Enzyme inhibitors to increase poly-3-hydroxybutyrate production by transgenic tobacco

Chemical regulation of secondary-metabolite synthesis was investigated through the improvement of poly-3-hydroxybutyrate (PHB) production in transgenic tobacco plants by the use of enzyme inhibitors. Two tobacco lines, BC3 and rCAB8, that produce PHB in both the cytosol and plastids were used. An acetyl-CoA carboxylase inhibitor, D-(+)-Quizalofop-ethyl, increased PHB accumulation in both lines 2-fold. The accumulation rate of plastidial PHB in the rCAB8 line was 2.5-fold higher than that of cytosolic PHB in the BC3 line. A specific inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, mevastatin, also increased PHB accumulation but only in the BC3 line. These results indicated that chemical regulation of the native metabolic flows by the specific enzyme inhibitors increased secondary-metabolite production in the transgenic tobacco plants we used.     

Hygrolidin induces p21 expression and abrogates cell cycle progression at G1 and S phases

Hygrolidin family antibiotics showed selective cytotoxicity against both cyclin E- and cyclin A-overexpressing cells. Among them, hygrolidin was the most potent and inhibited growth of solid tumor-derived cell lines such as DLD-1 human colon cancer cells efficiently more than that of hematopoietic tumor cells and normal fibroblasts. FACS analysis revealed that hygrolidin increased cells in G1 and S phases in DLD-1 cells. While hygrolidin decreased amounts of cyclin-dependent kinase (cdk) 4, cyclin D, and cyclin B, it increased cyclin E and p21 levels. Hygrolidin-induced p21 bound to and inhibit cyclin A-cdk2 complex more strongly than cyclin E-cdk2 complex. Furthermore, hygrolidin was found to increase p21 mRNA in DLD-1 cells, but not in normal fibroblasts. Thus, hygrolidin inhibited tumor cell growth through induction of p21. In respect to p21 induction, inhibition of vacuolar-type (H+)-ATPase by hygrolidin was suggested to be involved.     

Somatic cell mutation in pediatric patients undergoing allogeneic bone marrow transplantation

In order to examine whether bone marrow transplantation (BMT) has genotoxic effects in vivo, mutant frequencies (Mfs) at the hypoxanthine-guanine phosphoribosyl transferase (Hprt) locus were evaluated. Thirty-seven pediatric patients who had received allogeneic BMT for various hematologic or immunologic disorders were enrolled. Nine out of the 37 patients (24.3%) were found to have Hprt-Mfs exceeding the 99% confidence limits calculated from observation of healthy controls. Among factors including gender, primary disease of the patient, donor-recipient histocompatibility relationship, age of donor, and total body irradiation as conditioning regimen, none was associated with an increased Hprt-Mf. In three patients who had chimerism in their peripheral blood after BMT, Hprt mutant clones turned out to be of donor- or recipient-origin. Mfs at the T-cell receptor (TCR) locus were examined in 28 patients. Four patients (14.3%) were found to have increased TCR-Mfs. However, there were not any patients who showed elevation of both Hprt-and TCR-Mfs. These data, taken together, suggest that BMT may cause genotoxicity in vivo in some patients.     

A RelA-SpoT homolog (Cr-RSH) identified in Chlamydomonas reinhardtii generates stringent factor in vivo and localizes to chloroplasts in vitro

A gene encoding a putative guanosine 3′,5′-bispyrophosphate (ppGpp) synthase–degradase, designated Cr-RSH, was identified in the unicellular photosynthetic eukaryote Chlamydomonas reinhardtii. The encoded Cr-RSH protein possesses a putative chloroplast-targeting signal at its NH₂-terminus, and translocation of Cr-RSH into chloroplasts isolated from C.reinhardtii was demonstrated in vitro. The predicted mature region of Cr-RSH exhibits marked similarity to eubacterial members of the RelA–SpoT family of proteins. Expression of an NH₂-terminal portion of Cr-RSH containing the putative ppGpp synthase domain in a relA, spoT double mutant of Escherichia coli complemented the growth deficits of the mutant cells. Chromatographic analysis of ³²P-labeled cellular mononucleotides also revealed that expression of Cr-RSH in the mutant bacterial cells resulted in the synthesis of ppGpp. SpoT, which catalyzes (p)ppGpp degradation, is dispensable in E.coli only if cells also lack RelA, which possesses (p)ppGpp synthase activity. The complementation analysis thus indicated that Cr-RSH possesses both ppGpp synthase and degradase activities. These results represent the first demonstration of ppGpp synthase–degradase activities in a eukaryotic organism, and they suggest that eubacterial stringent control mediated by ppGpp has been conserved during evolution of the chloroplast from a photosynthetic bacterial symbiont.     

Short tandem repeat polymorphism in the flanking region of the human phosphoglycerate kinase gene in a Japanese population

The human phosphoglycerate kinase (PGK1) gene is located within Xqll-Xql3 and is closely linked to the androgen receptor gene within a region implicated in a number of X-chromosome-linked urologic disorders. A polymorphism of a TATC short tandem repeat (STR) is present downstream from the PGK1 3' nuclease-sensitive site. We present the PGK1 flanking STR sequence and population genetic data for 190 Japanese males and 83 Japanese females. Ten STR alleles and 29 genotypes were identified in the population. Five alleles--*10, *11, *12, *13, and *14--were common in the Japanese with frequencies greater than 10%. No significant deviations from Hardy-Weinberg equilibrium were established. The power of discrimination was 0.993 for females and 0.819 for males; heterozygosity was 0.759 for females; and the polymorphic information content was 0.936. These data indicate that this STR locus shows a high degree of polymorphism in this Japanese population and may prove to be a useful genetic marker in forensic medicine, in determining the clonality of neoplasms, and potentially in studying predisposition to prostate cancer and other urologic diseases.     

Molecular characterization of mammalian dicarbonyl/L-xylulose reductase and its localization in kidney

In this report, we first cloned a cDNA for a protein that is highly expressed in mouse kidney and then isolated its counterparts in human, rat hamster, and guinea pig by polymerase chain reaction-based cloning. The cDNAs of the five species encoded polypeptides of 244 amino acids, which shared more than 85% identity with each other and showed high identity with a human sperm 34-kDa protein, P34H, as well as a murine lung-specific carbonyl reductase of the short-chain dehydrogenase/reductase superfamily. In particular, the human protein is identical to P34H, except for one amino acid substitution. The purified recombinant proteins of the five species were about 100-kDa homotetramers with NADPH-linked reductase activity for alpha-dicarbonyl compounds, catalyzed the oxidoreduction between xylitol and l-xylulose, and were inhibited competitively by n-butyric acid. Therefore, the proteins are designated as dicarbonyl/l-xylulose reductases (DCXRs). The substrate specificity and kinetic constants of DCXRs for dicarbonyl compounds and sugars are similar to those of mammalian diacetyl reductase and l-xylulose reductase, respectively, and the identity of the DCXRs with these two enzymes was demonstrated by their co-purification from hamster and guinea pig livers and by protein sequencing of the hepatic enzymes. Both DCXR and its mRNA are highly expressed in kidney and liver of human and rodent tissues, and the protein was localized primarily to the inner membranes of the proximal renal tubules in murine kidneys. The results imply that P34H and diacetyl reductase (EC ) are identical to l-xylulose reductase (EC ), which is involved in the uronate cycle of glucose metabolism, and the unique localization of the enzyme in kidney suggests that it has a role other than in general carbohydrate metabolism.     

Retrotransposon-mediated restoration of Chlorella telomeres: accumulation of Zepp retrotransposons at termini of newly formed minichromosomes

To elucidate the contribution of LINE-like retrotransposon Zepp elements to the formation and maintenance of chromosomal telomeres, newly formed minichromosomes in irradiated Chlorella vulgaris cells were isolated and structurally characterized. A minichromosome (miniV4) of ~700 kb in size contained a Zepp cluster taking the place of the telomeric repeats on one terminus, whereas the other end of this chromosome consisted of canonical telomeric repeats. The Zepp copies in this cluster were in a tandem array with their poly(A) tails towards the centromere. Another minichromosome Y32 (~400 kb in size) was shown to have several copies of Zepp elements on both termini. On the right arm terminus, two copies of Zepp were found in a tandem array with poly(A) tracts facing towards the chromosomal end. The poly(A) tail and the 3′-end of ~400 bp of the distal copy were replaced by the telomeric repeats. On the 5′-side of the proximal copy was another Zepp element in the reverse orientation. These newly formed telomeric structures are very similar to those previously found in the left arm of chromosome I and the terminus of an unidentified chromosome and support the model of Zepp-mediated restoration and maintenance of Chlorella telomeres.     

Cloning and characterization of the gene encoding RNA polymerase sigma factor sigma(54) of deep-sea piezophilic Shewanella violacea

We have recently reported that a sigma(54)-like factor recognizes a DNA element, designated as region A, upstream of a pressure-regulated operon in piezophilic Shewanella violacea strain DSS12 (Nakasone et al., FEMS Microbiology Lett. 176 (1999) 351-356). In this study, we isolated and characterized the rpoN gene of this piezophilic bacterium. The rpoN gene was found to encode a putative protein consisting of 492 amino acid residues with a predicted molecular mass of 55359 Da. Significant homology was evident comparing the rpoN sequence of S. violacea with that of Escherichia coli (62.8% identity), Vibrio anguillarum (61.7% identity) and Pseudomonas putida (57.0% identity). The DNA-binding domain at the C-terminus of sigma(54) is well conserved in the case of the S. violacea rpoN gene product and the helix-turn-helix motif and the RpoN box are also present. In addition, the conserved glutamine-rich domain is present at the N-terminus. sigma(54) in S. violacea was expressed at a relatively constant level under various growth conditions as determined by both primer extension and Western blotting analyses. By means of a recombinant plasmid, a hexahistidine-tagged derivative of the sigma(54) from strain DSS12 was overexpressed in Escherichia coli and purified to near homogeneity. An electrophoretic mobility shift assay demonstrated that the purified sigma(54) protein specifically recognizes region A in the above-mentioned pressure-regulated operon.     

Roles of His-79 and Tyr-180 of D-xylose/dihydrodiol dehydrogenase in catalytic function

Mammalian dimeric dihydrodiol dehydrogenase is identical with d-xylose dehydrogenase and belongs to a protein family with prokaryotic proteins including glucose-fructose oxidoreductase. Of the conserved residues in this family, either His-79 or Tyr-180 of d-xylose/dihydrodiol dehydrogenase has been proposed to be involved in the catalytic function. Site-directed mutagenesis was used to examine the roles of the two residues of the monkey enzyme. A mutant, Y180F, was almost inactive, but, similarly to the wild-type enzyme, exhibited high affinity for NADP(H) and fluorescence energy transfer upon binding of NADPH. The H79Q mutation had kinetically largest effects on K(d) (>7-fold increase) and K(m) (>25-fold increase) for NADP(H), and eliminated the fluorescence energy transfer. Interestingly, the dehydrogenase activity of this mutant was potently inhibited with a 190-fold increase in the K(m) for NADP(+) by high ionic strength, which activated the activity of the wild-type enzyme. These results suggest a critical role of Tyr-180 in the catalytic function of this class of enzymes, in addition to functions of His-79 in the coenzyme binding and chemical steps of the reaction.