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T Mitamura

Publications and source records attributed to T Mitamura.

At least 19 recordsLinked to original sources
PubMed2006

Intraerythrocytic Plasmodium falciparum utilize a broad range of serum-derived fatty acids with limited modification for their growth.

Plasmodium falciparum causes the most severe form of malaria. Utilization of fatty acids in serum is thought to be necessary for survival of this parasite in erythrocytes, and thus characterization of the parasite fatty acid metabolism is important in developing a new strategy for controlling malaria. Here, we examined which combinations of fatty acids present in human serum support the continuous culture of P. falciparum in serum-free medium. Metabolic labelling and gas chromatography analyses revealed that, despite the need for particular fatty acids for the growth of intraerythrocytic P. falciparum, it can metabolize a broad range of serum-derived fatty acids into the major lipid species of their membranes and lipid bodies. In addition, these analyses showed that the parasite's overall fatty acid composition reflects that of the medium, although the parasite has a limited capacity to desaturate and elongate serum-derived fatty acids. These results indicate that the Plasmodium parasite is distinct from most cells, which maintain their fatty acid composition by coordinating de novo biosynthesis, scavenging, and modification (desaturation and elongation).

Animals↗
PubMed2005

High-temperature and high-pressure in situ SCC device for synchrotron radiation diffraction experiments and application using an austenitic stainless steel.

Suppressing the stress corrosion cracking (SCC) by reducing the carbon content in austenitic stainless steels is apparently not effective on core shrouds used in boiling water reactors in Japan: trans-granular cracking was found in the shrouds. To clarify the mechanism of the cracking, in situ stress measurements on specimens under stretched conditions in hot water have been attempted in the present study. An in situ device for diffraction measurements at synchrotron radiation facilities has been developed, and in situ experiments have been carried out at SPring-8. The SUS316L steel specimen was solution heat-treated, surface-ground and then placed in the in situ device. Sapphire windows were used for the light path in the device. A sufficient diffracted beam intensity was obtained through two sapphire windows and water. The side-inclination method was used for measuring the stress exerted on the specimen. A 2theta-sin2psi plot showed that a tensile stress was induced. The measured stress value is considered to be the summation of stresses owing to pre-straining, in situ loading and residual stress owing to surface grinding.

Elasticity↗
PubMed2000

Vacuolar H(+)-ATPase localized in plasma membranes of malaria parasite cells, Plasmodium falciparum, is involved in regional acidification of parasitized erythrocytes.

Recent biochemical studies involving 2',7'-bis-(2-carboxyethyl)-5, 6-carboxylfluorescein (BCECF)-labeled saponin-permeabilized and parasitized erythrocytes indicated that malaria parasite cells maintain the resting cytoplasmic pH at about 7.3, and treatment with vacuolar proton-pump inhibitors reduces the resting pH to 6.7, suggesting proton extrusion from the parasite cells via vacuolar H(+)-ATPase (Saliba, K. J., and Kirk, K. (1999) J. Biol. Chem. 274, 33213-33219). In the present study, we investigated the localization of vacuolar H(+)-ATPase in Plasmodium falciparum cells infecting erythrocytes. Antibodies against vacuolar H(+)-ATPase subunit A and B specifically immunostained the infecting parasite cells and recognized a single 67- and 55-kDa polypeptide, respectively. Immunoelectron microscopy indicated that the immunological counterpart of V-ATPase subunits A and B is localized at the plasma membrane, small clear vesicles, and food vacuoles, a lower extent being detected at the parasitophorus vacuolar membrane of the parasite cells. We measured the cytoplasmic pH of both infected erythrocytes and invading malaria parasite cells by microfluorimetry using BCECF fluorescence. It was found that a restricted area of the erythrocyte cytoplasm near a parasite cell is slightly acidic, being about pH 6.9. The pH increased to pH 7.3 upon the addition of either concanamycin B or bafilomycin A(1), specific inhibitors of vacuolar H(+)-ATPase. Simultaneously, the cytoplasmic pH of the infecting parasite cell decreased from pH 7.3 to 7.1. Neither vanadate at 0.5 mm, an inhibitor of P-type H(+)-ATPase, nor ethylisopropylamiloride at 0.2 mm, an inhibitor of Na(+)/H(+)-exchanger, affected the cytoplasmic pH of erythrocytes or infecting parasite cells. These results constitute direct evidence that plasma membrane vacuolar H(+)-ATPase is responsible for active extrusion of protons from the parasite cells.

Acids↗
PubMed2000

Effects of mifepristone (RU486) treatment on the development of uterine adenomyosis induced by pituitary grafting in mice.

To evaluate the effects of mifepristone (RU486) on the development of uterine adenomyosis induced by pituitary grafting (PG), 3 groups of mice receiving pituitary grafts at 7 weeks of age were given RU486 in food (20 mg/kg chow) from 3-14 (RU486-3 group) or 10-14 (RU486-10 group) weeks of age, or were given no further treatment (PG control group), respectively. All the mice were killed at 14 weeks of age. The uterine weight was significantly decreased in both RU486-treated groups compared with the PG control group. The incidence of adenomyosis was also decreased significantly in both the RU486-3 group (0/10 mice) and RU486-10 group (2/10 mice) compared with the PG control group (7/9 mice). To look for vascular changes in the uterine tissues, which have been reported to be related to the development of adenomyosis, immunohistochemical staining of von Willebrand factor in the blood vessels was performed. The mean surface area and minor axis of blood vessels in the uterus were thereby found to be significantly decreased in the RU486-10 group compared to the PG control group. The results clearly indicated that RU486, a potent antiprogestin, could inhibit the genesis of uterine adenomyosis in mice, and at the same time caused shrinkage of the vascular system. As in humans, progesterone as well as the vascular system therefore appear to be important factors in the pathogenesis of uterine adenomyosis in this mouse model.

Adenomyoma↗
PubMed2000

Importance of the major extracellular domain of CD9 and the epidermal growth factor (EGF)-like domain of heparin-binding EGF-like growth factor for up-regulation of binding and activity.

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family of growth factors. The membrane-anchored form of HB-EGF (proHB-EGF) is mitogenically active to neighboring cells as well as being a precursor of the soluble form. In addition to its mitogenic activity, proHB-EGF has the property of binding to diphtheria toxin (DT), serving as the specific receptor for DT. Tetramembrane-spanning protein CD9, a member of the TM4 superfamily, is physically associated with proHB-EGF at the cell surface and up-regulates both mitogenic and DT binding activities of proHB-EGF. To understand this up-regulation mechanism, we studied essential regions of both CD9 and proHB-EGF for up-regulation. Immunoprecipitation experiments revealed that not only CD9 but also other TM4 proteins including CD63, CD81, and CD82 associate with proHB-EGF on the cell surface. However, these TM4 proteins did not up-regulate DT binding activity of proHB-EGF. Transfection of a series of chimeric constructs comprising CD9 and CD81 showed that the major extracellular domain of CD9 is essential for up-regulation. Assays of DT binding activity and juxtacrine mitogenic activity of the deletion mutants of proHB-EGF and chimeric molecules, derived from proHB-EGF and TGF-alpha, showed that the essential domain of proHB-EGF for up-regulation is the EGF-like domain. These results indicate that the interaction of the extracellular domains of both molecules is important for up-regulation.

Animals↗
PubMed2000

Neutral sphingomyelinase activity dependent on Mg2+ and anionic phospholipids in the intraerythrocytic malaria parasite Plasmodium falciparum.

Sphingolipid metabolism and metabolites are important in various cellular events in eukaryotes. However, little is known about their function in plasmodial parasites. Here we demonstrate that neutral sphingomyelinase (SMase) involved in the sphingomyelin (SM) catabolism is retained by the intraerythrocytic parasite Plasmodium falciparum. When assayed in a neutral pH buffer supplemented with Mg(2+) and phosphatidylserine, an activity for the release of the phosphocholine group from SM was detected in parasite-infected, but not in uninfected, erythrocyte ghosts. The SMase activity in the parasite-infected erythrocyte ghosts was enhanced markedly by anionic phospholipids including unsaturated but not saturated phosphatidylserine. Mn(2+) could not substitute for Mg(2+) to activate SMase in parasite-infected erythrocyte ghosts, whereas both Mn(2+) and Mg(2+) activated mammalian neutral SMase. The specific activity level of SMase was higher in isolated parasites than in infected erythrocyte ghosts; further fractionation of lysates of the isolated parasites showed that the activity was bound largely to the membrane fraction of the parasites. The plasmodial SMase seemed not to hydrolyse phosphatidylcholine or phosphatidylinositol. The plasmodial SMase, but not SM synthase, was sensitive to scyphostatin, an inhibitor of mammalian neutral SMase, indicating that the plasmodial activities for SM hydrolysis and SM synthesis are mediated by different catalysts. Our finding that the malaria parasites possess SMase activity might explain why the parasites seem to have an SM synthase activity but no activity to synthesize ceramide de novo.

Amides↗
PubMed2000

Serum factors governing intraerythrocytic development and cell cycle progression of Plasmodium falciparum.

Malaria is clinically manifested only when the human malaria parasites in the genus Plasmodium enter the obligatory intraerythrocytic life cycle. Elucidation for the roles of the serum, the key nutrient, and its components is then deemed essential for thorough understanding of the proliferation of Plasmodium cells at the erythrocytic stage. Fractionation and analysis of serum and its components was performed by chromatography, solvent extraction, and subsequent reconstitution experiments. Only fractions containing serum albumin (SA) from the serum and purified intact bovine serum albumin (BSA) showed comparable growth promoting activity with human serum (HS). Delipidated BSA can only effect parasite growth after reconstitution with lipids extracted from intact BSA. Fatty acid (FA) species in the neutral lipid fraction from intact BSA proved likewise when reconstituted with delipidated BSA. Furthermore, the involved FA species have to come in a pair of one saturated and one unsaturated, with palmitic and oleic acids as the best combination. The results were further substantiated by morphological analysis as well as biochemical analysis of the DNA synthesis during the intraerythrocytic development. This study can be a basis to explore the molecular mechanism of lipid traffic within the parasitized red blood cell (RBC), which can be an important adjunct to the development of drugs for malaria therapy.

Animals↗
PubMed2000

Preventive effects of a herbal medicine on bone loss in rats treated with a GnRH agonist.

The study was designed to evaluate the effects of a traditional Chinese herbal medicine Hochu-ekki-to (Bu-zong-yi-qi-tang), which was composed of 10 herbal medicines and had been used for the treatment of oligospermia and as a postoperative medication in Japan, on bone loss in rats treated with a gonadotropin-releasing hormone (GnRH) agonist. Female rats at 40 weeks of age were divided into 4 groups of 8 rats each. In the three experimental groups, each animal received subcutaneous injections of the long-acting GnRH agonist, buserelin acetate, once every four weeks throughout the experiment. Beginning at 48 weeks of age, the experimental groups were given diets containing conjugated estrogens or Hochu-ekki-to for 8 weeks. The administration of the GnRH agonist reduced the bone mineral density in the whole femur to 91.0% of that in the control group. However, administration of conjugated estrogens and Hochu-ekki-to increased the serum concentrations of estradiol 16.8- and 5.3-fold respectively compared with concentrations in the GnRH agonist-treated group, resulting in the augmentation of the bone mineral density to 110.3% and 106.2% respectively. These findings indicate that Hochu-ekki-to enhances the reduced bone mineral density and causes a slight elevation of the serum estradiol levels in the chemically castrated rats.

Animals↗
PubMed1999

Prevention of osteopenia induced with a gonadotropin-releasing hormone agonist in rats.

We investigated the effects of conjugated estrogens as an add-back replacement drug, incadronate sodium as a bisphosphonate, and alfacalcidol as a vitamin D(3) analog on femoral bone mineral density (BMD) and bone mineral content (BMC) in female rats chronically treated with the gonadotropin-releasing hormone (GnRH) agonist leuprorelin acetate. The chemical castration of the rats by the administration of GnRH agonist for 16 weeks reduced the BMD values to 92.3%, 91.3%, and 93.3% of those of the normal control animals in the whole femur, metaphysis, and diaphysis of the femur, respectively. The BMC value was decreased to 91.0% of that of the normal control animals by the chronic GnRH agonist treatment. However, a simultaneous 8-week administration of conjugated estrogens, bisphosphonate, and vitamin D(3) analog markedly augmented the BMC values to 110.3%, 110.1%, and 114.4%, respectively, of those in the rats treated with the GnRH agonist alone. These findings indicate that antiosteoporotic agents could be useful for preventing induced osteopenia under the careful monitoring of biochemical markers of osteoblastic activity or bone resorption and BMD or BMC in patients undergoing GnRH treatment.

Animals↗
PubMed1999

Antibodies reactive with the N-terminal domain of Plasmodium falciparum serine repeat antigen inhibit cell proliferation by agglutinating merozoites and schizonts.

The serine repeat antigen (SERA) is a vaccine candidate antigen of Plasmodium falciparum. Immunization of mice with Escherichia coli-produced recombinant protein of the SERA N-terminal domain (SE47') induced an antiserum that was inhibitory to parasite growth in vitro. Affinity-purified mouse antibodies specific to the recombinant protein inhibited parasite growth between the schizont and ring stages but not between the ring and schizont stages. When Percoll-purified schizonts were cultured with the affinity-purified SE47'-specific antibodies, schizonts and merozoites were agglutinated. Indirect-immunofluorescence assays with unfixed parasite cells showed that SE47'-specific immunoglobulin G (IgG) bound to SERA molecules on rupturing schizonts and merozoites but the IgG did not react with the schizont-infected erythrocytes (RBC). Furthermore, double-fluorescence staining against SE47'-specific IgG and anti-human RBC membrane IgG showed that the RBC membrane disappeared from SE47'-specific-IgG-bound schizonts after cultivation. These observations suggest that the SE47'-specific antibodies inhibit parasite growth by cross-linking SERA molecules that are associated with merozoites in rupturing schizonts with partly broken RBC and parasitophorous vacuole membranes, blocking merozoite release.

Agglutination Tests↗
PubMed1999

Cycloprodigiosin hydrochloride obtained from Pseudoalteromonas denitrificans is a potent antimalarial agent.

Cycloprodigiosin hydrochloride (cPrG*HCl) is a stable fluorescent red pigment obtained from the marine bacterium Pseudoalteromonas denitrificans. It was found that the compound was incorporated into Plasmodium falciparum cells upon incubation and exhibited a potent antimalarial activity with the concentration required for 50% of the activity being 11 nM, which is stronger than that of chloroquine, a well-known antimalarial agent. The compound did not affect growth rate of mammalian cells. Antimalarial activity of cPrG*HCl was also observed in vivo. These results indicate that cPrG*HCl is a potent antimalarial drug.

Animals↗
PubMed1998

Plasmodium falciparum produces prostaglandins that are pyrogenic, somnogenic, and immunosuppressive substances in humans.

Plasmodium falciparum causes the most severe form of human malaria, which kills approximately 1.5-2.7 million people every year, but the molecular mechanisms underlying the clinical symptoms and the host-parasite interaction remain unclear. We show here that P. falciparum produces prostaglandins (PGs) D2, E2, and F2alpha. After incubation with 1 mM arachidonic acid (AA), cell homogenates of P. falciparum produced PGs as determined by enzyme immunoassay and gas chromatography-selected ion monitoring. PG production in the parasite homogenate was not affected by the nonsteroidal antiinflammatory drugs aspirin and indomethacin, and was partially heat resistant, whereas PG biosynthesis by mammalian cyclooxygenase was completely inhibited by these chemicals and by heat treatment. Addition of AA to the parasite cell culture markedly increased an ability of the parasite cell homogenate to produce PGs and of parasitized red blood cells to accumulate PGs in the culture medium. PGD2 and PGE2 accumulated in the culture medium at the stages of trophozoites and schizonts more actively than at the ring stage. These findings are the first evidence of the direct involvement of a malaria parasite in the generation of substances that are pyrogenic and injurious to the host defenses. We will discuss a possible contribution of the parasite-produced PGs to pathogenesis and host-parasite interaction of P. falciparum.

Animals↗
PubMed1998

Additive effects of medroxyprogesterone acetate and 5-fluorouracil derivative on 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors.

Chronic oral administration of 1-(2-tetrahydrofuryl)-5-fluorouracil in combination with uracil suppressed thymidylate synthetase (TS) gene expression followed by reduction of TS activity in rat mammary tumors induced with 7,12-dimethylbenz[a]anthracene. Addition of medroxyprogesterone acetate (MPA) to the anticancer drug caused an additional decrease in TS and thymidine kinase activities in the tumor growth and restoration of bone loss. These results suggest that the simultaneous administration of MPA and anticancer drugs causes increased inhibition of mammary tumor growth and also diminishes the bone loss.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1997

Cataract development induced by repeated oral dosing with FK506 (tacrolimus) in adult rats.

FK506 (tacrolimus), a potent immunosuppressant, is used for inhibiting allograft rejection in the organ transplantation field. In a preclinical toxicity study in rats, FK506 induced various toxicities, including renal and pancreatic injuries. One of these toxic findings was cataract, and we have found that cataract appeared in rats dosed orally with FK506 for 13 weeks and more. Therefore, to better elucidate the onset mechanism of FK506-induced cataract, we measured biochemical parameters, such as sorbitol, Na,K-ATPase and glutathione in the lens of rats. Rats were dosed with FK506 in oral daily doses of 0.2, 1 or 5 mg/kg for 13 weeks, the lowest dose of which approximated the expected clinical dosage. Cataract developed in the 5-mg/kg/day group, with an incidence of 25%, whereas no cataract formation was observed in the 0.2- or 1-mg/kg/day groups. Five mg/kg/day led an increase of sorbitol and a decrease of reduced type glutathione, but did not affect Na,K-ATPase activity of the lens. FK506 is known to have diabetogenicity mediated through pancreatic injury, which appears as vacuolation of islet cell in rats. Five mg/kg/day of FK506 induced an elevation of blood glucose associated with glucose intolerance, and decrease of both basal insulin level and insulin content in the pancreas, and the changes were in parallel with the cataract development in the present study. On the other hand, diabetic parameters did not change in the 0.2- or 1-mg/kg/day groups. These observation suggest that diabetes developed in the rats dosed with 5 mg/kg/day of FK506. Coadministration of a novel aldose reductase inhibitor, Zenarestat, at an oral dose of 50 mg/kg/day resulted in a reduction of incidence of the FK506-induced cataract and a decrease of sorbitol levels in the lens when compared to that in the lens of rats dosed with 5 mg/kg/day of FK506. These results suggest that FK506-induced cataract in rats is due to an accumulation of sorbitol in the lens, secondary to the diabetogenic effect of FK506. FK506 treatment at the doses of 0.2 and 1 mg/kg/day neither affected parameters indicative of diabetes nor induced cataract in rats, suggesting that the cataract would not develop with FK506 if diabetic parameters were kept under control.

Administration, Oral↗
PubMed1997

Structure-function analysis of the diphtheria toxin receptor toxin binding site by site-directed mutagenesis.

Diphtheria toxin (DT) binds to the epidermal growth factor (EGF)-like domain of human membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF), the human DT receptor (DTR). DT does not bind to mouse proHB-EGF because of amino acid substitutions within the EGF-like domain. We made 10 independent mutants, replacing a single amino acid within the EGF-like domain of human DTR/proHB-EGF with the corresponding amino acid residue in mouse proHB-EGF. The mutant proteins were transiently expressed in mouse L cells either expressing or not expressing DRAP27/CD9, and DT binding was measured. DT binding activity of GST fusion proteins containing the mutated EGF-like domain was also determined by a cell-free binding assay. The largest effect was seen with E141H, and second largest effects were seen with F115Y and L127F in all of the assay systems. We conclude that Phe115, Leu127, and Glu141 are critical amino acid residues for DT binding. A computer model of the tertiary structure of the EGF-like domain of human DTR/proHB-EGF was made. The model predicts that three amino acid residues critical for DT binding activity, Phe115, Leu127, and Glu141, are all located on the same face of the EGF-like domain, suggesting that this face of DTR/proHB-EGF interacts with the receptor-binding domain of DT.

Amino Acid Sequence↗
PubMed1996

Amino-terminal processing of cell surface heparin-binding epidermal growth factor-like growth factor up-regulates its juxtacrine but not its paracrine growth factor activity.

Human heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) expressed on Chinese hamster ovary (CHO) cells is synthesized as a 19-kDa major, and 22- and 27-kDa minor, membrane-anchored precursors (proHB-EGF). In contrast, the 27-kDa species is major and the 19- and 22-kDa ones are minor in mouse proHB-EGF. The juxtacrine growth factor activities of human and mouse proHB-EGFs on CHO cells toward EP170.7 cells in co-culture are significantly different. To investigate the relationship between the juxtacrine growth factor activities and the molecular species, we prepared human-mouse chimeras. Chimeras that have the human amino-terminal sequence with a mouse EGF-like domain showed approximately 8-fold up-regulation of the juxtacrine growth factor activity and the predominance of a 19-22-kDa major species. In contrast, chimeras that have the mouse amino-terminal sequence with a human EGF-like domain showed approximately 5-fold down-regulation of the juxtacrine activity and the predominance of the 27-kDa major species. A Gly32.HB-EGF (117-amino acid form), which is amino-terminally extended, induced the same mitogenic activity as that of Arg73.HB-EGF (75-amino acid form), which is amino-terminally truncated. These results strongly suggested that amino-terminal processing of human proHB-EGF would be required for up-regulation of its juxtacrine growth factor activity, but not for its paracrine activity.

Amino Acid Sequence↗