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S Ohno

Publications and source records attributed to S Ohno.

At least 577 records · Page 32Linked to original sources
PubMed1993

Evidence for a new, high-molecular weight isoform of protein kinase C in rat hippocampus.

We describe a new form of protein kinase C (PKC) with a molecular weight of 97 kDa, higher than the known forms of vertebrate PKC. This putative new high-molecular weight isoform, which we are calling PKC (HMW), is increased in the membrane fraction either upon application of phorbol esters or with afferent synaptic stimulation of Schaffer collaterals in hippocampal slices. The protein cross-reacts on immunoblot with affinity-purified polyclonal antiserum raised against a peptide derived from the carboxy-terminus of PKC eta; it does not cross-react, however, with antiserum against the amino-terminal region of PKC eta. In the tissues examined, PKC(HMW) is localized primarily in brain, in contrast to PKC eta, which is found predominantly in lung and skin.

Amino Acid Sequence↗
PubMed1993

Can a protein influence the fate of its own coding sequence?: the amino- and carboxyl-terminal regions of H1 histone.

The amino- and carboxyl-terminal regions of H1 histone are afforded an opportunity to bind to their own coding segments. While such binding is of no consequence to the type of H1 histone that is expressed only in terminally differentiated cells (here referred to as H5), the same binding occurring in H1 of proliferating cells might affect the course of evolution of its amino- and carboxyl-terminal regions. With the above in mind, amino acid sequences of H5 from three species were compared with those of H1 from five species. The spherical core was more conserved in H1 than in H5, whereas the amino- and carboxyl-terminal regions of both were hypervariable. A distinct evolutionary pattern, however, was observed only in these regions of H1 but not of H5. Tandem repeats of short palindromes often generated nearly identical long palindromes in distantly related species. Thus, as far as the 37-residue-long amino-terminal region was concerned, the greatest homology was between the rat and human rather than between the rat and the mouse. With regard to the last 25 residues in the carboxyl-terminal region, the greatest homology was between the chicken and the rainbow trout; the next was between the mouse and the trout. Successions of long palindromes populating the amino- and carboxyl-terminal regions of H1 might be the direct consequence of the autologous binding between peptides and their coding sequence.

Amino Acid Sequence↗
PubMed1993

Unique expression pattern of protein kinase C-theta: high mRNA levels in normal mouse testes and in T-lymphocytic cells and neoplasms.

A 2.2-kb cDNA that contains the entire coding region of mouse protein kinase C-theta (PKC-theta) was cloned from skeletal muscle mRNA using reverse transcription and the polymerase chain reaction (PCR). This clone was used as a probe to study the expression of this PKC isoform in normal and transformed hemopoietic cells and other normal tissues. By far the highest steady-state level of PKC-theta mRNA was found as a 2.8-kb transcript on a Northern blot of poly(A)+ RNA from testes. High levels were also found in skeletal muscle, spleen, T lymphomas and purified normal T lymphocytes, but these tissues and cells expressed two transcripts, 3.3 kb and 3.8 kb. Lower levels of similar size transcripts were found in normal brain, B lymphocytes and B-lymphocytic tumors and cell lines.

Animals↗
PubMed1993

A clinical trial of FK506 in refractory uveitis.

We performed a clinical open trial to evaluate the efficacy and the adverse side effects of a single therapy with FK506 in refractory uveitis as a multicenter study in Japan. Fifty-three patients (41 patients with Bechçet's disease, five with Vogt-Koyanagi-Harada disease, four with idiopathic retinal vasculitis, and three with other forms of uveitis) were enrolled in the study. FK506 was given orally for 12 weeks. Treatment with FK506 exhibited therapeutic effects in a dosage-dependent manner: the effectiveness was 38% in patients treated with an initial dosage of 0.05 mg/kg of body weight per day, 60% with 0.10 mg/kg of body weight per day, 83% with 0.15 mg/kg of body weight per day, and 79% with 0.20 mg/kg of body weight per day. Overall efficacy with dosage adjustment when needed was 76.5% at the conclusion of the study at the end of the 12th week. The FK506 therapy induced a variety of adverse side effects, the incidence of which depended on the dosage. The major side effects were renal impairment (28.3%, 15 of 53 patients), neurologic symptoms (20.8%, 11 of 53 patients), gastrointestinal symptoms (18.9%, ten of 53 patients), and hyperglycemia (13.2%, seven of 53 patients). The trough level of FK506 in the whole blood correlated with both the efficacy of the therapy and with the incidence of adverse effects. It is recommended to maintain the trough level between 15 and 25 ng/ml. On the basis of these results, a daily dosage of 0.10 to 0.15 mg/kg of body weight per day was suggested as an appropriate therapeutic dosage for refractory uveitis.

Administration, Oral↗
PubMed1993

The beta isoform of protein kinase C stimulates human melanogenesis by activating tyrosinase in pigment cells.

We have investigated the role of protein kinase C (PKC) in human melanogenesis. The level of PKC activity paralleled the total melanin content in cultured newborn melanocytes. Activation of PKC by treatment with 5 x 10(-7) M phorbol dibutyrate acutely caused a doubling in the activity of tyrosinase, the rate-limiting enzyme in melanogenesis, known to correlate directly with melanin synthesis in these cells. When PKC was depleted to 5-10% of initial levels, there was a 40-50% parallel reduction in tyrosinase activity; and regeneration of PKC activity was associated with the recovery of tyrosinase activity. By Northern blot analysis, the alpha and beta but not the gamma isoforms were detectable in melanocytes. By Western blot analysis, the racially determined pigment level in cultured melanocytes correlated with PKC-beta protein expression. In a pigmented human melanoma line (P-MM4, 20-30 ng melanin/micrograms protein)and its nonpigmented subclone (NP-MM4, undetectable melanin), PKC-alpha mRNA was expressed in both, whereas PKC-beta mRNA was detectable only in P-MM4 cells. Tyrosinase protein level was comparable in both cell lines. When NP-MM4 cell lysate was incubated with melanocyte lysate known to contain PKC-beta, tyrosinase activity per microgram of melanocyte protein in the combined lysate increased, consistent with activation of the previously inactive tyrosinase of NP-MM4 origin. Moreover, NP-MM4 cells transiently transfected with PKC-beta cDNA increased tyrosinase activity from undetectable to detectable levels. These combined data show that PKC-beta regulates human melanogenesis by activating tyrosinase.

Blotting, Northern↗
PubMed1993

Transfer RNAs with complementary anticodons: could they reflect early evolution of discriminative genetic code adaptors?

In accordance with the hypercycle theory of M. Eigen and P. Schuster [(1979) Hypercycle: A Principle of Natural Self-Organization (Springer, New York)], the ancestors of modern tRNAs appear to have emerged via the shortest possible way, both complementary strands of a short symmetrical double helix serving as pre-tRNAs with complementary anticodons. This conclusion is based upon results of comparative sequence analysis of the 17-base-long anticodon loop and stem of tRNAs totaling 896 and especially of 22 pairs of consensus tRNAs with complementary or quasi-complementary anticodons. With regard to the anticodon loop and stem of pairs of consensus tRNAs, complementary distances were considerably less than direct distances--i.e., antiparallel pairing invariably yielded fewer mismatches than direct pairing. Furthermore, the smallest complementary distance was detected when two antiparallel sequences formed irregular G-U bonds in their anticodon triplets. The above implies that pre-tRNAs in peribiotic times were long hairpin structures having 73 bases or more, the middle base of an anticodon being the center of symmetry. Accordingly, each pair of pre-tRNAs with complementary anticodons should have been almost identical with each other except for their three central bases. The above situation appears to have dictated the early establishment of direct links between anticodons and the type of amino acids with which tRNAs are to be charged. This direct link is still maintained between modern aminoacyl-tRNA synthetases and anticodons. Replication of the double helices concertedly generated new codons for the same pair of amino acids. Thus, occurrence of synonymous as well as certain "palindromic" features of the genetic code table might have been determined by this mechanism.

Anticodon↗
PubMed1993

Functional divergence of protein kinase C (PKC) family members. PKC gamma differs from PKC alpha and -beta II and nPKC epsilon in its competence to mediate-12-O-tetradecanoyl phorbol 13-acetate (TPA)-responsive transcriptional activation through a TPA-response element.

We have established an assay system where overexpression of a specific protein kinase C (PKC) type caused by introduction of the respective cDNA results in the enhancement of a cell response: the transcriptional activation of a set of genes in response to PKC activators such as 12-O-tetradecanoylphorbol 13-acetate (TPA). When monitored by the expression of a reporter gene containing the chloramphenicol acetyltransferase gene fused downstream of a synthetic TPA response element (TRE) or a serum response element (SRE), the overexpression of cPKC alpha and -beta II or nPKC epsilon all resulted in the enhancement of transcriptional activation through both TRE and SRE. On the other hand, PKC gamma activates TRE only very weakly, although it activates SRE in a similar manner to the other PKC members examined. The overexpression of cPKC alpha and -beta II or nPKC epsilon, but not cPKC gamma, resulted in the enhanced expression of the endogenous c-jun gene, which contains TRE in the 5'-upstream, promoter region. The gel mobility shift assay showed that the activation of PKC gamma, as well as PKC alpha and -beta II and nPKC epsilon, causes the increase in TRE-binding proteins, suggesting that transcriptional activation through TRE requires an additional step, which is not activated by PKC gamma, such as a qualitative change in TRE-binding or in TRE-associating proteins. This finding provides not only a rationale to explain the presence of multiple PKC family members, but also permits the dissection of the complex cellular signaling cascade involving PKC family members.

Amino Acid Sequence↗
PubMed1993

A new protein kinase C, nPKC eta', and nPKC theta are expressed in human platelets: involvement of nPKC eta' and nPKC theta in signal transduction stimulated by PAF.

We have detected in human platelets two protein kinase C isozymes that have not been reported previously. Using an anti-nPKC theta antibody and Western blotting, we calculated the molecular weight of platelet nPKC theta as 79K. This molecular weight is identical to that described for nPKC theta in skeletal muscle and in COS cells transfected with the nPKC theta-cDNA. Using an anti-nPKC eta antibody, we determined the molecular weight of an immunoreactive protein, which we called nPKC eta', to be 95K. This molecular weight is higher than that of nPKC eta found in lung and skin tissue of 82K and 78K, and it is higher than nPKC eta of COS cells transfected with the nPKC eta-cDNA expression plasmid. Together with previous reports, these findings make the total number of PKC isozymes in human platelets equal to six. These are the PKC isozymes: alpha, beta, delta and zeta, which have been previously described, and eta' and theta which we describe here. To assess the functionality of these new PKC isoforms, we stimulated platelets with PAF. We found a 200% and 175% increase in the levels of membrane-bound nPKC eta' and nPKC theta, respectively, in human platelets stimulated by PAF. A concomitant decrease in the level of these isoforms in the cytoplasm was observed. This PAF-induced translocation was time-dependent, and it reached its peak after a 1 minute incubation of human platelets with PAF for nPKC theta and 30 seconds for nPKC eta'.

Amino Acid Sequence↗
PubMed1993

Cross-reactivity of IgM- and IgG-secreting B cells in autoimmune mice.

OBJECTIVE: To determine the frequency with which in vivo-activated B cells secrete cross-reactive antibodies in lupus-prone mice. METHODS: Analysis of freshly isolated splenic lymphocytes by chamber enzyme-linked immunospot assay. RESULTS: Young New Zealand black, New Zealand black x New Zealand white, and MRL/lpr mice expressed repertoires in which 16-20% of IgM-secreting cells and 0-2% of IgG-secreting cells were cross-reactive. By comparison, 21-31% of IgM- and 4-14% of IgG-secreting cells in adult animals with active lupus were cross-reactive (P < 0.01). CONCLUSION: Cross-reactive B cells constitute an abnormally large proportion of the repertoire expressed in mice with active autoimmune disease.

Animals↗
PubMed1993

Anionic sites in glomerular basement membrane of rats with serum sickness nephritis: quick-freezing and deep-etching study.

Serum sickness nephritis was induced in male Fisher 344/JCL rats by injecting egg albumin into the foot pads and peritoneal cavity. The alteration of anionic sites in the glomerular basement membrane (GBM) of the rats with significant proteinuria was studied with a quick-freezing and deep-etching method using polyethyleneimine as a cationic probe. In control rats, anionic sites were located around the fibrils of the lamina rara externa, which radiated perpendicularly from the lamina densa to podocyte cell membranes. In the glomeruli of proteinuric rats, many electron-dense deposits were observed in the subepithelial side of the GBM, where the fibrils of the lamina rara externa were usually obscured and anionic sites around them could not be recognized. However, in some areas, a clear boundary could be observed between deposits and the lamina densa. Electron micrographs of freeze-fractured deposits showed that the fibrils radiated perpendicularly from the lamina densa and that anionic sites around them had been preserved. These results suggest that some of the deposits simply passed through the GBM and masked transiently the fibril structures of the GBM, but others probably destroyed these fibril structures, including anionic sites.

Animals↗
PubMed1993

Ultrastructural study of Cryptococcus neoformans by quick-freezing and deep-etching method.

The three-dimensional ultrastructure of Cryptococcus neoformans was studied by quick-freezing and deep-etching (QF-DE) method. C. neoformans, strain CDC551, was cultured on agar. The viable yeast cells (10(7) cells) were inoculated into each mouse from the tail vein. Three weeks after the inoculation, the brains of the mice were perfused with fixatives, quickly frozen, freeze-fractured, deeply etched and rotary shadowed with platinum and carbon. In addition, the viable cells of C. neoformans on agar were picked up and quickly frozen, and replica membranes were prepared as described above. The ultrastructure of C. neoformans was three-dimensionally demonstrated by the QF-DE method. The capsule was composed of fine meshworks of microfibrils (10-13 nm in diameter), which were directly attached to the cell walls. The capsule of the in vivo yeasts (yeast cells in the brain lesion) was thicker than that of the in vitro yeasts (yeast cells on agar culture). At the outer part of the cell wall, a particle-accumulating layer was observed. This layer in vivo was thicker than that in vitro. Occasionally, the yeast cells were ingested by phagocytes in the mouse brain. Although the cytoplasm of such yeast cells was destroyed, the capsular meshworks were well preserved. The ultrastructure of the capsule was the same both in cultured and phagocytized yeasts in the cystic lesions of the brains. This lack of morphological changes of the capsular meshworks suggests that they are resistant to the digestion by phagocytes. This stability of capsular structures may provide one of the important pathogenic factors in cystic lesions by C. neoformans.

Animals↗
PubMed1993

Effect of trimebutine maleate on emptying of stomach and gallbladder and release of gut peptide following a solid meal in man.

We investigated the effect of orally administered trimebutine maleate on gastric and gallbladder emptying and on the release of gut peptide, pancreatic polypeptide (PP), and gastrin in humans for 120 min after ingestion of a solid meal. Gastric emptying was measured by a radionuclide technique. Gallbladder emptying was estimated by real-time ultrasonography. The oral administration of 200 mg of trimebutine maleate significantly shortened the lag time in starting gastric emptying (P < 0.05). Considering gallbladder emptying, trimebutine significantly inhibited the fasting emptying induced by neural reflex. Postprandially, there was a tendency toward an accelerated gallbladder emptying in the early phase. Neither the maximal percentage of gallbladder emptying nor the time of peak gallbladder emptying were affected. Trimebutine significantly blunted the post-prandial PP response in the cephalic and gastric phases, reflecting a vagal-cholinergic activity (P < 0.05). The PP response in the intestinal phase was also blunted. Gastrin release was significantly augmented only during the period of fasting after drug administration (P < 0.05). The major effect of trimebutine maleate appears to be a shortening of the lag time at the start of gastric emptying probably via its anticholinergic activity.

Administration, Oral↗
PubMed1993

On concerted origin of transfer RNAs with complementary anticodons.

Pairs of antiparallely oriented consensus tRNAs with complementary anticodons show surprisingly small numbers of mispairings within the 17-bp- long anticodon stem and loop region. Even smaller such complementary distances are shown by illegitimately complementary anticodons, i.e. those with allowed pairing between G and U bases. Accordingly, we suppose that transfer RNAs have emerged concertedly as complementary strands of primordial double helix-like RNA molecules. Replication of such molecules with illegitimately complementary anticodons might generate new synonymous codons for the same pair of amino acids. Logically, the idea of tRNA concerted origin dictates very ancient establishment of direct links between anticodons and the type of amino acids with which pre-tRNAs were to be charged. More specifically, anticodons (first of all, the 2nd base) could selectively target 'their' amino acids, reaction of acylating itself being performed by another non-specific site of pre-tRNA or even by another ribozyme. In all, the above findings and speculations are consistent to the hypercyclic concept (Eigen and Schuster, 1979), and throw new light on the genetic code origin and associated problems. Also favoring this idea are data on complementary codon usage patterns in different genomes.

Amino Acids↗
PubMed1993

Immunocytochemical study of membrane skeletons in abnormally shaped erythrocytes as revealed by a quick-freezing and deep-etching method.

Ultrastructures of membrane skeletons in spherocytic and elliptocytic erythrocytes were investigated immunocytochemically. Erythrocytes obtained from patients with hereditary spherocytosis (HS) and hereditary elliptocytosis (HE) were split open mechanically to obtain exposed cytoplasmic sides of erythrocyte membranes and were immunostained with anti-spectrin antibody. Replica membranes were prepared by a quick-freezing and deep-etching method and were checked by electron microscopy. The in situ membrane skeletons of normal erythrocytes consisted mainly of reticular patterns of spectrin filaments, which formed networks on the cytoplasmic sides of the cell membrane. In contrast, the membrane skeletons of abnormally shaped erythrocytes (HS and HE) were much less filamentous and more granular than those of normal erythrocytes. This abnormal organization in erythrocyte membrane skeletons may be one of the factors that induce abnormally shaped erythrocytes in HS and HE patients.

Adult↗
PubMed1993

PKC gamma gene expression is delayed in postnatal central nervous system of mi/mi mice.

In the central nervous system (CNS), the expression of protein kinase C (PKC) genes is strictly controlled by the developmental stage. We have examined the expression of PKC genes (cPKC alpha, beta, gamma, and nPKC delta, epsilon) in the process of the postnatal development in normal (+/+) C57BL/6 and microphthalmic (mi/mi) C57BL/6 mouse brains by Northern blotting and in situ hybridization. By Northern blotting, the expression level of cPKC gamma mRNA in mi/mi mice was significantly lower than that in +/+ littermates at d 9, 13, and 17. By in situ hybridization analysis, cPKC gamma mRNA-positive cells were detected in hippocampal and Purkinje cells in +/+ and mi/mi mice, but the magnitude of the signals in mi/mi mice was lower than that of +/+ mice, and the number of positive cells was smaller, whereas other isozymes (cPKC alpha, beta, and nPKC delta, epsilon) showed no significant difference between normal and mi/mi mice. The neuronal morphometric analysis by anti-P400 antibody revealed the same number and expression level of P400 protein in cerebellar Purkinje cells compared with +/+ mice. These results indicate that the deficiency of mi gene product causes the delayed expression of the cPKC gamma gene.

Animals↗
PubMed1993

Ultrastructural study of glomerular basement membrane in diabetic rats by quick-freezing and deep-etching method.

The three-dimensional ultrastructure of glomerular basement membrane (GBM) in streptozotocin (STZ)-induced diabetic rats was examined by quick-freezing and deep-etching method. In three layers of the GBM of control rats, the outer and inner layers were formed by files of perpendicular fibrils, which connected the epithelial or endothelial cell surfaces with meshwork structures of the middle layer. In the diabetic rats, the inner layer was diffusely enlarged and the meshwork structure of the middle layer became markedly irregular due to the rupture of fine fibrils and thickening of material adherent to the fibrils. These ultrastructural changes correspond to those of subendothelial oedema, lamellation of lamina densa and fluffy material in the GBM, as revealed on conventional ultra-thin sections. It is suggested that the initial morphological change of STZ-induced diabetic nephropathy is disruption of matrix fibrils in the GBM, seemingly indicating a disturbance of size and/or charge barriers.

Animals↗