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Biomedical subjects

H Shima

Publications and source records attributed to H Shima.

At least 127 records · Page 7Linked to original sources
PubMed1995

Analysis by in vitro mutagenesis of PP2A alpha okadaic acid responsive sequences.

It has been shown that the protein serine/threonine phosphatase type 2A alpha catalytic subunit (PP2A alpha) has a mutation consisting of a glycine substitution for cysteine at 269 in the okadaic acid resistant variant of CHO cells, and that the mutant protein is resistant to okadaic acid (Shima, H. et al. Proc. Natl. Acad. Sci. USA 91, 9267-9271, 1994). In this study we analyzed okadaic acid responsive sequences in PP2A alpha by introducing a mutation at around codon 269 of the cDNA strand. The recombinant mutant PP2A alpha proteins Y265F, C266G, Y267G and C269Y were resistant to okadaic acid, with IC50s of 10, 24, 20 and 10 nM, respectively, as opposed to the recombinant wild PP2A alpha protein which had an IC50 of 0.24 nM. This observation suggests that not only cysteine at 269, but also other YCY amino acids, between 265-267, are necessary for the high sensitivity of PP2A to okadaic acid.

Amino Acid Sequence↗
PubMed1995

Neurofilament-associated protein phosphatase 2A: its possible role in preserving neurofilaments in filamentous states.

Neurofilament phosphatase (NF-phosphatase) activity, which dephosphorylates NF proteins phosphorylated by cyclic AMP-dependent protein kinase (A-kinase), was detected in NF fractions prepared from bovine spinal cords. This phosphatase was suggested to be associated with NFs by gel filtration and sedimentation analysis and was further demonstrated by dephosphorylation-dependent binding assay of NFs to microtubules. The NF-associated NF-phosphatase was identified as a type of protein phosphatase 2A (PP2A) by (i) its complete inhibition with 100 nM okadaic acid, at which concentration the purified type 1 protein phosphatase (PP1) was inhibited only 25%; (ii) the absence of effect of inhibitor-2, a specific inhibitor of PP1, on the NF-phosphatase activity; and (iii) the detection of 38-kDa catalytic and 65-kDa regulatory subunits of PP2A by immunoblotting. The NF-associated PP2A was partially solubilized from NFs by a high concentration of MgSO4, and the solubilized PP2A was suggested by gel filtration to be a dimeric holoenzyme consisting of a 38-kDa catalytic and a 65-kDa regulatory subunit. Phosphorylated NF-L, which is assembly incompetent, was induced to assemble into filaments by dephosphorylation with PP2A. These results suggest a role of NF-associated PP2A in preserving filamentous forms of NF in neurons.

Actin Cytoskeleton↗
PubMed1995

cDNA cloning of BR gamma, a novel brain-specific isoform of the B regulatory subunit of type-2A protein phosphatase.

A cDNA clone for a novel isoform of the B regulatory subunit of type 2A protein phosphatase (BR) was isolated from a rat brain cDNA library by hybridization under low-stringency conditions with the BR beta cDNA fragment as a probe. The novel cDNA encodes a protein of 447 amino acids. The identities of amino acid sequences of the novel isoform with those of rat BR alpha and BR beta were 80% and 85%, respectively, with a unique sequence at the C-terminal. Since this isoform is thought to be encoded by a third gene for the B regulatory subunit, it was named BR gamma. The mRNA for BR gamma was 4.2 kb. The expression level of BR gamma was high in the brain and spinal cord of rats, and its expression was also detected in mouse and human brains, but in contrast to BR beta, it was not detected in the testes of rats or mice. Thus BR gamma is though to be a brain-specific isoform of the B regulatory subunit.

Amino Acid Sequence↗
PubMed1995

Translocation of protein phosphatase 1 catalytic subunits during 1,25-dihydroxyvitamin D3-induced monocytic differentiation of HL-60 cells.

To elucidate the roles of protein phosphatases type 1 (PP1) and type 2A (PP2A) in 1,25-dihydroxy-cholecalciferol [1,25(OH)2D3]-induced differentiation of HL-60 cells into monocytes, we examined the enzyme activity and the protein and gene expressions of PP1 and PP2A in these cells. Calyculin-A augmented the 1,25(OH)2D3-induced differentiation of the cells. Treatment of the cells with 1,25(OH)2D3 led to a decrease in PP1-like activity in the cytosol fraction, with a concomitant increase in the membrane and nuclear PP1-like activity, as determined when protein phosphatase activity was assayed using myosin light chain as substrate in the presence of 5 nM okadaic acid. Western blot analysis with antibodies specific for PP1 catalytic subunit isozymes (PP1 alpha, PP1 gamma, and PP1 delta) showed that all three PP1 isozymes were expressed but were differentially distributed in each cellular fraction. Subcellular redistribution of PP1-like activity during 1,25(OH)2D3-induced differentiation was mainly attributed to PP1 gamma and PP1 alpha proteins. In contrast, the localizations of PP1 delta and PP2A catalytic and regulatory subunits were not significantly affected by 1,25(OH)2D3 treatment. The gene expressions of PP1 alpha and PP1 gamma appeared to be constant during processes of monocytic differentiation. The correlation between phenotypic and functional changes of HL-60 cells on the one hand and subcellular redistribution of PP1-like activity on the other suggest that the translocations of PP1 alpha and PP1 gamma isozymes may contribute to the 1,25(OH)2D3-induced monocytic differentiation of HL-60 cells.

Calcitriol↗
PubMed1995

Enhanced expression of catalytic subunit isoform PP1 gamma 1 of protein phosphatase type 1 associated with malignancy of osteogenic tumor.

The expressions of the three catalytic subunits of protein phosphatase (PP) type 1 and 2A, PP1 alpha, PP1 gamma 1, and PP2AC, were examined in 14 cases of three types of osteogenic tumor using immunohistochemical analysis. The percentage of tumor cells stained positively with antiserum against PP1 catalytic subunit-isoform PP1 gamma 1 was significantly higher in malignant osteogenic tumors than in benign osteogenic tumors. Furthermore, malignant osteogenic tumor showed markedly high S-phase fraction in the cell cycle of tumor cells, as compared to benign osteogenic tumors. These results suggest that PP1 gamma 1 is involved in the accelerated growth of malignant cells in osteogenic tumors.

Adolescent↗
PubMed1995

Sandwich enzyme immunoassay for endothelin with monoclonal antibodies and its application.

The development of a sensitive enzyme immunoassay for endothelin is described. This assay is based on a sandwich method using two different monoclonal antibodies against endothelin-1. A monoclonal antibody, which reacted to the C-terminal region of endothelin, was selected as an immobilized antibody. The Fab' fragment of another monoclonal antibody, which might recognize the N-terminal rigid region of endothelin, was used as a horseradish peroxidase-labeled detector antibody. The assay measures endothelin-1 and endothelin-2 with a sensitivity of 1 fmol/ml. We have determined that cultured endothelial cells actually produced endothelin in significant amounts in a time-dependent manner. The levels of plasma endothelin extracted with Sep-Pak tC18 light cartridges could also be monitored. A basal endothelin level was about 0.3 fmol/ml of plasma, and a transient increase was observed 4 h after starting blood collection under in vivo experimentation in the rat. This enzyme immunoassay will facilitate the investigation of physiological roles of endothelin.

Animals↗
PubMed1995

Protein serine/threonine phosphatases as binding proteins for okadaic acid.

Recently, many potent inhibitors of protein serine/threonine phosphatases (PPs) have been found. Some of them have proven to be tumor promoters in mouse skin two-step carcinogenesis and rat liver medium-term tests. Among these inhibitors, okadaic acid (OA) selectively inhibits PP2A, and its use has therefore been proposed to facilitate analysis of biological roles of this phosphatase. OA shows bimodal effects on in vitro transformation and, in addition to such epigenetic changes, also induces marked genetic changes. OA treatment for more than 1 week flattened NIH 3T3 transformants irreversibly, with loss of the transfected genes. It is also known to induce diphtheria toxin-resistant mutations in Chinese hamster lung cells and sister chromatid exchanges (SCEs) in Chinese hamster ovary cells and human lymphocytes. To analyze roles of protein phosphatases in gene stability, we isolated OA-resistant mutants. They were proven to have a mutation in the PP2A alpha catalytic subunit, in which cysteine 269 had been substituted for glycine; and it was demonstrated that this region interacts with OA. The recombinant mutant protein was 4 approximately 9-fold more resistant to OA than the wild type. Although the OA resistant mutants of CHO cells expressed high levels of P-glycoprotein, inhibition of PP2A itself was suggested to lead to SCE induction. However, the number of molecular species of PP which are known to be sensitive to OA continues to increase, and we have isolated cDNA for a novel type of OA sensitive PP. Our studies indicate that the fact that the roles of PP2A cannot be elucidated using only OA is of crucial importance.

3T3 Cells↗
PubMed1995

Immunohistochemical localization of protein phosphatase isoforms in the rat cerebellum.

Protein phosphatase isoforms, PP1 gamma 1, PP1 delta, PP1 alpha and PP2A (alpha and/or beta), were immunohistochemically localized in the rat cerebellum. Purkinje cell perikarya, dendrites and spines were very PP1 gamma 1 immunoreactive. PP1 delta and PP1 alpha were perinuclear in all neurons, PP1 alpha also revealed a new cell type and PP2A was homogeneous in Purkinje cell soma and large dendrites. PP1 gamma 1 seems to be dominant for dephosphorylation at the dendritic synapses of Purkinje cells.

Animals↗
PubMed1995

Protein phosphatase mRNA expression in Purkinje cells of staggerer and reeler mutant mice.

We used in situ hybridization to search for the expression of three protein phosphatase (PP) mRNAs in Purkinje cells of normal mice, and staggerer (sg/sg) and reeler (rl/rl) mutant mice, two strains with known Purkinje cell disorders. The expression of the mRNAs was comparable in the normal and rl/rl Purkinje cells, but considerably reduced in the sg/sg. We interpret this finding as indicating: (a) the staggerer mutant gene may directly affect the phosphatase component of the protein phosphorylation-dephosphorylation cycle in the sg/sg Purkinje cells; or (b) the reduced mRNA expression may be a secondary phenomenon, resulting from abnormal Purkinje cell function due to the lack of synaptic input.

Animals↗
PubMed1995

Increased calcium sequestration by sarcoplasmic reticulum in small muscular arteries in young spontaneously hypertensive rats.

1. Sarcoplasmic reticulum (SR) in small resistance arteries plays a role in the regulation of the cytosolic free calcium concentration by sequestration of calcium from cytoplasm. 2. To examine the contribution of calcium (Ca2+) sequestration by the SHR to both contraction and relaxation in young spontaneously hypertensive rats (SHR), we measured evoked tension before and after depletion of SR Ca2+ stores in the rings of the first branch of superior mesenteric artery in 5 week old SHR and age-matched Wistar-Kyoto (WKY) rats. Contractile responses were induced by 40 mmol/L potassium and 10 mmol/L caffeine before and after the treatment with ryanodine or thapsigargin, which depletes SR calcium stores. 3. The magnitude of potassium-induced contraction was almost the same in both strains. 4. Ryanodine and thapsigargin did not affect the resting tension and almost decreased caffeine-induced contraction in both strains. 5. After the treatment with ryanodine or thapsigargin, the magnitude and the rate of potassium-induced contraction were augmented greatly in SHR, but not in WKY. 6. The relative relaxation rate after exposure of potassium was significantly slowed in SHR by ryanodine or thapsigargin, but only slightly in WKY. 7. These results suggest that Ca2+ sequestration by the SR in SHR was greater than in WKY. Therefore, it is concluded that SR plays an important role in preventing the development of hypertension in SHR via a buffering effect on the elevation of cytosolic free Ca2+.

Animals↗
PubMed1995

Role of protein kinase C in relationship between Ca2+ and contractile elements in rat alpha-toxin-permeabilized mesenteric artery.

Phorbol ester, which activates protein kinase C (PKC), modulates vasoconstrictor-induced tension in vascular smooth muscle. Recently, Staphylococcal aureus alpha-toxin, which produces too small pores in the plasma membrane to allow passage of proteins, such as PKC, is used to investigate the signal transduction system in vascular smooth muscle cells. In order to elucidate the role of PKC on vascular smooth muscle contraction, we examined whether PKC activation influences the relationship between intracellular Ca2+ ([Ca2+]i) and tension in Wistar rat superior mesenteric artery (SMA) using vascular smooth muscle permeabilized with Staphylococcal alpha-toxin. [Ca2+]i was clamped at specified values (10(-8.5)-10(-4) mol/L) using EGTA-Ca2+ buffer. In alpha-toxin non-treated rings of SMA, isometric tension was evoked by 10 mmol/L caffeine and 10-30 mmol/L external potassium (high K+) in the absence or presence of phorbol 12, 13-dibutyrate (PDBu), a PKC activator, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and staurosporine (PKC inhibitors). PDBu significantly augmented caffeine- and high K(+)-evoked contractions. H-7 and staurosporine significantly attenuated caffeine- and high K(+)-evoked contractions augmented by PDBu. Moreover, H-7 significantly suppressed high K(+)-induced contraction in the absence of PDBu. In alpha-toxin permeabilized artery, PDBu shifted the [Ca2+]i-force relationship curve to the left. These results suggest that PKC activates vascular smooth muscle contraction by increasing the sensitivity of the contractile apparatus to Ca2+.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

[Review of the practice of care in a health unit: Experience developed in an integration project between school and service].

The paper shows a program concerning the review of the assistance practice in a Health Unit at the target region of the School-Service Integration Project between the College of Nursing at Ribeirão Preto/University of São Paulo and São Paulo city. The majority of the health workers took part in the program which was developed in 8 meetings where they analysed their own working practice and elaborated several proposals aiming at changing it. The greatest merit of this program was to allow the establishment of a collective and participative process of discussion and reflection.

Education, Nursing, Baccalaureate↗
PubMed1995

Decreased expression of protein phosphatase type 2A in HL-60 variant (HL-60RAr) cells resistant to induction of cell differentiation by all-trans retinoic acid.

To evaluate the molecular basis for susceptibility of the cell differentiation induced by all-trans retinoic acid (ATRA), we examined biochemical activities and expression of protein phosphatases type 1 (PP1) and type 2A (PP2A) from HL-60 cells that are susceptible to differentiation induced by ATRA and HL-60RAr cells, HL-60 variant cells that are resistant to such induction. One nM of calyculin-A (CAL-A) achieved the enhancement of granulocytic differentiation in ATRA-treated HL-60 (1 microM) cells. ATRA exerted no differential action in HL-60RAr cells, but when used in combination with CAL-A, the differential activity was partly resumed at functional and phenotypic levels without change in morphology. The phosphatase activity in the cytosol from HL-60RAr cells was 50% of that from parental HL-60 cells, but the enzyme activities in either membrane or nuclear fractions showed similar values. The decreased phosphatase activity in the cytosol of HL-60RAr cells was mainly due to the decreased expression of the PP2A catalytic subunit. This low level of PP2A protein was reflected at a relative deficiency in expression of the PP2A beta gene in HL-60RAr cells. The exposure to 1 microM ATRA resulted in downregulation of PP2A catalytic subunit protein in HL-60 cells, but ATRA did not affect PP2A expression in HL60RAr cells. Both cell lines expressed the proteins of each PP1 catalytic subunit isozyme (i.e., PP1 alpha, PP1 gamma, and PP1 delta) at comparable levels. ATRA treatment had no effect on the levels of PP1 isozymes. Our results show a correlation between the extent of PP2A expression and the response of HL-60 and HL-60RAr cells to the differentiative effects of ATRA.

Blotting, Northern↗
PubMed1995

A cytosolic endothelin converting activity in guinea-pig lung: purification of a novel metalloprotease.

A proteinous fraction that produces endothelin-1 (ET-1) from big ET-1 in guinea-pig lung cytosol is described. An active protein has been successfully purified to homogeneity by combinations of sequential column chromatographies. The purified enzyme was a metalloenzyme based upon its sensitivity to chelating agent, and a molecular mass of the enzyme was 38 kDa estimated by gel filtration and SDS-PAGE. Further investigations revealed that the enzyme activity was abolished by sulfhydryl modifier such as N-ethylmaleimide, but inhibited neither by phosphoramidon, by thiorphan nor by captopril. The enzyme actually produced ET-1 with the Km value of 14.5 microM for big ET-1. These results indicate that this enzyme seems to be a novel metalloprotease that converts big ET-1 to ET-1.

Animals↗
PubMed1995

[Pulmonary function abnormalities and respiratory manifestations in Sjögren's syndrome].

The respiratory system is the target organ of many autoimmune collagen diseases. In patients with Sjögren's syndrome (SS), the respiratory disorders are also common manifestations. Tracheal and pleural manifestations have been described (Strimlan 1976; Constantopoulos 1985). In the older literature (Shearn 1979), pneumonia and pleurisy were often reported but did not seem to constitute any serious problem. The symptoms are frequent and clinically important. They can present in different ways from dry cough secondary to desiccation of tracheobronchial mucosa (xerotrachea) to dyspnea from airway obstruction or hypersensitivity of trachea. The common respiratory manifestation of SS is small airway disease, and the detection of this involvement requires clinical, roentgenological and respiratory functional tests. In particular, the respiratory functional test is useful for the diagnosis of respiratory disorders in patients with SS.

Humans↗
PubMed1995

Isolation and characterization of jararaca GPIb-BP, a snake venom antagonist specific to platelet glycoprotein Ib.

A platelet glycoprotein Ib-binding protein (GPIb-BP) was isolated from the snake venom of Bothrops jararaca. Jararaca GPIb-BP showed a single band with M(r) of 30,000, and two distinct bands with M(r) of 17,000/13,000 under non-reducing and reducing conditions, respectively, on SDS-polyacrylamide gel electrophoresis. Jararaca GPIb-BP itself induced neither platelet aggregation nor serotonin release from platelets, but specifically bound to GPIb (40,629 +/- 2,521 molecules per normal platelet, with Kd 39.1 +/- 2.4 nM at saturation). The purified venom protein completely inhibited ristocetin- or botrocetin-induced von Willebrand factor (vWF) binding, and blocked the bovine vWF binding to GPIb, with IC50 values ranging from 28 to 42 nM, without affecting the platelet aggregation induced by ADP or alpha-thrombin. 125I-jararaca GPIb-BP binding to GPIb was not altered by the presence of human alpha-thrombin. Jararaca GPIb-BP at a final concentration of 104 nM totally abolished vWF-dependent shear-induced platelet aggregation (SIPA) at a high shear stress, but had no effect on SIPA at a low shear stress. Reduced and S-carboxyamido-methylated jararaca GPIb-BP lost its inhibitory activity on SIPA. The NH2-terminal amino acid sequences of the subunits revealed a high degree of homology with those of several Ca(2+)-dependent lectins, especially to those of two functionally opposite venom proteins, botrocetin (a vWF-modulator) and alboaggregin-B (a GPIb-modulator).

Amino Acid Sequence↗
PubMed1995

Endothelin-1 production and endothelin converting enzyme expression by guinea pig airway epithelial cells.

Significant amount of endothelin-1 (ET-1) production and endothelin converting enzyme (ECE) activity have been detected in cultured guinea pig airway epithelial cells. Subsequent screening of the cDNA library constructed from epithelial cells, a complete cDNA of guinea pig ECE and a partial cDNA encoding preproET-1 were newly isolated. The production of ET-1 from epithelial cells was influenced by some vasoactive or inflammatory mediators, especially LPS enhances ET-1 production. Northern blot analysis revealed that mRNA level of ET-1 was increased in the LPS stimulated epithelial cells, but the effect on the ECE mRNA expression was obscure. These data suggest that the ET-1 production in the airway epithelial cells is regulated by the level of ET-1 mRNA expression with a constitutive manner, and the level of ECE, which act at a key biosynthetic step of the conversion of big ET-1 to ET-2, seems not to make a major contribution to the regulation of ET-1 release.

Amino Acid Sequence↗