Biomedical subjects

S Ohno

Publications and source records attributed to S Ohno.

At least 19 recordsLinked to original sources

PubMed1992

Localization and developmental expression of a novel protein kinase C delta gene.

The expression and localization of a novel protein kinase C delta (nPKC delta) mRNA were investigated using Northern blotting and in situ hybridization in the developmental process of mouse brain. In adult mice, nPKC delta was abundantly expressed in the thalamus, moderately in the pons and the cerebellum, but faintly in the cerebral cortex and the spinal cord. By in situ hybridization, the signals were observed specifically at the sensory and motor relay nuclei of the thalamus, the dorsal cochlear nuclei of the pons, and the molecular layer of the cerebellum. When developmental changes in the expression of nPKC delta gene were analyzed by in situ hybridization, it was not detectable in embryonic and neonatal brains, very weakly expressed in the thalamus in the first week, and highly expressed at two weeks of age. These results suggest that the gene expression of nPKC delta is strictly controlled by both the cell type and the developmental process.

Animals↗

PubMed1992

Estrogen modulates Ca(2+)-independent lipid-stimulated kinase in the rabbit corpus luteum of pseudopregnancy. Identification of luteal estrogen-modulated lipid-stimulated kinase as protein kinase C delta.

Rabbit corpora lutea were tested for the presence of phosphorylative responses sensitive to estrogen. Luteal Ca(2+)-independent lipid-stimulated kinase activity was detected by phosphorylation of the endogenous substrate, p76. Estrogen treatment, by way of estradiol-17 beta implant, increased levels of the lipid-stimulated phosphoprotein 2-3-fold throughout pseudopregnancy. Midpseudopregnant rabbit luteal extracts were further evaluated to determine the identity of the lipid-stimulated kinase. Results of low pH-activated phosphorylation were consistent with the identification of p76 as an autophosphorylated member of the protein kinase C (PKC) family. Partial purification of the luteal lipid-stimulated kinase was performed using sequential DEAE-cellulose/hydroxylapatite chromatographies and using gel filtration. Western immunoblot with type-specific anti-PKC delta antiserum showed coelution of kinase p76 activity with immunoreactive PKC delta. Immunoblot analysis confirmed that luteal levels of PKC delta were increased by estrogen treatment.

Animals↗

PubMed1992

Increased therapeutic efficacy induced by tumor necrosis factor alpha combined with platinum complexes and whole-body hyperthermia in rats.

This study examined the effect of a trimodality therapy of the combination of recombinant human tumor necrosis factor alpha (TNF), whole-body hypertheria (WBH), and cis-diamminedichloroplatinum(II) (CDDP) or cis-diammine-1,1-cyclobutane dicarboxylate platinum(II) (CBDCA) on a fibrosarcoma and normal tissue in F344 rats. TNF (1 x 10(5) units/kg) increased the antitumor effect of both CDDP (1.5 mg/kg) + WBH (2 h at 41.5 degrees C) and CBDCA (30 mg/kg) + WBH. Tumor growth delay, which was 1.9 days for CDDP + WBH and 2.7 days for CBDCA + WBH (P less than 0.01 compared to control), was significantly increased to 2.9 days with TNF + CDDP + WBH and 5.4 days with TNF + CBDCA + WBH (P less than 0.05). WBH, TNF, CDDP or CBDCA alone, TNF + CDDP, TNF + CBDCA, or TNF + WBH had no significant effect on tumor growth. In contrast, administration of TNF did not enhance the CDDP- or CBDCA-mediated dose limiting normal tissue toxicity. CDDP + WBH-mediated acute renal injury and CBDCA + WBH-mediated acute myelosuppression, as determined by blood urea nitrogen and peripheral blood cell counts, respectively, were not increased with the addition of TNF to either dual modality therapy. Histopathologically, addition of TNF produced no significant alterations in the kidney and the bone marrow as compared to CDDP + WBH or CBDCA + WBH. These data show that TNF enhanced the platinum + WBH-mediated antitumor effect without increasing normal tissue toxicity, suggesting that TNF may increase the therapeutic efficacy of CDDP or CBDCA combined with WBH.

Animals↗

PubMed1992

Pig testicular 20 beta-hydroxysteroid dehydrogenase exhibits carbonyl reductase-like structure and activity. cDNA cloning of pig testicular 20 beta-hydroxysteroid dehydrogenase.

cDNA inserts encoding 20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53) were, for the first time, isolated and cloned from a pig testis cDNA library using synthetic oligonucleotides deduced from the partially determined amino acid sequences. The cDNA contains an open reading frame predicted to encode 289 amino acid residues. Surprisingly, it has 85% amino acid homology to human carbonyl reductase. The purified enzyme exhibited carbonyl reductase activity. Adenine-rich sequence was located in the 3'-untranslated nine-rich sequence was located in the 3'-untranslated region, which may mean that the gene originates by retroposition. RNA transcripts of 1.3, 3, and 6 kilobases were detected in poly(A)+ RNA extracted from pig testis by Northern blot hybridization. The steady-state level of the RNA species increased to a maximum in testes from 10-day-old pigs, but rapidly declined thereafter to the same levels found in testes of mature animals.

Alcohol Oxidoreductases↗

PubMed1992

Increased expression of protein kinase C alpha plays a key role in retinoic acid-induced melanoma differentiation.

Differentiation of B16 mouse melanoma cells induced by retinoic acid (RA) is preceded by a large increase in protein kinase C alpha (PKC alpha) mRNA and protein. To determine the role of PKC alpha in the differentiation program, we stably transfected B16-F1 cells with a plasmid containing the full length PKC alpha cDNA driven by an SV40 promoter. Two out of thirty-two colonies screened were determined to overexpress PKC by 2-4-fold according to Western blot analysis and PKC enzyme activity. When compared to control cells (wild-type cells and cells transfected only with the neomycin resistance gene), PKC alpha overexpressing clones displayed longer doubling times, diminished anchorage-independent growth, and increased melanin production. RA treatment of control cells mimicked these phenotypic characteristics. When injected subcutaneously into syngeneic mice, PKC alpha overexpressing clones produced smaller tumors and had longer latencies than control cells. These findings, combined with the fact that phorbol esters down-regulate PKC and antagonize RA action suggest that PKC alpha plays a key role in the RA-induced melanoma differentiation.

Animals↗

PubMed1992

Transcriptional activation of the gene for the large subunit of human m-calpain by 12-o-tetradecanoyl-phorbol-13-acetate.

The effect of the treatment of HeLa cells with a tumor-promoting phorbol ester, 12-o-tetradecanoyl-phorbol-13-acetate (TPA) on the expression of the genes for the calpain family has been examined. Among the mRNAs for the calpain family, only the mRNA for the large subunit of human m-calpain (calpain mL) was specifically induced by treatment of cells with TPA, suggesting its specific function in response to cellular stimuli. The effect of TPA on the expression of the calpain mL gene was further examined using fusion genes containing the promoter/enhancer region of the calpain mL gene fused upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene, showing that the promoter/enhancer sequence of the calpain mL gene contains a cis-acting element which responds to TPA and activates transcription of the downstream sequence.

Calcium-Binding Proteins↗

PubMed1992

How cytotoxic T cells manage to discriminate nonself from self at the nonapeptide level.

Class I major histocompatibility complex (MHC) antigens are confronted with an apparently insurmountable dilemma. Each should show a binding preference to a common enough variety of nonapeptides, so that one relevant nonapeptide can be found in at least every other viral protein to provoke a cytotoxic T-cell response. By so doing, however, the chance of that viral T epitope being self is greatly increased. Examination of human and viral nonapeptides preferred by HLA-B27 led to the following conclusions. (i) In normal cells, peptide fragments originating from 5000 or more diverse proteins vie for a finite number of class I MHC sites. Consequently, only those nonapeptides having the optimal binding affinity to a given class I MHC antigen can gain access to the plasma membrane. (ii) Tolerance is rendered only to those host nonapeptides with the optimal binding affinity. (iii) Because of the above noted tolerance, viral nonapeptides with the optimal binding affinity are invariably ignored. (iv) Viral T epitopes actually chosen are always second-echelon nonapeptides that are endowed with slightly less than the optimal binding affinity to a given class I MHC antigen. (v) Since such second-echelon nonapeptides would not gain access to the plasma membrane in normal cells, the issue of self or nonself is rendered irrelevant by this choice. (vi) Since viral T epitopes are of this type, cytotoxic T-cell responses against infected cells are expected to be effective only when a few viral proteins are made in large amounts at the expense of host proteins.

Amino Acid Sequence↗

PubMed1992

Positive and negative regulation of the transcription of the human protein kinase C beta gene.

To analyze the mechanism of the cell type-specific expression of protein kinase C beta (PKC beta), we isolated the 5'-portion of the human gene for PKC beta and identified multiple positive and negative regulatory sequences that regulate its transcription. S1 nuclease mapping as well as primer extension analysis of the 5'-end of the PKC beta mRNA identified a putative transcriptional initiation site (position +1) 484 base pairs (bp) upstream of the first ATG codon. The 5'-upstream sequence contains a CCAAT sequence at position -110, but no TATA box. The transcriptional activities of various 5'-deletion mutants of the PKC beta gene upstream region, fused to the chloramphenicol acetyltransferase structural gene, were examined in terms of chloramphenicol acetyltransferase expression after transfection into three kinds of rodent cell lines: P19 and GH4C1, which are positive for the expression of PKC beta mRNA; and 3Y1, which is negative. Mutants containing a 5'-flanking sequence longer than 1.9 kilobases (kb) showed chloramphenicol acetyltransferase activities of the same order as the expression of the endogenous gene. This indicates that this region contains sequences regulating the cell-type specificity of PKC beta gene expression and that the specificity is determined at least partially at the level of transcription. The 1.9-kb sequence contains at least three positive elements: P1 (-56 to -234 bp), P2 (-234 to -411 bp), and PN (-1.4 to -1.9 kb). PN is active only in P19 cells, P1 in GH4C1 and P19 cells, and P2 in all three cell lines. In addition to these positive elements, there are negative elements: N1 (-411 to -674 bp), which is active in all three cell lines; and PN, which is active only in GH4C1 cells. These results suggest the presence of multiple trans-acting factors that act on these positive and negative cis-acting elements and regulate the cell type-specific expression of the PKC beta gene.

Amino Acid Sequence↗

PubMed1992

Purification and characterization of protein kinase C epsilon from rabbit brain.

Protein kinase C epsilon was chromatographically purified from rabbit brain to electrophoretic homogeneity. We identified the enzyme as the epsilon species of novel-type protein kinase C (nPKC epsilon), originally discovered and defined by cDNA cloning [Ohno, S., et al. (1988) Cell 53, 731-741], on the basis of the following observations: (i) the enzyme reacts specifically with an antipeptidic antiserum to nPKC epsilon but not with antisera to any of the other molecular species of PKC thus far known; (ii) it exhibits enzymatic behavior essentially identical to that of a recombinant nPKC epsilon purified from transfected COS cells [Konno, Y., et al. (1989) J. Biochem. 106, 673-678] and distinct from that of conventional PKC (alpha, beta I/II, and gamma) in its dependence on magnesium concentration and cofactors such as phospholipids, calcium, and phorbol ester; and (iii) it has an apparent molecular weight of 95.7K +/- 0.4K on SDS-PAGE, significantly greater than the other conventional and novel PKCs thus far identified. Notably, calcium exhibits a complex effect, both positive and negative, on the kinase activity of epsilon depending on the kind of substrate and the coexisting phospholipid, calling for a modification of the current notion that epsilon is a kinase unresponsive to calcium. The amount of epsilon species in the brain was estimated to be comparable to that of each conventional species, indicating that epsilon stands as one of the major PKC family members in brain. Furthermore, the enzyme shows a broader substrate spectrum than conventional PKC when examined with endogenous substrates, implying that it may cover a wider or different range of physiological functions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Generation of plasmacytomas with the chromosomal translocation t(12;15) in interleukin 6 transgenic mice.

The mechanisms through which pristane or mineral oil can induce plasmacytomas in BALB/c or NZB mice are not fully understood, but involvement of interleukin 6 (IL-6), a growth factor for plasmacytomas and myelomas, has been strongly suggested. To clarify the role of IL-6 in plasmacytomagenesis, a human IL-6 cDNA was introduced into mouse germ lines under the transcriptional control of the murine major histocompatibility complex class I (H-2Ld) promoter. IL-6 transgenic mice of C57BL/6 origin developed a massive plasmacytosis but not plasmacytomas. However, introduction of BALB/c genetic background into IL-6 transgenic mice could generate monoclonal transplantable plasmacytomas with the chromosomal translocation t(12;15). These results provide firm evidence of the critical role of IL-6 in the plasmacytoma development.

Animals↗

PubMed1992

"Self" to cytotoxic T cells has to be 1,000 or less high affinity nonapeptides per MHC antigen.

In order to serve as the effective target of a relevant cytotoxic T-cell receptor, the same peptide fragment has to occupy at least 0.1% of the class I major histocompatibility complex (MHC) antigen sites on the plasma membrane. Because of this need, I content that the thymic educator cell of "self" to cytotoxic T cells can suppress autoreactive T-cell clones only with regard to at the most, 1,000 self nonapeptides per a given allelic form of class I MHC antigens; e.g., HLA-A2. Each allelic form of class I MHC antigen apparently developed the preferential binding affinity toward a specific set of nonapeptides. The requirement for preferential binding can either be permissive or stringent. In the case of human HLA-A2, those nonapeptides having either Leu or Met at the second position and mainly Val, but occasionally Leu at the ninth position are preferred. Since both Leu and Val are very common residues, the typical somatic cell type readily supplies nearly 3000 high affinity host nonapeptides preferred by HLA-A2. Of those, the tolerance can be induced, at the most, to only 1,000 nonapeptides. In view of this, permissive class I MHC antigens such as HLA-A2 carefully avoid high affinity nonapeptides in viral proteins, for their status as to self or nonself is uncertain, and they choose second choice nonapeptides as T epitopes. In sharp contrast to human HLA-A2, mouse H-2Db represents the stringent class I MHC antigens. In order to show the high binding affinity toward H-2Db, nonapeptides are required to carry Asn at position 5 and Met or Ile at the equally critical position 9. Inasmuch as Asn and Met are rare residues and Ile, too, is not a common residue, the typical somatic cell type can supply only several hundred host nonapeptides having the high binding affinity toward H-2Db. Under the circumstance, there is no problem in memorizing the selfness of all of them. Accordingly, T epitopes are almost invariably chosen from the high affinity nonapeptides that are present in their viral proteins.

Amino Acid Sequence↗

PubMed1992

Of palindromes and peptides.

On the average, 30% of the residues in a protein are members of peptidic palindromes, tripeptidic and longer. This percentage may go up to 50% in histones and certain other DNA binding proteins. The longest peptidic palindrome encountered thus far was 14 residues in length. However, there is every reason to expect even longer peptidic palindromes in other proteins not yet analyzed.

Amino Acid Sequence↗

PubMed1992

Immunocytochemical study of dystrophin in cultured mouse muscle cells by the quick-freezing and deep-etching method.

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, is deficient in patients with DMD and in mdx mice. It is immunocytochemically localized in skeletal muscle sarcolemma. However, little is known about the three-dimensional ultrastructural localization of dystrophin and its relationship with other cytoskeletal proteins. We found that dystrophin is localized irregularly, just underneath the plasma membrane in normal cultured mouse myotubes, by using the quick-freezing and deep-etching (QF-DE) method; it was found to be closely linked to actin-like filaments (8-10 nm in diameter), most of which were decorated with myosin subfragment 1, and was attached to the cytoplasmic side of the plasma membrane. These results suggest that dystrophin might play an important role in the preservation of cell membrane stability by connecting actin cytoskeletons with the cytoplasmic side of the plasma membrane.

Animals↗

PubMed1992

Localization of laminin in nephritic glomeruli as revealed by a quick-freezing and deep-etching method with immunohistochemistry.

The three-dimensional localization of laminin in rat glomeruli at the chronic phase of Masugi nephritis was investigated by a quick-freezing and deep-etching method combined with immunohistochemistry. Light-microscopically, laminin was localized in increased mesangial matrix and thickened glomerular basement membrane. The quick-freezing and deep-etching method revealed that the increased mesangial matrix, which was newly formed in axial portions and areas of mesangial interposition, was composed of fine fibrillar networks. They were revealed with the 3,3'-diaminobenzidine tetrahydrochloride (DAB) reaction products of peroxidase-labelled secondary antibody following anti-laminin antibody. However, these reaction products were not uniformly distributed in the newly formed matrix. Although the fibrils organizing lamina densa were also immunostained with anti-laminin antibody, the fibrils connected to mesangial cells, podocytes and endothelial cells had smaller amounts of DAB reaction products for laminin. These results indicate that one of the components of fibrils in the mesangial matrix and lamina densa is laminin, which is heterogeneously distributed in the newly formed matrix.

3,3'-Diaminobenzidine↗

PubMed1992

Congenital absence of the portal vein.

Absence of the terminal portion of the portal vein and its intrahepatic branches was demonstrated in a 22-year-old woman with focal nodular hyperplasia (FNH) of the liver. Various imaging studies and angiography showed that the portal vein entered into the inferior vena cava just above the renal veins. The clinical and radiological features of this patient and nine previously reported cases of this entity are herein reviewed.

Adult↗

PubMed1992

Ultrastructural study of the glomerular slit diaphragm in fresh unfixed kidneys by a quick-freezing method.

In normal kidneys fixed by perfusion with tannic acid and glutaraldehyde, glomerular slit diaphragms have been reported to consist of highly ordered and isoporous substructures with a zipper-like configuration. We have re-evaluated the ultrastructure of fixed or unfixed glomeruli using quick-freezing and deep-etching (QF-DE) and freeze-substitution (QF-FS) methods. In the fixed slit diaphragms, zipper-like substructures were often observed by the QF-DE method. In contrast, in fresh unfixed glomeruli the slit diaphragms mainly consisted of non-porous substructures. The slit diaphragms were more widely opened in the fixed glomeruli, as examined by the QF-FS method. These results suggest that the foot processes shrink during tissue preparation by conventional methods with chemical fixatives, and that the broadening of slit diaphragms and zipper-like substructures are formed by the pulling apart of adjacent foot processes due to shrinkage.

Animals↗

PubMed1992

Three-dimensional study of epithelioid cells by a quick-freezing and deep-etching method in muramyl dipeptide-induced granulomas.

The three-dimensional ultrastructure of epithelioid cells was studied by the quick-freezing and deep-etching (QF-DE), as well as the freeze-substitution (QF-FS) methods. The granulomas were induced in rats by injecting muramyl dipeptide (MDP) into the hind footpads. At 3 weeks after the injection, the footpads were perfused with a fixative, excised, and quickly frozen to prepare the replica membranes. Some unfixed footpads were also quickly frozen and freeze-substituted. Dense networks of intermediate filaments, connected with the nuclei, mitochondria and other vesicular cell organelles, were observed throughout the cytoplasm of epithelioid cells by the QF-DE method. A few actin filaments were located in filopodia and just beneath the cell membranes. Interdigitation of the cell membranes between adjacent cells was clearly demonstrated by the QF-FS method and clathrin-coated pits were identified at the base of interdigitating filopodia. In addition, the exact moment of fusion between endosomes and lysosomes was ascertained by the same method. These results suggest that the cytoskeletal organization of epithelioid cells resembles that of epithelial cells rather than actively motile macrophages.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1992

Ontogeny of testicular steroid dehydrogenase enzymes in pig (3 alpha/beta-, 20 alpha- and 20 beta-): evidence for two forms of 3 alpha/beta-hydroxysteroid dehydrogenase.

Three enzymatic activities (3 alpha/beta-hydroxysteroid dehydrogenase, 20 beta- and 20 alpha-hydroxysteroid dehydrogenases) were measured in testes of pigs as a function of age. Earlier studies reported a highly purified 20 beta-hydroxysteroid dehydrogenase from neonatal pig testes that also showed strong 3 alpha/beta-hydroxysteroid dehydrogenase activity [Ohno et al., J. Steroid Biochem. Molec. Biol. 38 (1991) 787-794]. We report here that neonatal pigs testis is rich in 3 alpha/beta- and 20 beta-hydroxysteroid dehydrogenase activities, both of which fall to low levels (measured as specific activity) at 60 days. Thereafter the activity of 3 alpha/beta-reduction rises to high levels whereas 20 beta-reduction remains low. Activity of 20 alpha-reduction is of intermediate level in the neonate, falls to a nadir at 60 days and rises to high levels in the mature animal. Western blots of cytosolic proteins show that the bifunctional enzyme (3 alpha/beta-plus 20 beta-hydroxysteroid dehydrogenase) is high in neonatal testes and falls to low levels at maturity. It is proposed that the neonatal testis possesses the bifunctional enzyme which is replaced by a second enzyme at maturity, that is a 3 alpha/beta-hydroxysteroid dehydrogenase without 20 beta-reductase activity. The possible functional significance of these changes is considered.

20-Hydroxysteroid Dehydrogenases↗