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Biomedical subjects

H Sakai

Publications and source records attributed to H Sakai.

At least 1,081 records · Page 60Linked to original sources
PubMed1987

Tyrosine phosphorylation of common and specific sets of cellular proteins rapidly induced by insulin, insulin-like growth factor I, and epidermal growth factor in an intact cell.

KB cells respond to insulin and insulin-like growth factor I (IGF-I) in a closely similar way (induction of membrane ruffling, stimulation of pinocytosis, and amino acid transport) but respond to epidermal growth factors (EGF) in a similar but distinct way. In the KB cells, using phosphotyrosine-specific antibody we have found that: the receptors for insulin (beta subunit), IGF-I (beta subunit), and EGF undergo tyrosine phosphorylation as early as 10 s after addition of their respective ligands; a 185-kDa protein is rapidly (less than 10 s) tyrosine phosphorylated by insulin and IGF-I through their respective receptor kinases but not EGF; tyrosine phosphorylation of a 190-kDa glycoprotein is rapidly (less than 10 s) induced by EGF through EGF receptor kinase; and tyrosine phosphorylation of a 240-kDa protein is stimulated within 30 s by all three growth factors. These patterns of tyrosine phosphorylation could be causally related to biological responses induced by the three growth factors.

Carcinoma, Squamous Cell↗
PubMed1987

A Mr = 190,000 glycoprotein phosphorylated on tyrosine residues in epidermal growth factor stimulated KB cells is the product of the c-erbB-2 gene.

In human epidermoid carcinoma KB cells, a glycoprotein of Mr = 190,000 (gp190) has been shown to be phosphorylated on tyrosine residues upon EGF stimulation (Kadowaki et al., 1987, J. Biol. Chem. in press). Using a specific antibody to the c-terminal portion of the human c-erbB-2 gene product, we have found that gp190 is the human c-erbB-2 gene product which is structurally closely related to the epidermal growth factor (EGF) receptor. Since monoclonal antibody specific for the EGF receptor abolished both EGF binding to its receptor and tyrosine phosphorylation of the c-erbB-2 gene product, we have concluded that activation of EGF receptor tyrosine kinase activity upon EGF binding leads to the phosphorylation of the c-erbB-2 gene product on its tyrosine residues.

Epidermal Growth Factor↗
PubMed1987

Limited chymotryptic digestion of bovine adrenal 190,000-Mr microtubule-associated protein and preparation of a 27,000-Mr fragment which stimulates microtubule assembly.

A heat stable microtubule-associated protein of Mr 190,000 (190-kDa MAP) has been purified from bovine adrenal cortex (Murofushi, H., Kotani, S., Aizawa, H., Hisanaga, S., Hirokawa, N., and Sakai, H. (1986) J. Cell Biol. 103, 1911-1919). Limited chymotryptic digestion of 190-kDa MAP produced a fragment of Mr 27,000 (27-kDa fragment), which bound to microtubules reconstituted in the presence of taxol. This fragment was purified with the aid of cosedimentation with microtubules. The purified 27-kDa fragment showed an ability to stimulate tubulin polymerization in the absence of taxol. Electron microscopic observation of microtubules reconstituted from purified 27-kDa fragment and tubulin revealed that the microtubules were in the form of thick bundles and that lateral projections which can be seen in microtubules reconstituted from intact 190-kDa MAP and tubulin were not observed. These results indicate that 27-kDa fragment includes or is a part of microtubule-binding domain of 190-kDa MAP and that this fragment is active in stimulating microtubule assembly. Amino acid analysis revealed that the 27-kDa fragment was rich in lysine, proline, and alanine, the sum of these three being about 45% of the total amino acids and that the contents of methionine, tyrosine, phenylalanine, and histidine were very low. These data suggest that the microtubule binding domain of the 190-kDa MAP comprises an unique structure.

Adrenal Cortex↗
PubMed1987

A mitochondrial frameshift-suppressor (+1) [corrected] of the yeast S. cerevisiae maps in the mitochondrial 15S rRNA locus.

The first case of a +1 "extrageneic" frameshift suppressor (MF1), mapping in the yeast mitochondrial 15S rRNA gene is reported. The suppressor was identified by genetic analyses in a leaky mitochondrial oxil frameshift mutant and the respective wild-type strain 777-3A of the yeast S. cerevisiae. This is in accordance with the finding that all mitochondrial frameshift mutants isolated from this strain tend to be leaky to a variable degree. MF1 does not suppress known nonsense mutations created by a direct basepair exchange in strain 777-3A. These mutants exhibit a non-leaky phenotype (Weiss-Brummer et al. 1984).

Chromosome Deletion↗
PubMed1987

Nasu-Hakola's disease (membranous lipodystrophy).

An autopsy case of Nasu-Hakola's disease (membranous lipodystrophy) is reported. A 43-year-old Japanese man, whose parents were not consanguineous, had been suffering from frequent long bone fractures since the age of 10. Neuropsychiatric symptoms, which were characterized by euphoria, disturbance of attention and dementia, appeared at his thirties and generalized and/or localized seizures and apallial syndrome at the later stage. The neuropathology revealed diffuse leukoencephalopathy of the cerebrum. The peculiar aspects in this case were membranocystic changes in the lungs [Yagishita et al. Virchows Arch [A] 408:211-217 (1985)], diffuse degeneration of the cerebral cortex, chiefly in frontal and temporal lobes, and many axonal spheroids throughout the cerebral cortex. The ultrastructure of spheroids in the cerebral cortex demonstrated aggregations of mitochondria, dense bodies and minute concentric bodies and a small amount of neurofilaments.

Adult↗
PubMed1987

A high molecular weight actin binding protein: its localization in the cortex of the sea urchin egg.

From the Triton-treated cortex fraction of sea urchin eggs, a high molecular weight actin binding protein (260K protein) was solubilized by a high salt solution and purified. A cosedimentation assay revealed that the 260K protein binds to actin filaments in a concentration-dependent manner. The low-shear viscosity of actin solutions largely increased in a concentration-dependent manner after addition of 260K protein. Electron microscopy showed that this protein induces the formation of large curled bundles of actin filaments. Different from fascin-induced actin bundles, no clear striations were observed within the actin bundles formed by the 260K protein. Antibodies against the 260K protein were raised in a rabbit and affinity purified. Immunoblotting analysis of Triton-solubilized cortex and various subcellular fractions showed that first only a single band reacted with the antibody and second that the 260K protein exclusively localized in the cortex fraction. Indirect immunofluorescence microscopy localized the protein in the cortex and the region of the cleavage furrow. After double staining, the fluorescence images for actin filaments and the 260K protein well correlate with each other.

Animals↗
PubMed1987

Replication origin (oric) on the complementary DNA strand of Escherichia coli phage G4: biological properties of mutants.

Phage G4 origin of complementary DNA strand synthesis (oric) consists of three stable secondary loop structures. In a cloned 274-bp DNA fragment that is active as an ori in the filamentous phage cloning vector R199, insertion mutants have been constructed by introducing EcoRI and HindIII linkers at the base of loop III. The in vivo activity of these oric mutants (conversion of single-strand form to replicative form in the presence of rifampicin) was significantly reduced (50-70%) but not completely abolished. Nucleotide sequences and/or potential secondary structure of loop III centered at the AvaII site are therefore an important functional part of oric.

Base Sequence↗
PubMed1987

Effect of surgical repair on respiratory mechanics in congenital diaphragmatic hernia.

To determine whether surgical repair of congenital diaphragmatic hernia (CHD) results in improvement in respiratory mechanics, we measured respiratory system compliance in nine patients (five survivors and four nonsurvivors) before and after operation. In all nine infants, CHD was diagnosed within 6 hours of life, and surgical repair was through an abdominal approach after a period of stabilization. Measurements were made noninvasively, using the passive expiratory flow-volume technique. In only one of the nine infants did compliance immediately improve after surgical repair, and in another it showed no change. Both of these infants survived, with an uneventful postoperative course. In the remaining seven infants, however, postoperative compliance immediately decreased to 10% to 77% from the preoperative value. The four infants with more than 50% decrease in compliance died with increasing hypoxemia and acidosis. These results suggest that respiratory mechanics in CHD, far from improving, frequently deteriorate as a result of repair of the hernia. The role of urgent surgery in this malformation should be reevaluated.

Hernia, Diaphragmatic↗
PubMed1987

Physical characteristics of various intraocular lenses.

Physical characteristics of 15 posterior chamber lenses from 15 companies were examined for residual ethylene oxide gas; the content varied from 13 ppm to 78 ppm. Seven lenses met the FDA regulation of less than 25 ppm. Scanning electron microscopic study revealed that molded-type lenses had the smoothest optic surface and cast-molded lenses had a less smooth surface than the plano side. The lathe-cut lens with barrel polishing also had a smooth optic surface. Neodymium: YAG laser shots were delivered to the optic and the spots were examined by phase contrast microscopy. The lenses made of high molecular weight polymethylmethacrylate were more resistant to YAG laser damage. It was concluded that lathe-cut posterior chamber lenses with barrel polishing made of high molecular weight polymethylmethacrylate would be preferable but further studies of polishing compounds are necessary.

Ethylene Oxide↗
PubMed1987

Cofilin is a component of intranuclear and cytoplasmic actin rods induced in cultured cells.

Incubation of cultured cells under specific conditions induces a dramatic change in the actin organization: induction of intranuclear and/or cytoplasmic actin rods (actin paracrystal-like intracellular structures). We have found that cofilin, a 21-kDa actin-binding protein, is a component of these rods. Antibodies directed against cofilin labeled intranuclear actin rods induced in cells treated with dimethyl sulfoxide or exposed to heat shock and also labeled cytoplasmic actin rods induced in cells incubated in specific salt buffers. Moreover, we found that these actin rods are not stained with fluorescent phalloidin derivatives at all and appear to be right-handed helices, different from straight bundles of F-actin such as stress fibers. In vitro experiments revealed that cofilin and phalloidin compete with each other for binding to F-actin. Since cofilin and phalloidin have the ability to stoichiometrically bind actin molecule in the filament in vitro, the above results seem to suggest that cofilin directly binds to actin molecule in nearly an equimolar ratio in these rods. We call these rods "actin/cofilin rods."

Actin Depolymerizing Factors↗
PubMed1987

Evidence for two activated forms of pyruvate kinase from Bacillus stearothermophilus in the presence of ribose 5-phosphate.

Allosteric activation of pyruvate kinase from a thermophilic bacterium, Bacillus stearothermophilus, by ribose 5-phosphate (R5P) was kinetically examined. Two activated forms of this enzyme could be distinguished, depending on the R5P concentration. One form (Form I) was observed at about 10(-5) M R5P. It showed a slightly negative cooperativity for phosphoenolpyruvate (PEP). The other form (Form II) was observed at more than 10(-3) M R5P and showed Michaelis-Menten kinetics for PEP. The PEP and ADP concentrations that yield half-maximal velocity were essentially identical for the two forms (about 0.1 and about 0.5 mM, respectively), but Form I had a larger Vmax value than Form II. In the absence of R5P, the enzyme showed a homotropic positive cooperativity for PEP; the concentration required for the half-maximal velocity was about 2 mM and that of ADP was about 1.6 mM. The enzyme was more susceptible to protease digestion in the presence of R5P than in the absence of it. The concentration of R5P required for the enzyme to be susceptible to protease digestion was approximately identical with that required to generate Form I. With more than 10(-3) M R5P, the thermostability of the enzyme was greatly increased. The concentration of R5P required for the enzyme to be thermostable was in good agreement with that required to generate Form II. These results indicate that the two activated forms distinguished kinetically differ in their conformations, too. The saturating level of PEP did not cause such a change in the thermostability or the susceptibility to protease.

Adenosine Diphosphate↗
PubMed1987

Comparison of a major heat-stable microtubule-associated protein in HeLa cells and 190-kDa microtubule-associated protein in bovine adrenal cortex.

A heat-stable microtubule-associated protein (MAP) with a molecular weight of 190,000, termed 190-kDa MAP, has been purified from bovine adrenal cortex (Murofushi, H. et al. (1986) J. Cell Biol. 103, 1911-1919). Immunoblotting experiments using an antibody against this MAP revealed that several kinds of culture cells derived from human tissues contain proteins with an apparent molecular weight of 180,000 reacting with the antibody. Indirect immunofluorescence microscopic observation of HeLa cells showed that the immunoreactive protein co-exists with microtubules, indicating that the protein is one of the HeLa MAPs. A heat-stable MAP with a molecular weight of 180,000, termed here HeLa 180-kDa MAP, was purified by the taxol-dependent procedure (Vallee, R.B. (1982) J. Cell Biol. 92, 435-442) and successive co-polymerization with brain tubulin. This protein was the most abundant MAP in HeLa cells, suggesting that the MAP is identical to the major HeLa MAP previously reported by Bulinski and Borisy (Bulinski, J.C. & Borisy, G.G. (1980) J. Biol. Chem. 255, 11570-11576) and Weatherbee et al. [1980) Biochemistry 19, 4116-4123). It was shown that, like bovine adrenal 190-kDa MAP, yet distinct from brain MAP2 and tau, purified HeLa 180-kDa MAP does not interact with actin filaments. This common characteristic of the two MAPs along with the same heat-stability strongly suggests that they are members of the same group of MAPs. The fact that HeLa 180-kDa MAP reacts with an antibody against bovine adrenal 190-kDa MAP means that they share common epitopes, in other words, common local amino acid sequences. However, the limited proteolytic patterns of the two MAPs with S. aureus V8 protease and chymotrypsin were distinct from each other, suggesting the presence of large differences in the overall primary structures between bovine adrenal 190-kDa MAP and HeLa 180-kDa MAP.

Actins↗
PubMed1987

Purification and characterization of phospholipase A2 from Trimeresurus gramineus venom.

Phospholipase A2 was purified to homogeneity from the venom of Trimeresurus gramineus (the Green Habu snake) via three steps consisting of Sephadex G-75, DEAE-cellulose, and DEAE-Toyopearl 650M column chromatographies. Molecular weight determinations showed that the enzyme consists of a single polypeptide chain with a molecular weight of approximately 14,000. The isoelectric point was 4.5. The enzyme is characterized by high contents of acidic amino acids, glycine, and half-cystine. Calcium ion was essential for eliciting activity. The enzyme was inactivated by alkylation of a single histidine residue with p-bromophenacyl bromide following pseudo first-order kinetics, and the rate of the inactivation was depressed in the presence of Ca2+. The N-terminal sequence of this enzyme determined to the 40th residue was found to be highly homologous to that of Trimeresurus okinavensis phospholipase A2 but not to that of Trimeresurus flavoviridis phospholipase A2. The phenylalanine residue at the 27th position of T. gramineus phospholipase A2 is noteworthy because all other phospholipases A2, with only two exceptions, contain a tyrosine residue at this position.

Acetophenones↗
PubMed1987

Identification of a temperature-resistant bacteriophage phi X174 mutant.

Bacteriophage phi X174am3trD, a high temperature-resistant mutant of phi X174am3, was 10(4) times more stable than phi X174am3 as judged by its survival ratio after heat treatment at 54 degrees C for 120 min. Complementation tests showed an involvement of gene G. Sequence analysis of this gene revealed three mutation sites, one transition and two insertions. The first was a silent mutation and the others brought about a change in one amino acid and an addition of another, respectively, in the gene G protein. These changes in amino acid sequence resulted in a change in the secondary structure of the protein. A beta-turn region in part of the gene G protein of phi X174am3 was changed to an alpha-helix in phi X174am3trD. These results indicate that the temperature resistance of phi X174am3trD may be caused by elevated hydrophobicity in the mutated region or by strong interaction between the mutated gene G protein and other capsid proteins.

Amino Acid Sequence↗
PubMed1987

Studies on glomerular immune solubilization by complement in patients with IgA nephropathy.

A study of the solubilization of glomerular immune deposits by serum or complement in patients with IgA nephropathy is described. Renal biopsy specimens were obtained from 15 patients with IgA nephropathy. These specimens were incubated with fresh and heated sera from healthy adults or with lyophilized complement components, i.e., C3 and C4, at 37 degrees C for one hour in plastic tubes. The sections were then stained with fluorescein isothiocyanate (FITC)-labelled anti-human IgA antisera and examined by fluorescence microscopy. Normal sera showed a marked capacity to solubilize the glomerular immune deposits characteristic of IgA nephropathy. The solubilization capacity was reduced after inactivation and absorption of sera with anti-human C3 antiserum. Lyophilized C3 or C4 did not show any ability to solubilize such deposits. It was concluded that the solubilization of glomerular immune deposits may require whole active (fresh) components of complement related to the alternative pathway.

Complement C3↗