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T Imagawa

Publications and source records attributed to T Imagawa.

At least 91 records · Page 5Linked to original sources
PubMed1994

Cloning, nucleotide sequence and expression of a hemolysin gene of Clostridium septicum.

A genomic library of Clostridium septicum NCTC547 strain was made in Escherichia coli by means of lambda gt10. The DNA insert of a hemolysin-positive (Hly+) lambda-clone was transferred into pUC19. The resulting plasmid, pCS21, confers a Hly+ phenotype on E. coli. Crude lysates of E. coli (pCS21) possessed a strong lytic activity on human erythrocytes and also a lethal effect on mice, characteristic of an alpha toxin. Nucleotide sequence analysis revealed that the insert DNA (5.2 kb) in pCS21 included at least one open reading frame of 1380 bp. The coding frame for hemolysin was predicted to be 1329 bp in size and to encode a protein of 49.8 kDa. It coincided with the molecular mass (48 kDa) of the alpha toxin secreted by C. septicum. Taken together, the data indicated that plasmid pCS21 indeed encoded an alpha toxin gene of C. septicum.

Amino Acid Sequence↗
PubMed1994

Anatomical and histological re-examination of Appendices colli in the goat.

Appendices colli (App. colli) were investigated by anatomical and histological methods in the goat. App. colli were composed of elastic cartilage located at central and the skin covering the cartilage, which included arterioles along the cartilage and nerve bundles. Three types of muscles connected to App. colli; superficial muscle bundles, a branch of the omohyoideus muscle, and a muscle arising from the pharyngeal raphe (appendico-pharyngeal muscle). The latter two muscles were connected to the root of the App. colli where the muscle fibers transformed into the perichondrium of the elastic cartilage. The appendico-pharyngeal muscle was innervated by branches from the glossopharyngeal nerve which were composed of myelinated nerve fibers. The subcutaneous area of the App. colli was supplied by cutaneous rami of the vagus and the second cervical nerves. The innervation and the musculature confirmed that the cartilage of the App. colli were derived from third and fourth branchial arches.

Animals↗
PubMed1994

Ultrastructure of blood vessels in the head kidney of the carp, Cyprinus carpio.

The ultrastructural features of blood vessels were examined by transmission and scanning electron microscopy in the perfused and nonperfused head kidney of the carp. They consisted mainly of the sinusoidal capillaries, arterioles, veins and portal veins. The sinusoidal capillaries were composed of endothelial cells that frequently exhibited large pores and an indistinct basal lamina. Some endothelial cells were trabecular in shape and protruded into the lumen. The reticular cells frequently attached to the outside of the endothelial cells, and macrophages were located on the luminal and abluminal surfaces of the endothelial cells. The arterioles possessed continuous endothelial cells with a thick basal lamina, continuous smooth muscle layers and a thick adventitia with collagen fibres and reticular cells. The veins were characterised by fenestrated endothelial cells and thin connective tissue. The portal veins were composed of continuous electron-lucent endothelial cells and thick layers of reticular cells and collagen fibres. The differences between the endothelial cells in each blood vessel probably reflect the various functions of the carp head kidney. The ultrastructural features of the sinusoids and the portal veins resemble those of lymphatic vessels of mammals and appear to relate to macrophage and lymphoid cell migration and proliferation in the carp head kidney.

Animals↗
PubMed1993

Cloning of the rat aortic smooth muscle Na+/Ca2+ exchanger and tissue-specific expression of isoforms.

The amino acid sequences of two isoforms of the rat aortic smooth muscle Na+/Ca2+ exchanger have been deduced by cloning and sequencing the cDNAs. These isoforms are identical in nucleotide sequence except that one has a 23-amino acid insertion at amino acid position 570. They are highly homologous to the canine cardiac exchanger except for the NH2-terminal portion and part of the large central hydrophilic domain (amino acid residues 570-631). They are 902 and 925 (with the insertion) amino acid long with calculated molecular masses of 100,676 and 103,200 (with the insertion), respectively, if the NH2-terminal 32-amino acid residues are eliminated as a cleaved signal sequence. Amplification of the variable region (amino acids 570-631) of the exchanger by means of the reverse transcriptase-polymerase chain reaction and DNA sequencing revealed that many isoforms of the exchanger are expressed in different rat tissues. The two clones isolated in this study are the predominant isoforms expressed in aorta, stomach, liver, and kidney. In cardiac and skeletal muscles, another isoform is dominant, which is equivalent to the canine cardiac exchanger. In brain, a third type is predominantly expressed. Alignment of the nucleotide sequences of these isoforms and Southern blot analysis of rat genomic DNA suggested that each isoform is generated through alternative splicing of the primary transcript.

Alternative Splicing↗
PubMed1993

Increased mechanical extraction of T-tubule/junctional SR from cardiomyopathic hamster heart.

We determined the contents of L-type calcium channels (LCC) and other membrane proteins in ventricular homogenates and microsomes prepared from hearts of 30- to 70-day-old Syrian cardiomyopathic (Bio 14.6) and normal hamsters. Quantitative immunoblot assay revealed that myopathic microsomes, as compared with normal controls, were enriched about twofold with the alpha 1-subunit of LCC, the ryanodine receptor calsequestrin, and Na(+)-K(+)-adenosinetriphosphatase (ATPase), whereas the contents of these proteins in ventricular homogenates were not different. In contrast, Na(+)-H+ antiporter and sarcoplasmic reticulum (SR) Ca(2+)-ATPase showed no difference in their contents in both homogenates and microsomes. Radioligand binding assay further showed no significant difference in the number of binding sites for [3H]prazosin, [125I]iodocyanopindolol, and [3H]saxitoxin between myopathic and normal microsomes. These result suggest that whereas membrane densities of LCC and the other proteins examined are not increased in myopathic cardiomyocytes, T-tubule/junctional SR membranes are more easily extracted from them by mechanical disruption. This, together with 1.5-fold higher yield of microsomal fractions from myopathic heart muscle, shows that abnormality exists in the mechanical property of cell membrane in the myopathic heart.

Animals↗
PubMed1992

Phosphorylation of ryanodine receptors in rat myocytes during beta-adrenergic stimulation.

We studied beta-adrenergic agonist-stimulated phosphorylation of the ryanodine receptor in rat cardiac myocytes. The ryanodine receptor solubilized from myocytes and immunoprecipitated by a monoclonal antibody against canine cardiac ryanodine receptor was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (PKA). Incubation of saponin-permeabilized myocytes with [gamma-32P]ATP also induced ryanodine receptor phosphorylation, which was enhanced significantly in the presence of isoproterenol. This stimulating action of isoproterenol was suppressed by the beta-adrenergic antagonist, propranolol. On the other hand, exogenously added cAMP caused a much larger stimulation of phosphorylation of the ryanodine receptor in permeabilized myocytes. The beta-agonist-induced phosphorylation of the ryanodine receptor was also observed in intact myocytes from the newborn rat heart. These results suggest that the ryanodine receptor is phosphorylated by PKA during beta-adrenergic stimulation of cardiac myocytes.

Adrenergic beta-Agonists↗
PubMed1992

Expression of Ca(2+)-induced Ca2+ release channel activity from cardiac ryanodine receptor cDNA in Chinese hamster ovary cells.

We constructed an expression plasmid (pMAMCRR51) that carried the entire protein-coding sequence of the rabbit cardiac ryanodine receptor cDNA, linked to the dexamethasone-inducible mouse mammary tumor virus promoter and Escherichia coli xanthine-guanine phosphoribosyltransferase (gpt). Chinese hamster ovary (CHO) cells were transfected with pMAMCRR51 and mycophenolic acid-resistant cells showing caffeine-induced intracellular Ca2+ transients were selected. Immunoprecipitation with a monoclonal antibody against the canine cardiac ryanodine receptor revealed that the cell clones thus selected exhibited Ca(2+)-dependent [3H]ryanodine binding activity, which was stimulated by 5 mM ATP or 1 M KCl. The apparent dissociation constant (Kd) for [3H]ryanodine was 6.6 nM in 1 M KCl, which was similar to the Kd obtained with cardiac microsomes. Immunoprecipitation also demonstrated that these cell clones expressed a protein indistinguishable in M(r) from the ryanodine receptor in canine cardiac microsomes. The ryanodine binding activity expressed in CHO cells increased significantly after dexamethasone induction. In saponin-skinned CHO cells transfected with pMAMCRR51, micromolar Ca2+ or millimolar caffeine evoked rapid Ca2+ release from the intracellular Ca2+ stores. In skinned control CHO cells, we did not observe such Ca2+ release activity. These results clearly demonstrate that the cardiac ryanodine receptor is stably expressed in internal membranes of CHO cells and functions as Ca(2+)-induced Ca2+ release channels.

Animals↗
PubMed1992

An activity which restores theta toxin activity in some theta toxin-deficient mutants of Clostridium perfringens.

Group a mutants of Clostridium perfringens are deficient in theta toxin but release a dialyzable substance ("substance A"), which restores theta toxin activity to group b mutants, into a culture supernatant; group b mutants are defective in "substance A" release. "Substance A" activity appeared in the exponentially growing phase of group a mutants and disappeared in the stationary phase. "Substance A" activity was most stable at pH 5.0 and 0 C and even increased threefold in the first 5 hr, but gradually decreased during the following 15 hr. It was quickly inactivated at neutral and higher pHs at 0 C.

Bacterial Proteins↗
PubMed1992

Cyclic AMP-dependent phosphorylation of the rat brain ryanodine receptor.

Phosphorylation of the rat brain ryanodine receptor was studied using a monoclonal antibody, Ry-1, against the cardiac ryanodine receptor. A large polypeptide with the same SDS-PAGE mobility as that of the canine cardiac receptor was detected in rat brain membranes by immunoblotting. The brain ryanodine receptor was solubilized from the microsomal membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), and more than 85% of the solubilized receptor was immunoprecipitated by Ry-1. Immunoprecipitated receptors were phosphorylated by cAMP-dependent protein kinase. The ryanodine receptor was also expressed in cultured fetal rat brain neurons and was phosphorylated by treating the cells with dibutyryl cAMP. The number of cells showing a caffeine-induced Ca2+ transient was increased significantly in the phosphorylating condition. These results suggest that the Ca channel activity of the brain ryanodine receptor is regulated by cAMP-dependent phosphorylation.

Animals↗
PubMed1991

Regulation of the cardiac ryanodine receptor by protein kinase-dependent phosphorylation.

The exogenous addition of the catalytic subunit of cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), or calmodulin (CaM) induced rapid phosphorylation of the ryanodine receptor (Ca2+ release channel) in canine cardiac microsomes treated with 1 mM [gamma-32P]ATP. Added protein kinase C (PKC) also phosphorylated the cardiac ryanodine receptor but at a relatively slow rate. The observed level of PKA-, PKG-, or PKC-dependent phosphorylation of the ryanodine receptor was comparable to the maximum level of [3H]ryanodine binding in cardiac microsomes, whereas the level of CaM-dependent phosphorylation was about 4 times greater. Phosphorylation by PKA, PKG, and PKC increased [3H]ryanodine binding in cardiac microsomes by 22 +/- 5, 17 +/- 4, and 15 +/- 9% (average +/- SD, n = 4-5), respectively. In contrast, incubation of microsomes with 5 microM CaM alone and 5 microM CaM plus 1 mM ATP decreased [3H]ryanodine binding by 38 +/- 14 and 53 +/- 15% (average +/- SD, n = 6), respectively. Phosphopeptide mapping and phosphoamino acid analysis provided evidence suggesting that PKA, PKG, and PKC predominantly phosphorylate serine residue(s) in the same phosphopeptide (peptide 1), whereas the endogenous CaM-kinase phosphorylates serine residue(s) in a different phosphopeptide (peptide 4). Photoaffinity labeling of microsomes with photoreactive 125I-labeled CaM revealed that CaM bound to a high molecular weight protein, which was immunoprecipitated by a monoclonal antibody against the cardiac ryanodine receptor. These results suggest that protein kinase-dependent phosphorylation and CaM play important regulatory roles in the function of the cardiac sarcoplasmic reticulum Ca2+ release channel.

Affinity Labels↗
PubMed1991

Growth inhibition of tumour cells by a liposome-encapsulated, mycolic acid-containing glycolipid, trehalose 2,3,6'-trimycolate.

In vivo growth of syngeneic tumour cells in the peritoneal cavity was strongly inhibited by intraperitoneal injection of a liposome-encapsulated, mycolic acid-containing glycolipid, trehalose 2,3,6'-trimycolate (TTM), derived from a non-pathogenic, acid-fast bacterium. Gordona aurantiaca. Peritoneal macrophages from mice after this treatment lysed tumour cells in vitro at a low effector/target ratio, and their culture supernatant inhibited tumour cell growth. The supernatant inhibited growth of not only tumour necrosis factor (TNF)-sensitive tumour cells, but also TNF-insensitive tumour cells. This inhibitory activity was enhanced by addition of lipopolysaccharide (LPS) to the culture medium of the macrophages. The macrophages released more superoxide (O2-), TNF and interleukin-1 (IL-1) on LPS triggering, and the releases of these compounds were further increased by addition of recombinant interferon-gamma (IFN-gamma) to the medium. Moreover, splenic T cells of TTM liposome-primed mice were found to produce eight times more IFN-gamma upon stimulation with LPS. These results indicated that priming with TTM liposomes resulted in strong activation of macrophages, which lysed tumour cells directly and also inhibited tumour cell growth by released factors.

Animals↗
PubMed1990

Primary structure and functional expression from cDNA of the cardiac ryanodine receptor/calcium release channel.

The sequence of 4968 (or 4976 with an insertion) amino acids composing the ryanodine receptor from rabbit cardiac sarcoplasmic reticulum has been deduced by cloning and sequencing the cDNA. This protein is homologous in amino acid sequence and shares characteristic structural features with the skeletal muscle ryanodine receptor. Xenopus oocytes injected with mRNA derived from the cardiac ryanodine receptor cDNA exhibit Ca2(+)-dependent Cl- current in response to caffeine, which indicates the formation of functional calcium release channels. RNA blot hybridization analysis with a probe specific for the cardiac ryanodine receptor mRNA shows that the stomach and brain contain a hybridizable RNA species with a size similar to that of the cardiac mRNA. This result, in conjunction with cloning and analysis of partial cDNA sequences, suggests that the brain contains a cardiac type of ryanodine receptor mRNA.

Amino Acid Sequence↗
PubMed1990

Does fluorescence of 4-nitrobenzo-2-oxa-1,3-diazole incorporated into sarcoplasmic reticulum ATPase monitor putative E1-E2 conformational transition?

When a purified preparation of sarcoplasmic reticulum Ca2(+)-ATPase was labeled with 0.3 mM 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in 1 mM AMPPNP and 1 mM CaCl2 at 25 degrees C and pH 7.0 for 60 min and then was treated with 10 mM dithiothreitol for 7 min, about 1 mol of NBD was incorporated per mol of the enzyme, and this inhibited the enzyme activity by 90 to 95%. The modified residue was identified as Cys-344 that is located near the phosphorylation site of the ATPase, Asp-351. The NBD-inhibition of enzyme activity could be reversed by treatment with membrane-acting agents such as C12E8, suggesting that Cys-344 is not directly involved in enzyme catalysis. A detailed study of partial reactions of ATP hydrolysis by the modified enzyme and associated changes in the fluorescence intensity of the incorporated NBD label revealed that a predominant effect of the NBD-modification was the inhibition of Ca2+ release from the ADP-sensitive phosphoenzyme intermediate and that two major fluorescent states of the enzyme alternated during ATP hydrolysis. The latter fluorescent data are consistent with the E1-E2 model of Ca2(+)-ATPase reaction.

4-Chloro-7-nitrobenzofurazan↗
PubMed1990

Protein kinase-dependent phosphorylation of cardiac sarcolemmal Ca2(+)-ATPase, as studied with a specific monoclonal antibody.

Phosphorylation of the Ca2(+)-pump ATPase of cardiac sarcolemmal vesicles by exogenously added protein kinases was examined to elucidate the molecular basis for its regulation. The Ca2(+)-pump ATPase was isolated from protein kinase-treated sarcolemmal vesicles using a monoclonal antibody raised against the erythrocyte Ca2(+)-ATPase. Protein kinase C (C-kinase) was found to phosphorylate the Ca2(+)-ATPase. The stoichiometry of this phosphorylation was about 1 mol per mol of the ATPase molecule. The C-kinase activation resulted in up to twofold acceleration of Ca2+ uptake by sarcolemmal vesicles due to its effect on the affinity of the Ca2+ pump for Ca2+ in both the presence and absence of calmodulin. Both the phosphorylation and stimulation of ATPase activity by C kinase were also observed with a highly-purified Ca2(+)-ATPase preparation isolated from cardiac sarcolemma with calmodulin-Sepharose and a high salt-washing procedure. Thus, C-kinase appears to stimulate the activity of the sarcolemmal Ca2(+)-pump through its direct phosphorylation. In contrast to these results, neither cAMP-dependent protein kinase, cGMP-dependent protein kinase nor Ca2+/calmodulin-dependent protein kinase II phosphorylated the Ca2(+)-ATPase in the sarcolemmal membrane or the purified enzyme preparation, and also they exerted virtually no effect on Ca2+ uptake by sarcolemmal vesicles.

Animals↗
PubMed1990

Effects of pinealectomy on the mitotic activity of adrenomedullary chromaffin cells in relation to time of day.

The frequency of mitoses of adrenaline (A) cells and noradrenaline (N) cells in the adrenal medulla of nonoperated (NO), sham-operated (SPX), and pinealectomized (PX) male, 53-day-old Holtzman rats (n = 133) was investigated by means of light microscopy. Animals were killed at eight time points during a standardized 24-h light-dark (12:12) cycle 14 days after surgery. Mitotic indices (n/1,000) were determined in sections of adrenal medulla fixed with glutaraldehyde and OsO4. Overall frequency of mitoses was extremely low (mitotic index: 0.73 = 115/157,223). Daily mean mitotic index was maximum in A cells (0.83) and minimum in N cells (0.52) of PX group but did not show statistically significant differences between cell types or experimental groups. Neither cell type in NO animals showed 24-h changes in mitotic index, but cells in SPX animals did, with highest value in the late dark phase and lowest in the late light phase, when values of two cell types were combined (P less than 0.01-0.001). In PX animals, mitotic index followed a similar but more distinct 24-h change in A cells (P less than 0.009), but not in N cells, resulting in different time-of-day changes between two types of cells (P less than 0.01-0.05). The mitotic index was higher in PX than in control (NO and SPX) animals in the middark phase (P less than 0.05) and lower in operated (SPX and PX) than in nonoperated (NO) animals from late light to the early dark phase, suggesting that the latter was possibly due to a residual effect of the surgery. These results are consistent with the interpretation that the pineal has an inhibitory action on A cells and may coordinate the two types of cells in their mitotic activity, especially in the middark phase of the daily cycle.

Adrenal Medulla↗
PubMed1989

Physicochemical properties of dexamethasone palmitate, a high fatty acid ester of an anti-inflammatory drug: polymorphism and crystal structure.

Two polymorphic crystalline forms of dexamethasone palmitate were obtained from acetone (Form A) and n-heptane (Form B), and characterized by X-ray powder patterns and IR spectra. The crystal structure of Form B was further analyzed by the X-ray diffraction method. The molecular conformation of the hydrocarbon chain was shown to be fully extended and nearly at right angles to the dexamethasone ring. A definite separation between the lipophilic and hydrophilic rows, which consist of palmityl and dexamethasone layers, respectively, was evident in the crystal structure. Utilizing the conformational and molecular packing similarities between dexamethasone palmitate and phospholipid molecules, a possible interaction mode between them is proposed and outlined in this paper.

Chemical Phenomena↗