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Y Sakai

Publications and source records attributed to Y Sakai.

At least 451 records · Page 25Linked to original sources
PubMed1995

A novel formaldehyde oxidation pathway in methylotrophic yeasts: methylformate as a possible intermediate.

A considerable amount of methylformate accumulated in the culture medium of methanol-grown methylotrophic yeasts. Methylformate is considered as an intermediate in a novel formaldehyde oxidation pathway. Through investigations with Pichia methanolica, methylformate formation was found to be catalysed by a new type of alcohol dehydrogenase, which was named methylformate synthase. When cells were grown on a relatively high concentration of methanol or exposed to a high concentration of formaldehyde, formation of methylformate was enhanced and the level of methylformate synthase in the cells increased. How methylformate synthase is involved in formaldehyde oxidation and formaldehyde detoxification is discussed.

Alcohol Dehydrogenase↗
PubMed1995

Mapping the mouse ZP3 combining site for sperm by exon swapping and site-directed mutagenesis.

During fertilization in mice, sperm bind to mouse ZP3 (mZP3), a M(r) approximately 83,000 glycoprotein present in the ovulated egg extracellular coat, or zona pellucida. Sperm recognize and bind to specific serine/threonine-linked (O-linked) oligosaccharides present at the mZP3 combining site for sperm. Binding to mZP3 induces sperm to undergo a form of exocytosis, the acrosome reaction. To map the mZP3 combining site for sperm, we examined the effect of exon swapping and site-directed mutagenesis on the glycoprotein's two activities, sperm binding and induction of the acrosome reaction. Stably transfected embryonal carcinoma cell lines were established that synthesized recombinant glycoproteins and secreted them into the culture medium. The glycoproteins were partially purified from culture medium and assayed for sperm-binding and acrosome reaction-inducing activities. Results of these assays suggest that glycosylation of one or more of five serine residues, clustered together in a polypeptide region encoded by mZP3 gene exon 7, is required for activity. Interestingly, this polypeptide region exhibits considerable sequence divergence during evolution and may be related to the proposed role for oligosaccharides in species-specific gamete adhesion during mammalian fertilization.

Amino Acid Sequence↗
PubMed1995

Expression of Saccharomyces adenylate kinase gene in Candida boidinii under the regulation of its alcohol oxidase promoter.

The methylotrophic yeast, Candida boidinii, was investigated as a new efficient host for heterologous gene expression. The Saccharomyces cerevisiae adenylate kinase gene (ADK1) was used as the first example for heterologous enzyme production in C. boidinii. C. boidinii cells were transformed with plasmids harboring the S. cerevisiae ADK1 gene under the alcohol oxidase (C. boidinii AOD1) promoter. The chromosome-integrant strains produced adenylate kinase protein corresponding to 22%-28% of the total soluble proteins in an enzymatically active form. When the three-copy integrative transformant was grown for 60 h on methanol-glycerol medium in a 1.5-l jar fermentor, adenylate kinase was produced intracellularly with a yield of up to 2 milligrams culture medium. As the expression of the S. cerevisiae ADK1 in C. boidinii was under similar regulation to that of the C. boidinii AOD1, the previously cloned 1.7-kb AOD1 promoter fragment was proved to harbor sufficient cis elements for AOD1 regulation and found to be an efficient promoter for heterologous gene expression.

Adenylate Kinase↗
PubMed1995

Isolation of cDNAs for mouse phenol and bilirubin UDP-glucuronosyltransferases and mapping of the mouse gene for phenol UDP-glucuronosyltransferase (Ugtla1) to chromosome 1 by restriction fragment length variations.

The mouse gene for phenol UDP-glucuronosyltransferase (UDPGT; Ugtla1) was mapped at 42 cM on chromosome 1, a position identical to that of the gene for bilirubin UDPGT (Ugtla1), from linkage analysis of a three-point cross test with Idh-1, En-1, and Ugtla1 as marker genes. The cDNAs for mouse phenol and bilirubin UDPGTs, isolated after amplification by PCR, shared an identical 3'-half region. Our results strongly suggest that mouse bilirubin and phenol UDPGTs are expressed from a single gene and involve alternative splicing events. We also detected duplication of the gene for phenol UDPGT in all mouse strains examined with the exception of MOL-MIT and SUB-SHH.

Animals↗
PubMed1995

Sprouting of non-sympathetic myelinated and unmyelinated fibres in response to chronic sympathetic denervation in the pineal gland of the Chinese hamster, Cricetulus griseus.

We have examined the effects of chronic sympathetic denervation on non-sympathetic myelinated and unmyelinated fibres in the superficial pineal gland of the Chinese hamster (Cricetulus griseus), using LM, EM and immunohistochemistry. The results suggest that non-sympathetic, myelinated and unmyelinated fibres enter the superficial pineal gland at its distal portion by way of the nervi conarii, and that these fibres are immunoreactive for calcitonin gene-related peptide or substance P. Non-sympathetic, myelinated and unmyelinated fibres in the superficial pineal gland increased in number following chronic superior cervical ganglionectomy. The number of unmyelinated fibres in the nervi conarii also increased in ganglionectomized animals. Thus, the numerical increase of calcitonin gene-related peptide or substance P fibres found in the superficial pineal gland after long-term sympathectomy may be due to sprouting of these fibres. It is speculated that the growth of non-sympathetic, myelinated and unmyelinated fibres and myelination of the former fibres occurring after sympathectomy are caused by nerve growth factor-related mechanisms.

Animals↗
PubMed1995

Effect of dehydroepiandrosterone on glucose uptake in cultured human fibroblasts.

Dehydroepiandrosterone (DHEA) and its sulfate derivative (DHEA-S) reportedly have antidiabetic and antiobesity effects. The effect of DHEA on glucose uptake in cultured human fibroblasts was examined. Incubation of cells with supraphysiologic concentrations of DHEA (10(-5) mol/L) for > or = 10 hours enhanced 2-deoxyglucose (2-DG) uptake significantly (P < .05). Supraphysiologic concentrations of insulin (10(-7) mol/L) increased the sensitivity of glucose uptake to DHEA. Conversely, the sensitivity of glucose uptake to insulin was increased by incubating cells with 10(-6) mol/L DHEA. Both the abundance of transcripts encoding glucose transporter-1 (Glut-1) and the maximal velocity (Vmax) of 2-DG transport were increased in cultured fibroblasts incubated with DHEA. Cultured fibroblasts expressed a specific binding factor with low affinity for [3H]DHEA (maximal number of binding sites, 18,496 sites per cell; Kd, 298 nmol/L). Other androgen hormones exerted a less-marked effect on glucose uptake; DHEA-S had no effect. These results suggested that DHEA increases Glut-1 mRNA through binding to a specific factor in cultured human fibroblasts and thereby stimulates glucose uptake in these cells.

Adult↗
PubMed1995

Type 1 angiotensin II receptors of adrenal tumors.

The present study was designed to clarify the transcriptional regulation of the human type 1 angiotensin II receptor (AT1) gene and its pathophysiological roles in steroidogenesis by adrenal tumors. A cDNA encoding type 1 angiotensin II receptor (AT1) was isolated from a human liver cDNA library encoding a protein of 359 amino acids with seven transmembrane segments. It is very likely that human has only one type of AT1 receptor, in contrast with rodents. A genomic clone containing 217 bp of exon 1 and 2558 bp of the 5'-flanking region of human AT1 receptor gene was isolated. Its proximal promoter region contained putative TATA and GC boxes, CRE and AP1 sites. Aldosterone-producing adenoma (APA) contained significantly higher levels of mRNA for AT1 and ACTH receptors than normal tissues adjacent to APA. There were no mutations within the cytoplasmic third loops of AT1 and ACTH receptors in APAs examined. APA showed increased expression of the mRNA for P450c11 and decreased expression of the mRNA for P450c17. These results suggest that renin-independent overproduction and clinically observed ACTH-dependent production of aldosterone in APAs may results from the enhanced transcription of P450c11 and ACTH receptor genes. The mechanism of the discordantly increased expression of AT1 receptor in APA remains to be clarified.

Adenoma↗
PubMed1995

Expression of the retinoblastoma gene product in human hepatocellular carcinoma.

Using the mouse anti-human retinoblastoma gene product (pRB) monoclonal antibody, PMG3-245, pRB was detected immunohistocytochemically in human hepatocellular carcinoma (HCC) tissues and a human HCC cell line, designated OCUH-16, recently established in our laboratory. This antibody reacted with human pRB and yielded a single band of approximately 110 kd from cultured OCUH-16 cells. The granules that stained for pRB were found mostly in the HCC cell nuclei, with a few granules observed in the rough endoplasmic reticulum by electron microscopy. Most of the stained granules were located in the euchromatin-rich areas. The percentage of OCUH-16 cells that expressed pRB or DNA polymerase alpha (DNA-PA) decreased over time as the number of OCUH-16 cells increased. The number of HCC cells that stained for pRB in the biopsy specimens from 11 patients varied and pRB expression was well maintained in early and advanced HCC. The level of pRB expression did not correlate with the differentiation of HCC cells or the clinical prognosis. The expression of pRB statistically correlated with that of DNA-PA (P < .01; r = .92). Some sinusoidal cells also stained for pRB. These findings imply that large deletions in the pRB gene are rare in the initiation or promotion of HCC. The correlation between pRB and DNA-PA may suggest that stained pRB participates in the proliferation of both HCC and non-HCC cells.

Adult↗
PubMed1995

Comparable sensitivities for detection of HIV-1 reverse transcriptase (RT) and other polymerases by RT assays requiring no radioisotopic materials.

An improved non-radioisotopic (Non-RI) reverse transcriptase (RT) assay with a template-primer-immobilized microtiter plate is described, which has greater sensitivity than the former Non-RI RT assay previously described. Non-RI and commercially available non-radioactive (Non-RA) RT assays were compared for their ability to detect various polymerases. Two RTs from Rous-associated virus 2 (RAV-2) and avian myeloblastosis virus (AMV), one polymerase from Escherichia coli (Pol-I) and one recombinant RT of human immunodeficiency virus type 1 (HIV-1) were assessed. Two HIV-1 samples in a culture supernatant and pelleted virion suspended in Triton X-100 solution were measured. The Non-RI RT assay was one hundred times more sensitive by RAV-2 and Pol-I polymerases, and one thousand times more sensitive by the Non-RA assay than by the AMV RT. The Non-RI RT assay was 10, 16 and 64 times more sensitive than the Non-RA assay for measuring recombinant HIV-1 RT, pelleted virus and virus suspended in culture medium, respectively. To explain the discrepancy, it is shown that free biotin, such as in culture medium, disturbs the assay system of the Non-RA RT assay, but not the Non-RI assay. The present assay can be used to clarify the inhibitory mechanism of an anti-HIV-1 substance.

Animals↗
PubMed1995

Aromatase in bone cell: association with osteoporosis in postmenopausal women.

To clarify the possible action of adrenal androgen on bone cell, the existence, characteristics and regulation of aromatase in human osteoblast-like osteosarcoma cells (HOS) and primary cultured osteoblast-like cells from normal human bones (HO) were examined in this study. Significant positive correlation between bone mineral density (BMD) and serum dehydroepiandrosterone sulfate (DHEA-S) was found in 120 postmenopausal women (51-99 years old) but no correlation was seen between BMD and serum estradiol (E2). In subset analysis, strongly positive correlation of serum DHEA-S and estrone (E1) with BMD was observed in postmenopausal women aged less than 69 years old. Administration of DHEA to ovariectomized rat significantly increased BMD and decreased relative osteoid volume in femur. These in vivo findings strongly suggested that serum adrenal androgen may be converted to estrogen in peripheral organ, especially, osteoblast and be important steroids to maintain BMD. [3H]DHEA was converted to [3H]androstenedione and [3H]androstenedione to [3H]estrone in primary cultured human osteoblast. Osteoblast-like cells showed aromatase activity, and an apparent Km and the Vmax were 4.74 +/- 0.78 nM (mean +/- SD, n = 3) and 0.83 +/- 0.79 fmol/mg protein/h for HOS, and 4.6 +/- 2.9 nM and 279 +/- 299 fmol/mg protein/h (mean +/- SD, n = 19) for HO, respectively. The aromatase activity was significantly increased by dexamethasone in a dose-dependent manner. Reverse transcription-polymerase chain reaction analysis revealed that dexamethasone increased the transcript of P450AROM gene. Osteoblast-specific promoters were also determined. Dexamethasone and 1 alpha,25-dihydroxyvitamin D3 synergistically enhanced aromatase activity and P450AROM mRNA expression. These results demonstrate that adrenal androgen, DHEA, is converted to E1 in osteoblast by P450AROM which is positively regulated by glucocorticoid and 1 alpha,25-dihydroxyvitamin D3 and important to maintain BMD in the 6 to 7th decade, after menopause.

Aged↗
PubMed1995

Functioning free muscle transplantation to the lower leg.

Many authors have reported on transplantation of functioning free muscle to the upper extremities, but few reports are available on functioning free muscle transfers to the lower extremities, where injuries have hitherto been treated mainly with tendon transfers. However, under suitable conditions, better results can be obtained using a functioning free musculocutaneous flap transfer to treat extensor muscle loss in the lower leg. The authors reconstructed the anterior extensors in two patients suffering from lower-leg necrosis. Ankle-joint active dorsiflexion was restored, and dorsiflexion in the ankle joint of about 0 degree was achieved; however, recovery of ankle-joint range of motion was limited by contracture of the ankle joint, due to associated injury, even when the strength and excursion of the grafted muscle were sufficient.

Adult↗
PubMed1995

Harvest of sural nerve grafts using the endoscope.

Endoscopic surgical techniques were employed in 5 patients to harvest the sural nerve atraumatically with a minimal number of skin incisions. The medial sural nerve was harvested by two 2-cm-wide transverse incisions made under endoscopic-magnified visualization, which allowed the surgeon to see the detailed structure of the nerve and surrounding tissue. This method requires minimal traction of the nerve during nerve dissection, and postoperative pain and swelling were less than encountered with conventional methods, because the method we have employed is less invasive and atraumatic.

Endoscopy↗
PubMed1995

Endoscopic nasofrontal disjunction.

Disjunction of the nasofrontal junction was performed under endoscopic control in three patients who underwent a monoblock nasal framework osteotomy and two patients who underwent a modified Le Fort type II osteotomy through a small midscalp incision, a stub wound at the root of the nose, and an intercartilaginous incision. A bilateral intercartilaginous incision and nasal root stub wound were used in one case. An osteotomy of the nasal root was performed using a specially designed detachable osteotomy consisting of a thin staff and large handle. The end of the staff was inserted through the access incision and pulled through a stub wound created in the nasal root. The staff was then attached to the handle, and the osteotomy was performed under endoscopic control. This approach minimizes the surgical scar, the area of the subperiosteal dissection, and postoperative swelling of the face, in comparison with the coronal approach.

Adolescent↗
PubMed1995

Approaching the zygoma with an endoscope.

A hemicoronal or coronal incision is required in combination with a buccal incision to expose the zygoma, including zygomatic process of the temporal bone, and lateral orbital rim under direct visualization. An endoscope was used to expose zygoma in selected cases to reduce the length of the incision. The zygomatic bone and arch were exposed and corrected using an endoscope in eight cases of zygomatic fracture and one case of zygomatic reduction through a small incision just behind the temporal hairline together with a buccal incision. The results obtained were satisfactory with no complications. Clear, magnified endoscopic visualization made it possible to correct some deformities located in this area without using a coronal incision. Endoscopic procedures result not only in dramatically less postoperative scarring because of the small access incisions, but they also cause less damage to vascular and lymphatic circulation and, therefore, are associated with less postoperative edema. However, the fixation of the zygomatic arch using the percutaneous method with an endoscope is a complicated procedure that requires more time than conventional procedures.

Adolescent↗
PubMed1995

Plasma thrombomodulin levels in coronary sinus blood in patients with coronary artery disease.

Plasma thrombomodulin (TM) levels in coronary sinus blood were measured in 15 patients with coronary artery disease who underwent elective PTCA (group A). TM was also measured in eight patients with coronary artery disease (group B) and six patients without coronary artery disease (group C), who only underwent diagnostic coronary angiography. The baseline level was 21.6 +/- 3.4 U/ml in group A, and 21.0 +/- 4.9 in group B, and both levels were slightly higher (P < 0.05) than those in group C (15.1 +/- 3.3). In group A, the level increased to 27.5 +/- 8.3 (P < 0.01) immediately after PTCA and then returned to 22.2 +/- 5.1 by 24 h. In groups B and C, levels showed no change immediately after angiography. In group A, the percentage increase in the TM level immediately after PTCA from that before the procedure correlated with the number of balloon inflations (r = 0.76, P < 0.01). In conclusion, the plasma TM level in coronary sinus blood might indicate endothelial damage due to atherosclerotic coronary artery disease and an increase in the level after PTCA might directly indicate vascular injury by balloon inflation.

Aged↗
PubMed1995

Versatility of a microsurgical free-tissue transfer from the forearm in treating the difficult nose.

Ten selected patients with nasal defects in the covering, lining, or framework were treated by a vascularized free-tissue transfer from the forearm. A vascularized skin flap, fascia, and split radius were transferred in accordance with the shape of the defects. All flaps "took" perfectly, and satisfactory results were obtained. The grafted bones were stable over a follow-up period ranging from 1 to 6 years; however, some problems did arise in the course of treatment, including poor matches between the flap and surrounding facial skin, postoperative drooping of the flap used in the lining, and conspicuous donor sites. However, despite these drawbacks, microvascular free-tissue transfer from the forearm has proven to be a good choice for the restoration of a nose that is difficult to reconstruct by more conventional methods.

Adolescent↗
PubMed1995

Low-count organisms concealed by dominant uropathogenic organisms in urine of patients with asymptomatic bacteriuria.

We compared organisms isolated by selective culture media with those isolated by routine bacteriological methods using the same urine specimens. Urine was obtained by suprapubic bladder aspiration from 84 patients suspected of having asymptomatic bacteriuria, including 31 catheterized patients. The selective media included mannitol salt agar, Enterococcus faecalis agar, nalidixic acid cetrimide agar, and desoxycholate hydrogen sulfide lactose agar with polymyxin B or cefsulodin. Colony counts on the selective media approximated to those on heart infusion agar, except for desoxycholate hydrogen sulfide agar with polymyxin B, where the number of organisms was underestimated. In 24 patients the selective media detected a total of 32 strains not reported by the hospital laboratory. The unreported strains included coagulase-negative staphylococci (41%), other cocci (38%), enterobacteriaceae (9%) and glucose-nonfermenting gram-negative bacilli (9%) and fungi (3%), with the number of organisms being mostly < 10(5)/ml. The majority of the unreported strains coexisted with dominant gram-negative bacilli or enterococci. These results indicate that low-count organisms coexisting with dominant organisms tend to be missed by the routine bacteriological methods.

Adolescent↗