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T Shimizu

Publications and source records attributed to T Shimizu.

At least 19 recordsLinked to original sources
PubMed1999

Critical duration of intracellular Ca2+ response required for continuous translocation and activation of cytosolic phospholipase A2.

When cells are exposed to certain external stimuli, arachidonic acid (AA) is released from the membrane and serves as a precursor of various types of eicosanoids. A Ca2+-regulated cytosolic phospholipase A2 (cPLA2) plays a dominant role in the release of AA. To closely examine the relation between Ca2+ response and AA release by stimulation of G protein-coupled receptors, we established several lines of Chinese hamster ovary cells expressing platelet-activating factor receptor or leukotriene B4 receptor. Measurement of intracellular Ca2+ concentration ([Ca2+]i) demonstrated that cell lines capable of releasing AA elicited a sustained [Ca2+]i increase when stimulated by agonists. The prolonged [Ca2+]i elevation is the result of Ca2+ entry, because this elevation was blocked by EGTA treatment or in the presence of Ca2+ channel blockers (SKF 96365 and methoxyverapamil). cPLA2 fused with a green fluorescent protein (cPLA2-GFP) translocated from the cytosol to the perinuclear region in response to increases in [Ca2+]i. When EGTA was added shortly after [Ca2+]i increase, the cPLA2-GFP returned to the cytosol, without liberating AA. After a prolonged [Ca2+]i increase, even by EGTA treatment, the enzyme was not readily redistributed to the cytosol. Thus, we propose that a critical time length of [Ca2+]i elevation is required for continuous membrane localization and full activation of cPLA2.

Animals↗
PubMed1999

Crystal structure of neuropsin, a hippocampal protease involved in kindling epileptogenesis.

Neuropsin is a novel serine protease, the expression of which is highly localized in the limbic areas of the mouse brain and which is suggested to be involved in kindling epileptogenesis and hippocampal plasticity. The 2.1-A resolution crystal structure of neuropsin provides the first three-dimensional view of one of the serine proteases highly expressed in the nervous system, and reveals a serine protease fold that exhibits chimeric features between trypsin and nerve growth factor-gamma (NGFgamma), a member of the kallikrein family. Neuropsin possesses an N-glycosylated "kallikrein loop" but forms six disulfide bonds corresponding to those of trypsin. The ordered kallikrein loop projects proline toward the active site to restrict smaller residues or proline at the P2 position of substrates. Loop F, which participates in forming the S3/S4 sites, is similar to trypsin rather than NGFgamma. The unique conformations of loops G and H form an S1 pocket specific for both arginine and lysine. These characteristic loop structures forming the substrate-binding site suggest the novel substrate specificity of neuropsin and give a clue to the design of its specific inhibitors.

Amino Acid Sequence↗
PubMed1999

Thalidomide increases both REM and stage 3-4 sleep in human adults: a preliminary study.

Polysomnography was used to assess the effect of thalidomide on human sleep. This compound significantly increased the time spent in REM and stage 3-4 sleep as compared with placebo. On the other hand, thalidomide significantly decreased the time spent in stage 1, while the time spent in stage 2 was unchanged. The effect of thalidomide on REM and stage 3-4 sleep is unique as compared with other hypnotics. Although the mode of action of this compound is unknown, further studies on thalidomide should help in our understanding of the mechanisms of sleep regulation.

Adult↗
PubMed1999

Splenic artery aneurysm associated with systemic lupus erythematosus: report of a case.

We herein report on a 64-year-old Japanese female patient who presented with a splenic artery aneurysm (SAA) associated with systemic lupus erythematosus (SLE). The saccular aneurysm, which measured 3 cm in diameter, was located in the proximal third of the splenic artery from the pancreas with a portosystemic shunt. A double ligation of the splenic artery (the distal and proximal sides of the aneurysm) was performed without a splenectomy. The postoperative course showed acute pancreatitis without either splenic infarction or portal thrombus. To our knowledge, the closed association of SLE with an aneurysmal dilatation of the splenic artery has not been previously reported. Both the pathogenesis and the management of SAA associated with SLE are discussed following the presentation of this case. This is the first reported case of SAA associated with SLE.

Aneurysm↗
PubMed1999

Characterization of the leukotriene B4 receptor in porcine leukocytes. Separation and reconstitution with heterotrimeric GTP-binding proteins.

Leukotriene B4 (LTB4) is a potent chemoattractant derived from arachidonic acid. When cDNAs for LTB4 receptor (BLT) were cloned it was found that they belong to a guanine nucleotide-binding regulatory protein (G-protein)-coupled receptor superfamily. However, purification of BLT from inflammatory cells and reconstitution with various types of G-proteins have not been successful. In the present study, BLT from porcine leukocytes was solubilized, separated from associated G-proteins by Ricinus communis agglutinin (RCA) 120 chromatography, and reconstituted with several endogenous and exogenous G-proteins, in combination with the fraction which contained endogenous phospholipids and Gbeta gamma. Kinetic studies of LTB4 were performed to determine the association with G-proteins. A partially purified BLT fraction (retained on an RCA120 column) free of G-proteins showed a lower affinity for LTB4 (Kd = 500 nm), but reconstitution of the BLT fraction with a G-protein-rich fraction (flow-through of an RCA column) increased the affinity for LTB4 10-fold (Kd = 50 nm). The partially purified BLT fraction was also reconstituted with exogenous G-proteins such as a heterotrimeric Gi2 purified from bovine brain or recombinant alpha subunits of Gi1, Gi2, Gi3, and Go expressed in Spodoptera frugiperda-9 cells. These increases in LTB4 bindings demonstrate that the BLT of porcine leukocytes can interact with pertussis toxin-sensitive G-proteins in vitro. The method is useful for the purification and reconstitution of other, as yet unisolated, G-protein-coupled receptors.

Animals↗
PubMed1999

Ultraviolet B radiation upregulates the production of macrophage migration inhibitory factor (MIF) in human epidermal keratinocytes.

Human epidermal cells are capable of secreting various cytokines with immunologic, inflammatory, and proliferative properties. In a previous study, by reverse transcription-polymerase chain reaction and immunohistochemical analysis, we have shown that human epidermal keratinocytes express macrophage migration inhibitory factor and identified its presence in the cytoplasm. In this study, we detected an increased serum macrophage migration inhibitory factor level by enzyme-linked immunosorbent assay after a single total-body ultraviolet B exposure in vivo, indicating that human keratinocytes respond and release this cytokine in response to ultraviolet B irradiation. Moreover, we evaluated the effect of ultraviolet B on migration inhibitory factor production in cultured human epidermal keratinocytes and epidermal sheets. The results of enzyme-linked immunosorbent assay and northern blot analyses showed that migration inhibitory factor production of cultured keratinocytes was increased by ultraviolet B exposure. During the past few years, migration inhibitory factor was found to have a variety of biologic functions, such as being essential for T cell activation and induction of inflammatory cytokines. In this context, these results should encourage further investigation on the pathophysiologic role of migration inhibitory factor in cutaneous inflammatory reactions and immune responses.

Cell Survival↗
PubMed1999

Ubiquitous localization of leukotriene A4 hydrolase in the rat nephron.

BACKGROUND: Leukotriene (LT) B4 is a well-known inflammatory mediator and is implied to play some roles in glomerulonephritis. Although LTA4 hydrolase, a final-step key enzyme to produce LTB4, is located in glomerular mesangial cells, as well as in leukocytes, platelets, and endothelial cells, its precise distribution in the kidney other than in mesangial cells remains unknown. Therefore, we have investigated the localization of mRNA, protein, and enzyme activity of LTA4 hydrolase in the rat kidney. METHODS: Microdissection reverse transcriptase-polymerase chain reaction was used for the determination of LTA4 hydrolase mRNA. The enzyme protein was detected by Western blot, and immunohistochemistry was performed. Finally, LTA4 hydrolase activity and LTB4 were assayed in kidney tissues. RESULTS: LTA4 hydrolase mRNA was detectable in all microdissected nephron segments of the cortex and outer medulla. The corresponding size of approximately 70 kDa protein was shown in descending order in the inner medullary > outer medullary >/= cortical homogenates. The immunohistochemical study demonstrated the ubiquitous presence of the enzyme in all nephron segments of cortex, outer medulla, and inner collecting tubules. LTA4 hydrolase activity was detected in the inner medullary >/= outer medullary >/= cortical tissue homogenates. LTB4 was demonstrated in the inner medullary > outer medullary >/= cortical tissues during the basal condition, and was time-dependently increased by stimulation with arachidonic acid and ionomycin in the cytosolic fraction from outer medulla and in the glomerular suspension. CONCLUSIONS: These results strongly suggest that renal tubular cells as well as glomerular cells have an LTB4-forming potency, which may participate in physiological and pathophysiological roles in the kidney.

Animals↗
PubMed1999

Using hydroxylapatite ceramic buttons covered with periosteum to prevent cerebrospinal fluid leakage: technical note.

BACKGROUND: Many attempts have been made to prevent cerebrospinal fluid (CSF) leakage, but the procedures proposed to date are troublesome and not promising. We describe a method of completely preventing CSF leakage using hydroxylapatite ceramics. METHODS: Exposed frontal sinus, mastoid air cells, and frontal base defects caused by fracture are covered with periosteum or fascia and plugged with a trimmed hydroxylapatite ceramic button. RESULTS: There was no CSF leakage, postoperative meningitis, or other complication related to the technique in a consecutive series of 25 patients treated using this procedure. CONCLUSION: The use of this easy and quick technique may prevent CSF leakage completely.

Ceramics↗
PubMed1999

Therapeutic effects of benzoxazinorifamycin KRM-1648 administered alone or in combination with a half-sized secretory leukocyte protease inhibitor or the nonsteroidal anti-inflammatory drug diclofenac sodium against Mycobacterium avium complex infection in mice.

The effects of half-sized secretory leukocyte protease inhibitor or diclofenac sodium administered alone or in combination with the benzoxazinorifamycin KRM-1648 on the therapeutic efficacy of KRM-1648 against Mycobacterium avium complex (MAC) in mice were studied. Neither of the two anti-inflammatory drugs affected the efficacy of KRM-1648, while they exerted significant modulating effects on tumor necrosis factor alpha production by MAC-infected macrophages.

Animals↗
PubMed1998

Autoxidation rates of neuronal nitric oxide synthase: effects of the substrates, inhibitors, and modulators.

Autoxidation rates of the full-length neuronal nitric oxide synthase (nNOS) were analyzed and found to be composed of three phases, 60 s(-1) (28%), 5.5 s(-1) (11%) and 0.048 s(-1) (61%). Addition of L-Arg, N(G)-hydroxy-L-Arg (NHA), and N(G)-monomethyl-L-Arg markedly decreased the rate constants for the first and second phases down to 12-20 s(-1) and 0.32-2.6 s(-1), respectively. Addition of (6R)-5,6,7,8-tetrahydro-L-biopterin (H4B) increased the amplitude of the second phase up to 29% of the total. Addition of NHA decreased the rate of the first phase by 4.4-fold in the presence of H4B, whereas addition of L-Arg and other modulators did not significantly affect the rates under the same conditions. Thus, we deduce that (1) L-Arg stabilizes the O2-bound ferrous complex for efficient O-O bond cleavage to occur; (2) H4B influences the O2-bound ferrous complex in a fashion different from L-Arg; and (3) NHA induces a characteristic distal-site structure in the presence of H4B, reflecting a difference in the mechanism of activation of O2 in the first step (monooxygenation of L-Arg) and the second step (monooxygenation of NHA).

Animals↗
PubMed1998

Conditional expression of the dual-specificity phosphatase PYST1/MKP-3 inhibits phosphorylation of cytosolic phospholipase A2 in Chinese hamster ovary cells.

Stimulation of platelet-activating factor (PAF) receptor induces activation of extracellular signal-regulated kinase (ERK) and cytosolic phospholipase A2 (cPLA2) and release of arachidonic acid in Chinese hamster ovary cells. To determine whether the dual-specificity protein phosphatase PYST1/MKP-3 inhibits phosphorylation of cPLA2, we have generated a cell line that conditionally expresses PYST1 under the control of a tetracycline-regulated inducible system. We found that induction of PYST1 suppressed phosphorylation and activation of cPLA2 as well as ERK. Arachidonic acid release was also reduced by about 30%. Pretreatment of cells with an MEK inhibitor, PD98059, had similar effects on PAF-induced cPLA2 phosphorylation and arachidonic acid release. These experiments demonstrate that expression of PYST1 prevents phosphorylation of a cytoplasmic substrate for ERK. Thus, this inducible system may offer a valuable means of investigating physiological roles of ERK in vivo.

Animals↗
PubMed1998

Detection of O-mannosyl glycans in rabbit skeletal muscle alpha-dystroglycan.

alpha-Dystroglycan, which is a cell surface component of dystroglycan complex, is known to bind laminin in basal lamina of muscle cells and Schwann cells. We found previously that a novel O-glycan, Siaalpha2-3Galbeta1-4GlcNAcbeta1-2Man, is the major oligosaccharide in bovine peripheral nerve alpha-dystroglycan, and that this structure might mediate the binding of laminin. In order to determine whether this structure is specific for peripheral nerve alpha-dystroglycan or present on different forms of alpha-dystroglycan, we analyzed the structures of the sialylated O-glycans of rabbit skeletal muscle alpha-dystroglycan. Their structures were elucidated to be a mixture of a core 1 O-glycan and the same O-mannosyl glycan that we found in bovine peripheral nerve. These results indicate that alpha-dystroglycan in different species and tissues share a common structure of its major O-linked acidic carbohydrate, suggesting its relevance to the basic functional role of alpha-dystroglycan.

Animals↗
PubMed1998

Spectroscopic studies of the ncd motor domain.ADP complex: CD spectrum of ADP induced by binding to the motor domain of ncd.

Previously, we reported that the nucleotide-free ncd motor domain exhibited a near-UV CD spectrum different from that of the ordinary ncd motor domain.ADP complex [Shimizu and Morii, (1996) J. Biochem. 120, 1176-1181]. In the present study, we exchanged the bound nucleotide ADP with N6-methylADP (MeADP) which has a UV absorption spectrum different from that of ADP. The resultant ncd motor domain. MeADP complex gave a near-UV CD spectrum different from that of the ordinary ncd motor domain with bound ADP. This result indicates that the bound nucleotide contributes to the near-UV CD spectra to a considerable extent although ADP or MeADP free in solution gives a spectrum with negligible peaks and troughs. In addition, the absorption intensity of ADP or MeADP at the peak wavelengths decreased to a considerable extent upon binding to the nucleotide-free ncd motor domain. It is suggested that interaction between adenine moiety and chromophore(s) of the protein contributed to the spectral changes of ADP. A candidate chromophore is Tyr442 which is stacked with the adenine moiety at a distance of 0.43 nm. On the other hand, we detected an intensity decrease of tryptophanyl fluorescence upon binding of a nucleotide to the nucleotide-free ncd motor domain, while at the same time tyrosyl fluorescence increased. The fluorescence changes, as well as the UV absorption change described above, gave similar rates upon addition of a nucleotide to the nucleotide-free ncd motor domain. Therefore, they are likely to originate from the same conformational change of the protein.

Adenosine Diphosphate↗
PubMed1998

Cyclic GMP-dependent cytoprotection against ethanol-induced damage in rabbit isolated gastric parietal cells.

Prostaglandin E2 stimulates a nitric oxide/cyclic GMP (NO/cGMP) pathway which activates basolateral Cl- channels in rabbit gastric parietal cells. We examined whether the NO/cGMP pathway protects parietal cells from ethanol (EtOH)-induced cytotoxicity, using a parietal cell-rich suspension purified from rabbit gastric mucosa. Cytotoxicity was assayed by measuring the release of a fluorescent dye from the cells. N2,O2-dibutyryl guanosine 3',5'-cyclic monophosphate (DBcGMP) showed a concentration-dependent protective effect against EtOH-induced cytotoxicity. The half-maximal effect of DBcGMP was observed at 24 microM. DBcGMP in a concentration-dependent manner opened the basolateral Cl- channels of parietal cells, the EC50 value being 44 microM. The EtOH-induced cytotoxicity decreased as the Cl- concentration of medium decreased. A 30-s treatment with 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), an inhibitor of the Cl- channel, had a cytotoxic effect which was not prevented by pre-incubation with DBcGMP. The cytotoxic effects of EtOH and NPPB were additive and the NPPB effects did not depend on the medium Cl- concentration. The present study showed that cGMP protects the gastric parietal cell from EtOH-induced cytotoxicity, and this cytoprotection is related to basolateral Cl- channel activity in the plasma membrane via an unknown mechanism(s).

Animals↗
PubMed1998

Evidence for existence of multiple conformations of kinesin and ncd motor domains in solution revealed by 31P-NMR of the tightly bound ADP.

The motor domains of kinesin and ncd contain ADP bound tightly at the enzymatic active sites even after purification. The microenvironments surrounding the phosphates of the tightly bound ADP molecules in these motor domains were studied using 31P-NMR. The alpha-phosphate and beta-phosphate of the tightly bound ADP molecules gave 31P-NMR signals at positions shifted to higher and lower field, respectively, from those of free MgADP in solution. On the other hand, the peak areas of the signals of the alpha-phosphates and beta-phosphates of bound ADP were much smaller than those of free MgADP. The temperature dependence of these signals was investigated to reveal that each signal of bound MgADP exhibited a large temperature dependence in the peak area from 0 to 20 degrees C with both proteins. This result implied that these motor domains would take multiple conformations in solution; one of these conformations gives the signals of bound MgADP observed by 31P-NMR as above, while another makes the signals of MgADP very broad and, hence, invisible in the 31P-NMR spectra. A possible reason for invisible signals would be strong chemical shift anisotropy effects of the phosphorus nuclei. In addition, we found a difference between kinesin and ncd motor domains in the chemical shift of the alpha-phosphate of bound MgADP, indicating that the electrostatic or magnetic microenvironments of this site of these motor domains differed from each other.

Adenosine Diphosphate↗
PubMed1998

Marked enhancement in the reductive dehalogenation of hexachloroethane by a Thr319Ala mutation of cytochrome P450 1A2.

Mutation of the conserved Thr319 residue to Ala of cytochrome P4501A2 (CYP1A2) increased the value of Vmax 9-fold for reductive dehalogenation of hexachloroethane in the reconstituted system under anaerobic conditions. The Thr319Ala mutation also increased the elimination over substitution product ratio by 5-fold. The addition of aliphatic alcohols increased by 22-fold the activity obtained with the wild type and varied the elimination over substitution product ratio. Increasing pH increased the ratio of elimination over substitution by primarily affecting the rate of elimination.

Alcohols↗
PubMed1998

The cholesterol metabolite cholest-4-en-3-one and its 3-oxo derivatives suppress body weight gain, body fat accumulation and serum lipid concentration in mice.

Based on the findings that cholest-4-en-3-one, an intestinal metabolite of cholesterol, has an anti-obesity effect on animals, the structure-effect relationship of its 3-oxo derivatives and related compounds were investigated. Cholesten-3-ones, which possesses an enone structure with a carbonyl group at C3, markedly inhibit body weight gain and body fat accumulation, as well as the levels of serum triglyceride and cholesterol in animals without any clinical abnormalities.

Adipose Tissue↗