Biomedical subjects

H Shima

Publications and source records attributed to H Shima.

At least 145 records · Page 8Linked to original sources

PubMed1994

Characterization of the PP2A alpha gene mutation in okadaic acid-resistant variants of CHO-K1 cells.

Okadaic acid (OA)-resistant variants of Chinese hamster ovary cells, clones CHO/OAR6-6 and CHO/OAR2-3, were isolated from a CHO-K1 culture. These variant cells were 17- to 26-fold more resistant to OA than the parental cells. The phosphorylase phosphatase activity of the variant cell extracts was 2- to 4-fold more resistant to OA than that of the parental cells in the presence of inhibitor 2, a specific inhibitor of type 1 protein serine/threonine phosphatase (PP1). Nucleotide sequencing of PP2A alpha (an isotype of PP2A catalytic subunit) cDNA demonstrated that both variants have a T-->G transversion at the first base of codon 269 (805 nt), which results in substitution of glycine for cysteine. We expressed in COS-1 cells a mutant PP2A alpha tagged with the influenza hemagglutinin epitope. The recombinant mutant PP2A alpha protein immunoprecipitated with an anti-influenza hemagglutinin antibody was more resistant than the wild type to OA, their IC50 values being 0.65 nM and 0.15 nM, and their IC80 values being 4.0 nM and 0.45 nM, respectively. The cysteine at residue 269 present only in highly OA-sensitive protein serine/threonine phosphatase catalytic subunit isozymes, PP2A alpha, PP2A beta, and PPX, is suggested to be involved in the binding of OA. CHO/OAR6-6 and CHO/OAR2-3 cells also overexpressed the P-glycoprotein, and the efflux of OA was more rapid. It is suggested that the PP2A alpha mutation in cooperation with a high level of P-glycoprotein makes the CHO-K1 variants highly resistant to OA.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Chinese hamster ovary cells resistant to okadaic acid express a multidrug resistant phenotype.

Two Chinese hamster ovary cell clones resistant to okadaic acid (OA) were isolated. The OA-resistance was associated with resistance to colchicine, Vinca alkaloids and inhibitors of DNA topoisomerase (topo) II. Drug accumulation assays showed that the intracellular levels of OA, vinblastine and vincristine, but not the topo II inhibitor etoposide, were significantly lowered in the OA-resistant mutants than in the parental cells. These results, together with the finding of an increased level of P-glycoprotein (P-gp) in the mutant cells, indicate that the resistances to OA, Vinca alkaloids and colchicine are due to a P-gp-mediated mechanism. Resistance to topo II inhibitors, however, was associated with reduced activity of topo II. Thus, at least two events, overexpression of P-gp and reduction of topo II activity, occurred in a single OA-resistant cell line, contributing to expression of the MDR phenotype.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Expression of the catalytic and regulatory subunits of protein phosphatase type 2A may be differentially modulated during retinoic acid-induced granulocytic differentiation of HL-60 cells.

To elucidate the regulation of protein phosphatases types 1 (PP1) and 2A (PP2A) during all-trans retinoic acid (ATRA)-induced granulocytic differentiation of HL-60 cells, the phosphatase activity, proteins, and gene expressions of PP1 and PP2A were examined. Treatment with 1 microM ATRA caused an 85% decrease in the PP2A activity in extracts from HL-60 cells, while the PP1 activity was constant. This reduction in PP2A activity appeared to parallel phenotypic and functional changes of HL-60 cells induced by ATRA. Western blot analysis showed that the level of PP2A catalytic subunit (PP2A-C) decreased during the course of ATRA-induced differentiation, whereas expressions of A and B (M(r) 55,000) regulatory subunits of PP2A were relatively unaltered. Expressions of PP1 catalytic subunit isozymes (PP1 alpha, PP1 gamma, and PP1 delta) were not significantly affected by ATRA treatment. Northern blot analysis revealed that mRNA levels of PP2A-C beta and A alpha regulatory subunits were decreased following treatment with ATRA, while levels of PP2A-C alpha and B (M(r) 55,000) alpha regulatory subunit transcripts were relatively constant. Selective down regulation of PP2A-C beta preceded the granulocytic maturation induced by ATRA. Expressions of PP2A-C isoforms and A and B regulatory subunits may be differentially modulated during ATRA-induced granulocytic differentiation of HL-60 cells.

Blotting, Northern↗

PubMed1994

A mutant of protein phosphatase-1 that exhibits altered toxin sensitivity.

A chimeric mutant was constructed in which a 4-amino acid region (GEFD, residues 274-277) of rabbit muscle protein phosphatase-1 was replaced with the sequence YRCG corresponding to residues 267-270 of rabbit protein phosphatase-2A. This was based on the findings of a gene mutation in okadaic acid-resistant cells which results in a Cys-->Gly conversion in protein phosphatase-2A. The YRCG mutant of protein phosphatase-1 was expressed and purified. The properties of the mutant enzyme were investigated in terms of its sensitivity toward several toxin inhibitors (okadaic acid, microcystin, nodularin, calyculin A, and cantharidic acid), as well as inhibitor-2. The mutant enzyme exhibited a gain of function in the form of a 10-fold increased sensitivity toward okadaic acid that suggests this region is involved in toxin binding. Significant changes in sensitivity to inhibitor-2 and several of the other toxins were also observed, indicating that these may have a common binding region.

Amino Acid Sequence↗

PubMed1994

A 28 kDa-protein with disintegrin-like structure (jararhagin-C) purified from Bothrops jararaca venom inhibits collagen- and ADP-induced platelet aggregation.

A 28 kDa-protein with inhibitory activity on collagen- and ADP-induced platelet aggregation was purified from the venom of the snake Bothrops jararaca. Its complete amino acid sequence corresponded to the carboxyl-terminal region consisting of disintegrin-like and cysteine-rich domains of jararhagin, a high molecular weight hemorrhagic metalloprotease. Sequence homology of the protein to other disintegrins and disintegrin-like proteins from various snake venoms is also presented.

Adenosine Diphosphate↗

PubMed1994

A regulatory subunit of smooth muscle myosin bound phosphatase.

The relationship between two putative myosin-binding subunits of smooth muscle myosin phosphatase was investigated. A monoclonal antibody (MoAb) to the 58 kD component of smooth muscle myosin-bound phosphatase (MBP) cross-reacted with a 130 kD protein in extracts of fresh chicken gizzards. The MoAb in combination with protein A immunoprecipitated from gizzard extracts a complex of the 130 kD protein plus the 38 kD catalytic subunit of the type 1 delta protein phosphatase. It is proposed that the 130 kD component is a native subunit of MBP and that the 58 kD protein is its proteolytic degradation product. The distribution of the 130 kD component in chicken tissues was screened using the MoAb. An immunoreactive band of appropriate mass was detected in all tissues except liver and skeletal muscle. Higher concentrations of the 130 kD component were evident in the smooth muscle samples.

Animals↗

PubMed1994

PP1 gamma 2, a testis-specific protein-serine/threonine-phosphatase type 1 catalytic subunit, is associated with a protein having high sequence homology with the 78-kDa glucose-regulated protein, a member of the 70-kDa heat shock protein family.

Protein phosphatase 1 gamma 2 (PP1 gamma 2) is a testis-specific isotype of the protein-serine/threonine-phosphatase type 1 catalytic subunit. Three native forms of PP1 gamma 2 were detected in a crude fraction of rat testis by electrophoresis in a nondenaturing polyacrylamide gel. We purified a major native form of PP1 gamma 2 to homogeneity by successive column chromatography on Mono Q-Sepharose, EAH-agarose, protamine-agarose, and G3000SW and by electrophoresis in a nondenaturing polyacrylamide gel. The G3000SW-purified PP1 gamma 2 native form had an apparent molecular mass of 170 kDa. The purified holoenzyme from nondenaturing polyacrylamide gel was composed of the catalytic subunit and two noncatalytic subunits, of 78 kDa and 55 kDa. Partial amino acid sequence analysis of the 78-kDa protein suggested that it is the 78-kDa glucose-regulated protein, a member of the 70-kDa heat shock protein family. The 78-kDa protein may possibly function as a chaperone or by confining substrate specificity of PP1 gamma 2.

Amino Acid Sequence↗

PubMed1994

Localization of mRNA for protein phosphatase 2A in the brain of adult rats.

The gene expression for alpha and beta isoforms of type 2A protein phosphatase (PP2A-alpha and -beta) in the adult rat brain was examined by in situ hybridization analysis. No marked difference in the gene expression was discerned between the two isoforms in large portions of brain, except for the thalami in which the expression level for the alpha isoform was similar to that in the cerebral neocortex whereas that for the beta was lower than that in the neocortex. The gene expression was observed intensely in the piriform cortex, the cerebellar Purkinje and granule cell layers, and the hippocampal pyramidal and dentate granule cell layers, and the locus ceruleus, whereas the moderate levels of its expression were observed in the olfactory mitral cells and the pontine nuclei. The cerebral neocortex expressed the mRNA moderately to weakly without any laminar patterns, whereas the expression level in the caudate-putamen was very low. This expression pattern is basically similar to that of PP2C reported previously, except for the plexus choroideus and ependyma having no significant expression for PP2A.

Animals↗

PubMed1994

Calyculin A and okadiac acid inhibit human platelet aggregation by blocking protein phosphatases types 1 and 2A.

Two potent inhibitors of protein phosphatase type 1 (PP1) and type 2A (PP2A), calyculin A (CAL-A) and okadaic acid (OKA), inhibited human platelet aggregation induced by thrombin, collagen and 9,11-epithio-11,12-methano-thromboxane A2 (STA2). IC50 values of CAL-A and OKA for STA2-induced aggregation were 53 nM and 3.5 microM, respectively. These drugs also inhibited thrombin-induced [14C]serotonin secretion of platelets. CAL-A and OKA elicited phosphorylation of certain proteins with an apparent M(r) (x 10(-3) of 200, 60, 50 and 20 light chain of myosin (MLC). Agonist-induced 47,000 M(r) protein phosphorylation was strongly inhibited by these compounds, whereas phosphorylation of 20,000 M(r) MLC was enhanced. The increase in 50,000 M(r) protein phosphorylation by CAL-A and OKA was observed in the presence of agonists, and the 50,000 M(r) phosphorylation may be involved in the inhibition of platelet activation by these compounds. Subcellular analysis of the phosphatase activity in human platelets showed that MLC phosphatase activity was present mainly (approx. 78%) in the cytosolic fraction. Chromatography of human platelet extract on heparin-Sepharose resolved two peaks of MLC phosphatase activity: PP2A in 0.1 M NaCl eluate and PP1 in 0.5 NaCl eluate. PP2A and PP1 isozymes (PP1 alpha, PP1 gamma and PP1 delta) have also been identified in human platelets, by cross-reactivity with polyclonal antibodies against PP2A and PP1 isozymes, respectively. These results suggest that PP1 and/or PP2A may play an important role in the process of platelet activation by regulating levels of phosphorylation of certain proteins.

Blood Platelets↗

PubMed1994

5 alpha-reductase activity in developing urogenital tracts of fetal and neonatal male mice.

5 alpha-Reductase activity in testes and urogenital tract tissues from male mice at 14.5 days gestation and 0 days of age (birth) was assessed by measurement of metabolites of testosterone released into the medium in a serum-free culture system. In male mice at 14.5 days gestation, the testis and the cranial portion of the urogenital ridge (UGR), which develops into the epididymis, had little 5 alpha-reductase activity. The caudal portion of the UGR, which develops into the seminal vesicle (SV), and the cranial portion of the urogenital sinus (UGS) had 12-fold higher activity than the cranial portion of the UGR. 5 alpha-Reductase activity in the middle portion of UGR was intermediate between that of its cranial and caudal portions. The highest levels of 5 alpha-reductase activity were detected in the caudal portion of UGS, even though this higher value was not significantly different from those in the caudal UGR or the cranial UGS. In 0-day-old male mice, 5 alpha-reductase activity of testis remained low at a level similar to that observed at 14.5 days gestation. The epididymis also exhibited low 5 alpha-reductase activity in 0-day-old mice, even though this activity had increased 2.2-fold relative to that observed in the cranial UGR at 14.5 days gestation. 5 alpha-Reductase activity of the cranial portion of UGS (prostatic rudiment) was relatively high on day 0 and similar to that in the same region at 14.5 days gestation. The SV and bulbourethral gland (BUG) on day 0 had high 5 alpha-reductase activity, which had increased 1.4- and 1.3-fold, respectively, compared to their 14.5 day embryonic anlage, although the increase was not significant. Through examination of 5 alpha-reductase activity regionally in the developing urogenital tract, it is evident that the concept that testosterone is the active androgen during Wolffian development is overly simplified. The present study suggests the importance of spatial/regional factors in Wolffian duct development in regard to the role of dihydrotestosterone. 5 alpha-reductase activity in the caudal Wolffian duct (SV rudiment) equals or exceeds that in the developing prostate, and at birth, dihydrotestosterone production is virtually identical in the SV and prostate. This suggests that 5 alpha-reduced androgens play a prominent role in the development of SV.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1994

A case of orthostatic and postexertional hypotension with ischemic electrocardiographic changes.

A case of orthostatic and postexertional hypotension with ischemic electrocardiographic changes is reported. The etiology was considered to be the partial dysfunction of efferent sympathetic nerve endings. Peripheral adrenergic receptors (alpha and beta) were up-regulated, which might have caused pseudoischemic electrocardiographic changes and abnormal vasodilation after exercise.

Autonomic Nervous System Diseases↗

PubMed1994

A male case of synchronous double cancers of the breast and prostate.

A male case of synchronous double cancers of the breast and prostate is reported. An 84-year-old male was admitted to the hospital complaining of general malaise, anorexia and weight loss. A tumor 3 cm in diameter was noted in his left breast, which was removed by mastectomy, and was diagnosed as papillotubular carcinoma. An induration of his prostate and elevated prostate specific antigen, gamma-seminoprotein and prostatic acid phosphatase levels were also noted. Needle biopsy of his prostate revealed adenocarcinoma. Among the previous 18 reported cases of this combination of cancers, those with no prior estrogen therapy were very rare, the present case being the third ever reported.

Adenocarcinoma↗

PubMed1994

[Adrenal hepatoid carcinoma producing alpha-fetoprotein: a case report].

A case of left adrenal hepatoid carcinoma is reported. The patient was a 57-year-old male and his chief complaint was general fatigue. Preoperative laboratory data showed markedly increased levels of alpha-fetoprotein (AFP) (30,500 ng/ml) and PIVKA-II (3.01 AU/ml). Both computed tomography (CT) and magnetic resonance computed tomography (MRCT) showed left adrenal tumor (8 x 5 cm). Angiography showed hypervascular tumor over the upper pole of the left kidney. Thoracoabdominal nephro-adrenalectomy was performed. Histological examination demonstrated hepatoid carcinoma of the left adrenal gland. AFP was positive in the tumor cells. The levels of both AFP and PIVKA-II dropped to the normal range postoperatively. Hepatoid carcinoma in the urological field is very rare. As hepatoid carcinoma in the Japanese literature in the urological field, there were only 4 cases of renal tumor, 2 cases of renal pelvic tumor, 2 cases of retroperitoneal space and 1 case of urachal tumor. This is the first report of adrenal hepatoid carcinoma in Japan.

Adenocarcinoma↗

PubMed1994

Endothelin converting enzymes in guinea-pig lung membrane fractions: purifications and characterizations.

We have identified neutral proteolytic activities that convert big endothelin-1 (big ET-1) to ET-1 in guinea-pig lung membrane fraction. The active proteins have been solubilized and two distinct enzymes have been purified by combinations of sequential column chromatographies. One purified enzyme was a metalloenzyme based upon its sensitivity to chelating agent with a molecular mass of 108 kDa on SDS-PAGE. Another enzyme was also a metalloenzyme with a molecular mass of 162 kDa. Further investigations revealed that the 108 kDa enzyme was inhibited by phosphoramidon and also by thiorphan, and produced ET-1 with the Km value of 5.7 microM for big ET-1. The 162 kDa enzyme was also inhibited by phosphoramidon, but neither by thiorphan nor by captopril, and quantitatively produced ET-1 from big ET-1 with a Km value of 2.1 microM. These results indicate that the 108 kDa enzyme probably seems to be a neutral-endopeptidase (EC 3. 4. 24. 11.) or similar one, but the 162 kDa enzyme is a unique metalloprotease that converts big ET-1 to ET-1.

Animals↗

PubMed1993

Serum tissue inhibitor of metalloproteinases in patients with chronic liver disease and with hepatocellular carcinoma.

To examine the clinical significance of serum level of tissue inhibitor of metalloproteinases (TIMP) in chronic liver disease and in hepatocellular carcinoma, we measured serum TIMP concentration by a sandwich enzyme immunoassay in 79 patients with chronic liver disease and 49 patients with hepatocellular carcinoma. Serum TIMP concentration was 164 +/- 20 ng/ml in healthy controls, and was 10% higher than control in chronic persistent hepatitis, 36% higher in chronic active hepatitis, 62% higher in liver cirrhosis and 30% higher in primary biliary cirrhosis. Serum TIMP level was closely correlated with serum level of type IV collagen 75 domain and with the histological degree of liver fibrosis in chronic liver disease. Serum TIMP level in hepatocellular carcinoma was increased 2.3-fold compared with that in controls, and was significantly higher than in liver cirrhosis. Serum TIMP level increased with tumor size, and significantly correlated with serum alpha-fetoprotein level. Gel filtration on Sephadex G-75 showed that the TIMP in serum was present as an enzyme-complexed form. These results suggest that the measurement of serum TIMP concentration is useful in the clinical assessment of liver fibrosis in chronic liver disease and of the development of hepatocellular carcinoma.

Adult↗

PubMed1993

Protein phosphatase 1 gamma 2 is associated with nuclei of meiotic cells in rat testis.

We have shown that there are at least 4 isotypes of the catalytic subunit of type 1 protein phosphatase (PP1), PP1 alpha, PP1 gamma 1, PP1 gamma 2 and PP1 delta by rat cDNA cloning. Among these, PP1 gamma 2 mRNA was found to be abundant in rat testis. To study the roles of PP1 gamma 2 in rat spermatogenesis, we raised an antibody against a synthetic 21 mer peptide corresponding to the predicted amino acid sequence of the C-terminal region of the PP1 gamma 2. The antibody interacted with a testicular protein of 39-kDa prepared from rats after puberty (25 days after birth). However, the antibody did not interact with proteins extracted from the immature testes or from other tissues examined. Indirect immunofluorescent staining of PP1 gamma 2 of the seminiferous tubules from rats of various ages and costaining with DNA showed that the protein is located in the nuclei of late spermatocytes and of early spermatids. We suggest that PP1 gamma 2 plays some role in meiotic division during spermatogenesis.

Amino Acid Sequence↗

PubMed1993

Expression of PP2A B regulatory subunit beta isotype in rat testis.

We isolated a rat cDNA encoding part of the beta-isotype of the B regulatory subunit (BR beta) of protein phosphatase 2A (PP2A). The isolated cDNA encoded the region corresponding to amino acids positions 8(R) to 177(N) of human BR beta. The identities of the nucleotide and amino acid sequences of the rat and human BR beta s were 95.7% and 100%, respectively. The BR beta mRNA was specifically expressed in rat brain and testis, the lengths of mRNAs in these two organs being different. In the testis, the BR beta mRNA was first detected 40 days after birth, increasing gradually thereafter, and was expressed specifically in elongated spermatids, while mRNA of the alpha-isotype (BR alpha) was expressed equally in all spermatogenic cells. After meiosis, round spermatids change morphologically to elongated spermatids. BR beta may regulate the activity of the PP2A catalytic subunit in spermatids, and be involved in spermatogenic maturation, especially spermatid elongation.

Aging↗

PubMed1993

Identification of PP1 catalytic subunit isotypes PP1 gamma 1, PP1 delta and PP1 alpha in various rat tissues.

cDNA clones for 4 rat protein phosphatase 1 (PP1) catalytic subunit isotypes, PP1 alpha, PP1 gamma 1, PP1 gamma 2 and PP1 delta, were isolated (K. Sasaki et al., Jpn. J. Cancer Res. 81, 1272-1280, 1990). Antibodies were raised to determine the physiological functions of 4 isotypes. Among these isotypes, the PP1 gamma 2 protein was detected specifically in rat testis (H. Shima et al., Adv. Prot. Phosphatases, vol. 7, in press). On the other hand, PP1 gamma 1, PP1 delta and PP1 alpha were detected as 36kDa, 37kDa and 38kDa proteins in several rat organs by immunoblot analysis. All three isotypes were found in both soluble and particulate fractions. PP1 gamma 1 was predominant in the brain and PP1 delta in the brain, lung and small intestine, but the levels of both were low in the liver and muscle. It became evident that PP1 alpha was not a predominant isotype in any tissue examined.

Amino Acid Sequence↗