at 37 degrees C. Flow cytometric analysis showed that the internalization of XG by freshly isolated hepatocytes was stronger than preincubated hepatocytes with 20 mm galactose. The concentration of XG in AL/XG capsules to perform the best liver-specific functions was 0.5 mg/ml, where the highest albumin secretion rates were obtained. The albumin secretion, ammonia elimination rates and cell viability of hepatocytes were slowly decreased with culture time in AL/XG capsules, whereas those were rapidly decreased in AL capsules, indication of the more rapid formation of hepatocyte spheroids in AL/XG capsules than in AL capsules. More than 70% of the seeded hepatocytes in AL/XG capsules participated in spheroid formation after 2 days, whereas most hepatocytes in AL capsules remained as single cells and only a few cells began to form aggregates after 3 days. Intercellular molecule genes, such as connexin (Cx) 32 and E-cadherin, of hepatocyte spheroids in AL or AL/XG capsules were detected by reverse transcriptase-polymerase chain reaction. Cx32 and E-cadherin genes in AL/XG capsules were more rapidly reexpressed and expressed, respectively, than in AL ones. The results suggest that the multicellular spheroid formation of hepatocytes can enhance the liver-specific functions in the three-dimensional space in the presence of XG as a new synthetic ECM owing to the specific interaction between the galactose moieties of XG and asialoglycoprotein receptors of hepatocytes.
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PMID:Alginate microcapsules prepared with xyloglucan as a synthetic extracellular matrix for hepatocyte attachment. 1562 Dec 51

Diesel exhaust particles (DEPs) at three concentrations (5, 35, and 50 mg/kg body weight) were instilled into rats intratracheally. We studied gene expression at 1, 7, and 30 days postexposure in cells obtained by bronchoalveolar lavage (BAL) and in lung tissue. Using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), we measured the mRNA levels of eight genes [interleukin (IL)-1beta, IL-6, IL-10, iNOS (inducible nitric oxide synthase), MCP-1 (monocyte chemoattractant protein-1), MIP-2 (macrophage inflammatory protein-2), TGF-beta1 (transforming growth factor-beta1), and TNF-alpha (tumor necrosis factor-alpha )] in BAL cells and four genes [IL-6, ICAM-1 (intercellular adhesion molecule-1), GM-CSF (granulocyte/macrophage-colony stimulating factor), and RANTES (regulated upon activation normal T cell expressed and secreted)] in lung tissue. In BAL cells on day 1, high-dose exposure induced a significant up-regulation of IL-1beta, iNOS, MCP-1, and MIP-2 but no change in IL-6, IL-10, TGF-beta1, and TNF-alpha mRNA levels. There was no change in the mRNA levels of IL-6, RANTES, ICAM-1, and GM-CSF in lung tissue. Nitric oxide production and levels of MCP-1 and MIP-2 were increased in the 24-hr culture media of alveolar macrophages (AMs) obtained on day 1. IL-6, MCP-1, and MIP-2 levels were also elevated in the BAL fluid. BAL fluid also showed increases in albumin and lactate dehydrogenase. The cellular content in BAL fluid increased at all doses and at all time periods, mainly due to an increase in polymorphonuclear leukocytes. In vitro studies in AMs and cultured lung fibroblasts showed that lung fibroblasts are a significant source of IL-6 and MCP-1 in the lung.
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PMID:Time course of gene expression of inflammatory mediators in rat lung after diesel exhaust particle exposure. 1586 72

The detection of prostate-specific antigen (PSA) mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) in the bloodstream of prostate cancer patients has been hypothesized as a prognostic marker, however little data are available concerning the association between this molecular marker and other laboratory values of potential importance. In this study, in patients with hormone-refractory prostate cancer (HRPC), relationships were determined between PSA RT-PCR positivity, survival, and various relevant markers including serum PSA, LDH, albumin, alkaline phosphatase and hemoglobin. A total of 19/30 HRPC patients were positive for PSA by RT-PCR. Positivity was significantly linked to serum PSA (P=0.004) and serum alkaline phosphatase (P=0.026) but not to the other laboratory variables. Median survival time for RT-PCR-positive patients was 9 months, compared to 19 months for RT-PCR-negative patients (P=0.035). Median survival time for patients with a hemoglobin>or=11 g/dL was 12 months, compared to 9 months for patients with <11 g/dL (P=0.005). Dichotomized (>or=or<median) serum PSA, LDH, alkaline phosphatase, and albumin were not significantly associated with survival in univariate analyses. In multivariate analysis, only dichotomized hemoglobin (<11 g/dL vs. >or=11 g/dL) remained statistically significant (P=0.019), indicating that RT-PCR had no independent association with survival after controlling for hemoglobin status in this study.
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PMID:Relationships between reverse transcriptase-polymerase chain reaction for prostate specific antigen, survival, and various prognostic laboratory factors in patients with hormone refractory prostate cancer. 1590 15

Differentiation of adult bone marrow (BM) cells into nonhematopoietic cells is a rare phenomenon. Several reports, however, suggest that human umbilical cord blood (hUCB)-derived cells give rise to hepatocytes after transplantation into nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. Therefore, we analyzed the hepatic differentiation potential of hUCB cells and compared the frequency of newly formed hepatocyte-like cells in the livers of recipient NOD-SCID mice after transplantation of hUCB versus murine BM cells. Mononuclear cell preparations of hUCB cells or murine BM from enhanced green fluorescent protein transgenic or wild-type mice were transplanted into sublethally irradiated NOD-SCID mice. Liver regeneration was induced by carbon tetrachloride injury with and without subsequent hepatocyte growth factor treatment. By immunohistochemistry and reverse transcriptase-polymerase chain reaction, we detected clusters of hepatocyte-like cells in the livers of hUCB-transplanted mice. These cells expressed human albumin and Hep Par 1 but mouse CK18, suggesting the formation of chimeric hepatocyte-like cells. Native fluorescence microscopy and double immunofluorescence failed to detect single hepatocytes derived from transplanted enhanced green fluorescent protein-transgenic mouse BM. Fluorescent in situ hybridization rarely revealed donor-derived hepatocyte-like cells after cross-gender mouse BM transplantation. Thus, hUCB cells have differentiation capabilities different from murine BM cells after transplantation into NOD-SCID mice, demonstrating the importance of further testing before hUCB cells can be used therapeutically.
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PMID:Human cord blood stem cells generate human cytokeratin 18-negative hepatocyte-like cells in injured mouse liver. 1604 39

Carboxypeptidase D (CPD) functions in the processing of proteins that transit the secretory pathway, and is present in all vertebrates examined as well as Drosophila. Several forms of CPD mRNA were previously found in Drosophila that resulted from differential splicing of the gene. In the present study, Northern blot, reverse transcriptase PCR, and Western blot analysis showed that each splice variant occurs in a single cell type, the Drosophila-derived Schneider 2 (S2) cell line. The short forms containing a single carboxypeptidase domain were secreted from the S2 cells while the long forms containing three carboxypeptidase domains, a transmembrane domain, and one of two different cytosolic tails were retained in the cell. To investigate the role of the two different C-terminal tail sequences (tail-1 and tail-2) that result from the differential splicing within exon 8, constructs containing a reporter protein (albumin) attached to the transmembrane domain and tail-1 or tail-2 of CPD were expressed in S2 cells and a mouse pituitary cell line (AtT20 cells). Immunofluorescence analysis revealed different intracellular distributions of the two constructs, with the tail-2 construct showing considerable overlap with a Golgi marker. The two C-terminal tail sequences also resulted in different internalization efficiencies from the cell surface in both cell lines. Interestingly, the distribution and routing of the tail-2 form of Drosophila CPD in the AtT20 cells are similar to the previously characterized endogenous mouse CPD protein, indicating that the elements for this trafficking have been conserved between Drosophila and mammals.
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PMID:Drosophila S2 cells produce multiple forms of carboxypeptidase D with different intracellular distributions. 1667 61

Embryonic stem (ES) cells can replicate indefinitely and differentiate into all cell types, including hepatocytes. Research using primate ES cells is considered to be important for studies of potential cell therapies. Recently, we established cynomolgus monkey ES cells designated as CMK6. The CMK6 cell line is a useful tool for investigating the mechanism of differentiation in primate ES cells and developing cell therapies, because of its biological similarity to human ES cells. To examine whether cynomolgus monkey ES cells differentiate into hepatocytes, CMK6 cells were cultured with or without acidic fibroblast growth factor (aFGF). Evaluation of the hepatic differentiation was performed by analysis of the mRNA expression in early hepatic marker genes using the reverse transcriptase-polymerase chain reaction (RT-PCR). The protein expression of albumin (ALB) was also studied by immunocytochemistry. RT-PCR analyses revealed mRNA expressions of alpha-fetoprotein, transthyretin, and ALB in the presence of aFGF at 3 wk of differentiation, whereas no mRNA expression of these genes was detected in cells without aFGF. The protein expression of ALB in the presence of aFGF at 3 wk of differentiation was also confirmed by immunocytochemistry. However, tyrosine aminotransferase, which is a mature hepatic marker, was not detected in the presence or absence of aFGF at any stage of differentiation. These results suggested that aFGF successfully promoted in vitro differentiation of cynomolgus monkey ES cells to an early hepatic lineage.
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PMID:Acidic fibroblast growth factor promotes hepatic differentiation of monkey embryonic stem cells. 1675 53

Since 1999, numerous articles have reported the generation of hepatocytes from different types of extrahepatic stem or precursor cells. This opens exciting new possibilities for pharmacology and toxicology, as well as for cell therapy. Hepatocyte marker expression, including albumin, cytokeratin 18, c-met, alpha-fetoprotein and cytochrome P450 3A4 and -2B6, has been observed after transplantation of different types of human stem cells into the liver of laboratory animals or in vitro after incubation with cytokines. These intriguing observations have prompted scientists to classify stem cell-derived cell populations as hepatocytes. However, this conclusion may be premature. It has been shown that factors of the liver microenvironment can induce expression of a limited number of hepatocyte marker genes in nonhepatic cell types. To conclude on the grounds of a limited number of markers that these cells are true hepatocytes is not indicated. In this case one should carefully evaluate crucial hepatocyte-defining enzymatic properties. The present article: i) reviews studies describing the fate of extrahepatic human stem and precursor cells in livers of laboratory animals, including the possibility of cell fusion; and ii) critically discusses the phenotype of stem cells after application of various differentiation protocols aimed at generating human hepatocytes. In addition, the necessary criteria needed for defining a true hepatocyte are suggested. Establishing the necessary properties for stem cell-derived hepatocytes is timely and reasonable, and thus avoids further misleading semantic confusion. Finally, it is essential to understand that the definition of a bona fide hepatocyte should not be limited to qualitative assays, such as reverse transcriptase polymerase chain reaction and immunohistochemistry, but has to include a quantitative analysis of enzymatic activities, which allows direct comparison with primary hepatocytes. Although the stem cell-derived-hepatocyte does not yet exist there is a good chance that this aim may be achieved in the future.
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PMID:Generation of human hepatocytes by stem cell technology: definition of the hepatocyte. 1692 53


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