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)

Effective approaches using array technologies are critical to understand the molecular bases of human diseases. The results obtained using such procedures require analysis and validation procedures that are still under development. In the context of Alzheimer's disease, in which the identification of molecular mechanisms of underlying pathologies is vital, we describe a robust assay that is the first real-time reverse transcriptase-polymerase chain reaction-based high-throughput approach that can simultaneously quantitate the expression of a large number of genes at the copy number level from a minute amount of starting material. Using this approach within the human brain, we were able to quantitate as many as 19 genes at a time with only one type of fluorescent probe. The number of genes included can be considerably increased. Examples of consistent changes in Alzheimer's disease within these 19 candidate genes included reductions in targets related to the dendritic and synaptic apparatus. These changes were specific to Alzheimer's disease when compared with Parkinson's disease cases. We also present comparison data with microarray analysis from the same brain region and the same patients. The high sensitivity and reproducibility of this technology coupled with appropriate multivariate analysis is proposed here to form a biotechnology platform that can be widely used for diagnostic purposes as well as basic research.
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PMID:Single-channel quantitative multiplex reverse transcriptase-polymerase chain reaction for large numbers of gene products differentiates nondemented from neuropathological Alzheimer's disease. 1498 34

Identification of reliable markers to predict drug-related adverse events (DRAEs) is an important goal of the pharmaceutical industry and others within the healthcare community. We have used genetic polymorphisms, including the most frequent source of variation (single nucleotide polymorphisms, SNPs) in the human genome, in pharmacogenetic approaches designed to predict DRAEs. Three studies exemplify the principles of using polymorphisms to identify associations in progressively larger genomic regions: polymorphic repeats within the UDP-glucuronysltransferase I (UGT1A1) gene in patients experiencing hyperbilirubinemia after administration of tranilast, an experimental drug to prevent re-stenosis following coronary revascularization; high linkage disequilibrium within the Apolipoprotein E (ApoE) gene in patients with Alzheimer Disease (AD); and the polymorphic variant HLA-B57 in patients with hypersensitivity reaction after administration of abacavir, a nucleoside reverse transcriptase inhibitor for the treatment of HIV. Together, these studies demonstrate in a stepwise manner the feasibility of using pharmacogenetic approaches to predict DRAEs.
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PMID:Pharmacogenetics to predict drug-related adverse events. 1520 98

Alzheimer's disease (AD) phenotype complexity raises the question whether genetic features remain unknown. Although a few percentage of patients are familial cases linked to mutations in amyloid precursor protein, presenilin 1 or presenilin 2 genes, the remainder are considered mainly sporadic late-onset cases with a complex etiology. However, changes in gene expression or other genetic features of the individual can clearly contribute to develop the illness. Consequently, in this paper we have focused on the identification of new genes, the expression of which is altered in AD. We used the technique of differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) in order to study the gene expression differences in brain tissue from patients in an advanced stage of AD. After studying medial septum and hippocampus brain areas, we found an inhibition of the KIAA0471 gene expression in three out of six AD patients, including one with a presenilin 1 gene mutation. This gene encodes for a large protein that presents, in its predicted form, 95% homology with IDN4-GGTR sequences. These results may provide significant clues for understanding the molecular mechanisms underlying septohippocampal neurodegeneration. In addition, they may open a new area of research for diagnostic and therapeutic tools, the relevance of which is also considered.
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PMID:Reduced KIAA0471 mRNA expression in Alzheimer's patients: a new candidate gene product linked to the disease? 1536 92

Oxidative modification of cytoplasmic RNA in vulnerable neurons is an important, well documented feature of the pathophysiology of Alzheimer disease. Here we report that RNA-bound iron plays a pivotal role for RNA oxidation in vulnerable neurons in Alzheimer disease brain. The cytoplasm of hippocampal neurons showed significantly higher redox activity and iron(II) staining than age-matched controls. Notably, both were susceptible to RNase, suggesting a physical association of iron(II) with RNA. Ultrastructural analysis further suggested an endoplasmic reticulum association. Both rRNA and mRNA showed twice the iron binding as tRNA. rRNA, extremely abundant in neurons, was considered to provide the greatest number of iron binding sites among cytoplasmic RNA species. Interestingly, the difference of iron binding capacity disappeared after denaturation of RNA, suggesting that the higher order structure may contribute to the greater iron binding of rRNA. Reflecting the difference of iron binding capacity, oxidation of rRNA by the Fenton reaction formed 13 times more 8-hydroxyguanosine than tRNA. Consistent with in situ findings, ribosomes purified from Alzheimer hippocampus contained significantly higher levels of RNase-sensitive iron(II) and redox activity than control. Furthermore, only Alzheimer rRNA contains 8-hydroxyguanosine in reverse transcriptase-PCR. Addressing the biological significance of ribosome oxidation by redox-active iron, in vitro translation with oxidized ribosomes from rabbit reticulocyte showed a significant reduction of protein synthesis. In conclusion these results suggest that rRNA provides a binding site for redox-active iron and serves as a redox center within the cytoplasm of vulnerable neurons in Alzheimer disease in advance of the appearance of morphological change indicating neurodegeneration.
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PMID:Ribosomal RNA in Alzheimer disease is oxidized by bound redox-active iron. 1576 56

Thrombin is a serine protease that is generated by proteolytic cleavage of its precursor, prothrombin. We previously showed that thrombin proteolyses the microtubule-associated protein tau and that phosphorylation of tau inhibits this process. To characterize further the role of thrombin in the brain, we investigated prothrombin and thrombin expression in cultured brain cells and in brains of control, Alzheimer disease (AD) and parkinsonism-dementia complex of Guam (PDCG). We show by reverse transcriptase-polymerase chain reaction that prothrombin mRNA is expressed in brain tissues, neuroblastoma cells, and cultured human astrocytes, oligodendrocytes, and microglial cells. We also show by immunohistochemistry that the proteins prothrombin and thrombin are present in brain using specific monoclonal and polyclonal antibodies for both proteins. All antibodies stained residual serum in blood vessels, as well as normal pyramidal neurons and their processes, and some astrocytes. Additionally, in AD and PDCG cases, all antibodies stained extra- and intracellular neurofibrillary tangles (NFTs), senile plaques, and reactive microglial cells. The ubiquitous expression of prothrombin and thrombin in brain cells suggests that thrombin plays an important physiological role in normal brain. The accumulation of thrombin and prothrombin in NFTs supports the hypothesis that thrombin may be involved in tau proteolysis and that failure to metabolize tau may lead to its aggregation in neurodegenerative diseases.
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PMID:Thrombin and prothrombin are expressed by neurons and glial cells and accumulate in neurofibrillary tangles in Alzheimer disease brain. 1641 Jul 45

Dopaminergic cell loss in the mesencephalic substantia nigra is the hallmark of Parkinson's disease and may be associated with abnormal oxidative metabolic activity. However, the delicate balance underlying dopamine decline and oxidative stress is still a matter of debate. The aim of this study was to analyze the possible modulation of D2 agonists and antagonists on MPP+ (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium ion) -induced cellular death in differentiated and undifferentiated PC12 cells. Using colorimetric assays, western blots and reverse transcriptase-PCR, we demonstrated that two D2 agonists, bromocriptine and quinpirole, consistently increased MPP+ -induced cytotoxicity in both differentiated and undifferentiated PC12 cells, whereas D2 antagonists do not modulate cell death. However, this increase in cellular death was reversed when bromocriptine or quinpirole were used in presence of D2 antagonists. On the other hand, 1-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-4-(3-phenylpropyl)piperazine (GBR 12909), a potent inhibitor of the dopamine transporter, partially reversed MPP+ -induced cellular death and completely abolished the increase of cellular death induced by bromocriptine. Dopamine agonists and antagonists also modulate the expression of the dopamine transporter in PC12 cells; in particular, bromocriptine may alter MPP+ uptake by increasing DAT expression We also show that, in our cellular paradigm, D2 receptor mRNA levels are more abundant that D3 mRNA levels and MPP+ and /or bromocriptine could not modulate D2 gene expression while D3 gene expression clearly decrease after MPP+ and /or bromocriptine treatment.
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PMID:Dopamine D2 agonists, bromocriptine and quinpirole, increase MPP+ -induced toxicity in PC12 cells. 1700 Apr 68

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) have been recently identified in families with autosomal-dominant late-onset Parkinson disease. We report that by reverse transcriptase-polymerase chain reaction, the mRNA of LRRK2 is expressed in soluble extracts of human brain, liver, and heart and in cultured human astrocytes, microglia, and oligodendroglia as well as in human neuroblastoma cell lines. We find by Western blotting using a polyclonal antibody of the leucine-rich repeat kinase 2 protein (Lrrk2) specific for C-terminal residues 2,511-2,527 that an apparent full-length protein and several of its fractions are expressed in soluble extracts of normal human brain. By immunocytochemistry, the antibody recognizes neurons, and more weakly astrocytes and microglia, in normal brain tissue. It intensely labels Lewy bodies in Parkinson disease and related neurodegenerative disorders. It also labels a subset of neurofibrillary tangles in Alzheimer disease and the Parkinsonism dementia complex of Guam (PDCG). It labels thorn-shaped astrocytes and oligodendroglial coiled bodies in PDCG; oligodendroglial inclusions in multiple system atrophy; Pick bodies in Pick disease; nuclear and cytoplasmic inclusions in Huntington disease; and intraneuronal and glial inclusions in amyotrophic lateral sclerosis. In summary, LRRK2 is constitutively expressed in neurons and also in glial cells of human brain. It strongly associates with pathological inclusions in several neurodegenerative disorders.
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PMID:LRRK2 expression in normal and pathologic human brain and in human cell lines. 1702

Neurodegeneration in Alzheimer's disease and various experimental lesion paradigms are associated with an unscheduled upregulation of cell cycle-related proteins, indicating a link between cell cycle reactivation and neuronal death. Recent evidence, however, suggests that at least some of the canonical cell cycle regulators are constitutively expressed in differentiated neurons of the adult brain. Systematic investigations on the constitutive expression of cell cycle regulators in differentiated neurons in vivo, providing the basis for further insights into their potential role under pathological conditions, however, have not been carried out. Here, we demonstrate a constitutive neuronal expression of Cdks 1, 2, and 4; their activators cyclins D, A, B, and E; and their inhibitors p15(Ink4b), p16(Ink4a), p18(Ink4c), p19(Ink4d), p21(Waf1/Cip1), p27(Kip1), and p57(Kip2) within the neocortex of adult mice by western blot and immunocytochemistry. Expression was verified by single-cell reverse transcriptase-polymerase chain reaction applied to individual microscopically identified neurons captured with laser dissection. Immunoprecipitation and in vitro kinase assays revealed that Cdks 1, 2, and 4 are properly complexed to cyclins and exhibit kinase activity. This physiological expression of positive cell cycle regulators in adult neurons is clearly not related to neuronal proliferation. Taken together, our findings demonstrate a constitutive expression of functionally active cyclin-dependent kinases and their regulators in differentiated neurons suggesting a noncanonical role of cell cycle regulators potentially linked to neuronal plasticity and/or stability.
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PMID:Constitutive expression of functionally active cyclin-dependent kinases and their binding partners suggests noncanonical functions of cell cycle regulators in differentiated neurons. 1705 Jun 46

The expression of the purinergic receptor subtype P2X(7)R, a nonselective cationic channel activated by high levels of adenosine triphosphate (ATP), has been studied in adult microglia obtained from Alzheimer disease (AD) and nondemented (ND) brains, in fetal human microglia exposed to Abeta(1-42) peptide and in vivo in Abeta(1-42)-injected rat hippocampus. Semiquantitative reverse transcriptase-polymerase chain reaction showed enhanced expression (increase of 70%) of P2X(7)R in AD microglia compared with ND cells (analysis of 6 AD and 8 ND cases). Immunohistochemical analysis showed prominent P2X(7)R expression in association with Abeta plaques and localized to HLA-DR-immunoreactive microglia. In cultured fetal human microglia, cells exposed to Abeta(1-42) (5 microM for 18 hours) had significantly elevated levels of P2X(7)R (by 106%) compared with untreated cells. Amplitudes of Ca(2+) responses in these cells, induced by the selective P2X(7)R agonist BzATP, were increased by 145% with Abeta(1-42) pretreatment relative to control (no peptide pretreatment) and were largely blocked if the P2X(7)R inhibitor-oxidized ATP (oxATP) was added with peptide in pretreatment solution. In vivo, double immunostaining analysis showed considerable P2X(7)R colocalized with microglia after injection of Abeta(1-42) (1 nmol) into rat hippocampus. The overall results suggest roles of P2X(7)R in mediating microglial purinergic inflammatory responses in AD brain.
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PMID:Upregulated expression of purinergic P2X(7) receptor in Alzheimer disease and amyloid-beta peptide-treated microglia and in peptide-injected rat hippocampus. 1708 6

The gamma-synuclein protein is involved in breast carcinogenesis and has also been implicated in other forms of cancer and in ocular diseases. Furthermore, gamma-synuclein is believed to have a role in certain neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. This work reports the cloning and characterization of the porcine (Sus scrofa) gamma-synuclein cDNA (SNCG). The SNCG cDNA was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The porcine SNCG cDNA codes for a protein of 126 amino acids which shows a high similarity to bovine (90%), human (87%) and mouse (83%) gamma-synuclein. A genomic clone containing the entire porcine SNCG gene was isolated and its genomic organization determined. The gene is composed of five exons, the general structure being observed to be very similar to that of the human SNCG gene. Expression analysis by quantitative real-time RT-PCR revealed the presence of SNCG transcripts in all examined organs and tissues. Differential expression was observed, with very high levels of SNCG mRNA in fat tissue and high expression levels in spleen, cerebellum, frontal cortex and pituitary gland. Expression analysis also showed that porcine SNCG transcripts could be detected in different brain regions during early stages of embryo development. The porcine SNCG orthologue was mapped to chromosome 14q25-q29. The distribution of recombinant porcine gamma-synuclein was studied in three different transfected cell lines and the protein was found to be predominantly localized in the cytoplasm.
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PMID:Porcine gamma-synuclein: molecular cloning, expression analysis, chromosomal localization and functional expression. 1846 69


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