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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mycoplasma hominis, an opportunistic pathogenic bacterium of humans, has a small genome of 700 kb. Despite this, multiple copies of gene sequences with similarities to the structural gene (lmp1) of a 135-kDa surface-located membrane protein (Lmp1) have been identified on the genome of M. hominis PG21 (lmp2, lmp3, and lmp4). The distance between the lmp1-lmp2 region and the lmp3-lmp4 region was more than 110 kb. lmp3-lmp4 of M. hominis PG21 was sequenced and found to contain two putative genes. The gene region of 6.5 kb contained a 5' unique region and a 3' unique region separated by 9 0.5-kb repeats with 51 to 90% similarity to 10 similar repeats found in the lmp1-lmp2 region. The 0.5-kb DNA repeats thus comprised about 1% of the entire genome. In both regions, a base change in one of the repeats gave rise to a stop codon, and thereby lmp2 and lmp4 occurred. By PCR amplification of reverse-transcriptase-generated cDNA it was shown that all four genes were transcribed. By use of Lmp-specific antibodies we showed that both lmp1 and lmp3 were translated into proteins (Lmp1 and Lmp3). Each of the four lmp genes represented by their unique cloned segments was used as a probe to analyze the presence, distribution, and organization of the genes within the genome in 13 M. hominis isolates. The repetitive element was detected at one or two locations on the chromosome for all isolates. The lmp3-specific element was present in all isolates, and lmp1- and lmp2-specific elements were present in all but one isolate. The lmp4-specific element was present in about half the isolates tested. For five M. hominis isolates the chromosomal location of the lmp genes was mapped.
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PMID:Analysis of 0.5-kilobase-pair repeats in the Mycoplasma hominis lmp gene system and identification of gene products. 863 64

A cDNA fragment (HSD-1) coding for part of a human sperm membrane protein (hSMP-1) was previously isolated from a human testis cDNA expression library, with the serum from an infertile patient used as a probe. By rescreening human testis cDNA libraries with the HSD-1 insert and using rapid amplification of cDNA ends, the complete cDNA of 2482 bp was identified and sequenced. An open reading frame of 1572 bp encodes 523 amino acid residues with a computed molecular mass of 55.08 kDa. This protein sequence does not match any other sequence in the databases, indicating that it represents a novel sperm antigen. Northern blot analysis of human and rat testis poly(A) mRNA detected a band of approximately 2.5 kb in both species. Reverse transcriptase polymerase chain reaction analysis showed that hSMP-1 mRNA was present in human testis but was not in either kidney or liver. When the cDNA was expressed in Escherichia coli under the control of the T7 promoter, the expressed protein accumulated to a level of about 50% of the total cellular protein. The expressed protein, which contained an N-terminal poly(his) sequence tag, was purified by chromatography on an nitrilo-tri-acetic acid affinity resin. Approximately 10 mg of pure protein was obtained from a 500-ml culture, purified, and used as antigen to generate a polyclonal antiserum in rabbits. Western blot analysis of human sperm extracts showed a single specific band at 55.5 kDa. Immunofluorescence data showed that hSMP-1 was localized to the head of human sperm. The fluorescent staining formed a cap-shaped pattern that was similar in morphology to the human sperm acrosome. The availability of large amounts of recombinant hSMP-1 and its antiserum will facilitate studies on the function and expression of the protein during spermatogenesis and the assessment of its potential value as a contraceptive immunogen.
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PMID:Expression and characterization of a novel human sperm membrane protein. 878 82

1. [125I]-[LTT]SRIF-28 and [125I]-SMS 201-995 were used to identify and characterize somatostatin (SRIF) receptors localized in rat lung tissue. In vitro autoradiography of rat lung tissue sections showed the existence of specific, high affinity binding sites for [125I]-[LTT]SRIF-28 without any significant specific binding of the sst2/sst5-receptor selective ligand [125I]-SMS 201-995. 2. In radioligand binding studies, specific binding of [125I]-[LTT]SRIF-28 to membranes of rat lung was linearly related to the concentration of membrane protein used with only a small portion of nonspecific binding. With [125I]-SMS 201-995 no specific binding could be observed up to a membrane concentration of 0.1 mg of protein/assay tube. 3. [125I]-[LTT]SRIF-28 bound rapidly to rat lung membranes with an apparent association rate constant (kapp) of 1.8 +/- 0.1 h-1 (n = 3). The equilibrium of specific binding was reached after an incubation period of approximately 90 min at room temperature and remained constant for the next 3 h. The association rate constant (k1) was calculated to be 3.7 x 10(10) M-1 h-1. The dissociation reaction followed first order kinetics with a dissociation rate constant (k-1) = 0.44 +/- 0.07 h-1 corresponding to a half-time of 95 +/- 15 min (n = 3). From these kinetic experiments an equilibrium dissociation constant (KD) for the binding of [125I]-[LTT]SRIF-28 was calculated to be 11.9 pM. 4. Saturation binding of [125I]-[LTT]SRIF-28 revealed an equilibrium dissociation constant (KD) of 50.1 pM (pKD = 10.3 +/- 0.1; n = 3) and a receptor density (Bmax) of 78 +/- 3 fmol mg-1 protein. A Hill coefficient not significantly different from 1 indicated saturable binding to a single class of high affinity binding sites. 5. Specific binding of [125I]-[LTT]SRIF-28 to rat lung membranes was inhibited by SRIF-14, SRIF-28 and different SRIF analogues. SRIF and different synthetic short chain SRIF analogues exhibited the following rank order of potency: SRIF-28 > SRIF-14 > CGP 23996 >> RC 160 > BIM 23014 > SMS 201- 995 > BIM 23056 > MK 678. 6. The binding affinities for SRIF and the various SRIF analogues determined using rat lung tissue were in close correlation to those obtained with Chinese hamster ovary (CHO) cells stably expressing sst, (r = 0.92) and sst4 (r = 0.95) receptors, respectively. 7. Reverse transcriptase--polymerase chain reaction (RT-PCR) showed the predominant expression of mRNA specific for sst4 receptors as well as some weak sst1 mRNA expression. 8. The findings suggest that sst4 receptor expression is the predominant form of the somatostatin receptors identified in rat lung tissue. In this study we demonstrated for the first time the existence of sst4 receptors in mammalian tissue.
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PMID:Identification and pharmacological characterization of somatostatin receptors in rat lung. 922 54

The hepatitis C virus is the major causative agent of nonA-nonB hepatitis worldwide. Although this virus cannot be cultivated in cell culture, several of its features have been elucidated in the past few years. The viral genome is a single-stranded, 9.5kb long RNA molecule of positive polarity. The viral genome is translated into a single polyprotein of about 3000 amino acids. The virally encoded polyprotein undergoes proteolytic processing by a combination of cellular and viral proteolytic enzymes in order to yield all the mature viral gene products. The gene order of HCV has been determined to be C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. The mature structural proteins, C, E1 and E2 have been shown to arise from the viral polyprotein via proteolytic processing by host signal peptidases. Conversely, generation of the mature nonstructural proteins relies on the activity of viral proteases. Thus, cleavage at the NS2/NS3 junction is accomplished by a metal-dependent autoprotease encoded within NS2 and the N-terminus of NS3. The remaining cleavages downstream from this site are effected by a serine protease contained within the N-terminal region of NS3. Besides the protease domain, NS3 also contains an RNA helicase domain at its C-terminus. NS3 forms a heterodimeric complex with NS4A. The latter is a membrane protein that has been shown to act as a cofactor of the protease. Whereas the NS5B protein has been shown to be the viral RNA-dependent RNA polymerase, no function has yet been attributed to NS4B and NS5A. The latter is a cytoplasmic phosphoprotein and appears to be involved in mediating the resistance of the hepatitis C virus to the action of interferon.
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PMID:The nonstructural proteins of the hepatitis C virus: structure and functions. 922 25

Although bile salts are toxic to the liver at high plasma concentrations, the effects of physiological concentrations of bile salts on normal hepatic function are poorly understood. We examined the effect of taurocholate (TC) on the basolateral uptake of [3H]TC in WIF-B cells, a hybrid cell line stably exhibiting in vitro the structural and functional polarity of hepatocytes. Cells were grown in the absence or presence of TC (50 micromol/L) over 12 days, and then incubated with [3H]TC concentrations ranging from 1 to 250 micromol/L. For both control and TC-grown cells, uptake of [3H]TC was linear over 2 minutes. In control cells, the Km for [3H]TC Na+-dependent uptake over 1 minute was 6 +/- 5 micromol/L, and the Vmax was 45 +/- 6 pmol TC/mg protein/min (+/- SEM). TC-grown cells exhibited no significant change in Km but showed a doubling of Vmax to 87 +/- 6 pmol TC/mg protein/min (P < .005). In both control and TC-grown cells, maximal uptake of [3H]TC occurred following 10 to 12 days in culture, with TC-grown cells consistently showing greater rates of [3H]TC uptake from 4 to 14 days in culture. Western blots immunostained for the basolateral Na+-dependent plasma membrane protein, ntcp, revealed the appropriate approximately 50-kd band in control and TC-grown cells, and confocal immunofluorescence microscopy demonstrated staining along the basolateral plasma membrane. Northern blots hybridized with a cDNA probe directed against ntcp indicated a modest TC-induced increase in mRNA levels. Reverse-transcriptase polymerase chain reaction (RT-PCR) using RNA isolated from WIF-B cells and oligonucleotide primers specific for rat ntcp or human NTCP transcripts revealed only the presence of the rat ntcp transcript. We conclude that bile salts, at concentrations normally found in mammalian portal blood, may be capable of promoting enhanced hepatocellular bile salt uptake via an increase in basolateral Na+-dependent plasma membrane transport capacity.
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PMID:Enhanced Na+-dependent bile salt uptake by WIF-B cells, a rat hepatoma hybrid cell line, following growth in the presence of a physiological bile salt. 942 37

Insulin-like growth factor-I (IGF-I) is implicated in the development, survival and maintenance of function of sympathetic and sensory neurons. These neurons are affected at an early stage during the course of diabetes. Reverse transcriptase polymerase chain reaction (RT-PCR) based assay revealed that rat superior cervical ganglia (SCG) express mRNA transcripts for IGF-I and its receptor. Moreover, specific membrane protein binding sites for IGF-I within the SCG have also been demonstrated using competition-inhibition and affinity cross-linking techniques. An induction of diabetes with streptozotocin (STZ, 55 mg/kg, i.v.) produced a marked decrease in the SCG levels of mRNA transcripts for IGF-I and its receptor. Concentrations of circulating IGF-I and its receptor protein within the SCG were also reduced in this disease state. Insulin treatment partially prevented diabetes-related alterations in circulating IGF-I and the SCG-IGF-I system. Overall, the data described in this study may be of value in understanding the pathogenetic mechanism(s) responsible for the development of diabetic sympathetic neuropathy.
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PMID:Diabetes-induced suppression of IGF-1 and its receptor mRNA levels in rat superior cervical ganglia. 948 70

Endoglin/CD105 is a membrane protein involved in the TGF-beta receptor signalling pathway. Endoglin expression has been reported to be selective for a few cell types, in particular endothelial cells, although a number of conflicting reports have been published. In this study, we performed a detailed analysis of endoglin expression in human lung tumors and different tumor and endothelial cell lines, employing reverse-transcriptase-polymerase-chain reaction as well as immunoblotting and immunohistochemistry using verified antibodies to endoglin. Our data show a clearly preferential expression of both endoglin mRNA and protein in endothelial cells. In tumors, endoglin expression was strongly elevated in the angiogenic endothelium at the tumor edges. In agreement with this observation, we find a clear correlation between endoglin expression and markers of proliferation, such as cyclin A and Ki-67, suggesting that endoglin expression is linked to cell-cycle regulation. These findings not only resolve some of the discrepancies in the literature, but also provide the basis for further applications making use of its selective localization and expression in the tumor vasculature.
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PMID:Elevated expression of endoglin, a component of the TGF-beta-receptor complex, correlates with proliferation of tumor endothelial cells. 1022 46

Hepatitis C virus (HCV) NS5A is a phosphoprotein that possesses a cryptic trans-activation activity. To investigate its potential role in viral replication, we searched for the cellular proteins interacting with NS5A protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a newly discovered soluble N-ethylmaleimide-sensitive factor attachment protein receptor-like protein termed human vesicle-associated membrane protein-associated protein of 33 kDa (hVAP-33). In vitro binding assay and in vivo coimmunoprecipitation studies confirmed the interaction between hVAP-33 and NS5A. Interestingly, hVAP-33 was also shown to interact with NS5B, the viral RNA-dependent RNA polymerase. NS5A and NS5B bind to different domains of hVAP-33: NS5A binds to the C-terminus, whereas NS5B binds to the N-terminus of hVAP-33. Immunofluorescent staining showed a significant colocalization of hVAP-33 with both NS5A and NS5B proteins. hVAP-33 contains a coiled-coil domain followed by a membrane-spanning domain at its C-terminus. Cell fractionation analysis revealed that hVAP-33 is predominantly associated with the ER, the Golgi complex, and the prelysosomal membrane, consistent with its potential role in intracellular membrane trafficking. These interactions provide a mechanism for membrane association of the HCV RNA replication complex and further suggest that NS5A is a part of the viral RNA replication complex.
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PMID:Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein. 1054 80

Infection with the hepatitis C virus (HCV) is the major cause of nonA-nonB hepatitis worldwide. Although this virus cannot be cultivated in vitro, several of its key features have been elucidated in the past few years. The viral genome is a positive-sense, single-stranded, 9.6 kb long RNA molecule. The viral genome is translated into a single polyprotein of about 3000 amino acids. The viral polyprotein is proteolytically processed by the combination of cellular and viral proteinases in order to yield all the mature viral gene products. The genomic order of HCV has been shown to be C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. C, E1 and E2 are the virion.structural proteins. The function of p7 is currently unknown. These proteins have been shown to arise from the viral polyprotein via proteolytic processing by the host signal peptidases. Generation of the mature nonstructural proteins, NS2 to NS5B, relies on the activity of viral proteinases. Cleavage at the NS2/NS3 junction is accomplished by a metal-dependent autocatalytic proteinase encoded within NS2 and the N-terminus of NS3. The remaining cleavages downstream from this site are effected by a serine proteinase also contained within the N-terminal region of NS3. NS3 also contains an RNA helicase domain at its C-terminus. NS3 forms a heterodimeric complex with NS4A. The latter is a membrane protein that has been shown to act as a cofactor of the proteinase. While no function has yet been attributed to NS4B, it has recently been suggested that NS5A is involved in mediating the resistance of the hepatitis C virus to the action of interferon. Finally, the NS5B protein has been shown to be the viral RNA-dependent RNA polymerase.
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PMID:Molecular virology of the hepatitis C virus. 1062 60

LALP70 is a novel lysosomal membrane protein belonging to the apyrase protein family. The apyrase protein family comprises enzymes capable of cleaving nucleotide tri- and diphosphates in a calcium- or magnesium-dependent manner, not being altered by P-type, F-type, or V-type NTPase inhibitors. In this study we have cloned and sequenced the human LALP70 gene to determine the genomic structure. The gene is organized in 11 introns and 12 exons covering a genomic region of approximately 16 kilobase pairs. By fluorescence in situ hybridization analysis, the hLALP70 gene was mapped to the human chromosome 8p21.1-p21.3. We further show that there is at least one alternatively spliced variant, hLALP70v, which can be generated via an alternative splice side at the 3'-end of exon 7, leading to a protein variant differing in 8 amino acids (VSFASSQQ). This is the first splice variant that has been described in the apyrase protein family. Reverse transcriptase polymerase chain reaction analysis showed an ubiquitous expression of both variants, with different relative mRNA expression levels in different tissues. Comparison of the enzymatic properties of the splice variants revealed a broader substrate specificity for hLALP70v with CTP, UDP, CDP, GTP, and GDP as preferred substrates, while hLALP70 utilized UTP and TTP preferentially. Furthermore, enzyme activity of hLALP70v was equally dependent on Ca(2+) and Mg(2+), being saturated already at 1 mm concentration. In contrast, hLALP70 enzymatic activity were unsaturated up to 10 mm Ca(2+), while Mg(2+) showed a saturation at already 1 mm concentration with 2-3-fold lower enzymatic activity as observed with Ca(2+). Our data suggest that the presence or absence of the 8-amino acid motif VSFASSQQ provoke differences in substrate specificity and divalent cation dependence of hLALP70/hLALP70v.
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PMID:First apyrase splice variants have different enzymatic properties. 1085 52


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