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Y Wada

Publications and source records attributed to Y Wada.

At least 19 recordsLinked to original sources
PubMed1992

Structure of serum transferrin in carbohydrate-deficient glycoprotein syndrome.

The structure of the defective transferrin in carbohydrate-deficient glycoprotein syndrome was characterized. Structurally abnormal sugar chains were not found in reversed phase chromatograms of pyridylaminated derivatives from the transferrin of two patients in different families. Electrospray ionization mass spectrometry of the whole transferrin molecules revealed an abnormal species that was smaller than normal tetrasialotransferrin by 2,200 daltons, just the size of the disialylated biantennary sugar chain. These data indicated that the disialotransferrin specifically found in this syndrome is missing either of two N-linked sugar chains, suggesting a metabolic error in the early steps of protein glycosylation.

Carbohydrate Metabolism, Inborn Errors↗
PubMed1992

In vitro proliferation and the cytotoxic specificity of a cryopreserved cytotoxic T cell clone reacting against human autologous tumor cells.

Proliferation and functional maintenance of CTL after cell cryopreservation often proves to be quite difficult. We developed an improved method for proliferating cryopreserved CTL, and for gaining their specific cytotoxic function. T cells were cryopreserved at -180 degrees C in RPMI 1640 containing 50% FCS and 10% DMSO. The cryopreserved T cells were well recovered by culturing in a medium containing the supernatant of primary cultures with TIL and autologous tumor cells, in addition to a high concentration (350 U/ml) of rIL-2. Furthermore, these cells were proliferated more efficiently when MMC-treated autologous tumor cells were used in vitro as a feeder and an antigenic stimulant. However, such a high dose IL-2 cultivation resulted in the loss of cytotoxic reactivity of CTL clone. In contrast, the withdrawal of rIL-2 from in vitro cultivation for 24 h prior to the cytotoxic assays conferred the specificity of cytotoxicity on CTL. By these methods, one can obtain a large number of CTL, and pursue the physiologic detail of the specific cytotoxic mechanism of CTL against autologous human tumor cells.

Antigens, Surface↗
PubMed1992

Genes for directing vacuolar morphogenesis in Saccharomyces cerevisiae. I. Isolation and characterization of two classes of vam mutants.

We identified nine VAM genes (for vacuolar morphology) by genetic analyses on mutants with defective vacuolar morphologies and assembly in the yeast Saccharomyces cerevisiae. The nine VAM genes were classified into two classes according to the mutant phenotypes. The class I vam mutants (vam1, vam5, vam8, and vam9) show a few small vesicles that are stained with histochemical markers for the vacuolar compartment. They also have defects in the maturation of vacuolar marker proteins, and their growth is hypersensitive to high concentrations of CaCl2 or a temperature of 37 degrees C. There are apparent genetic overlaps among the class I vam mutations and other mutations including cls, end, pep, and vps, which have been shown to be involved in the expression of the vacuolar functions. The class II vam mutants (vam2, vam3, vam4, vam6, and vam7) contain numerous small vesicles stained with the vacuolar histochemical markers and mature forms of the vacuolar proteins and do not show any apparent growth defects in the presence of CaCl2 or at 37 degrees C.

Blotting, Western↗
PubMed1992

Genes for directing vacuolar morphogenesis in Saccharomyces cerevisiae. II. VAM7, a gene for regulating morphogenic assembly of the vacuoles.

VAM7 gene function has shown to be required for proper morphogenesis of the vacuole in yeast. The DNA fragments that complemented the defective vacuolar morphology of the vam7-1 mutation were isolated from a yeast genomic library. An overlapping 2.5-kilobase BglII-HindIII region was found to be sufficient for complementation of the vam7-1 phenotype. This fragment was integrated at the chromosomal VAM7 locus, indicating that it contained an authentic VAM7 gene. On nucleotide sequencing of the VAM7 gene, an open reading frame of 948 base pairs, coding for a hydrophilic polypeptide of 316 amino acid residues, was identified. The deduced amino acid sequence of the carboxyl-terminal region of the VAM7 gene product has heptad repeats and shows potential ability to form a coiled-coil structure. Disruption of VAM7 was not lethal; cells with a disrupted VAM7 gene did not, however, have a prominent large vacuoles but rather numerous small compartments that accumulated the histochemical marker molecule of the vacuolar compartment. They contained mature forms of the vacuolar marker proteins carboxypeptidase Y and vacuolar glycoprotein vgp72. A mutant with both vam7 and vam5 null mutations was constructed and shown to have neither vacuolar structures stained with ade-related fluorochrome nor mature forms of vacuolar marker proteins. These findings suggested that the VAM7 gene product functions in the process of morphogenic assembly of the vacuolar compartment but is not involved in the protein sorting and delivery to the vacuole.

Amino Acid Sequence↗
PubMed1992

Chloride transport of yeast vacuolar membrane vesicles: a study of in vitro vacuolar acidification.

Effects of various solutes on acidification inside the vacuolar membrane vesicles of the yeast Saccharomyces cerevisiae were examined. ATP-dependent acidification was stimulated by the presence of chloride salts. There was essentially no difference in the stimulatory effects of NaCl, KCl, LiCl, and choline chloride. The membrane potential across the vacuolar membrane was reduced by the presence of Cl- salts. Transport of 36Cl- is driven by the protonmotive force across the vacuolar membrane. Kinetic analyses have revealed that the stimulatory effect of Cl- on internal acidification depends on two distinct components. One shows linear dependency on chloride concentration and is inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid (DIDS). The other exhibits saturable kinetics with an apparent Km for chloride of 15-20 mM. We conclude that the vacuolar membrane of yeast is equipped with Cl- transport systems contributing to the formation of a chemical gradient of protons across the vacuolar membrane by shunting the membrane potential generated by proton translocation.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Role of serotonin receptor subtype in seizures kindled from the feline hippocampus.

This study assessed the effects of a serotonin (5-HT)1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT2 agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and a 5-HT2 antagonist ketanserin on fully kindled seizures from the hippocampus. 8-OH-DPAT produced a marked suppression in hippocampal kindled seizures, while producing behavioral signs similar to those seen in the 5-HT syndrome. Although DOI and ketanserin did not affect kindled focal epileptic activity, DOI shortened and ketanserin prolonged the latency to onset of generalized convulsions. Our data suggest that 5-HT1A receptors play an inhibitory role in the generation of hippocampal seizures, whereas 5-HT2 receptors may participate in kindled seizure generalization from this region.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1992

Sphingosine inhibits factor Xa-catalyzed prothrombin activation on the surface of cultured calf pulmonary artery endothelium perturbed by hydrogen peroxide.

The effects of hydrogen peroxide on factor Xa-catalyzed prothrombin activation on the surface of vascular endothelial cells, and the inhibitory effect of sphingosine on prothrombin activation were studied in cultured calf pulmonary artery endothelial cells (CPAEC). Hydrogen peroxide enhanced factor Xa-catalyzed prothrombin activation on the cell surface in a time and dose-dependent manner. Sphingosine, a potent inhibitor of protein kinase C and the extrinsic pathway of blood coagulation, showed a partial inhibition of prothrombin activation on the surface of CPAEC. When cells were perturbed by hydrogen peroxide, sphingosine inhibited prothrombin activation and displaced factor Xa bound to the cell surface.

Amino Acid Sequence↗
PubMed1992

Behavioral and electroencephalographic effects of a serotonin receptor agonist (5-methoxy-N,N-dimethyltryptamine) in a feline model of photosensitive epilepsy.

The effects of a serotonin (5-HT) receptor agonist, 5-methoxy-N,N-dimethyltryptamine (5-MeODMT), on epileptic photosensitivity were studied in the lateral geniculate-kindled cat. 5-MeODMT at 4 mg/kg significantly suppressed photically induced myoclonus, but not paroxysmal EEG activity, at 0.5-1 h after injection. This antiepileptic effect was seen in association with the appearance of behavioral signs similar to those seen in the 5-HT syndrome. The present data provide further evidence that 5-HT plays an important role in photosensitive epilepsy, and suggest that the inhibitory effect of 5-MeODMT on photosensitivity results from its agonist action at 5-HT1 receptors.

Animals↗
PubMed1992

Site-directed mutagenesis in hemoglobin: functional and structural role of inter- and intrasubunit hydrogen bonds as studied with 37 beta and 145 beta mutations.

In order to clarify the functional and structural role of intra- and intersubunit hydrogen bonds in human hemoglobin (Hb A), we prepared two artificial beta chain mutant hemoglobins by site-directed mutagenesis. The mutant Hb Phe-37 beta, in which Trp-37 beta is replaced by Phe to remove the intersubunit hydrogen bond between Asp-94 alpha and Trp-37 beta at the alpha 1-beta 2 interface in deoxy Hb A, showed a markedly increased oxygen affinity and almost completely diminished Bohr effect and cooperativity. However, 1H-NMR data indicated that the structure of deoxy Hb Phe-37 beta is rather similar to that of deoxy Hb A. The enhanced tetramer-to-dimer dissociation previously observed in Hb Hirose (Trp-37 beta----Ser) together with our observation of the effects of organic phosphate on the structure and function of Hb Phe-37 beta suggested that a large part of the abnormal properties of Hb Phe-37 beta observed for dilute solutions appears to result from partial dissociation into alpha beta dimers rather than direct destabilization of the T-quaternary structure in the deoxygenated state. Thus, the primary and direct role of the hydrogen bond between Asp-94 alpha and Trp-37 beta is to stabilize the tetrameric assembly, and thereby this hydrogen bond indirectly contributes to stabilization of the T-quaternary structure. The other mutant Hb Phe-145 beta has a Phe residue at the 145 beta site and lacks the intrasubunit hydrogen bond formed between Tyr-145 beta and the carbonyl group of Val-98 beta in deoxy Hb A.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Identification of immuno-reactive lipocortin 1-like molecules in serum and plasma by an enzyme immunoassay for lipocortin 1.

A two-site enzyme immunoassay for lipocortin 1 (LC1) has been developed. The detection limit of LC1 was 0.2 ng/tube and the optimal assay range was 1 to 100 ng/tube. The assay system enabled us to identify immunoreactive lipocortin 1-like molecules (IR-LC1) in human serum and plasma. Normal human serum and plasma IR-LC1 concentrations were 44.6 ng/ml for males and 43.1 ng/ml for females with no significant difference between both sexes. The age-related analysis among nine age groups from newborn to 69 years old revealed that the serum or plasma level was high in infants (77.5 ng/ml for newborn and 75.6 ng/ml for 1 month-1 year group), and the 40-year-old (52.2 ng/ml) and 50-year-old (51.3 ng/ml) groups. The major population of plasma IR-LC1 was of 70 kDa in size corresponding to that of the LC1 homodimer. The present enzyme immunoassay system is sufficiently sensitive for the clinical study of LC1 in human body fluids, tissues and organs.

Adolescent↗
PubMed1992

Mass spectrometry in the integrated strategy for the structural analysis of protein variants.

A significant proportion of genetic disorders are caused by point mutations in proteins, and many variants of haemoglobin have been characterized by mass spectrometry. The strategy employs fast atom bombardment and/or tandem mass spectrometry of enzymatically digested peptides for elucidating the type and position of the mutations. Electrospray ionization mass spectrometry is useful for detecting mutations in the intact protein molecule, but the competence diminishes with increasing mass or increasing number of 13C atoms. Recent developments of DNA techniques requires us to re-evaluate the status of mass spectrometry in this field. The role of mass spectrometry in the structural analysis of protein variants is discussed, and is compared with chemical methods and DNA strategies.

Amino Acid Sequence↗
PubMed1992

Experimental and clinical evaluation by flow cytometry for the mechanism of combination therapy (cisplatin and peplomycin).

Pharmacologic effects of cisplatin (CDDP) and peplomycin (PEP) on tumor cell kinetics were studied both in vitro and in vivo with the aid of flow cytometry (FCM). Double staining with propidium iodide (PI) and fluorescein isothiocyanate (FITC)-labeled bromodeoxyuridine (BrdU) was used to analyze the cell cycle, and the number of viable cells was determined with fluorescein diacetate (FDA). Effects of combining the 2 agents were also studied to establish the most effective method of combination therapy. Furthermore, these agents were tried clinically on the basis of experimental results. Results showed that CDDP exerted its action at the S and G2M phases in the cell cycle and PEP at the G2M phase. Among the combination regimens in the experiments with CDDP, PEP, and CDDP + PEP as analyzed by FCM, the strongest block on the G2M phase was shown in the one at a 2-day interval, resulting in the most effective killing of the tumor cells. Clinical trial of the combination therapy showed the same results as the in vitro experiment; the therapy proved useful for improving the patient's clinical condition and the results obtained with CT imaging and pathology.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1992

Electrospray ionization mass spectra of hemoglobin and transferrin by a magnetic sector mass spectrometer. Comparison with theoretical isotopic distributions.

Electrospray ionization mass spectra of human hemoglobin chains and of transferrin were acquired on a magnetic sector mass spectrometer. The observed molecular ion for each hemoglobin chain was in good agreement with the theoretical isotopic distribution at a reasonable resolution of 2000. The clear separation of a variant beta-chain in admixture with the normal counterpart at mass 15,867 that differed from it by 14 Da (0.09%) ensured that a smaller mass difference could be detected. The molecular ions for human transferrin were too broad compared with the theoretical shape to determine the molecular mass accurately, probably due to the heterogeneity of the carbohydrate moiety. A decrease in mass by neuraminidase digestion, however, determined the average number of sialic acids in the molecule.

Hemoglobins↗
PubMed1992

Anticonvulsant effect of allopurinol on hippocampal-kindled seizures.

This study assessed the anticonvulsant effect of allopurinol (5 and 50 mg/kg, IP) on seizures kindled from the feline hippocampus. Allopurinol at a higher dose significantly reduced the behavioral seizures stage, but not afterdischarge duration, without producing any behavioral toxicity. The present results lend experimental support to the contention that allopurinol possesses anticonvulsant efficacy in the treatment of human epilepsy.

Allopurinol↗
PubMed1992

The NMDA receptor antagonist MK-801 has a dissociative effect on seizure activity of hippocampal-kindled cats.

This study assessed the behavioral and electrographic effects of (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclohepten-5,10-imine maleate (MK-801) (0.1 and 0.5 mg/kg, IP), a noncompetitive antagonist of the NMDA receptors, in hippocampal (HIP)-kindled cats. MK-801 at a higher dose significantly reduced the afterdischarge duration, but not the behavioral seizure stage, of HIP-kindled seizures. This anticonvulsant effect occurred in association with the appearance of severe behavioral toxicity and paradoxical worsening of background electroencephalogram characterized by profound spike and wave discharges. The present data suggest the dissociative effect of MK-801 on seizure activity and limitations of its clinical utility as an antiepileptic agent.

Animals↗
PubMed1992

Neonatal cerebral infarction: symptoms, CT findings and prognosis.

In a retrospective multi-center study, we investigated eighteen infants with unilateral cerebral infarctions confirmed by computed tomography (CT) scans. The initial symptoms were observed in all the patients between 0 and 3 days of age. Convulsions or apneic attacks were the initial symptoms in all but one. Only 4 patients had complicated obstetric histories and none showed polycythemia or electrolyte abnormalities. All of the initial CT scans revealed unilaterally localized hypodense areas. In 10, the initial CT scans were performed within 24 hours after the clinical onset. In 16, the lesions were within the territory of the middle cerebral artery, 9 of which also involved the cortico-spinal tract (CST). In the remaining 2 patients, the lesions were located within the territory of the posterior cerebral artery. None of the 9 patients without CST involvement developed hemiplegia, whereas 5 (56%) of the 9 with CST involvement had hemiplegia, which is a fairly low incidence compared with that in adult cases. This difference was thought to be related to neonatal brain plasticity.

Cerebral Infarction↗
PubMed1992

Atrial contractility affects phasic blood flow velocity of atrial small vessels in the dog.

OBJECTIVES: The aim was to evaluate the relative contribution of atrial muscle contraction and atrial pressure to the phasic patterns of left atrial arterial and venous flows. METHODS: Using a laser Doppler velocimeter, blood velocities were measured in the atrial small arteries and veins (outer diameter: 150-500 microns) in anaesthetised open chest dogs (n = 21). The velocity sensor was fixed on the vessel surface with a drop of cyanoacrylate glue when good quality Doppler signals were consistently observed. Left atrial pressure and the contractility of the left atrium were changed by premature ventricular contraction and by intracoronary injection of isoprenaline (0.5 microgram), respectively. RESULTS: Premature ventricular contraction increased left atrial pressure significantly during arterial velocity measurements from 8.1(SD 2.7) to 16.4(1.3) mm Hg and during venous measurements from 8.2(1.2) to 14.3(3.7) mm Hg. However, premature ventricular contraction did not change the blood velocity patterns, the maximum deceleration rate of the systolic velocity wave in arteries, or the maximum acceleration rate of the systolic velocity wave in veins. Although isoprenaline did not change the left atrial pressure, it decreased minimum arterial blood velocity during atrial systole, from 3.3(3.4) to -2.5(3.2) cm.s-1, and increased maximum venous blood velocity from 15.9(5.5) to 19.2(7.4) cm.s-1. Isoprenaline also increased both the maximum arterial systolic velocity deceleration rate, from 90(45) to 234(143) cm.s-2, and the maximum venous systolic velocity acceleration rate from 356(230) to 763(366) cm.s-2. CONCLUSIONS: (1) Left atrial pressure is not a major determinant of the blood flow patterns of the atrial arteries and veins, and therefore it may not closely reflect pressure around mural vessels. (2) Atrial contractility affects the blood flow patterns of the atrial arteries and veins.

Animals↗