Search PubMed⌕ Search

Biomedical subjects

Y Wada

Publications and source records attributed to Y Wada.

At least 631 records · Page 35Linked to original sources
PubMed1994

"Module" substitution in hemoglobin subunits. Preparation and characterization of a "chimera beta alpha-subunit".

In the genes of alpha- and beta-subunits of hemoglobin, Go showed that modules F1, F2 + F3, and F4 correspond to exons 1, 2, and 3, respectively (Go, M. (1981) Nature 291, 90). The analysis of the correlation of function with its exon pattern showed that the residues associated with the defined function are concentrated in the specific exons encoding the "module" (Eaton, W.A. (1980) Nature 284, 183). To investigate the functional and structural significance of the "modular structure," we engineered a "chimera" subunit, in which module F4 of the beta-subunit was replaced by that of the alpha-subunit by use of mutagenesis. The NMR and resonance Raman spectra of the isolated "chimera beta alpha-subunit" have revealed that it has a beta-subunit-like heme environmental structure. However, the gel chromatography and NMR spectra of mixtures of the chimera and native subunits clearly showed that the chimera beta alpha-subunit binds specifically to the beta-subunit to form a heterotetramer, not to the alpha-subunit. These results led us to conclude that the predominant role of the module F4 is the subunit association and suggest that the modules are structural and functional units that have advantages in producing stable functional proteins.

Carboxyhemoglobin↗
PubMed1994

Effect of glycosidase inhibitors on the biosynthesis of alpha 2-plasmin inhibitor and antithrombin III in Hep G2 cells.

We studied the effect of the glucosidase I inhibitor, N-methyl-1-deoxynojirimycin (MdN) and the mannosidase inhibitor, 1-deoxymannojirimycin (dMM) on the biosynthesis and secretion of alpha 2-plasmin inhibitor (alpha 2-PI) and antithrombin III (ATIII) in cultures of human hepatoma (Hep-G2) cells. Incubation with 1 mM MdN decreased secreted alpha 2-PI activity and antigen levels by about 40%, whereas those of ATIII were not affected. Neither inhibitor affected the messenger RNA levels as determined by Northern blotting. Pulse-chase studies using [35S]-methionine showed that MdN decreased alpha 2-PI and ATIII secretion rates. By the 18 h chase, MdN had decreased secreted alpha 2-PI to 50-60%, with little effect on ATIII. Intracellular forms of alpha 2-PI or ATIII synthesized by cells treated with 1 mM MdN were sensitive to endoglycosidase H (Endo H), whereas almost all the secreted forms were resistant, suggesting the presence of complex-type oligosaccharides. In the presence of 1 mM dMM, cells synthesized Endo H-sensitive alpha 2-PI and ATIII with similar secretion rates. These results suggest that retention of glucose on N-linked oligosaccharides not only retards the exit of alpha 2-PI and ATIII, but also changes the catabolic rate of alpha 2-PI in the endoplasmic reticulum.

1-Deoxynojirimycin↗
PubMed1994

Site-directed mutagenesis in hemoglobin: functional and structural role of the penultimate tyrosine in the alpha subunit.

The penultimate tyrosine in the hemoglobin subunit is considered to be one of the most important residues for the normal structure and function of hemoglobin. To elucidate the functional and structural role of the penultimate residue in the alpha-subunit, we prepared new artificial mutants; Hb Y140 alpha Q, in which Tyr-140 alpha is replaced by a nonaromatic residue, Gln, and Hb Y140 alpha F, which loses its hydrogen bond to Val-93 alpha by the substitution of Phe for Tyr. HB Y140 alpha Q exhibited a markedly increased oxygen affinity and almost completely diminished cooperativity, whereas Hb Y140 alpha F showed similar but less extensively impaired function, indicating that the aromatic residue at the penultimate position in the alpha-subunit contributes to the stabilization of the T-quaternary structure as does the corresponding residue in the beta-subunit. However, the deoxygenated forms of these mutants bear significant T-state character in their spectroscopic properties observed at high protein concentrations. The tetramer-dimer equilibrium data of the mutants suggested that a significant part of the functional alterations observed for dilute solution appears to result from partial dissociation into alpha beta dimers rather than direct destabilization of the T-quaternary structure in the deoxygenated form. Therefore, we can conclude that the penultimate tyrosine in the alpha-chain plays a key role not only in the stabilization of the T-state but also in the subunit assembly.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1994

Deficiency of the E1 beta subunit in the branched-chain alpha-keto acid dehydrogenase complex due to a single base substitution of the intron 5, resulting in two alternatively spliced mRNAs in a patient with maple syrup urine disease.

A patient with maple syrup urine disease (MSUD) associated with a E1 beta subunit deficiency of the branched-chain alpha-keto acid dehydrogenase (BCKDH) complex was investigated at the molecular level. The defect responsible for the deficiency of the E1 beta subunit protein was identified by analysis of cDNA and genomic DNA by polymerase chain reaction. Total RNA isolated from lymphoblastoid cells was transcribed into cDNA and amplified using a set of primers located within exon 3 and exon 9 of the E1 beta gene. Agarose gel electrophoresis of cDNA amplification products revealed two shortened bands as well as a faint band of normal size. Nucleotide sequencing of the shortened cDNA amplification products showed that sequences corresponding to exon 5 and both exons 5 and 6 were absent. Nucleotide sequencing of the proband's amplified genomic DNA corresponding to this region of the E1 beta gene revealed a single base substitution from G to T of the invariant GT dinucleotides at 5' splice site of the intron 5. Analysis of family members using primer-specified restriction map modification showed that the patient is homozygous for this mutation. We postulate that this mutation leads to the skipping of either exon 5 or both exons 5 and 6, thus producing two shortened E1 beta mRNA. The percentage of normal and two shortened transcripts was estimated to be 9, 71 and 20%, respectively. To our best knowledge, this is the first documented example of exon skipping in the E1 beta gene as the cause of MSUD and the novel mutation of the invariant G at the 5' splice site which results in two alternatively spliced mRNA due to the skipping of the preceding exon as well as both preceding and following exon.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1994

cDNA cloning and chromosomal mapping of human N-acetylglucosaminyltransferase V+.

Human N-acetylglucosaminyltransferase V (GnT-V, EC 2.4.1.155) cDNA was isolated from a human fetal liver cDNA library. Oligonucleotide primers for polymerase chain reaction were designed according to the amino acid sequence of human GnT-V. Screening for the cDNA was carried out by plaque hybridization using PCR products of about 500 bp. Human GnT-V has 741 amino acids and six putative N-glycosylation sites. The homology to rat GnT-V is 88% at the nucleotide level and is 97% at the amino acid level, and there is one amino acid insertion. Using the cDNA clones as probe, five overlapping genomic clones have been isolated from a human phagemid DNA library. The GnT-V gene has been mapped to chromosome 2q21 using fluorescence in situ hybridization.

Amino Acid Sequence↗
PubMed1994

The effect of different routes of administration on the metabolism of morphine: the disposition of morphine and its metabolites after topical application.

The disposition of morphine (MOR) and its metabolites in the rabbit was measured after topical administration of its hydrochloride salt (MOR.HCl), and their time course was compared with those after intravenous and oral administration. The area under the plasma concentration-time curve (AUC) ratio of metabolites/MOR after the topical application of MOR.HCl was similar to that after intravenous injection, but differed from that after oral administration. Pharmacokinetic parameters of the disposition of MOR and its metabolites were obtained by a general curve fitting of the time course of plasma concentrations of these compounds after intravenous injection of MOR.HCl and its metabolites, respectively. On the other hand, the time courses of plasma concentrations of the metabolites after intravenous, oral, and topical administration of MOR.HCl were simulated using a simple compartment model without consideration of enterohepatic circulation and the pharmacokinetic parameters obtained as above. The resulting curves of the metabolites agreed well with the observed values except for those after oral administration. These results suggest that no first-pass metabolism of MOR.HCl occurs after percutaneous administration, and that topical administration of this salt is more advantageous than oral administration in terms of bioavailability.

Administration, Oral↗
PubMed1994

Diagnosis of carbohydrate-deficient glycoprotein syndrome by matrix-assisted laser desorption time-of-flight mass spectrometry.

Serum transferrin from patients with carbohydrate-deficient glycoprotein syndrome was analysed by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF mass spectrometry). Abnormal molecular species with a defect of one or two oligosaccharide chains of 2200 Da were clearly identified. MALDI-TOF mass spectrometry will serve to diagnose this aetiologically unknown, metabolic disease.

Carbohydrate Metabolism, Inborn Errors↗
PubMed1994

Electrospray ionization mass spectrometry of pyridylaminated oligosaccharide derivatives: sensitivity and in-source fragmentation.

Oligosaccharide chains fluorescence-labelled at their reducing end with 2-aminopyridine were analysed by electrospray ionization mass spectrometry (ESIMS). Protonated molecules of complex type oligosaccharides were detectable at a level of 1 pmol. Fragmentation at the glycosidic bonds occurred in the electrospray ion source when the voltage between the capillary nozzle and the first skimmer was elevated, referred to as "in-source fragmentation". Most fragment ions observed had retained the pyridylaminated (PA) site of the molecule and gave significant information on the saccharide sequence. In conjunction with the accumulating data of reversed-phase chromatography of PA oligosaccharides, ESIMS of PA oligosaccharide will be useful as a sensitive method in the structural study of oligosaccharides from various biological sources.

Carbohydrate Sequence↗
PubMed1994

Chemiosmotic coupling of ion transport in the yeast vacuole: its role in acidification inside organelles.

Acidification inside the vacuo-lysosome systems is ubiquitous in eukaryotic organisms and essential for organelle functions. The acidification of these organelles is accomplished by proton-translocating ATPase belonging to the V-type H(+)-ATPase superfamily. However, in terms of chemiosmotic energy transduction, electrogenic proton pumping alone is not sufficient to establish and maintain those compartments inside acidic. Current studies have shown that the in situ acidification depends upon the activity of V-ATPase and vacuolar anion conductance; the latter is required for shunting a membrane potential (interior positive) generated by the positively charged proton translocation. Yeast vacuoles possess two distinct Cl- transport systems both participating in the acidification inside the vacuole, a large acidic compartment with digestive and storage functions. These two transport systems have distinct characteristics for their kinetics of Cl- uptake or sensitivity to a stilbene derivative. One shows linear dependence on a Cl- concentration and is inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). The other shows saturable kinetics with an apparent Km for Cl- of approximately 20 mM. Molecular mechanisms of the chemiosmotic coupling in the vacuolar ion transport and acidification inside are discussed in detail.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Quantitative EEG analysis at rest and during photic stimulation in drug-naive patients with first-episode paranoid schizophrenia.

In the present study, quantitative EEG analysis was performed at rest and during 10 Hz photic stimulation in 14 drug-naive patients with first-episode paranoid schizophrenia and 20 sex- and age-matched control subjects. Compared with the normal controls, the patients had significantly lower alpha-2 band amplitude in the resting EEG over all recording regions. No significant group differences were found in other frequency bands. In addition, EEG analysis during photic stimulation demonstrated that the patients had a rather uniform topographic profile in EEG amplitude for the alpha band, with significant group differences being confined to the posterior regions in the left hemisphere. There were no significant group differences in the amplitude for the frequency bands harmonically related to the stimulus frequency. These findings provide further evidence that schizophrenic patients have abnormal EEG activity in both non-stimulus and stimulus conditions, and suggest a dysfunction in the mechanisms underlying EEG alpha generation in schizophrenia.

Adult↗
PubMed1994

Analysis of growth hormone release from rat anterior pituitary cells by reverse hemolytic plaque assay: influence of interleukin-1.

Interleukin-1 (IL-1) has been suggested to directly affect pituitary growth hormone (GH) release, although other investigators have failed to observe this effect. We examined the effects of IL-1 beta on GH secretion from single somatotrophs by means of reverse hemolytic plaque assay (RHPA). Anterior pituitary cells of adult male rats were enzymatically dispersed and subjected to RHPA. IL-1 beta at 100 pM and 1 nM, increased both the mean plaque area and the fraction of somatotrophs forming large plaques. IL-1 beta did not increase the mean plaque area in the presence of the IL-1 receptor antagonist (IL-1ra). IL-1 beta (1 nM) added together with GH-releasing hormone (GHRH; 10 nM), showed no additive effect on GHRH-induced GH release. The stimulatory action of IL-1 beta on the release of GH was suppressed by somatostatin. In conclusion, our data show that IL-1 beta stimulates GH-secretion through direct action on the pituitary.

Animals↗
PubMed1994

Changes of ampulla pressure in the semicircular canal of pigeons by caloric stimulation.

Still now several hypotheses about the mechanisms of the caloric nystagmus have been in conclusive. In this study we confirmed the convection effect and the volume change effect of the endolymph in horizontal semicircular canal following the caloric stimulation using pigeons (Columba livia). Although the direction of the caloric nystagmus depended on the head position and the stimulus site of calorization, the caloric nystagmus disappeared after plugging of horizontal semicircular canal. On the other hand, the ampulla pressure increased by cold calorization and decreased by hot calorization and these pressure changes had no relation to the head position. These results show that the main role of the mechanisms of the caloric nystagmus under 1G is the convection effect but the volume change effect may act on the caloric nystagmus not only under 1G but also under microgravity.

Animals↗
PubMed1994

Telescience testbed experiments for biomedical studies: fertilization potential recording of amphibian eggs using tele-manipulation under stereoscopic vision.

The telescience testbed experiments were carried out to test and investigate the tele-manipulation techniques in the intracellular potential recording of amphibian eggs. Implementation of telescience testbed was set up in the two separated laboratories of the Tsukuba Space center of NASDA, which were connected by tele-communication links. Manipulators respective for a microelectrode and a sample stage of microscope were moved by computers, of which command signals were transmitted from a computer in a remote control room. The computer in the control room was operated by an investigator (PI) who controlled the movement of each manipulator remotely. A stereoscopic vision of the microscope image were prepared by using a head mounted display (HMD) and were indispensable to the intracellular single cell recording. The fertilization potential of amphibian eggs was successfully obtained through the remote operating system.

Animals↗
PubMed1994

Changes of endolymphatic pressure in the semicircular canal of pigeon by caloric stimulation.

It gets into difficult to explain the mechanism of caloric nystagmus only by convection theory from results of microgravity experiments. One of the other theories is an occurrence of a relative volume change due to a temperature change. Since the volume change must lead to a pressure change after caloric stimulation, we tried to measure the ampulla pressure of the horizontal semicircular canal in pigeons (Columba livia) using an improved servo micropipette system. The main result was that the ampulla pressure increased by cooling and decreased by heating. The changes of the ampulla pressure depended on the temperature change but were not influenced by the pigeon's head position.

Animals↗
PubMed1994

Candidiasis caused by Candida glabrata in the forestomachs of a calf.

A female Japanese black calf suffering from diarrhoea was treated with six different antibiotics for 9 days. The diarrhoea worsened and the calf was killed when 17 days old. At necropsy, the mucosal surface of the forestomachs showed diffuse haemorrhagic necrosis and pseudomembrane formation. Candida glabrata infection was diagnosed histologically, immunohistochemically, and by isolation and examination of the yeast. The intensive antibiotic treatment was considered to have been a predisposing or exacerbating factor. This is the first report of C. glabrata infection of the bovine forestomachs.

Animals↗
PubMed1994

Monoallelic expression of normal mRNA in the PIT1 mutation heterozygotes with normal phenotype and biallelic expression in the abnormal phenotype.

The combined deficiency of thyrotropin, growth hormone and prolactin, caused by PIT1 abnormality manifests in the homozygous or heterozygous state. We studied a patient having an allele with Arg271Trp mutation, which produces clinical symptoms in heterozygotes by a dominant-negative effect. However, in the family, her father, grandmother and aunts had the same mutation without clinical symptoms, although the proband had typical phenotypic expression. We analyzed the PIT1 transcript in peripheral lymphocytes by reverse transcription-polymerase chain reaction and found monoallelic expression of normal allele in the father and grandmother and skewed pattern of biallelic expression in the proband. The phenotypic expression of PIT1 abnormality may depend on different transcription of the PIT1 gene.

Alleles↗