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Biomedical subjects

K Yajima

Publications and source records attributed to K Yajima.

At least 37 records · Page 2Linked to original sources
PubMed1998

Relationship between an increase in thermostability and amino acid substitutions in N-carbamyl-D-amino acid amidohydrolase.

For the production of D-amino acids using stable N-carbamyl-D-amino acid amidohydrolase (DCase) in an immobilized form, the DCase gene of Agrobacterium sp. KNK712 was mutagenized to increase its enzymatic thermostability. In a search for thermostability-related amino acid sites besides the two known sites of DCase, i.e., the 57th and 203rd amino acids, the new mutant enzyme found, in which the 236th amino acid, valine, had been changed to alanine, showed a 10 degrees C increase in thermostability. These known three thermostability-related amino acids were changed to other amino acids by the PCR technique, and it was proved that the thermostability of the DCase increased when the 57th amino acid of DCase, histidine, was changed to leucine, the 203rd amino acid, proline, to asparagine, glutamate, alanine, isoleucine, histidine, or threonine, and the 236th amino acid, valine, to threonine or serine, in addition to the known mutations.

Amidohydrolases↗
PubMed1998

Immobilization of N-carbamyl-D-amino acid amidohydrolase.

N-Carbamyl-D-amino acid amidohydrolase (DCase), produced with recombinant Escherichia coli cells using a cloned gene from Agrobacterium sp. strain KNK712, has been immobilized for use in the production of D-amino acids. The porous polymers, Duolite A-568 and Chitopearl 3003, were much better than other resins for the activity and stability of the adsorbed enzyme. The activity of DCase expressed on Duolite A-568 and Chitopearl 3003 amounted to 96 units/g-wet-resin and 91 units/g-wet-resin, respectively. DCase immobilized on Duolite A-568 was found to be most stable at about pH 7, and it was further stabilized by reductants such as dithiothreitol, L-cysteine, cysteamine, and sodium hydrosulfite. The stability during the repeated batch reactions was greatly improved when dithiothreitol was in the reaction mixture, and the higher crosslinking degree with glutaraldehyde also stabilized the immobilized enzyme. After 14 times repeated reactions, the remaining activity of the immobilized enzyme cross-linked with 0.1% and 0.2% of glutaraldehyde, and 0.2% of glutaraldehyde with dithiothreitol in the reaction mixture was 12%, 18%, and 63%, respectively. DCase produced with Pseudomonas sp. strain KNK003A and Pseudomonas sp. strain KNK505, which are thermotolerant soil bacteria, and that with Agrobacterium sp. strain KNK712 were also immobilized on Duolite A-568. The stability of the enzymes of thermotolerant bacteria during reactions was superior to that of Agrobacterium sp. strain KNK712, though the activity was lower than that of strain KNK712.

Amidohydrolases↗
PubMed1998

Isolation of Agrobacterium sp. strain KNK712 that produces N-carbamyl-D-amino acid amidohydrolase, cloning of the gene for this enzyme, and properties of the enzyme.

Agrobacterium sp. strain KNK712, which produced N-carbamyl-D-amino acid amidohydrolase (DCase) was isolated from soil. The bacterium had D-specific hydantoinase activity also. Both enzymes are suitable for use in the production of D-amino acids. The DCase gene from Agrobacterium sp. strain KNK712 was cloned into Escherichia coli. The cloned DNA fragment contained one open reading frame, predicted to encode a peptide of 304 amino acids, with a calculated molecular weight of 34,285. The DCase gene was overexpressed under the control of the lac promoter, and DCase accounted for 50% of the soluble protein in the cells. The enzyme was purified and some properties were investigated. Both the optimum pH and the pH that gave greatest stability were about pH 7.0. The optimum temperature was 65 degrees C, and the enzyme was stable at 55 degrees C. The enzyme had strict specificity toward N-carbamyl-D-amino acids, and was inhibited by thiol reagents, Cu2+, Hg2+, Ag+, and ammonia.

Amidohydrolases↗
PubMed1998

Screening, characterization, and cloning of the gene for N-carbamyl-D-amino acid amidohydrolase from thermotolerant soil bacteria.

For the production of D-amino acids, thermotolerant bacteria producing N-carbamyl-D-amino acid amidohydrolase were isolated from soil by enrichment culture at 45 degrees C with N-carbamyl-D-amino acids as the sole nitrogen source. The enzyme activities and substrate specificities of these strains were examined by the resting cells reaction. One of the enzymes, produced by Pseudomonas sp. strain KNK003A, was purified and characterized, and the amino acids of its N-terminal region were sequenced. A DNA fragment containing the gene for a thermostable N-carbamyl-D-amino acid amidohydrolase was then cloned into Escherichia coli. The gene encoded a peptide of 312 amino acids, with a calculated molecular weight of 35,000. The similarity of the deduced amino acid sequences of this enzyme and a related enzyme from a mesophile, Agrobacterium sp. strain KNK712, was 60%. A database was searched for similar sequences.

Amidohydrolases↗
PubMed1995

Brain uptake and metabolism of [1-11C]octanoate in rats: pharmacokinetic basis for its application as a radiopharmaceutical for studying brain fatty acid metabolism.

The uptake of octanoate in rat brain and its metabolism were investigated by means of intravenously injecting [1-11C] or [1-14C]octanoate as a tracer. The radioactivity in the cerebrum was increased by an injection of [1-11C]octanoate, and reached its peak level (0.33% ID/g) in about 2 to 5 min, and then decreased slowly. The cerebrum-to-blood ratio of the radioactivity increased with time over a period of 30 min. At 30 sec, [1-11C]octanoate that remained unchanged in the cerebrum accounted for only 8% of the total radioactivity, in spite of there being about 90% in the blood. By means of an injection of [1-14C]octanoate, more than 70% of the total radioactivity in the cerebrum was found to be attributable to radiolabeled glutamate and glutamine at each time point measured between 30 sec and 30 min. The results show that [1-11C]octanoate enters rat brain easily and is trapped in the cerebrum, probably in the form of glutamate and glutamine, and the usefulness of [1-11C]octanoate as a radiopharmaceutical for studying brain fatty acid metabolism by positron emission tomography is therefore suggested.

Animals↗
PubMed1994

Determination of nadolol diastereomers in dog plasma using chiral derivatization and reversed-phase high-performance liquid chromatography with fluorescence detection.

A stereospecific high-performance liquid chromatographic method has been developed for the determination of four diastereomers of nadolol in plasma. After the nadolol diastereomers were extracted from plasma using an Extrelut-1 solid-phase extraction cartridge, they were derivatized with (R)-(-)-1-(1-naphthyl)ethylisocyanate to form urea derivatives. These derivatives were then separated on a YMC-AM-303 ODS column using water-acetonitrile (60:40, v/v). The calibration curves of (SR)-, (RS)-, (SS)-, and (RR)-nadolol were linear over the range 2.5-200 ng/ml, and the correlation coefficient (r) of the curves were higher than 0.9991 for each diastereomer. The limit of quantification was 2.5 ng/ml for each diastereomer in plasma. This method was used for a pharmacokinetic study in four dogs after oral administration of nadolol (1 mg/kg). The plasma concentrations of nadolol diastereomers showed no significant differences in Cmax, Tmax or AUC values. The assay appears to be readily applicable to the study of diastereoselective nadolol pharmacokinetics in animals and humans.

Animals↗
PubMed1994

Effect of head up tilt on cerebral circulation.

This study was performed to study the effect of the head up tilt (HUT) on cerebral circulation across the time course (60 degrees HUT for 15 minutes) and across the different angles of HUT (15, 30, 45, 60 degrees HUT for 15 minutes). Cerebral circulation was continuously monitored during 15 minutes of HUT by the carotid Doppler flow meter, the transcranial Doppler flow meter, and the near infra-red spectrophotometer. The results show that the cerebral blood flow decreased during HUT and that the cerebral blood volume decreased initially and then gradually increased. And the magnitude of the effect may have the relationship with the angles of the HUT.

Blood Pressure↗
PubMed1994

Human cardiovascular and vestibular responses in long minutes and low +Gz loading by short arm centrifuge.

1.4 G, 1.7 G, and 2.0 G of +Gz and 60 minutes centrifugation was adopted to 20 healthy male subjects using 1.8 m radius centrifuge equipped to Nihon University School of Medicine. G was applied from lower G, considering G training effect for the subjects. Effects on performance decline and side effects of such a short-arm centrifugation were especially observed in the experiments, because this size of centrifuge could be used in space station in future for a strong countermeasure of cardiovascular deconditioning, demineralization from bone, etc. G training effect was observed same as higher and rapid G acceleration in fighter pilot. Subjects suffered from many types of discomfort; such as sensation of heaviness of diaphragm, cold sweat, nausea, irritable feeling, arrhythmia, tachycardia, rapid decrease of blood pressure, which sometimes caused interruption of G load. As 2.0 G and 60 minutes centrifugation seemed very tough load to the subjects, there should be necessary some G suit or other countermeasure, if we apply a higher G and/or longer G duration. Performance decline due to the load commonly continued for 1 hour or so. Side effects were observed in relation to neuro-vestibular, cardio-vascular, and autonomic nervous system.

Adult↗
PubMed1994

Artificial G-load and chemical changes of saliva.

Effectiveness of artificial G load is expected when it is used as a countermeasure against various bio-medical mishaps which are taking place in the micro-gravity environment, and probably more importantly, to prepare re-entry to the earth 1-G environment. Regarding the salivary chemical analysis, it has been known that, in case of (experimental) motion sickness, symptomatic outputs are delivered through the autonomic nervous system, and changes in salivary chemical components occur. In this study, accordingly, quantitative analyses of salivary potassium, sodium, chloride and total protein were performed before and after various levels of G loads which were produced by our short-radius human centrifuge. When the results were compared, the subjects who failed 60 minutes ride exhibited significantly higher post-load levels of potassium and sodium than those of successful 60 minutes riders. However, the differences between two groups were less clear in chloride and total protein. The result of present study indicates that salivary potassium and sodium are good indicators to represent autonomic nervous system function under the stressful condition produced by an artificial G load.

Adult↗
PubMed1994

Individual differences of cerebrovascular responses to gravitational stress--prediction of orthostatic intolerance.

This experiment was designed to investigate the individual differences in the cerebrovascular responses to orthostatic stress. Seven male volunteers were exposed to head-up tilt (HUT) at 60 degrees for 15 min and to lower body negative pressure (LBNP) at 30 mmHg for 25 min. We measured the flow velocity of the middle cerebral artery and the quantity of the oxygenated and deoxygenated hemoglobin (oxy-Hb and deoxy-Hb) in the brain. Based upon oxy-Hb change during exposure to HUT, we classified the subjects into two groups: 1) the "good responder" group in which the oxy-Hb gradually increased from the 5th to 10th minute of HUT; 2) the "bad responder" group in which the oxy-Hb stabilized at lower levels after the initial decrease. The oxy-Hb changes between the two groups were significantly different during exposure to LBNP. During exposure to HUT and LBNP, no significant difference was observed in blood pressure and heart rate between the two groups. Our results suggest that there might be an individual difference in the cerebrovascular responses to orthostatic stress.

Adult↗
PubMed1994

Recombinant variants of tissue-type plasminogen activator containing amino acid substitutions in the fibronectin finger-like domain and the kringle 1 domain.

Tissue-type plasminogen activator (t-PA) is a fibrin-specific agent which is used to treat acute myocardial infarction. Pharmacokinetically, t-PA is characterized by a rapid clearance from the circulation. In a previous study, we constructed variant forms of t-PA with genetic modifications at the fibronectin finger-like domain (finger domain) or at the kringle 1 domain (K1 domain). The finger modified variant, t-PA N37S.S38V.G39V.R40E. A41F.Q42S had about a 6.0-fold higher plasma half-life in vivo than wild-type t-PA. Two variants with modifications in the K1 domain, t-PA G161R.K162R.S165W and t-PA N115P, showed an improved kinetic parameters and a 2.2-fold higher plasma half-life in vivo than wild-type t-PA, respectively. To create a recombinant variant of t-PA with a higher enzymatic activity and a further prolonged half-life in vivo, the genes containing each modifications were joined and expressed in animal cells. The two variants, t-PA N37S.S38V.G39V.R40E.A41F.Q42S.G16 1R.K162R.S165W and t-PA N37S.S38V.G39V.R40E.A41F.Q42S.N11 5P, were purified from conditioned media and their biochemical, pharmacokinetic and thrombolytic profiles were investigated. Although the variant t-PA N37S.S38V.G39V.R40E.A41F.Q42S.G16 1R.K162R.S165W demonstrated an impaired enzymatic activity compared to the wild-type t-PA, the half-life of the variant, t-PA N37S.S38V.G39V.R40E.A41F.Q42S. N115P, following intravenous bolus injection in rabbits was considerably longer than that of finger-domain modified variants.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

The presence of CD5LOW+NK cells in normal controls and patients with pulmonary tuberculosis.

CD5 antigen is present on all normal alpha beta T cells and some B cells. Human NK cells do not usually express CD5 antigen, but we found a subset of CD5LOW+ (low density of CD5) NK cells in some patients with pulmonary tuberculosis. Unlike CD5-NK cells, most CD5LOW+NK cells had HLA-DR. We observed few CD5LOW+NK cells in the normal controls and some in the large granular lymphocyte (LGL) population purified by Percoll density centrifugation. Sorted CD5LOW+NK populations were LGL. The CD5LOW+NK cells had high lytic activity on K562 cells in a 4-h 51chromium release assay. Our results indicate that there is a previously unidentified subset of NK cells.

Adult↗
PubMed1993

Effects of oral administration of chlormadinone acetate on canine prostatic hypertrophy.

Seven of the 25 dogs (4 to 15 years old) with benign prostatic hypertrophy (BPH) at the teaching hospital of Nippon Veterinary and Animal Science University from 1989 through 1990 were treated orally with chlormadinone acetate (CMA), a synthetic anti-androgen, 2 mg/kg/day, twice a day for 3 or 4 weeks. Prostatic size was measured by radiography and ultrasonography before and after CMA treatment, and prostatic volume was calculated from the images. Semen quality was examined in 3 of the dogs with BPH. Peripheral blood samples were collected and plasma levels of LH, 4-androstenedione, 5 alpha-dihydrotestosterone, testosterone and estradiol-17 beta were measured. Clinical signs disappeared within 10 days after the start of CMA treatment. The mean (+/- S.E.) prostatic volume 3 or 4 weeks after treatment decreased to 39 +/- 4% of the pretreatment volume. The mean level of plasma testosterone in the dogs with BPH before treatment was significantly lower than that in the normal dogs (P < 0.01). In 6 of the CMA-treated dogs the levels of all hormones examined, except LH, were significantly lower after CMA treatment than before (P < 0.05). The sperm count and motility decreased and the percentage of abnormal sperms increased after CMA treatment. These results demonstrate that, although CMA treatment in dogs with BPH inhibits testicular function, the prostatic volume is markedly reduced and clinical signs disappear in a short period after the therapy. However, a relapse of BPH was observed approximately 6 months after oral CMA treatment in some dogs.

Administration, Oral↗
PubMed1993

Effect of transdermally administered scopolamine on the vestibular system in humans.

In order to elucidate the effect of scopolamine on the vestibular system in humans, various experimentally-induced forms of nystagmus, i.e. caloric nystagmus, rotational nystagmus, optokinetic nystagmus, visual-vestibular interaction and optokinetic after nystagmus, were evaluated before and after the administration of two pieces of Scopoderm-TTS or placebo patches retro-auricularly. Scopolamine reduced the responses of both the caloric and optokinetic after nystagmus compared with the placebo. The possible action site of this drug is discussed.

Administration, Cutaneous↗
PubMed1993

Effect of lower body negative pressure on cerebral circulation.

This experiment was performed to study the effects of lower body negative pressure (LBNP) on cerebral circulation. Cerebral hemodynamics were monitored continuously and noninvasively in eight subjects at 30 mmHg of LBNP for 25 minutes by following items: 1) the carotid Doppler flowmeter which measures the carotid blood flow, 2) the transcranial Doppler sonography which measures the flow velocity of the middle cerebral artery, 3) the near infrared spectrophotometer which measures the oxygenated and deoxygenated hemoglobin in the brain. The mean flow velocity of the middle cerebral artery and the blood flow of the common carotid artery significantly decreased during LBNP, even though the mean arterial blood pressure was well maintained. The oxygenated hemoglobin was significantly increased during LBNP, while the deoxygenated hemoglobin was not changed significantly. Our results suggest that exposure to moderate LBNP (30 mmHg) decreased the cerebral blood flow with the vasodilation on the arterial side of the brain.

Adult↗
PubMed1993

[Chronic hypothermia and a disordered thermoregulation in a child with severe brain damage and malnutrition].

Patients with brain damage, especially under malnutrition, sometimes show a disordered thermoregulation. Our case was an 11 year-3-month-old boy with severe brain damage probably of prenatal origin, who was physically extremely small in size for age and showed difficulty of taking food. Low body temperature ranging from 34.0 degrees C to 36.0 degrees C became outstanding since 9 years of age. Continuous rectum temperature monitoring was starged, when he was admitted in our department of pediatrics in Tokyo Women's Medical College Hospital for the therapy of acute infection. Before hospitalization, he took orally only 150 to 200 kcal/day. After the recovery from the infection, a higher calorie diet appropriate for his body weight was given through a nasal tube. Dramatic improvement was observed in terms of sleep pattern, body temperature and thermoregulation after the administration of higher calorie nutrients. It is suggested that the circadian rhythm of the body temperature would be disturbed by chronic malnutrition in children with brain damage.

Body Temperature Regulation↗
PubMed1993

Hemodynamic and hormonal correlates with exposure to lower body negative pressure after 12 hours head-down tilt.

Hemodynamic and hormonal responses to lower body negative pressure (LBNP) after 12-h head-down tilt (HDT) were examined to investigate the mechanism of orthostatic intolerance following spaceflight. Ten young healthy male volunteers participated in the experiment. After 6 degrees HDT for 12 h, the tolerance to 1 h continuous -30 mm Hg LBNP was tested. Variables were measured before and after HDT, during LBNP, and 1 and 10 min after LBNP. Significant reduction in leg volume and plasma volume were observed at the end of 12-h HDT (500 ml and 5.5%, respectively). In the tolerant group, stroke volume index (SI) was decreased by 35% after 15 min LBNP, and carotid arterial blood flow (CBF), by 27% at the end of LBNP compared to the pre-LBNP value. Of the 10 subjects, 5 developed presyncope during LBNP; they exhibited remarkable reduction in SI, CBF, and heart rate, and increased secretion of ADH and adrenaline during or just prior to presyncope. This non-tolerant group tended to be hemo-concentrated from the start of the experiment. However, it will require a prospective study to determine if this variable is a reliable predictor of the outcome of the post-HDT LBNP test from data collected at the end of HDT.

Adaptation, Physiological↗