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Y Ohsumi

Publications and source records attributed to Y Ohsumi.

At least 19 recordsLinked to original sources
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

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

Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction.

For determination of the physiological role and mechanism of vacuolar proteolysis in the yeast Saccharomyces cerevisiae, mutant cells lacking proteinase A, B, and carboxypeptidase Y were transferred from a nutrient medium to a synthetic medium devoid of various nutrients and morphological changes of their vacuoles were investigated. After incubation for 1 h in nutrient-deficient media, a few spherical bodies appeared in the vacuoles and moved actively by Brownian movement. These bodies gradually increased in number and after 3 h they filled the vacuoles almost completely. During their accumulation, the volume of the vacuolar compartment also increased. Electron microscopic examination showed that these bodies were surrounded by a unit membrane which appeared thinner than any other intracellular membrane. The contents of the bodies were morphologically indistinguishable from the cytosol; these bodies contained cytoplasmic ribosomes, RER, mitochondria, lipid granules and glycogen granules, and the density of the cytoplasmic ribosomes in the bodies was almost the same as that of ribosomes in the cytosol. The diameter of the bodies ranged from 400 to 900 nm. Vacuoles that had accumulated these bodies were prepared by a modification of the method of Ohsumi and Anraku (Ohsumi, Y., and Y. Anraku. 1981. J. Biol. Chem. 256:2079-2082). The isolated vacuoles contained ribosomes and showed latent activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase. These results suggest that these bodies sequestered the cytosol in the vacuoles. We named these spherical bodies "autophagic bodies." Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle. Genetic analysis revealed that the accumulation of autophagic bodies in the vacuoles was the result of lack of the PRB1 product proteinase B, and disruption of the PRB1 gene confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.

Acetates↗
PubMed1992

Purification and characterization of dipeptidyl peptidase IV in rat liver lysosomal membranes.

Dipeptidyl peptidase IV (m-DPP IV) in rat liver lysosomal membranes was purified about 50-fold over the lysosomal membranes with 38% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The enzyme amounts to about 3% of lysosomal membrane protein constituents. The purification procedures included: extraction of lysosomal membranes by Triton X-100, WGA-Sepharose affinity chromatography, hydroxylapatite chromatography, ion exchange chromatography, and preparative polyacrylamide gel electrophoresis. The enzyme (M(r) 240,000) is composed of two identical subunits with an apparent molecular weight of 110,000. The enzyme contains about 12.4% carbohydrate and the carbohydrate moiety was composed of mannose, galactose, fucose, N-acetylglucosamine, and neuraminic acid in a molar ratio of 14:17:2:24:11. Susceptibility to neuraminidase and immunoreactivity of the enzyme in intact tritosomes were examined to study the topology of the enzyme in tritosomal membranes. Neuraminidase susceptibility and immunoreactivity of the enzyme were not observed in the intact tritosomes until the tritosomes had been disrupted by osmotic shock. This result indicated that both the oligosaccharide chains and the main protein portion of the enzyme are on the inside surface of the tritosomal membranes. Subcellular localization of DPP IV was determined by means of enzyme immunoassay, which indicated that bile canalicular membranes and lysosomal membranes are the major sites of localization, and DPP IV activity in lysosomes was separated into a membrane bound form (60%) and a soluble form (40%). Immunoelectron microscopy clearly confirmed that DPP IV occurs not only in the bile canalicular domain but also in the lysosomes of rat liver.

Animals↗
PubMed1992

Intravenous recombinant tissue plasminogen activator in acute carotid artery territory stroke.

To determine the effect of intravenous recombinant tissue plasminogen activator (rt-PA) on vascular and neurologic outcomes, we enrolled 31 patients with acute carotid artery-territory ischemic stroke within 6 hours from symptom onset in a randomized, double-blind, placebo-controlled study. We gave either rt-PA (duteplase at the dose of 20 or 30 mega-international units [MIU]) or placebo intravenously for 60 minutes in patients randomly assigned to the three groups. A comparison between the baseline and postinfusion angiograms showed that complete or partial reperfusion occurred in 50% (5/10) of patients treated with 30 MIU rt-PA, 44% (4/9) of those treated with 20 MIU rt-PA, and 17% (2/12) in the control group. In patients with middle cerebral artery occlusions, reperfusion occurred in 71% (5/7) of the 30-MIU group, in 67% (4/6) of the 20-MIU group, and in 13% (1/8) of the control group. Patients treated with 30 MIU rt-PA showed a significantly early and better clinical improvement, as measured by the neurologic scale, than did those treated with placebo. Parenchymal hemorrhage occurred in one patient in each group, and frequency of clinically insignificant hemorrhagic infarction was comparable among the treatment groups. No major systemic complications occurred in any group. These results support the efficacy of intravenous infusion of rt-PA soon after the onset of stroke in producing rapid thrombolysis and neurologic recovery; it may be of particular value in patients with thromboembolic occlusion in the middle cerebral artery.

Acute Disease↗
PubMed1992

[A case of acute multiple sclerosis mimicking tumor on the neuro-imaging studies].

A 31-year-old woman with a history of suspected optic neuritis was admitted with weakness of right-sided extremities and confusion. On admission general physical examination revealed no abnormality. Neurological examination revealed central facial palsy, mild hemiparesis and hemisensory deficit on the right side. Cerebrospinal fluid analysis showed monocytosis of 29 cells, protein of 82 mg/dl, glucose of 62 mg/dl and myelin basic protein of 6.8 ng/ml. No oligoclonal bands were seen. X-ray computed tomographic (CT) scans showed multiple homogeneous contrast-enhancing lesions in the white matter of bilateral parietal lobes without distinct edema or mass effect. Follow-up CT scans showed ring-enhanced lesions. Magnetic resonance image (MRI) showed multiple Gadolinium-enhanced lesions. Additionally, 123I-IMP SPECT [Gamma view-SPCT 2000 H-20 (Hitachi Co.)] was performed at 30 minutes and 5 hours after intravenous administration of IMP (3 mCi). It showed high IMP uptake corresponding to the CT and MRI lesions. Cerebral angiography was considered to be normal. Other laboratory findings were within normal limits. A biopsy was performed. Histological examination showed spongiosis, gliosis and perivascular cuffing. The histological diagnosis was acute demyelinating disease. After therapy with methylprednisolone, she improved gradually. Enhanced lesions in CT and MRI may correspond to active demyelination at acute MS. High uptake of SPECT may also appear in acute stage, although it has not been reported. We should perform neuro-imaging studies including SPECT on acute MS.

Adult↗
PubMed1991

Dimeric structure of H(+)-translocating pyrophosphatase from pumpkin vacuolar membranes.

Vacuolar membrane H(+)-translocating pyrophosphatase (H(+)-PPase) was purified from pumpkin seedlings. Its enzymatic properties including molecular size of constituting polypeptide (75 kDa) were very similar to those of mung bean H(+)-PPase [(1989) J. Biol. Chem. 264, 20068-20073]. The native, functional molecular size of the pumpkin H(+)-PPase was estimated to be 135-139 kDa from gel permeation HPLC of the purified enzyme in the presence of detergent and from radiation inactivation of the enzyme in vacuolar membranes. It is concluded that native, functional pumpkin H(+)-PPase, and also probably H(+)-PPases from other plants, is a dimer of 75 kDa subunits.

Biological Transport, Active↗
PubMed1991

Binding specificity of D-mannose 6-phosphate receptor of rabbit alveolar macrophages.

The existence of terminal D-mannosyl 6-phosphate groups (Man-6-P) was required (for an inhibitor) to exert a strong inhibitory potency against the binding of bovine serum albumin (BSA) conjugated with 17 molecules of penta-D-mannose 6-phosphate [(M5P)17-BSA] to the Man-6-P receptor in rabbit alveolar macrophages. In addition, the underlying oligosaccharide structures, such as linkage mode between the nonreducing sugar group and the penultimate sugar residue, and the length of sugar chain also affected the inhibitory potency in this system. In general, the oligosaccharides with an alpha-(1----2)-linked Man-6-P unit gave stronger inhibitory potencies than those with an alpha-(1----3)- or alpha-(1----6)-linked Man-6-P unit. Trisaccharides containing a terminal Man-6-P group were more potent inhibitors than disaccharides. A synthetic, branched, and divalent ligand, which does not have a penultimate sugar residue, gave about the same level of inhibitory potency as Man-6-P itself. The "cluster effect" was observed in this system, i.e., as the number of Man-6-P units conjugated to BSA [(Man-6-P)5,5,8, and 46-BSA] increased, the stronger inhibitory potencies were observed with decreasing I50 values of 1.93, 1.36, and 0.0345 microM, respectively. Synthetic divalent oligosaccharides also showed higher inhibitory potencies than the corresponding monovalent oligosaccharides.

Animals↗
PubMed1990

Biogenesis of vacuolar membrane glycoproteins of yeast Saccharomyces cerevisiae.

To investigate the biogenesis of the yeast vacuole, we have sought novel marker proteins localized to the vacuolar membrane. Glycoproteins were prepared from vacuolar membrane vesicles by concanavalin A-Sepharose column chromatography and used to raise monoclonal antibodies. The antibodies obtained recognize several vacuolar proteins that have N-linked oligosaccharide chains. A set of the antibodies reacts with a vacuolar glycoprotein with a major molecular species of 72 kDa (vgp72), which appears to associate peripherally with the vacuolar membrane. The biosynthesis of vgp72 has been examined in detail by pulse-chase experiments and by analyses using various secretory mutants (sec18, sec7, and sec1) and a vacuolar protease mutant (pep4). vgp72 first appears in the endoplasmic reticulum as a 74-kDa species and is quickly modified in the Golgi apparatus to two distinct species: a 79-kDa form, and a heterogeneously glycosylated form (90-150 kDa). Subsequently, both species are proteolytically processed in the vacuole giving rise to a 72-kDa species as well as heavily glycosylated form. Thus, the biogenesis of vgp72 utilizes the early part of the secretory pathway as is the case of vacuolar soluble enzymes. A unique feature is that two species that are different in the extent of glycosylation appear to follow the same destination to the vacuolar membrane.

Acetylglucosaminidase↗
PubMed1990

Archaebacterial ATPases: relationship to other ion-translocating ATPase families examined in terms of immunological cross-reactivity.

Immunological cross-reactivity among three types of H(+)-ATPases, that is, three archaebacterial ATPases, the F1-ATPase from thermophilic bacterium PS3 (TF1) and the vacuolar membrane ATPase from Saccharomyces cerevisiae, was examined by means of immunoblot analyses. The three archaebacterial ATPases were very similar in immunological cross-reactivity, suggesting that they belong to the same family of ATPases. Cross-reaction was also observed between the ATPase from Sulfolobus acidocaldarius, one of the three archaebacteria, and TF1. S. cerevisiae vacuolar ATPase reacted with the antibodies prepared against each of the three archaebacterial ATPases, but did not react with the antibody against TF1. Electron microscopic examination revealed that the oligomeric structure of Sulfolobus ATPase was very similar to that of F1-ATPase. These results, taken together, suggest that the archaebacterial ATPases share close structural similarities with the vacuolar ATPases, and, to a lesser degree, with the F0F1-ATPases.

Adenosine Triphosphatases↗
PubMed1989

Functional molecular masses of vacuolar membrane H+-ATPase from Saccharomyces cerevisiae as studied by radiation inactivation analysis.

The functional molecular masses of the vacuolar membrane H+-ATPase in Saccharomyces cerevisiae under two kinetic conditions for ATP hydrolysis were measured by radiation inactivation. When vacuolar membrane vesicles were exposed to gamma-rays from 60Co, the activities catalyzing a single-cycle and multi-cycles of ATP hydrolysis both decreased as single-exponential functions of the radiation dosage. By applying the target theory, the functional molecular masses for single- and multi-cycle hydrolyses of ATP were determined to be approx. 0.9-1.1 X 10(5) and 4.1-5.3 X 10(5) Da, respectively. N,N'-Dicyclohexylcarbodiimide (DCCD) did not inhibit the former reaction but strongly inhibited the latter. It is suggested that the ATPase with a minimal composite of subunits a and b, in which subunit c is not necessarily involved operationally, can catalyze single-cycle hydrolysis of ATP, whereas for multi-cycle hydrolysis of ATP, the ATPase requires a properly organized oligomeric structure with subunits a-c, which may direct a positive cooperative mechanism of ATP hydrolysis and coupled H+ translocation in a DCCD-sensitive manner.

4-Chloro-7-nitrobenzofurazan↗
PubMed1989

Serial section reconstruction using a computer graphics system: applications to intracellular structures in yeast cells and to the periodontal structure of dogs' teeth.

A computer graphics system for reconstruction from serial section micrographs was applied to intracellular details of a yeast target cell (Saccharomyces cerevisiae cell) induced by the alpha factor mating pheromone and was also applied to a periodontal structure of a dog tooth moved orthodontically. In the former, intracellular organelles and a distribution of vesicles could be clearly observed through the cell membrane using the transparent display method in which the smoothing of the reconstructed outer cell membrane surface by computer processing was applied to the transparent display. In the latter case, by cutting through a reconstructed dog tooth and its periodontal tissues, labiolingual and mesiodistal cut surfaces of the tooth and of adjacent alveolar bone could be observed with fine details (232 sections were used).

Animals↗
PubMed1989

A case of primary malignant lymphoma of the brain with high uptake of 123I-IMP.

In general the 123I-IMP SPECT image of a brain tumor is visualized as a defect. Tumor cases with high IMP uptake have been rarely reported. We encountered a case of primary malignant lymphoma of the brain with high IMP uptake on both early and late scans corresponding to the CT lesions. Previously reported positive cases seemed to be confined to the early scan. For the present the exact behaviour of IMP is incompletely understood, but the mechanism of high uptake has been accounted for by increased extraction and increased amine receptors at the tumor sites. We need further information to establish whether these SPECT findings are specific for primary malignant lymphoma of the brain.

Aged↗
PubMed1989

Three-dimensional analysis of morphogenesis induced by mating pheromone alpha factor in Saccharomyces cerevisiae.

Ultrastructural analyses of cytoplasmic changes in Saccharomyces cerevisiae X2180-1A (MATa) that had been treated with alpha factor were performed by using the freeze-substitution fixation method. After alpha factor treatment, cells exhibited a pointed projection, which is a unique pattern of oriented cell surface growth. The relationship between projection formation and intracellular organelles was examined using serial thin sections and computer-aided three-dimensional reconstructions. Using these analyses membrane vesicles and other organelles were detected, and studies on their dynamic structural reorganization became feasible. Production of membrane vesicles (average 65 nm in diameter) was induced upon exposure of the cells to alpha factor before projection emergence. The total number of membrane vesicles increased at the early stage and decreased at the late stage of projection formation. Three-dimensional analysis indicated that the vesicles were at first dispersed throughout the cell, then accumulated at the site where the projection formed. Morphological changes and multiplication of the Golgi body were seen during the process of projection formation. Other intracellular organelles (nucleus, vacuole, rough endoplasmic reticulum and mitochondria) were also rearranged, showing a polar organization of the cytoplasm during projection formation.

Mating Factor↗
PubMed1989

Predictability of left heart dysfunction from right heart performance--cardiac index-venous pressure plots and cardiac index-mean pulmonary artery wedge pressure plots at rest and their shift during dynamic exercise.

In an attempt to examine the extent to which the right heart performance can predict the left heart performance in heart diseases primarily affecting the left heart, we recorded cardiac index-venous pressure (CI-VP) plot and cardiac index-mean pulmonary artery wedge pressure (CI-PAW) plot at rest and investigated the shift of CI-VP plot and CI-PAW plot that occurred during mild dynamic exercise of the lower limbs. Six patients had normal heart function and 20 patients had heart diseases primarily affecting the left heart. The sensitivity, specificity, positive predictive value and negative predictive value in the estimation of the left heart function with delta CI/delta VP were 80%, 100%, 89% and 100%, respectively, when delta CI/delta PAW was regarded as the golden standard for the estimation of the left heart function. The sensitivity, specificity, positive predictive value and negative predictive value in estimating the left heart function with delta CI/delta RA were 86%, 93%, 92% and 86%, respectively. When the left heart function was estimated by delta VP alone without measuring delta CI, the sensitivity, specificity positive predictive value and negative predictive value were 40%, 100%, 73% and 100%, respectively. In short, it was possible to predict the left heart dysfunction with delta CI/delta VP or delta CI/delta RA, in the presence of the right heart dysfunction. It was also possible to predict a steep heart slope from normal delta CI/delta VP with error of 2/18 (11%), when steep left heart slope was predicted, based on the presence of steep right heart slope. In comparison, delta VP alone was a less sensitive index of the performance of the left heart.

Adolescent↗
PubMed1989

Quantitative estimation of compliance of human systemic veins by occlusion plethysmography with radionuclide--methodology and the effect of nitroglycerin.

Volume-pressure relationship and compliance of human systemic veins were estimated quantitatively and noninvasively using radionuclide. The effect of nitroglycerin (NTG) on these parameters was examined. Plethysmography with radionuclide (RN) was performed using the occlusion method on the forearm in 56 patients with various cardiac diseases after RN angiocardiography with 99mTc-RBC. The RN counts-venous pressure curve was constructed from (1) the changes in radioactivity from region of interest on the forearm that were considered to reflect the changes in the blood volume of the forearm, and (2) the changes in the pressure of the forearm vein (fv) due to venous occlusion. The specific compliance of the forearm veins (Csp.fv; (1/V).(delta V/delta P] was obtained graphically from this curve at each patient's venous pressure (Pv). Csp.fv was 0.044 +/- 0.012 mmHg-1 in class I (mean +/- SD; n = 13), 0.033 +/- 0.007 mmHg-1 in class II (n = 30), and 0.019 +/- 0.007 mmHg-1 in class III (n = 13), of the previous NYHA classification of work tolerance. There were significant differences in Csp.fv among the three classes. The systemic venous blood volume (Vsv) was determined by subtracting the central blood volume, measured by RN-angiocardiography, from total blood volume, measured by the indicator dilution method utilizing 131I-human serum albumin. Systemic venous compliance (Csv) was calculated from Csv = Csp.fv.Vsv. The Csv was 127.2 +/- 24.8 ml.mmHg-1 (mean +/- SD) in class I, 101.1 +/- 24.1 ml.mmHg-1 in class II and 62.2 +/- 28.1 ml.mmHg-1 in class III. There were significant differences in Csv among the three classes. The class I Csv value was calculated to be 127.2 +/- 24.8 ml.mmHg-1 and the Csv/body weight was calculated to be 2.3 +/- 0.7 ml.mmHg-1.kg-1 of body weight, which was very close to the values for Csv reported previously by other investigators in animal experiments. The administration of NTG caused the RN counts - venous pressure curve to become steeper and shift upward. It increased Csv significantly in all cases.

Adult↗