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Biomedical subjects

A Nakai

Publications and source records attributed to A Nakai.

At least 73 records · Page 4Linked to original sources
PubMed1997

The immunosuppressant drug FK506 ameliorates secondary mitochondrial dysfunction following transient focal cerebral ischemia in the rat.

Recirculation following 2 h of focal ischemia due to transient middle cerebral artery (MCA) occlusion has previously been found to be accompanied by an initial, partial recovery of the cellular bioenergetic state and of mitochondrial respiratory functions, with secondary deterioration during the first 2-4 h of reflow. Both the free radical spin trap alpha-phenyl-N-tert-butyl nitrone (PBN) and the immunosuppressant drug FK506 ameliorate the damage incurred by the 2-h period of focal ischemia, even when given 1-3 h after the start of the recirculation. The primary objective of this study was to find out if FK506, like PBN, prevents the secondary deterioration of mitochondrial function, as this can be studied in vitro. Since this proved to be the case, we addressed the question of whether the secondary mitochondrial dysfunction and bioenergetic failure were related to a secondary compromise of microcirculation and cellular oxygen delivery. Six groups of male Wistar rats were studied for measurement of mitochondrial respiratory activity (total, n = 36). One group was used as control (n = 6). In the other groups of animals, MCA occlusion of 2 h duration was induced by an intraluminal filament technique, Neocortical focal and perifocal ("penumbra") tissues were sampled after 2 h of ischemia (n = 6) and after 1 h (n = 6), 2 h (n = 6 with vehicle), and 4 h (n = 6 with vehicle; n = 6 with FK506) of recirculation. The vehicle or 1.0 mg.kg-1 of FK506 was injected intravenously after 1 h of recirculation. Homogenates were prepared, and stimulated (+ADP), nonstimulated (-ADP), and uncoupled respiratory rates were measured polarographically. The uncoupling agent used was carbonyl cyanide m-chlorophenylhydrazone. Local CBF and tissue oxygen tension were evaluated by laser-Doppler flowmetry and PO2 microelectrodes, respectively, throughout the whole periods of 2 h of ischemia and 4 h of recirculation, using a remote MCA occlusion technique. After 2 h of ischemia, the penumbra showed a moderate decrease and the focus a marked decrease in ADP-stimulated and uncoupled respiratory rates, with a marked fall in the respiratory control ratio, defined as ADP-stimulated divided by nonstimulated respiration. Recirculation (1 h) brought about partial recovery, but continued reflow (2 and 4 h) was associated with a secondary deterioration of respiratory functions. The secondary deterioration was prevented by FK506. The results thus confirm previous findings showing that secondary mitochondrial dysfunction occurs following transient focal cerebral ischemia and demonstrate that FK506, like PBN, improves the in vitro performance of mitochondria in focal and penumbral areas. Following MCA occlusion, local CBF in a penumbral area and tissue PO2 in a focal area decreased to about 30 and 5% of control, respectively. However, recirculation brought about rapid recovery of blood flow and oxygen delivery. During the whole 4-h period of recirculation, local CBF and tissue PO2 were maintained close to 100% and at about 160% of the preischemic level, respectively. The results make it highly unlikely that the secondary bioenergetic failure during recirculation is due to a compromised microcirculation. It follows that oxygen delivery is not rate-limiting for recovery events. Very likely, FK506 (and PBN) acts at the cellular level to improve mitochondrial energy functions.

Adenosine Diphosphate↗
PubMed1997

Alteration of platelet aggregation in patients with thyroid disorders.

To determine whether Graves' disease or primary hypothyroidism influence platelet function, we evaluated platelet aggregation in the platelet-rich plasma (PRP) from such patients. Platelet aggregation induced by adenosine diphosphate (ADP) in blood obtained from patients with untreated Graves' disease was significantly lower than normal, whereas that in patients with untreated primary hypothyroidism was relatively increased. The magnitude of platelet aggregation induced by collagen in both groups of patients resembled that induced by ADP. However, significant differences were evident between the two diseases (P < .05). In addition, we observed a significant inverse correlation between the extent of platelet aggregation and plasma levels of thyroid hormones (triiodothyronine [T3], thyroxine [T4], and free T3). Platelet aggregation returned to normal when the euthyroid condition was obtained in the patients following administration of antithyroid drugs or thyroid hormone. The findings are consistent with the possibility that thyroid hormones influence platelet aggregation partly via inhibition of myosin light-chain kinase (MLCK).

Adenosine Diphosphate↗
PubMed1997

Changes in free radical scavengers and lipid peroxide in thyroid glands of various thyroid disorders.

To clarify whether the changes of free radicals and its scavengers are induced by thyroid disorders, we measured levels of free radical scavengers and checked O2 radical generating systems in the human thyroid gland. Thyroid specimens from patients with Graves' disease, follicular adenoma, and papillary and follicular carcinomas contained significantly higher concentrations of xanthine oxidase (XOD) and gluthathione peroxidase (GSH-PX), compared to those in the normal thyroid tissue. Catalase concentration was significantly lower in thyroid specimens from patients with Graves' disease and significantly lower in thyroid specimens from patients with follicular adenoma, compared to those in the normal thyroid tissue. Cu/Zn superoxide dismutase (Cu/Zn SOD) concentration was significantly lower in the specimens from follicular adenoma and papillary carcinoma and Mn SOD concentration was significantly higher in the specimens from papillary carcinoma than those in the normal thyroid tissue. The lipid peroxide concentration, expressed as malondialdehyde (MDA) concentration, was significantly higher in the specimens from papillary carcinoma than those in the normal thyroid tissue. These findings suggest that the levels of free radicals are increased and are scavenged and catalyzed in the thyroid of Graves' disease, whereas free radicals and lipid peroxide are not completely scavenged in papillary carcinoma tissues, suggesting that these substances affect some role in cell function of thyroid tumors.

Adenocarcinoma, Follicular↗
PubMed1997

Identification of genetic mutations in Japanese patients with fructose-1,6-bisphosphatase deficiency.

Fructose-1,6-bisphosphatase (FBPase) deficiency is an autosomal recessive inherited disorder and may cause sudden unexpected infant death. We reported the first case of molecular diagnosis of FBPase deficiency, using cultured monocytes as a source for FBPase mRNA. In the present study, we confirmed the presence of the same genetic mutation in this patient by amplifying genomic DNA. Molecular analysis was also performed to diagnose another 12 Japanese patients with FBPase deficiency. Four mutations responsible for FBPase deficiency were identified in 10 patients from 8 unrelated families among a total of 13 patients from 11 unrelated families; no mutation was found in the remaining 3 patients from 3 unrelated families. The identified mutations included the mutation reported earlier, with an insertion of one G residue at base 961 in exon 7 (960/961insG) (10 alleles, including 2 alleles in the Japanese family from our previous report [46% of the 22 mutant alleles]), and three novel mutations--a G-->A transition at base 490 in exon 4 (G164S) (3 alleles [14%]), a C-->A transversion at base 530 in exon 4 (A177D) (1 allele [4%]), and a G-->T transversion at base 88 in exon 1 (E30X) (2 alleles [9%]). FBPase proteins with G164S or A177D mutations were enzymatically inactive when purified from E. coli. Another new mutation, a T-->C transition at base 974 in exon 7 (V325A), was found in the same allele with the G164S mutation in one family (one allele) but was not responsible for FBPase deficiency. Our results indicate that the insertion of one G residue at base 961 was associated with a preferential disease-causing alternation in 13 Japanese patients. Our results also indicate accurate carrier detection in eight families (73%) of 11 Japanese patients with FBPase deficiency, in whom mutations in both alleles were identified.

Amino Acid Substitution↗
PubMed1997

HSF4, a new member of the human heat shock factor family which lacks properties of a transcriptional activator.

Heat shock transcription factors (HSFs) mediate the inducible transcriptional response of genes that encode heat shock proteins and molecular chaperones. In vertebrates, three related HSF genes (HSF1 to -3) and the respective gene products (HSFs) have been characterized. We report the cloning and characterization of human HSF4 (hHSF4), a novel member of the hHSF family that shares properties with other members of the HSF family yet appears to be functionally distinct. hHSF4 lacks the carboxyl-terminal hydrophobic repeat which is shared among all vertebrate HSFs and has been suggested to be involved in the negative regulation of DNA binding activity. hHSF4 is preferentially expressed in the human heart, brain, skeletal muscle, and pancreas. Transient transfection of hHSF4 in HeLa cells, which do not express hHSF4, results in a constitutively active DNA binding trimer which, unlike other members of the HSF family, lacks the properties of a transcriptional activator. Constitutive overexpression of hHSF4 in HeLa cells results in reduced expression of the endogenous hsp70, hsp90, and hsp27 genes. hHSF4 represents a novel hHSF that exhibits tissue-specific expression and functions to repress the expression of genes encoding heat shock proteins and molecular chaperones.

Amino Acid Sequence↗
PubMed1997

The calmodulin antagonist trifluoperazine in transient focal brain ischemia in rats. Anti-ischemic effect and therapeutic window.

BACKGROUND AND PURPOSE: This study was performed to assess the efficacy and the therapeutic window for the calmodulin antagonist trifluoperazine in experiments involving transient middle cerebral artery (MCA) occlusion. METHODS: Male Wistar rats were subjected to transient (2 hours) MCA occlusion by an intraluminal filament technique. Trifluoperazine (5.0 mg.kg-1) was injected intraperitoneally 5 minutes, 1 hour, or 2 hours after the induction of ischemia. Drug administration was repeated 24 hours after the first injection. Neurological scores and infarct volumes were evaluated at 48 hours of reperfusion. The effect of trifluoperazine on cortical blood flow was studied with continuous laser-Doppler flowmetry. RESULTS: The median value of neurological scores in the control rats (n = 7) was 3, while those in the treated groups were 1 (5-minute group; n = 7, P < .05) and 2 (1-hour and 2-hour groups; each n = 7). The percentage of infarct volume in the control rats was 34.8 +/- 4.9% (mean +/- SD), while those in the treated groups were 11.3 +/- 12.3% (5-minute group; P < .01), 24.8 +/- 15.1% (1-hour group), and 28.8 +/- 8.3% (2-hour group). Trifluoperazine, given at 5 minutes after ischemia, had no influence on blood flow in the neocortical penumbra during and after ischemia. CONCLUSIONS: The results demonstrate that trifluoperazine markedly reduces infarct volume after 2 hours of MCA occlusion when given 5 minutes after the induction of ischemia. However, the therapeutic window for trifluoperazine seems narrow since the drug had no significant effect when given after 1 or 2 hours.

Animals↗
PubMed1997

Heat shock protein 72 level decreases during sleep in patients with obstructive sleep apnea syndrome.

Patients with obstructive sleep apnea syndrome (OSAS) suffer from stresses related to repetitive apneas during sleep. To examine whether the level of 72 kDa heat shock protein (HSP72) increases during sleep, 11 OSAS patients (apnea-hypopnea index: 63.5 +/- 36.1, mean +/- SD) underwent polysomnography and their peripheral blood mononuclear cells (PBMC) were isolated before, during, and after sleep. HSP72 level was determined by Western blotting and hsp72 mRNA level was quantified by Northern blotting. Nine normal subjects without OSAS were examined as normal controls. HSP72 level decreased progressively during sleep and its level at 8:00 A.M. was 78.0 +/- 17.5% of that at 8:00 P.M. (p < 0.01). No such decrease was seen in normal subjects. When OSAS patients received nasal continuous positive airway pressure (NCPAP) therapy, HSP72 level did not decrease significantly. In untreated OSAS patients, hsp72 mRNA level decreased during sleep (p < 0.01). When OSAS patients were treated with NCPAP therapy, the decrease in hsp72 mRNA level was not observed. HSP72 level before sleep in OSAS patients was higher than that in normal subjects (p < 0.01). We concluded that repetitive apneas caused high HSP72 level before sleep in OSAS patients and that NCPAP therapy had significant effect on HSP72 levels during sleep.

Blotting, Northern↗
PubMed1997

Enhanced myopathy following administration of hypolipidemic agents under urethane anesthesia.

The enhanced effect of urethane anesthesia on the serum creatine kinase (CPK) level following administration of hypolipidemic agents was examined to develop a convenient experimental screening method for drug-induced myopathy. After oral administration of a hypolipidemic agent to rats, 25% urethane solution was infused intravenously at a very low rate using a microinfusion pump. Blood samples were collected 7 h after drug administration and the risk of myopathy was evaluated based on the CPK level. When bezafibrate (BF), simvastatin (SV), or pravastatin (PV) (50-500 mg/kg) was orally administered under urethane infusion, enhanced elevation of the serum CPK level was observed dose dependently for BF and SV, but not for PV. The elevation of serum CPK was much higher with BF than with SV or PV. In addition, when SV or PV (50-500 mg/kg) was coadministered with 50 mg/kg of BF, there was a striking increase in the serum CPK level as compared with the drug alone. Without urethane infusion, no significant elevation in serum CPK level was observed even at a high dose of these hypolipidemic agents. These phenomena suggest that the urethane anesthesia enhanced the elevation of the serum CPK level following administration of hypolipidemic agents. We propose that this method is a simple and speedy screening test for drug-induced myopathy.

Anesthesia↗
PubMed1997

Preparation and characterization of polylactic acid microspheres containing bovine insulin by a w/o/w emulsion solvent evaporation method.

The objective of this study was to produce polylactic acid (PLA) microspheres containing bovine insulin as a sparingly water soluble model drug using a water-in-oil-in-water (w/o/w) emulsion solvent evaporation method. The preparative conditions were optimized. Employment of smaller internal aqueous phase volume (50 microliters or 100 microliters) in the manufacturing process, resulted in the high loading efficiency (over 95% of theoretical insulin loading efficiency). The addition of 10% (w/v) NaCl to the external aqueous phase (0.5% polyvinyl alcohol solution) reduced loading efficiency compared to the case where no NaCl was added to the external phase. The mean volume diameter for prepared PLA microspheres was in the region of 15-25 microns in all cases. PLA microspheres containing 5% and 10% insulin theoretically exhibited burst release in the initial stage. After a three week dissolution test, the surface of the microspheres became more porous due to the degradable characteristics of PLA polymer itself. Nevertheless, about 80% of the insulin still remained undegraded in PLA microspheres. Finally, insulin-loaded PLA microspheres (corresponding to 4 I.U. insulin) were administered to normal rats subcutaneously, and the pharmacological effect (a decrease in serum glucose level) was demonstrated.

Animals↗
PubMed1996

Reperfusion causes significant activation of heat shock transcription factor 1 in ischemic rat heart.

BACKGROUND: The myocardial protective role of heat shock protein (HSP) has been demonstrated, and there has been increasing interest in stress response in the heart. We examined the DNA-binding activity of heat shock transcription factor (HSF), by which the transcription of heat shock genes is mainly regulated, during heat shock or ischemia/reperfusion in isolated rat heart. METHODS AND RESULTS: Rat hearts were isolated and perfused with Krebs-Henseleit buffer by the Langendorff method. Whole-cell extracts were prepared for gel mobility shift assay using oligonucleotides containing the heat shock element, which is present upstream of all heat shock genes. Induction of mRNAs for HSP70, HSP90, and GRP78 (glucose-regulated protein) was examined by Northern blot analysis. Although the activation of HSF during global ischemia was weak and rapidly attenuated, postischemic reperfusion induced a significant activation of HSF. In addition, although HSP70 mRNA was hardly induced during ischemia, its burst induction was detected during postischemic reperfusion. Supershift assays using specific antisera for HSF1 and HSF2 revealed that ischemia/reperfusion as well as heat shock induced the activation of HSF1 in hearts. Although the expression of HSP70 mRNA during heat shock was more vigorous than the expression during ischemia/reperfusion, the induction of HSP90 mRNA in postischemic reperfusion was significantly greater than that in heat shock. CONCLUSIONS: Our findings demonstrated that reperfusion causes a significant activation of HSF1 in ischemia-reperfused heart. The striking contrast between the induction of HSP70 mRNA and that of HSP90 mRNA suggests the presence of regulatory mechanisms other than HSF.

Animals↗
PubMed1996

Short stature due to growth hormone deficiency associated with Cushing's disease and ulcerative colitis.

We report a rare case of Cushing's disease associated with ulcerative colitis in a patient primarily treated with growth hormone due to short stature. At the age of fifteen years, the patient had a short stature due to GHD and was treated with the extracted GH for a short period. At the age of twenty-one years, his body weight gradually increased and, based on the results of several tests, he was diagnosed with Cushing's disease and GHD was observed in our patient. The excess secretion attenuation of pituitary hormones with reduced secretion periods as well as the relation between ulcerative colitis and elevated plasma cortisol concentrations is briefly discussed in this paper.

Adult↗
PubMed1996

Prenatal diagnosis of organic acidemias based on amniotic fluid levels of acylcarnitines.

Acylcarnitines in amniotic fluid samples were analyzed for the prenatal diagnosis of propionic acidemia, methylmalonic aciduria, isovaleric acidemia, and glutaric aciduria by electrospray tandem mass spectrometry. Although the levels of the specific acylcarnitine between affected and unaffected cases showed an overlap, the ratios of propionylcarnitine to 4-carbon acylcarnitine levels for propionic acidemia and methylmalonic aciduria, those of isovalerylcarnitine to propionylcarnitine for isovaleric acidemia, and those of glutarylcarnitine to propionylcarnitine for glutaric aciduria type I were shown to be reliable indicators in the prenatal diagnosis. In addition, it is suggested that the combination of the ratios of glutarylcarnitine, isovaleryl-carnitine, and hexanoylcarnitine to propionylcarnitine may be useful for the prenatal diagnosis of glutaric aciduria type II.

Acetylcarnitine↗
PubMed1996

Drug interaction between simvastatin and cholestyramine in vitro and in vivo.

The interaction between simvastatin (SV), a prodrug lactone, HMG-CoA reductase inhibitor which converts to the active hydroxy acid form (SVH) in vivo, and cholestyramine (CT), an anionic exchange resin, was evaluated both in vitro and in vivo. In an in vitro SV-stability study, it was shown that SV degraded gradually to SVH in an aqueous solution at pH 2 and 7. To evaluate the binding ability of SV or SVH to CT, the incubation of 5 micrograms/ml of SV or SVH solution with 200 mg of CT in various pH (2.0, 5.0 and 7.0) solutions was performed at 37 degrees C for 10 min. After incubation, the concentration of SV decreased by 59.02% (pH 2), 63.90% (pH 5) and 67.36% (pH 7), respectively, and an interaction between SV and CT was suggested. The values were much larger than those expected from the stability test of SV in the absence of CT. SVH was found to bind more strongly to CT. The binding ability of SVH to CT was 66.71% (pH 2), 87.44% (pH 5) and 92.11% (pH 7), respectively. Judging from these results, SV was considered to interact with CT by the following procedure: SV underwent hydrolysis to SVH in aqueous solution, then CT activated the hydrolysis by binding the formed SVH, resulting in a significant reduction in concentration of SV. On the other hand, an in vivo animal experiment also demonstrated a significant reduction (about 50% with AUC) in the concentration of SVH in plasma following the coadministration of SV (500 mg/kg p.o.) and CT (600 mg/kg p.o.), compared with the administration of SV alone. This phenomenon suggested that a combination therapy using SV and CT might result in a smaller cholesterol-lowering effect of SV.

Animals↗
PubMed1996

A case of myocarditis associated with IDDM.

We report a case of diabetic ketoacidosis (DKA) complicated by acute myocarditis, which was confirmed by cardiac biopsy. A 26-year-old man was hospitalized with severe DKA. On admission, nonspecific ST-T change was noted on the electrocardiogram (ECG). The patient's levels of creatine phosphokinase (CPK) and glutamic oxaloacetic transaminase were slightly elevated, but he did not complain of chest discomfort or symptoms of heart disease. On the first day after admission, ST-T elevation was noted on ECG during treatment of DKA. By cardiac angiography and cardiac biopsy, coronary heart disease was ruled out and postmyocarditic change was histologically confirmed. An episode of upper respiratory viral infection before the onset of acute diabetes suggested that the patient suffered from viral-induced myocarditis and consequent development of IDDM. This possibility was confirmed by the clinical course of ECG change, with elevated CPK and lactate dehydrogenase and a slightly elevated antibody titer for echovirus.

Adult↗
PubMed1995

Activation of heat shock factor 1 in rat brain during cerebral ischemia or after heat shock.

Recently, many studies have demonstrated the induction of stress proteins in the mammalian nervous system under various pathological conditions. These altered genetic programs may function to protect individual cells against stressful conditions. However, little is known about the molecular mechanisms regulating these stress responses in animals. We report here the activation of a heat shock factor (HSF) in the rat brain during cerebral ischemia or after heat shock. Gel mobility shift assays revealed an increase in DNA binding activity to the heat shock element (HSE) during the early phases of ischemia. Supershift experiments using specific antisera against HSF1 and HSF2 showed that the ischemia-induced HSE-binding activity was mainly due to HSF1. In the heat-shocked brain, HSF1 was also activated, and the HSE-binding activity was higher in the cerebellum than in the cerebral cortex or hippocampus; Western blot analysis also showed that HSF1 was more abundant in the cerebellum than in the other two brain regions. Our results indicate that heat shock gene transcription is regulated by the activation of HSF1 in both cerebral ischemia and heat shock, and that different brain regions display differential sensitivities in their stress response. The cellular signals for heat shock gene transcription under in vivo pathological conditions will also be discussed.

Animals↗
PubMed1995

Cloning and expression of murine high molecular mass heat shock proteins, HSP105.

We have shown that the 105-kDa heat shock protein (HSP105) and the 42 degrees C-specific heat shock protein (42 degrees C-HSP) constitute high molecular mass heat shock proteins. To elucidate the structure of these heat shock proteins, we have screened a cDNA library constructed with poly(A)+ RNA derived from mouse FM3A cells preheated at 42 degrees C for 2 h using an antibody against murine HSP105. Two full-length cDNA clones were obtained: the pB105-1 insert encoded an 858-amino acid protein, and the pB105-2 insert encoded an 814-amino acid protein and lacked 44 amino acids found in pB105-1. The two clones contained the amino acid sequence found in the 17-kDa polypeptide fragments from HSP105 and 42 degrees C-HSP by lysylendopeptidase digestion. In vitro translation products of the RNA transcripts from pB105-1 and pB105-2 migrated to the same positions of HSP105 and 42 degrees C-HSP, respectively, on SDS-polyacrylamide gel electrophoresis. Northern blot analysis showed that the transcript was approximately 4 kilobases in murine FM3A cells and was strongly induced by heat shock and by treatment with arsenite or an amino acid analog. By reverse transcription-polymerase chain reaction analysis using primers by which deletion of 132 nucleotides in pB105-2 could be detected, the polymerase chain reaction product corresponding to pB105-2 was increased only after heat shock at 42 degrees C, whereas the product corresponding to pB105-1 was induced by heat shock at either 42 or 45 degrees C and also by other stresses. Thus, the cDNA clones pB105-1 and pB105-2 encode HSP105 and 42 degrees C-HSP, respectively, and HSP105 and 42 degrees C-HSP (a short form of HSP105) are suggested to be produced by alternative splicing. Here, HSP105 and 42 degrees C-HSP are renamed HSP105 alpha and HSP105 beta, respectively. A protein sequence homology search revealed that HSP105 shares 54, 34, and 25% amino acid identity with human HSP70RY, the sea urchin egg receptor for sperm, and murine inducible HSP70, respectively. Furthermore, by Northern blot analysis, HSP105 mRNA was revealed to be present in most murine tissues and to be highly expressed in the brain.

Amino Acid Sequence↗
PubMed1995

Identification of a genetic mutation in a family with fructose-1,6- bisphosphatase deficiency.

Fructose-1,6-bisphosphatase deficiency is an inheritable disorder of gluconeogenesis. Sequence analysis of the cDNA of the fructose-1,6-bisphosphatase mRNA isolated from monocytes from a girl with this disease and her consanguineous parents revealed that the patient and her parents were a homozygote and heterozygotes for an insertion of one G residue at G957GGGG961, respectively. This mutation resulted in translation of a truncated enzyme protein, and the mutant protein showed no fructose-1,6- bisphosphatase activity in an overexpression experiment in Escherichia coli. However, this mutation is located in a region of the amino acid sequence which is not well conserved among mammals. A mutagenized clone was prepared from the normal clone. The extents of substitutions and deletions of the amino acid sequence were predicted to be less in the mutagenized protein than in the mutant protein. This mutagenized clone also expressed no fructose-1,6-bisphosphatase activity, although both of two normal clones from control monocytes and a control liver sample expressed an apparently normal level of fructose-1,6-bisphosphatase activity. Thus, this mutation is concluded to be responsible for fructose-1,6-bisphosphatase deficiency in this patient.

Amino Acid Sequence↗