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)

The expression of pS2 (TFF1) has been previously shown to identify patients with improved response to anti-hormonal therapy and more favorable outcome. In the current study, 100 human breast carcinoma samples obtained from the Manitoba Breast Tumor Bank were analyzed for pS2 mRNA using a quantitative, competitive reverse transcriptase-polymerase chain reaction (qcRT-PCR) assay. A pS2/beta-actin cut-off criterion of 0.010 was established to classify tumors as either pS2 positive or pS2 negative. pS2 mRNA levels were positively associated with both ER and PR, with the majority of ER+ (59%) and PR+ (60%) tumors also being positive for pS2. In addition, a significant linear correlation was observed between the amount of pS2 mRNA and ER (p < 0.0001) and PR (p < 0.0001) protein. pS2 mRNA levels also exhibited an inverse association with tumor size and histological grade, consistent with the observation that pS2 is primarily expressed in small (T < 2.0 cm), but well differentiated tumors (Grades I and II). No associations were observed with tumor cell type, patient age, or lymph node status. The strong correlation displayed between pS2 and a number of currently used breast cancer prognostic markers supports the clinical use of pS2 to further assess tumor status and patient outcome.
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PMID:pS2 (TFF1) levels in human breast cancer tumor samples: correlation with clinical and histological prognostic markers. 1057 16

The treatment of difficult wounds remains a considerable clinical challenge. The goal of this study was to determine whether genetic augmentation of dermal cells on resorbable matrices can stimulate the healing process, leading to increased tissue repair in a rat full-thickness excisional wound repair model. The human platelet-derived growth factor B (PDGF-B) gene was the initial gene chosen to test this hypothesis. The human PDGF-B gene was obtained from human umbilical vein endothelial cells (HUVEC) by reverse transcriptase-polymerase chain reaction, cloned into retroviral vectors under control of either the cytomegalovirus promoter or the rat beta-actin promoter, and introduced into primary rat dermal cells. In vitro results demonstrate that rat dermal cells are transduced and selected readily using retroviral vectors, and engineered to secrete PDGF-B at a steady-state level of approximately 2 ng per milliliter culture per 1 million cells per 24 hours. Seeding of the gene-modified cells onto polyglycolic acid (PGA) scaffold matrices and introduction into the rat model resulted in substantially increased fibroblast hypercellularity over control wounds at both 7 and 14 days posttreatment. Our results demonstrate that gene augmentation of rat dermal fibroblasts with the PDGF-B gene introduced into this animal model via PGA matrices modulates wound healing and suggests that experimentation with additional genes for use separately or in combination with PDGF-B for additional, improved wound healing is warranted.
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PMID:Gene-enhanced tissue engineering: applications for wound healing using cultured dermal fibroblasts transduced retrovirally with the PDGF-B gene. 1059 24

The cortical collecting duct (CCD) is a major site of regulation of K+ homeostasis in the fully differentiated mammalian kidney. CCDs isolated from adult rabbits and microperfused in vitro secrete K+ into the tubular fluid at high rates. However, CCDs dissected from newborn animals show no significant net K+ secretion until the 3rd wk of life, at least in part because of a paucity of conducting apical secretory K+ (SK) channels. To determine whether the abundance of genes encoding the SK channel is developmentally regulated, we used reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis to test for the presence of mRNA encoding rat outer medullary K+ channel (ROMK), considered to be a major subunit of the SK channel, in kidney and single CCDs isolated from maturing rabbits. Using rat ROMK-specific primers, RT-PCR of rabbit kidney yielded an amplification product of expected size and sequence. Northern blot analysis identified a single band at approximately 2.9 kb in kidney at all ages. Densitometric analysis revealed a progressive increase in steady state expression of ROMK message in kidney after birth. RT-PCR of individual CCDs yielded a single band of predicted size for ROMK in all segments isolated from animals > or =3 wk old. In contrast, transcripts were not detected in any CCD samples obtained from 1-wk-old animals and were identified in only 30% of CCD samples isolated from 2-wk-old rabbits. In all of the latter tubular samples, a specific PCR product of correct size for beta-actin mRNA was detected. These results suggest that an increase in steady state expression of ROMK mRNA contributes to the developmental appearance of conducting secretory K+ channels in the CCD.
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PMID:Developmental expression of ROMK mRNA in rabbit cortical collecting duct. 1062 82

Gap junction coupling between neurons is important for the temporal and spatial co-ordination of neocortical development and can be visualised by dye-coupling. Neuronal dye-coupling in the rat neocortex is extensive during the first 2 postnatal weeks and diminishes rapidly thereafter. We used RT (reverse transcriptase)-PCR to investigate the time-related changes in mRNA expression for the connexins (Cx) Cx 26, Cx 30, Cx 32, Cx 36, Cx 37, Cx 40, Cx 43, Cx 45 and Cx 46 as well as for beta-actin and GAPDH in rat neocortex during the first 6 postnatal weeks. The time courses for mRNA expression for GAPDH, Cx 30, Cx 36 and Cx 43 were also investigated by northern blotting. Cx 30 and Cx 45 mRNA abundance showed no time-dependent changes during the early postnatal period. The relative abundance of Cx 32, Cx 43 and Cx 46 mRNA increased significantly during the first 2-3 weeks and then remained relatively constant during weeks 3-6. The relative abundance of Cx 26, Cx 36, Cx 37 and Cx 40 mRNA also increased significantly during the first 10-15 postnatal days but then declined significantly from their peak values during weeks 3-6. beta-actin mRNA expression showed no time-related changes but GAPDH mRNA expression increased significantly during the first postnatal week, then remained constant. The time-dependent changes in mRNA relative abundance for GAPDH, Cx 36 and Cx 43 determined by northern blotting corroborate the results from the RT-PCR study. None of the Cx exhibited time-dependent changes in mRNA expression in homogenates of rat neocortex which parallel the changes in neuronal dye-coupling during postnatal development.
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PMID:Time-related changes in connexin mRNA abundance in the rat neocortex during postnatal development. 1064 78

To analyze the regulation of water channels in the peritoneum, we tried to establish a primary mesothelial cell culture system. Male Sprague-Dawley rats weighing about 250 g were anesthetized, and 10 mL of phosphate-buffered saline (PBS) containing 0.25% trypsin and 1 mmol/L ethylenediamine tetraacetic acid (EDTA) was infused into the peritoneal cavity for 15 minutes. Sediments from the recovered fluid were cultured in medium M199 supplemented with 10% fetal bovine serum (FBS). The culture was succeeded 4-6 times before experiments commenced. After exposure to the test medium, RNA was extracted and subjected to reverse transcriptase polymerase chain reaction (RT-PCR) for 10-19 cycles, then was measured by Southern blot analysis with a digoxin-labeled probe. Cultured cells were positively stained with mouse monoclonal anti-cytokeratin antibody, confirming their characteristics as mesothelial cells. Aquaporin-1 (AQP-1) message in the cultured cells increased with increases in glucose and mannitol concentrations when beta-actin message was used as an internal control. Tranexamic acid effected no change in AQP-1 message in the cultured mesothelial cells. This system offers potential as a simple approach to test the effects of osmolytes, cytokines, and vasoactive hormones on aquaporin expression and water transport in the peritoneum.
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PMID:Water channel AQP-1 in the primary cell culture of rat peritoneum. 1068 62

We describe a reverse transcriptase-polymerase chain reaction method for the semiquantitative detection of mRNAs encoding the human heat shock proteins alphaB-crystallin, Hsp27, and Hsp60. The method involves the coamplification of cellular mRNA-derived cDNA with a dilution series of a competitor fragment (internal standard), using 1 primer pair common to both templates. Internal standards were based on cellular-derived cDNA engineered to be slightly smaller to differentiate between the target and the standard on electrophoretic separation. Initial cDNA quantitations can be corrected for possible variations during cDNA synthesis by standardizing to the levels of beta-actin-encoding cDNA. We show that the coamplified templates accumulate in a parallel manner with the cellular-derived cDNA throughout both the exponential and the nonexponential phase of amplification. Furthermore, we illustrate the utility of this technique by quantifying increased expression of alphaB-crystallin, Hsp27, and Hsp60 mRNA in astroglioma cells on heat shock.
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PMID:The stress kit: a new method based on competitive reverse transcriptase-polymerase chain reaction to quantify the expression of human alphaB-crystallin, Hsp27, and Hsp60. 1070 37

Receptors for 1,25(OH)2vitaminD3 are found in most immune cells and important immunological effects have been described in vitro, reflected by its capacity to prevent autoimmunity and to prolong graft survival. The aim of this study was to examine the presence and nature of the enzyme responsible for final activation of the molecule, 1-alpha-hydroxylase, in murine macrophages and to analyse its regulation and possible role in the immune system. Peritoneal macrophages from C57Bl/6 mice were incubated with lipopolysaccharide (LPS; 100 microg/ml), interferon-gamma (IFN-gamma; 500 U/ml) or a combination of both. By quantitative reverse transcriptase-polymerase chain reaction, using primers based on the murine renal cDNA sequence, low levels of 1-alpha-hydroxylase mRNA were detected in freshly isolated cells (18 +/- 7 x 10-6 copies/beta-actin copies). Analysis of the cDNA sequence of the gene revealed identical coding sequences for the macrophage and renal enzymes. mRNA levels rose three-fold with LPS (NS), but a six-fold increase was seen after IFN-gamma stimulation (P < 0.05). Combining LPS and IFN-gamma did not result in a major additional increase, but addition of cyclosporin A further increased levels 2.5-fold both in IFN-gamma- and combination-stimulated cells (P < 0.05). Time course analysis revealed that up-regulation of 1-alpha-hydroxylase was a late phenomenon, preceded by the up-regulation of activating macrophage products such as IL-1 and tumour necrosis factor-alpha. Finally, a defect in 1-alpha-hydroxylase up-regulation by immune stimuli was found in autoimmune non-obese diabetic mice. In conclusion, we propose that the up-regulation of 1-alpha-hydroxylase in activated macrophages, resulting in the synthesis of 1,25(OH)2D3, might be a negative feedback loop in inflammation. A defect in this system might be an additional element in tipping the balance towards autoimmunity.
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PMID:Identification and immune regulation of 25-hydroxyvitamin D-1-alpha-hydroxylase in murine macrophages. 1075 75

To determine the ease and feasibility of amplifying the beta-actin gene in fish by the polymerase chain reaction (PCR), genomic DNAs of several fish (Rivulus, Southern top mouth minnow, common fat minnow, oily bitterling, carp, Far Eastern catfish, medaka, and European flounder) were extracted and used as a template with conserved primers, designed on the basis of high amino acid homology (approximately 98% or more). Among them, the self-fertilizing hermaphroditic fish Rivulus marmoratus was chosen for further characterization. After amplification of the Rivulus beta-actin PCR product with Taq polymerase, PCR product was subcloned to pCRII vector. After restriction enzyme mapping of Rivulus beta-actin gene, the amplified insert was sequenced using ALF Express automatic DNA sequencer with conserved internal primers. The R. marmoratus beta-actin gene consists of 1763 bp encoding 375 amino acids including 5 exons and 4 introns. The splicing and acceptance sites of the exon and intron boundaries of the Rivulus beta-actin gene were highly conserved with consensus sequences (GT/AG). The amino acid homology of R. marmoratus beta-actin to other species was high: 98.93% to human; 98.93%, Atlantic salmon; 98.93%, common carp; 98.93%, grass carp; 98.93%, zebrafish; 98.67%, medaka; and 98.40%, sea bream. To determine the expression of the R. marmoratus beta-actin gene in liver and ovary, reverse transcriptase-polymerase chain reaction was carried out with internal primers. In conclusion, these universal primers are successful in the rapid cloning of the fish beta-actin gene by PCR, based on a high homology of the beta-actin gene conserved through evolution. This approach will be applicable to the isolation of other beta-actin homologues in the investigation of phylogenetic comparisons of fish species, along with a possible application to cloning strategy in other conserved genes.
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PMID:The Internally Self-fertilizing Hermaphroditic Teleost Rivulus marmoratus (Cyprinodontiformes, Rivulidae) beta-Actin Gene: Amplification and Sequence Analysis with Conserved Primers. 1081 55

Human ejaculate spermatozoa contain multiple mRNA species carried over from earlier stages in spermatogenesis. To date, gene-specific RT-PCR or in situ hybridization has detected transcripts for beta-actin, heat shock proteins (HSP) 70 and 90, protamines (PRM) 1 and 2, transition protein (TNp) 2, HLA II, beta-integrins, and, most recently, phosphodiesterase subtypes. We have further evidence for a complex population of transcripts based on screening a human testis cDNA library with a heterologous spermatozoal probe. High levels of transcribed repetitive sequences are present in human spermatozoa, including medium reiteration repeats (MERs) and short and long nuclear interspersed repeats (SINES and LINES). Both SINES and LINES belong to the retroposon class of repeat elements, which are thought to proliferate via an intermediate RNA that is converted to DNA by an RNA-dependent DNA polymerase (reverse transcriptase). We have circumstantial evidence for the presence of an RT in ejaculate sperm based on the detection of transcripts for ORF2 of LINE 1 encoding such an enzyme. Our data suggests the following: 1. Ejaculate spermatozoa may be a very useful tool in the identification of genes linked to an infertile phenotype. 2. Spermatozoa (or spermatids) may express a reverse transcriptase, the role of which is unknown. 3. RNA isolated from spermatozoa or washed semen samples may facilitate the detection of mutations and deletions in testis-expressed AZF-linked genes.
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PMID:Analysis and significance of messenger RNA in human ejaculated spermatozoa. 1082 80

This study investigates the volume-sensitive KCI cotransporter (KCC) in various types of human cervical epithelial cell, testing the hypothesis that cervical malignancy is accompanied by differential expression of volume-sensitive KCC. Normal human cervical epithelial cells have KCCs which are quiescent in normal physiological conditions and are relatively refractory to hypotonic stress. By contrast, cervical cancer cells have KCCs which are also nearly quiescent in normal physiological conditions but high transport rates are observed in response to hypotonic challenge. Using isoform-specific primers, mRNA transcripts of KCC1, KCC3 and KCC4 were identified by reverse transcriptase polymerase chain reaction (RT-PCR) in several types of cervical cell, and confirmed by digestion with specific restriction endonucleases. By semiquantitative RT-PCR with beta-actin as the internal standard, the results indicate that cervical carcinogenesis is accompanied by the up-regulation of mRNA transcripts in KCC1, KCC3 and KCC4. [(Dihydroindenyl)oxy] alkanoic acid (DIOA), a KCC inhibitor, blocked both the regulatory volume decrease (RVD) process and volume-sensitive 86Rb+ efflux from cervical cancer cells in a dose-dependent manner. The volume-sensitive 86Rb+ efflux from cervical cancer cells was also blocked by two protein phosphatase inhibitors, calyculin A and okadaic acid, with IC50 values of 0.8 and 6 nM, respectively. Conversely, protein kinase inhibitors, chelerythrine and staurosporine, increased Cl- dependent 86Rb+ efflux. NEM (1 mM) led to a fivefold stimulation of 86Rb+ efflux which was totally Cl- dependent in cervical cancer cells. Hypotonicity could not stimulate any further 86Rb+ efflux after NEM treatment. These results indicate that the volume-sensitive KCC in cervical cancer cells plays a significant role in volume regulation and that the activities are modulated by a phosphorylation cascade. Taken together with our previous studies, we suggest the volume-regulatory ion channels and the co-transport systems work synergistically for volume regulation in human cervical cancer cells.
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PMID:Volume-sensitive KCI cotransport associated with human cervical carcinogenesis. 1100 18


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