Identification and characterization of kraken, a gene encoding a putative hydrolytic enzyme in Drosophila melanogaster

BACKGROUND AND AIMS OF THE STUDY: The study aimed to determine the clinical performance of bovine pericardial aldehyde-treated products alone or in combination with aortic leaflets of porcine origin. These included a composite porcine stentless aortic valve attached to a scalloped pericardial tube (BSAV), and valved and non-valved bovine pericardial conduits for use in left-sided heart lesions (BPG). METHODS: For BSAV grafts, between January 1990 and August 1996, 163 patients (119 males) had their aortic valves replaced by SJM Biocor BASV. Mean age was 37.9 +/- 17.6 years (range: 1 to 76 years). Rheumatic heart disease sequelae (n = 72) and replacement of a prosthetic heart valve (n = 46) were predominant. Preoperative NYHA functional class showed 90 patients (55.2%) in class III and 50 (30.7%) in class IV. BPVC and NVPC grafts were used in 166 patients: acute aortic dissection was the main indication in 52 (31.3%) and chronic in 36 (21/7%). The ascending aorta was involved in 141 patients (84.9%); grafts were seldom used at other sites. In most patients the graft implanted was either a non-valved (n = 79) or a valved (n = 75) pericardial conduit. Twelve patients had a localized lesion and required a patch repair. RESULTS: For BASV grafts, the non-valve-related hospital mortality rate was 4.9%. There were 14.7% non-fatal complications with full recovery of all patients. Mean follow up in 141 patients was 3.0 +/- 1.4 years (range: 1 month to 7.2 years); 14 patients were lost to follow up. Late, non-conduit-related, mortality occurred in seven patients (4.9%). Eight patients underwent reoperation. The current clinical follow up of 127 patients has shown 118 (92.9%) with competent valves and nine (7.0%) with mild stable aortic insufficiency. For BPVC and NVPC grafts, hospital mortality rate was 16.9%, death being related to poor preoperative clinical condition. Postoperative follow up was accomplished in 125 patients; reoperation was necessary in seven patients. Histology showed good tissue preservation up to five years; echocardiography revealed satisfactory findings. No valved conduit had to be reoperated for valve or pericardial tissue wear. CONCLUSIONS: Clinical results of left-sided heterologous pericardial grafts have shown excellent performance over time. The BASV (over seven years) and BPVC and NVPC (eight years) have demonstrated superior results as aortic valves alone or in combination with a pericardial conduit.     

Saccharomyces cerevisiae GNA1, an essential gene encoding a novel acetyltransferase involved in UDP-N-acetylglucosamine synthesis

The Saccharomyces cerevisiae gene, YFL017C, for a putative acetyltransferase was characterized. Disruption of YFL017C was lethal, leading to a morphology similar to those caused by the depletion of AGM1 or UAP1, the genes encoding phospho-N-acetylglucosamine mutase and UDP-N-acetylglucosamine pyrophosphorylase, respectively. This implies the involvement of YFL017C in UDP-N-acetylglucosamine synthesis. The recombinant protein for YFL017C displayed phosphoglucosamine acetyltransferase activities in vitro and utilized glucosamine 6-phosphate as the substrate. When incubated with Agm1p and Uap1p, the Yfl017c protein produced UDP-N-acetylglucosamine from glucosamine 6-phosphate. These results indicate that YFL017C specifies glucosamine-6-phosphate acetyltransferase; therefore, the gene was designated GNA1 (glucosamine-6-phosphate acetyltransferase). In addition, whereas bacterial phosphoglucosamine acetyltransferase and UDP-N-acetylglucosamine pyrophosphorylase activities are intrinsic in a single polypeptide, they are encoded by distinct essential genes in yeast. When the sequence of ScGna1p was compared with those of other acetyltransferases, Ile97, Glu98, Val102, Gly112, Leu115, Ile116, Phe142, Tyr143, and Gly147 were found to be highly conserved. When alanine was substituted for these amino acids, the enzyme activity for the substituted Phe142 or Tyr143 enzymes was severely diminished. Although the activity of Y143A was too low to perform kinetics, F142A displayed a significantly increased Km value for acetyl-CoA, suggesting that the Phe142 and Tyr143 residues are essential for the catalysis.     

Identification of a poxvirus gene encoding a uracil DNA glycosylase

An open reading frame, BamHI D6R, from the central highly conserved region of the Shope fibroma virus (SFV) genome was sequenced and found to have significant homology to that of uracil DNA glycosylases from a number of organisms. Uracil DNA glycosylase catalyzes the initial step in the repair pathway that removes potentially mutagenic uracil from duplex DNA. The D6R polypeptide was expressed in reticulocyte lysates programmed with RNA transcribed from an expression vector containing the T7 RNA polymerase promoter. A highly specific ethidium bromide fluorescence assay of the in vitro translation product determined that the encoded protein does indeed possess uracil DNA glycosylase activity. Open reading frames from other poxviruses, including vaccinia virus (HindIII D4R) and fowlpox (D4), are highly homologous to D6R of SFV and are predicted to encode uracil DNA glycosylases. Identification of the SFV uracil DNA glycosylase provides evidence that this poxviral protein is involved in the repair of the viral DNA genome. Since this enzyme performs only the initial step required for the removal of uracil from DNA, creating an apyrimidinic site, we suggest that other, possibly virus-encoded, repair activities must be present in the cytoplasm of infected cells to complete the uracil excision repair pathway.     

In-vitro metabolism of YM17E, an inhibitor of acyl coenzyme A:cholesterol acyltransferase, by liver microsomes in man

Because YM17E (1,3-bis[[1-cycloheptyl-3-(p-dimethylaminophenyl) ureido]methyl]benzene dihydrochloride) inhibits acyl coenzyme A:cholesterol acyltransferase (ACAT) it has potential application in the treatment of hypercholesterolaemia. In man and animals YM17E is extensively metabolized, via N-demethylation, to five active metabolites (M1, M2-a, M2-b, M3 and M4). The main objectives of this study were to examine inhibition of YM17E metabolism by the products and identify the cytochrome P450 isoforms in liver microsomes which catalyse in-vitro YM17E metabolism in man. In microsomes in man N-demethylation of YM17E to M1 occurred enzymatically; for up to 45 s the rate was linearly proportional to the microsomal protein concentration. This reaction was inhibited by metabolites M2-a, M2-b, M3 and M4. Further, N-demethylation of [14C]-YM17E was also inhibited by its product, M1. These results showed that primary metabolism of YM17E was inhibited by its products, and supported the finding that the non-linear increase in plasma concentration of the parent drug and metabolites observed in an in-vivo study was due to inhibition by these products. Metabolic activity in microsomes from ten individual human livers demonstrated that YM17E N-demethylase activity correlated closely with testosterone 6 beta-hydroxylase activity. When cytochrome P450 isozyme-specific substrates and chemical inhibitors were used to inhibit YM17E N-demethylase activity, CYP3A-specific substrate and inhibitors such as nifedipine, ketoconazole and triacetyloleandomycin strongly inhibited this activity, whereas CYP1A-specific substrate or inhibitor, ethoxyresorufin and alpha-naphthoflavone, inhibited weakly. Other CYP inhibitors, in contrast, had few or no effects. An inhibition study using anti-rat CYP1A1, CYP2B1, CYP2C11, CYP2E1 and CYP3A2 antibodies demonstrated that only anti-rat CYP3A2 antibody inhibited YM17E metabolism, to 40% of control level, with no other antibodies showing an inhibitory effect. Of seven cDNA-expressed P450 isoforms in man (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP2E1 and CYP3A4), CYP3A4, CYP2D6 and CYP1A2 isozyme exhibited substantial catalytic activity of N-demethylation of YM17E. These results indicate the predominant role of CYP3A4 in liver metabolism of YM17E in man.     

Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain

The generation of pacemaker activity in heart and brain is mediated by hyperpolarization-activated cation channels that are directly regulated by cyclic nucleotides. We previously cloned a novel member of the voltage-gated K channel family from mouse brain (mBCNG-1) that contained a carboxy-terminal cyclic nucleotide-binding domain (Santoro et al., 1997) and hence proposed it to be a candidate gene for pacemaker channels. Heterologous expression of mBCNG-1 demonstrates that it does indeed code for a channel with properties indistinguishable from pacemaker channels in brain and similar to those in heart. Three additional mouse genes and two human genes closely related to mBCNG-1 display unique patterns of mRNA expression in different tissues, including brain and heart, demonstrating that these channels constitute a widely expressed gene family.     

Identification of the gene encoding scHelI, a DNA helicase from Saccharomyces cerevisiae

The gene encoding scHelI, a previously characterized DNA helicase from Saccharomyces cerevisiae, has been identified as YER176w, an open reading frame on chromosome V. The gene has been named HEL1 to indicate the DNA helicase activity of the gene product. HEL1 was identified by screening a lambda gt11 yeast protein expression library with antiserum to purified scHelI. Several independent immunopositive clones were isolated and shown to contain portions of HEL1 either by sequencing or by hybridization to a probe containing HEL1 sequences. The HEL1 open reading frame includes the seven conserved helicase motifs, consistent with the DNA helicase activity of scHelI, and the predicted size of the protein is in agreement with the size of purified scHelI. Partially purified cellular extracts from a hel1 deletion mutant strain did not contain scHelI activity. Homology searches revealed protein sequence homology between HEL1 and two previously identified and biochemically characterized yeast helicases, encoded by the DNA2 and UPF1 genes. Haploid hel1 deletion strains were constructed and shown to be viable with growth rates equivalent to those of parental strains. These strains did not differ from the parental strains in ultraviolet light sensitivity or the generation of petite colonies. Furthermore, these haploid deletion strains were capable for mating, the resultant diploid homozygous mutants were viable, capable of sporulation, and the spores displayed no reduction in viability.     

Identification of a Streptococcus pneumoniae gene locus encoding proteins of an ABC phosphate transporter and a two-component regulatory system

The Escherichia coli Pst system belongs to the family of ABC transporters. It is part of a phosphate (PHO) regulon which is regulated by extracellular phosphate. Under conditions of phosphate limitation, the response regulator PhoB is phosphorylated by the histidine kinase PhoR and binds to promoters that share a consensus PHO box. Under conditions of phosphate excess, PhoR, Pst, and PhoU downregulate the PHO regulon. Screening of a library of pneumococcal mutants with defects in exported proteins revealed a putative two-component regulatory system, PnpR-PnpS, and a downstream ABC transporter, similar to the Pst system in E. coli including a gene encoding a PhoU protein. Similar to E. coli, mutagenesis of the ATP-binding cassette gene, pstB, resulted in decreased uptake of phosphate. The effects of the loss of the pneumococcal Pst system extended to decreased transformation and lysis. Withdrawal of phosphate led to transformation deficiency in the parent strain R6x but not to penicillin tolerance, suggesting that reduced bacterial death was independent of phosphate. None of these phenotypes was observed in the pneumococcal loss-of-function mutant phoU. By using a lacZ reporter construct, it was demonstrated that expression of the two-component regulatory system PnpR-PnpS was not influenced by different concentrations of phosphate. These results suggest a more complex role of the Pst system in pneumococcal physiology than in that of E. coli.     

Cloning, sequencing, and expression of the gene encoding cyclic 2, 3-diphosphoglycerate synthetase, the key enzyme of cyclic 2, 3-diphosphoglycerate metabolism in Methanothermus fervidus

Cyclic 2,3-diphosphoglycerate synthetase (cDPGS) catalyzes the synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate. cDPG is known to be accumulated to high intracellular concentrations (>300 mM) as a putative thermoadapter in some hyperthermophilic methanogens. For the first time, we have purified active cDPGS from a methanogen, the hyperthermophilic archaeon Methanothermus fervidus, sequenced the coding gene, and expressed it in Escherichia coli. cDPGS purification resulted in enzyme preparations containing two isoforms differing in their electrophoretic mobility under denaturing conditions. Since both polypeptides showed the same N-terminal amino acid sequence and Southern analyses indicate the presence of only one gene coding for cDPGS in M. fervidus, the two polypeptides originate from the same gene but differ by a not yet identified modification. The native cDPGS represents a dimer with an apparent molecular mass of 112 kDa and catalyzes the reversible formation of the intramolecular phosphoanhydride bond at the expense of ATP. The enzyme shows a clear preference for the synthetic reaction: the substrate affinity and the Vmax of the synthetic reaction are a factor of 8 to 10 higher than the corresponding values for the reverse reaction. Comparison with the kinetic properties of the electrophoretically homogeneous, apparently unmodified recombinant enzyme from E. coli revealed a twofold-higher Vmax of the enzyme from M. fervidus in the synthesizing direction.     

Identification of stsC, the gene encoding the L-glutamine:scyllo-inosose aminotransferase from streptomycin-producing Streptomycetes

Eight new genes, strO-stsABCDEFG, were identified by sequencing DNA in the gene cluster that encodes proteins for streptomycin production of Streptomyces griseus N2-3-11. The StsA (calculated molecular mass 43.5 kDa) and StsC (45.5 kDa) proteins - together with another gene product, StrS (39.8 kDa), encoded in another operon of the same gene cluster - show significant sequence identity and are members of a new class of pyridoxal-phosphate-dependent aminotransferases that have been observed mainly in the biosynthetic pathways for secondary metabolites. The aminotransferase activity was demonstrated for the first time by identification of the overproduced and purified StsC protein as the L-glutamine:scyllo-inosose aminotransferase, which catalyzes the first amino transfer in the biosynthesis of the streptidine subunit of streptomycin. The stsC and stsA genes each hybridized specifically to distinct fragments in the genomic DNA of most actinomycetes tested that produce diaminocyclitolaminoglycosides. In contrast, only stsC, but not stsA, hybridized to the DNA of Streptomyces hygroscopicus ssp. glebosus, which produces the monoaminocyclitol antibiotic bluensomycin; this suggests that both genes are specifically used in the first and second steps of the cyclitol transamination reactions. Sequence comparison studies performed with the deduced polypeptides of the genes adjacent to stsC suggest that the enzymes encoded by some of these genes [strO (putative phosphatase gene), stsB (putative oxidoreductase gene), and stsE (putative phosphotransferase gene)] also could be involved in (di-)aminocyclitol synthesis.     

Identification, genomic organization, and alternative splicing of KNSL3, a novel human gene encoding a kinesin-like protein

Despite increasing importance of molecular genetics, electromyography has preserved its place as a valuable tool in the diagnostic procedure of myopathies. Conventional electromyography allows the assessment of spontaneous activity, motor unit action potentials and interference patterns. In myopathies, fibrillations and positive sharp waves can be found in the majority of the cases. Motor unit action potentials are of short duration, low amplitude and may show increased polyphasia and number of satellite potentials. The interference pattern may be of low amplitude and compact already at submaximal contraction. Compared to conventional electromyography, automatic interference pattern analysis provides quantitative results and has the higher sensitivity and specificity. Normal conventional or automatic electromyography does not exclude a myopathy. For diagnostic purposes, electromyography will be followed by muscle biopsy and DNA analysis in most of the cases.     

Identification of a rat testis-specific gene encoding a potential rat outer dense fibre protein

Histamine H2-receptor antagonistic properties of the anti-ulcer agent T-593, (+/-)-(E)-1-[2-hydroxy-2-(4-hydroxyphenyl)ethyl] -3-[2[[[5-(methylamino)methyl-2-furyl]methyl]thio] ethyl] -2-(methylsulfonyl)guanidine, were investigated on [14C]aminopyrine accumulation in isolated canine gastric mucosal cells and compared with those of ranitidine and famotidine. The potency of T-593-inhibition of [14C]aminopyrine accumulation stimulated by 10(-4) M histamine, with an IC50 value of 1.85 x 10(-6) M, was approximately 5 times greater than that of ranitidine, but half that of famotidine. T-593 did not affect [14C]aminopyrine accumulation stimulated by carbachol or dibutyryl-cAMP. T-593 depressed the maximal response of the histamine concentration-response curve with a dose-related displacement to the right, indicating that the nature of the H2-receptor antagonism of T-593 was insurmountable and included non-competitive inhibition. The inhibitory efficacy of T-593 was time-dependent and was retained after the cells were washed. The inhibitory potency of (-)-S-T-593, one of the enantiomers, on the [14C]aminopyrine accumulation stimulated by histamine was approximately twice that of racemic T-593 and it also behaved as an insurmountable H2-receptor antagonist. However, the potency of (+)-R-T-593 was markedly weak. These results suggest that T-593 has H2-receptor antagonism that is insurmountable and this agent slowly associates and dissociates with the receptor in isolated canine gastric mucosal cells and that the specific substance causing H2-receptor antagonism is (-)-S-T-593.     

Cloning, sequence and expression of the gene encoding the malolactic enzyme from Lactococcus lactis

Many lactic acid bacteria can carry out malolactic fermentation. This secondary fermentation is mediated by the NAD- and Mn(2+)-dependent malolactic enzyme, which catalyses the decarboxylation of L-malate to L-lactate. The gene we call mleS, coding for malolactic enzyme, was isolated from Lactococcus lactis. The mleS gene consists of one open reading frame capable of coding for a protein with a calculated molecular mass of 59 kDa. The amino acid sequence of the predicted MleS gene product is homologous to the sequences of different malic enzymes. Bacterial and yeast cells expressing the malolactic gene convert L-malate to L-lactate.     

Cloning and characterization of a gene from Escherichia coli encoding a transketolase-like enzyme that catalyzes the synthesis of D-1-deoxyxylulose 5-phosphate, a common precursor for isoprenoid, thiamin, and pyridoxol biosynthesis

For many years it was accepted that isopentenyl diphosphate, the common precursor of all isoprenoids, was synthesized through the well known acetate/mevalonate pathway. However, recent studies have shown that some bacteria, including Escherichia coli, use a mevalonate-independent pathway for the synthesis of isopentenyl diphosphate. The occurrence of this alternative pathway has also been reported in green algae and higher plants. The first reaction of this pathway consists of the condensation of (hydroxyethyl)thiamin derived from pyruvate with the C1 aldehyde group of D-glyceraldehyde 3-phosphate to yield D-1-deoxyxylulose 5-phosphate. In E. coli, D-1-deoxyxylulose 5-phosphate is also a precursor for the biosynthesis of thiamin and pyridoxol. Here we report the molecular cloning and characterization of a gene from E. coli, designated dxs, that encodes D-1-deoxyxylulose-5-phosphate synthase. The dxs gene was identified as part of an operon that also contains ispA, the gene that encodes farnesyl-diphosphate synthase. D-1-Deoxyxylulose-5-phosphate synthase belongs to a family of transketolase-like proteins that are highly conserved in evolution.     

Identification of a structural gene encoding a metallothionein-like domain that includes a putative regulator protein for Streptomyces protease gene expression

An open reading frame (termed ORF-PR) encoding a metallothionein-like domain-including protein was found upstream of a previously identified Streptomyces chymotrypsin-type protease gene (sam-P20). Promoter and terminator activities of ORF-PR were detected using the promoterless Streptomyces tyrosinase gene as a reporter gene and expression of ORF-PR was supposed to occur before that of sam-P20 gene. Frameshift mutation analysis showed that the ORF-PR product might act as a repressive regulator of the sam-P20 gene.     

Cloning and characterization of a maize cDNA encoding phytoene desaturase, an enzyme of the carotenoid biosynthetic pathway

The work presents the results of researches of binding and degradation of 125I-Insulin by erythrocyte receptors in the patients with essential hypertension and healthy patients after glucose intake. In order to obtain full representation of the pattern of changes the serum IRI and glucose concentrations were assayed. Binding and degradation of 125I-Insulin by erythrocyte receptors were determined with the method described by Gambhir (1977), modified by the authors. The modification consisted in usage of constant concentrations of iodized insulin (0.9 pg/0.1 ml) and bovine insulin (2.4 I.U./0.1 ml). Before administration of glucose and in 30, 60 and 120 minutes after, venous blood was collected from ulnar vein. All examined persons were in sitting position during the trial of glucose intake. Obtained results show, that blood insulin level in the patients with essential hypertension is statistically significantly higher than in healthy persons of similar anthropometric characteristics. Binding of 125I-Insulin to erythrocyte receptors in fasting state is statistically significantly lower. Degradation after glucose intake in the patients shows decreasing tendency, while in healthy persons--growing tendency.     

Identification and characterization of phoN-Sf, a gene on the large plasmid of Shigella flexneri 2a encoding a nonspecific phosphatase

A gene encoding a nonspecific phosphatase, named PhoN-Sf, was identified on the large virulence plasmid (pMYSH6000) of Shigella flexneri 2a YSH6000. The phosphatase activity in YSH6000 was observed under high-phosphate conditions. However, it was found that low-phosphate conditions induced a slightly higher level of activity. The nucleotide sequence of the phoN-Sf region cloned from pMYSH6000 possessing the phoN-Sf gene encoded 249 amino acids with a typical signal sequence at the N terminus. The deduced amino acid sequence of the PhoN-Sf protein revealed significant homology to sequences of nonspecific acid phosphatases of other bacteria, such as Providencia stuartii (PhoN, 83.2%), Morganella morganii (PhoC, 80.6%), Salmonella typhimurium (PhoN, 47.8%), and Zymomonas mobilis (PhoC, 34.8%). The PhoN-Sf protein was purified, and its biochemical properties were characterized. The apparent molecular mass of the protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was calculated to be 27 kDa. The 20 amino acids at the N terminus corresponded to the 20 amino acid residues following the putative signal sequence of PhoN-Sf protein deduced from the nucleotide sequence. The PhoN-Sf activity had a pH optimum of 6.6, and the optimum temperature was 37 degrees C. The enzymatic activity was inhibited by diisopropyl fluorophosphate, N-bromosuccinimide, or dithiothreitol but not by EDTA. The subcellular localization of the PhoN-Sf protein in YSH6000 revealed that the protein was found predominantly in the periplasm. Examination of Shigella and enteroinvasive Escherichia coli strains for PhoN-Sf production by immunoblotting with the PhoN-specific antibody and for the presence of phoN-Sf DNA by using a phoN-Sf probe indicated that approximately one-half of the strains possessed the phoN-Sf gene on the large plasmid and expressed the PhoN-Sf protein. The Tn5 insertion mutants of YSH6000 possessing phoN-Sf::Tn5 still retained wild-type levels of invasiveness, as well as the subsequent spreading capacity in MK2 epithelial cell monolayers, thus suggesting that the PhoN-Sf activity is not involved in expression of the virulence phenotypes of Shigella strains under in vitro conditions.