Biomedical subjects

H Yokota

Publications and source records attributed to H Yokota.

At least 73 records · Page 4Linked to original sources

PubMed2000

Regional distribution and plasma concentration of peptide YY in sheep.

Peptide YY (PYY)-positive cells are distributed in the mucosa of the ileum, cecum, colon, and rectum of sheep, but not in other layers of these regions. By radioimmunoassay, mucosal content of PYY in the ovine large intestine was much less than that in the rat intestine. The plasma concentration of immunoreactive PYY did not significantly fluctuate over a 48-h period in conscious sheep, even after ingestion of roughage and concentrate. Intraluminal nutrients into the ileum and i.v. CCK8 also did not raise the plasma level of PYY. Therefore, PYY seems unlikely to play a role as "ileal brake" in sheep.

Animals↗

PubMed2000

Adsorption of endotoxin on glass in the presence of rhIL-11.

Poor recovery of spiked endotoxin in the Limulus amebocyte lysate assay (LAL assay) was observed in the presence of recombinant human interleukin-11 (rhIL-11), a cationic, hydrophobic protein. Detection of endotoxin activity remaining in the empty glass tubes in which endotoxin and rhIL-11 mixtures were incubated indicated adsorption of endotoxin on glass. At low concentrations of rhIL-11, a correlation between endotoxin adsorbed on glass and a decrease of endotoxin in solution was observed. Adsorption of rhIL-11 on glass correlated with adsorption of endotoxin, which indicates that rhIL-11 mediates adsorption of endotoxin on glass. Consequently, adsorption of endotoxin on glass may occur in the presence of other substances which bind to both of endotoxin and glass.

Adsorption↗

PubMed2000

Smooth muscle layer-dependent distribution of 5-hydroxytryptamine(7) receptor in the porcine myometrium.

1. To analyse the mechanisms of muscle layer-dependent inhibition of porcine myometrial contractility by 5-hydroxytryptamine (5-HT), the effects of 5-HT, 5-carboxamidotryptamine(5-CT), 5-methoxytryptamine (5-MeOT), forskolin and cyclic adenosine 3', 5'-monophosphate (cyclic AMP) analogues on spontaneous and stimulant-induced contractions were examined in longitudinal (LM) and circular muscles (CM). In addition, accumulation of cyclic AMP by 5-HT and distribution of 5-HT(7) receptors in LM and CM layers were compared using biochemical and molecular approaches. 2. 5-HT receptor agonists inhibited the spontaneous contractions of LM and CM (5-CT>5-HT>5-MeOT), but CM was more sensitive than was LM. The inhibition by the agonists was antagonized by methiothepin (100 nM). 3. Carbachol-, high-K(+)-, histamine- and Ca(2+)-induced contractions were inhibited by 5-HT with different responses (CM>LM). Even in the presence of 3-isobutyl-1-methylxanthine (IBMX), the inhibition by 5-HT in the CM was still more conspicuous than that in the LM. 4. Compared with the CM, the inhibition of spontaneous contraction by forskolin, dibutyryl-cyclic AMP and 8-bromo-cyclic AMP was marked in the LM. 5. 5-HT (1 nM - 1 microM) increased the cyclic AMP in both muscle layers, but the increment in the CM was higher than that in the LM whether IBMX was present or not. 6. LM and CM layers contained a single class of [(3)H]-5-CT binding sites with a similar K(d) value (0.21 - 0.24 nM). However, B(max) (5-HT(7) receptor concentration) in the CM (120.6 fmol mg(-1) protein) was higher than that in the LM (30.4 fmol mg(-1) protein). 7. The molecular study (reverse transcription polymerase chain reaction) demonstrated the expression of 5-HT(7) receptor mRNA in the CM was higher than that in the LM. 8. These results suggest that the muscle layer-dependent difference in inhibition by 5-HT is not restricted to spontaneous contraction but applies to various contractions in the porcine myometrium. Different inhibition of the contractility by 5-HT is caused by muscle layer-related accumulation of cyclic AMP (CM>LM), due to smooth muscle-layer dependent distribution (CM>LM) of 5-HT(7) receptors.

5-Methoxytryptamine↗

PubMed2000

Future opportunities for life science programs in space.

Most space-related life science programs are expensive and time-consuming, requiring international cooperation and resources with trans-disciplinary expertise. A comprehensive future program in "life sciences in space" needs, therefore, well-defined research goals and strategies as well as a sound ground-based program. The first half of this review will describe four key aspects such as the environment in space, previous accomplishments in space (primarily focusing on amphibian embryogenesis), available resources, and recent advances in bioinformatics and biotechnology, whose clear understanding is imperative for defining future directions. The second half of this review will focus on a broad range of interdisciplinary research opportunities currently supported by the National Aeronautics and Space Administration (NASA), National Institute of Health (NIH), and National Science Foundation (NSF). By listing numerous research topics such as alterations in a diffusion-limited metabolic process, bone loss and skeletal muscle weakness of astronauts, behavioral and cognitive ability in space, life in extreme environment, etc., we will attempt to suggest future opportunities.

Animals↗

PubMed2000

Measurements of cortical cellular pH by intracranial tonometer in severe head injury.

OBJECTIVE: To evaluate the cortical cellular damage in acute severe head injury, we measured the cortical cellular pH by using an intracranial tonometer made in our institution. DESIGN: Prospective, 3.5-yr data collection. SETTING: University hospital trauma intensive care unit. PATIENTS: Severely head-injured patients (n = 29) with Glasgow Coma Scale score <8. INTERVENTION: Routine emergency neurologic procedure. MEASUREMENTS AND MAIN RESULTS: We made 98 measurements of cortical cellular pH by intracranial tonometer in 29 severely head-injured patients in the acute phase. Each patient's intracranial pressure was recorded, and in 16 patients, the saturation of jugular venous oxygen was monitored. The outcome at 6 months after injury was significantly better in patients having a cortical cellular pH of >7.2 than those with <7.2. The cerebral perfusion pressure and cortical cellular pH correlated significantly (p < .0001). CONCLUSIONS: Our study suggests the usefulness of measurement of cortical cellular pH by intracranial tonometer for evaluating the severity of focal anaerobic cerebral metabolism and predicting patient prognosis.

Acid-Base Equilibrium↗

PubMed2000

Single-molecule imaging of interaction between dextran and glucosyltransferase from Streptococcus sobrinus.

Using total internal reflection fluorescence microscopy, we directly observed the interaction between dextran and glucosyltransferase I (GTF) of Streptococcus sobrinus. Tetramethylrhodamine (TMR)-labeled GTF molecules were individually imaged as they were associating with and then dissociating from the dextran fixed on the glass surface in the evanescent field. Similarly dynamic behavior of TMR-labeled dextran molecules was also observed on the GTF-fixed surface. The duration of the stay on the surface (dwell time) was measured for each of these molecules by counting the number of video frames that had recorded the image. A histogram of dwell time for a population of several hundred molecules indicated that the GTF-dextran interaction obeyed an apparent first-order kinetics. The rate constraints estimated for TMR-labeled GTF at pH 6.8 and 25 degrees C in the absence and presence of sucrose were 9.2 and 13.3 s(-1), respectively, indicating that sucrose accelerated the dissociation of GTF from dextran. However, the accelerated rate was still much lower than the catalytic center activity of GTF (> or = 25 s(-1)) under comparable conditions.

Dextrans↗

PubMed2000

[Assessment of functional severity on in vivo hepatic 31P-MRS in diffuse hepatic disease: comparative studies with 99mTc-GSA].

The purpose of this study was to compare the assessment of functional severity on in-vivo hepatic 31P-MRS in diffuse hepatic disease with functional severity assessed with 99mTc galactosyl serum albumin (99mTc-GSA). 31P-MRS was performed in 10 healthy control subjects and 16 patients with diffuse hepatic disease. Data were expressed as peak area ratios: PME/beta-ATP, PDE/beta-ATP, PME/PDE, Pi/beta-ATP, and PME/Pi. The functional severity of hepatic damage was evaluated visually and quantitatively (HH15, LHL15) by 99mTc-GSA in the group of patients with diffuse hepatic disease. Visual evaluation was classified into four grades based on anterior images of cardiac blood-pool and liver. We studied the correlation of spectral metabolic ratios and functional severity by 99mTc-GSA. We found statistically significant differences (ANOVA) among the classifications of Grade I, Grade II, and Grade III with both PME/beta-ATP and PME/PDE. A statistically significant direct correlation was found between HH15 and both PME/beta-ATP and PME/PDE. A significant inverse correlation was also seen between LHL15 and both PME/beta-ATP and PME/PDE. The studies comparing 31P-MRS with functional severity assessed by 99mTc-GSA showed that PME/beta-ATP and PME/PDE were useful for the assessment of functional severity in patients with diffuse hepatic disease.

Adult↗

PubMed2000

[The genetic diagnosis of hematopoietic malignancy by polymerase chain reaction method].

Since 1994, chimeric gene test for major-BCR/ABL, minor-BCR/ABL, AML1/MTG8, PML/RARa, DEK/CAN, CBFb/MYH11, MLL/LTG9, E2A/PBX1, TEL/AML1, and MLL/LTG4 by reverse transcription-nested-polymerase chain reaction (RT-nested-PCR) have been routinely performed in our laboratory. The frequently requested tests were those for major-BCR/ABL, minor-BCR/ABL, AML1/MTG8, PML1/RARa, and TEL/AML1 chimeric genes, being the test for major-BCR/ABL the most frequent one, accounting for more than 50% of the total orders. By the high sensitivity for minimal residual disease(MRD) detection, these methods are extremely useful for the therapeutic control of chemotherapy and bone marrow transplantation, as well as for prediction of prognosis. However, in the most cases a well-controlled monitoring could not be obtained, due to the 6-months interval between the tests, the minimal interval allowed by the insurance. The following points should be carefully observed. (1) The setting site of the primer can affect the sensitivity and the specificity of the test; (2) For the detection of chimeric genes with multiple translocation breakpoints, a genetic DNA sequencing is necessary to confirm them; (3) In patients receiving chemotherapy or bone marrow transplantation, that show extremely low blood cell counts, false-negative tests dependent on the quality of the extracted RNA and the low volume of DNA, should be avoided by careful managements. For improvements of the MRD detection tests, the quantification of the expression levels of chimeric gene and WT1 mRNA is necessary.

Hematologic Neoplasms↗

PubMed1999

Spin-stretching of DNA and protein molecules for detection by fluorescence and atomic force microscopy.

We have developed a rapid and efficient way of stretching DNA and denatured protein molecules for detection by fluorescence microscopy and atomic force microscopy (AFM). In the described method, a viscous drag created by transient rotational flow stretches randomly coiled DNA molecules or denatured proteins. Stretching is achieved by dispensing a droplet of sample solution containing DNA or denatured protein on a MgCl2-soaked mica surface. We present fluorescent images of straightened lambdaDNA molecules and AFM images of stress-shared, reduced von Willebrand factor as well as straightened lambdaDNA. The described quick and reliable spin-stretching technique will find wide applications in the analysis of single biopolymer molecules.

DNA↗

PubMed1999

Novel rabphilin-3-like protein associates with insulin-containing granules in pancreatic beta cells.

A novel rabphilin-3-like gene, granuphilin, has been identified in pancreatic beta cells by comparing genes expressed in pancreatic alpha and beta cell lines using mRNA differential display. The domain structure of the protein products of the granuphilin gene contains an amino-terminal zinc-finger motif and carboxyl-terminal C(2)-domains, similar to that of the rabphilin-3 gene. There are two isoforms: the larger isoform, granuphilin-a, has two C(2)-domains, whereas the smaller one, granuphilin-b, contains only the first C(2)-domain. Granuphilin is specifically expressed in pancreatic beta cells and the pituitary gland, but not in pancreatic alpha cells, the adrenal gland, or other major organs such as the brain. A portion of granuphilin associates with insulin-containing dense-core granules, but not with synaptic-like microvesicles in beta cells. Thus, its distribution pattern presents a striking contrast with that of rabphilin-3, which associates with small synaptic vesicles in neurons. The first C(2)-domain of granuphilin binds phospholipids in a Ca(2+)-independent manner, whereas the second one does not. These distinctive characteristics of granuphilin suggest that it is not a simple counterpart of rabphilin-3 in endocrine cells and that it has a unique role in the regulated exocytosis of dense-core granules in endocrine tissues.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Four-helical-bundle structure of the cytoplasmic domain of a serine chemotaxis receptor.

The bacterial chemotaxis receptors are transmembrane receptors with a simple signalling pathway which has elements relevant to the general understanding of signal recognition and transduction across membranes, how signals are relayed between molecules in a pathway, and how adaptation to a persistent signal is achieved. In contrast to many mammalian receptors which signal by oligomerizing upon ligand binding, the chemotaxis receptors are dimeric even in the absence of their ligands, and their signalling does not depend on a monomer-dimer equilibrium. Bacterial chemotaxis receptors are composed of a ligand-binding domain, a transmembrane domain consisting of two helices TM1 and TM2, and a cytoplasmic domain. All known bacterial chemotaxis receptors have a highly conserved cytoplasmic domain, which unites signals from different ligand domains into a single signalling pathway to flagella motors. Here we report the crystal structure of the cytoplasmic domain of a serine chemotaxis receptor of Escherichia coli, which reveals a 200 A-long coiled-coil of two antiparallel helices connected by a 'U-turn'. Two of these domains form a long, supercoiled, four-helical bundle in the cytoplasmic portion of the receptor.

Chemotaxis↗

PubMed1999

Glucuronidation of the environmental oestrogen bisphenol A by an isoform of UDP-glucuronosyltransferase, UGT2B1, in the rat liver.

Bisphenol A, an environmental oestrogenic chemical, was found to conjugate highly with glucuronic acid in male rat liver microsomes studied in vitro. In the various isoforms tested (1A1, 1A3, 1A5, 1A6, 1A7 and 2B1), glucuronidation of bisphenol A and of diethylstilboestrol, a synthetic crystalline compound possessing oestrogenic activity and known to be glucuronidated by liver microsomes, was catalysed by an isoform of UDP-glucuronosyltransferase (UGT), namely UGT2B1, which glucuronidates some endogenous androgens. UGT activity towards bisphenol A in liver microsomes and in UGT2B1 expressed in yeast AH22 cells (22.9 and 0.58 nmol/min per mg of microsomal proteins respectively) was higher than that towards diethylstilboestrol (75.0 and 4.66 pmol/min per mg of microsomal proteins respectively). UGT activities towards both bisphenol A and diethylstilboestrol were distributed mainly in the liver but were also observed at substantial levels in the kidney and testis. Northern blot analysis disclosed the presence of UGT2B1 solely in the liver, and about 65% of the male rat liver microsomal UGT activities towards bisphenol A were absorbed by the anti-UGT2B1 antibody. These results indicate that bisphenol A, in male rat liver, is glucuronidated by UGT2B1, an isoform of UGT.

Amino Acid Sequence↗

PubMed1999

Sharp DNA bends as landmarks of protein-binding sites on straightened DNA.

We have developed a fluorescence-based method for mapping single or multiple protein-binding sites on straightened, large-size DNA molecules (> 5 kbp). In the described method, protein-DNA complexes were straightened and immobilized on a flat surface using surface tension. A fraction of the immobilized complexes displayed a sharp DNA bend with two DNA segments extending from the apex. The presence of DNA-binding proteins at the apex was verified by atomic force microscopy. The position of protein binding relative to the ends of the DNA molecule was determined by measuring the length of two DNA segments using fluorescence microscopy. We demonstrate the potential of the fluorescence-based method to localize protein-binding sites on the DNA template and to evaluate relative binding affinity. The proposed protein-binding-site mapping technique is simple and easy to perform. Practical applications include screening for DNA-binding proteins and the localization of protein-binding sites on large segments of DNA.

Bacteriophage lambda↗

PubMed1999

Small intestinal UDP-glucuronosyltransferase sheUGT1A07: partial purification and cDNA cloning from sheep small intestine.

A phenol UDPglucuronosyltransferase (UGT) was partially purified, and the cDNA encoding the isoform was cloned and sequenced from sheep small intestine. The purified preparation containing a one major band (57 kDa) and one minor band (50 kDa) revealed high activities toward xenobiotics such as 1-naphthol (1-NA), 4-nitrophenol, and 4-methylumbelliferone. The preparation, however, had only little activity toward 4-hydroxybiphenyl and no activity toward bilirubin, suggesting that the preparation contains UGT1 isoforms. The NH2-terminal amino acid sequence of the major band was determined to be Gly-Lys-Leu-Leu-Val-Val-Pro-Met-Asp-Gly-Ser. A full-length UGT cDNA was obtained by reverse transcription-polymerase chain reaction with the degenerated 5'-primer from the NH2-terminal amino acid sequence of the purified major one and rapid amplification of cDNA ends from sheep small intestine. The cloned cDNA named sheUGT1A07 by amino acid similarity has a NH2-terminus sequence identical to that of the purified major one. Another phenol UGT cDNA named sheUGT1A6 was also cloned from sheep liver. sheUGT1A6 was expressed mainly in the liver, whereas sheUGT1A07 mRNA was expressed almost only in the alimentary organs, suggesting that sheUGT1A6 plays a role as a general drug metabolizing UGT isoform in the liver and sheUGT1A07 plays important role in the xenobiotics glucuronidation in the sheep small intestine.

Amino Acid Sequence↗

PubMed1999

The significance of amino acid residue Asp446 for enzymatic stability of rat UDP-glucuronosyltransferase UGT1A6.

Asp446 in rat UDP-glucuronosyltransferase (UGT), UGT1A6, is an essential amino acid residue for its enzymatic activity (H. Iwano et al. Biochem. J. 325, 587-591, 1997). The role of Asp446 in UGT1A6 was investigated by comparing some properties of UGT mutant proteins that have a single mutation (D446K, D446E, D446N, D446Q, D446A, and D446T). These mutants, except D446K, had catalytic activities toward 1-naphthol and 4-methylumbelliferone. The UGT activities of D446E and D446N were about half of that of the wild type, and the activities of the other mutants were only about 1/5-1/10 of that of the wild type. The Km values for 1-naphthol of these mutants were similar to that of the wild type, while the values for UDP-glucuronic acid were slightly higher. The mutants were unstable in a low-pH buffer solution and were dramatically inactivated by heat treatments. Interestingly, after 30 min of treatment at 37 degrees C in the presence of UDP-glucuronic acid, the UGT activities of all functional mutants were elevated. These results suggest that Asp446 is an indispensable residue for folding a functional conformation of rat UGT1A6 by cooperation with UDP-glucuronic acid.

Amino Acid Sequence↗

PubMed1999

Expression, purification and preliminary X-ray analysis of a fibrillarin homolog from Methanococcus jannaschii, a hyperthermophile.

Fibrillarin plays a central role in ribosome biogenesis as a ribosomal RNA-processing protein. A Methanococcus jannaschii homolog of fibrillarin has been overexpressed, purified and crystallized. Crystals belong to the C2 space group with unit-cell parameters a = 121.4, b = 43.2, c = 55.3 A, beta = 96.9 degrees. Under flash-frozen conditions and using synchrotron radiation, the crystals diffract to 1.8 A resolution. For structural determination, a selenomethionine derivative of the protein has also been crystallized.

Archaeal Proteins↗

PubMed1999

Endovascular irradiation prevents smooth muscle cell proliferation and neointimal hyperplasia in rabbits.

The present study examined the temporal responses and the efficacy of 192Ir-HDR endovascular irradiation for preventing smooth muscle cell proliferation of rabbit iliac arteries after PTA with a cutting balloon catheter. Endovascular irradiation with 12 Gy was randomly performed on the one side of iliac arterial segment with the unirradiated side serving as a control. Animals were euthanatized 1, 2, 3, 4, 8 and 12 week(s) after angioplasty. Histopathological and immunohistochemical studies were carried out. Histopathology showed repair of the dissection by cellular accumulation and a striking reduction in the amount of neointimal hyperplasia in the irradiated arteries as compared with control vessels. A peak of PCNA-positive ratio was in neointima of the control arterial segments at a week. 2-4 weeks after irradiation, the neointimal PCNA-positive ratio was still significantly increased in the control arterial segments compared with the irradiated arterial segments. After 8 weeks, PCNA-positive ratio was below 1% in both irradiated arterial segments and the control. Our results showed that the 192Ir-HDR afterloading irradiation with a dose of 12 Gy can be considered sufficient for inhibiting neointimal hyperplasia in angioplastized rabbit iliac arteries with cutting balloon catheter.

Angioplasty, Balloon, Coronary↗

PubMed1999

Immunohistochemical study of some cytoskeletal proteins in hereditary myopathy of the diaphragmatic muscles in Holstein-Friesian cattle.

We have investigated the expression, using immunohistochemical and Western blot methods, of some cytoskeletal proteins including desmin, vimentin, actin, alpha-actinin, and ubiquitin in hereditary myopathy of the diaphragmatic muscles in Holstein-Friesian cattle (the histochemical and electron microscopical aspects have been previously reported). Immunohistochemically, the expression of desmin was observed strongly in the subsarcolemmal regions, but was lacking or faint in the area corresponding to the core-like structures. Vimentin showed almost the same localization as desmin, but no activity could be observed in the core-like structures. In addition, the core-like structures showed strong immunoreactivity for actin and ubiquitin, but no immunoreactivity for alpha-actinin. F-actin stained with phalloidin-tetramethyl-rhodamine was strongly positive in irregular spots that corresponded to the core-like structures, but was negative for desmin-positive regions. Western blot analysis of the diseased muscles revealed a significant increase in the amount of desmin and vimentin immunoreactivities and similar amounts of actin and alpha-actinin compared with the control muscles. Two-dimensional electrophoresis revealed no isoforms of desmin, suggesting the absence of abnormal phosphorylated forms of desmin. Since the co-localization of desmin and vimentin and the absence of phosphorylated desmin suggest that the overexpression of desmin may be reflected in the reactive change or regenerating process, the present myopathy should be regarded as an entity separate from desmin-storage myopathy or desmin-related myopathies. We also discuss the possibility that the present myopathy could be considered as myofibrillar myopathy, a recently proposed nosological entity.

Actinin↗