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)

Neisseria gonorrhoeae is capable of utilizing host iron-binding proteins, such as transferrin, lactoferrin, and hemoglobin, as the sole source of iron. The receptor involved in transferrin iron acquisition is composed of two distinct transferrin-binding proteins, TbpA and TbpB. The genes that encode these proteins are linked on the chromosome in the order tbpB-tbpA but are separated by an inverted repeat of unknown function. In this study, we sought to understand the transcriptional organization and regulation of the tbp genes, using a combination of lacZ transcriptional fusion analysis and reverse transcriptase PCR (RT-PCR). First, we demonstrated that tbpB and tbpA are cotranscribed and coregulated from the common upstream promoter that precedes tbpB. Using beta-galactosidase activity as a surrogate for tbp-specific transcription, we found that tbpB-specific transcripts were more prevalent than tbpA-specific transcripts after 2 h of growth under iron stress conditions. We confirmed the results obtained by fusion analysis by using RT-PCR applied to native RNA isolated from wild-type gonococci. Three different varieties of RT-PCR were employed: relative, competitive, and real time quantitative. The results of all analyses indicated that tbpB-specific transcripts were approximately twofold more prevalent than tbpA-specific transcripts at steady state. In iron-stressed cultures, the ratio of tbpB- to tbpA-specific message was approximately 2; however, in iron-replete cultures, this ratio dropped to 1. Using these techniques, we also quantitated the effects of iron, external pH, and presence of ligand on tbp mRNA levels.
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PMID:Gonococcal genes encoding transferrin-binding proteins A and B are arranged in a bicistronic operon but are subject to differential expression. 1155 78

AIM:To investigate the characteristics of newly established four hepatocellular carcinoma cell lines (SNU-739, SNU-761, SNU-878 and SNU-886) from Korean hepatocellular cancer patients.METHODS:Morphologic and genetic studies were done.RESULTS:All four lines grew as a monolayer with an adherent pattern, and their doubling times ranged from 20 to 29 hours. The viability rate was relatively high (88%-94%).Neither mycoplasmal nor bacterial contamination was present.The lines showed different patterns in fingerprinting analysis. The hepatitis B virus (HBV) DNA was integrated in the genomes of all four lines, and in all of them HBx,HBc and HBs transcripts were detected by reverse transcriptase-PCR methods. Among the three cell lines used as control (Hep 3B, SK Hep1 and Hep G2),only Hep 3B showed HBx expression, and this line was used as a HBV integrated control.The RNA of albumin was detected in three lines (SNU-761, SNU-878 and SNU-886), that of transferrin in two lines (SNU-878, SNU-886), and that of IGF-II was detected in none of the cell lines.CONCLUSION:These well characterized cell lines may be very useful for studying the biology of hepatocellular carcinoma in association with the hepatitis Bvirus.
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PMID:Establishment and characterization of four human hepatocellular carcinoma cell lines containing hepatitis B virus DNA. 1181 50

LuxS is responsible for the production of autoinducer 2 (AI-2), which functions in Vibrio harveyi as a quorum-sensing signal that controls the cell density-dependent expression of the lux operon. In nonluminescent organisms, the physiologic role of AI-2 is not clear. We report that inactivation of luxS in Actinobacillus actinomycetemcomitans JP2 results in reduced growth of the mutant, but not the wild-type organism, under aerobic, iron-limited conditions. Stunted cultures of the luxS mutant A. actinomycetemcomitans JP2-12 grew to high cell density when subcultured under iron-replete conditions. In addition, the mutant strain grew to high cell density under iron limitation after transformation with a plasmid containing a functional copy of luxS. Results of real-time PCR showed that A. actinomycetemcomitans JP2-12 exhibited significantly reduced expression of afuA (eightfold), fecBCDE (10-fold), and ftnAB (>50-fold), which encode a periplasmic ferric transport protein, a putative ferric citrate transporter, and ferritin, respectively. The expressions of putative receptors for transferrin, hemoglobin, and hemophore binding protein were also reduced at more modest levels (two- to threefold). In contrast, expressions of sidD and frpB (encoding putative siderophore receptors) were increased 10- and 3-fold, respectively, in the luxS mutant. To better understand the mechanism of the AI-2 response, the A. actinomycetemcomitans genome was searched for homologs of the V. harveyi signal transduction proteins, LuxP, LuxQ, LuxU, and LuxO. Interestingly, ArcB was found to be most similar to LuxQ sensor/kinase. To determine whether arcB plays a role in the response of A. actinomycetemcomitans to AI-2, an arcB-deficient mutant was constructed. The isogenic arcB mutant grew poorly under anaerobic conditions but grew normally under aerobic iron-replete conditions. However, the arcB mutant failed to grow aerobically under iron limitation, and reverse transcriptase PCR showed that inactivation of arcB resulted in decreased expression of afuA and ftnAB. Thus, isogenic luxS and arcB mutants of A. actinomycetemcomitans exhibit similar phenotypes when cultured aerobically under iron limitation, and both mutants exhibit reduced expression of a common set of genes involved in the transport and storage of iron. These results suggest that LuxS and ArcB may act in concert to control the adaptation of A. actinomycetemcomitans to iron-limiting conditions and its growth under such conditions.
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PMID:luxS and arcB control aerobic growth of Actinobacillus actinomycetemcomitans under iron limitation. 1249 79

Experimental data suggest the antimicrobial peptide hepcidin as a central regulator in iron homeostasis. In this study, we characterized the expression of human hepcidin in experimental and clinical iron overload conditions, including hereditary hemochromatosis. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we determined expression of hepcidin and the most relevant iron-related genes in liver biopsies from patients with hemochromatosis and iron-stain-negative control subjects. Regulation of hepcidin mRNA expression in response to transferrin-bound iron, non-transferrin-bound iron, and deferoxamine was analyzed in HepG2 cells. Hepcidin expression correlated significantly with serum ferritin levels in controls, whereas no significant up-regulation was observed in patients with hemochromatosis despite iron-overload conditions and high serum ferritin levels. However, patients with hemochromatosis showed an inverse correlation between hepcidin transcript levels and the serum transferrin saturation. Moreover, we found a significant correlation between hepatic transcript levels of hepcidin and transferrin receptor-2 irrespective of the iron status. In vitro data indicated that hepcidin expression is down-regulated in response to non-transferrin-bound iron. In conclusion, the presented data suggest a close relationship between the transferrin saturation and hepatic hepcidin expression in hereditary hemochromatosis. Although the causality is not yet clear, this interaction might result from a down-regulation of hepcidin expression in response to significant levels of non-transferrin-bound iron.
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PMID:Expression of hepcidin in hereditary hemochromatosis: evidence for a regulation in response to the serum transferrin saturation and to non-transferrin-bound iron. 1263 25

Lung cells import iron across the plasma membrane as ferrous (Fe2+) ion by incompletely understood mechanisms. We tested the hypothesis that human bronchial epithelial (HBE) cells import non-transferrin-bound iron (NTBI) using superoxide-dependent ferri-reductase activity involving anion exchange protein 2 (AE2) and extracellular bicarbonate (HCO3-). HBE cells that constitutively express AE2 mRNA by reverse transcriptase-polymerase chain reaction and AE2 protein by Western analysis avidly transported NTBI after exposure to either Fe2+ or Fe3+, but reduction of Fe3+ to Fe2+ was first required. The ability of HBE cells to reduce Fe3+ and transport Fe2+ was inhibited by active extracellular superoxide dismutase (SOD). Similarly, HBE cells that overexpress Cu,Zn SOD after adenoviral infection with AdSOD1 showed diminished iron uptake. The role of AE2 in iron uptake was indicated by three lines of evidence: (i) lack of both iron reduction and iron transport in bicarbonate-free buffer at controlled pH, (ii) failure of HBE cells treated with stilbene AE inhibitors to reduce Fe3+ or transport iron, and (iii) inhibition of iron uptake in HBE cells by inhibition of AE2 protein expression with antisense oligonucleotides. We thus disclose a novel ferri-reductase mechanism of NTBI uptake by human lung cells that employs superoxide exchange for HCO3- by AE2 protein in the plasma membrane.
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PMID:Superoxide-dependent iron uptake: a new role for anion exchange protein 2. 1279 78

Usually thyroid cells isolated from tissue obtained by surgery or thyroid cell lines are used to investigate the pathogenesis of autoimmune thyroid diseases. Isolation and cultivation of thyrocytes from fine-needle aspiration biopsy (FNAB) has not yet been published. The aim of this study was to isolate and cultivate thyrocytes from samples of FNAB. FNAB samples were obtained from nine adults and nine children with Hashimoto's thyroiditis (HT). The aspiration material was filtered resulting in small samples of tissue on the surface of the filter membrane. These tissue fragments were digested by collagenase I and dispase II. The yielding cells were cultivated for 3 weeks in Ham's F12 Kaighn's Modification medium in presence of 1 mU/mL bovine thyrotropin (TSH), 10 microg/mL human insulin, 6 microg/mL transferrin, and 10(-8) M hydrocortisone. Finally, isolated thyroid cells were characterized by determination of gene expression of thyrotropin receptor (TSHR), thyroperoxidase (TPO), and thyroglobulin (Tg) using a nested reverse transcriptase-polymerase chain reaction (RT-PCR). Thyroid cells obtained by FNAB can be maintained over a time period of approximately 3 weeks. Depending on the sample size a final number of 1000-14,000 cells was gained per FNAB. In addition, all cells isolated by the described method expressed TPO mRNA. TSHR mRNA was found in 4 samples, whereas 15 samples were Tg mRNA-positive. There were no differences with respect to the expression TSHR and TPO mRNA between samples from adults and children. The isolation and cultivation of thyroid cells obtained by FNAB has been established. In contrast to surgical specimen, this technique provides an easy access to thyrocytes derived from individual patients allowing repeated sampling to investigate the time progression of the chronic disease or the effect of treatment over time.
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PMID:Isolation of thyroid cells obtained by fine-needle aspiration biopsy. 1618 6

The mechanisms of reproductive malfunction of male mammals caused by 2,2-bis(4-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE, hereafter DDE) remain unknown. To explore the effects of DDE on the expressions of transferrin (Tf) and androgen-binding protein (ABP), we isolated Sertoli cells from healthy immature rats (18-20 days SD rats), set up Sertoli cell cultures, evaluated the toxicity, and measured the expression levels of mRNA of Tf and ABP genes by the one-step reverse transcriptase polymerase chain reaction method after cultured Sertoli cells were in vitro exposed to DDE at different concentrations for 24 h. The results showed that the number and survival rate of Sertoli cells decreased sharply with increased doses of DDE. The expression level of Tf mRNA decreased, whereas ABP mRNA increased gradually with increased DDE doses. There existed an obvious dose-effect relationship between the concentration of DDE and the expression levels of Tf mRNA and ABP mRNA. These findings suggest that DDE may inhibit the expression of Tf and up-modulate expression of ABP in cultured rat Sertoli cells.
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PMID:Effects of p,p'-dichlorodiphenyldichloroethylene on the expressions of transferrin and androgen-binding protein in rat Sertoli cells. 1638 Jan 12

Chronic cadmium (Cd2+) exposure results in renal proximal tubular cell damage. Delivery of Cd2+ to the kidney occurs mainly as complexes with metallothionein-1 (molecular mass approximately 7 kDa), freely filtered at the glomerulus. For Cd2+ to gain access to the proximal tubule cells, these complexes are thought to be internalized via receptors for small protein ligands, such as megalin and cubilin, followed by release of Cd2+ from metallothionein-1 in endosomal/lysosomal compartments. To investigate the role of megalin in renal cadmium-metallothionein-1 reabsorption, megalin expression and dependence of cadmium-metallothionein-1 internalization and cytotoxicity on megalin were studied in a renal proximal tubular cell model (WKPT-0293 Cl.2 cells). Expression of megalin was detected by reverse transcriptase-polymerase chain reaction and visualized by immunofluorescence both at the cell surface (live staining) and intracellularly (permeabilized cells). Internalization of Alexa Fluor 488-coupled metallothionein-1 was concentration-dependent, saturating at approximately 15 microM. At 14.3 microM, metallothionein-1 uptake could be significantly attenuated by 30.9 +/- 6.6% (n = 4) by 1 muM of the receptor-associated protein (RAP) used as a competitive inhibitor of cadmium-metallothionein-1 binding to megalin and cubilin. Consistently, cytotoxicity of a 24-h treatment with 7.14 muM cadmium-metallothionein-1 was significantly reduced by 41.0 +/- 7.6%, 61.6 +/- 3.4%, and 26.2 +/- 1.8% (n = 4-5 each) by the presence of 1 microM RAP, 400 microg/ml anti-megalin antibody, or 5 microM of the cubilin-specific ligand, apo-transferrin, respectively. Cubilin expression in proximal tubule cells was also confirmed at the mRNA and protein level. The data indicate that renal proximal tubular cadmium-metallothionein-1 uptake and cell death are mediated at least in part by megalin.
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PMID:Megalin-dependent internalization of cadmium-metallothionein and cytotoxicity in cultured renal proximal tubule cells. 1669 Jul 19

While cellular micropatterning approaches are employed extensively in cell biology and tissue engineering, only a limited number of methods for analysis of local function in the context of a complex, microfabricated environment are currently available. The present study develops a novel strategy for analysis of local tissue-specific function in cellular micropatterns. Model hepatocytes (HepG2 cells) were seeded onto silane-modified glass slides containing robotically printed arrays of collagen type I. These model hepatocytes formed cell arrays with individual cell cluster dimensions (150 or 500 microm) corresponding in size to the printed collagen spots. Non-parenchymal cells (3T3 fibroblasts) were added to hepatocellular micropatterns to create heterotypic cocultures. Expression of hepatic phenotype in HepG2 cells was first verified by traditional techniques including intracellular staining and ELISA for albumin. In order to evaluate local liver function in the cellular microarray, individual array members composed of approximately 400 hepatocytes were retrieved using laser capture microdissection and analyzed with real-time reverse transcriptase (RT)-polymerase chain reaction (PCR). Hepatic function was assessed based on expression of four genes associated with differentiated liver phenotype: albumin, transferrin, alpha-fetoprotein, and alpha1-antitrypsin. "Titration" experiments, carried out to identify the smallest population of HepG2 cells yielding detectable mRNA levels and RT-PCR signals, showed that extraction area of 12,500 microm2 (corresponding to approximately 70 cells) provided detectable gene expression signals. All four liver-specific genes were routinely evaluated after extraction of approximately 400 HepG2 from the micropatterned surfaces. Significantly, selective retrieval and subsequent analysis of tissue-specific function was demonstrated for hepatic cells micropatterned alone and in coculture with non-parenchymal cells. In the future, methods described in this study will offer the possibility to investigate dynamic and reciprocal interactions between two or more cell types positioned on a microfabricated cell culture surface. We also envision the proposed approaches to be ideally suited for cell analysis in the context of combinatorial microenvironment.
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PMID:Analysis of local tissue-specific gene expression in cellular micropatterns. 1716 20

Turkey poults inoculated with plasma obtained from turkeys affected with lymphoproliferative disease (LPD) developed typical lymphoproliferative lesions in the pancreas, spleen and thymus, 3 and 6 weeks post infection. Virus-associated reverse transcriptase activity in plasma reached a significant level at 3 weeks and was further elevated at 6 weeks post infection, concomitantly with a marked increase in serum IgG, 7S-immuno-globulin level. There was no alteration in serum total iron binding capacity (transferrin) level in LPD virus-infected animals, as compared to controls. Natural field cases of LPD also demonstrated elevated serum IgG levels which persisted for more than 3 months, along with viraemia. There was a significant decrease in serum albumin concentration in about 30% of the infected animals, but in few of these turkeys was total serum protein elevated due to the very marked increase in gamma-globulins (IgG).
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PMID:Lymphoproliferative disease of turkeys: pathogenesis, viraemia and serum protein analysis following infection. 1876 66


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