Effects of transglucosidase on diabetes, cardiovascular risk factors and hepatic biomarkers in patients with type 2 diabetes: a 12-week, randomized, double-blind, placebo-controlled trial.

In this 12-week, randomized, double-blind, placebo-controlled trial, the efficacy and safety of transglucosidase (TGD) were compared with placebo in patients with type 2 diabetes mellitus (T2DM). At 12 weeks, TGD 300 mg/day and TGD 900 mg/day significantly reduced HbA1c (0.18 and 0.21%) and insulin concentration (19.4 and 25.0 pmol/l), respectively, vs. placebo. TGD 300 mg/day and TGD 900 mg/day also significantly reduced low-density lipoprotein cholesterol (0.22 and 0.17 mmol/l, respectively). TGD 900 mg/day significantly reduced triglyceride by 0.24 mmol/l and diastolic blood pressure by 8 mmHg. Placebo was associated with a significant increase from baseline in body mass index, alanine aminotransferase and aspartate aminotransferase (0.17 kg/m(2) , 3 and 2 U/l, respectively), whereas TGD was not. TGD 300 mg/day significantly increased high-molecular-weight adiponectin by 0.6 µg/ml. Adverse events did not differ significantly between the groups. TGD resulted in lowering of HbA1c and blood insulin level and improvements in metabolic and cardiovascular risk factors in T2DM.

Cloning of the RNase H genes from a metagenomic DNA library: identification of a new type 1 RNase H without a typical active-site motif.

AIMS: The study aimed to combine a metagenomics approach with complementary genetics to identify novel bacterial genes with orthologous functions, with the identification of novel RNase H genes as a test case. METHODS AND RESULTS: A metagenomic DNA library was prepared from leaf-and-branch compost and used to screen for the RNase H genes by their abilities to complement the temperature-sensitive growth phenotype of the rnhA mutant Escherichia coli strain MIC3001. Determination of the nucleotide sequences of the cloned DNA fragments allowed us to identify 12 different genes encoding type 1 RNases H. Eleven of them encode novel RNases H, which show 40-72% amino acid sequence identities to those available from database. One of them lacks a typical DEDD/E active-site motif, which is almost fully conserved in various RNases H. CONCLUSIONS: Functional screening of environmental DNA without cultivation of microbes is a useful procedure to isolate novel RNase H genes. SIGNIFICANCE AND IMPACT OF THE STUDY: One of the identified RNase H genes had no sequence similarity to a previously assumed conserved motif, suggesting multiple catalytic mechanisms exist. This test case illustrates that metagenomics combined with complementary genetics can identify novel genes that are orthologous without sequence similarity to those from cultivated bacteria.

Cis- and trans-acting factors for transcription of the adenovirus 12 E1A gene.

Cis- and trans-acting factors were analyzed for transcription of the adenovirus 12 E1A gene possessing two sites for transcription initiation. These sites are located at nucleotide positions 306 and 445 with respect to the left end of the viral genome as position 1. The template activity of DNAs with various deletions at the 5'-upstream region of the E1A gene was examined in a cell-free system using a nuclear extract of Ehrlich ascites tumor cells. A DNA region specifically stimulating transcription initiated at the site distal to the E1A coding sequence was found located between positions 1 and 166. No DNA sequence affecting transcription from a proximal start-site appeared to be present in the region between positions 1 and 378. DNaseI-footprinting indicated that factors present in the extract bind to two distinct DNA segments, both of which are located within a region stimulating distal transcription. Two footprints were observed, one between positions 19 and 55 and the other between 77 and 94. The former footprint was inhibited by synthetic oligonucleotides containing a sequence recognized by nuclear factor I and the latter contained a sequence similar to one present in the B-enhancer of polyoma virus. Competition of in vitro transcription with synthetic oligonucleotides indicated (a) nuclear factor(s) bound to the region between positions 19 and 55 to be responsible for stimulating distal transcription of the adenovirus 12 E1A gene.

Nuclear factor I stimulates transcription of the adenovirus 12 E1A gene in a cell-free system.

Binding to the cis-acting region of NF-I-like protein and/or NF-III-like protein was previously suggested to be responsible for the preferential stimulation of transcription from distal start-site of the adenovirus 12 E1A gene in a cell-free system. In this study, nuclear extracts of Ehrlich ascites tumor cells depleted of NF-I-like protein were found to lose activity to stimulate the E1A gene transcription. This activity was recovered when NF-I purified from HeLa cells with no contamination of NF-III was supplemented. It is thus evident that NF-I is involved in stimulating distal transcription of the adenovirus 12 E1A gene. Moreover, activities for both stimulating the E1A gene transcription and binding to a region recognized by NF-I did not apparently exist in nuclear extracts of a cell line expressing the adenovirus 12 E1A gene. These results suggest that transcription of the adenovirus 12 E1A gene may possibly be autoregulated at least in part through modulation of the activity of NF-I.

[Bactericidal action of lomefloxacin a new pyridonecarboxylic acid derivative].

Lomefloxacin (NY-198) [(+/-)-1-ethyl-6,8-difluoro-1,4-dihydro-7-(3-methyl-1-piperazinyl)-4-oxo -3- quinolinecarboxylic acid hydrochloride] strongly inhibited the growths of not only Gram-negative Escherichia coli but Gram-positive Staphylococcus aureus. In vivo and in vitro experiments showed deoxyribonucleic acid (DNA) synthesis was specifically inhibited by this drug in E. coli.

Interactions of factors bound at two different sites in the 5'-upstream region of the adenovirus 12 E1A gene.

A nuclear extract of Ehrlich ascites tumor cell contains factors binding to two distinct sites in the 5'-upstream region of the adenovirus 12 E1A gene. The gel shift assay was performed for characterization of the binding factors with oligo DNA probes containing sequences corresponding to these sites. The specific binding of a factor to one probe was enhanced when the other oligo DNA was present in excess in the binding reaction. Thus possibly, protein-protein interactions between factors may mutually prevent their binding to target sequences.

Molecular cloning of the gene for bilirubin oxidase from Myrothecium verrucaria and its expression in yeast.

Myrothecium verrucaria bilirubin oxidase (EC 1.3.3.5) is an enzyme catalyzing the oxidation of bilirubin to biliverdin and other substrates. We have purified bilirubin oxidase from the medium of M. verrucaria and determined its partial amino acid sequence and isolated cDNA fragment amplified by polymerase chain reaction using oligonucleotide primers designed on the basis of the partial amino acid sequence. The gene for bilirubin oxidase has been cloned from a genomic library using the cDNA fragment as a probe. The gene encodes a precursor of bilirubin oxidase consisting of 572 amino acid residues, which comprises the prepro-region of 38 amino acid residues and the mature enzyme of 534 amino acid residues containing one cysteine. Five introns were found within the coding region. Sequence comparison of bilirubin oxidase with other blue copper proteins (laccase, ascorbate oxidase, human ceruloplasmin, plastocyanin, and azurin) revealed the presence of four domains corresponding to potential copper ligands. We have expressed this bilirubin oxidase gene in Saccharomyces cerevisiae under the repressible acid phosphatase promotor and found an active recombinant bilirubin oxidase, establishing the functional identity of the gene.

Conformation of bilirubin oxidase in native and denatured states.

The conformation of bilirubin oxidase (EC 1.3.3.5) from Myrothecium verrucaria was studied by circular dichroism (CD). The far-UV CD spectrum showed a single minimum at 215 nm and a maximum near 198 nm, suggesting the dominance of beta-sheets. There was another negative band at 187 nm that is absent from the spectra of model alpha-helix or beta-sheet. CD analysis by the method of Chang et al. agreed well with the estimates based on the Chou and Fasman sequence-predictive method, but the Provencher-Glöckner method of CD analysis agreed well with the sequence-predictive method of Garnier et al. At pH 12 the 215- and 187-nm bands completely disappeared and the protein was denatured. This denaturation was accompanied by the appearance of a large positive band at 250 nm, probably due to ionization of tyrosine residues. In 20 mM sodium dodecyl sulfate the magnitude of the 215-nm band increased, but the spectrum transformed to that of partial helices after heating at 100 degrees C. In 6 M guanidine hydrochloride the far-UV CD spectrum was monotonic and became more negative at the lower wavelength limit (near 212 nm), suggesting that the secondary structure of the protein was disrupted. However, the near-UV CD spectrum retained residual aromatic bands even after heating at 100 degrees C. Thus, our denaturation studies suggest that bilirubin oxidase has a rigid tertiary structure.

Molecular cloning of the gene for microbial transglutaminase from Streptoverticillium and its expression in Streptomyces lividans.

The microbial transglutaminase (TGase)-producing strains S-8112 [Agric. Biol. Chem., 53, 2613-2617 (1989)] was identified as a variant of Streptoverticillium mobaraense. We amplified a partial gene fragment by polymerase chain reaction (PCR) using oligonucleotides synthesized from the amino acid sequence of TGase, and cloned the gene for TGase using the PCR amplified fragment as a probe. The gene encoded a precursor of TGase consisting of 406 amino acid residues, which comprised the prepro region of 75 amino acid residues and the mature region of 331 amino acid residues. We expressed the TGase gene in Streptomyces lividans under a tyrosinase promoter, and found an active and mature recombinant enzyme, indicating the processing of the gene product.

Chemical synthesis of the gene for microbial transglutaminase from Streptoverticillium and its expression in Escherichia coli.

The gene coding for microbial transglutaminase (TGase) from Streptoverticillium, which consists of 331 amino acids, was chemically synthesized. The codons have been substituted for those mainly favored in yeast. Our strategy involved the construction of the TGase gene in five sections (54 oligomers) that contained unique restriction enzyme sites at both ends, which could readily be ligated to form the full-length product. The chemically synthesized gene was inserted downstream from the ompA signal peptide of the E. coli expression vector, pIN-III-ompA, which carries lpp and lac promotors. The resultant plasmid directed the expression of TGase, with the activity being secreted mainly into the periplasmic space of E. coli. The induced gene product was identical with native TGase in size and in immunological properties, though the enzyme activity was low.

Transcription stimulation of the adenovirus type 12 E1a gene in vitro by the 266-amino-acid E1A protein.

We previously showed that the 13S but not the 12S mRNA product of the E1a gene of the highly oncogenic type 12 adenovirus (Ad12) stimulates the expression of its own gene. In this study, the mechanism for the autoregulation of the Ad12 E1a gene was investigated in vitro. The 266-amino-acid E1A protein of Ad12 was synthesized in yeast cells and purified as a 57-kDa polypeptide. The purified Ad12 E1A protein stimulated transcription from the proximal promoter of its own gene but had almost no effect on that from the distal promoter. A 35-bp upstream region including a TATA box for the proximal promoter seemed to be sufficient for transcription stimulation by the E1A protein. The Ad12 E1A protein formed a complex with a TATA box-binding protein (TBP), as does the E1A protein of nononcogenic Ad serotypes. Moreover, the E1A protein significantly reduced the binding of TBP to a TATA sequence, while it did not affect the DNA-binding activity of nuclear factor I, a stimulatory protein of the distal transcription of the Ad12 E1a gene. These results suggest that the 13S mRNA product of the Ad12 E1a gene regulates the transcription of its own gene by modulating the activity of TBP.

Analysis of the expression of two phosphoglycerate kinase genes in a mouse cultured cell line during activation and inactivation of the c-myc gene.

The effect of a change in the concentration of c-myc protein on the expression of genes for two phosphoglycerate kinase isozymes was investigated. The steady state levels of messenger ribonucleic acids (mRNAs) for sperm-type and non-sperm-type proteins were determined by blot hybridization using the RNA of the mouse cell line 38-2 containing the inducible rat c-myc gene cultured under various conditions. Without induction the c-myc gene. mRNA for non-sperm-type protein was detected at a level that remained essentially constant during both activation and inactivation of the c-myc gene. mRNA for sperm-type protein was not detected in 38-2 cells cultured under any conditions used. Change in the amount of c-myc protein alone does not appear to bring about a switch of the expression of the two phosphoglycerate kinase genes during spermatogenesis in mouse testis.

Transcription switch of two phosphoglycerate kinase genes during spermatogenesis as determined with mouse testis sections in situ.

In order to elucidate the mechanistic interpretations underlying differential expression of the two phosphoglycerate kinase (PGK) genes during mammalian spermatogenesis, localization of its mRNAs in mouse testis sections was determined by in situ hybridization. MRNA for nonsperm-type PGK-1 was identified in nongerminal Leydig and Sertoli cells, spermatogonia, and spermatocytes, but was not detected in spermatids. In contrast, mRNA for sperm-type PGK-2 was notable in leptotene spermatocytes, becoming most abundant in pachytene spermatocytes. It was amply present in spermatids only up to step 10, completely disappearing after step 12. It is possible to assume that a transcription switch of the two PGK genes ensued following the onset of meiosis. These findings taken together with previous observations indicate that differential expression of the two PGK genes during mammalian spermatogenesis is regulated at the transcriptional and post-transcriptional levels.