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)

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

Formation of metastasis is a multistep process involving attachment to the basement membrane, local proteolysis and migration into surrounding tissues, lymph or bloodstream. In the present study, we have analysed the correlation between in vitro invasion and presence of the epidermal growth factor receptor (EGFR) in a panel of 21 small-cell lung cancer (SCLC) cell lines. We have previously reported that ten of these cell lines expressed EGFR protein detected by radioreceptor and affinity labelling assays. In 11 small-cell lung cancer (SCLC) cell lines, EGFR mRNA was detected by Northern blot analysis. In vitro invasion in a Boyden chamber assay was found in all EGFR-positive cell lines, whereas no invasion was detected in the EGFR-negative cell lines. Quantification of the in vitro invasion in 12 selected SCLC cell lines demonstrated that, in the EGFR-positive cell lines, between 5% and 16% of the cells added to the upper chamber were able to traverse the Matrigel membrane. Expression of several matrix metalloproteases (MMP), of tissue inhibitor of MMP (TIMP) and of cathepsin B was evaluated by immunoprecipitation, Western blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). However, in vitro invasive SCLC cell lines could not be distinguished from non-invasive cell lines based on the expression pattern of these molecules. In six SCLC cell lines, in vitro invasion was also determined in the presence of the EGFR-neutralizing monoclonal antibody mAb528. The addition of this antibody resulted in a significant reduction of the in vitro invasion in three selected EGFR-positive cell lines. Our results show that only EGFR-positive SCLC cell lines had the in vitro invasive phenotype, and it is therefore suggested that the EGFR might play an important role for the invasion potential of SCLC cell lines.
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PMID:In vitro invasion of small-cell lung cancer cell lines correlates with expression of epidermal growth factor receptor. 974 4

To examine whether proteases are possibly involved in cellular migration and/or spermiation when developing germ cells translocate across the seminiferous epithelium during spermatogenesis, in situ hybridization was used to localize messenger RNA (mRNA) transcripts of cathepsin L, D, and S in the epithelium at different stages of the spermatogenic cycle in the rat. Cathepsin L mRNA was found to localize almost exclusively near the basal lamina of the epithelium. At stages VI and VII of the cycle before spermiation, the signal of cathepsin L mRNA was so intense that it formed a complete dark precipitate near the basal lamina encircling the entire tubule. At stage VIII, the expression of cathepsin L was completely abolished, and no staining of cathepsin L mRNA was seen in the epithelium. The mRNA of cathepsin D and S was found near the basal lamina, a finding consistent with their localization in Sertoli cells and possibly primary spermatocytes in almost all stages, but peaked at stages VII-IX and VII-VIII of the cycle, respectively, at the time before and during spermiation. These results illustrate the possible involvement of these proteases in facilitating germ cell movement and spermiation. To examine whether germ cells express any of these cathepsin genes, spermatocytes with a purity of greater than 95% were isolated from 15-day-old rat testes by Percoll gradient centrifugation for reverse transcriptase-polymerase chain reaction. It was found that primary spermatocytes expressed multiple cathepsin genes, including cathepsin B, C, D, H, L, and S. Furthermore, the expression of cathepsin L by germ cells isolated from 15-day-old rats (largely spermatocytes and spermatogonia) can be stimulated by Sertoli cell-enriched culture medium in a dose-dependent manner, but not by germ cell-conditioned medium. These results reveal that germ cell function can be regulated by Sertoli cells. Moreover, these results suggest that germ cells may play an active role in the overall testicular protease expression. Also, we present evidence suggesting there is cross-talk between Sertoli and germ cells, since the expression of cathepsin L in each cell type is regulated by one another via either soluble factors or cell-cell contact.
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PMID:Evidence for cross-talk between Sertoli and germ cells using selected cathepsins as markers. 987 20

The influence of the gene expression of critical components of the cytoplasmic and lysosomal proteolytic pathways on the rate of protein degradation was evaluated in the leg skeletal muscle of 8 severely traumatized patients. Muscle proteolysis was determined as the intramuscular phenylalanine rate of appearance by L-[ring-2H5]phenylalanine infusion and the leg arteriovenous catheterization technique combined with muscle biopsy. Muscle mRNA levels of UbB polyubiquitin and cathepsin B were determined by reverse transcriptase-competitive polymerase chain reaction and expressed as a percent of the mRNA level of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In the patients, individual values for UbB polyubiquitin mRNA levels directly correlated with the rate of muscle proteolysis (r = .76, P < .05), whereas no correlation (r = .10) was found between cathepsin B mRNA levels and proteolysis. Thus, after trauma, the rate of muscle proteolysis appears to be largely regulated by the ubiquitin-proteasome system at the level of gene transcription.
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PMID:Contribution of the ubiquitin-proteasome pathway to overall muscle proteolysis in hypercatabolic patients. 1087 90

Using serial analysis of gene expression on cultured human keratinocytes we found high expression levels of genes putatively involved in host protection and defense, such as proteinase inhibitors and antimicrobial proteins. One of these expressed genes was the recently discovered cysteine proteinase inhibitor cystatin M/E that has not been characterized so far at the protein level with respect to tissue distribution and additional biologic properties. Here we report that cystatin M/E has a tissue-specific expression pattern in which high expression levels are restricted to the stratum granulosum of normal human skin, the stratum granulosum/spinosum of psoriatic skin, and the secretory coils of eccrine sweat glands. Low expression levels were found in the nasal cavity. The presence of cystatin M/E in skin and the lack of expression in a variety of other tissues was verified both at the protein level by immunohistochemistry or western blotting, and at the mRNA level by reverse transcriptase polymerase chain reaction or northern blotting. Using biotinylated hexapeptide probes we found that cystatin M/E is an efficient substrate for tissue type transglutaminase and for transglutaminases extracted from stratum corneum, and that it acts as an acyl acceptor but not as an acyl donor. Western blot analysis showed that recombinant cystatin M/E could be cross-linked to a variety of proteins extracted from stratum corneum. In vitro, we found that cystatin M/E expression in cultured keratinocytes is upregulated at the mRNA and protein level, upon induction of differentiation. We demonstrate that cystatin M/E, which has a putative signal peptide, is indeed a secreted protein and is found in vitro in culture supernatant and in vivo in human sweat by enzyme-linked immunosorbent assay or western blotting. Cystatin M/E showed moderate inhibition of cathepsin B but was not active against cathepsin C. We speculate that cystatin M/E is unlikely to be a physiologically relevant inhibitor of intracellular lysosomal cysteine proteinases but rather functions as an inhibitor of self and nonself cysteine proteinases that remain to be identified.
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PMID:Cystatin M/E expression is restricted to differentiated epidermal keratinocytes and sweat glands: a new skin-specific proteinase inhibitor that is a target for cross-linking by transglutaminase. 1134 57

In this paper, we used the genetic manipulation technique known as RNA-interference to suppress the expression of a target, cathepsin B, gene in the platyhelminth parasite, Schistosoma mansoni. Parasites were cultured for 6 days in the presence of double stranded RNA derived from the cathepsin B cDNA sequence or from two control sequences. Relative to the controls, the cathepsin B double stranded RNA-treated group exhibited lower levels of cathepsin B as determined by immuno-staining and by enzyme activity measurements. Additionally, using the reverse transcriptase-PCR, suppression was seen in the inability to detect cathepsin B cDNA, using RNA obtained from those parasites. This ability to manipulate gene expression represents a powerful new tool for investigating gene function in these debilitating human parasites.
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PMID:Suppression of cathepsin B expression in Schistosoma mansoni by RNA interference. 1270 30

Osteosarcoma is the most frequent malignant bone tumor with a poor survival rate for patients with metastasis. Previous studies have shown that beside other proteases, distinct sets of cathepsins are involved in the process of metastasis of different tumors. In this study we investigated the expression of cathepsin proteases in human osteosarcoma metastasis. First, the mRNA expression of 14 human cathepsins was studied in SAOS-2 osteosarcoma cells and the highly metastatic LM5 and LM7 sublines by reverse transcriptase (RT)-polymerase chain reaction (PCR). The expression of cathepsin D, K, and L mRNA was found upregulated and that of cathepsin F, H, and V downregulated in the highly metastatic LM5 and LM7 cells. A subgroup of the cathepsin proteases was further studied at the protein level by Western blot analysis of cell extracts. The expression of cathepsin B and H was decreased and that of cathepsin D, K, and L was increased in the highly metastatic cell lines as compared to the SAOS-2 cell line. Diagnostic relevance of cathepsin K expression in osteosarcoma was revealed upon correlation of survival and metastasis with immunohistochemical cathepsin K staining of biopsies collected from 92 patients prior to chemotherapy. Patients with metastatic high-grade osteosarcoma and low cathepsin K expression at diagnosis had a better prognosis than those with high expression. Thus, it appears that cathepsin K expression is of predictive prognostic value for patients with high-grade tumors and metastasis at diagnosis.
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PMID:Cathepsins and osteosarcoma: Expression analysis identifies cathepsin K as an indicator of metastasis. 1768 65

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

Biomphalaria glabrata snails are known to display a wide range of susceptibility phenotypes to Schistosoma mansoni infection depending on the genetics of both the snail and the invading parasite. Evidence exists for a role of hydrolytic enzymes in the defense of molluscs against invading parasites. To elucidate the role of these enzymes in the outcome of infection in the snail, proteolysis was examined in parasite-resistant and -susceptible snails. Zymographs of extracts from the whole snail or hepatopancreas indicated higher proteolytic activity in resistant, compared with susceptible, snails. Lytic activity coincided with a high-molecular-weight smear (220 to 66 kDa) that was abrogated by the cysteine protease inhibitor trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane. Quantitative flourimetric assays showed 3.5-fold higher activity in resistant than in susceptible snails. From a hepatopancreas cDNA library, several cysteine protease encoding expressed sequence tags including the full-length cDNA for cathepsin B were identified. Sequence analysis revealed that this cathepsin B belonged to the C1A family of peptidases characterized by the presence of the catalytic cysteine-histidine dyad, the "occluding loop," signal sequence, and cleavage sites for the prepro and propeptides. Quantitative real-time reverse transcriptase-polymerase chain reaction showed higher up-regulation of cathepsin B transcript in resistant than in the susceptible snail after parasite exposure.
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PMID:Differences in cysteine protease activity in Schistosoma mansoni-resistant and -susceptible Biomphalaria glabrata and characterization of the hepatopancreas cathepsin B Full-length cDNA. 1860 96

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor neurons in the cerebral cortex, brain stem, and spinal cord. Most cases (90%) are classified as sporadic ALS (sALS). The remainder 10% are inherited and referred to as familial ALS, and 2% of instances are due to mutations in Cu/Zn superoxide dismutase (SOD1). Using cDNA microarray on postmortem spinal cord specimens of four sALS patients compared to four age-matched nonneurological controls, we found major changes in the expression of mRNA in 60 genes including increase of cathepsin B and cathepsin D (by the factors 2 and 2.3, respectively), apolipoprotein E (Apo E; factor 4.2), epidermal growth factor receptor (factor 10), ferritin (factor 2), and lysosomal trafficking regulator (factor 10). The increase in the expression of these genes was verified by quantitative reverse transcriptase polymerase chain reaction. Further analysis of these genes in hSOD1-G93A transgenic mice revealed increase in the expression in parallel with the deterioration of motor functions quantified by means of rotorod performance. The comparability of the findings in sALS patients and in the hSOD1-G93A transgenic mouse model suggests that the examined genes may play a specific role in the pathogenesis of ALS.
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PMID:Spinal cord mRNA profile in patients with ALS: comparison with transgenic mice expressing the human SOD-1 mutant. 1865 Dec 50


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