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)

The expression of a recombinant gene by yeasts seeded into soil samples was directly measured by analyzing transcripts and gene product occurrences in soil extracts. Two yeast species, Saccharomyces cerevisiae WHL292 and Hansenula polymorpha LR9-Apr4, both engineered by a synthetic gene sequence encoding the mammalian peptide aprotinin, produced and secreted this peptide in batch cultures at concentrations of 90 and 64 ng ml-1, respectively. In S. cerevisiae, the aprotinin gene was located on plasmid p707 and expressed constitutively. H. polymorpha carried the gene chromosomally integrated, and its expression was inducible by methanol. To detect aprotinin transcripts, cells were directly lysed in the soil samples and the crude lysates were hybridized to oligo(dT)-coated magnetized polystyrene beads (Dynabeads). After separation and purification in a magnetic field, aprotinin mRNA was detected by reverse transcriptase PCR with aprotinin gene-specific primers. Transcripts from 10 cells g of soil-1 were sufficient for detection. When 10(7) cells of S. cerevisiae were inoculated into soil, aprotinin mRNA was detectable during the first 4 days. Addition of methanol and a combined nutrient solution was necessary to induce aprotinin gene expression of H. polymorpha in soil. Aprotinin could be detected directly in soil extracts by an indirect enzyme-linked immunosorbent assay with monoclonal aprotinin-specific antibodies. The detection threshold was 45 pg g of soil-1. In presterilized soil inoculated with S. cerevisiae (10(6) CFU g-1), aprotinin accumulated during the first 10 days to 12 ng g of soil-1 and then remained constant.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Direct detection of recombinant gene expression by two genetically engineered yeasts in soil on the transcriptional and translational levels. 853 97

We studied the effects of serine proteases on cytokine gene expression by cultured normal human keratinocytes. In resting keratinocytes, steady-state mRNA levels for interleukins IL-1 alpha, IL-1 beta, IL-7, and IL-8, transforming growth factors alpha and beta, and tumor necrosis alpha were sufficient to be detected by our reverse transcriptase-polymerase clozin reaction method. Incubation of keratinocytes with 25 nM trypsin or 1 unit/ml thrombin for 24 hr selectively upregulated mRNA levels for granulocyte-macrophage colony-stimulating factor (GM-CSF) and Il-6 to detectable levels. Keratinocytes secreted GM-CSF and IL-6 protein in response to these proteases. Monensin did not inhibit the gene expression for the cytokines, thereby excluding the possibility of intervention by secreted molecules. Aprotinin and argatroban inhibited the effects of the proteases. SFLLRN and SLIGRL, tethered ligand receptor peptides for thrombin receptor and for proteinase-activated receptor 2 (PAR-2), respectively, duplicated the effects of the proteases on keratinocytes, which expressed mRNA for both receptors. Trypsin increased tyrosine phosphorylated proteins and intracellular free calcium concentrations. Tyrphostin, pertussis toxin, or H-7 suppressed trypsin- and thrombin-induced GM-CSF gene expression. Our results demonstrate that the serine proteases activate thrombin receptors and PAR-2 on keratinocytes, triggering intracellular signaling and then inducing the synthesis of GM-CSF. We speculate that serine proteases modulate the course of physiological and pathological processes in the skin by stimulating keratinocytes to produce the cytokines.
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PMID:Thrombin and trypsin induce granulocyte-macrophage colony-stimulating factor and interleukin-6 gene expression in cultured normal human keratinocytes. 906 88