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 cDNA encoding mouse phosphatidylinositol transfer protein (PI-TP) was isolated by means of reverse transcriptase polymerase chain reaction. The nucleotide sequence of this cDNA has a high similarity (98%) with that of rat PI-TP; the predicted amino acid sequence is 99.6% identical to that of rat PI-TP. The cDNA encoding mouse PI-TP was cloned into the expression vector pET3d and the Escherichia coli strain BL21(DE3) was transformed with the resulting plasmid. After induction of the bacteria with isopropyl-beta-D-thiogalactopyranoside, PI-TP was efficiently expressed in the E. coli strain. It was estimated that 5% of the total soluble cell protein consisted of PI-TP. The recombinant mouse PI-TP was purified from the bacterial lysate in four steps: ammonium sulphate precipitation, anion-exchange chromatography, heparin-Sepharose affinity and gel filtration chromatography. Fractionation on the heparin-Sepharose affinity column yielded two forms: PI-TP Hepa1 and Hepa2. These two proteins have the same molecular mass of 35 kDa, both contain a phosphatidylglycerol molecule and both are recognized by anti-PI-TP antibody. Both recombinant proteins have an isoelectric point of 5.4 as compared to 5.5 for bovine brain PI-TP. Sequence analysis of the first 25 N-terminal amino acid residues showed that both forms are identical, except that PI-TP Hepa1 contains the initiator methionine which is lacking from PI-TP Hepa2. The two PI-TP forms have similar phospholipid-binding and transfer activity, comparable to that of bovine brain PI-TP. Both forms and bovine brain PI-TP are phosphorylated equally well in a Ca2+/phospholipid-dependent way by protein kinase C.
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PMID:Characterization of mouse phosphatidylinositol transfer protein expressed in Escherichia coli. 804 44

Polyclonal antibodies have been raised against a C-terminal peptide of NaPi-1, a recently cloned Na-Pi cotransport system of rabbit kidney cortex with a predicted (unglycosylated) molecular mass of 52 kDa. By Western blot analysis using brush-border membranes isolated from rabbit kidney cortex, two proteins with apparent molecular masses of 64 kDa and 35 kDa were specifically recognized (peptide protectable) by the antiserum obtained. The 64-kDa protein was found to migrate in parallel with the luminal membrane during separation by free-flow electrophoresis of brush-border and basolateral membranes. In immunofluorescence studies using cryostat sections of rabbit kidney, specific binding of antibodies was observed in proximal tubules (including S1, S2 and S3 segments) of superficial and deep nephrons. Anti-(NaPi-1)-antibody-mediated fluorescence was restricted to the brush border of proximal tubular cells. No specific immunoreaction was observed in other tubular segments. The results suggest that the native NaPi-1-related protein (Na-Pi cotransport system) has an apparent molecular mass of 64 kDa and is uniformly expressed in the apical membrane of proximal tubules of all nephron generations in the rabbit kidney. Immunohistochemical localization of the Na-Pi cotransport system NaPi-1 confirms the segmental localization within the nephron of NaPi-1-related mRNA as revealed by the reverse transcriptase/polymerase chain reaction (see preceding paper).
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PMID:Localization of NaPi-1, a Na/Pi cotransporter, in rabbit kidney proximal tubules. II. Localization by immunohistochemistry. 841 8

Syntaxin 1/HPC-1 is an integral membrane protein, which is thought to be implicated in the regulation of synaptic neurotransmitter release. We investigated syntaxin 1 expression in pancreatic beta cells and the functional role of syntaxin 1 in the insulin release mechanism. Expression of syntaxin 1A, but not 1B, was detected in mouse isolated islets by the reverse transcriptase-polymerase chain reaction procedure. An immunoprecipitation study of metabolically labeled islets with an anti-rat syntaxin 1/HPC-1 antibody demonstrated syntaxin 1A protein with an apparent molecular mass of approximately 35 kDa. Immunohistochemistry of the mouse pancreas demonstrated that syntaxin 1/HPC-1 was present in the plasma membranes of the islets of Langerhans. In order to determine the functional role of syntaxin 1 in pancreatic beta-cells, rat syntaxin 1A or 1B was overexpressed in mouse beta TC3 cells using the transient transfection procedure. Transfection of beta TC3 cells with either syntaxin 1 resulted in approximately 7-fold increases in their immunodetectable protein levels. Glucose-stimulated insulin release by syntaxin 1A-overexpressing cells was suppressed to about 50% of the level in control cells, whereas insulin release by syntaxin 1B-overexpressing and control cells did not differ. Next, we established stable beta TC3 cell lines that overexpressed syntaxin 1A and used them to evaluate the effect of syntaxin 1A on the regulatory insulin release pathway. Two insulin secretogogues, 4-beta-phorbol 12-myristate 13-acetate or forskolin, increased insulin release by untransfected beta TC3 cells markedly, but their effects were diminished in syntaxin 1A-overexpressing beta TC3 cells. Glucose-unstimulated insulin release and the proinsulin biosynthetic rate were not affected by syntaxin 1A overexpression, indicating a specific role of syntaxin 1A in the regulatory insulin release pathway. Finally, in vitro binding assays showed that syntaxin 1A binds to insulin secretory granules, indicating an inhibitory role of syntaxin 1A in insulin exocytosis via its interaction with vesicular proteins. These results demonstrate that syntaxin 1A is expressed in the islets of Langerhans and functions as a negative regulator in the regulatory insulin release pathway.
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PMID:Expression and functional role of syntaxin 1/HPC-1 in pancreatic beta cells. Syntaxin 1A, but not 1B, plays a negative role in regulatory insulin release pathway. 855 45

We have previously shown by reverse transcriptase-PCR (rtPCR) that CML CD34+ HLA-DR- cells are enriched for BCR/ABL(-) hematopoietic progenitor cells (HPC) while leukemic HPC reside predominately within CML CD34+ HLA-DR+ cells. We investigated whether the 30/35 kDa fragment of fibronectin (FN) could be used to enhance retroviral-mediated gene transfer (RMGT) in chronic phase CML marrow HPC. CML CD34+ HLA-DR- and CD34+ HLA-DR+ cells were transduced with vector supernate containing the neomycin resistance gene on plates coated with either FN or bovine serum albumin (BSA) as control, then assayed for transduced HPC in progenitor cell assays in the presence or absence of G418. Transduction efficiency of CML CD34+ HLA-DR- cells over BSA ranged from 0.09 to 7.2% (mean 3.3 +/- 1.5%), while that over FN plates ranged from 3.8 to 23% (mean 11.0 +/- 4.5%) (n = 4). Transduction efficiencies of CML CD34+ HLA-DR+ cells ranged from 0.4 to 9.8% (mean 3.7 +/- 1.7%) and 6.0 to 26% (mean 17.3 +/- 4.5%) (n = 5) over BSA and FN, respectively. rtPCR analysis for BCR/ABL mRNA of individual G418-resistant HPC generated from CD34+ HLA-DR- cells revealed that normal BCR/ABL(-) HPC were successfully transduced under these experimental conditions. These results demonstrate the feasibility of transducing normal CML primitive HPC, and illustrate the potential clinical use of FN in the setting of gene therapy for CML, as well as other diseases.
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PMID:The 30/35 kDa chymotryptic fragment of fibronectin enhances retroviral-mediated gene transfer in purified chronic myelogenous leukemia bone marrow progenitors. 900 33

HIV-Nef protein supports viral infectivity prior to proviral integration. This requires Nef to be present before the expression of viral genes and suggests its delivery as part of the virion. We report here that the Nef proteins of HIV-2-HOM and HIV-2-ROD are associated with the virion. After the separation of pelleted virus in a 20-60% sucrose density gradient, both proteins cosedimented with the virion-associated reverse transcriptase (RT) activity at a density characteristic of retroviral particles. Whereas Nef-2-ROD was present in the virion only as the full-length protein, HIV-2-HOM appeared as 32 and 35 kDa isoforms. The smaller isoform is identical in molecular weight to the protein expected from proteolytic cleavage of full-length Nef-2-HOM by the virion-based protease. Virion-association of Nef helps to explain the recently redefined biological function of this regulatory protein.
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PMID:Evidence for the association of Nef protein with HIV-2 virions. 902 83

An endonuclease named DNase gamma has been purified from the nuclei of apoptotic rat thymocytes [Shiokawa, Ohyama, Yamada and Tanuma (1997) Biochem. J. 326, 675-681]. Here we report the molecular cloning of a cDNA encoding a 35 kDa precursor protein for rat DNase gamma. A 1.6 kb mRNA coding for the DNase gamma precursor is detected at high levels in spleen, lymph nodes, thymus and liver. By using reverse transcriptase-mediated PCR, expression of DNase gamma mRNA is observed in kidney and testis but not in brain or heart. Analysis of recombinant DNase gamma reveals that full-length DNase gamma, including the N-terminal precursor, is an inactive proenzyme. The mature form of recombinant DNase gamma, from which the N-terminal precursor has been removed, has the same properties as purified DNase gamma: requirement for divalent cations, dependence on pH, sensitivity to Zn2+, and cleavage of chromosome DNA to nucleosomal units. In HeLa S3 cells stably transfected with the DNase gamma cDNA, exogenously introduced DNase gamma is activated by apoptotic stimuli; enhancement of DNA fragmentation, chromatin condensation and nuclear collapse are observed. These findings provide evidence for the involvement of DNase gamma in DNA fragmentation and nuclear structural changes during apoptosis.
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PMID:Molecular cloning and expression of a cDNA encoding an apoptotic endonuclease DNase gamma. 962 Aug 74

A novel family of chloride channel proteins has recently been discovered including two bovine (Lu-ECAM-1, bCLCA1), one murine (mCLCA1), and two human (hCLCA1 and hCLCA2) members. Here, we describe the cloning, expression, and molecular characterization of a truncated human homolog, tentatively named hCLCA3. It was cloned from a human spleen cDNA library and is expressed in numerous tissues including lung, trachea, spleen, thymus, and mammary gland as determined by reverse transcriptase-polymerase chain reaction. Unlike all previously known CLCA family members which consistently encode an approximately 125-kDa transmembrane protein that is cleaved to form a heterodimer of two proteins of approximately 90 and 35 kDa, the 3.6-kb hCLCA3 mRNA encodes a 37-kDa glycoprotein that corresponds to the N-terminal extracellular domain of its homologs. Moreover, when expressed in human embryonic kidney 293 or Chinese hamster ovary cells, this 37-kDa glycoprotein is secreted into the culture supernatant. These observations suggest that hCLCA3 is a structurally divergent member of the CLCA family of proteins and that it does not act as a channel protein but has distinct, yet unidentified functions.
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PMID:Molecular cloning and biochemical characterization of a truncated, secreted member of the human family of Ca2+-activated Cl- channels. 1009 65

In plants, the pollen coat covers the exine wall of the pollen and is the outermost layer that makes the initial contact with the stigma surface during sexual reproduction. Little is known about the constituents of the pollen coat, especially in wind-pollinated species. The pollen coat was extracted with diethyl ether from the pollen of maize (Zea mays L.), and a predominant protein of 35 kDa was identified. On the basis of the N-terminal sequence of this protein, a cDNA clone of the Xyl gene was obtained by reverse transcriptase-polymerase chain reaction. The deduced amino acid sequence of the 35-kDa protein shared similarities with the sequences of many microbial xylanases and a barley aleurone-layer xylanase. The 35-kDa protein in the pollen-coat extract was purified to homogeneity by fast protein liquid chromatography and determined to be an acidic endoxylanase that was most active on oat spelt xylan. Northern and in situ hybridization showed that Xyl was specifically expressed in the tapetum of the anther after the tetrad microspores had become individual microspores. Southern hybridization and gene-copy reconstruction studies showed only one copy of the Xyl gene per haploid genome. Analyses of the genomic DNA sequence of Xyl and RNase protection studies with the transcript revealed many regulatory motifs at the promoter region and an intron at the 5' leader region of the transcript. The Xyl transcript had a 562-nucleotide (nt) 5' leader, a 54-nt sequence encoding a putative signal peptide, a 933-nt coding sequence, and a 420-nt 3'-untranslated sequence. The unusually long 5' leader had an open reading frame encoding a putative 175-residue protein whose sequence was most similar to that of a microbial arabinosidase. The maize xylanase is the first enzyme documented to be present in the pollen coat. Its possible role in the hydrolysis of the maize type II primary cell wall (having xylose, glucose, and arabinose as the major moieties) of the tapetum cells and the stigma surface is discussed.
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PMID:The predominant protein on the surface of maize pollen is an endoxylanase synthesized by a tapetum mRNA with a long 5' leader. 1042 75

In our study, surfactant protein (SP)-A was characterized in adult human trachea and bronchi. SP-A mRNA and protein were localized to serous cells in submucosal gland by in situ hybridization and immunohistochemistry, respectively. A 2.2 kb SP-A mRNA transcript was detected in tracheal tissues by Northern blot analysis. Primer extension analysis and gene-specific reverse transcriptase polymerase chain reaction (RT-PCR) revealed the predominance of SP-A2 mRNA. However, using nested PCR, we also detected low amounts of SP-A1 mRNA in the tracheal tissues. A approximately 35 kDa SP-A immunoreactive protein was detected in the tracheal tissues by immunoblot analysis and was shown to be modified by the addition of N-linked oligosaccharides. We conclude that submucosal glands in the conducting airways produce a novel SP-A protein with a molecular weight and post-translational modification similar to the SP-A produced in the distal lung. We speculate that this SP-A2 protein, like other serous secretions from airway submucosal glands, functions in local antimicrobial host defense mechanisms in the conducting airways.
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PMID:In situ hybridization of SP-A mRNA in adult human conducting airways. 1155 37

The roots of the sanchi ginseng, Panax notoginseng, were extracted with an aqueous buffer. The extract was chromatographed on a CM-cellulose column to remove extraneous unadsorbed proteins. The adsorbed fraction was dialyzed and chromatographed on Affi-gel blue gel. The adsorbed fraction was again collected, dialyzed and applied on a column of Mono S. The second peak was dialyzed and chromatographed on an FPLC-gel filtration Superdex 75 column. An antifungal protein with an N-terminal sequence similar to those of chitinases was isolated from the first peak which had a molecular mass of 35 kDa. The sequence was distinctive in that the third and ninth highly conserved N-terminal residues (C and G) were replaced by H and M, respectively. The protein inhibited mycelial growth in Coprinus comatus, Physalospora piricola, Botrytis cinerea, and Fusarium oxysporum with an IC 50 of 100 nM, 1 microM, 630 nM and 560 nM, respectively. It inhibited cell-free translation with an IC 50 of 630 nM. Its antifungal and translation-inhibitory activities were more potent than those of previously reported antifungal proteins. It inhibited human immunodeficiency virus-1 reverse transcriptase by 35.8 % at 12.6 microM and 24.7 % at 1.26 microM.
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PMID:Pananotin, a potent antifungal protein from roots of the traditional chinese medicinal herb Panax notoginseng. 1245 95


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