Search PubMed⌕ Search

Biomedical subjects

Y Shinohara

Publications and source records attributed to Y Shinohara.

At least 55 records · Page 3Linked to original sources
PubMed2001

Significant expression of functional human type 1 mitochondrial ADP/ATP carrier in yeast mitochondria.

As a first step to characterize the unknown functional properties of the human mitochondrial ADP/ATP carrier (AAC), we tried to express human type 1 AAC (hAAC1) in Saccharomyces cerevisiae. Expression of hAAC1 in yeast mitochondrial membrane was very low, although its transcript level was high. Its expression was improved greatly by replacement of its N-terminal region with the corresponding region of yeast type 2 AAC (yAAC2), as observed with the bovine type 1 AAC (bAAC1). This chimeric hAAC1 showed similar ADP transport activity to that of chimeric bAAC1, corresponding to the transport activity of bAAC1 in bovine heart mitochondria. These results suggested that the N-terminal region of yAAC2 is important for expression of the mammalian carriers in yeast mitochondria. Using the present expression system, studies on the functional properties of the human AAC isoforms in relation to their structures are now possible.

Animals↗
PubMed2001

Kinetics of intramolecular acyl migration of 1beta-O-acyl glucuronides of (R)- and (S)-2-phenylpropionic acids.

The stereoselective acyl migration of diastereomeric 1beta-O-acyl glucuronides of (R)- and (S)-2-phenylpropionic acid [(R)-1PG and (S)-IPG, respectively] in phosphate buffer (pH 7.4) at 310K was investigated using HPLC. The disappearance of (R)-1PG was faster than that of (S)-1PG according to pseudo first-order kinetics. A kinetic model describing the degradation reactions was constructed. The rate constant for acyl migration from the 1beta-O-isomer to the 2-O-acyl isomer (k12) was about one order magnitude larger than that for hydrolysis from 1beta-O-acyl isomer to aglycone (k10). The k12 of (R)-IPG (0.377 +/- 0.005 h(-1)) was about two times larger than that of (S)-IPG (0.184 +/- 0.003 h(-1)). The results indicated that the stereoselectivity in the degradation of 1PG was apparently governed by the acyl migration from 1-isomer to 2-isomer. The kinetic parameters for acyl migration from 1-isomer to 2-isomer were estimated from temperature-dependent experiments using the transition state theory. The value of the free energy of activation at 310 K for (R)-1PG (99.67 kJ/mol) was smaller than that of (S)-IPG (101.60kJ/mol), suggesting that (R)-IPG showed thermodynamically higher reactivity in acyl migration than (S)-1PG.

Acylation↗
PubMed2001

MRI study of talonavicular alignment in club foot.

We studied in vivo the talonavicular alignment of club foot in infants using MRI. We examined 26 patients (36 feet) with congenital club foot. The mean age at examination was 9.0 months (4 to 12). All analyses used MRI of the earliest cartilaginous development of the tarsal bones in the transverse plane, rather than the ossific nucleus. The difference in the mean talar neck angle (44.0 +/- 8.1 degrees) in club foot was statistically significant (p < 0.001) when compared with that of the normal foot (30.8 +/- 5.5 degrees). The difference between the mean angles in the group treated by operation (47.9 +/- 6.7 degrees) and those treated conservatively (40.1 +/- 7.5 degrees) was also statistically significant. The anatomical relationship between the head of the talus and the navicular was divided into two patterns, based on the position of the mid-point of the navicular related to the long axis of the head. In the operative group, 18 feet were classified as having a medial shift of the navicular and none had a lateral shift. In the conservative group, 12 showed a medial shift of the navicular and six a lateral shift. All nine unaffected normal feet in which satisfactory MRI measurements were made showed a lateral shift of the navicular. Club feet had a larger talar neck angle and a more medially deviated navicular when compared with normal feet. This was more marked in the surgical group than in the conservative group.

Bone Malalignment↗
PubMed2001

Detection of telomerase reverse transcriptase mRNA in biopsy specimens and bile for diagnosis of biliary tract cancers.

Human telomerase reverse transcriptase (hTERT), which codes for the catalytic subunit of telomerase, is essential for telomerase activity. Recent studies revealed that levels of hTERT mRNA as well as telomerase activity are high in neoplasm. The purpose of this study was to correlate the expression of hTERT mRNA with telomerase activity in biopsy specimens and bile from biliary tract cancers and to evaluate the potential diagnostic value of hTERT mRNA analysis for biliary malignancy. We analyzed hTERT mRNA and telomerase activity in biopsy specimens and exfoliated bile cells from patients with cholangiocarcinoma, gallbladder carcinoma and bile duct stones. hTERT was detected by either nested reverse transcriptase-polymerase chain reaction (PCR) or real-time PCR. Telomerase activity was examined by a fluorescence-based telomeric repeat amplification protocol assay. Six of 10 malignant biopsy specimens had detectable hTERT and 7 of 10 had telomerase activity. All cases with hTERT expression had telomerase activity. In bile, 7 of 10 malignant patients had detectable hTERT and 3 of 10 had telomerase activity. Importantly, there were no false positive results in tissue specimens or bile examined in 6 non-cancerous cases. In conclusion, the detection of hTERT mRNA in biopsy specimens and bile cells, in combination with routine histologic and cytologic examination may improve the diagnosis of biliary tract cancers.

Aged↗
PubMed2000

Specific labeling of the bovine heart mitochondrial phosphate carrier with fluorescein 5-isothiocyanate: roles of Lys185 and putative adenine nucleotide recognition site in phosphate transport.

The amine/SH-modifying fluorescein 5-isothiocyanate (FITC) specifically labeled Lys(185) in the putative membrane-spanning region of the phosphate carrier from both the cytosolic and matrix sides of bovine heart mitochondria at 0 degrees C and pH 7.2, and the labeling inhibited the phosphate transport. Nonmodifying fluorescein derivatives having similar structural features to those of ADP and ATP (Majima, E., Yamaguchi, N., Chuman, H., Shinohara, Y., Ishida, M., Goto, S., and Terada, H. (1998) Biochemistry 37, 424-432) inhibited the specific FITC labeling and phosphate transport, but the nonfluorescein phenylisothiocyanate did not inhibit FITC labeling, suggesting that there is a region recognizing the adenine nucleotides in the phosphate carrier and that this region is closely associated with the transport activity. The phosphate transport inhibitor pyridoxal 5'-phosphate inhibited the specific FITC labeling, possibly due to competitive modification of Lys(185). In addition, FITC inhibited the ADP transport and specific labeling of the ADP/ATP carrier with the fluorescein SH reagent eosin 5-maleimide. Based on these results, we discuss the structural features of the phosphate carrier in relation to its transport activity.

Adenosine Diphosphate↗
PubMed2000

Novel expression of equivocal messages containing both regions of choline/ethanolamine kinase and muscle type carnitine palmitoyltransferase I.

For characterization of the detailed gene structure of human muscle type carnitine palmitoyltransferase I (M-CPTI), we analyzed the 5'-upstream region of the M-CPTI transcripts. As a result, we found a cDNA clone containing a nucleotide sequence unexpected from the reported M-CPTI gene structure in the upstream region of its 5' end. Comparison of this nucleotide sequence with that of genomic DNA showed that this sequence was derived from the 3'-untranslated region of the gene encoding choline/ethanolamine kinase-beta (CK/EK-beta) located upstream of the M-CPTI gene. Southern blot analysis showed that there was no other region homologous to the CK/EK-beta gene in the whole human genome. Thus, the overlapping transcript was concluded to be produced from the functional genes of CK/EK-beta and M-CPTI. Furthermore, cDNAs containing both exons of these genes were detected by the polymerase chain reaction using the cDNA of human heart M-CPTI obtained by specific reverse transcription from its 3'-untranslated region as a template. From these results, the production and organization of these overlapping transcripts are discussed.

3' Untranslated Regions↗
PubMed2000

Bax and Bcl-xL independently regulate apoptotic changes of yeast mitochondria that require VDAC but not adenine nucleotide translocator.

Mitochondria play an essential role in apoptosis by releasing apoptogenic molecules such as cytochrome c and AIF, and some caspases, which are all regulated by Bcl-2 family proteins. Pro-apoptotic Bax and Bak have been shown to induce cytochrome c release and loss of membrane potential (Deltapsi) leading to AIF release in the isolated mitochondria. We have previously shown that Bax and Bak open the voltage-dependent anion channel (VDAC) allowing cytochrome c to pass through the channel, and Bcl-xL closes the channel. However, it has been reported that it is adenine nucleotide translocator (ANT) with which Bax/Bcl-xL interacts that modulate the channel activity. Here, we investigated the role of ANT and VDAC in the changes of isolated mitochondria triggered by Bax and by chemicals that induce permeability transition (PT). In rat and yeast mitochondria, Bax did not affect the ADP/ATP exchange activity of ANT. VDAC-deficient but not ANT-deficient yeast mitochondria showed resistance to cytochrome c release, Deltapsi loss, and swelling caused by Bax and PT inducers. Bcl-xL showed similar inhibition of all these changes in ANT-deficient and wild type yeast mitochondria. Furthermore, Bax induces cytochrome c release in wild type yeast cells but not VDAC1-deficient yeast cells. These data indicate that VDAC, but not ANT, is essential for apoptotic mitochondrial changes. The data also indicate that Bcl-xL and Bax possess an ability to regulate mitochondrial membrane permeability independently of other Bcl-2 family members.

Adenosine Triphosphate↗
PubMed2000

Stable isotope dilution analysis of human urinary metabolites of 17alpha-methyltestosterone.

A method based on gas chromatography-mass spectrometry-selected-ion monitoring was developed to measure the main metabolites of 17alpha-methyltestosterone, 17alpha-methyl-5alpha-androstan-3alpha,17beta-di ol and 17alpha-methyl-5beta-androstan-3alpha,17beta-dio l, in human urine. 17alpha-Methyl-[(2)H3]-5alpha-androstan-3alpha,1 7beta-diol and 17alpha-methyl-[(2)H3]-5beta-androstan-3alpha,17 beta-diol were used as internal standards. The methods involved purification using a Sep-Pak C(18) cartridge, hydrolysis by beta-glucuronidase from Ampullaria and derivatization with N-methyl-N-trimethylsilyl-trifluoroacetamide/dithioerythriol/ammon ium iodide. Quantitation was achieved by selected-ion monitoring of the characteristic fragment ions ([(M+H)-2xTMSOH]+) of the di-TMS derivatives on the chemical ionization mode. The method provides a specific, sensitive and reliable technique to determine the urine levels of 17alpha-methyl-5alpha-androstan-3alpha,17beta-di ol and 17alpha-methyl-5beta-androstan-3alpha,17beta-dio l, and can be applied to pharmacokinetic studies of 17alpha-methyltestosterone.

Calibration↗
PubMed2000

Reduced cerebral glucose metabolism in subjects with incidental hyperintensities on magnetic resonance imaging.

OBJECTIVE: To clarify the significance of incidental and asymptomatic hyperintensities on T(2)-weighted magnetic resonance images (MRI) in adults, we examined the relationship between a variety of these lesions and cerebral metabolism evaluated by positron emission tomography (PET) with (18)F-labeled fluorodeoxyglucose ([(18)F]FDG). SUBJECTS AND METHODS: Two hundred and thirty-one persons with hyperintensities on T(2)-weighted MRI but without overt neurological disease (mean age 60+/-9 years) were studied. MR hyperintensities were classified into deep and/or subcortical white matter hyperintensities (DSWMHs), periventricular hyperintensities (PVHs) and hyperintensities in the basal ganglia and/or thalamus (HBGTs). The relationship between these lesions and cerebral metabolic rate of glucose (CMRgl) measured by [(18)F]FDG-PET was investigated. RESULTS: The CMRgl values in white matter and cerebral cortex in the group with severe PVHs were lower than those in the group with mild PVHs (P<0.0001 and P<0.005). Although the severity of PVHs was associated with the numbers of DSWMHs and HBGTs, the results of multivariate analysis showed a significant relationship of PVHs to glucose metabolism in cerebral cortex and white matter. CONCLUSIONS: We conclude that increasing severity of MRI hyperintensities in adults is associated with a deterioration of cerebral metabolism. In particular, involvement of PVHs may be a marker of widespread deterioration of cortical metabolism.

Adult↗
PubMed2000

Specific elevation of transcript levels of particular protein subtypes induced in brown adipose tissue by cold exposure.

To understand the difference in metabolic flow in rat brown adipose tissue (BAT) from that in white adipose tissue (WAT) at the molecular level, we examined the steady-state transcript levels of 39 proteins in both adipose tissues with and without cold exposure by Northern blot analysis. In addition to the transcript levels of uncoupling protein isoforms, those of proteins involved in the transport and catabolism of fatty acids and glucose in BAT were elevated by cold exposure, suggesting the stimulation of utilization of fatty acids and glucose as fuels in BAT. As to these changes, the muscle-type subtypes were remarkable; and therefore, they were suggested to be responsible for the cold exposure-induced acceleration of energy expenditure in BAT. Furthermore, of the isoforms of beta-adrenergic receptor (beta-AR) and CCAAT enhancer binding protein (C/EBP), transcript levels of beta(1)-AR and C/EBPbeta in BAT were increased by the cold exposure. Possible roles of these proteins in energy metabolism in BAT were discussed.

Adipose Tissue, Brown↗
PubMed2000

Nitrotyrosine generation via inducible nitric oxide synthase in vascular wall in focal ischemia-reperfusion.

Nitrotyrosine produced by NO-mediated reaction is a possible marker for cytotoxicity in brain ischemia. In this study, we aimed to determine whether iNOS is responsible for the nitrotyrosine formation and which type of cell is predominantly nitrated. Fifty-eight wild-type and 28 iNOS knockout male mice were used. Under halothane anesthesia the left middle cerebral artery was occluded for 2 h and reperfused for 0.5 or 15 h. The ratio of nitrotyrosine to total tyrosine (%NO2-Tyr) was measured by means of a hydrolysis/HPLC. After 0.5-h reperfusion, %NO2-Tyr in the ischemic cortex of wild-type and knockout mice amounted to 0.037 +/- 0.040% (n = 8) and 0.064 +/- 0.035% (n = 6), respectively, being significantly higher than that in the sham operation group (n = 7) (P < 0.05). After 15-h reperfusion, nitrotyrosine was detected only in wild-type mice (0.039 +/- 0.025%, n = 7), not in knockout or sham-operated mice (P < 0.05). Immunohistochemical reaction for nitrotyrosine was seen predominantly in the vascular wall in the peri-infarct region of the cerebral cortex in wild-type mice after 15-h reperfusion, but not in corresponding knockout mice. Our data suggest that iNOS is responsible for nitrotyrosine formation in the later phase of reperfusion, and that vascular endothelium is the major site of this reaction, at least in the case of 15-h reperfusion.

Acidosis↗
PubMed2000

Nuclear cyclin D1 overexpression is a critical event associated with cell proliferation and invasive growth in gallbladder carcinogenesis.

Cyclin D1 overexpression is remarkably frequent in several human carcinomas and is believed to be a critical event in oncogenesis. We examined cyclin D1 expression, p53 expression, and the Ki-67 labeling index by immunostaining in human gallbladder mucosa in conditions varying from normal to malignant tissue. We also examined K-ras codon 12 mutations in these tissues with a two-step polymerase chain reaction. Nuclear cyclin D1 overexpression was observed in 48% of carcinomas occurring independently of adenoma, but not in adenomas, carcinomas arising in adenomas, or nonneoplastic lesions. Cytoplasmic cyclin D1 overexpression was observed in about 15% of abnormal specimens, irrespective of the type of epithelial abnormality. Carcinomas showing nuclear cyclin D1 overexpression had significantly higher Ki-67 labeling indexes than those with no overexpression. Moderately to poorly differentiated adenocarcinomas showed a higher incidence of nuclear cyclin D1 overexpression than papillary to well differentiated carcinomas. Specimens with cyclin D1 overexpression showed a high incidence of lymph permeation, venous permeation, and lymph node metastasis. We conclude that nuclear cyclin D1 overexpression is a critical event importantly associated with cell proliferation and invasive growth in gallbladder carcinogenesis, and that cyclin D1 immunostaining may become a useful marker for evaluating gallbladder carcinomas.

Adenocarcinoma↗
PubMed2000

Prenatal exposure to diethylstilbestrol decreases the number of estrogen receptor alpha-containing neurons innervating the ovary in rat celiac ganglion.

Recently, we reported that, in rats, transplacental exposure to diethylstilbestrol, a potent synthetic estrogen, decreases the density of the ovarian sympathetic nerve network compared to that in control rats not exposed to diethylstilbestrol. To clarify the mechanism of the decrease in the density, we performed a series of experiments using rats prenatally exposed to diethylstilbestrol and unexposed control rats. First, a retrograde tract tracer, Fast Blue, was microinjected into the ovaries of both groups of rats at four months of age, and the number of Fast Blue-positive neurons in the celiac ganglion was counted. The number of neurons in the ganglion was smaller in diethylstilbestrol-exposed rats than in the controls. Second, double labeling of the neurons with antibody against estrogen receptor alpha and Fast Blue in the celiac ganglion was carried out in both groups of rats. The results showed that estrogen receptor alpha-containing neurons in the ganglion innervated the ovary, and their number was decreased selectively by prenatal diethylstilbestrol exposure. Finally, a decrease in the celiac ganglion volume in rats prenatally exposed to diethylstilbestrol was also detected on day 23 of gestation, as compared to the volume in the control rats. From these observations, we propose that prenatal exposure to diethylstilbestrol can induce a loss of estrogen receptor alpha-containing neurons innervating the ovary during development, resulting in paucity of the neural network in the ovary.

Amidines↗
PubMed2000

Giant negative T waves in Guillain-Barré syndrome.

A Guillain-Barré syndrome patient showed giant negative T waves on electrocardiography at the height of the disease, with large left ventricular hypokinesis on echocardiography and extensive defects on 123I-meta-iodobenzylguanidine myocardial scintigraphy. Gamma-globulin improved the neurological symptoms, and the above abnormalities resolved. We speculate that cardiac sympathetic nerve endings were transiently damaged, with consequent myocardial injury, due to norepinephrine toxicity.

3-Iodobenzylguanidine↗
PubMed2000

Characterization of porin isoforms expressed in tumor cells.

Mitochondria from malignant tumor cell lines show a higher capability for hexokinase binding than those from normal liver. To explore possible differences in hexokinase binding sites of mitochondria between tumor cells and normal liver, we characterized porin isoforms expressed in tumor cells. Cloning experiments on the three porin isoforms, VDAC1, VDAC2 and VDAC3 from malignant tumor cell line AH130 clearly showed that their primary structures were completely identical to those of the corresponding VDACs of normal liver cells. Possible expression of the fourth porin isoform in AH130 cells was excluded by degenerate primer-based RT-PCR. However, the transcript levels of the three VDAC isoforms in AH130 cells were significantly higher than those in normal liver. These results suggest that the high hexokinase-binding capability of malignant tumor cell mitochondria was not due to any structural difference, but due to a quantitative difference in binding sites.

Alternative Splicing↗