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Biomedical subjects

T Beppu

Publications and source records attributed to T Beppu.

At least 289 records · Page 16Linked to original sources
PubMed1986

Nucleotide sequence of afsB, a pleiotropic gene involved in secondary metabolism in Streptomyces coelicolor A3(2) and "Streptomyces lividans".

The nucleotide sequence of afsB from Streptomyces coelicolor A3(2), a pleiotropic gene which positively controls the biosynthesis of A-factor and the pigmented antibiotics actinorhodin and undecylprodigiosin in S. coelicolor A3(2) and "Streptomyces lividans," was determined. The determinant of the afsB gene, which includes the putative AfsB protein consisting of 243 amino acids, was mapped functionally by tests for A-factor and pigment production in "S. lividans" and S. coelicolor A3(2) after introduction of recombinant plasmids containing various restriction endonuclease fragments on the vector plasmids pIJ41 and pIJ702. The putative AfsB protein contains two regions separated by 167 residues which resemble conserved domains of known DNA-binding proteins. High-resolution nuclease S1 protection mapping revealed that the afsB mRNA, approximately 1,300 base pairs (bp) long, which was determined by Northern blot hybridization, had its start point 340 bp upstream of the putative methionine start codon. The Northern hybridization experiment also suggested that the afsB gene was constitutively transcribed throughout growth. Also shown by the Northern hybridization was the presence of an unidentified gene with an extraordinary amount of 880-bp mRNA located downstream from afsB. Dot hybridization with the brown pigment production genes, possibly involved in polyketide biosynthesis, as the probe suggested that the afsB gene did not stimulate transcription of the pigment production genes. In Southern blot DNA-DNA hybridization analysis with the afsB sequence as the probe, sequences exhibiting various degrees of homology were found in several Streptomyces spp. A DNA sequence showing strong homology to the afsB in Streptomyces griseus FT-1, a high streptomycin producer, behaved like an extrachromosomal element, homologous to the afsA gene, a structural gene for A-factor biosynthesis.

4-Butyrolactone↗
PubMed1986

Cloning and characterization of the streptothricin-resistance gene which encodes streptothricin acetyltransferase from Streptomyces lavendulae.

The streptothricin-resistance gene of Streptomyces lavendulae No. 1080 was cloned in S. lividans using pIJ41 as a vector. From subcloning experiments, the 1.6 kb BamH I fragment was determined to encode the structural gene. The cell extracts of S. lividans carrying the gene on the plasmid pKS7 had activity to inactivate streptothricin in the presence of S-acetyl coenzyme A, indicating that the gene product was streptothricin acetyltransferase.

Acetyltransferases↗
PubMed1986

[Combination of radiation and PVB chemotherapy for intracranial malignant germ cell tumor].

Intracranial malignant germ cell tumors such as embryonal carcinoma, endodermal sinus tumor, and choriocarcinoma are neoplasms of poor prognosis in the pediatric age group. Recently evidences of effectiveness of combination chemotherapy using cisplatin, vinblastine, and bleomycin (PVB therapy) have been reported. The authors experienced two cases with these tumors treated with radiation therapy and PVB therapy. PVB therapy was performed along with irradiation in hoping the effect of radiation sensitizer of cisplatin. Both patients showed no signs of tumor recurrence and no re-elevation of tumor makers even in the cerebrospinal fluid after more than ten months from the onset. Cisplatin has radiation sensitizing effect besides antitumor activity, but its permeability through the blood-CSF barrier is very poor. Also true is that germ cell tumors often disseminate in the cerebrospinal fluid. From these points, combination of irradiation and chemotherapy using cisplatin seems reasonable, though the superiority of this radiochemotherapy to simple PVB therapy can not be concluded from the present experience. Side effects of this combination therapy were the same as PVB therapy alone and tolerable.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1985

Auramine O as a fluorescent probe for the binding of basic drugs to human alpha 1-acid glycoprotein (alpha 1-AG). The development of a simple fluorometric method for the determination of alpha 1-AG in human serum.

A cationic fluorescent dye, auramine O (AO), exhibited an intense increase in fluorescence after binding to human alpha 1-acid glycoprotein (alpha 1-AG). The interaction between AO and the protein was studied by fluorescence spectroscopy and by equilibrium dialysis. AO binds to the protein via a single site with a dissociation constant of 24 microM. Various basic drugs such as chlorpromazine, imipramine, desipramine, quinidine, propranolol and lidocaine, which are known to bind to the protein, competitively inhibited the AO binding to the protein. The dissociation constants of these basic drugs obtained from such inhibitory experiments were comparable to those obtained with other methods (equilibrium dialysis, quenching of protein intrinsic fluorescence, and the difference spectrophotometric method) and from the literature. It is concluded that AO may be a useful fluorescent probe that binds to a single basic drug binding site on alpha 1-AG. In addition, a simple fluorometric method for the determination of alpha 1-AG in serum was developed using AO, and the validity of this method was confirmed by comparing it with the conventional radial immunodiffusion method.

Adult↗
PubMed1985

Serum concentrations of glucuronidated and sulfated bile acids in children with cholestasis.

Serum concentrations of nonglucuronidated-nonsulfated, glucuronidated, and sulfated bile acids in 9 control children and 16 children with cholestasis were quantitated by mass fragmentography. Total bile acid levels in control children were 19.55 +/- 2.78 mumol/liter (mean +/- SEM), and glucuronidated and sulfated bile acids comprised 2.6 +/- 0.5 and 17 +/- 3.1%, respectively. In 9 patients with congenital biliary atrasia, total bile acid levels were 167.34 +/- 11.18 mumole/liter of which 2.1 +/- 0.3% were glucuronidated and 15 +/- 1.4% were sulfated. Lithocholic and 3 beta-hydroxy-5-cholenoic acids, which have hepatotoxic effects, were presented in only small amounts in cholestatic children, and they were almost all glucuronidated or sulfated. The percentages of glucuronidated bile acids in control and cholestatic children were lower than in healthy and cholestatic adults, which may be explained by the lower activity of UDP-glucuronyltransferase in neonatal liver.

Adolescent↗
PubMed1985

Determination of 3 beta-hydroxy-5-cholenoic acid in serum of hepatobiliary diseases--its glucuronidated and sulfated conjugates.

3 beta-Hydroxy-5-cholenoic acid in the serum of control subjects and 62 patients with various hepatobiliary diseases was quantitated by mass fragmentography after separation into nonglucuronidated-nonsulfated, glucuronidated, and sulfated fractions. Deuterium-labeled deoxycholic acid and its glucuronide and sulfate were used as internal standards. Mean concentrations of total 3 beta-hydroxy-5-cholenoic acid in serum (mumole/liter) were as follows: Control subjects (14), 0.184; obstructive jaundice (15), 6.783; liver cirrhosis, compensated (12), 0.433, and decompensated (12), 1.636; chronic hepatitis (12), 0.241; and acute hepatitis (11), 2.364. Most of the 3 beta-hydroxy-5-cholenoic acid was glucuronidated or sulfated. Only in patients with obstructive jaundice did glucuronidation (60 +/- 14%) exceed sulfation (31 +/- 14%), sulfation exceeding glucuronidation in the others. The UDP-glucuronyltransferase might have different substrate specificities for 3 beta-hydroxy-5-cholenoic acid and other common bile acids, especially in the cholestatic state.

Adult↗
PubMed1985

Subterminal hydroxylation of fatty acids by a cytochrome P-450-dependent enzyme system from a fungus, Fusarium oxysporum.

The cell-free extract of a cytochrome P-450-producing fungus, Fusarium oxysporum, was found to catalyze the hydroxylation of fatty acids. Three product isomers were formed from a single fatty acid. The products from lauric acid were identified by mass-spectrometry as 9-, 10-, and 11-hydroxydodecanoic acids, and those from palmitic acid as 13-, 14-, and 15-hydroxyhexadecanoic acids. The ratio of the isomers formed was 50 : 36 : 14 in the case of laurate hydroxylation, and 37 : 47 : 16 in the case of palmitate. The reaction was dependent on both NADPH (or NADH) and molecular oxygen,and was strongly inhibited by carbon monoxide, menadione, or the antibody to purified Fusarium P-450. Further, lauric acid induced a type I spectral change in purified Fusarium P-450. Further, lauric acid induced a type I spectral change in purified Fusarium P-450 with an apparent Kd of 0.3 mM. The hydroxylase activity together with cytochrome P-450 could be detected in both the soluble and microsome fractions, and the activity was almost proportional to the amount of cytochrome P-450 reducible with NADPH. It can be concluded from these results that Fusarium P-450 reducible with NADPH. It can be concluded from these results that Fusarium P-450 is involved in the (omega-1)-, (omega-2)-, and (omega-3)-hydroxylation of fatty acids catalyzed by the cell-free extract of the fungus.

Antibodies↗
PubMed1985

The reaction mechanisms and substrate-stereospecificities of L-alloisocitrate dehydrogenase and oxalosuccinate decarboxylase.

A sequential reaction was suggested for the conversion of L-alloisocitrate to alpha-oxoglutarate by an enzyme complex of L-alloisocitrate dehydrogenase and oxalosuccinate decarboxylase from Pseudomonas strain No. 2, during which oxalosuccinate was not released from the enzyme-substrate complex. The stereochemistry of oxalosuccinate formed by L-alloisocitrate dehydrogenase and decarboxylated by oxalosuccinate decarboxylase was opposite to that of the substrate for D-isocitrate dehydrogenase. Incubation of L-alloisocitrate with the dehydrogenase and decarboxylase in deuterium oxide provided [3-2H]-alpha-oxoglutarate, the configuration of which turned out to be the same as that produced by D-isocitrate dehydrogenase from D-isocitrate. The data suggested that enol form of alpha-oxoglutarate was involved as an intermediate in decarboxylation of oxalosuccinate by oxalosuccinate decarboxylase. L-Alloisocitrate dehydrogenase was shown to react with pro-S proton of NADH.

Alcohol Oxidoreductases↗
PubMed1985

Construction and application of a promoter-probe plasmid that allows chromogenic identification in Streptomyces lividans.

We cloned a Streptomyces coelicolor A3(2) DNA fragment which directed synthesis of a brown pigment, presumably a shunt product in the actinorhodin biosynthetic pathway, on the plasmid vector pIJ41 in Streptomyces lividans. The pigment production was observed only when the DNA fragment was inserted downstream from a functional promoter sequence. By subcloning the fragment together with in vitro manipulation, a promoter-probe plasmid vector (pARC1) with a unique BamHI cloning site was constructed that allows chromogenic identification of transcriptional control signals in Streptomyces lividans based on the expression of the cloned pigment gene(s). The Escherichia coli tac (trp-lac hybrid) promoter, consisting of 92 base pairs and a promoter region including the leader sequence of erythromycin resistance gene (ermC) on staphylococcal plasmid pE194, when ligated in the correct orientation in the BamHI site of pARC1, promoted expression of the cloned pigment gene(s) in Streptomyces lividans, whereas the Saccharomyces cerevisiae GAL7 promoter did not. In the case of the ermC, induction of the pigment production by the addition of either erythromycin or lincomycin, but not virginiamycin, was observed. The system was also shown to be useful and convenient in isolating transcriptional control signals of Streptomyces chromosomal DNA and estimating their activities.

Cloning, Molecular↗
PubMed1985

Profiles of bile acids and their glucuronide and sulphate conjugates in the serum, urine and bile from patients undergoing bile drainage.

Bile acid profiles in serum, urine, and bile from patients undergoing bile drainage and the changes of serum bile acids after bile drainage were studied. Bile acids were separated into non-glucuronidate-non-sulphate, glucuronidated, and sulphated fractions and were measured by mass fragmentography using conjugates of deuterium labelled bile acids as internal standards. Glucuronidated and sulphated bile acids contribute 14-32% and 16-44% of serum bile acids, 4-11% and 61-82% of urine bile acids and 0.2-1% and 0.3-2% of biliary bile acids respectively. After bile drainage the concentration of serum non-glucuronidated-non-sulphated bile acids decreased more rapidly than glucuronidated and sulphated bile acids. There was little biliary excretion of the glucuronidated and sulphated bile acids. Such conjugation appears to have a role in facilitating bile acid excretion by the urinary route.

Aged↗
PubMed1985

Demalonyl derivatives of guanidylfungin A and copiamycin: their synthesis and antifungal activity.

Guanidylfungin A was chemically modified by alkylation, reduction and/or demalonylation. Demalonylmethylguanidylfungin A became soluble in water and showed approximately eight-fold higher activity against fungi and Gram-positive bacteria than guanidylfungin A along with strongly fungicidal effect. Similarly, copiamycin was converted to demalonylmethylcopiamycin, which also showed higher antifungal activity than copiamycin itself.

Antifungal Agents↗
PubMed1985

Leptomycins A and B, new antifungal antibiotics. III. Mode of action of leptomycin B on Schizosaccharomyces pombe.

Mode of action of leptomycin B (LMB), a new antifungal antibiotic, was studied with Schizosaccharomyces pombe. A low concentration of LMB caused inhibition of cell division, producing elongated cells with morphologically altered nuclei and several cell plates, while it inhibited nucleic acid synthesis in intact cells at 100-fold higher concentration. Addition of LMB during G2 phase in synchronous culture blocked following events in cell cycle. Analysis of the effect of LMB on cdc mutants suggested the antibiotic inhibited some specific step, possibly in M phase just prior to nuclear division.

Antifungal Agents↗
PubMed1985

The cloned Streptomyces bikiniensis A-factor determinant.

By cleavage with restriction endonucleases and cloning the resultant fragments, the A-factor determinant cloned from streptomycin-producing Streptomyces bikiniensis (Horinouchi et al., J. Bacteriol. 158: 481 approximately 487, 1984) was narrowed down to a 1.1-kilobase (kb) fragment. A hybrid multicopy plasmid (pAFB15) carrying the 1.1-kb fragment conferred A-factor production in a large quantity to A-factor-deficient mutants of S. bikiniensis and Streptomyces griseus as well as afsA mutants of Streptomyces coelicolor A3(2). A transcriptional control signal in the 1.1-kb fragment was identified by using a promoter-probe vector pARC 1. Plasmid pAFB15 also caused A-factor production with a marked gene dosage effect in four different Streptomyces strains which originally had no ability to produce A-factor and no DNA sequence homologous to the S. bikiniensis A-factor determinant, suggesting that precursors of A-factor are common metabolites in streptomycetes and that acquisition of only a single key enzyme encoded by the 1.1-kb fragment is sufficient for Streptomyces strains to synthesize A-factor.

4-Butyrolactone↗