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Biomedical subjects

H Tokunaga

Publications and source records attributed to H Tokunaga.

At least 19 recordsLinked to original sources
PubMed2001

In vivo protection of a water-soluble derivative of vitamin E, Trolox, against methylmercury-intoxication in the rat.

Methylmercury (MeHg) is a well-known neurotoxicant. MeHg-intoxication causes a disturbance in mitochondrial energy metabolism in skeletal muscle and apoptosis in cerebellum. We report the first in vivo effectiveness of antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carhoxylic acid), a water soluble vitamin E analog, against the MeHg-induced cellular responses. Treatment with Trolox (6-hydroxy-2.5,7,8-tetramethylchroman-2-carboxylic acid) clearly protects MeHg-treated rat skeletal muscle against the decrease in mitochondrial electron transport system enzyme activities despite the retention of MeHg. Tdt-mediated dUTP nick-end-labeling method clarified that Trolox is effective for protecting cerebellum from MeHg-induced apoptosis. These data indicate that MeHg-mediated oxidative stress plays an important role in the in vivo pathological process of MeHg intoxication. Trolox may prevent some of clinical manifestations of MeHg-intoxication in humans.

Animals↗
PubMed2001

NaCl-activated nucleoside diphosphate kinase from extremely halophilic archaeon, Halobacterium salinarum, maintains native conformation without salt.

Enzymes from extremely halophilic archaea are readily denatured in the absence of a high salt concentration. However, we have observed here that a nucleoside diphosphate kinase prepared from Halobacterium salinarum was active and stable in the absence of salt, though it has the amino acid composition characteristic of halophilic enzymes. Recombinant nucleoside diphosphate kinase expressed in Escherichia coli requires salt for activation in vitro, but once it acquires the proper folding, it no longer requires the presence of salts for its activity and stability.

Amino Acid Sequence↗
PubMed2001

Efficient expression, purification and characterization of mouse salivary alpha-amylase secreted from methylotrophic yeast, Pichia pastoris.

We constructed a secretion vector of mouse salivary alpha-amylase, pPAM, using the AOX1 promoter-terminator and the secretion signal of 128 kDa pGKL killer protein, for an alternative yeast, Pichia pastoris. Taking advantage of multicopy insertion of the expression cassette and optimized growth conditions, we succeeded in highly efficient extracellular production (approximately 240 microg/ml) of mouse alpha-amylase in the 10 ml scale by conventional flask culture: this efficiency was about 90-fold higher than that of Saccharomyces cerevisiae. Growth temperature of cells was critical for efficient production of alpha-amylase. P. pastoris transformants secreted both core-glycosylated and non-glycosylated alpha-amylase molecules with a glycosylated:non-glycosylated ratio of about 20:80. Both glycosylated and non-glycosylated alpha-amylases were purified separately to apparent homogeneity. The signal sequence was correctly processed in both species, and the molecular masses of glycosylated and non-glycosylated alpha-amylase were determined to be 58 600 and 56 300, respectively, by mass spectrometry. We further studied the outer chain glycosylation of engineered mouse alpha-amylase secreted by P. pastoris.

Amino Acid Sequence↗
PubMed2001

Expression and purification of cytokine receptor homology domain of human granulocyte-colony-stimulating factor receptor fusion protein in Escherichia coli.

Direct expression of the cytokine receptor homology (CRH) domain of granulocyte-colony-stimulating factor (G-CSF) receptor is lethal to Escherichia coli. For the efficient and stable production of an active CRH domain in E. coli, we fused the CRH domain with different proteins, such as maltose-binding protein (MalE), glutathione S-transferase, and thioredoxin (Trx). Among these, Trx appeared to be the best in terms of the protein expression level, purification efficiency by affinity chromatography, and binding activity to its ligand, G-CSF. The yield of active Trx-CRH fusion protein increased about 200-fold compared to that of previously reported MalE-CRH fusion.

ATP-Binding Cassette Transporters↗
PubMed2001

Expression, purification, and characterization of the active immunoglobulin-like domain of human granulocyte-colony-stimulating factor receptor in Escherichia coli.

We succeeded in the expression, purification, and refolding of the immunoglobulin-like (Ig) domain of human granulocyte-colony-stimulating factor (G-CSF) receptor with amino-terminal His-tag in Escherichia coli. The refolded Ig domain bound to a G-CSF affinity column and could be eluted with free G-CSF as a receptor-ligand complex, demonstrating that the Ig domain has the information necessary for binding its ligand, G-CSF. The eluted His-Ig/G-CSF complex could be separated from excess G-CSF by Ni-NTA column chromatography. The yield of this active recombinant His-Ig protein is about 0.72 mg per liter of culture. Its small size and the ease of production make this receptor fragment a useful reagent for the structural analysis of its complex with G-CSF.

Amino Acid Substitution↗
PubMed2001

Endothelin receptor antagonist prevents parathyroid cell proliferation of low calcium diet-induced hyperparathyroidism in rats.

Secondary hyperparathyroidism, one of the most frequently encountered disorders of the calcium homeostasis, is characterized by an increase in parathyroid epithelial (PT) cell number, which is crucial from a functional viewpoint. However, it is still unknown what factors are involved in PT cell proliferation. Endothelin-1 (ET-1), a vasoconstrictive peptide, has been shown to act as a mitogen in a variety of cell types. Rat PT cells are reported to synthesize ET-1 and possess its receptors. To test the hypothesis that ET-1 plays a role in PT cell proliferation, we used rat test subjects fed a low calcium diet for 8 weeks (low Ca rats). The number of the proliferating PT cells, measured by proliferating cell nuclear antigen immunostaining, was significantly increased, with striking immunoreactivity of ET-1 in the low Ca rats. An endothelin receptor antagonist, bosentan (100 mg/kg.day), prevented any increase in the proliferation of PT cells in the low Ca rats (14.3 +/- 2.7/1000 PT cells with no bosentan; 2.1 +/- 1.3 with bosentan; P < 0.01). These results indicate that ET-1 is involved in PT cell proliferation in vivo and suggest that blocking of ET receptors may become one of the important therapeutic strategies for preventing secondary hyperparathyroidism.

Animals↗
PubMed2001

An association between an antibody against Chlamydia pneumoniae and common carotid atherosclerosis.

OBJECTIVE: Chlamydia pneumoniae (C. pneumoniae) is an important pathogen for infections of the respiratory tract, and there are recently also a number of reports suggesting its relation with atherosclerosis. This study was performed to clarify the relation between C. pneumoniae infection and sclerotic lesions of the common carotid arteries. METHODS AND PATIENTS: We evaluated sclerotic lesions of common carotid arteries by ultrasonography in 147 in-patients (mean age, 70 years; 95% confidence interval, 68-72) in the internal medicine ward, and studied the relation of the known risk factors for atherosclerosis including C. pneumoniae infection. An ultrasonograph and 7.5 MHz linear type B-mode probe were used by a specialist to evaluate sclerotic lesions of common carotid arteries. C. pneumoniae infection was determined by measuring anti-C. pneumoniae IgG specific antibody level (IgG index) using enzyme-linked immunosorbent assay (ELISA) method with serum of fasting blood, which had been preserved at -70 degrees C. RESULTS: IgG index (p=0.0263), from multiple regression analysis using various risk factors as explanatory variables, was a significant independent contributing factor (R2=0.3465, p<0.0001) along with known risk factors such as male (p=0.0289), age (p=0.0007), Brinkman index (p=0.0067), hypertension (p=0.0443) and T-Chol (p=0.0220). CONCLUSION: This study confirmed that the observations of an association between antibody against C. pneumoniae and common carotid atherosclerosis in Western nations is also present in Japan. Our results suggests that C. pneumoniae infection is also an important risk factor for common carotid atherosclerosis.

Adult↗
PubMed2000

Functional anatomical study of psychogenic amnesia.

Psychogenic amnesia is characterized by an inability to recall information already stored in the patient's memory. It is usually related to a stressful or traumatic event that cannot be explained by manifest brain damage. To examine the underlying functional disturbance of brain areas in this condition, we performed a positron emission tomography (PET) activation study on a psychogenic amnesic patient and on 12 normal control subjects. A task requiring explicit retrograde memory of faces was compared with a control task. To assess functional modifications associated with the processes of recovery, a second PET study was performed on the patient 12 months after onset. During the task, activation of the right anterior medial temporal region including the amygdala was increased in the psychogenic amnesic patient. Activation of the bilateral hippocampal regions increased only in the control subjects. During recovery, the right anterior medial temporal region became less active while the right hippocampal region became more active. Activation levels also differed in the anterior cingulate cortex, prefrontal cortex and some other cortical regions between control subjects and the patient. These findings suggest that the changes in these limbic and limbic-cortical functions are related to symptoms of the psychogenic amnesia.

Adult↗
PubMed2000

Expression of bone morphogenetic protein receptors type-IA, -IB and -II correlates with tumor grade in human prostate cancer tissues.

Bone morphogenetic proteins (BMPs) are potential regulators of prostate cancer cell growth and metastasis that signal through an interaction with BMP membrane receptors (BMPRs) type I and type II. In the present study, Western blot and immunohistochemical analysis of BMPRs were carried out in benign and malignant human prostate tissues to explain the loss of BMP response in human prostate cancer cells. The results demonstrated that the benign prostate specimens expressed high levels of all three BMPRs. In normal prostate, BMPRs were localized predominantly to epithelial cells. Among prostate cancer specimens, well-differentiated cancers were positive for the expression of BMPR-II, BMPR-IA, and BMPR-IB, for the most part. In contrast, only 1 of 10 poorly differentiated prostate cancer cases was positive for each of the three BMPRs (P < 0.005 for all three receptors). Taken together, these results indicate that human prostate cancer cells frequently exhibit loss of expression of BMPRs and suggest that loss of BMPRs may play an important role during the progression of prostate cancer.

Blotting, Western↗
PubMed2000

The neural basis of perceptual and conceptual word priming--a PET study.

Positron emission tomography scans were obtained in 13 normal subjects during perceptual and conceptual word priming tasks with the aim to investigate the neural system specific to the two priming conditions. In the prescan phase, subjects were primed perceptually or conceptually with two separate procedures, while in the scan phase, they performed the same stem completion task. Therefore we could compare the results of the two priming tasks in a direct manner. A fixation control task and a baseline task (completion of stems that did not correspond to previously seen words) were also given. A specific blood flow decrease was found in the left inferior temporal cortex in the perceptual word priming condition and in the left superior temporal / inferior parietal cortex in the conceptual word priming condition. Each blood flow change may reflect transient changes in the cortical areas subserving the processing of the perceptual and conceptual components of word priming.

Adult↗
PubMed2000

Growth hormone secreting adenoma with unusual extension: coexisting pituitary cyst and its clinical significance.

A 58 year old man showed acromegalic features. The serum growth hormone (GH) level was 7.3 ng/ml and SMC (somatomedin-C) 637 U/ml. Triple stimulation test showed abnormal response compatible with a GH secreting tumour. The conventional enhanced MRI revealed a less enhanced hemisphere-shaped lesion at the right corner of the sella turcica. In addition, dynamic MRI demonstrated an elongated lesion extending to the left beyond the midline. The patient underwent transsphenoidal surgery. Besides the soft and suckable tumour at the right corner, we entered into a small cavity loosely filled with the tumour, which was subsequently also removed. The operative finding corresponded to the lesion shown in dynamic MRI. Postoperative GH and SMC levels became 2.3 ng/ml and 326 U/ml respectively. Incidental pituitary cystic lesions in autopsied cases have been reported to be 6-33%. This case had a GH secreting adenoma with coexisting pituitary cyst. The coexisting pituitary cyst supposedly influenced the unusual shape and extension of the pituitary adenoma. Coexistence of such lesion should be kept in mind for microadenoma on neuroradiological evaluation and on intraoperative inspection surrounding the tumour.

Adenoma↗
PubMed2000

Secretion of hen egg white lysozyme from Kluyveromyces lactis.

Hen egg white (HEW) lysozyme was correctly processed and efficiently secreted from an alternative yeast, Kluyveromyces lactis. We constructed secretion vectors using PHO5, PGK, and LAC4 promoters, and found that the highest secretion was obtained under the direction of the PGK promoter in non-selective rich medium. K. lactis secreted HEW lysozyme with two-fold higher efficiency than S. cerevisiae, estimated by using a K. lactis-S. cerevisiae shuttle vector.

Animals↗
PubMed2000

Sodium-dependent calcium release from vascular smooth muscle mitochondria.

Interest in mitochondrial calcium (Ca2+) uptake and release waned as it became apparent that sarcoplasmic reticulum calcium stores dominate the control of cytoplasmic calcium concentration. Our recent demonstration of a very large rise in vascular smooth muscle (VSM) cytoplasmic sodium (Na+) concentration after inhibition of the sodium, potassium-ATPase (sodium pump) led us to several questions. Do VSM mitochondria show Na(+)-dependent Ca2+ release? Are the documented changes in cytoplasmic Na+ concentration sufficient to cause Ca2+ release? Do features of the cardiac mitochondrial exchange system, including differential sensitivity to a number of calcium antagonists and cation specificity, apply to VSM? We isolated mitochondria from bovine aorta and mesenteric arteries and employed arsenazo III as the Ca2+ indicator. Mitochondria from arterial vessels accumulated added calcium (up to 50 nmol Ca2+/mg protein) and released Ca2+ on exposure to Na+. This concentration-dependent relationship was linear from 0 to 10 mM of Na+, and it plateaued between 20 mM and 40 mM of Na+. VSM mitochondria exposed to 20 mM Na+ released 118 +/- 25 nmol Ca2+ per mg mitochondrial protein in 20 min, when a new equilibrium was reached. Lithium (Li+), in contrast to Na+, produced much smaller amounts of Ca2+ release from the VSM mitochondria. Na+-dependent Ca2+ release was antagonized in a concentration-dependent manner by diltiazem (0-320 microM) with a Ki of 10.2 microM. Nifedipine had a lesser effect, and verapamil produced almost no inhibition. VSM mitochondria responses resemble those from heart mitochondria in that Na+-dependent Ca2+ release is present with a similar range of sensitivity to Na+ and a similar pattern of influence of diltiazem, nifedipine and verapamil. However, the influence of Li+ on Ca2+ release was much smaller and the amount of the Ca2+ released was much greater for VSM mitochondria compared with that reported for heart mitochondria. The large amount of Ca2+ released and the range of Na+ concentration that provoked Ca2+ release being within the physiologically achievable range raise the interesting possibility that these mechanisms may modify intramitochondrial cytosolic Ca2+ concentration, and hence could potentially contribute to the contractile response that follows inhibition of the sodium pump.

Animals↗
PubMed2000

[The correlation between the exacerbation of bronchial asthma and picornavirus (human rhino virus) infection in throat gargles by RT-PCR].

Viral infection is one of important factors to cause the exacerbation of bronchial asthma. We have investigated 167 adults of asthmatics to clarify the correlation between viral infection and exacerbation of asthma. Patients were classified to four group by the symptoms of common cold and asthma attack. Furthermore, we have examined Picornavirus and Human rhino virus RNA from throat gargles of patients using RT-PCR (reverse transcription--polymerase chain reaction) method. Forty of 65 (61.5%) asthmatics with common cold revealed asthma attack and common cold was significantly associated with acute exacerbation of asthma (p < 0.01). We identified Picornavirus RNA, which include 113 of Human rhino virus serotypes and enterovirus, from the samples of 16 of 52 (30.8%) patients who had acute exacerbation. It was significantly higher than the detection rate of viral RNA from patient without asthma attack. Furthermore, we analyzed Human rhino virus RNA from the same samples by RT-PCR and 93.7% of Picornavirus were identified as Human rhino virus. Taken together, these findings suggest that common cold is significantly associated with the exacerbation of bronchial asthma. Human rhino virus infection might be one of important virus in this procedure.

Adult↗
PubMed2000

[VEGF is an essential molecule for glomerular endothelial cells and its excretion in urine might be a unique marker of glomerular injury].

A glomerulus is a functional unit of the kidney, and endothelial cells in the glomerulus are often exposed to more than 5 times higher pressure than peripheral capillaries. Glomerular development proceeds through angiogenesis and VEGF was shown to mediate the angiogenesis. VEGF is constitutively expressed in the glomerulus from the embryo to adults. When VEGF signal was blocked by the antibody, glomerular endothelial cells were swollen and capillary lumen was interrupted. Changes were more prominent in the juxta-medullary than in the cortical glomerulus. A major VEGF receptor, Flk-1/KDR, is specifically localized to the glomerular endothelial cell among tissues and more predominantly in the juxta-medullary than in the cortical glomerulus. As capillary pressure is higher in the juxta-medullary than in the cortical glomeruli, endothelial cells in the former are exposed to more tension than those in the latter. VEGF might be a protective molecule for endothelial cells against tension. The effect of VEGF on the repair of an impaired glomerulus was evaluated in the rat Thy-1 glomerulonephritis. VEGF inhibited early endothelial injury and accelerated consequent remodeling of the glomerulus. In the patient study, VEGF excretion in the urine was independent from its serum or plasma level, but increased as renal function decreased. VEGF signaling is essential in glomerular development, maintenance and repair. VEGF excreted in the urine might reflect its generation in the kidney and be a unique marker of renal function.

Animals↗
PubMed1999

Different neural substrates for Kanji and Kana writing: a PET study.

To investigate the neural substrate underlying the mechanisms of Kanji and Kana writing, we conducted a PET activation study during mental writing task in eight right-handed normal Japanese subjects. During scans subjects were required to mentally write a Kanji or three Kana letters with their right hand, for each stimulant word presented auditorily. The direct comparisons between Kanji writing and Kana writing revealed that the left posterior inferior temporal gyrus was activated in Kanji writing while the left angular gyrus was activated in Kana writing. In addition, more extensive areas were activated in Kanji writing compared with Kana writing. These results suggest that different respective neural substrates are involved in Kanji and Kana writing respectively.

Adolescent↗
PubMed1999

Liquid chromatography-fast atom bombardment mass spectrometry for detection and determination of pentazocine in human tissues.

A reliable and sensitive method was developed for the detection and determination of pentazocine in human solid tissues using liquid chromatography-dynamic fast atom bombardment (FAB) mass spectrometry, combined with a three-step liquid-liquid extraction procedure. Levallorphan tartrate served as an internal standard. The extract was evaporated to dryness and dissolved in the mobile phase, acetonitrile-10 mM ammonium acetate solution (20:80, pH 4.0) containing 0.5% glycerol as FAB matrix. The eluent was pumped at a flow rate of 25 microl/min and split before introduction to FAB mass spectrometer. Quantitative analysis was carried out by means of monitoring quasi-molecular ions with m/z 286 for pentazocine and m/z 284 for levallorphan. The lower limit of detection of pentazocine in each tissue tested was 1 ng/g with scan mode and 0.1 ng/g with SIM mode. Using this method, the concentrations of pentazocine were determined in the tissues of an autopsied individual to perform toxicological evaluation.

Aged↗