Biomedical subjects

S Yamano

Publications and source records attributed to S Yamano.

At least 91 records · Page 5Linked to original sources

PubMed1994

Cloning and expression of the gene encoding alpha-acetolactate decarboxylase from Acetobacter aceti ssp. xylinum in brewer's yeast.

Acetobacter aceti ssp. xylinum genomic library was constructed using cosmid pJB8 in Escherichia coli. The gene encoding alpha-acetolactate decarboxylase (ALDC) was isolated from the library by direct measurement of ALDC activity. The ALDC gene was expressed by its own promoter in E. coli. The nucleotide sequence was determined, and an open reading frame which may encode a protein composed of 304 amino acids with a molecular weight of 33,747 was found. A brewer's yeast was transformed with the YEp-type plasmid containing the ALDC gene placed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The laboratory-scale growth test confirmed that the total diacetyl concentration was considerably reduced by the transformant. The analysis of the wort indicates that the Acetobacter ALDC reduces the concentration of diacetyl more effectively than that of 2,3-pentanedione.

Amino Acid Sequence↗

PubMed1994

Construction of a brewer's yeast having alpha-acetolactate decarboxylase gene from Acetobacter aceti ssp. xylinum integrated in the genome.

alpha-Acetolactate decarboxylase (ALDC) gene from Acetobacter aceti ssp. xylinum has several possible initiation codons in the N-terminus. To determine the initiation codon of the ALDC giving the highest expression levels, glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter was linked just upstream of each possible initiation codon. The ALDC whose translation starts 130 bp downstream from the first ATG codon had the highest activity in yeast cells. When expression levels of the ALDC gene were compared using three strong yeast promoters of glycolytic genes, alcohol dehydrogenase I (ADC1), phosphoglycerate kinase (PGK) and GPD, the GPD promoter was the strongest. The ALDC gene was integrated in a ribosomal RNA gene of a brewer's yeast by co-transformation with an expression plasmid of G418-resistance gene. The laboratory-scale growth test confirmed that the total diacetyl concentration was reduced in wort.

Base Sequence↗

PubMed1994

Cloning of the Acetobacter xylinum cellulase gene and its expression in Escherichia coli and Zymomonas mobilis.

A DNA fragment corresponding to carboxymethylcellulase activity of Acetobacter xylinum IFO 3288 was isolated and cloned in Escherichia coli, and the DNA sequence was determined. The DNA fragment sequenced had an open-reading frame of 654 base pairs that encoded a protein of 218 amino acid residues with a deduced molecular mass of 23,996 Da. The protein encoded in the DNA fragment expressed in E. coli hydrolyzed a carboxymethylcellulose. This gene was subcloned into the shuttle vector [pZA22; Misawa et al. (1986) Agric Biol Chem 50:3201-3203] between Zymomonas mobilis and E. coli. The recombinant plasmid pZAAC21 was introduced into Z. mobilis IFO 13756 by electroporation. The carboxymethylcellulase gene was efficiently expressed in both bacteria, although the level of expression in Z. mobilis was ten times greater than that in E. coli. Approximately 75% of the total carboxymethylcellulase activity detected in Z. mobilis was located in the periplasmic space (outside of the cytoplasmic space). Enzyme activity was not detected in the periplasmic space, but in the cytoplasm of E. coli.

Acinetobacter↗

PubMed1994

Successful freezing of unfertilized mouse oocytes and effect of cocultures in oviducts on development of in vitro fertilized embryos after thawing.

PURPOSE: To establish a freeze-thawing method for unfertilized oocytes with a high success rate, we examined several conditions for freeze-thawing. The effects of EDTA and cocultures in oviducts on the development of embryos fertilized in vitro after thawing were also studied. RESULTS: In the first experiment, unfertilized oocytes that were frozen in 1.5 M dimethylsulfoxide (DMSO) supplemented with 0.2 M sucrose by a slow freeze-thawing method showed the best results (fertilization rate, 71.9%; blastocyst rate per frozen oocyte, 18.8%). The proportion of embryos that developed to blastocysts was significantly higher when DMSO was added at 4 degrees C than at room temperature (39.4 vs 19.4%; P < 0.01). The addition of EDTA (10 microM) to the culture medium did not promote embryo development after fertilization in vitro. However, the rate of development of in vitro fertilized embryos to blastocysts after thawing was significantly higher when the embryos were cultured in oviducts in vitro than the rates in control cultures and those cultured with EDTA (blastocyst rate from fertilized oocytes, 71.4 vs 51.0 and 52.8%, respectively; P < 0.01). CONCLUSION: Unfertilized mouse oocytes can be cryopreserved successfully by a slow freeze-thawing method with the addition of 1.5 M DMSO and 0.2 M sucrose at low temperatures, and coculture with oviducts enhances the development of embryos that are fertilized in vitro after thawing.

Animals↗

PubMed1994

Metabolic engineering for production of beta-carotene and lycopene in Saccharomyces cerevisiae.

We have engineered a conventional yeast, Saccharomyces cerevisiae, to confer a novel biosynthetic pathway for the production of beta-carotene and lycopene by introducing the bacterial carotenoid biosynthesis genes, which are individually surrounded by the promoters and terminators derived from S. cerevisiae. beta-Carotene and lycopene accumulated in the cells of this yeast, which was considered to be a result of the carbon flow for the ergosterol biosynthetic pathway being partially directed to the pathway for the carotenoid production.

Carotenoids↗

PubMed1994

[A longitudinal study of brain atrophy and its relation with background factors and common carotid hemodynamics].

This longitudinal study was performed to clarify the relation between brain atrophy and common carotid hemodynamics. A total of 147 patients, including 70 males and 77 females, who had neither neurologic deficits nor organic lesions on computed tomography of the brain, were enrolled in this study. The ages of the patients ranged from 47 to 76 years (mean: 62 years) at the first diagnosis of brain atrophy. The patients were divided into three groups: 66 controls without hypertension or diabetes mellitus (Group I), 60 with hypertension (Group II) and 21 with both hypertension and diabetes mellitus (Group III). Brain atrophy was diagnosed by caudate head index (CHI). Mean blood flow volume (BF) at the right common carotid artery was measured by an ultrasonic quantitative flow measurement system. Both CHI and BF were measured twice at a mean interval 6.5 years. Increment in CHI per year (delta CHI) and decrement in mean blood flow volume per year (delta BF) were calculated. delta CHI of Group I and Group II had a significant relation with aging. delta CHI of Group III showed a larger increase than that of both Group I and Group II in subjects in the sixties. delta CHI had a significant relation with delta BF in each group. These results indicate that complication of both HT and DM, or decrement in mean blood flow volume may cause brain atrophy to progress.

Aged↗

PubMed1993

Functional expression of the Erwinia uredovora carotenoid biosynthesis gene crtl in transgenic plants showing an increase of beta-carotene biosynthesis activity and resistance to the bleaching herbicide norflurazon.

Among the enzymes involved in carotenoid biosynthesis, phytoene desaturase is considered to be a rate-limiting enzyme in this pathway and is also the target of many bleaching herbicides. This enzyme shows diversity concerning its function and amino acid homology among various organisms. The phytoene desaturase gene crtl of Erwinia uredovora was expressed, the 5'-region of which was fused to the sequence for the transit peptide of a pea Rubisco small subunit, in tobacco plants under the control of the CaMV 35S promoter. This chimeric gene product was targeted into chloroplasts and processed in the transgenic plants. The production and processing of the corresponding protein could be demonstrated by Western blotting. Immunogold localization showed that the location of the gene product Crtl was preferentially in the thylakoids. A radioactive labeling study using the leaves demonstrated enhanced activity for the synthesis of beta-carotene. In addition, the transgenic tobacco acquired elevated resistance to the bleaching herbicide norflurazon.

Amino Acid Sequence↗

PubMed1992

Expression in Escherichia coli, purification, and reactivation of the recombinant Erwinia uredovora phytoene desaturase.

A plasmid has been constructed by cloning the complete crtI gene encoding phytoene desaturase from Erwinia uredovora behind the lac Z promoter of pUC18 resulting in a reading frame for the full polypeptide with additional 9 amino acids at the N terminus. This plasmid mediated the overexpression of phytoene desaturase in transformed Escherichia coli. The overexpressed enzyme was sequestrated into inclusion bodies requiring urea treatment for solubilization. Purification to homogeneity was subsequently performed on a DEAE-cellulose column and by SDS-polyacrylamide gel electrophoresis. The purification scheme allowed the isolation of 5.3 mg of homogeneous desaturase protein from 100 ml of E. coli cell suspension. On SDS-polyacrylamide gel electrophoresis an apparent molecular mass of 56.2 kDa was determined. An antiserum raised against phytoene desaturase cross-reacted with the expressed protein and was employed to monitor the isolation steps. Upon removal of urea, desaturase activity was restored. The isolated desaturase catalyzed the conversion of 15-cis-phytoene to trans-lycopene as well as to bisdehydrolycopene. FAD was involved in desaturation, whereas NAD and NADP were inhibitory. This is the first time that a membrane-integrated carotenogenic enzyme has been purified and finally obtained in an active state.

Amino Acid Sequence↗

PubMed1992

Relationship among serum levels of luteinizing hormone, estradiol, and progesterone during follicle stimulation and results of in vitro fertilization and embryo transfer (IVF-ET).

Eighty-eight IVF-ET cycles were classified into four groups according to the results of IVF-ET (Group A-conceptional cycles, 10 cycles; Group B-cycles with cleaved oocytes, 58 cycles; Group C-cycles with fertilized oocytes, 9 cycles; Group D-cycles without fertilization, 11 cycles). Serum luteinizing hormone (LH), estradiol (E2), and progesterone (P) levels during follicle stimulation were studied in these groups. Patients participated in our IVF-ET program due to irreparable tubal damage. Follicle development was stimulated with a clomiphene-human menopausal gonadotropin (hMG)-human chorionic gonadotropin (hCG) regimen. Group C showed a low E2 response to follicle stimulation. Groups B and D showed significantly higher serum P levels on day 0 (the day of hCG injection) than Group A (Group A, 0.73 +/- 0.11, vs Groups B and D, 1.43 +/- 0.15 and 2.17 +/- 0.42 ng/ml; P less than 0.01). The effects of serum P and LH levels on the fertilization and pregnancy rates were studied. The pregnancy rate was not affected by the serum LH level but was only 2.7% in cycles in which serum P was 1.2 ng/ml on day 0, which was significantly lower than that in cycles in which serum P was less than 1.2 ng/ml on day 0 (19.1%) (P less than 0.05). The fertilization rate was significantly lower in the cycles with higher levels of serum P and/or LH than in cycles in which serum P was less than 1.2 ng/ml and serum LH was normal (50.5 vs 78.8%; P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Clomiphene↗

PubMed1992

Silver-induced frog skeletal muscle contraction and its modulation by calcium antagonists nifedipine and felodipine and calcium agonist Bay K 8644.

1. Ag+ induces one phase of a transient contracture in frog skeletal muscle (0.3-10 microM) and potentiates twitch tension when the fiber is given continuous stimulation. 2. The potentiation of fiber contraction by Ag+ is similar to the effect of Ca2+ antagonists nifedipine and felodipine. 3. Bay K 8644 (100 nM) potentiates and accelerates Ag(+)-induced tension development and the inactivation occurs more rapidly than in the control (Ag+ alone). 4. Two factors can be considered to be essential for the induction of Ag+ contracture: (1) a certain number of Ag+ ions must bind to free SH groups of the voltage sensor; and (2) the binding must occur within a limited time to raise the mechanical threshold to induce contracture. 5. All results suggest that Ag+ binding to crucial SH groups on the Ca2+ channel may be responsible for the activation of muscle contraction, potentiation, and the inhibition of excitation-contraction coupling in skeletal muscle.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1992

Effect of sperm-immobilizing antibodies on the acrosome reaction of human spermatozoa.

OBJECTIVE: To study by a triple stain technique the effect of sperm-immobilizing antibodies on the acrosome reaction of human spermatozoa. DESIGN: The spermatozoa were allowed to swim up and culture in a medium containing 7.5% (vol/vol) serum with sperm-immobilizing antibodies or control serum up to 6 hours. Sperm mobility was analyzed, and the percentage of live acrosome reacted spermatozoa was determined. SETTING: Samples were collected from patients referred to university hospital infertility clinics. MATERIALS: Serum samples were drawn from seven patients with sperm-immobilizing antibodies. All the sera were heat activated and stored at -40 degrees C until use. Semen samples were taken from two healthy donors. RESULTS: During culture for 6 hours, the percentage of live sperm showing the acrosome reaction increased significantly (P less than 0.01) in the control group but not in the sperm-immobilizing antibodies group. However, the inhibitory effect of sperm-immobilizing antibodies on the acrosome reaction was reversed when sperm was reincubated in medium with control serum (P less than 0.01). CONCLUSIONS: Sperm-immobilizing antibodies block fertilization at least in part by inhibiting the acrosome reaction of human spermatozoa.

Acrosome↗

PubMed1991

Lectin histochemistry of feline polyglucosan bodies.

Lectin histochemistry was conducted to identify sugar residues of the polyglucosan bodies (PGB) in the feline brain. PGB stained with Concanavalia ensiformis agglutinin (Con A) and peanut agglutinin, but not with soybean agglutinin, wheat germ agglutinin, Dolichos biflorus agglutinin, Ricinus communis agglutinin or Ulex europaeus agglutinin. In addition, PGB bound to Con A after periodate oxidation. These results suggest that feline PGB contain mannose and galactose in addition to glucose and that the partial component of PGB is derived from rough endoplasmic reticulum and the Golgi apparatus.

Aging↗

PubMed1991

Production of beta-carotene in Zymomonas mobilis and Agrobacterium tumefaciens by introduction of the biosynthesis genes from Erwinia uredovora.

The Erwinia uredovora crtB, crtE, crtI, and crtY genes required for beta-carotene biosynthesis were introduced by conjugal transfer into an ethanol-producing bacterium, Zymomonas mobilis, and a phytopathogenic bacterium, Agrobacterium tumefaciens, in which no carotenoid is synthesized. The transconjugants of Z. mobilis and A. tumefaciens carrying these genes appeared as yellow colonies and produced 220 and 350 micrograms of beta-carotene per g of dry weight, respectively, in the stationary phase in liquid culture.

Carotenoids↗

PubMed1991

Effect of prolactin in serum and follicular fluid on fertilization and cleavage of human oocyte.

To assess the effect of PRL on fertilization and cleavage of oocytes, PRL levels in serum and follicular fluid of normoprolactinemic women who had undergone in vitro fertilization and embryo transfer (IVF-ET) were determined, and the relationship between PRL levels and fertilization and cleavage rates was studied. A transient increase in serum PRL at the ovulatory phase was found in about half of the IVF-ET patients during clomiphene stimulation, and increased levels of PRL in serum and follicular fluid at the ovulatory phase may suppress the fertilization and cleavage of oocytes.

Cell Division↗

PubMed1991

Vascular effects of isosorbide dinitrate on the common carotid artery in human volunteers.

The effects of 20 mg of orally administered isosorbide dinitrate (ISDN, Isocoronal R; CAS 87-33-2) on cerebral hemodynamics were investigated in 14 normal healthy volunteers (average age 32.6 years). The common carotid artery hemodynamics in the right supraclavicular region were measured using an ultrasonic quantitative flow measurement system. 2 h after drug administration, systolic blood pressure (15.5 mmHg, p less than 0.001) and mean blood pressure (7.2 mmHg, p less than 0.02) decreased significantly, but diastolic blood pressure did not change. A decrease in blood flow volume (2.85 ml/s, p less than 0.001) and blood flow velocity (7.00 cm/s, p less than 0.001), and an increase in mean vessel diameter (0.19 mm, p less than 0.05), and volume elasticity (0.68 x 10(5) dyn/cm2, p less than 0.02) were observed with ISDN. Mean vessel diameter increased gradually and reached its maximum at 4 h. However, the other maximal effects of ISDN were exerted at about 2 h. The ultrasonic quantitative flow measurement system was found to be useful in the case of repetitive measurements of the common carotid artery hemodynamics. It has been demonstrated that ISDN (20 mg/subject, p.o.) increases the diameter of the common carotid artery, although it decreases the blood flow in the common carotid artery.

Adult↗

PubMed1990

The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes.

Three cDNAs, designated IIA3, IIA3v, and IIA4, coding for P450s in the CYP2A gene subfamily were isolated from a lambda gt11 library prepared from human hepatic mRNA. Only three nucleotide differences and a single amino acid difference, Leu160----His, were found between IIA3 and IIA3v, indicating that they are probably allelic variants. IIA4 displayed 94% amino acid similarity with IIA3 and IIA3v. The three cDNAs were inserted into vaccinia virus, and recombinant viruses were used to infect human hepatoma Hep G2 cells. Only IIA3 was able to produce an enzyme that had a reduced CO-bound spectrum with a lambda max at 450 nm. This expressed enzyme was able to carry out coumarin 7-hydroxylation (turnover number of 15 min-1) and ethoxycoumarin O-deethylation. cDNA-expressed IIA3v and IIA4 failed to incorporate heme and were enzymatically inactive. Analysis of IIA proteins in human liver microsomes, using antibody against rat IIA2, revealed two proteins of 49 and 50 kDa, the former of which appeared to correlate with human microsomal coumarin 7-hydroxylase activity. A more striking correlation was found between IIA mRNA and enzyme activity. The rat antibody was able to completely abolish coumarin 7-hydroxylase activity in 12 liver samples. In addition, kinetics of coumarin metabolism in two livers were monophasic over the substrate concentration tested. Km values obtained from human liver (2.3 microM) were similar to those obtained from lysates of hepatoma cells expressing IIA3 (3.6-7.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1990

Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B1.

Twelve forms of human hepatic cytochrome P450 were expressed in hepatoma cells by means of recombinant vaccinia viruses. The expressed P450s were analyzed for their abilities to activate the potent hepatocarcinogen aflatoxin B1 to metabolites having mutagenic or DNA-binding properties. Five forms, P450s IA2, IIA3, IIB7, IIIA3, and IIIA4, activated aflatoxin B1 to mutagenic metabolites as assessed by the production of His revertants of Salmonella typhimurium in the Ames test. The same P450s catalyzed conversion of aflatoxin B1 to DNA-bound derivatives as judged by an in situ assay in which the radiolabeled carcinogen was incubated with cells expressing the individual P450 forms. Seven other human P450s, IIC8, IIC9, IID6, IIE1, IIF1, IIIA5, and IVB1, did not significantly activate aflatoxin B1 as measured by both the Ames test and the DNA-binding assay. Moreover, polyclonal anti-rat liver P450 antibodies that crossreact with individual human P450s IA2, IIA3, IIIA3, and IIIA4 each inhibited aflatoxin B1 activation catalyzed by human liver S-9 extracts. Inhibition ranged from as low as 10% with antibody against IIA3 to as high as 65% with antibody against IIIA3 and IIIA4. These results establish that metabolic activation of aflatoxin B1 in human liver involves the contribution of multiple forms of P450.

Aflatoxin B1↗

PubMed1990

Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli.

The most important function of carotenoid pigments, especially beta-carotene in higher plants, is to protect organisms against photooxidative damage (G. Britton, in T. W. Goodwin, ed., Plant Pigments--1988, 1988; N. I. Krinsky, in O. Isler, H. Gutmann, and U. Solms, ed., Carotenoids--1971, 1971). beta-Carotene also functions as a precursor of vitamin A in mammals (G. A. J. Pitt, in I. Osler, H. Gutmann, and U. Solms, ed., Carotenoids--1971, 1971). The enzymes and genes which mediate the biosynthesis of cyclic carotenoids such as beta-carotene are virtually unknown. We have elucidated for the first time the pathway for biosynthesis of these carotenoids at the level of enzyme-catalyzed reactions, using bacterial carotenoid biosynthesis genes. These genes were cloned from a phytopathogenic bacterium, Erwinia uredovora 20D3 (ATCC 19321), in Escherichia coli and located on a 6,918-bp fragment whose nucleotide sequence was determined. Six open reading frames were found and designated the crtE, crtX, crtY, crtI, crtB, and crtZ genes in reference to the carotenoid biosynthesis genes of a photosynthetic bacterium, Rhodobacter capsulatus; only crtZ had the opposite orientation from the others. The carotenoid biosynthetic pathway in Erwinia uredovora was clarified by analyzing carotenoids accumulated in E. coli transformants in which some of these six genes were expressed, as follows: geranylgeranyl PPiCrtB----prephytoene PPiCrtE----phytoeneCrtI---- lycopeneCrtY----beta-caroteneCrtZ----zeaxanthinCrtX--- -zeaxanthin-beta- diglucoside. The carotenoids in this pathway appear to be close to those in higher plants rather than to those in bacteria. Also significant is that only one gene product (CrtI) for the conversion of phytoene to lycopene is required, a conversion in which four sequential desaturations should occur via the intermediates phytofluene, zeta-carotene, and neurosporene.

Amino Acid Sequence↗