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)

Isolation and cloning of abundant reverse transcriptase-polymerase chain reaction (RT-PCR) products from the filarial nematode Brugia malayi using the conserved nematode spliced leader sequence and poly A as amplification targets has allowed us to identify abundant, stage specific transcripts from infective and post-infective larvae. The predicted protein products of the most prominent full-length transcripts from mosquito-derived L3 parasites are: (i) Bm-ALT-1, a homologue of a Dirofilaria immitis abundant larval protein: (ii) Bm-CPI-1, a cystatin-type cysteine protease inhibitor; (iii) Bm-ALT-3, a novel predicted 6 kDa glycine/tyrosine-rich protein; and (iv) Bm-TPH-1, a homologue of a mammalian translationally-controlled tumour protein. Some transcripts were not full-length but had mis-primed at A-rich stretches of coding sequence: the most abundant of these was Bm-col-3, a which encodes a collagen homologous to Bp-COL-1 of Brugia pahangi. Similar analysis of abundant spliced leader (SL)/oligo-dT products from fourth-stage larvae 9 days post-infection yielded two dominant transcripts: (i) Bm-cdd-1, which encodes a protein with homology to cytidine deaminase, differing at only one amino acid position from its homologue described in Brugia pahangi; and (ii) the same truncated form of Bm-col-3 found in L3 preparations. Expression of the major transcripts was assessed by PCR amplification of cDNA libraries derived from each stage of the life cycle. alt1, alt-3 and cpi-1 were all found to be specific to the L3 stage, while cdd-1 was found only in the L4 cDNA library. Expression of these larval-specific transcripts was not detected in either microfilarial or adult libraries.
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PMID:Differentially expressed, abundant trans-spliced cDNAs from larval Brugia malayi. 923 76

Proteolytic activity present in the excreted/secreted (ES) material of newly excysted juvenile (NEJ) Fasciola hepatica was biochemically analyzed. By gelatin substrate SDS-PAGE, only one region of activity was observed in the NEJ ES material at a molecular mass of 29 kDa. Both the secreted cathepsin L from adult fluke and the 29-kDa proteolytic activity of NEJ ES show a common pH optimum of 7.5, a cysteine protease inhibition profile, and preference for the N-benzyloxycarbonyl (Z)-Phe-Arg-NHMec fluorogenic substrate over Z-Arg-Arg-NHMec and Z-Arg-NHMec. In vitro analysis revealed that the NEJ protease activity digested sheep immunoglobulin heavy chain and bovine serum albumin but not bovine hemoglobin. Amino-terminal protein sequence analysis of the 29-kDa NEJ protease band revealed two sequences with homology to the cathepsin B family of proteases. Using degenerate oligonucleotides designed from the N-terminal sequence, reverse transcriptase polymerase chain reaction with NEJ RNA amplified a cDNA sequence encoding the first 236 amino acids of mature cathepsin B. Using this cDNA fragment an overlapping cDNA was isolated from a LambadaZAP cDNA library constructed with poly(A)+ RNA from immature 5-week-old liver fluke. Together with the N-terminal sequence, these cDNAs predict a mature cathepsin B sequence of 254 amino acids which shows 48-51% sequence identity to mammalian and Schistosoma mansoni cathepsin B. We conclude that, in contrast to the major proteases released by adult fluke, the major secreted protease of NEJ of F. hepatica is of the cathepsin B class.
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PMID:Fasciola hepatica: characterization and cloning of the major cathepsin B protease secreted by newly excysted juvenile liver fluke. 953 62

The cystatin superfamily of cysteine protease inhibitors consists of three major families, including the stefins, cystatins and kininogens. However, the recent identification of several genes that possess sequence similarity with the cystatins but have different gene or protein structures indicates that several new cystatin families or subgroups of families might exist. We previously identified the cystatin-related epididymal spermatogenic (Cres) gene, which is related to the family 2 cystatins but exhibits highly tissue-specific expression in the reproductive tract. In the studies presented here, an analysis of gene structure as well as chromosomal mapping studies suggest that the Cres gene might represent a new subgroup within the family 2 cystatins. Although the Cres gene possesses an additional exon encoding 5' untranslated sequences, its coding exons are similar in size to the three coding exons of the cystatin family 2 genes, and the Cres exon/intron splice junctions occur in identical locations as in the cystatin C gene. Furthermore, chromosomal mapping studies show that the Cres gene co-segregates with the cystatin C gene on mouse chromosome 2. Similar to the cystatin family 2 proteins, the Cres protein possesses the type A and B disulphide loops that are necessary for cystatin folding. Interestingly, Cres protein also possesses half of a type C disulphide loop. Although probably related to the cystatin genes, the Cres gene is distinct in that its promoter contains consensus motifs typical of regulated genes. Finally, reverse transcriptase-mediated PCR studies and the identification of new Cres cDNA clones indicate that the Cres mRNA is alternatively spliced, resulting in two Cres mRNAs that might be involved in the regulation of Cres function.
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PMID:Structure, alternative splicing and chromosomal localization of the cystatin-related epididymal spermatogenic gene. 1022 62

Calpain, a Ca2+-activated cysteine protease, has been implicated in apoptosis of immune cells. Since central nervous system (CNS) is abundant in calpain, the possible involvement of calpain in apoptosis of CNS cells needs to be investigated. We studied calpain expression in rat C6 glioma cells exposed to reactive hydroxyl radical (.OH) [formed via the Fenton reaction (Fe2++H2O2+H+-->Fe3++H2O+.OH)], interferon-gamma (IFN-gamma), and calcium ionophore (A23187). Cell death, cell cycle, calpain expression, and calpain activity were examined. Diverse stimuli induced apoptosis in C6 cells morphologically (chromatin condensation as detected by light microscopy) and biochemically [DNA fragmentation as detected by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay]. Oxidative stress arrested a population of C6 cells at the G2/M phase of cell cycle. The levels of mRNA expression of six genes were analyzed by the reverse transcriptase-polymerase chain reaction (RT-PCR). Diverse stimuli did not alter beta-actin (internal control) expression, but increased calpain expression, and the upregulated bax (pro-apoptotic)/bcl-2 (anti-apoptotic) ratio. There was no significant increase in expression of calpastatin (endogenous calpain inhibitor). Western blot analysis showed an increase in calpain content and degradation of myelin-associated glycoprotein (MAG), a calpain substrate. Pretreatment of C6 cells with calpeptin (a cell-permeable calpain inhibitor) blocked calpain overexpression, MAG degradation, and DNA fragmentation. We conclude that calpain overexpression due to.OH stress, IFN-gamma stimulation, or Ca2+ influx is involved in C6 cell death, which is attenuated by a calpain-specific inhibitor.
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PMID:Diverse stimuli induce calpain overexpression and apoptosis in C6 glioma cells. 1035 May 26

Cathepsin K is a cysteine protease involved in degradation of human type I collagen and plays a primary role in bone resorption. We have cloned rhesus monkey cathepsin K by reverse transcriptase-polymerase chain reaction (RT-PCR) from rhesus ovary poly A+ RNA. The sequence for the rhesus enzyme is 98% identical to that of the human with 100% identity within the mature active form of cathepsin K. Rhesus monkey cathepsin K was transiently expressed in Chinese hamster ovary (CHO) cells and found to be secreted as the proenzyme in the culture media and 50% activated to the mature form intracellularly. The substrate specificity preference of aminomethylcoumarin and rhodamine peptide substrates was Leu > Phe > Pro in the P2 position when tested with constant arginine at P1. The enzyme activity expressed in CHO cell extracts was sensitive to inhibition by E-64 and cystatin with IC50s of 3.5 nmol/L and 13 ng/mL, respectively. The apparent second order rate constants of inactivation by E-64 were 66,000 M(-1) s(-1) and 130,000 M(-1) s(-1) for the recombinantly expressed rhesus monkey and human cathepsin K, respectively. The high similarity between the sequences and the kinetic properties of rhesus monkey and human cathepsin K establishes this monkey species as a suitable animal model for development of novel cathepsin K inhibitors as antiresorptive agents.
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PMID:Cloning and expression of rhesus monkey cathepsin K. 1045 86

Calpain, a Ca(2+)-dependent cysteine protease, has been implicated in cytoskeletal protein degradation and neurodegeneration in the lesion and adjacent areas following spinal cord injury (SCI). To attenuate apoptosis or programmed cell death (PCD) in SCI, we treated injured rats with E-64-d, a cell permeable and selective inhibitor of calpain. SCI was induced on T12 by the weight-drop (40 g-cm force) method. Within 15 min, E-64-d (1 mg/kg) in 1.5% DMSO was administered i.v. to the SCI rats. Following 24 h treatment, a 5-cm long spinal cord section with the lesion in the center was collected. The spinal cord section was divided equally into five 1-cm segments (S1: distant rostral, S2: near rostral, S3: lesion or injury, S4: near caudal and S5: distant caudal) for analysis. Determination of mRNA levels by reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that ratios of bax/bcl-2 and calpain/calpastatin were increased in spinal cord segments from injured rats compared to controls. Degradation of the 68-kD neurofilament protein and internucleosomal DNA fragmentation were also increased. All of these changes were maximally increased in the lesion and gradually decreased in the adjacent areas of SCI rats, while largely undetectable in E-64-d treated rats and absent in sham controls. The results indicate that apoptosis in rat SCI appears to be associated with calpain activity which can be attenuated by the calpain inhibitor E-64-d.
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PMID:E-64-d prevents both calpain upregulation and apoptosis in the lesion and penumbra following spinal cord injury in rats. 1083

Caspase-3 is a member of the cysteine protease family, which plays a crucial role in apoptotic pathways by cleaving a variety of key cellular proteins. Caspase-3 can be activated by diverse death-inducing signals, including the chemotherapeutic agents. The purpose of this study was to determine the levels of caspase-3 expression in breast tumor samples and to determine whether alterations in its expression can affect their ability to undergo apoptosis. Primary breast tumor and normal breast parenchyma samples were obtained from patients undergoing breast surgery and the expression of caspases-3 was studied. Similarly, normal mammary epithelial cells and several established mammary cancer cell lines were studied for caspases-3 expression by reverse transcriptase-polymerase chain reaction, Northern blot analysis, and Western blot analysis. Approximately 75% of the tumor as well as morphologically normal peritumoral tissue samples lacked the caspase-3 transcript and caspase-3 protein expression. In addition, the caspases-3 mRNA levels in commercially available total RNA samples from breast, ovarian, and cervical tumors were either undetectable (breast and cervical) or substantially decreased (ovarian). Despite the complete loss of caspase-3, the expression levels of other caspases, such as caspase-8 and caspase-9, were normal in all of the tumor samples studied. The sensitivity of caspase-3-deficient breast cancer (MCF-7) cells to undergo apoptosis in response to doxorubicin and other apoptotic stimuli could be augmented by reconstituting caspase-3 expression. These results suggest that the loss of caspases-3 expression may represent an important cell survival mechanism in breast cancer patients.
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PMID:Down-regulation of caspase 3 in breast cancer: a possible mechanism for chemoresistance. 1248 36

Retrotransposons commonly encode a reverse transcriptase (RT), but other functional domains are variable. The acquisition of new domains is the dominant evolutionary force that brings structural variety to retrotransposons. Non-long-terminal-repeat (non-LTR) retrotransposons are classified into two groups by their structure. Early branched non-LTR retrotransposons encode a restriction-like endonuclease (RLE), and recently branched non-LTR retrotransposons encode an apurinic/apyrimidinic endonuclease-like endonuclease (APE). In this study, we report a novel non-LTR retrotransposon family Dualen, identified from the Chlamydomonas reinhardtii genome. Dualen encodes two endonucleases, RLE and APE, with RT, ribonuclease H, and cysteine protease. Phylogenetic analyses of the RT domains revealed that Dualen is positioned at the midpoint between the early-branched and the recently branched groups. In the APE tree, Dualen was branched earlier than the I group and the Jockey group. The ribonuclease H domains among the Dualen family and other non-LTR retrotransposons are monophyletic. Phylogenies of three domains revealed the monophyly of the Dualen family members. The domain structure and the phylogeny of each domain imply that Dualen is a retrotransposon conserving the domain structure just after the acquisition of APE. From these observations, we discuss the evolution of domain structure of non-LTR retrotransposons.
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PMID:An extraordinary retrotransposon family encoding dual endonucleases. 1607 10

Aphids exclusively feed on plant phloem sap that contains much sugar and some nonessential amino acids but is poor in lipids and proteins. Conventionally, it has been believed that aphids substantially have no intestinal digestion of proteins. However, we here report an unexpected finding that cysteine protease genes of the family cathepsin B are massively amplified in the lineage of aphids and that many of the protease genes exhibit gut-specific overexpression. By making use of expressed sequence tag data, sequenced cDNAs, and genomic trace sequences of the pea aphid Acyrthosiphon pisum, we identified a total of 28 cathepsin B-like gene copies in the genome of A. pisum. Phylogenetic analyses of all the cathepsin B genes in aphids revealed that genic expansion has continuously proceeded with basal, intermediary, and recent duplications. Estimation of molecular evolutionary rates indicated that major alterations of the rates often occurred after duplications. For example, a gene copy ("348") was shown to be slow evolving and close to genes of other insects like Drosophila melanogaster, whereas the other gene copies appeared to have evolved faster with higher ratios of nonsynonymous to synonymous substitutions. We identified a number of gene copies (16 in A. pisum) that contained a replacement at the site required for catalytic activity of the protease. Among these, 2 copies were pseudogenes, whereas the remaining copies were structurally intact and possibly acquired new functions. For example, a cluster of such gene copies ("1674") has been subjected to positive selection. Quantitative reverse transcriptase-polymerase chain reaction analyses revealed that the more conserved gene copy ("348") showed a constitutive expression, whereas 5 other forms ("84," "16," "16D," "1874," and "2744") were preferentially expressed in the gut of A. pisum. Putative biological roles of the diversified cathepsin B-like gene copies in aphids are discussed in relation to their nutritional physiology specialized for plant sap feeding lifestyle.
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PMID:Large gene family expansion and variable selective pressures for cathepsin B in aphids. 1793 9

Identifying parasitism genes encoding proteins secreted from nematodes is the key to understanding the molecular basis of nematode parasitism to insects. In this paper, a cDNA with two introns and three exons encoding a cysteine protease inhibitor was identified by screening a cDNA subtractive library constructed from the nematode, Steinernema carpocapsae, induced by Galleria mellonella hemolymph. The full-length cDNA contains an open reading frame encoding a 139-amino acid protein, designated Sc-cys, with a 19-residue signal peptide. The mature protein was predicted to have a molecular weight of 12,531.59 Da, a pI of 9.44, one disulfide bond, and three conserved domains believed to be important for the inhibition of cysteine proteases. In Basic Local Alignment and Search Tool analyses, the putative protein precursor displayed 26-42% identities to a multitude of cystatins or cystatin-like proteins. Phylogenetic analysis suggested the novel cystatin is likely a new member of the family 2 cystatins. Reverse northern blot, semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR), and real-time RT-PCR analyses showed that the expression level of Sc-cys was upregulated substantially after induction by insect hemolymph. The specific analysis of genes encoding secretory proteins is providing a profile of putative parasitism genes expressed in S. carpocapsae throughout the parasitic cycle.
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PMID:Identification, characterization of functional candidate genes for host-parasite interactions in entomopathogenetic nematode Steinernema carpocapsae by suppressive subtractive hybridization. 1854


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