scholarly journals Isolation and characterization of the monkey UGT2B30 gene that encodes a uridine diphosphate-glucuronosyltransferase enzyme active on mineralocorticoid, glucocorticoid, androgen and oestrogen hormones

2002 ◽  
Vol 365 (1) ◽  
pp. 213-222 ◽  
Author(s):  
Caroline GIRARD ◽  
Olivier BARBIER ◽  
David TURGEON ◽  
Alain BÉLANGER
Keyword(s):  
Cynomolgus Monkey ◽  
Genomic Organization ◽  
Human Kidney ◽  
Relative Activity ◽  
Pcr Analysis ◽  
Uridine Diphosphate ◽  
Lipophilic Compounds ◽  
Isolation And Characterization ◽  
Hormonal Steroids

The present study reports the genomic organization and the characterization of a novel cynomolgus monkey UDP-glucuronosyltransferase (UGT) enzyme, UGT2B30. UGT enzymes are microsomal proteins that catalyse the transfer of the glucuronosyl group from UDP-glucuronic acid (UDPGA) to a wide variety of lipophilic compounds, namely hormonal steroids. The 15kb UGT2B30 gene amplified by PCR showed a genomic organization similar to those encoding UGT2B human enzymes. The cDNA encoding UGT2B30 was isolated from a cynomolgus monkey prostate cDNA library, and the deduced amino acid sequence showed an identity of 94% with UGT2B19, a monkey isoform previously characterized. Stable expression of UGT2B30 protein in human kidney 293 (HK293) cells was assessed by Western-blot analysis and its conjugating activity was screened using 39 potential substrates. The UGT2B30 enzyme is active on many compounds of different classes, including testosterone, dihydrotestosterone, 5α-androstane-3α,17β-diol, androsterone, oestradiol, tetrahydroaldosterone and tetrahydrocortisone, with glucuronidation efficiencies (Vmax/Km ratios) ranging from 0.6 to 8.8μl·min−1·mg of protein−1. Reverse-transcriptase-PCR analysis revealed that the UGT2B30 transcript is expressed in several tissues, including prostate, testis, mammary gland, kidney, adrenals and intestine. The relative activity of UGT2B30 in comparison with other simian UGT2B isoforms, as well as its large variety of substrates, strongly suggest that this enzyme is essential to inactivation of several steroids.

Genomic organization of the 5′ end of human β-ENaC and preliminary characterization of its promoter

2002 ◽  
Vol 282 (5) ◽  
pp. F898-F909 ◽  
Author(s):  
Christie P. Thomas ◽  
Randy W. Loftus ◽  
Kang Z. Liu ◽  
Omar A. Itani
Keyword(s):  
Genomic Organization ◽  
Human Kidney ◽  
Β Subunit ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Nuclear Extracts ◽  
Flanking Region ◽  
Rapid Amplification ◽  
Flanking Regions

The mRNA for the β-subunit of the epithelial Na+ channel (β-ENaC) is regulated developmentally and, in some tissues, in response to corticosteroids. To understand the mechanisms of transcriptional regulation of the human β-ENaC gene, we characterized the 5′ end of the gene and its 5′-flanking regions. Adaptor-ligated human kidney and lung cDNA were amplified by 5′ rapid amplification of cDNA ends, and transcription start sites of two 5′ variant transcripts were determined by nuclease protection or primer extension assays. Cosmid clones that contain the 5′ end of the gene were isolated, and analysis of these clones indicated that alternate first exons ∼1.5 kb apart and ∼ 45 kb upstream of a common second exon formed the basis of these transcripts. Genomic fragments that included the proximal 5′-flanking region of either transcript were able to direct expression of a reporter gene in lung epithelia and to bind Sp1 in nuclear extracts, confirming the presence of separate promoters that regulate β-ENaC expression.

Type IV Collagens: Isolation and Characterization of 7S Collagen from Human Kidney, Liver and Lung

1981 ◽  
Vol 1 (6) ◽  
pp. 549-556 ◽  
Author(s):  
S.N. Dixit ◽  
J.M. Stuart ◽  
J.M. Seyer ◽  
J. Risteli ◽  
R. Timpl ◽  
...  
Keyword(s):  
Human Kidney ◽  
Type Iv ◽  
Isolation And Characterization ◽  
Kidney Liver ◽  
7S Collagen

scholarly journals Isolation and Characterization of Genes Responsible for Naphthalene Degradation from Thermophilic Naphthalene Degrader, Geobacillus sp. JF8

2019 ◽  
Vol 8 (1) ◽  
pp. 44 ◽  
Author(s):  
Daisuke Miyazawa ◽  
Le Thi Ha Thanh ◽  
Akio Tani ◽  
Masaki Shintani ◽  
Nguyen Hoang Loc ◽  
...  
Keyword(s):  
Amino Acid ◽  
Thermophilic Bacterium ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Pcr Analysis ◽  
Naphthalene Degradation ◽  
Isolation And Characterization ◽  
Dihydrodiol Dehydrogenase ◽  
Reading Frames

Geobacillus sp. JF8 is a thermophilic biphenyl and naphthalene degrader. To identify the naphthalene degradation genes, cis-naphthalene dihydrodiol dehydrogenase was purified from naphthalene-grown cells, and its N-terminal amino acid sequence was determined. Using a DNA probe encoding the N-terminal region of the dehydrogenase, a 10-kb DNA fragment was isolated. Upstream of nahB, a gene for dehydrogenase, there were two open reading frames which were designated as nahAc and nahAd, respectively. The products of nahAc and nahAd were predicted to be alpha and beta subunit of ring-hydroxylating dioxygenases, respectively. Phylogenetic analysis of amino acid sequences of NahB indicated that it did not belong to the cis-dihydrodiol dehydrogenase group that includes those of classical naphthalene degradation pathways. Downstream of nahB, four open reading frames were found, and their products were predicted as meta-cleavage product hydrolase, monooxygenase, dehydrogenase, and gentisate 1,2-dioxygenase, respectively. A reverse transcriptase-PCR analysis showed that transcription of nahAcAd was induced by naphthalene. These findings indicate that we successfully identified genes involved in the upper pathway of naphthalene degradation from a thermophilic bacterium.

scholarly journals Genomic organization of the 3′-region of the human MUC5AC mucin gene: additional evidence for a common ancestral gene for the 11p15.5 mucin gene family

1998 ◽  
Vol 332 (3) ◽  
pp. 729-738 ◽  
Author(s):  
Marie-Pierre BUISINE ◽  
Jean-Luc DESSEYN ◽  
Nicole PORCHET ◽  
Pierre DEGAND ◽  
Anne LAINE ◽  
...  
Keyword(s):  
Gene Family ◽  
Genomic Organization ◽  
Splice Sites ◽  
Ancestral Gene ◽  
Remarkable Similarity ◽  
Mucin Gene ◽  
Isolation And Characterization ◽  
Entire Sequence ◽  
Chromosome 11P15.5

The human mucin gene MUC5AC is mapped clustered with MUC2, MUC5B and MUC6 on chromosome 11p15.5. We report here the isolation and characterization of a genomic cosmid clone, designated ELO9, spanning the 3´-region of MUC5AC and the 5´-region of MUC5B, allowing us to conclude that MUC5AC and MUC5B have the same transcriptional orientation. We determined the genomic organization and the entire sequence of the 3´-region of MUC5AC. The comparative molecular analysis of MUC5AC and MUC5B points to a remarkable similarity in the size and the distribution of exons, and in the type of splice sites, supporting the notion that MUC5AC and MUC5B have evolved from a single common ancestral gene. The derivation of the four genes of the 11p15.5 mucin gene family from a single ancestral gene is discussed.

Isolation and characterization of Escherichia coli O157:H7 and shiga toxin-converting bacteriophages from strains of human, bovine and porcine origin

2002 ◽  
Vol 2 (3) ◽  
pp. 29-38 ◽  
Author(s):  
E.E. Müller ◽  
M.B. Taylor ◽  
W.O.K. Grabow ◽  
M.M. Ehlers
Keyword(s):  
Escherichia Coli ◽  
Pcr Analysis ◽  
Sewage Sample ◽  
Natural Environments ◽  
Escherichia Coli O157 ◽  
Electron Microscopic ◽  
E Coli ◽  
Isolation And Characterization ◽  
Faecal Samples

Toxin-converting bacteriophages encoding the Stx2 gene were induced from strains of Escherichia coli O157:H7 isolated from sewage, bovine and porcine faeces. Toxin synthesis can be stimulated by the induction of integrated toxin-converting phages from the host E. coli O157:H7 organism by ultra-violet (UV) exposure. The UV-mediated DNA damage of E. coli O157:H7 triggers a bacterial SOS response resulting in phage release. Free ranging phages outside their E. coli O157:H7 hosts were detected but could not be isolated directly from environmental samples such as sewage and river water. E. coli O157:H7 colonies carrying the genes coding for Stx2 were isolated from 1 sewage sample (0.76% of positive samples), 8 cattle faecal samples (16.67% of positive samples) and 2 pig faecal samples (14.28% of positive samples). Characterization of E. coli O157:H7 was done by repetitive sequence analysis using ERIC-PCR to determine the relationships between the individual E. coli O157:H7 strains. The ERIC-PCR analysis revealed distinct patterns for all E. coli O157:H7 strains with some small differences between the strains. DNA sequencing of some of the E. coli O157:H7 positive isolates carrying the Stx2 genes were performed confirming the amplified DNA nucleotide sequences of Stx2. Electron microscopic analysis revealed, for the first time in South Africa, that Stx2-converting phages induced from E. coli O157:H7 have different morphologies to that of phage lambda which was previously described. The role of the induced integrated Stx2 phages in natural environments such as river and dam water remains unclear. With the induction of Stx2-converting phages from environmental E. coli O157:H7 isolates, it is now possible to determine the potential of these phages to convert non-pathogenic E. coli strains and other enterobacteriaciae into pathogenic strains.

scholarly journals Isolation and Characterization of Transformed Human T-Cell Lines Infected by Epstein-Barr Virus

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4521-4530 ◽  
Author(s):  
Hervé Groux ◽  
Françoise Cottrez ◽  
Claire Montpellier ◽  
Brigitte Quatannens ◽  
Jean Coll ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Pcr Analysis ◽  
Barr Virus ◽  
Isolation And Characterization ◽  
Human T Cell ◽  
Ebv Dna ◽  
Epstein Barr ◽  
T Cell Lines

Abstract Epstein-Barr virus (EBV) is a human lymphotropic virus whose main targets have traditionally been described as B lymphocytes and epithelial cells. Here we report the isolation and characterization of largely monoclonal transformed human T-cell lines infected by EBV. The transformed T cells expressed CD2, CD3, and either CD4 or CD8 surface molecules and more generally displayed the phenotype of naive T cells with a complete and clonal rearrangement of the T-cell receptor. None of the cell lines expressed B cells, natural killer, or myeloid antigens or had immunoglobulins genes rearrangement. They grew in the absence of growth factor; however, they all secreted interleukin-2 after mitogenic activation. Polymerase chain reaction (PCR) analysis showed the presence of EBV DNA in all these cell lines. Moreover, Southern blot analysis of one of these cell lines shows the presence of circular episomic EBV DNA, and by Northern blot or reverse transcriptase-PCR analysis, only the expression of Epstein-Barr nuclear antigen-1 (EBNA-1) and latent membrane protein-1 (LMP-1) genes was detected. Finally, the complete transformed phenotype of this T-cell line was shown by its injection into nude or recombination activating gene 2 (RAG2)-deficient mice that led to the formation of solid tumors.

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