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)

We describe a technique, called reverse transcriptase (RT) in situ PCR, whereby RNA may be nonisotopically detected in fixed cells when amplified by PCR after cDNA synthesis by RT. RT in situ PCR using primers specific for the measles virus generated an intense signal in most measles-infected HeLa cells, as compared to the weak signal generated in few cells using standard in situ hybridization analysis. The viral RNA that localized to the nucleus spared the nucleoli, was most evident when the RT step used the primer complementary to the negative genomic strand, and was demonstrated in all multinucleated cells and the majority of uninucleate cells. A hybridization signal was evident with standard RNA in situ hybridization using the human megakaryocyte cell line Dami and a probe for glycoprotein IIB (GIIB) mRNA but not a probe for amyloid precursor protein (APP) or gelsolin (GEL) mRNA. After RT in situ PCR, signals were evident for each target localizing to the nucleolus for APP and to perinucleolar and cytoplasmic locations for GEL and GIIB. The latter findings suggest that mRNAs may follow different geographic pathways as they progress from premessage to transcriptionally active message.
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PMID:In situ localization of PCR-amplified human and viral cDNAs. 128 36

The expression of beta-amyloid precursor protein (BAPP) and its mRNAs was studied in fibroblasts obtained from patients afflicted with Alzheimer's disease (AD) and age-matched controls. Using reverse transcriptase-polymerase chain reaction (RT-PCR), transcripts corresponding to 770, 751, 714, and 695 amino acids were detected in both AD and control fibroblasts. Antibody 22C11 against BAPP (Boehringer Mannheim) labeled an intracellular protein, specifically localized to the intermediate filament network. In addition to bands of the predicted molecular weights for BAPP (120-135 kDa), Western blotting revealed a 57 kDa band which was not evident in samples of human brain. As cytoskeletal elements are vital in maintaining cellular architecture and various cell interactions, localization of BAPP or a related molecule to the cytoskeleton suggests a possible structural role for this protein within the cell.
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PMID:Antibody to beta-amyloid precursor protein recognizes an intermediate filament-associated protein in Alzheimer's and control fibroblasts. 145 84

C1 inhibitor was identified in human brain tissue by Western blotting and by immunohistochemistry using multiple antibodies to the native protein. The presence of C1 inhibitor mRNA was identified by reverse transcriptase-polymerase chain reaction analysis of brain mRNA extracts. The mRNA was also detected in cultured postmortem human microglia and in the IMR-32 human neuroblastoma cell line. Immunohistochemically, the native protein was detected in residual serum of capillaries and pyramidal neurons of both control and Alzheimer disease cases, as well as in occasional senile plaques of Alzheimer tissue. The reacted protein was detected on dystrophic neurites and neuropil threads in Alzheimer tissue by 4C3 monoclonal antibody, which recognizes a neoepitope following suicide inhibition. These data indicate that C1 inhibitor, a regulatory molecule controlling multiple inflammatory proteolytic cascades, is produced in normal brain. In Alzheimer disease, C1 inhibitor undergoes a prominent reaction in abnormal neuronal processes, such as dystrophic neurites and neuropil threads.
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PMID:Complement C1 inhibitor is produced by brain tissue and is cleaved in Alzheimer disease. 779 55

Three alternative splicing products of amyloid precursor protein (APP), APP770, 751 and 695, were detected in mouse embryonal carcinoma (EC) P19 cells by reverse transcriptase RNA polymerase chain reaction (RT-PCR). Alternative splicing of APP pre-mRNA in P19EC cells was remarkably changed by c-jun transformation. The relative ratio of APP770 encoding exons 7 and 8, non-neuron type, was increased by c-jun transformation, while that of APP 695 not encoding exons 7 and 8, neuron-specific one, was decreased. These results suggested that skipping of exons 7 and 8 was specifically blocked in c-jun transformed cells. APP 695, which increases in P19 EC cells under the culture conditions that induce the neuronal differentiation, did not increase in C2C5 cells under the same conditions, suggesting that c-jun transformed cells were not in the neuronal cell lineage and lost the ability to differentiate into neurons.
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PMID:c-jun inhibited the alternative splicing of neuron-specific amyloid precursor protein, but stimulated the non-neuron type one in P19 EC cells. 783 92

The amyloid precursor protein (APP), which is localized on both human chromosome 21 and its murine counterpart, chromosome 16 and which is involved in the formation of deposits in Alzheimer's disease, could be shown to bind effectively to a glytolytic enzyme: rat glyceraldehyde 3-phosphate dehydrogenase (Gapdh). We report here the isolation of a cDNA of murine Gapdh from mouse chromosome 16 (MMU16) originating from microclones of the distal part of MMU16 and the use of homologous genomic DNA sequences to further screen a cDNA phage library. The cDNA was sequenced, confirmed by polymerase chain reaction following reverse transcriptase (RT-PCR) and the open reading frame was expressed in vitro. The possible localization of Gapdh on MMU16--which may provide a mouse model for Down's syndrome and Alzheimer's disease--may lead to new insights into glycolysis and its role in the two syndromes.
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PMID:Isolation of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) cDNA from the distal half of mouse chromosome 16: further indication of a link between Alzheimer's disease and glycolysis. 789 98

To establish a cell line expressing enhanced levels of beta-amyloid precursor protein (beta-APP), we constructed plasmid DNAs expressing beta-APP-751 mRNA and transfected them into COS-1 cells. Using a modified version of the reverse transcriptase polymerase chain reaction which is RNA sensitive to study the beta-APP iso-RNAs, we have made the unexpected observation that enhanced expression of beta-APP-751 mRNA resulted in a significant reduction of beta-APP-770 and -695 mRNA levels. Suppression of beta-APP-770 and -695 was also observed in cells expressing truncated and chimeric beta-App-751 mRNAs. Similar observations were made in P19 cells expressing a chimeric beta-APP-751 mRNA where endogenous beta-APP-751 mRNA levels also were decreased. Also, suppression of beta-APP-770 and -751 mRNAs was observed in human kidney cells expressing exogenous beta-APP-695 mRNA.
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PMID:Suppression of Alzheimer amyloid precursor protein (APP) expression by exogenous APP mRNA. 861 Oct 30

We reported earlier that the levels of Ca2+-dependent metalloproteinases are increased in Alzheimer's disease (AD) specimens, relative to control specimens. Here we show that these enzymes are forms of the matrix metalloproteinase MMP-9 (EC3.4.24. 35) and are expressed in the human hippocampus. Affinity-purified antibodies to MMP-9 labeled pyramidal neurons, but not granular neurons or glial cells. MMP-9 mRNA is expressed in pyramidal neurons, as determined with digoxigenin-labeled MMP-9 riboprobes, and the presence of this mRNA is confirmed with reverse transcriptase PCR. The cellular distribution of MMP-9 is altered in AD because 76% of the total 100 kDa enzyme activity is found in the soluble fraction of control specimens, whereas only 51% is detectable in the same fraction from AD specimens. The accumulated 100 kDa enzyme from AD brain is latent and can be converted to an active form with aminophenylmercuric acetate. MMP-9 also is detected in close proximity to extracellular amyloid plaques. Because a major constituent of plaques is the 4 kDa beta-amyloid peptide, synthetic Abeta1-40 was incubated with activated MMP-9. The enzyme cleaves the peptide at several sites, predominantly at Leu34-Met35 within the membrane-spanning domain. These results establish that neurons have the capacity to synthesize MMP-9, which, on activation, may degrade extracellular substrates such as beta-amyloid. Because the latent form of MMP-9 accumulates in AD brain, it is hypothesized that the lack of enzyme activation contributes to the accumulation of insoluble beta-amyloid peptides in plaques.
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PMID:Matrix metalloproteinase-9 (MMP-9) is synthesized in neurons of the human hippocampus and is capable of degrading the amyloid-beta peptide (1-40). 898 19

It has been shown previously that mobilization of caffeine-sensitive intracellular calcium (Ca2+i) stores increased the release of amyloid beta-peptide (Abeta) from transfected human embryonic kidney cells (HEK293) [Querfurth, Jiang, Geiger and Selkoe (1997) J. Neurochem. 69, 1580-1591]. The present study was to test the hypothesis that the caffeine/Abeta responses were due to interactions with specific subtypes of ryanodine receptors (RyR) using [3H]ryanodine receptor binding, epifluorescence imaging of Ca2+i, immunocytofluorescence, immunoprecipitation and PCR techniques. [3H]Ryanodine bound to a single class of high-affinity caffeine-sensitive sites (Kd=9.9+/-1.6 nM, Bmax=25+/-4 fmol/mg of protein). RyRs were immuno-decorated in a punctate reticulo-linear pattern. Results from SDS/PAGE and reverse transcriptase-PCR demonstrated endogenous expression of type 1 (skeletal) and type 2 (cardiac) RyRs. HEK293 cell RyRs were functionally active, because (i) [Ca2+]i increased 2.8-fold over baseline following applications of 5-15 mM caffeine, (ii) repetitive spiked increases in [Ca2+]i were observed, and (iii) evidence for a use-dependent block was obtained. Some of these findings were extended to include HeLa and human fibroblast cell lines, suggesting a broader applicability to cells of epithelioid lineage. Implications for the processing of the beta-amyloid precursor protein in Alzheimer's disease and for calcium channel research using transfected HEK293 cells are discussed.
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PMID:Expression of ryanodine receptors in human embryonic kidney (HEK293) cells. 969 5

To understand the mechanism underlying cognitive deficits in AIDS patients, we examined the influence of gp41 peptides on the expression and the secretion of Alzheimer's amyloid precursor protein (APP) in human astroglial cell line T98G. Western blotting analyses demonstrated that treatment of glial cells with a putative immunosuppressive domain (aa 583-599) of gp41 remarkably downregulated the interleukin 1beta- (IL-1beta) induced elevation of the secreted form of APP (sAPP alpha) containing Kunitz-type protease inhibitor (KPI) domain without significant changes of the expression pattern of APP mRNAs as revealed by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Recombinant gp41 protein encoding for ectodomain, including aa 583-599 residues, also elicited a similar dose-dependent inhibitory effect, whereas the control peptides resulted in little change. The molecular mechanism underlying this gp41-mediated reduction of sAPP alpha secretion appears not to be owing to the difference in the function of extracellular proteases based on the finding of similar proteolytic activities responsible for APP metabolism in vitro present in the conditioned media from the cultures treated with or without gp41 peptide. However, the known PKC inhibitors such as H-7 or staurosporine, partially inhibited the elevation of sAPP alpha secretion in response to protein kinase C (PKC) agonist phorbol 12,13-dibutyrate (PdBu) as well as to IL-1beta, mimicking the immunosuppressive gp41 peptide. These observations implicate that part of the neurodegenerative cascade in AIDS brains may involve the inhibitory effect of gp41 on secretion of sAPP alpha, a potent glial neurotrophic factor, through impaired PKC response.
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PMID:Effect of HIV-1 gp41 peptides on secretion of beta-amyloid precursor protein in human astroglial cell line, T98G. 1052 58

The molecular, biochemical and cellular events that result in synaptic dysfunction and neuronal degeneration in the brain in Alzheimer's disease (AD) are becoming known. Age-related increases in cellular oxidative stress, and impairment of energy metabolism, result in disruption of neuronal calcium homeostasis and increased vulnerability of neurons to excitotoxicity and apoptosis. Inherited forms of AD that result from mutations in the beta-amyloid precursor protein (APP) and presenilins accelerate the neurodegenerative cascade by increasing production and deposition of neurotoxic forms of amyloid beta-peptide and by perturbing calcium homeostasis. Dietary restriction (DR; reduced calorie intake with maintained nutrition) extends life span of rodents and (probably) humans. DR increases resistance of neurons to dysfunction and degeneration, and improves behavioral outcome, in experimental models of AD and other age-related neurodegenerative disorders by a mechanism involving a mild stress response. Telomerase, a specialized reverse transcriptase, has been proposed to possess anti-aging properties. The catalytic subunit of telomerase (TERT) is expressed in neurons throughout the brain during development, but is absent from neurons in the adult brain. TERT exhibits neuroprotective properties in experimental models of neurodegenerative disorders suggesting that manipulations that induce telomerase in neurons may protect against age-related neurodegeneration. Finally, the exciting and exploding field of stem cell research suggests methods for replacing damaged or lost brain cells in an array of neurological disorders.
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PMID:Emerging neuroprotective strategies for Alzheimer's disease: dietary restriction, telomerase activation, and stem cell therapy. 1095 37


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