Biomedical subjects

K Okazaki

Publications and source records attributed to K Okazaki.

At least 217 records · Page 12Linked to original sources

PubMed1995

A novel Ca2+/calmodulin-dependent protein kinase lacking autophosphorylation activity in the rabbit heart.

We report the discovery, semi-purification and characterization of a novel Ca2+/calmodulin-dependent protein kinase (peak I kinase) using syntide 2 as a substrate from the rabbit heart. In the study of dependence of peak I kinase on the concentration of calmodulin, half-maximal activation was obtained at approx. 2.0 x 10(-7) M calmodulin. Peak I kinase did not undergo autophosphorylation. This kinase phosphorylates the synthetic peptides such as syntide 2, autocamtide-2, site 3 in a Ca2+/CaM-dependent manner, but not myosin light chain-peptide, gamma-peptide, and cAMP Response Element Binding Protein (CREB) peptide. Elongation Factor-2, alpha-casein and histone-IIIs were not phosphorylated. These data indicate that this CaM kinase is different from other identified Ca2+/calmodulin-dependent protein kinases and therefore constitutes a novel protein kinase.

Animals↗

PubMed1995

Immunohistochemical localization of neurocalcin in the rat inner ear.

Localization in the rat inner ear of neurocalcin, a three EF-hand calcium-binding protein, was examined immunohistochemically. Neurocalcin-like immunoreactivity was restricted to neurons in neuroepithelial receptor organs, while hair cells and supporting cells showed no such immunoreactivity. In the organ of Corti, both afferent and efferent nerve terminals, which formed synapses on both inner and outer hair cells, showed distinct immunoreactions. Spiral ganglion neurons and cochlear nerves were immunopositive. In the cristae ampullaris, macula utriculi and macula sacculi, afferent nerve terminals forming nerve calices or terminal boutons were strongly immunopositive. Efferent nerve terminals making synapses either on nerve calices or on hair cells showed an intense immunoreactivity. Vestibular ganglion neurons were strongly immunopositive. In electron microscopy, immunoreaction products were diffuse in the cytoplasm of ganglion neurons and nerve terminals. Neurocalcin-like immunoreactivity occurred in association with microtubules, outer mitochondrial membranes, synaptic vesicles and synaptic membranes. It is thus likely that neurocalcin is involved in neural functions in each type of afferent and efferent transmission in the inner ear.

Animals↗

PubMed1995

Neurocalcin-immunoreactive receptor cells in the rat olfactory epithelium and vomeronasal organ.

Occurrence of neurocalcin, a calcium-binding protein with three EF hand motifs, was examined immunohistochemically in the rat olfactory epithelium and vomeronasal organ epithelium. Immunoreactivity was detected in receptor cells in these epithelia. Immunoreactions were distributed in cytoplasm associated with outer mitochondrial membrane, endoplasmic reticulum and microtubules. Olfactory and vomeronasal nerve fibers in the lamina propria exhibited immunoreactivity. Neurocalcin may participate in calcium signalling and cytoskeletal arrangement in receptor cells.

Animals↗

PubMed1995

MAP kinase activation is essential for oncogenic transformation of NIH3T3 cells by Mos.

The c-mos proto-oncogene product, Mos, is a serine/threonine protein kinase that controls the meiotic cell cycle in vertebrate oocytes. Both in vivo and in vitro, Mos can activate mitogen-activated protein kinase (MAPK) most probably by direct phosphorylation of MAPK kinase (MAPKK). In many cell types transformed by diverse oncogene products such as Raf, MAPK is constitutively activated, suggesting that the MAPK pathway may mediate oncogenic signalling by many oncogene products. Using mouse NIH3T3 cells, we examined whether oncogenic transformation by Mos is mediated by MAPK activation. Coexpression of a kinase-defective (dominant-negative) mutant of Mek1, one of the MAPKK isoforms, completely suppressed transformation by Mos. By contrast, coexpression of wild-type Mek1 markedly enhanced the transforming efficiency of Mos. Moreover, overexpression of the dominant-negative Mek1 reverted the transformation phenotype of Mos-transformed cells. These results indicate that in NIH3T3 cells the Mek1/MAPK pathway is necessary and sufficient for transformation (and its maintenance) by Mos. Transformation of NIH3T3 cells by Raf or Ras was also suppressed by the dominant-negative Mek1, but significantly less efficiently than that by Mos, suggesting the existence of multiple signalling pathways for Raf and Ras oncoproteins.

3T3 Cells↗

PubMed1995

S100 beta is a target protein of neurocalcin delta, an abundant isoform in glial cells.

To clarify the function of neurocalcin delta, an isoform found abundantly in glial cells, we attempted to find its target proteins by using neurocalcin delta-affinity chromatography and the 125I-neurocalcin delta gel-overlay method. The 10, 14, 27, 36 and 50 kDa bands found on SDS/PAGE bound to 125I-neurocalcin delta, and 10, 11, 19, 24, 26, 50 and 70 kDa proteins were eluted from a neurocalcin delta-affinity column in a Ca(2+)-dependent manner. Sequence analysis of proteolytic peptides revealed the following identities: S100 beta (10 kDa), S100 alpha (11 kDa), myelin basic protein (19 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa) and tubulin beta-chain (50 kDa). A zero-length cross-linking study indicated that 1 mol of S100 beta bound to 1 mol of neurocalcin delta. With the gel-overlay method, purified S100 beta protein and calcyclin bound to 125I-neurocalcin delta whereas calgizarrin and calvasculin, other members of the S100 family, did not. These findings suggest that S100 beta is one of the target proteins of neurocalcin delta, and the neurocalcin delta-S100 beta complex may be involved in Ca(2+)-signalling in the glial cell.

Amino Acid Sequence↗

PubMed1995

Localization of neurocalcin-like immunoreactivity in rat cranial motoneurons and spinal cord interneurons.

Neurocalcin (NC) is a calcium-binding protein with at least three putative calcium-binding domains called EF-hands. In this study, the distribution of neurocalcin-like immunoreactivity (LI) was examined in the rat motor system. Motoneurons in the III, IV and VI cranial nerve nuclei were NC-immunoreactive (IR) and strong labelling was seen in the nerve bundles and in the myoneural junctions in all extraocular muscles. In the ventral horn of the spinal cord, interneurons were NC-IR, whereas motoneurons, identified by Fluorogold tracing, were unlabelled. A large number of NC-IR neurons was present in the dorsal horn. NC-IR nerve fibers were seen in the ventral roots and, more abundantly, in the dorsal roots. The present results demonstrate NC-LI in the supraspinal motoneurons and spinal cord interneurons, both of which are fast-firing neurons. Provided neurocalcin regulates the concentration of free intracellular Ca2+, it may participate in several cellular functions in the fast-firing neurons.

Animals↗

PubMed1995

Involvement of capsaicin-sensitive neurons in gastrin release provoked by intragastric administration of bile salts in the rat.

To clarify the mechanism of gastrin release provoked by the reflux of bile juice into the stomach, we studied the effects of tetrodotoxin (0.08 mg/kg), atropine sulfate (0.5 mg/kg), truncal vagotomy, and chemical denervation of afferent sensory neurons with capsaicin (100 mg/kg) on gastrin release induced by bile salts in the rat (n = 6 per group). Sodium taurocholate and deoxycholate (> 2.5 mM) significantly increased the serum levels of gastrin. However, sodium tauroursodeoxycholate had no effect. The levels of serum gastrin before and 1 h after administration of 2.5 mM sodium taurocholate were 94.6 +/- 10.7 and 211.0 +/- 21.1 pg/ml, respectively. Tetrodotoxin and atropine sulfate completely inhibited this sodium taurocholate induced-gastrin increase, while truncal vagotomy was without effect. Capsaicin markedly reduced the increasing effects of sodium taurocholate. These findings suggested that the neuronal pathways involved in gastrin release are probably an intragastric local circuit originating from capsaicin-sensitive afferent sensory neurons and terminating in muscarinic receptors in the postsynaptic efferent cholinergic neuron system.

Animals↗

PubMed1995

Perpetuation of influenza A viruses in Alaskan waterfowl reservoirs.

To provide information on the mechanism of perpetuation of influenza viruses among waterfowl reservoirs in nature, virological surveillance was carried out in Alaska during their breeding season in summer from 1991 to 1994. Influenza viruses were isolated mainly from fecal samples of dabbling ducks in their nesting places in central Alaska. The numbers of subtypes of 108 influenza virus isolates were 1 H2N3, 37 H3N8, 55 H4N6, 1 H7N3, 1 H8N2, 1 H10N2, 11 H10N7, and H10N9. Influenza viruses were also isolated from water samples of the lakes where they nest. Even in September of 1994 when the most ducks had left for migration to south, viruses were still isolated from the lake water. Phylogenetic analysis of the NP genes of the representative isolates showed that they belong to the North American lineage of avian influenza viruses, suggesting that the majority of the waterfowls breeding in central Alaska migrate to North America and not to Asia. The present results support the notion that influenza viruses have been maintained in waterfowl population by water-borne transmission and revealed the mechanism of year-by-year perpetuation of the viruses in the lakes where they breed.

Alaska↗

PubMed1995

Pancreatic involvement in patients with Sjögren's syndrome and primary biliary cirrhosis.

Serum pancreatic enzyme activities, exocrine pancreatic function, and pancreatic ductal morphology were evaluated in patients with one or both of Sjögren's syndrome and primary biliary cirrhosis. Ten of 20 patients with Sjögren's syndrome (50%), 6 of 17 patients with primary biliary cirrhosis (35%), and 4 of 11 patients with both diseases (36%) had an elevated level of at least one pancreatic enzyme, including elastase-1, lipase, and trypsin. Diminished excretion of N-benzoyl-L-tyrosyl-para-aminobenzoic acid was observed in 3 of 17 patients with Sjögren's syndrome (18%), 4 of 16 with primary biliary cirrhosis (25%), and none of 7 with both diseases. Endoscopic retrograde pancreatograms demonstrated an abnormal pancreatic ductal configuration in 3 of 11 patients with Sjögren's syndrome (27%), 2 of 9 with primary biliary cirrhosis (22%), and 3 of 4 with both diseases (75%). Only minimal changes in branches of the pancreatic duct were observed in the pancreatogram. Finally, 9-30% of patients with Sjögren's syndrome and/or primary biliary cirrhosis had a mild and intermittent abdominal pain. These findings support the concept of a disease complex, "autoimmune exocrinopathy," in patients with Sjögren's syndrome, primary biliary cirrhosis, and chronic pancreatitis.

4-Aminobenzoic Acid↗

PubMed1995

Characterization of murine PACAP mRNA.

A murine PACAP precursor cDNA was isolated by screening a brain cDNA library. The amino acid sequence of the precursor was highly similar (from 81% to 93% similarity) to its rat, human, and ovine counterparts. The primary structure of murine PACAP is identical with those from sheep, humans, and rats, indicating that the mature PACAP is well conserved among mammals. Northern blot analysis revealed that the approximately 2.4 kb transcript for the PACAP precursor is expressed in murine brain. The verification that murine PACAP is identical to its human counterpart provides a rationale for physiological and pathophysiological studies of PACAP in mice.

Amino Acid Sequence↗

PubMed1995

Hippocalcin in rat retina. Comparison with calbindin-D28k, calretinin and neurocalcin.

The post-natal developmental expression in rat retina of four calcium-binding proteins belonging to the calmodulin-troponin-C family was investigated by immunohistochemistry using anti-calbindin-D28k, anti-calretinin, anti-hippocalcin and anti-neurocalcin polyclonal antibodies on paraffin sections from Wistar rat retinae aged from post-natal days 1 (P1), 5 (P5), 10 (P10), 20 (P20) to adulthood (8 weeks). Immunoblot using anti-hippocalcin and homogenates proteins from retina, cerebellar cortex, hippocampus and cerebellum was also performed. Hippocalcin immunoreactivity in adult rat retina was demonstrated by both immunohistochemistry and Western blot. During post-natal development, calbindin-D28k, calretinin and neurocalcin immunoreactivity were detected at P1 in ganglion cells, whereas hippocalcin immunoreactivity was seen later at P5 in this cell layer. In the amacrine cell layer, neurocalcin immunoreactivity was detected at P5 and hippocalcin at P10. Calbindin-D28k was labelling the immature horizontal cell, calretinin was detected in nearly all ganglion cells and in some amacrine cells since P1. These three calcium-binding proteins do not seem to play a role in synaptogenesis which takes place later. We confirmed that calbindin-D28k appeared to be a good marker for horizontal cells. The presence of hippocalcin, a myristoylated calcium-binding protein belonging to the recovering subfamily and previously localized in few brain areas has been detected for the first time in retina.

Animals↗

PubMed1995

The structure of calcyclin reveals a novel homodimeric fold for S100 Ca(2+)-binding proteins.

The S100 calcium-binding proteins are implicated as effectors in calcium-mediated signal transduction pathways. The three-dimensional structure of the S100 protein calcyclin has been determined in solution in the apo state by NMR spectroscopy and a computational strategy that incorporates a systematic docking protocol. This structure reveals a symmetric homodimeric fold that is unique among calcium-binding proteins. Dimerization is mediated by hydrophobic contacts from several highly conserved residues, which suggests that the dimer fold identified for calcyclin will serve as a structural paradigm for the S100 subfamily of calcium-binding proteins.

Amino Acid Sequence↗

PubMed1995

K+ channel-opening properties of a novel compound, NIP-121, in guinea pig myocardium as compared with those of cromakalim.

Myocardial effects of NIP-121, a novel compound with potent vasorelaxant activity, were examined in comparison with those of cromakalim in isolated tissue and cells from guinea pig hearts. NIP-121 and cromakalim concentration-dependently reduced the action potential duration (APD) of isolated papillary muscle; the effect was antagonized by glibenclamide. In isolated ventricular tissue, NIP-121 and cromakalim decreased the contractile force concentration dependently. In these two experiments, the potency of NIP-121 was approximately 20 times higher than that of cromakalim. The effects of NIP-121 and cromakalim on membrane currents were examined in voltage-clamped ventricular myocytes. NIP-121 (10(-6) M) and cromakalim (3 x 10(-5) M) increased the steady-state outward currents. The normal inwardly rectifying current-voltage relationship changed to a linear relationship that reversed at the K+ current reversal potential. The current activated by NIP-121 and cromakalim was inhibited either by glibenclamide or by increased intracellular ATP concentration. NIP-121, at this concentration, had little effect on the calcium current. Thus, NIP-121 was demonstrated to produce AP shortening and decrease in contractile force through activation of ATP-sensitive K+ currents in cardiac muscle, with a potency approximately 20 times higher than that of cromakalim.

Action Potentials↗

PubMed1995

Degradation of c-Fos by the 26S proteasome is accelerated by c-Jun and multiple protein kinases.

c-Fos is associated with c-Jun to increase the transcription of a number of target genes and is a nuclear proto-oncoprotein with a very short half-life. This instability of c-Fos may be important in regulation of the normal cell cycle. Here we report a mechanism for degradation of c-Fos. Coexpression of c-Fos and c-Jun in HeLa cells caused marked increase in the instability of c-Fos, whereas v-Fos, the retroviral counterpart of c-Fos, was stable irrespective of the coexpression of c-Jun. Interestingly, deletion of the C-terminal PEST region of c-Fos, which is altered in v-Fos by a frameshift mutation, greatly enhanced its stability, with loss of the effect of c-Jun on its stability. c-Fos synthesized in vitro was degraded by the 26S proteasome in a ubiquitin-dependent fashion. Simple association with c-Jun had no effect on the degradation of c-Fos, but the additions of three protein kinases, mitogen-activated protein kinase, casein kinase II, and CDC2 kinase, resulted in marked acceleration of its degradation by the proteasome-ubiquitin system, though only in the presence of c-Jun. In contrast, v-Fos and c-Fos with a truncated PEST motif were not degraded, suggesting that they escaped from down-regulation by breakdown. These findings indicate a new oncogenic pathway induced by acquisition of intracellular stability of a cell cycle modulatory factor.

Amino Acid Sequence↗

PubMed1995

Purification and properties of two chitinases from Streptomyces sp. J-13-3.

Two chitinases (Chi-A and Chi-B) purified from Streptomyces sp. J-13-3 had the same molecular weights (31,000) and enzymatic properties (optimum pH and temperature of pH 6.0 and 45 degrees C) but had significantly different isoelectric points (3.9 for Chi-A, 3.5 for Chi-B). Chi-A and -B had identical N-terminal amino acid sequences (ADXAAAWNASSVYTGGGSASYNGHN), similar amino acid compositions, and immunological cross-reactivities. A concomitant decrease of Chi-A and increase of Chi-B was observed in their productions during cultivation.

Amino Acid Sequence↗

PubMed1995

[Effects of arterial carbon dioxide (PaCO2 not equal to 60 mmHg) on regional myocardial tissue oxygen tension and metabolism].

We investigated the effects of increased arterial carbon dioxide (PaCO2 not equal to 60 mmHg) on myocardial tissue oxygen tension and metabolism in anesthetized dogs. Ten adult mongrel dogs weighing 15.3 +/- 4.1 kg were anesthetized with 0.5% isoflurane in 50% oxygen and ventilated mechanically to maintain normocapnia. After thoracotomy, regional myocardial tissue PO2 was measured using a monopolar polarographic needle electrode inserted in the myocardium. Electromagnetic blood flow probes were applied on the left anterior descending artery and the circumflex artery. A 23-gauge catheter was inserted into a coronary vein to obtain coronary venous blood for measuring oxygen content, lactate and pyruvate. After control normocapnic ventilation, hypercapnia (PaCO2 62.7 +/- 3.2 mmHg) was induced by adding 10% carbon dioxide to the inspired gas for 20 minutes. As a result, the coronary blood flow and myocardial oxygen tension increased during hypercapnia. The myocardial lactate extraction and coronary venous lactate were unchanged and excess lactate was kept below zero, although coronary venous L/P ratio increased during hypercapnia. These results indicate that hypercapnia (PaCO2 not equal to 60 mmHg) increases coronary blood flow and myocardial oxygen tension, while myocardial aerobic metabolism is not impaired under hypercapnia.

Animals↗

PubMed1995

Analysis of a Hind III site polymorphism in the type II collagen gene: it's location and frequencies in the Japanese population.

Hind III restriction fragment length polymorphism (RFLP) in the cartilage-specific type II collagen gene was studied in a sample of 135 persons randomly selected from the Japanese population. Localization of the Hind III site was determined by the dideoxy method. Based on this sequence, a pair of primers was designed and a polymerase chain reaction (PCR) was carried out. The Hind III allele had a frequency of 0.52 in the Japanese. The genotype digested by Hind III, (++) genotype was present with a frequency of 16.9%, (+-) was present in 70.0% and (--) was present in 13.1%, showing a lower rate of the (--) genotype in the Japanese compared to the Finnish. The difference was statistically significant, tested by chi 2 analysis (p < 0.0001). The present study is the first to report a race-dependent variance in Hind III RFLP on the type II collagen gene.

Asian People↗

PubMed1995

Differential association of three actin-bundling proteins with microfilaments in Dictyostelium amoebae.

Three actin-bundling proteins have been isolated from Dictyostelium discoideum cells. To investigate whether these proteins might play different roles in Dictyostelium cells, the distribution of these proteins was examined in cells of the slime mold at various developmental stages by agar-overlay immunofluorescence. ABP-30a was localized in pseudopods and projections, and regions of intercellular contacts, where filaments of actin were present at high levels. ABP-30a was not concentrated in the cleavage furrow of dividing cells or in the tail cortical regions of actively moving cells even though actin was present at high levels in both regions. Some ABP-50 was concentrated at the leading edges of pseudopods and at the distal ends of projections, but most ABP-50 was distributed diffusely in the cytoplasm. ABP-30b was not concentrated in any restricted regions and was diffusely distributed in the cytoplasm. When developed cells were stimulated with cAMP, fluorescence due to ABP-30a and ABP-50 increased in the cortical region 5 to 10 sec after stimulation. Quantitative analysis of levels of both proteins in Triton-insoluble cytoskeletons after stimulation by cAMP also showed that levels of both proteins increased within 5 to 10 sec. These observations indicate that levels of ABP-30a and AMP-50 are regulated by signal transduction during chemotaxis and that the three actin-bundling proteins play different roles in Dictyostelium cells.

Agar↗