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)

Two forms of the monocyte chemoattractant protein-1 receptors (the type A monocyte chemoattractant protein 1 (MCP-1) receptor CCR-2A and the type B MCP-1 receptor (CCR-2B) have been recently cloned and found to differ only in their terminal carboxyl tails. Here, we report that the two isoforms are alternatively spliced variants of a single MCP-1 receptor gene. Sequencing of the gene revealed that the 47-amino acid carboxyl tail of CCR2B was located in the same exon as the seven transmembrane domains of the receptor, and the 61-amino acid tail of CCR2A was in a downstream exon. Examination of freshly isolated human monocytes by reverse transcriptase-polymerase chain reaction revealed that CCR2B was the predominant isoform and that message levels of both CCR2A and CCR2B decreased as the monocytes differentiated into macrophages. In stably transfected cell lines, CCR2B trafficked well to the cell surface, but CCR2A was found predominantly in the cytoplasm. Equilibrium binding studies revealed that those CCR2A receptors that successfully trafficked to the cell surface bound MCP-1 with high affinity (Kd = 310 pM), similar to CCR2B. In signaling studies, both CCR2A and CCR2B mediated agonist-dependent calcium mobilization, as well as inhibition of adenylyl cyclase. Creation of chimeras between CCR2A and the human thrombin receptor revealed that the cytoplasmic retention of CCR2A was due to its terminal carboxyl tail. Progressive truncation of the carboxyl tail indicated that a cytoplasmic retention signal(s) was located between residues 316 and 349. These data indicate that the alternatively spliced form of the human MCP-1 receptor (CCR2A) binds MCP-1 with high affinity and is a functional receptor and that expression at the cell surface is controlled by amino acid sequences located in the terminal carboxyl tail.
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PMID:Organization and differential expression of the human monocyte chemoattractant protein 1 receptor gene. Evidence for the role of the carboxyl-terminal tail in receptor trafficking. 899

GluR2 is the key subunit of heteromeric AMPA-preferring glutamate receptors. GluR2 mRNA has been shown by in situ hybridization histochemistry to be decreased in the hippocampal formation in schizophrenics. Here, a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method was used to investigate GluR2 expression further and to examine the relative abundance of its alternatively spliced mRNA isoforms ('flip' and 'flop') in 11 schizophrenics and 11 matched controls. Compared to the controls, schizophrenics showed reduced expression of both isoforms relative to cyclophilin mRNA, but a greater loss of the flop isoform led to a higher flip:flop ratio. These differences were observed having controlled for the confounding effects of brain pH and age upon the mRNAs. We also found that the abundance of GluR2 mRNA correlates with that of the encoded subunit. This study has confirmed that, in schizophrenia, hippocampal GluR2 mRNA is reduced, and indicates that GluR2 subunits are composed of a higher proportion of the flip variant. These data extend the evidence for glutamatergic dysfunction in the disease. They suggest that signal transduction through hippocampal AMPA receptors is impaired in schizophrenia both by an overall loss of GluR2 expression, and by the change in flip:flop ratio which is predicted to alter the desensitization kinetics of the remaining GluR2 subunits.
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PMID:GluR2 glutamate receptor subunit flip and flop isoforms are decreased in the hippocampal formation in schizophrenia: a reverse transcriptase-polymerase chain reaction (RT-PCR) study. 903 Jul 2

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) gamma-subunits were cloned from a porcine aortic smooth muscle cDNA library resulting in identification of alternatively spliced CaM kinase II gammaB- and gammaC-subunits and a novel gamma-subunit variant predicted to encode a 60.2-kDa polypeptide, which was designated the gammaG-subunit. A clone predicted to encode a 62. 2-kDa gamma-subunit, designated as gammaE, was isolated with a variable domain structure similar to a gammaB-subunit but with a 114-nucleotide insertion in the conserved "association" domain of CaM kinase II subunits. A full-length gammaE-subunit construct expressed in COS cells resulted in multimeric CaM kinase II holoenzymes (470 kDa) with activation and autoregulatory properties similar to expressed holoenzymes composed of gammaB-, gammaC-, or gammaG-subunits. Expression of gammaE and related gamma-subunit mRNAs containing the 114-base insertion was documented in porcine tissues by reverse transcriptase-polymerase chain reaction. CaM kinase II subunits containing the 38-amino acid insert were identified by Western analysis of partially purified CaM kinase II from carotid arterial smooth muscle and brain using a sequence-specific anti-peptide antibody. Immunoprecipitations of tissue homogenates indicated a comparatively high level of expression of subunits containing the insert in brain and provided evidence for their co-assembly with other more abundant subunits into CaM kinase II heteromultimers. Our analyses indicate the following patterns of gamma-subunit expression: vascular smooth muscle, gammaB > gammaC > gammaE,G; heart, gammaB > gammaE,C > gammaG; brain, gammaE and related subunits >> gammaA,B,C,G.
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PMID:Novel Ca2+/calmodulin-dependent protein kinase II gamma-subunit variants expressed in vascular smooth muscle, brain, and cardiomyocytes. 908 77

CD44 is a widely expressed cell surface glycoprotein which is involved in both cell-matrix and cell-cell interactions which regulate a variety of processes, including leucocyte migration and activation. Therefore, we examined the expression of CD44, and its major ligand hyaluronan, during the induction and progression of experimental glomerulonephritis. Antibody staining of normal rat kidney showed constitutive CD44 expression by resident glomerular macrophages, parietal epithelial cells, medullary and occasional cortical tubules. There was a marked increase in CD44 expression over days 1, 7 and 21 of rat crescentic anti-glomerular basement membrane (GBM) glomerulonephritis. Infiltrating monocytes and lymphocytes were CD44+, with ultrastructural studies showing high levels of CD44 expressed on the surface of lymphocytes adherent to activated endothelium. Marked hyaluronan deposition was seen in areas of fibrosis on days 7 and 21, such as glomerular crescents and the periglomerular area. Hyaluronan deposition was accompanied by the presence of many CD44+ cells. Double immunohistochemistry showed that both CD44+ED1+ macrophages and CD44+ myofibroblasts (identified by expression of alpha-smooth muscle actin) were present in areas of fibrosis. There was also a dramatic increase in cortical tubular CD44 expression, which was most evident in areas of tubular damage. Although tubular epithelial cells expressed CD44 upon both the basolateral and luminal surface, CD44 expression was most prominent within tightjunctions, suggesting a role for CD44-CD44 interactions in cell-cell adhesion within the tubule. Analysis of CD44 isoforms by reverse transcriptase-polymerase chain reaction (RT-PCR) showed that the standard form of CD44 predominated in both normal and diseased kidney. However, a series of alternatively spliced CD44 isoforms was also detected, whose expression was markedly increased during disease. At least seven isoforms containing the v6 domain were identified, with the smallest form representing activated T cells. In conclusion, CD44 is constitutively expressed in normal kidney and is dramatically up-regulated in rat anti-GBM disease, suggesting possible roles for the CD44-hyaluronan interaction in leucocyte recruitment, renal fibrosis and tubular cell-matrix and cell-cell interactions during the induction and progression of crescentic glomerulonephritis.
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PMID:CD44 and hyaluronan expression in the development of experimental crescentic glomerulonephritis. 909 14

Melanomas tend to become less pigmented in the course of malignant progression. Thus, as proliferation increases, the tumors are decreasingly characterized by the tissue-specific phenotype of normally differentiated melanocytes. To learn whether the decline in melanization is associated with a shift from constitutive to alternative splicing of some pigment gene pre-mRNAs, melanomas were collected from Tyr-SV40E transgenic mice of the standard C57BL/6 strain. The mRNAs of the tyrosinase gene, which has a key role in melanogenesis, were analyzed by quantitative reverse transcriptase-PCR in 34 samples from 16 cutaneous tumors and 9 metastases. The cutaneous tumors included some cases with distinct melanotic and amelanotic zones, which were separately analyzed. All tyrosinase transcripts found in the melanomas were also found in normal skin melanocytes. However, the Delta1b and Delta1d alternatively spliced transcripts, due to deletions within the first exon, were specifically augmented in most of the tumors over their very low levels in skin; the exceptions were some all-amelanotic tumors in which no tyrosinase transcripts were detected. The level of Delta1b rose as high as 11.3% of total tyrosinase mRNAs as compared with 0.6% in skin; Delta1d reached 4.0% as compared with 0. 8% in skin. Expression of these splice variants was highest in the melanotic components of zonal primary tumors, relatively lower in their amelanotic components, and still lower in all-amelanotic primary tumors and amelanotic metastases. The increase in Delta1b and Delta1d transcripts may be predicted to increase the levels of unusual peptides, which could have antigenic potential in the tumors, especially in the relatively early phases of malignancy. Analyses of the alternative transcripts of other pigment genes may identify additional candidate antigens, ultimately enabling melanoma cells in all phases of the disease to be represented as a basis for immune intervention.
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PMID:Selective increase in specific alternative splice variants of tyrosinase in murine melanomas: a projected basis for immunotherapy. 914 37

Complementary DNA clones of mRNAs for sheep and goat NADPH-ferredoxin reductases (ferredoxin NADP+ oxidoreductase, EC 1.18.1.2) were isolated by the reverse transcriptase polymerase chain reaction method, and the complete nucleotide sequences of the coding and 3'-flanking regions of these cDNA clones were determined. Comparative analysis using the deduced amino acid sequences of NADPH-ferredoxin reductases clarified the interspecific conservation of the ferredoxin-binding and flavin adenine dinucleotide (FAD)-binding regions, confirming the results reported previously. During this study, we happened to identify an alternatively spliced mRNA that completely lacks exon 3, just adjacent to the FAD-binding region of the sheep NADPH-ferredoxin reductase cDNA clone. In the screening of other alternatively spliced mRNAs of NADPH-ferredoxin reductases derived from several steroidogenic organs, such as adrenocortices, testes, and ovaries of sheep and goats, only one kind of alternatively spliced mRNA as described above was detected in sheep adrenocortices. Then, we constructed Escherichia coli expression systems for these two forms of mRNA and analyzed their enzymatic properties. We found that the ability of the alternatively spliced NADPH-ferredoxin reductase protein to transfer electrons to ferredoxin is completely abolished because FAD binding is inhibited.
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PMID:Molecular cloning of sheep and goat ferredoxin reductase messenger ribonucleic acids, and identification of an alternatively spliced form of sheep ferredoxin reductase. 916 Jul 36

Interleukin (IL)-15 is a four-helix bundle cytokine sharing several biological properties with IL-2. By reverse transcriptase-polymerase chain reaction analysis, human cancer cell lines of different histotypes are shown to express two IL-15 amplification products: a 524-bp band corresponding to the IL-15 mRNA found in macrophages, and another of 643 bp corresponding to an alternatively spliced mRNA including a 119-bp alternative exon. IL-15 was undetectable in the supernatant of tumor cell lines expressing either one or both of the mRNA isoforms as evaluated by a bioassay or by ELISA, indicating that IL-15 is not secreted. However, IL-15 could be detected intracellularly in some tumor cells by confocal microscopy analysis. Since the pre-proteins encoded by the two mRNA isoforms differ in the signal peptide sequence, we have analyzed the characteristics of these signal peptides and their possible role in controlling secretion. The two IL-15 cDNA isoforms, expressed in COS-7 cells, induced very low levels of IL-15 secretion. However, substitution of the sequence encoding natural signal peptide(s) with the one from IgV kappa chain in the IL-15 cDNA results in a significantly higher secretion of biologically active IL-15 (15-30-fold) upon cDNA transfection. A poor efficiency of natural signal peptides may represent one of the mechanisms involved in the control of IL-15 secretion.
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PMID:Expression of two interleukin-15 mRNA isoforms in human tumors does not correlate with secretion: role of different signal peptides. 917 91

Steroidogenic factor 1, a member of the fushi tarazu factor 1 (FTZ-F1) subfamily of nuclear receptors, is a key regulator in mammalian reproduction. From an embryonic complementary DNA library, the zebrafish homolog of FTZ-F1 (zFF1A) and an alternatively spliced variant (zFF1B) were isolated. zFF1B represented a C-terminally truncated version of zFF1A. Whole mount in situ hybridization and reverse transcriptase-PCR analysis revealed that both zFF1A and B transcripts were present in the developing pituitaries, adult fish brain, gonads, and liver, albeit zFF1B messenger RNA was absent in testis. Comparison of the primary sequences of zFF1 with those of other FTZ-F1 subfamily members showed a close structural relationship between the mouse liver receptor homolog, which activated the alpha1-fetoprotein gene in rodent liver. However, similar to mouse steroidogenic factor 1, zFF1A regulated chinook salmon gonadotropin IIbeta subunit gene expression. On the contrary, zFF1B, which could bind a consensus gonadotrope-specific element with an affinity similar to that of zFF1A, lacked both the trans-activation function and synergistic interaction with the estrogen receptor. Furthermore, cotransfection studies in HeLa cells showed that zFF1B was a strong competitor for the action of zFF1A on the chinook salmon gonadotropin IIbeta subunit gene promoter. Our investigation suggests that 1) zFF1 represents an ancestor protein of the vertebrate FTZ-F1 homologs; 2) the antagonistic relationship between zFF1A and -B may dictate the expression of the FTZ-F1 target genes in a variety of tissues, including the pituitary; and 3) the naturally occurring zFF1B provides evidence that the C-terminal portion of zFF1A (80 amino acid residues) contains a major trans-activation function and a protein-protein interface.
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PMID:Teleost FTZ-F1 homolog and its splicing variant determine the expression of the salmon gonadotropin IIbeta subunit gene. 917 48

We review the current understanding of the myosin heavy chain (MHC) isoforms and show that the mRNA levels of smooth muscle (SM)1 and SM2 mimic the expressed levels of SM1 and SM2 protein. The reverse transcriptase-polymerase chain reaction technique has been shown to be sufficiently sensitive to examine SM-MHC expression at the single cell level. Most single smooth muscle cells isolated from adult rabbit carotid express both SM1 and SM2. However, expression of these SM-MHC isoforms at the cellular level is nonuniform and highly variable. This work provides a foundation for future investigations as to the possible unique functional characteristics of the SM-MHC isoforms, SM1 and SM2. This methodology may also prove useful when used with mechanical studies to determine the physiological significance of the alternatively spliced myosin isoforms, including the SM-MHC-head and LC17 isoforms.
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PMID:Myosin isoform heterogeneity in single smooth muscle cells. 918 12

HuD, one of the Hu antigens (HuD and HuC), was recognized in the sera of small cell lung cancer (SCLC) patients with antibody-associated paraneoplastic encephalomyelitis/peripheral sensory neuropathy (PEM/PSN). Three forms of HuD mRNA, 197, 156, 110 nucleotides are made by alternative splicing at 868-909 residues and an additional 3'-splice site. To determine the diagnostic value of the HuD expression for small cell lung cancer, we examined 4 SCLC cell lines, 9 surgically resected SCLCs, and 12 surgically resected non-SCLCs using the reverse transcriptase-polymerase chain reaction with the HuD-specific primer pairs that spanned the putative alternative 3'-splicing site and direct DNA sequencing. None of the patients were associated with PEM/PSN. A single RNA transcript (156 nucleotides) among three forms (110, 156, 197 nucleotides) of the HuD gene was an alternatively spliced at 868-909 residues in SCLC cell lines. Expression of the HuD gene was stronger in three classic cell lines, but not in a variant cell line. Two of 9 SCLCs (22%) and 3 of 12 non-SCLCs (25%) expressed only the major RNA transcript (156 nucleotides) of the HuD gene, which was alternatively spliced in the same fashion as the cell line. These results revealed that no aberrant alternative splicing occurred in SCLC not associated with PEM/PSN and the expression of HuD gene was not specific for a particular histologic subtype of human lung cancer.
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PMID:Expression of HuD (a paraneoplastic encephalomyelitis antigen) mRNA in lung cancer. 928 29


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