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Biomedical subjects

A Watabe

Publications and source records attributed to A Watabe.

At least 19 recordsLinked to original sources
PubMed1999

Target-cell specificity of fusogenic liposomes: membrane fusion-mediated macromolecule delivery into human blood mononuclear cells.

Fusogenic liposome, a unique vector prepared by fusing ultraviolet-inactivated Sendai virus and liposome, is known to efficiently deliver content into various animal cells through membrane fusion. In this study, we examined the target-cell specificity of fusogenic liposome (FL)-mediated macromolecule delivery into human blood cells using diphtheria toxin fragment A (DTA) as a probe. Among the peripheral blood mononuclear cells (PBMC), FL was able to deliver its encapsulates into CD14+ monocytes and CD4-/CD8- T-cells, but not into CD19+ B-lymphocytes, CD4+ T-cells or CD8+ T-cells. The susceptibility of human leukemia cell lines to FL was similar to that of PBMC; the order of the reactivity was U937 (monoblastic leukemia)>MOLT4, Jurkat (T-lymphoma)>Daudi, BALL1 (B-lymphoma)>K562 (erythroblastic leukemia). Interestingly, FL showed similar binding activity to all of these leukemia cell lines. These findings indicate that, among blood cells, monocytes, monoblastic leukemia cells, CD4-/CD8- T-cells and T-lymphoma cells are preferable targets for FL-mediated macromolecule delivery. This is the first demonstration of the existence of non-permissive cells against FL. Our results also suggest that some molecules on target-cells other than the binding targets of SV-derived protein may participate in fusion between FL and cells.

Diphtheria Toxin↗
PubMed1998

Gene transfer vectors based on Sendai virus.

A gene delivery system is a fundamental technology used in human gene therapy. In order to treat patients suffering from incurable metabolic diseases, we must be able to deliver genes efficiently in situ and induce stable gene expression in non-dividing tissue cells. However, none of the current gene transfer systems (both viral and non-viral) satisfies this goal. In order to develop a novel gene delivery system that is free from the defects of existing gene transfer vectors, we analyzed natural biological phenomena that involve gene transfer and expression, and made artificial components that mimic the functioning of these systems. Our recent results shed light on three major aspects of gene transfer and expression: (1) the direct delivery of DNA into cytoplasm using fusogenic liposomes, (2) the transfer of DNA from cytoplasm to nucleus with a nuclear localization signal, and (3) the stabilization of DNA in the nucleus as an independent replicon. The possible development of a hybrid vector by combining these components is discussed.

Cell Nucleus↗
PubMed1997

Characterization of plasmin-induced platelet aggregation.

This study was undertaken to determine if reocclusion after treatment of myocardial infarction with a tissue-plasminogen activator (t-PA) may be due to plasmin-induced platelet aggregation. t-PA caused platelet aggregation by conversion of plasminogen to plasmin under certain conditions. Plasmin-induced platelet aggregation was inhibited by serine protease inhibitors, aprotinin and bdellin, and a lysine binding site inhibitor, epsilon-aminocaproic acid (EACA). Extracellular [Ca2+], and RGDS sequence-dependent steps were involved in the aggregation process. The action of plasmin was inhibited by large thrombin antagonistic molecules such as argatroban-inactivated thrombin or anti-thrombin receptor peptide antibodies but not by small molecules like thrombin receptor antagonist peptides. This suggests that target molecules of plasmin on the surface of platelets may not be thrombin receptors but may exist very close to thrombin receptors. Binding experiments using FITC-labeled plasmin showed that plasmin has its binding sites on platelets. Flow cytometric analyses with four types of anti-plasmin(ogen) monoclonal antibodies suggested that plasmin might bind to platelets through the N-terminal region. The binding of plasmin to platelets was suppressed by aprotinin and EACA, furthermore indicating that protease catalytic sites and lysine binding regions are involved in interaction of plasmin to platelet.

Aminocaproic Acid↗
PubMed1995

Characterization of the signal transduction of prostaglandin E receptor EP1 subtype in cDNA-transfected Chinese hamster ovary cells.

We examined the signal transduction of mouse prostaglandin E receptor EP1 subtype using Chinese hamster ovary cells stably expressing the cloned EP1. Sulprostone, an EP1 agonist, induced a rapid increase in intracellular Ca2+ concentration in the EP1-expressing cells. Most of the increase was abolished by removal of extracellular Ca2+, and was insensitive to U-73122, a phospholipase C inhibitor. Sulprostone stimulated phosphatidylinositol hydrolysis, but this stimulation was abolished by removal of extracellular Ca2+, indicating that EP1-stimulated phosphatidylinositol hydrolysis is the result of extracellular Ca2+ influx. Thus, the signal transduction of EP1 is extracellular Ca2+ entry through a pathway independent of phospholipase C activation. We further examined the regulation of the signal transduction of EP1 having potential phosphorylation sites for either protein kinase C or protein kinase A. Short-term exposure of the cells to 12-O-tetradecanoylphorbol 13-acetate (TPA) completely suppressed the sulprostone-induced increase in intracellular Ca2+ concentration, while forskolin or dibutyryl cAMP did not affect it, suggesting that protein kinase C but not protein kinase A is involved in the regulation of the EP1 signal transduction. Furthermore, long-term exposure to TPA decreased PGE2 protein kinase A is involved in the regulation of the EP1 signal transduction. Furthermore, long-term exposure to TPA decreased PGE2 binding activity of EP1 due to the reduction of the EP1 mRNA level. Protein kinase C induces short- and long-term desensitization of EP1.

Animals↗
PubMed1994

[A 10-year field surveillance in Hinohara Village of Tokyo Prefecture from 1981 to 1990].

We conducted a surveillance to clarify the relationship between risk factors for diseases of adulthood and lifestyle in a Japanese rural community, Hinohara Village, a small village outside of Tokyo. The survey, carried out from 1981 to 1990 among residents aged 40 and over, comprised physical examination and blood chemistry with a questionnaire about dietary intake. Mean systolic blood pressure significantly decreased (p < 0.0001) from 140.9 mmHg in 1981 to 132.3 mmHg in 1990, whereas mean serum total cholesterol, mainly of male examinees, increased (p < 0.0001) from 181.4 mg/dl in 1981 to 191.7 mg/dl in 1990. Dietary salt intake significantly decreased (p < 0.0001) from 14.3 g/day in 1981 to 12.1 g/day in 1990. Adjusted mortality rate per 1,000 residents from cerebrovascular disease in this village decreased from 1.80 in 1981 to 0.50 in 1990. In contrast to its decline, the mortality rates from heart disease, bronchitis/pneumonia and neoplasms were 0.40, 0.35 and 0.55 in 1981 and increased to 1.25, 1.10 and 0.64 in 1990. The prevailing practice of maintaining a low-salt diet might cause the decrease of systolic blood pressure, which in turn was thought to decrease the mortality rate from cerebrovascular diseases. Although our previous study before 1981 suggested that total cholesterol was one of the preventive factors against cerebrovascular disease, in the present study a preventive effect of cholesterol was not substantiated. In contrast, cholesterol is a possible risk factor for ischemic heart disease. Thus, a changing pattern of risk factors of diseases of adulthood was observed in this village.

Adult↗
PubMed1993

Third isoform of the prostaglandin-E-receptor EP3 subtype with different C-terminal tail coupling to both stimulation and inhibition of adenylate cyclase.

A functional cDNA clone for a third isoform of the mouse prostaglandin-E-receptor EP3 subtype, derived by alternative RNA splicing, named the EP3 gamma receptor, was obtained in addition to those for the two other isoforms, EP3 alpha and EP3 beta. The three isoforms are only different in the amino acid sequence of the putative cytoplasmic carboxy-terminal tail. When expressed, EP3 gamma shows identical ligand-binding properties to these of the other isoforms. The EP3-selective agonist, M&B 28767, increased the basal cAMP level and inhibited the forskolin-induced increase in the cAMP level in EP3 gamma, while it decreased both the basal and forskolin-elevated cAMP levels in EP3 alpha and EP3 beta. The M&B 28767-stimulated GTPase activity consisted of pertussis-toxin-sensitive and cholera-toxin-sensitive portions in the EP3 gamma-expressing cell membrane, suggested that EP3 gamma is coupled to both guanine nucleotide-binding inhibitory and stimulatory proteins. These results indicate that EP3 gamma is coupled to both stimulation and inhibition of adenylate cyclase, but that EP3 alpha and EP3 beta are exclusively coupled to inhibition of adenylate cyclase. Thus, alternative splicing produces a third isoform with a different carboxy-terminal tail, which differs from the other two isoforms in the specificity of coupling to a signal-transduction pathway.

Adenylyl Cyclase Inhibitors↗
PubMed1993

Cloning and expression of cDNA for a mouse EP1 subtype of prostaglandin E receptor.

A functional cDNA clone encoding a mouse EP1 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse thromboxane A2 receptor cDNA. The clone isolated encodes a protein consisting of 405 amino acid residues with putative seven-transmembrane domains. [3H]PGE2 specifically bound to the membrane of Chinese hamster ovary cells expressing this clone. The binding to the membrane was displaced with unlabeled PGs in the order of PGE2 > iloprost (a prostacyclin analogue) > PGE1 > PGF2 alpha > U-46619 (a thromboxane A2 analogue) > PGD2. The binding was also inhibited by 17-phenyl trinor PGE2 (an EP1 agonist) and sulprostone (an EP1 and EP3 agonist) but not by 11-deoxy PGE1 (an EP2 and EP3 agonist) and butaprost (an EP2 agonist). PGE2 induced a rapid increase in intracellular Ca2+ concentration in Chinese hamster ovary cells expressing the receptor. These results suggest that this receptor belongs to EP1 subtype of PGE receptor. Northern blot analysis demonstrated that the mRNA of this receptor is expressed abundantly in kidney and in a lessor amount in lung.

Amino Acid Sequence↗
PubMed1993

Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2 subtype.

A functional cDNA clone encoding mouse EP2 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse EP3 subtype PGE receptor cDNA. The mouse EP2 receptor consists of 513 amino acid residues with putative seven-transmembrane domains. In contrast to EP3 receptor, this receptor possesses long third intracellular loop and carboxyl-terminal tail. [3H] PGE2 specifically bound to the membrane of mammalian COS cells transfected with the cDNA. The binding to the membrane was displaced with unlabeled PG in the order of PGE2 = PGE1 >> iloprost > or = PGF2 alpha > or = PGD2. The binding was also inhibited by misoprostol, an EP2 and EP3 agonist, but not by sulprostone, an EP1 and EP3 agonist, and SC-19220, an EP1 antagonist. PGE2 markedly increased cAMP level in COS cells transfected with the cDNA. These results suggest that this receptor is EP2 subtype. Northern blot analysis demonstrated that the EP2 mRNA is widely expressed in various tissues, the abundant expression being observed in ileum, thymus, and mastocytoma P-815 cells.

Amino Acid Sequence↗
PubMed1993

Functional interaction of prostaglandin E receptor EP3 subtype with guanine nucleotide-binding proteins, showing low-affinity ligand binding.

The functional interaction of prostaglandin E (PGE) receptor EP3 subtype with GTP-binding proteins (G proteins) was characterized in the membranes prepared from mouse EP3 receptor cDNA-transfected Chinese hamster ovary cells. PGE2 inhibited forskolin-stimulated adenylate cyclase activity in CHO cells expressing EP3 receptor and this inhibition was abolished by pertussis toxin (PT) treatment. The PGE2 binding to the membranes was increased by GTP gamma S, and PT treatment also increased the binding activity to the same level as that increased by GTP gamma S, but the sensitivity of GTP gamma S was lost. Reconstitution with PT-sensitive G proteins into the ADP-ribosylated membranes reduced the PGE2 binding activity with the following preference: Gi1 = Gi2 > Gi3 > GO, but GTP gamma S completely blocked the reduction by G proteins. The G-protein-induced reduction of the binding was due to the increase in Kd without the change of Bmax, and due to suppression of association rate. [3H]PGE2-bound EP3 receptor solubilized from the ADP-ribosylated membranes in the presence or absence of GTP gamma S was eluted at the position of M(r) = approx. 60 kDa, similar to the relative molecular mass of EP3 receptor deduced from its amino acid sequence. In contrast, [3H]PGE2-bound receptor solubilized from Gi2-reconstituted membranes was eluted at the position of M(r) = approx. 130 kDa, corresponding to the M(r) of the complex of EP3 receptor and Gi2, but GTP gamma S shifted the position of its elution from M(r) = 130 to 60 kDa. Furthermore, addition of PGE2 stimulated the GDP release from G proteins reconstituted into the ADP-ribosylated membranes, and PGE2 inhibited forskolin-stimulated adenylate cyclase activity in G-protein-reconstituted membranes with a selectivity order of Gi1 = Gi2 > Gi3 > GO. These results indicate that EP3 receptor can functionally couple to PT-sensitive G proteins and unusually the complex form with G proteins has low affinity for the ligand but the form not associated with G proteins has high affinity.

Adenosine Diphosphate↗
PubMed1993

Two isoforms of the EP3 receptor with different carboxyl-terminal domains. Identical ligand binding properties and different coupling properties with Gi proteins.

Functional cDNA clones for two isoforms of the mouse prostaglandin E receptor EP3 subtype derived from alternative RNA splicing were obtained. The two isoforms are only different in the sequence of the putative cytoplasmic carboxyl-terminal tail and their hydrophobicity; one isoform, named EP3 alpha, has a hydrophilic tail, and the other, named EP3 beta, has a hydrophobic tail. When expressed, the two receptors displayed identical ligand binding properties but different responses to guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Without a change in the Bmax value, GTP gamma S increased Kd for prostaglandin E2 of EP3 beta and decreased that of EP3 alpha. These effects were abolished by the treatment of membranes with pertussis toxin and restored by the addition of Gi2. Although both isoforms exerted inhibition of forskolin-induced cAMP accumulation, three orders lower concentrations of agonists were required for EP3 alpha than EP3 beta for 50% inhibition of cAMP formation. A similar difference in agonist potency was observed also for agonist-induced stimulation of GTPase activity in membranes. Thus, the two receptors with different carboxyl-terminal tails show different coupling to the Gi protein, leading to the opposite responses to GTP in the ligand binding affinity and to different affinities of the agonist-occupied receptors to the G proteins.

Adenylyl Cyclases↗
PubMed1992

Mouse thromboxane A2 receptor: cDNA cloning, expression and northern blot analysis.

A cDNA clone for the mouse thromboxane A2 receptor was isolated from mouse lung cDNA library. The cDNA has a 1,023 base pair open reading frame which encodes a protein of 341 amino acid residues. STA2 and U-46619 induced inward current in Xenopus laevis oocytes injected with the transcript of the clone. Specific binding of [3H]S-145 was found in membranes of COS-1 cells transfected with the cDNA (Kd = 3.3 nM) and was displaced with unlabeled prostaglandins and thromboxane analogues in the order of S-145 greater than STA2 greater than U-46619 greater than PGD2 greater than PGF2 alpha = PGE2. Northern blot analysis demonstrated that thromboxane A2 receptor mRNA is expressed abundantly in thymus, spleen and lung.

Amino Acid Sequence↗
PubMed1992

Purification and properties of L-histidine decarboxylase from mouse stomach.

L-Histidine decarboxylase was purified to electrophoretic homogeneity from mouse stomach according to a procedure described previously [Ohmori E, Fukui T, Imanishi N, Yatsunami K and Ichikawa A, J Biochem (Tokyo) 107: 834-839, 1990]. The purified enzyme exhibited a specific activity of 750 nmol histamine formed per min per mg protein, which constituted a 37,500-fold purification compared to the crude extract, with a 1.6% yield. The molecular mass of the enzyme was estimated to be 54 kDa by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 100 kDa by gel filtration. The isoelectric point of the enzyme was determined to be pH 5.4. The Km value for L-histidine was estimated to be 0.29 mM. The single mRNA encoding the amino acid sequence of the mouse stomach enzyme was examined and its size was found to be 2.7 kb. These molecular and catalytic property values of the L-histidine decarboxylase of mouse stomach are quite similar to those of the enzyme from mouse mastocytoma P-815 cells.

Animals↗
PubMed1991

Gas transport in serpentine microporous tubes under steady and pulsatile blood flow conditions.

A serpentine gas exchange unit was built with cylindrical tubular microporous membranes featuring periodic arcs with a fixed curvature ratio (ratio of tube radius to radius of curvature) of 1/14 and circular angles between 30 and 360 deg. Oxygen transfer was measured under steady and pulsatile blood flow conditions in vitro and ex vivo to assess the design features which most effectively augment gas transfer. Under steady blood flow conditions, oxygen transfer increased with circular angles beyond 70 deg. Under pulsatile conditions, a wide range of geometrical and fluid mechanical parameters could be combined to enhance gas transfer performance, which eventually depended upon the secondary Reynolds number and the Womersley parameter.

Animals↗
PubMed1986

Iodine content of various meals currently consumed by urban Japanese.

Various meals being currently consumed by urban Japanese were determined for iodine. The meal samples were collected in 1982 and 1984. The habitual daily home meals of 4 middle-aged Japanese living in urban areas contained 45-1,921 micrograms (mean; 362, 361, 429 and 1,023 micrograms, respectively) of iodine per day. The regular meals served in two university hospitals contained 95-287 micrograms (mean; 195 micrograms) and 89-4,746 micrograms (mean; 1,290 micrograms) of iodine per day, respectively, and the diets for diabetes mellitus contained 59-144 micrograms (mean; 96 micrograms) of iodine per day. In the daily meals containing iodine exceeding ca. 300 micrograms, some kinds of seaweeds and, in some cases, several foods containing a red food color with low iodine bioavailability, erythrosine, provided a large portion of iodine. The iodine contents of refectory meals in a university were 47-203 micrograms (mean; 113 micrograms) per meal and those of lunches in two elementary schools were 25-31 micrograms (mean; 27 micrograms) and 18-43 micrograms (mean; 36 micrograms) per lunch, respectively. These results suggest that the current daily iodine intake of urban Japanese is not great and that erythrosine elevates the iodine content of meals.

Child↗