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 enantiomers of carbovir, a carbocyclic analog of 2',3'-dideoxyguanosine, were compared with respect to their phosphorylation and the phosphorylation of their nucleotides by mammalian enzymes. 5'-Nucleotidase catalyzed the phosphorylation of (-)-carbovir, which is active against HIV (human immunodeficiency virus), but did not phosphorylate (+)-carbovir. (-)-Carbovir monophosphate was 7,000 times more efficient as a substrate for GMP kinase than was (+)-carbovir monophosphate. Pyruvate kinase, phosphoglycerate kinase, and creatine kinase phosphorylated both enantiomers of carbovir diphosphate at similar rates. Nucleoside-diphosphate kinase preferentially phosphorylated the (-)-enantiomer. Both enantiomers of carbovir triphosphate were substrates and alternative substrate inhibitors of HIV reverse transcriptase. Thus, the contrasting HIV-inhibitory activities of carbovir enantiomers were due to differential phosphorylation by cellular enzymes and not due to enantioselectivity of HIV reverse transcriptase.
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PMID:Phosphorylation of carbovir enantiomers by cellular enzymes determines the stereoselectivity of antiviral activity. 138 19

The mutations in one-third of both Duchenne and Becker muscular dystrophy patients remain unknown because they do not involve gross rearrangements of the dystrophin gene. Here we report the first example of multiple exon skipping during the splicing of dystrophin mRNA precursor encoded by an apparently normal dystrophin gene. A 9-year-old Japanese boy exhibiting excessive fatigue and high serum creatine kinase activity was examined for dystrophinopathy. An immunohistochemical study of muscle tissue biopsy disclosed faint and discontinuous staining of the N-terminal and rod domains of dystrophin but no staining at all of the C-terminal domain of dystrophin. The dystrophin transcript from muscle tissue was analyzed by the reverse transcriptase polymerase chain reaction. An amplified product encompassing exons 67-79 of dystrophin cDNA was found to be smaller than that of the wild-type product. Sequence analysis of this fragment showed that the 3' end of exon 70 was directly connected to the 5' end of exon 75 and, thus, that exons 71-74 were completely absent. As a result, a truncated dystrophin protein lacking 110 amino acids from the C-terminal domain should result from translation of this truncated mRNA, and the patient was diagnosed as having Becker muscular dystrophy at the molecular level. Genomic DNA was analyzed to identify the cause of the disappearance of these exons. Every exon-encompassing region could be amplified from genomic DNA, indicating that the dystrophin gene is intact. Furthermore, sequencing of these amplified products did not disclose any particular nucleotide change that could be responsible for the multiple exon skipping observed. Considering that exons 71-74 are spliced out alternatively in some tissue-specific isoforms, to suppose that the alternative splicing machinery is present in the muscle tissue of the index case and that it is activated by an undetermined mechanism is reasonable. These results illustrate a novel genetic anomaly that results in dystrophinopathy.
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PMID:A case of Becker muscular dystrophy resulting from the skipping of four contiguous exons (71-74) of the dystrophin gene during mRNA maturation. 886 44

Latissimus dorsi muscle (LDM) transformation following chronic stimulation is the critical requirement for its use in cardiac assist procedures. In order to identify one or two molecular markers that can be used to effectively monitor the LDM transformation, the modulation in the expression of creatine kinase (CK) and phospholamban (PLB) genes by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was examined. Continuous in situ stimulation of left LDM was performed in four dogs for a period of 10 weeks after a vascular delay period of 2 weeks following surgery. For RT-PCR, gene-specific radiolabeled primers and equal amounts of cDNA synthesized from total RNA extracted from the LDM biopsies obtained at 4, 7, and 10 weeks of stimulation were used. A 2.6-fold increase in creatine kinase (brain type) (CK-B) mRNA was observed at transformed LDM compared to the control (P = 0.004) following 10 weeks of stimulation. On the contrary, a 30% decline was observed in creatine kinase (muscle type) (CK-M) mRNA level. An increase up to eight-fold was also observed in PLB mRNA in stimulated LDM compared to the contralateral muscle (P = 0.002). The PLB mRNA level in transformed LDM reached plateau and became comparable to that of normal heart after 7 weeks of stimulation. However, a sustained increase in CK-B mRNA level was observed until 10 weeks of stimulation. The level of beta-actin mRNA used as control remained the same in both stimulated and control samples. Thus the increase in CK-B and PLB mRNA and downregulation of CK-M mRNA in transformed LDM, demonstrated here by RT-PCR, indicate a switch from anaerobic to aerobic potential of transformed LDM along with a change towards slow-twitch phenotype and provide valuable markers to monitor the effectiveness of muscle transformation in cardiomyoplasty.
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PMID:Activation of creatine kinase-B and phospholamban gene expression in transformed latissimus dorsi muscle: evaluation of mRNA by polymerase chain reaction. 889 49

Though some mechanisms of photoreception have been well characterized, others remain obscure. Presumably, most, if not all, of the major players in photoreceptor-specific functions are present in large amounts in the photoreceptor layer, and a catalog of these proteins will prove a useful tool for vision researchers. As a first step toward a complete catalog of photoreceptor cells, we have developed a novel method for isolating the photoreceptor cell monolayer from bovine retina. Electron microscopic studies of both the photoreceptor layer and the residual retina from which the photoreceptor layer had been removed, indicate that the preparation contains the photoreceptor outer segments and the majority of the inner segments. Proteins were extracted from the isolated photoreceptor cell layer as well as the rest of the retina with isoelectric focusing lysis buffer, and the protein components were separated by two-dimensional gel electrophoresis. The obtained protein maps reveal several classes of proteins that appear to be expressed more abundantly or specifically in the photoreceptor layer than in the rest of the retina. Four of these protein spots were excised and in-gel digested with trypsin, and the digests were extracted with solvent. The mixture of peptides digested from each protein was analyzed by high performance liquid chromatography interfaced with electrospray ionization tandem quadrupole mass spectrometry or by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Some of the peptides were isolated and their sequences were determined by gas phase Edman degradation. RNA transcripts extracted from the photoreceptor layer or the whole retina were subjected to Northern blot analysis as well as to reverse transcriptase-polymerase chain reaction amplification of probes for the successful selection of cDNA clones. These data permit both the identification of virtually any protein detectable on a two-dimensional gel, and also enable the corresponding cDNA clone to be selected. We have validated this approach by identifying aspartate aminotransferase and creatine kinase from the populations of abundant photoreceptor layer proteins. Both aspartate aminotransferase and creatine kinase are of mitochondrial origin and are thought to play crucial roles in photoreceptor functions by producing glutamate and ATP, respectively. We also identified two photoreceptor layer specific proteins: an acidic and high molecular weight protein, interphotoreceptor retinoid-binding protein, and an acidic and small molecular weight protein, recoverin.The technique presented here will allow vision researchers to discover and identify the proteins that are expressed specifically or abundantly in the photoreceptor cell as well as the proteins that undergo post-translational modification or modulation in expression under a defined biological condition. With the use of this technology, we anticipate that a researcher who knows only the 2-D gel position of a protein of interest can identify the protein, isolate a cDNA clone, and move into molecular genetic studies. Moreover, this streamlined technology will enable one to assemble a catalog of photoreceptor proteins using a minute amount of materials in a short period of time. We believe that such a catalog will serve as a valuable resource for vision investigators and will accelerate the rate of research progress.
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PMID:Initiating ocular proteomics for cataloging bovine retinal proteins: microanalytical techniques permit the identification of proteins derived from a novel photoreceptor preparation. 1043 56

We report two siblings with a relatively severe limb-girdle muscular dystrophy. The elder sister presented at 8 years of age with inability to climb and abnormal gait. At 12 years she was barely ambulant. Her sister followed a similar course. Serum creatine kinase was 8500-10000 IU (N 25-200) in the elder sister and 17000-19000 IU in the younger sister. Muscle biopsy of the elder sister at 8 years showed chronic myopathic changes with loss of muscle fibres, active necrosis and regeneration. Immunocytochemistry demonstrated normal spectrin and dystrophin, reduced alpha-sarcoglycan and absent gamma-sarcoglycan--indicating a gamma-sarcoglycanopathy. Haplotype analysis for the markers D13S115, D13S232, D13S292, D13S787, D13S1243 and D13S283 internal to and flanking the gamma-sarcoglycan gene showed the affected sisters shared haplotypes, indicating it was possible they were suffering from a gamma-sarcoglycanopathy. Non-inheritance of paternal alleles for D13S232, D13S292 and D13S1243 suggested the inheritance of a deletion, which was confirmed by FISH, using a genomic probe from the gamma-sarcoglycan gene. The gamma-sarcoglycan cDNA was amplified by reverse transcriptase PCR from the muscle biopsy of the elder sister and sequenced. A missense mutation changing codon 69 from GGC glycine to CGC arginine was identified. HhaI digestion of exon 3 genomic PCR products showed the two affected sisters were hemizygous for the mutation, while the mother and grandmother were heterozygotes. The mutation, identified by SSCP analysis, was not observed in 116 unrelated, unaffected individuals. Previously, only two other missense mutations, the Cys283Tyr missense mutation in Gypsies and the Leu193Ser mutation in a Dutch family, have been described in the gamma-sarcoglycan gene. The fact that the affected individuals in the current and Gypsy families are gamma-sarcoglycan negative may indicate that codons 69 and 283 are important in gamma-sarcoglycan function.
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PMID:Severe gamma-sarcoglycanopathy caused by a novel missense mutation and a large deletion. 1071 84

Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease with a significant morbidity and mortality. The major clinical features include persistent fever, chills/rigor, myalgia, malaise, dry cough, headache, and dyspnoea. Older subjects may present without the typical febrile response. Common laboratory features include lymphopenia, thrombocytopenia, raised alanine transaminases, lactate dehydrogenase, and creatine kinase. The constellation of compatible clinical and laboratory findings, together with certain characteristic radiological features and lack of clinical response to broad spectrum antibiotics, should arouse suspicion of SARS. Measurement of serum RNA by real time reverse transcriptase-polymerase chain reaction technique has a detection rate of 75%-80% in the first week of the illness.
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PMID:Severe acute respiratory syndrome (SARS): epidemiology and clinical features. 1525

Seven neonates required intensive care at our institution with enterovirus myocarditis, 2001-2003. Presentation was at a median age of 9 days. All had ischaemic electrocardiograms, poor ventricular function, raised creatine kinase, and enterovirus RNA detected by reverse transcriptase polymerase chain reaction. Four survived. Enterovirus myocarditis may be an under recognised cause of neonatal collapse.
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PMID:Enterovirus myocarditis as a cause of neonatal collapse. 1532 71

In yeast, mitochondrial dysfunction activates a specific pathway, termed retrograde regulation, which alters the expression of specific nuclear genes and results in increased replicative life span. In mammalian cells, the specific nuclear genes induced in response to loss of mitochondrial function are less well defined. This study characterizes responses in nuclear gene expression to loss of mitochondrial DNA sequences in three different human cell types: T143B, an osteosarcoma-derived cell line; ARPE19, a retinal pigment epithelium cell line; and GMO6225, a fibroblast cell population from an individual with Kearns-Sayre syndrome (KSS). Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to measure gene expression of a selection of glycolysis, TCA cycle, mitochondrial, peroxisomal, extracellular matrix, stress response, and regulatory genes. Gene expression changes that were common to all three cell types included up-regulation of GCK (glucokinase), CS (citrate synthase), HOX1 (heme oxygenase 1), CKMT2 (mitochondrial creatine kinase 2), MYC (v-myc myelocytomatosis viral oncogene homolog), and WRN (Werner syndrome helicase), and down-regulation of FBP1 (fructose-1, 6-bisphosphatase 1) and COL4A1 (collagen, type IV, alpha 1). RNA interference experiments show that induction of MYC is important in rho0 cells for the up-regulation of glycolysis. In addition, a variety of cell type-specific gene changes was detected and most likely depended upon the differentiated functions of the individual cell types. These expression changes may help explain the response of different tissues to the loss of mitochondrial function due to aging or disease.
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PMID:Common and cell type-specific responses of human cells to mitochondrial dysfunction. 1556 Nov 7

Eastern equine encephalitis (EEE) was diagnosed in a flock of African penguins. Diagnosis was based on history and clinical signs and confirmed via serologic testing, virus isolation, reverse transcriptase-polymerase chain reaction (RT-PCR) assay, and histologic examination. Clinical signs in penguins included anorexia, behavior changes, depression, regurgitation, ataxia, recumbency, and seizures, and some penguins did not have any clinical signs. Mean +/- SD number of days that affected penguins had clinical signs was 12 +/- 5 days. Abnormalities initially detected on CBC included heterophilic leukocytosis and anemia; lymphocytosis and monocytosis were detected later. Plasma biochemical abnormalities included high activities of aspartate amino-transferase and creatine kinase, hyponatremia, hypochloremia, hyperglycemia, and high concentrations of globulin, triglycerides, and cholesterol. Mean +/- SD number of days required for resolution of CBC and plasma biochemical abnormalities was 67 +/- 24 days after the onset of clinical signs. Treatment consisted of supportive therapy. All penguins survived with the exception of one that was euthanatized; histopathologic findings were consistent with encephalitis. Results of RT-PCR assays performed on tissue from the right cerebrum of the penguin that was euthanatized were positive for EEE viral RNA. An inability to isolate virus several weeks after illness suggested successful viral clearance in recovered penguins. To the authors' knowledge, EEE infection in any penguin species has not been reported.
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PMID:Eastern equine encephalitis in a flock of African penguins maintained at an aquarium. 1598 91

Voltage-gated chloride channels (ClCs) are important mediators of cellular ion homeostasis and volume regulation. In an earlier study, we used immunohistochemical, Western blot, and reverse transcriptase PCR (RT-PCR) approaches to identify ClC-K variants in types II, IV, and V fibrocytes of the rodent spiral ligament. We have now confirmed the expression of ClC-K2 in these cells by in situ hybridization. All three of these fibrocyte subtypes are thought to be involved in cochlear K(+) recycling; thus, it is important to understand the precise mechanisms regulating their membrane conductance and the role played by ClCs in this process. In this study, we report the characterization of a secondary cell line derived from explants from the region of the rat spiral ligament underlying and inferior to the spiral prominence. The cultured cells were immunopositive for vimentin, Na,K/ATPase, Na,K,Cl-cotransporter, carbonic anhydrase isozyme II, and creatine kinase isozyme BB, but not for cytokeratins or Ca/ATPase, an immunostaining profile indicative of the type IV subtype. Evaluation of the cultures by RT-PCR and Western blot analysis confirmed the presence of both ClC-2 and -K2. Whole-cell patch clamp recordings identified two biophysically distinct Cl(-) currents in the cultured cells. One, an inwardly rectifying Cl(-) current activated by hyperpolarization or decreasing extracellular pH corresponded with the properties of ClC-2. The other, a weak outwardly rectifying Cl(-) current regulated by extracellular pH, Cl(-), and Ca(2+) resembled the channel characteristics of ClC-K2 when expressed in Xenopus oocytes. These findings suggest that at least two functionally different chloride channels are involved in regulating membrane anion conductance in cultured type IV spiral ligament fibrocytes.
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PMID:Identification of ClC-2 and CIC-K2 chloride channels in cultured rat type IV spiral ligament fibrocytes. 1733 50


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