Biomedical subjects

J Nakao

Publications and source records attributed to J Nakao.

At least 37 records · Page 2Linked to original sources

PubMed1989

The conformation of mature human alpha-amylase conditions its secretion from yeast.

The yeast Saccharomyces cerevisiae expresses the cloned cDNA (Amy) encoding human salivary alpha-amylase (Amy) under control of the yeast PHO5 promoter, and secretes the active enzyme into the culture medium. Two approaches were utilized to define the moiety of Amy, which is required for proper secretion and glycosylation. In one approach, chimeras were constructed with a variety of secretion signal sequences (yeast mating factor precursor sequence, yeast acid phosphatase signal sequence and human gastrin signal sequence) fused to the secretion signal-deleted Amy cDNA. The other approach involved analysis of a set of deletion series and a set of point mutations in the Amy-encoding region. The results showed that heterologous signal sequences were sufficient for proper secretion in yeast, irrespective of the insertion of some extra amino acids. In most cases, enzymes with deletions and Cys-465 substitution were not secreted, even though they had complete secretion signal sequences. Instead, they accumulated in the cell in a glycosylated form. Thus, proper secretion seems to require an appropriate conformation in the polypeptide moiety to be secreted.

Amino Acid Sequence↗

PubMed1988

Formation of gamma-amino-beta-hydroxybutyric acid from 2-hydroxyputrescine in rat brain.

gamma-Amino-beta-[3H]hydroxybutyric acid ([3H]-GABOB) was formed in rat brain from 2-[3H]-hydroxyputrescine that had been chemically synthesized from 2-oxoputrescine and [3H]sodium borohydride. After the injection of 2-[3H]hydroxyputrescine into the lateral ventricle of a rat brain, the rat was killed and then the brain was removed. [3H]GABOB in the brain was identified by a combination of ion-exchange chromatography, high-voltage paper electrophoresis, and recrystallization of the radioactive compound with authentic GABOB.

Animals↗

PubMed1987

Determination of putrescine in brain tissue using gas chromatography-mass spectrometry.

We used gas chromatography-mass spectrometry to assay putrescine in minute regions of single rat brains. Acid extraction, partial purification on Amberlite CG 120, and derivatization with pentafluoropropionic anhydride preceded the gas chromatography-mass spectrometry. A moving-needle solventless system and a direct inlet system were also used to increase sensitivity. Putrescine was measured accurately at the picomole level; the mean concentration of this polyamine in five regions of rat brain found by this method was 2.7-3.8 times higher than reported by other researchers.

Animals↗

PubMed1987

Expression of hepatitis B virus middle and large surface antigen genes in Saccharomyces cerevisiae.

The hepatitis B virus genome carries the surface antigen (SAg) gene and an open reading frame that encodes two SAg-related polypeptides: SAg with a 55-amino-acid N-terminal extension polypeptide and SAg with a 174-amino-acid N-terminal extension polypeptide. These are termed middle S and large S, respectively. These polypeptides or their glycosylated derivatives have been detected in Dane particles, but their chemical and biological properties have remained largely unknown because of their limited availability. We attempted to produce these proteins in Saccharomyces cerevisiae by placing the coding regions under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Yeast cells carrying middle S and large S coding sequences produced 33,000- and 42,000-dalton products, respectively, each of which reacted with anti-S antibody and bound to polymerized human serum albumin, in accordance with the known properties of pre-S proteins from particles in human sera (K. H. Heermann, U. Goldmann, W. Schwartz, T. Seyffarth, H. Baumgarten, and W. H. Gerlich, J. Virol. 52:396-402, 1984; A. Machida, S. Kishimoto, H. Ohnuma, K. Baba, Y. Ito, H. Miyamoto, G. Funatsu, K. Oda, S. Usuda, S. Togami, T. Nakamura, Y. Miyakawa, and M. Mayumi, Gastroenterology 86:910-918, 1984). The middle S polypeptide is glycosylated and can be assembled into particles whose size and density are similar to those of SAg. However, this polypeptide was highly susceptible to proteolytic degradation into 29,000- and 26,000-dalton polypeptides, of which only the former retained the binding activity to polymerized albumin. The large S polypeptides are nonglycosylated, relatively stable, and do not seem to assemble into particles by themselves.

Cloning, Molecular↗

PubMed1987

Hypothermia caused by antipsychotic drugs in a schizophrenic patient.

In a schizophrenic patient, hypothermia was caused by combined treatment with zotepine, biperiden, and fluphenazine, although combined treatment with zotepine and biperiden had caused no side effects. Other side effects closely resembled those in neuroleptic malignant syndrome.

Adult↗

PubMed1986

The effect of L-erythro-dihydroxyphenylserine injected into the lateral ventricle and the hypothalamus on the locomotor activity.

The effect of dihydroxyphenylserine (DOPS) on locomotor activity was studied using the Animex activity meter. One microgram of L-erythro-DOPS, a precursor of d-noradrenaline, was injected into the lateral ventricle once a day for one week or into the anterior hypothalamic area (AHA) once. After the intraventricular injection, the total locomotor activity (from 8:00 p.m. to 8:00 a.m.) of the rats injected with DOPS was significantly less than that of the rats injected with an artificial cerebrospinal fluid (5-ion). Analysis of the locomotor activity in consecutive 2-hr periods showed that the activity of the DOPS group during the time intervals of 10 p.m.-12 a.m., 12 a.m.-2 a.m. and 2 a.m.-4 a.m. was significantly less than that of the control group. After injection of DOPS or 5-ion into AHA, the total activity of the DOPS group was significantly less than that of the control. Analysis of the activity of the DOPS group for each 2-hr period between 10 p.m.-4 a.m. was also significantly less than that of the control. On the basis of these findings, the effect of DOPS in the brain noradrenergic system are discussed.

Animals↗

PubMed1986

Spontaneous release of gamma-aminobutyric acid formed from putrescine and its enhanced Ca2+-dependent release by high K+ stimulation in the brains of freely moving rats.

The spontaneous release of [3H] gamma-aminobutyric acid ([3H]GABA) in various areas of rat brain injected with [3H]putrescine was examined using a push-pull perfusion technique. The release in a 25-min perfusate was highest in the caudate-putamen. The effect of high K+ stimulation on the release of [3H]GABA formed from [3H]putrescine was examined in the caudate-putamen. The release was enhanced by high K+ solution in a Ca2+-dependent manner.

Animals↗

PubMed1986

Saccharomyces cerevisiae PHO5 promoter region: location and function of the upstream activation site.

Saccharomyces cerevisiae repressible acid phosphatase (PHO5) is induced when inorganic phosphate in the culture medium is depleted. To study the mechanism of this regulation, we constructed various deletions in the 5'-flanking region of the PHO5 gene. Two elements were revealed by this analysis: an upstream activation site (UAS) and a downstream element, both playing parts in the expression of this gene. The UAS is located between -384 and -292 upstream of the initiation codon and activates expression of the gene when inorganic phosphate is depleted. It consists of two homologous regions (UAS I and UAS II) that contain CTGCACAAATG and an adenine-plus-thymine-rich sequence, either one of which suffices for the effect. The downstream element includes a putative TATA box at -100 from the ATG codon and is necessary for efficient transcription and expression of the normal-sized PHO5 transcript. The distance between the UAS and the downstream element can be altered without causing loss of expression efficiency, and the action of the UAS is not affected by its orientation. These results are consistent with a model wherein UAS acts as a site of activation for transcription by interaction with a protein factor(s) that becomes active when inorganic phosphate is depleted from the culture medium.

Base Sequence↗

PubMed1986

Ultrastructural appearances of subcultured aortic smooth muscle cells migrating into Millipore filters.

The ultrastructures of vascular smooth muscle cells migrating in Millipore filters were examined using a modified Boyden chamber. The cells (seventh and eighth subculture) were obtained from explants of rat aorta. Very small numbers of bipolar-shaped cells migrated deeply into the filter toward a platelet suspension added to the lower compartment. The migrating cells were richer in intermediate filaments and microtubules than the cells on the filter. The cytoskeleton was oriented along the elongated cell body, and the cortical microfilamentous mat was disorganized along the entire cell periphery. These data are discussed.

Animals↗

PubMed1985

Enhancement of endogenous production of 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid in aortic smooth muscle cells by platelet-derived growth factor.

Platelet-derived growth factor (PDGF) has a chemotactic effect on smooth muscle cells, which is inhibited by lipoxygenase inhibitor caffeic acid. In order to study the role of endogenous lipoxygenase products of arachidonic acid on the chemotactic action of PDGF, effects of PDGF on the lipoxygenase pathway in smooth muscle cells were examined. Lipoxygenase products were analyzed by high-performance liquid chromatography. 15-, 5- and 12-lipoxygenase activities, in order of magnitude, were found in smooth muscle cell homogenate. However, when the lipoxygenase products were analyzed using intact cells prelabelled with [14C]arachidonic acid, only 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) was found to be produced endogenously. In addition, 12-HETE was not released into the medium. Treatment of the cells with PDGF increased the endogenous production of 12-HETE. The amounts of intracellular 12-HETE in PDGF-treated cells were 126, 132 and 146% at 1, 3, and 10 hr's after the initiation of PDGF treatment, respectively, when control value at each time point was considered as 100%. Caffeic acid (10(-4) M) completely inhibited the PDGF effect on 12-HETE production. However, PDGF treatment did not significantly alter the 12-lipoxygenase activity. These results suggest that the stimulatory effect of PDGF on 12-HETE production was not mediated by the activation of 12-lipoxygenase activity. Since 12-HETE itself is a potent chemoattractant for smooth muscle cells, the present dat strongly suggest that 12-HETE could be an important intracellular mediator of the chemotactic action of PDGF on aortic smooth muscle cells.

Animals↗

PubMed1985

Long-term treatment of postmenopausal osteoporosis with active vitamin D3, 1-alpha-hydroxycholecalciferol (1 alpha-OHD3) and 1, 24 Dihydroxycholecalciferol (1, 24(OH)2D3).

The effects of active vitamin D3 analogues on radial mineral content (RMC) in postmenopausal osteoporosis were examined. Seventy eight subjects with postmenopausal osteoporosis were divided into 5 groups; Group 1 (n = 23) as the control group and Group 2 (n = 27), Group 3 (n = 8), Group 4 (n = 9) and Group 5 (n = 11) which were given 1 microgram of 1, 24(R) (OH)2D3 per day, 1 microgram of 1, 24(S)(OH)2D3 per day, 0.5 and 1 microgram of 1 alpha-OHD3 per day for 6 to 24 months, respectively. After 3-months administration of these drugs, RMC values were significantly increased in Groups 2 (102.8 +/- 1.8%), 4 (103.9 +/- 3.3%) and 5 (114.2 +/- 3.6%), when compared with the controls (97.9 +/- 2.4%). RMC in Group 3 (97.9 +/- 2.4%) was not significantly different from the control value. The administration of 1 alpha-OHD3 caused in increase in RMC in a dose-related manner. A rapid decrease in RMC was observed after withdrawal of the treatment in Groups 2, 4, and 5. A subsequent increase in RMC was observed after re-administration of 1 alpha-OHD3 and 1, 24(R)(OH)2D3. Serum Ca levels were increased in the group treated with 1, 24(R)(OH)2D3 and were decreased after the discontinuation of 1 alpha-OHD3 administration. Serum A1-P activity was decreased by treatment with 1 alpha-OHD3 (1 microgram per day) and a subsequent increase was observed in both groups treated with 1, 24(R)(OH)2D3 and 1 alpha-OHD3. Serum PTH levels were decreased by the administration of 1, 24(R)(OH)2D3 and 1 alpha-OHD3. In the group treated with 1 microgram of 1 alpha-OHD3 per day, hypercalcemia (2 out of 11 cases and these patients took calcium tablets) and an increase in BUN (1 out of 2 hypercalcemic patients) were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗

PubMed1985

Stimulatory effect of insulin on aortic smooth muscle cell migration induced by 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid and its modulation by elevated extracellular glucose levels.

In investigations on the role of insulin on migration of rat aortic smooth muscle cells, migration of the cells was measured by a modified Boyden chamber technique with 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) as a chemoattractant. Insulin itself was not a chemoattractant for these cells, and insulin added just before the migration assay did not affect cell migration in the presence or absence of 12-HETE. Cells pretreated with insulin in culture dishes for a long period, however, showed a significant increase in migration induced by 12-HET, and the increase depended on the insulin concentration: concentrations of insulin of greater than 50 microIU/ml caused about twofold increase in cell migration. On the other hand, long-term incubation with various concentrations of insulin (0.15-1000 microIU/ml) did not affect nonspecific cell migration in the absence of 12-HETE. The stimulatory effect of insulin on cell migration gradually increased with the duration of insulin treatment, reaching a plateau after 4 days. Thus, insulin stimulated 12-HETE-induced smooth muscle cell migration in a time- and dose-dependent manner. When the extracellular D-glucose concentration in the Boyden chamber was increased from 100 to 300 mg/dl, the stimulatory effect of insulin on 12-HETE-induced cell migration was augmented. This modulation by D-glucose was not due to an increase in the osmotic pressure of the medium, since addition of mannitol to increase the osmotic pressure did not enhance the effect of insulin on cell migration.(ABSTRACT TRUNCATED AT 250 WORDS)

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1985