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 used reverse transcriptase-polymerase chain reaction and Western blotting techniques to measure the levels of complement mRNAs and their protein products in Alzheimer's disease (AD) brain compared with non-AD brain. mRNAs for C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9 were detected in the 11 regions of brain that were investigated. The mRNA levels were markedly up-regulated in affected areas of AD brain. In the entorhinal cortex, hippocampus, and midtemporal gyrus, which had dense accumulations of plaques and tangles, C1q mRNA was increased 11- to 80-fold over control levels, and C9 mRNA 10- to 27-fold. These levels were substantially higher than in the livers of the same cases. Western blot analysis of AD hippocampus established the presence of all of the native complement proteins as well as their activation products C4d, C3d, and the membrane attack complex. These data indicate that high levels of complement are being produced in affected areas of AD brain, that full activation of the classical complement pathway is continuously taking place, and that this activation may be contributing significantly to AD pathology.
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PMID:Up-regulated production and activation of the complement system in Alzheimer's disease brain. 1007 71

We used adenoviral-mediated gene transfer of human amyloid precursor proteins (h-APPs) to evaluate the role of various h-APPs in causing neuronal cell death. We were able to infect PC12 cells with very high efficiency because approximately 90% of the cells were cytochemically positive for beta-galactosidase activity when an adenoviral vector containing LacZ cDNA was used to infect cells. Cells infected with adenovirus containing h-APP cDNA showed high-level transcription and expression of h-APP as measured by reverse transcriptase-polymerase chain reaction and Western immunoblot analyses, respectively. Intracellular and extracellular levels of h-APP were elevated approximately 17-and 24-fold in cultures infected with recombinant adenovirus containing wild-type mutant and 13- and 17-fold with V642F mutant. No elevation in h-APP was seen in cultures infected with antisense h-APP or null adenovirus. H-APP levels were maximal 3 days after infection. Overexpression of V642F mutant h-APP in PC12 cells and hippocampal neurons resulted in about a twofold increase in death compared with overexpression of wild-type h-APP. These results demonstrate the usefulness of recombinant adenoviral mediated gene transfer in cell culture studies and suggest that overexpression of a familial Alzheimer's disease mutant APP may be toxic to neuronal cells.
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PMID:Death of PC12 cells and hippocampal neurons induced by adenoviral-mediated FAD human amyloid precursor protein gene expression. 1008 85

Alzheimer's disease (AD) has been hypothesized to be associated with oxidative stress. In this study, the expression of key oxidative stress-handling genes was studied in hippocampus, inferior parietal lobule, and cerebellum of 10 AD subjects and 10 control subjects using reverse transcriptase-polymerase chain reaction (RT-PCR). The content of Mn-, Cu,Zn-superoxide dismutases (Mn- and Cu,Zn-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase (GSSG-R) mRNAs, and the "marker genes" (beta-actin and cyclophilin) mRNAs was determined. This study suggests that gene responses to oxidative stress can be significantly modulated by the general decrease of transcription in the AD brain. To determine if the particular oxidative stress handling gene transcription was induced or suppressed in AD, the "oxidative stress-handling gene/beta-actin" ratios were quantified and compared with control values in all brain regions studied. The Mn-SOD mRNA/beta-actin mRNA ratio was unchanged in all regions of the AD brain studied, but an increase of the Cu,Zn-SOD mRNA/beta-actin mRNA ratio was observed in the AD inferior parietal lobule. The levels of peroxidation handling (CAT, GSHPx, and GSSG-R) mRNAs normalized to beta-actin mRNA level were elevated in hippocampus and inferior parietal lobule, but not in cerebellum of AD patients, which may reflect the protective gene response to the increased peroxidation in the brain regions showing severe AD pathology. The results of this study suggest that region-specific differences of the magnitude of ROS-mediated injury rather than primary deficits of oxidative stress handling gene transcription are likely to contribute to the variable intensity of neurodegeneration in different areas of AD brain.
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PMID:The expression of key oxidative stress-handling genes in different brain regions in Alzheimer's disease. 1009 42

We used the techniques of reverse transcriptase-polymerase chain reaction, Western blotting and immunohistochemistry to evaluate the expression of cyclooxygenase (COX)-1 and -2 in brain and peripheral organs of Alzheimer disease (AD) and control cases. We found both COX-1 and COX-2 to be constitutively expressed in all organs tested, i.e., brain, heart, liver, kidney, spleen and intestine. COX-2 was substantially upregulated in affected areas of AD brain and in infarcted areas of human heart. COX-1 was only mildly upregulated in AD brain. Immunohistochemically, COX-2 was strongly expressed in the perinuclear, dendritic and axonal areas of pyramidal neurons, with enhanced staining in AD. These data suggest a special role for COX-2 in neuronal function.
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PMID:Distribution of cyclooxygenase-1 and cyclooxygenase-2 mRNAs and proteins in human brain and peripheral organs. 1036 79

Proteins characteristic of activated complement are associated with Alzheimer disease (AD) lesions. The classical complement pathway can be activated only when the influence of such endogenous regulators as C1-inhibitor (C1-inh) and CD59 are overcome. We used the techniques of reverse transcriptase-polymerase chain reaction and Western blotting to assess the mRNA and protein levels of C1-inh and CD59 in AD and control brains in comparison with levels of the complement components with which they interact. The inhibitors were only slightly upregulated and then only in heavily affected areas of AD brain such as the entorhinal cortex, hippocampus, midtemporal gyrus and midfrontal gyrus. The ratio of AD to control mRNAs in these four areas was 1.17 for C1-inh and 1.12 for CD59, compared to 3.06 for C1r, 2.67 for C1s, 2.35 for C5, 2.56 for C6, 2.42 for C7, 5. 08 for C8 and 16.3 for C9. Peripheral organ expression of C1-inh and CD59 mRNAs was no different in AD than controls but was slightly upregulated in infarcted heart tissue. Again, the increase was small compared with that of the competitive complement components. These data indicate that the forces which upregulate and activate complement in AD and myocardial infarction are not effectively suppressed by the endogenous regulators, C1-inh and CD59.
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PMID:Complement regulators C1 inhibitor and CD59 do not significantly inhibit complement activation in Alzheimer disease. 1037 8

Acetylcholine is an important regulator of local cerebral blood flow. There is, however, limited information available on the possible sites of action of this neurotransmitter on brain intraparenchymal microvessels. In this study, a combination of molecular and functional approaches was used to identify which of the five muscarinic acetylcholine receptors (mAChR) are present in human brain microvessels and their intimately associated astroglial cells. Microvessel and capillary fractions isolated from human cerebral cortex were found by reverse transcriptase-polymerase chain reaction to express m2, m3, and, occasionally, m1 and m5 receptor subtypes. To localize these receptors to a specific cellular compartment of the vessel wall, cultures of human brain microvascular endothelial and smooth muscle cells were used, together with cultured human brain astrocytes. Endothelial cells invariably expressed m2 and m5 receptors, and occasionally the m1 receptor; smooth muscle cells exhibited messages for all except the m4 mAChR subtypes, whereas messages for all five muscarinic receptors were identified in astrocytes. In all three cell types studied, acetylcholine induced a pirenzepine-sensitive increase (62% to 176%, P<0.05 to 0.01) in inositol trisphosphate, suggesting functional coupling of m1, m3, or m5 mAChR to a phospholipase C signaling cascade. Similarly, coupling of m2 or m4 mAChR to adenylate cyclase inhibition in endothelial cells and astrocytes, but not in smooth muscle cells, was demonstrated by the ability of carbachol to significantly reduce (44% to 50%, P<0.05 to 0.01) the forskolin-stimulated increase in cAMP levels. This effect was reversed by the mAChR antagonist AFDX 384. The results indicate that microvessels are able to respond to neurally released acetylcholine and that mAChR, distributed in different vascular and astroglial compartments, could regulate cortical perfusion and, possibly, blood-brain barrier permeability, functions that could become jeopardized in neurodegenerative disorders such as Alzheimer's disease.
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PMID:Functional acetylcholine muscarinic receptor subtypes in human brain microcirculation: identification and cellular localization. 1041 35

The transglutaminase (TGase) family of enzymes, of which seven different members are known in the human genome, participate in many biological processes involving cross-linking proteins into large macromolecular assemblies. The TGase 2 enzyme is known to be present in neuronal tissues and may play a role in neuronal degenerative diseases such as Alzheimer's disease (AD) by aberrantly cross-linking proteins. In this paper, we demonstrate by reverse transcriptase-polymerase chain reaction and immunological methods with specific antibodies that in fact three members, the TGase 1, TGase 2, and TGase 3 enzymes, and are differentially expressed in various regions of normal human brain tissues. Interestingly, the TGase 1 and 3 enzymes and their proteolytically processed forms are involved in terminal differentiation programs of epithelial cell development and barrier function. In addition, we found that the levels of expression and activity of the TGase 1 and 2 enzymes were both increased in the cortex and cerebellum of AD patients. Furthermore, whereas normal brain tissues contain approximately 1 residue of cross-link/10,000 residues, AD patient cortex and cerebellum tissues contain 30-50 residues of cross-link/10,000 residues. Together, these findings suggest that multiple TGase enzymes are involved in normal neuronal structure and function, but their elevated expression and cross-linking activity may also contribute to neuronal degenerative disease.
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PMID:Differential expression of multiple transglutaminases in human brain. Increased expression and cross-linking by transglutaminases 1 and 2 in Alzheimer's disease. 1052 60

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

Apolipoprotein E (apoE) plays a central role in lipid metabolism from its ability to interact with lipoprotein receptors. Besides its role in cardiovascular diseases, apoE polymorphism contributes to susceptibility to neurodegenerative diseases, such as Alzheimer's disease. The statistical significance of the combined match scores obtained after apoE motif-based protein sequence database searches, the structural features of the deduced protein, and the phylogenetic analysis, support the evidence that a homologue to mammalian apoE can be found in teleost fish. Isolation and characterization of the first nonmammalian APOE revealed that the zebrafish gene spans 2555/2692 bp instead of 3597 bp in human and has the same splice junctions and exon/intron organization as found in mammals, except that there is an additional intron that splits the last exon (exon 4) into two exons (exons 4 and 5). Enlargement of APOE size in the mammalian lineage occurs mainly by Alu repeats insertion. The additional intron found in zebrafish gene was also identified at the same splicing site in trout APOE and is located in the corresponding linker region following the conserved low density lipoprotein receptor binding domain. Primer extension and reverse transcriptase PCR (RT-PCR) assays demonstrated that two transcription start sites are located 26 and 28 bp upstream of the first intron and 22 or 24 bp downstream from a canonical TATA box. Sequence inspection of the 5'-flanking region upstream of the TATA box revealed potential regulatory DNA elements. These results will serve as a basis for comparative studies on transcriptional and post-transcriptional mechanisms of APOE regulation in vertebrates.
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PMID:Conserved protein motifs and structural organization of a fish gene homologous to mammalian apolipoprotein E. 1063 25

Metallothionein (MT)-III, originally discovered as a growth inhibitory factor (GIF), is a brain specific isomer of MTs and is markedly reduced in the brain of patients with Alzheimer's disease (AD) or other neurodegenerative diseases. We analyzed the level and regulation of mRNA expression of MT-III in immortalized fetal mouse brain glial cells (VR-2g) by reverse transcriptase-polymerase chain reaction (RT-PCR). We have recently reported that dopamine (DA) increases the expression of MT-III mRNA in vitro. In this study, we investigated the mechanism of such increase by examining the effects of DA agonists (SKF38393 or bromocriptine) and DA antagonists (SCH23390 or sulpiride) on the expression of MT-III mRNA. MT-III mRNA did not change by either agonist and DA-increased MT-III mRNA was not inhibited by either antagonist. These results suggested that the induction of MT-III mRNA by DA was not mediated by stimulation of DA receptors. On the other hand, DA-induced MT-III mRNA expression was strongly inhibited by the addition of antioxidants (glutathione, vitamin E or ascorbic acid), indicating that DA-enhanced MT-III mRNA was mediated by reactive oxygen species. Our results suggest that oxidative stress may be one of the principle factors that modulate MT-III mRNA expression.
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PMID:Antioxidants protect against dopamine-induced metallothionein-III (GIF) mRNA expression in mouse glial cell line (VR-2g). 1064 Jun 28


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