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 pyrethroid-resistant strains of horn flies were found to be 17- and 688-fold more resistant to permethrin and 17- and 11,300-fold more resistant to cyhalothrin than a susceptible control strain. Synergism experiments with piperonyl butoxide showed that both target site insensitivity and metabolic resistance mechanisms were present in the Super Resistant strain. Using the reverse transcriptase-polymerase chain reaction (RT-PCR), a 0.9 kb fragment of the putative sodium channel gene from susceptible and resistant flies was cloned and sequenced. Two sequence variants were detected, presumably arising from alternative splicing of transcripts. The amino acid sequences deduced from the resistant and susceptible fly gene fragments were identical except for three amino acid substitutions, two of which have been associated with resistance in house flies. A leucine to phenylalanine substitution associated with knockdown resistance (kdr) was found in both resistant strains. A methionine to threonine substitution associated with super-kdr was found in the Super Resistant strain. Translation of poly(A)+ RNA followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) detected translation products whose concentrations increased in association with pyrethroid resistance. Random-amplified polymorphic DNA (RAPD)-PCR of genomic DNA with over 260 DNA oligomers yielded one resistance-associated marker, designated HF-77, which was not detected in any susceptible flies but was present in 16% of the resistant individuals.
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PMID:Toxicological and molecular characterization of pyrethroid-resistant horn flies, Haematobia irritans: identification of kdr and super-kdr point mutations. 944 75

A P1 cloned insert of about 85.5 kilobases (kb) was isolated, containing four members of the human growth hormone/chorionic somatomammotropin (GH/CS) gene family and the thyroid hormone receptor interacting protein (TRIP-1) gene. The presence of the CS-like, CS-A, GH-variant and, most downstream, CS-B gene was confirmed by DNA blotting and sequence analysis. The TRIP-1 gene was detected 40 kb downstream of the CS-B gene and in the reverse transcriptional orientation to all the GH/CS genes. The TRIP-1 gene is highly homologous to the SUG-1 gene in yeast and is evolutionarily conserved among several species. Based on the common location of the GH and TRIP-1 (or homologue) genes on the same chromosome in the human, pig and rat genomes, we suggest that these loci are physically linked. Previously, it was reported that a muscle-specific sodium channel (SCN4A) gene is located immediately upstream of the pituitary growth hormone (GH-N) gene, and is linked to the GH gene locus in both humans and rats. This suggests a further linkage between the SCN4A, GH and TRIP-1 loci. Also, deoxyribonuclease hypersensitive sites have been reported in and around these loci and were associated with an important locus control region for the GH/CS genes. Unlike the GH/CS genes, we show, using reverse transcriptase-polymerase chain reaction that the TRIP-1 gene is expressed ubiquitously and, through RNA blotting, as a 1.4-kb transcript. This implies an open and active chromatin structure. The possible effect of this structure on the adjacent human GH/CS gene locus is discussed.
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PMID:Physical linkage of the human growth hormone gene family and the thyroid hormone receptor interacting protein-1 gene on chromosome 17. 966 65

We examined the larvae of several organophosphate and pyrethroid-resistant Mexican strains of Boophilus microplus using biochemical and molecular tests to investigate the mechanisms conferring acaricide resistance. The electrophoretic profiles of esterase activity in protein extracts from coumaphos and permethrin-resistant strains compared to the susceptible strain revealed distinct differences, which inhibitor studies attributed to carboxylesterases. Esterase hydrolysis assays showed significant enhancement of both total and permethrin hydrolysis in one pyrethroid-resistant strain, with no enhancement in two other strains with very high resistance to pyrethroids. Sequence analysis of sodium channel mRNA fragments in all pyrethroid-resistant strains determined that they did not possess the classic kdr and super-kdr mutations known to confer pyrethroid resistance in several insect species. Using reverse transcriptase-polymerase chain reaction (RT-PCR) with degenerate primers designed from conserved regions of insect esterase amino acid sequences, a B. microplus larval cDNA fragment was isolated whose deduced amino acid sequence was significantly similar to esterases from a wide range of species. In Northern blot RNA analysis the cDNA hybridized to a 2.1 kb mRNA that was abundant in all resistant strains except one, in which a very low abundance could provide a marker for the mechanism conferring resistance in this strain.
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PMID:Molecular and biochemical survey of acaricide resistance mechanisms in larvae from Mexican strains of the southern cattle tick, Boophilus microplus. 1074 17

The sodium channel isoform NaCh6 is abundant in the adult rat brain and is expressed in both neurons and glia (Schaller et al. [1995] J. Neurosci. 15:3231-3242; Krzemien et al. [2000] J. Comp. Neurol. 20:70-83). With reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunolabeling, NaCh6 expression was investigated in the developing rat brain and spinal cord [embryonic day 15 (E15) through postnatal day 28 (P28)]. The relative abundance of the four major central nervous system NaCh subtypes was quantitated with RT-PCR. In all regions that were investigated (olfactory bulb, cortex, hippocampus, cerebellum, and spinal cord), each subtype had a unique pattern of expression. NaCh6 mRNA and protein were not detected in either brain or spinal cord at E15 and E18 by in situ hybridization and immunohistochemistry. Neurons in the hippocampus, cortex, and olfactory bulb began to express NaCh6 mRNA and protein shortly after birth. The mRNA signal peaked at P7-P14, and protein expression increased as development proceeded. NaCh6 mRNA was detected at P1 in the cerebellum, and a nonuniform distribution of NaCh6 immunoreactivity in both Purkinje cells and granule cells was observed by P7-P14. NaCh6 protein was expressed in granule cells as soon as they left the proliferative phase and began to migrate. Both NaCh6 mRNA and protein were detected in the spinal cord at P1 and were expressed clearly at P7 in motor neurons. The time course of appearance of NaCh6 in postnatal development is consistent with the development of neurologic symptoms in med and jolting mice, which have mutations in the mouse ortholog of NaCh6.
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PMID:Developmental and regional expression of sodium channel isoform NaCh6 in the rat central nervous system. 1074 21

The expression of the sole alpha-subunit of muscle or brain sodium channels in frog oocytes mediates currents with a bimodal inactivation with an abnormal slow mode that is strongly depressed only by co-expression of the beta1-subunit. In contrast, in the expression of the alpha-subunit in the human embryonic kidney cell line, HEK-293, the slow mode is almost absent, suggesting an endogenous expression of the beta1-subunit. We have tested this hypothesis by reverse transcriptase-polymerase chain reaction. We found an abundant expression of mRNA encoding the beta1A splicing of the putative regulatory sodium channel subunit but no mRNA encoding the beta1-subunit in HEK cells. This finding is consistent with the idea that the endogenous beta1A-subunit is sufficient for suppressing the slowly inactivating mode of sodium currents by co-assembly with alpha-subunits, and calls attention to the reliability of effects attributed in HEK cells to alpha-beta1 co-expression.
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PMID:Endogenous expression of the beta1A sodium channel subunit in HEK-293 cells. 1081 59

Gene expression, protein expression, and function of amiloride-sensitive sodium channels were examined in human lymphocytes from normal individuals and individuals with Liddle's disease. Using reverse transcriptase polymerase chain reactions, expression of all three cloned epithelial sodium channel (ENaC) subunits was detected in lymphocytes. Polyclonal antibodies to bovine alpha-ENaC bound to the plasma membrane of normal and Liddle's lymphocytes. A quantitative analysis of fluorescence-tagged ENaC antibodies indicated a 2.5-fold greater surface binding of the antibodies to Liddle's lymphocytes compared with normal lymphocytes. The relative binding intensity increased significantly (25%; p < 0.001) for both normal and Liddle's cells after treatment with 40 microM 8-CPT-cAMP. Amiloride-sensitive whole cell currents were recorded under basal and cAMP-treated conditions for both cell types. Liddle's cells had a 4.5-fold larger inward sodium conductance compared with normal cells. A specific 25% increase in the inward sodium current was observed in normal cells in response to cAMP treatment. Outside-out patches from both cell types under both treatment conditions revealed no obvious differences in the single channel conductance. The P(open) was 4.2 +/- 3.9% for patches from non-Liddle's cells, and 27.7 +/- 5.4% in patches from Liddle's lymphocytes. Biochemical purification of a protein complex, using the same antibodies used for the immunohistochemistry, yielded a functional sodium channel complex that was inhibited by amiloride when reconstituted into lipid vesicles and incorporated into planar lipid bilayers. These four independent methodologies yielded findings consistent with the hypotheses that human lymphocytes express functional, regulatable ENaC and that the mutation responsible for Liddle's disease induces excessive channel expression.
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PMID:Expression and regulation of normal and polymorphic epithelial sodium channel by human lymphocytes. 1111 30

Retinal bipolar neurons transmit visual information by means of graded synaptic potentials that spread to the synaptic terminal without sodium-dependent action potentials. Although action potentials are not involved, voltage-dependent sodium channels may enhance subthreshold depolarizing potentials in the dendrites and soma of bipolar cells, as they do in other CNS neurons. We report here that voltage-dependent sodium currents are observed in a subset of bipolar neurons from goldfish retina. Single-cell reverse transcriptase-PCR identified four different sodium channel alpha subunits in goldfish bipolar cells, putatively corresponding to the mammalian voltage-gated sodium channels Na(v)1.1, Na(v)1.2, Na(v)1.3, and Na(v)1.6. The amount of sodium current was largest in cells with smaller synaptic terminals, which probably represent cone bipolar cells. Localization of sodium channel immunoreactivity in goldfish retina confirmed the expression of voltage-gated sodium channels in cone bipolar cells of both ON and OFF types. Both immunocytochemical and physiological evidence suggests that the sodium channels are localized to the soma and dendrites where they may play a role in transmission of synaptic signals, particularly in the long, thin dendrites of cone bipolar cells.
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PMID:Voltage-dependent sodium channels are expressed in nonspiking retinal bipolar neurons. 1142 83

Although the canine atrium has proven useful in several experimental models of atrial fibrillation and for studying the effects of rapid atrial pacing on atrial electrical remodeling, it may not fully represent the human condition because of reported differences in functional ionic currents and ion channel subunit expression. In this study, we reassessed the molecular components underlying one current, the ultrarapid delayed rectifier current in canine atrium [IKur(d)], by evaluating the mRNA, protein, immunofluorescence, and currents of the candidate channels. Using reverse transcriptase-polymerase chain reaction, we found that Kv1.5 mRNA was expressed in canine atrium whereas message for Kv3.1 was not detected. Western analysis on cytosolic and membrane fractions of canine tissues, using selective antibodies, showed that Kv3.1 was only detectable in the brain preparations, whereas Kv1.5 was expressed at high levels in both atrial and ventricular membrane fractions. Confocal imaging performed on isolated canine atrial myocytes clearly demonstrated the presence of Kv1.5 immunostaining, whereas that of Kv3.1 was equivocal. Voltage- and current-clamp studies showed that 0.5 mmol/L tetraethylammonium had variable effects on sustained K+ currents, whereas a compound with demonstrated selectivity for hKv1.5 versus Kv3.1, hERG or the sodium channel, fully suppressed canine atrial IKur tail currents and depressed sustained outward K+ current. This agent also increased action potential plateau potentials and action potential duration at 20% and 50% repolarization. These results suggest that in canine atria, as in other species including human, Kv1.5 protein is highly expressed and contributes to IKur.
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PMID:Kv1.5 is an important component of repolarizing K+ current in canine atrial myocytes. 1450 Mar 35

Multiple voltage-gated sodium channels are the primary mediators of cell excitability. They are multimers that consist of the pore-forming alpha subunit and auxiliary beta subunits. Although ion permeability and voltage sensing are primarily determined by the alpha subunit, beta subunits are important modulators of sodium channel function. The purpose of this study was to assess the effect of axotomy on the expression of beta subunits (beta(1), beta(2) and beta(3)) and coexpression of Na(v)1.3 and beta(3) subunits in the dorsal root ganglion (DRG). We used sciatic nerve transection models or spared nerve injury (SNI) models in the rat. In reverse transcriptase-polymerase chain reaction analysis, there were no significant differences between contralateral and ipsilateral DRGs of beta(1) and beta(2) mRNA 3 days after axotomy. beta(3) mRNA expression in ipsilateral DRGs increased significantly compared with contralateral DRGs 3 days after axotomy. In in situ hybridization histochemistry, beta(1) mRNA was predominantly expressed in medium- to large-size neurons, whereas beta(2) mRNA was expressed in small- to large-size neurons. There were no significant differences in beta(1) and beta(2) mRNA between contralateral and ipsilateral DRGs 3 days after axotomy. In contrast, beta(3) mRNA was mainly expressed in small neurons and occasionally in medium- to large-size neurons, and beta(3) mRNA expression in small c-type neurons in ipsilateral DRGs was increased significantly compared with contralateral DRGs. We examined beta(3) mRNA expression with one of alpha subunits, Na(v)1.3-ir, in DRG neurons after axotomy using the double labeling method. We found a high percentage of coexpression in injured DRG neurons: 83.6+/-2.8% of neurons expressing beta(3) mRNA were labeled for Na(v)1.3-ir; 70.1+/-3.1% of Na(v)1.3-ir neurons expressed beta(3) mRNA. We also examined the expression of beta(3) mRNA in DRG neurons in the SNI model, a neuropathic pain model. We used activating transcription factor 3 to identify axotomized neurons, and found that beta(3) mRNA up-regulation occurred mainly in axotomized neurons in the neuropathic pain model. These data strongly suggest that beta(3) expression in injured DRG neurons following axotomy might be an important pathomechanism of post-nerve injury pain in primary sensory neurons.
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PMID:Expression of auxiliary beta subunits of sodium channels in primary afferent neurons and the effect of nerve injury. 1452 2

Glaucoma is a prevalent cause of blindness, resulting in the apoptotic death of retinal ganglion cells and optic nerve degeneration. The disease is often associated with elevated intraocular pressure, however, molecular mechanisms involved in ganglion cell death are poorly understood. To identify proteins contributing to this pathological process, we analysed the retinal gene expression of DBA/2J mice that develop an elevated intraocular pressure by the age of 6 months with subsequent ganglion cell loss. In this study, we identified subunits of the epithelial sodium channel (ENaC) family that are specifically expressed under elevated intraocular pressure. Using reverse transcriptase polymerase chain reaction we observed a significant increase of alpha-ENaC in the neuronal retina of DBA/2J mice when compared with control animals, while beta-ENaC and gamma-ENaC were not detectable in this tissue. Specific immune sera to ENaC subunits showed up-regulation of alpha-ENaC in synaptic and nuclear layers of the retina, and in the retinal pigment epithelium. Consistent with our polymerase chain reaction data, beta-ENaC was not detected by specific antibodies in the retina, while gamma-ENaC was only present in the retinal pigment epithelium under ocular hypertension. Finally, the increase of alpha-ENaC gene expression in the neuronal retina and the retinal pigment epithelium was not observed in other tissues of DBA/2J mice. Since the intraocular pressure is regulated by the transport of aqueous humour across epithelial structures of the eye that in turn is associated with ion flux, the specific up-regulation of ENaC proteins could serve as a protecting mechanism against elevated intraocular pressure.
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PMID:Subunits of the epithelial sodium channel family are differentially expressed in the retina of mice with ocular hypertension. 1595 55


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