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Biomedical subjects

K Takano

Publications and source records attributed to K Takano.

At least 91 records · Page 5Linked to original sources
PubMed2000

Effect of selective brain hypothermia on regional cerebral blood flow and tissue metabolism using brain thermo-regulator in spontaneously hypertensive rats.

To investigate the effect of selective hypothermia of the brain (brain cooling) on regional cerebral blood flow and tissue metabolism, we have developed a brain thermo-regulator. Brain temperature was modulated by a water-cooled metallic plate placed on the surface of the rats' scalp to get the appropriate brain temperature precisely with ease. Regional cerebral blood flow and brain temperature were measured simultaneously using a Teflon-coated platinum electrode and thermocouple probe inserted stereotaxically into the parietal cortex and thalamus in spontaneously hypertensive rats. Experimental forebrain ischemia was induced by the occlusion of bilateral common carotid artery under normo- and hypothermic brain condition, and the supratentorial brain tissue metabolites were measured enzymatically after 60 min of forebrain ischemia. When cortical temperature was set to hypothermia, cortical blood flow was significantly lowered by 40% at 30 degree C and 20% at 33 degree C as compared with that at 36 degree C (p < 0.0001 and p < 0.05, respectively). Thalamic blood flow was also significantly reduced by 20% when cortical temperature was set to 30 degree C as compared with 36 degree C (p < 0.05). There were no significant differences in arterial blood pressure and gas parameters throughout these experiments. In the rats with selective brain hypothermia (30 degree C) immediately after the induction of cerebral ischemia, the level of brain ATP concentration after 60 min of ischemia was significantly higher than that in normothermia rats (36 degree C) (p < 0.05). Our findings indicate that: 1) the metallic plate brain thermo-regulator is useful in small animal experiments; 2) regional brain temperature regulates regional cerebral blood flow; and 3) selective brain hypothermia, even started after the forebrain ischemia, ameliorates the derangement of brain metabolism, suggesting its effectiveness as a cytoprotective strategy.

Animals↗
PubMed2000

Distribution of c-fos mRNA in the brain following intracerebroventricular injection of nitric oxide (NO)-releasing compounds: possible role of NO in central cardiovascular regulation.

We injected nitric oxide (NO)-releasing compounds and NO synthase (NOS) inhibitors into the brains of conscious, freely moving rats and measured the effects on mean arterial blood pressure (MAP) and heart rate, as well as on the expression of c-fos mRNA, neuronal NOS (nNOS) mRNA and NADPH-diaphorase, an indicator of NOS activity. When administered i.c.v., the NO donor, NOC-18, caused a significant fall in MAP and heart rate, whereas the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), induced a significant rise in MAP. The same dose of NOC-18 or L-NAME when administered i.v. did not affect MAP and heart rate. Centrally administered NOC-18 induced c-fos mRNA expression in several regions of the brain involved in the baroreceptor response, including the nucleus of the solitary tract, the area postrema and the rostral ventrolateral medulla, as well as areas involved in the integration of autonomic, neuroendocrine and behavioural responses, including the medial preoptic area, the organum vasculosum lamina terminalis, the bed nucleus of stria terminalis, the paraventricular nucleus (PVN), the supraoptic nucleus (SON), the central nucleus of amygdala (CeA) and the locus coeruleus. Most of the areas that expressed c-fos also contained nNOS mRNA and/or NADPH-d-positive neurones and fibres. i.c.v. injection of L-NAME induced c-fos mRNA expression in PVN, SON, locus coeruleus and NTS, suggesting a tonic inhibition of neuronal activity by NO or stimulation of neuronal activity by endogenous NO. i.v. injection of NOC-18 or L-NAME did not induce any significant c-fos mRNA expression in rat brain. These results demonstrate that NO acts directly in the brain to reduce the systemic blood pressure, and that the endogenous NO pathway may play a role in cardiovascular and autonomic regulation by modulating neuronal activities in discrete regions of the brain.

Amygdala↗
PubMed2000

Clinical and in vitro evaluation of membrane humidifier that does not require addition of water.

It is well known that conventional bubbling humidifiers are capable of producing micro-aerosols contaminated with bacteria. We developed a unique humidifier, named a membrane humidifier, that does not require an external water supply. This new system obtains moisture from room air. We investigated the clinical and in vitro evaluation of the membrane humidifier. Ten patients with chronic pulmonary disease participated in the study. We evaluated the partial pressure of oxygen in arterial blood (PaO2) of 10 patients who used the new device. We conducted an in vitro study to determine whether the device could prevent the bacterial contamination of humidified-oxygen. We passed compressed air contaminated with Pseudomonas aeruginosa outside the hollow fibres of the membrane humidifier, and the humidified-oxygen passed inside the hollow fibres was sampled into nutrient broth periodically for 10 days. We also compared the relative humidity of oxygen humidified by a membrane humidifier with that of oxygen humidified by a bubbling humidifier. There was no significant difference between measured PaO2 while breathing oxygen humidified using a membrane humidifier and that while breathing oxygen humidified using a bubbling humidifier. Cultures of the humidified-oxygen passed through the hollow fibres were negative for bacteria. The membrane humidifier could produce good humidification. The new device appeared to prevent bacterial contamination, and may help to reduce the risk of infection in patients at hospital and home.

Aged↗
PubMed2000

Effect of extra N-terminal residues on the stability and folding of human lysozyme expressed in Pichia pastoris.

A human lysozyme expression system by Pichia pastoris was constructed with the expression vector of pPIC9, which contains the alpha-factor signal peptide known for high secretion efficiency. P. pastoris expressed the human lysozyme at about 300 mg/l broth, but four extra residues (Glu(-4)-Ala(-3)-Glu(-2)-Ala(-1)-) were added at the N-terminus of the expressed protein (EAEA-lysozyme). To determine the effect of the four extra residues on the stability, structures and folding of the protein, calorimetry, X-ray crystal analysis and GuHCl denaturation experiments were performed. The calorimetric studies showed that the EAEA-lysozyme was destabilized by 9.6 kJ/mol at pH 2.7 compared with the wild-type protein, mainly caused by the substantial decrease in the enthalpy change (DeltaH). On the basis of structural information on the EAEA-lysozyme, thermodynamic analyses show that (1) the addition of the four residues slightly affected the conformation in other parts far from the N-terminus, (2) the large decrease in the enthalpy change due to the conformational changes would be almost compensated by the decrease in the entropy change and (3) the decrease in the Gibbs energy change between the EAEA and wild-type human lysozymes could be explained by the summation of each Gibbs energy change contributing to the stabilizing factors concerning the extra residues.

Calorimetry, Differential Scanning↗
PubMed2000

Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells.

Although it is generally accepted that relatively high efficiencies of somatic cell cloning in mammals can be achieved by using donor cells from the female reproductive system (e.g., cumulus/granulosa, oviduct, and mammary gland cells), there is little information on the possibility of using male-specific somatic cells as donor cells. In this study we injected the nucleus of immature mouse Sertoli cells isolated from the testes of newborn (Days 3-10) males into enucleated mature oocytes in order to examine the ability of their nuclei to support embryonic development. After activation of the oocytes that had received the freshly recovered immature Sertoli cells, some developed into the morula/blastocyst stage, depending on the age of the donor cells (22.0-37.4%). When transferred into pseudopregnant females, 7 (3.3%, 7 of 215) developed into normal pups at term. Nuclear transfer of immature Sertoli cells after 1 wk in culture also produced normal pups after embryo transfer (3.1%, 2 of 65). Even after cryopreservation in a conventional cryoprotectant solution, their ability as donor cells was maintained, as demonstrated by the birth of cloned young (6.7%, 7 of 105). Immature Sertoli cells transfected with green fluorescent protein gene also supported embryo development into morulae/blastocysts, which showed specific fluorescence. This study demonstrates that immature Sertoli cells, male-specific somatic cells, are potential donors for somatic cell cloning.

Animals↗
PubMed2000

Hydralazine decreases blood pressure and endothelin-1 mRNA expression in tissues but not cardiac weight in SHR-SP/Izm rats.

Although evidence has been accumulated to support a role of endothelin-1 (ET-1) in cardiac hypertrophy, details of the pathophysiological significance of ET-1 in cardiac hypertrophy remain to be elucidated. In the present study, we investigated the effects of the vasodilator hydralazine on the blood pressure, cardiac hypertrophy and ET-1 gene expression in various tissues of spontaneously hypertensive rats (SHR-SP/Izm). Hydralazine (20 mg/kg/day) was administered orally from the age of 4 weeks for 8 weeks. Tissues of the kidney, heart, aorta and brain were obtained at the age of 12 weeks. Tissue expression of ET-1 mRNA was determined by reverse transcriptase polymerase chain reaction (RT-PCR) followed by Southern blot analysis. Administration of hydralazine resulted in a significant decrease in the blood pressure (156 +/- 1 mmHg vs 212 +/- 4 mmHg in controls) and an increase in the heart rate (470 +/- 20 bpm vs 402 +/- 23 bpm in controls). ET-1 mRNA expression was significantly decreased in the heart (x 1/2), kidney (x 1/4) and brain (x 1/2). There was no significant change of the cardiac weight (309 +/- 4 mg/100 g body weight vs 307 +/- 5 mg/100 g body weight in controls). The dissociation between ET-1 mRNA expression and cardiac hypertrophy in hydralazine-treated rats may suggest that the increased tissue ET-1 is not an indispensable factor of cardiac hypertrophy in hypertension. Sympathetic activation, as shown by the reactive tachycardia, may overcome the effects on the blood pressure and ET-1 expression.

Animals↗
PubMed2000

Augmented expression of tissue endothelin-1 messenger RNA is a common feature in hypertensive rats.

Recent studies revealed important roles for endothelin-1 (ET-1) in the pathogenesis of hypertension. Whether ET-1 could be a primary cause of hypertension or a secondary factor associated with hypertension, however, remains unknown. In this study, we determined plasma ET-1 levels and the expression of ET-1 mRNA in tissues of rats rendered hypertensive using distinct mechanisms: deoxycorticosterone acetate (DOCA)-salt hypertension: N(G)-nitro-L-arginine-methyl ester- (L-NAME) induced hypertension; and spontaneously hypertensive rats (SHR-SP). ET-1 mRNA expression level was determined by reverse transcriptase polymerase chain reaction (RT-PCR) followed by Southern blotting. There was no significant difference in plasma ET-1 levels between the hypertensive rats and normotensive rats. By contrast, ET-1 mRNA level was significantly increased in various tissues including the adrenal, lung, kidney and brain of these hypertensive rats compared with control rats. Thus, ET-1 gene expression was ubiquitously augmented in tissues of hypertensive rats irrespective of the cause of the hypertension. The results suggest that the increase in ET-1 expression is not the primary cause of hypertension but a secondary outcome which may further exacerbate the hypertension.

Animals↗
PubMed2000

Effects of acute and chronic cigarette smoking on the expression of endothelin-1 mRNA of the cardiovascular tissues in rats.

Although smoking has been suggested to be involved in the development of cardiovascular diseases, details of the mechanism still need to be revealed. We investigated the effects of cigarette smoking on the tissue mRNA expression of endothelin-1 (ET-1). Male Wistar rats of 4 weeks of age were exposed to smoke from six cigarettes for 30 min (acute exposure) and six cigarettes for 30 min/day, 5 days a week for 6 months (chronic exposure). Half of the rats exposed to 6 months smoking were kept in clean-air conditions for a further 3 months to clear the effects. Tissue expression of ET-1 mRNA in the kidney, aorta, heart and lung was determined by reverse transcriptase polymerase chain reaction (RT-PCR) followed by Southern blot analysis. There was no significant difference in body and organ weight of the heart and kidney between the control and smoking group in either the acute or chronic experiment. In the acute-exposure experiment, expression of ET-1 mRNA was increased in the heart and lung, while that in the kidney and aorta was unchanged. In the chronic-exposure experiment, however, there was no significant difference in the expression of ET-1 mRNA in all the tissues between the smoking and control groups. These results suggest that cigarette smoking could cause cardiovascular and pulmonary diseases by modulating ET-1 mRNA expression in the tissues.

Animals↗
PubMed2000

Hemodynamic and biochemical effects of endothelin-A- and -B-receptor antagonist TAK-044 in stroke-prone spontaneously hypertensive rats.

We have demonstrated previously that endothelin-1 (ET-1) mRNA expression is increased in hypertensive rats. The aim of the study reported here was to elucidate the effects of the endothelin (ET) receptor antagonist on the hemodynamic and biochemical parameters in stroke-prone spontaneously hypertensive rats (SHRSPs/Izm). The endothelin-A- and -B- (ETA/ETB) receptor antagonist (TAK-044, Takeda Chemical Industries, Osaka, Japan) was administered subcutaneously at a dose of 10 mg/kg/day from the age of 8 weeks for 4 weeks. Blood samples and tissues of the kidney, heart and brain were obtained at the age of 12 weeks. Tissue expression of ET-1 mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) followed by Southern blot analysis. Treatment with TAK-044 resulted in a significant decrease in systolic blood pressure (SBP), blood urea nitrogen (BUN), serum creatinine concentration, plasma aldosterone level, heart weight, and kidney weight. In addition, ET-1 contents and mRNA expression level in the kidney, heart and brain were significantly decreased by the treatment with TAK-044. These results suggest that the ET receptor antagonist TAK-044 is able to attenuate ET-1 gene expression in addition to its specific antagonism of the biological actions of ET via the receptors.

Animals↗
PubMed2000

Amyloid protofilament formation of hen egg lysozyme in highly concentrated ethanol solution.

Mutant human lysozymes (Ile56Thr & Asp67His) have been reported to form amyloid deposits in the viscera. From the standpoint of understanding the mechanism of amyloid formation, we searched for conditions of amyloid formation in vitro using hen egg lysozyme, which has been extensively studied from a physicochemical standpoint. It was found that the circular dichroism spectra in the far-ultraviolet region of the hen egg lysozyme changed to those characteristic of a beta-structure from the native alpha-helix rich spectrum in 90% ethanol solution. When the concentration of protein was increased to 10 mg/mL, the protein solution formed a gel in the presence of 90% ethanol, and precipitated on further addition of 10 mM NaCl. The precipitates were examined by electron microscopy, their ability to bind Congo red, and X-ray diffraction to determine whether amyloid fibrils were formed in the precipitates. Electron micrographs displayed unbranched protofilament with a diameter of approximately 70 A. The peak point of the difference spectrum for the Congo red binding assay was 541 nm, which is characteristic of amyloid fibrils. The X-ray diffraction pattern showed a sharp and intense diffraction ring at 4.7 A, a reflection that arises from the interstrand spacing in beta-sheets. These results indicate that the precipitates of hen egg lysozyme are amyloid protofilament, and that the amyloid protofilament formation of hen egg lysozyme closely follows upon the destruction of the helical and tertiary structures.

Amyloid↗
PubMed2000

The relationship between bone turnover and body weight, serum insulin-like growth factor (IGF) I, and serum IGF-binding protein levels in patients with anorexia nervosa.

Malnutrition is one of the risk factors for bone loss in patients with anorexia nervosa (AN). To clarify the effects of nutritional status on bone metabolism, we examined the relationship between serum levels of nutritional indicators [insulin-like growth factor I (IGF-I), IGF-binding protein-2 (IGFBP-2), and IGFBP-3] and markers for bone metabolism [serum osteocalcin and urinary excretion of C-terminal telopeptide of collagen type I (CrossLaps)] in 45 AN out-patients, including 8 severely malnourished patients who required hospitalization and iv hyperalimentation (IVH). Compared to healthy subjects, serum IGF-I and IGFBP-3 were lower, whereas IGFBP-2 was higher in out-patients who had a body mass index (BMI) less than 16.5 kg/m2. In these patients, urinary excretion of CrossLaps, a marker of bone resorption, was higher, whereas serum osteocalcin, a marker of bone formation, was lower than those in control subjects. All of these parameters were normal in patients whose BMI ranged from 16.5-18.5 kg/m2. Serum levels of osteocalcin correlated positively with BMI (r = 0.512; P<0.0001), IGF-I (r = 0.558; P<0.0001), and IGFBP-3 (r = 0.369; P<0.001) in AN out-patients. In the 8 severely malnourished AN patients, serum levels of IGF-I and osteocalcin significantly increased 3 and 7 days, respectively, after the start of a 5-week IVH therapy regimen and reached normal levels within 5 weeks, accompanied by still elevated urinary excretion of CrossLaps. The present study demonstrates that an improvement in nutritional status in AN patients during IVH therapy rapidly increases the serum IGF-I levels, followed by a progressive increase in osteocalcin, suggesting immediate start of bone formation. However, increased bone resorption appears to continue for at least 5 weeks.

Adolescent↗
PubMed2000

Two novel nodal-related genes initiate early inductive events in Xenopus Nieuwkoop center.

In vertebrates, Nodal-related protein plays crucial roles in mesoderm and endoderm induction. Here we describe two novel Xenopus nodal-related genes, Xnr5 and Xnr6, which are first zygotically expressed at the mid-blastula transition, in the dorsal-vegetal region including the Nieuwkoop center. Xnr5 and Xnr6 were isolated by expression screening of a library enriched with immediate-early-type transcripts, and are strong inducers of both mesoderm and endoderm. They also induce the other nodal-related genes in the animal cap. In embryos, cerberus-short (nodal-specific inhibitor) can inhibit Xnr1 and Xnr2 express to the same extent goosecoid, but not Xnr5 and Xnr6 transcription. Xnr5 and Xnr6 are regulated completely cell autonomously, differently from other Xnrs in the cell-dissociated embryos. The expression of Xnr5 and Xnr6 is regulated by maternal VegT and (beta)-catenin, but does not require TGF-(beta) signaling. Therefore, expression of Xnr5 and Xnr6 is controlled by different mechanisms from other Xnr family genes.

Amino Acid Sequence↗
PubMed2000

Adrenomedullin amidation enzyme activities in hypertensive patients.

Adrenomedullin (AM) is a potent vasodilating peptide secreted from the vasculature of various organs. It is biologically active when its C-terminus is amidated. Recently, an RIA method was developed for measurement of the active form of AM, or mature AM. We here employed this method to investigate the significance of amidation of AM in controlling cardiovascular function. Thirty-six patients under hemodialysis were recruited and divided into hypertensive (n = 25; 157/86 mmHg) and normotensive (n= 11; 116/66 mmHg) groups. Mature AM, immature AM and blood pressure were monitored during hemodialysis in all patients. There was a significant reduction in blood pressure during hemodialysis in both groups, although after hemodialysis blood pressure was still higher in hypertensives than in normotensives (139 +/-14.8/76 +/- 2.5 mmHg vs. 110 +/- 5.1/66.7 +/- 3.1 mmHg). Mature AM before hemodialysis were lower in hypertensives than normotensives and it decreased in both groups. Although mature AM decreased more in normotensives than in hypertensives (-27 +/- 8% vs. -17 +/- 5%), at the end point, its level was still higher in normotensives. The ratio of mature AM/immature AM decreased only in normotensives (-11.4 8.7%), whereas it remained stable in hypertensives (0.2 +/- 5.6%). Both groups showed similar changes in ANP, endothelin, catecholamines, cGMP, and NOx. The low level in mature AM level in hypertensives may have contributed to the higher blood pressure in this group. The attenuation of AM amidation in normotensives indicates that an unspecified amidative enzyme of AM was regulated in order to normalize blood pressure.

Adrenomedullin↗
PubMed2000

A defect of lipopolysaccharide-induced nitric oxide synthase gene expression in the paraventricular nucleus of Lewis rats.

The hypothalamo-pituitary-adrenal (HPA) axis activity of Lewis rats has been reported to be hyporesponsive to immune challenge, and nitric oxide (NO) has been demonstrated to be involved in the regulation of the HPA axis during the response to immune challenge. The present study investigates the effect of systemic injection of lipopolysaccharide (LPS) on NO production within the paraventricular nucleus (PVN) of immature female Fisher-344 (F344) and Lew/N rats (Lew/N), to clarify the pathophysiological role of NO in the dysregulation of the HPA axis in Lewis/N. Intraperitoneal injection of 25 mg/kg LPS significantly increased neuronal NO synthase (nNOS) mRNA expression in the PVN of F344 rats, but did not change nNOS mRNA levels in the PVN of Lew/N rats. CRF mRNA levels in the PVN significantly increased in response to LPS injection only in F344 rats. In contrast, inducible NOS (iNOS) mRNA increased similarly in both strains. These results demonstrated a defect of up-regulation of nNOS gene expression in the PVN of Lew/N rats following immune challenge. The defect appears to be associated with the dysfunction of the HPA axis in this strain. An increase in iNOS mRNA may partially restore NO production in the PVN.

Animals↗
PubMed2000

Alternate exon in the 5'-untranslated region of the human ACTH receptor gene.

The human ACTH receptor (ACTH-R) gene consists of two exons; the first encodes the 5'-untranslated region (UTR) and the second encodes part of the 5'-UTR, the entire coding region, and the whole of the 3'-UTR. While investigating the transcription initiation site of the human ACTH-R gene by 5'-rapid amplification of cDNA ends, we found a 157-bp alternate exon in the 5'-UTR. This newly identified exon was irrelevant to the alternate exon in the mouse ACTH-R gene which we reported previously.

5' Untranslated Regions↗