Cloning and expression of the cDNA for rat NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

Following interleukin-1 (IL1) stimulation, an IL1 receptor associated kinase (IRAK) is rapidly recruited to the receptor complex. However, it is not understood if IRAK is able to interact directly with the intracellular portion of the IL1-RI or if its recruitment is mediated by a different molecule. Using the yeast two-hybrid system, we have analysed possible protein-protein interactions between IRAK, IL1-RI and IL1-RAcP. We found that IRAK is able to interact with the equivalent cytoplasmic region of the IL1-RAcP but is unable to interact with the cytoplasmic region of the IL1-RI. Immunoprecipitation of the IL1-RAcP followed by Western blot analysis using anti-IRAK antibodies revealed that IRAK co-precipitated with the IL1-RAcP. We propose that, in non-stimulated cells, IRAK is bound to the IL1-RAcP and therefore, following IL1 stimulation, both molecules are recruited simultaneously to the ILI-RI complex.     

Sequence of a novel mRNA coding for a C-terminal-truncated form of human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase

We amplified, using the polymerase chain reaction (PCR) and NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (type-I 15-PGDH)-specific primers, RNA extracted from the HL-60 cell line. Two bands, differing in size by approx. 160 bp, were detected with ethidium bromide staining after electrophoresis of amplification products and hybridization with a 15-PGDH-specific probe. Sequencing these DNA bands revealed that the largest corresponded to the 15-PGDH cloned from human placenta [Ensor et al., J. Biol. Chem. 265 (1990) 14888-14891]. The smaller sequence coded for a predicted C-terminal-truncated form of 15-PGDH. This subtype of the type-I 15-PGDH mRNA was also found using RT-PCR in human liver, placenta and a cell line derived from a human medullary thyroid carcinoma (TT cells). Hybridization studies using specific probes indicated that this new mRNA form probably corresponded to the 3.4-kb mRNA, one of the two 15-PGDH mRNAs previously detected in Northern blot analysis.     

NAD(+)-dependent isocitrate dehydrogenase from Arabidopsis thaliana. Characterization of two closely related subunits

Two cDNA clones which appear to encode different subunits of NAD(+)-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.41) were identified by homology searches from the Arabidopsis EST database. These cDNA clones were obtained and sequenced; both encoded full-length messages and displayed 82.7% nucleotide sequence identity over the coding region. The deduced amino acid sequences revealed preprotein lengths of 367 residues, with an amino acid identity of 86.1%. Genomic Southern blot analysis showed distinct single-copy genes for both IDH subunits. Both IDH subunits were expressed as recombinant proteins in Escherichia coli, and polyclonal antibodies were raised to each subunit. The Arabidopsis cDNA clones were expressed in Saccharomyces cerevisiae mutants which were deficient in either one or both of the yeast NAD(+)-dependent IDH subunits. The Arabidopsis cDNA clones failed to complement the yeast mutations; although both IDH-I and IDH-II were expressed at detectable levels, neither protein was imported into the mitochondria.     

Cloning of cDNA and genomic structure of the mouse gamma-glutamyl transpeptidase-encoding gene

We have isolated and characterized cDNA and genomic clones containing the coding region for the mouse gamma-glutamyl transpeptidase (GGT). The sequences of the full-length cDNAs for three of the seven known mouse Ggt RNAs (types I, II and III) were determined and found to be identical in the coding region. Comparisons of the deduced amino-acid sequence of mouse GGT with that of rat and human reveal 95 and 79% overall identities, respectively. The mouse Ggt gene has 12 coding exon and spans approx. 12 kb. We have also re-analyzed rat genomic Ggt clones previously isolated by us and found that the rat and mouse genes share the same intron/exon boundaries. Our findings are of interest because they define the structure of the mouse and rat Ggt genes and will allow comparison with human GGT genes which, recent findings suggest, have diverged substantially from rodents.     

cDNA cloning and characterization of a new human microsomal NAD+-dependent dehydrogenase that oxidizes all-trans-retinol and 3alpha-hydroxysteroids

We report the cDNA sequence and catalytic properties of a new member of the short chain dehydrogenase/reductase superfamily. The 1134-base pair cDNA isolated from the human liver cDNA library encodes a 317-amino acid protein, retinol dehydrogenase 4 (RoDH-4), which exhibits the strongest similarity with rat all-trans-retinol dehydrogenases RoDH-1, RoDH-2, and RoDH-3, and mouse cis-retinol/androgen dehydrogenase (相似文献   

Structures stabilizing the dimer interface on human 11 beta-hydroxysteroid dehydrogenase types 1 and 2 and human 15-hydroxyprostaglandin dehydrogenase and their homologs

Human 11 beta-hydroxysteroid dehydrogenase-types 1 and 2 and human 15-hydroxyprostaglandin dehydrogenase belong to a large family of oxidoreductases that includes human dihydropteridine reductase and Streptomyces hydrogenans 20 beta-hydroxysteroid dehydrogenase, for which 3D structures are available. Almost all of these enzymes are either dimers or tetramers. The dimer interface of rat dihydropteridine reductase consists of alpha-helices E and F from each monomer arranged in a four alpha-helix bundle [Varughese et al. (1992) Proc. Natl. Acad. Sci. USA 89, 6080-6084]. Alpha-helix F contains tyrosine-146 and lysine-150, residues that are highly conserved in this protein superfamily and have been proposed to be at the catalytic site. We have examined the dimer interface between alpha-helix F in human and rat dihydropteridine reductase and Streptomyces hydrogenans 20 beta-hydroxysteroid dehydrogenase as well as modeled 3D structures of steroid and prostaglandin dehydrogenases and homologs for stabilizing interactions. We find a site in the middle of alpha-helix F that stabilizes the dimer. This anchor is adjacent to conserved lysine on alpha-helix F. Our analysis suggests that sequence variation in the anchor may be important in substrate specificity.     

Characterization of mouse Rt6.1 NAD:arginine ADP-ribosyltransferase

Rat RT6 proteins, and perhaps mouse Rt6, identify a set of immunoregulatory T lymphocytes. Rat RT6.1 (RT6.1) and rat RT6.2 (RT6. 2) are NAD glycohydrolases, which catalyze auto-ADP-ribosylation, but not ADP-ribosylation of exogenous proteins. Mouse Rt6.1 (mRt6.1) also catalyzes auto-ADP-ribosylation. The activity of mouse cytotoxic T lymphocytes is reportedly inhibited by ADP-ribosylation of surface proteins, raising the possibility that mRt6 may participate in this process. The reactions catalyzed by mRt6, would, however, need to be more diverse than those of the rat homologues and include the ADP-ribosylation of acceptors other than itself. To test this hypothesis, mRt6.1 and rat RT6.2 were synthesized in Sf9 insect cells and rat mammary adenocarcinoma (NMU) cells. mRt6.1, but not rat RT6.2, catalyzed the ADP-ribosylation of guanidino-containing compounds (e.g. agmatine). Unlike RT6.2, mRt6.1 was a weak NAD glycohydrolase. In the presence of agmatine, however, the ratio of [adenine-14C]ADP-ribosylagmatine formation from [adenine-14C]NAD to [carbonyl-14C]nicotinamide formation from [carbonyl-14C]NAD was approximately 1.0, demonstrating that mRt6.1 is primarily a transferase. ADP-ribosylarginine hydrolase, which preferentially hydrolyzes the alpha-anomer of ADP-ribosylarginine, released [U-14C]arginine from ADP-ribosyl[U-14C]arginine synthesized by mRT6.1, consistent with the conclusion that mRt6.1 catalyzes a stereospecific Sn2-like reaction. Thus, mRt6.1 is an NAD:arginine ADP-ribosyltransferase capable of catalyzing a multiple turnover, stereospecific Sn2-like reaction.     

Characterization of the human dihydropyrimidine dehydrogenase gene

Dihydropyrimidine dehydrogenase (DPD) catabolizes endogenous pyrimidines and pyrimidine-based antimetabolite drugs. A deficiency in human DPD is associated with congenital thymine-uraciluria in pediatric patients and severe 5-fluorouracil toxicity in cancer patients. The dihydropyrimidine dehydrogenase gene (DPYD) was isolated, and its physical map and exon-intron organization were determined by analysis of P1, PAC, BAC, and YAC clones. The DPYD gene was found to contain 23 exons ranging in size from 69 bp (exon 15) to 961 bp (exon 23). A physical map derived from a YAC clone indicated that DPYD is at least 950 kb in length with 3 kb of coding sequence and an average intron size of about 43 kb. The previously reported 5' donor splice site mutation present in pediatric thymine-uraciluria and cancer patients can now be assigned to exon 14. All 23 exons were sequenced from a series of human DNA samples, and three point mutations were identified in three racial groups as G1601A (exon 13, Ser534Asn), A1627G (exon 13, Ile543Val), and G2194A (exon 18, Val732Ile). These studies, which have established that the DPYD gene is unusually large, lay a framework for uncovering new mutations that are responsible for thymine-uraciluria and toxicity to fluoropyrimidine drugs.     

NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase from Thermoproteus tenax. The first identified archaeal member of the aldehyde dehydrogenase superfamily is a glycolytic enzyme with unusual regulatory properties

The hyperthermophilic archaeum Thermoproteus tenax possesses two glyceraldehyde-3-phosphate dehydrogenases differing in cosubstrate specificity and phosphate dependence of the catalyzed reaction. NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase catalyzes the phosphate-independent irreversible oxidation of D-glyceraldehyde 3-phosphate to 3-phosphoglycerate. The coding gene was cloned, sequenced, and expressed in Escherichia coli. Sequence comparisons showed no similarity to phosphorylating glyceraldehyde-3-phosphate dehydrogenases but revealed a relationship to aldehyde dehydrogenases, with the highest similarity to the subgroup of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenases. The activity of the enzyme is affected by a series of metabolites. All effectors tested influence the affinity of the enzyme for its cosubstrate NAD+. Whereas NADP(H), NADH, and ATP reduce the affinity for the cosubstrate, AMP, ADP, glucose 1-phosphate, and fructose 6-phosphate increase the affinity for NAD+. Additionally, most of the effectors investigated induce cooperativity of NAD+ binding. The irreversible catabolic oxidation of glyceraldehyde 3-phosphate, the control of the enzyme by energy charge of the cell, and the regulation by intermediates of glycolysis and glucan degradation identify the NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase as an integral constituent of glycolysis in T. tenax. Its regulatory properties substitute for those lacking in the reversible nonregulated pyrophosphate-dependent phosphofructokinase in this variant of the Embden-Meyerhof-Parnas pathway.     

Characterization of the human aldose reductase gene promoter

The promoter region of the human aldose reductase gene has been identified upstream of the translation start ATG codon. The promoter contains a TATA box, a CCAAT promoter element, and three Sp1 protein binding consensus sequences upstream of the capsite. A 640-base pair insert spanning +31 to -609 directs expression of the reporter gene chloramphenicol acetyltransferase in an orientation-specific manner in transfected Hep G2 cells. The promoter activity remained constant with deletions from base pairs -609 to -186. The TATA and the CCAAT consensus sequences show significant promoter activity, whereas the three Sp1 binding consensus sequences, individually, have no significant promoter activity. A GA-rich region (-186 to -146) contains two CGGAAA/G motifs, which show promoter activity and interaction with Hep G2 nuclear extract and GA-binding proteins (GABP alpha and GABP beta 1) as shown by mobility shift assays and DNase I footprinting. Similar cis-elements in herpes simplex virus type 1 interact with rat liver GABP and the viral VP16 protein to mediate the induction of immediate early viral genes. A GC-rich region (-87 to -31) is identified by mobility shift assay, and a consensus sequence of an androgen response element is present at -396 to -382. The human aldose reductase promoter, thus, has regulatory response elements that may be important during early development and puberty. These regulatory elements may play a significant role in the development of certain diabetic complications.     

Cloning, genomic structure, and expression of mouse ring finger protein gene Znf179

OBJECTIVE: H. pylori causes chronic gastritis, which may progress to peptic ulcer, gastric atrophy, or gastric cancer. However, little is known about the role of H. pylori infection in reflux esophagitis and the relationship between reflux esophagitis and atrophic gastritis needs to be clarified. We sought to identify the possible interrelationships among Helicobacter pylori infection, reflux esophagitis, and atrophic gastritis, to signal areas in which researchers should consider focusing their attention. METHODS: A broad-based Medline search was performed to identify all related publications addressing H. pylori infection, atrophic gastritis, gastroesophageal reflux disease (GERD), secretion of gastric acid, and gastric motility published between 1966 and July 1997. RESULTS: Whereas some studies have shown no significant association between H. pylori infection and reflux esophagitis, others have observed that the prevalence of H. pylori infection was lower in patients with GERD, implying a protective role. Eradication of H. pylori leads to occurrence of reflux esophagitis in some cases, but the mechanisms inducing posteradication reflux esophagitis are unknown. H. pylori infection may lead to atrophic gastritis (and hence hypochlorhydia) through both bacterial and host factors, although gastric atrophy and subsequent intestinal metaplasia are hostile to H. pylori because of hypochlorhydria. Although it has been reported that long-term proton pump inhibitor therapy for refractory reflux esophagitis may induce or enhance the development of gastric atrophy in H. pylori-infected patients, this relationship has been disputed. CONCLUSIONS: H. pylori infection may be negatively associated with reflux esophagitis, but this requires confirmation. Research then needs to focus on whether this is explained through motility- or acid-related mechanisms. The potential costs of maintenance antireflux therapy may need to be taken into account when evaluating the cost effectiveness of anti-H. pylori therapy.