Biomedical subjects

T Mitamura

Publications and source records attributed to T Mitamura.

At least 37 records · Page 2Linked to original sources

PubMed1996

Recombinant Plasmodium falciparum dihydrofolate reductase-based in vitro screen for antifolate antimalarials.

We describe the system for screening the effective antifolate antimalarials that uses the recombinant Plasmodium falciparum DHFR domain of the bifunctional DHFR-TS expressed in Escherichia coli, and were designed with amino acid alterations found in the DHFR genes of the antifolate resistant strains. The validity of the screen was verified by the subsequent examination of several substituted pyrrolo[2,3-d]pyrimidines for their antimalarial activity. Among the 120 chemical derivatives, 5 compounds were identified by their preferential inhibition of the drug sensitive pfDHFR to that of the mammalian isoenzyme. As compared to the sensitive enzyme, the decrease in response of the cycolguanil-resistant and pyrimethamine-resistant enzymes to the selected compounds were relatively moderate. This gave folds decrease in sensitivity of 0.8-7.5 and 3.6-29, respectively, while those for cycloguanil and pyrimethamine were 400 and 308. The compounds inhibited the growth of drug-sensitive cultured P. falciparum with 50% effective concentrations of the ranged 0.17-30 nM. As contrasted with the sensitive strain, the fold decrease in sensitivity of the resistant parasites were 0.9-2 and 15-50 in the case of the test compounds, while those for cycloguanil and pyrimethamine were 690 and 20,500. Moreover, the most selective pyrrolo-pyrimidine (P-1) showed in vivo activity against P. berghei in mice.

Animals↗

PubMed1996

Effects of lycopene, a carotenoid, on intrathymic T cell differentiation and peripheral CD4/CD8 ratio in a high mammary tumor strain of SHN retired mice.

We previously reported that lycopene, one of the carotenoids, significantly suppressed the development of spontaneous mammary tumors of mice and the contribution of lycopene to the maintenance of physical homeostasis was suggested. In this study, we investigated the effects of lycopene on the intrathymic and peripheral T cell subpopulation as immunoregulatory parameters. In tumorous control mice, inactivation of intrathymic T cell differentiation, an increase of the percentage of CD4+ CD8+ cells and a decrease of CD4+CD8+ cells were observed. On the other hand, in the lycopene-treated mice, the abnormal intrathymic T cell differentiation was recovered and brought to non-tumorous levels. The peripheral CD4/CD8 ratio was partially augmented by lycopene treatment which resulted from an increased CD4+ subpopulation. These observations suggest that one of the immunomodulating roles of lycopene is to normalize the change of intrathymic T cell differentiation caused by tumorigenesis.

Animals↗

PubMed1996

1-(2-Tetrahydrofuryl)-5-fluorouracil in combination with uracil suppresses mammary carcinogenesis and growth of tumors induced with 7,12-dimethylbenz[a]anthracene in rats.

The effects of 1-(2-tetrahydrofuryl)-5-fluorouracil in combination with uracil (UFT) on mammary carcinogenesis and growth of tumors induced with 7,12-dimethylbenz[a]anthracene (DMBA) were investigated in rats. Daily oral administration of UFT reduced the incidence and number of mammary tumors compared with those of the DMBA control group, resulting in lower activities in DNA synthesizing enzymes, thymidylate synthetase and thymidine kinase, and a reduction of bromodeoxyuridine-immunoreactive (S-phase) cells in mammary tumors of UFT-treated rats.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1996

Pharmacotherapeutic effects of toki-shakuyaku-san on leukorrhagia in young women.

Toki-shakuyaku-san is a traditional Chinese herbal prescriptions that is composed of 6 herbal plants, i.e., peony root, atractylodes lancea rhizome, alisma rhizome, hoelen, cnidium rhizome and Japanese angelica root. Administration with Toki-shakuyaku-san normalized irregular menstrual cycle, healed cervical pseudo-erosion and reduced leukorrhagia in young women who had insufficient luteal function.

Adult↗

PubMed1995

Diphtheria toxin binds to the epidermal growth factor (EGF)-like domain of human heparin-binding EGF-like growth factor/diphtheria toxin receptor and inhibits specifically its mitogenic activity.

The membrane anchored form of human heparin-binding epidermal growth factor-like growth factor (HB-EGF) acts as the diphtheria toxin (DT) receptor. Transfection of human HB-EGF cDNA into mouse LC cells, L cells stably expressing DRAP27, conferred sensitivity to DT, but transfection of mouse HB-EGF cDNA did not. To define the essential regions of HB-EGF that serve as the functional DT receptor, we examined the sensitivity to DT and DT binding of cells expressing several human/mouse HB-EGF chimeras. It was found that DT binds to the EGF-like domain of the human HB-EGF. However, mouse HB-EGF does not serve as a functional DT receptor due to non-conserved amino acid substitutions in this domain. In addition, CRM197, a non-toxic mutant of DT, inhibited strongly the mitogenic activity of the secreted form of human HB-EGF, but not of mouse HB-EGF and other EGF receptor-binding growth factors. These results confirmed further that DT interacts with the EGF-like domain of HB-EGF and that this interaction is specific for human HB-EGF.

Amino Acid Sequence↗

PubMed1995

Malignant hypercalcemia due to gastric endocrine cell carcinoma.

A 62-year-old man was admitted to our Neurology Unit due to consciousness disturbance. Laboratory data showed marked hypercalcemia and azotemia. Serum parathyroid hormone-related protein (PTHrP) level was extremely high. We performed intensive hemodialysis for renal failure, but his condition deteriorated rapidly. On day 10, he died of multiple organ failure. The autopsy revealed gastric undifferentiated adenocarcinoma with systemic dissemination. Immunohistological study showed positive PTHrP staining in carcinoid-like parts of the tumor. This is the first reported case of malignant hypercalcemia due to PTHrP-producing carcinoid or endocrine cell carcinoma of the stomach.

Adenocarcinoma↗

PubMed1994

Heparin-binding EGF-like growth factor, which acts as the diphtheria toxin receptor, forms a complex with membrane protein DRAP27/CD9, which up-regulates functional receptors and diphtheria toxin sensitivity.

DRAP27, the monkey homolog of human CD9 antigen (DRAP27/CD9) and diphtheria toxin receptor (DTR) were expressed in mouse L cells. L cells transfected transiently with both DRAP27/CD9 and DTR cDNA bound approximately 10 times more diphtheria toxin (DT) than cells transfected with DTR alone. Stable L cell transfectants expressing both DTR and DRAP27/CD9 (LCH-1 cells) had 15 times more cell surface DT-binding sites and were 20 times more sensitive to DT than were stable L cell transfectants expressing DTR alone (LH-1 cells). Increased DT-binding and DT sensitivity were not due to increased DTR transcription or increased cell surface DTR protein. Co-immunoprecipitation of DRAP27/CD9 with DTR and chemical cross-linking suggest a tight association of these membrane-bound proteins. In addition, the identity of DTR and a growth factor (HB-EGF) was established. Immobilized DT specifically adsorbed HB-EGF precursor solubilized from transfected L cells and [125I]DT bound to immobilized recombinant HB-EGF. We conclude that DRAP27/CD9 associates tightly with DTR/HB-EGF and up-regulates the number of functional DTRs and DT sensitivity, and that HB-EGF is identical to DTR.

Amino Acid Sequence↗

PubMed1994

Mechanism of FK506-induced glucose intolerance in rats.

To clarify the mechanism of glucose intolerance induced by FK506, a novel immunosuppressant, 5 or 10 mg/kg/day of FK506 was dosed orally to rats for 2 weeks, and 125I-insulin binding to the erythrocytes, plasma glucose and insulin levels, and pancreatic insulin content were examined. Insulin binding to the erythrocytes of rat dosed with FK506 was similar to that to erythrocytes of the placebo control; Scatchard analysis confirmed that FK506 did not cause damage to the insulin receptor of the erythrocytes. Contrarily, FK506 caused a clear decrease of pancreatic insulin content as well as a slight decrease of plasma insulin level. The results suggest that the glucose intolerance induced by FK506 is associated with a decrease of insulin secretion, but is not associated with impairment of the insulin receptor.

Animals↗

PubMed1994

Tacrolimus (FK506)-induced nephrotoxicity in spontaneous hypertensive rats.

To clarify the profile of the tacrolimus (FK506)-induced nephrotoxicity and its mechanism, 1, 2 and 4 mg/kg/day of tacrolimus was administered intramuscularly (i.m.) to spontaneous hypertensive rats (SHR) for 2 weeks, and biochemical and pathological parameters were studied in the animals. The acute nephrotoxicity of tacrolimus was characterized as increase of blood urea nitrogen (BUN) and plasma creatinine (P-Cr) levels in the groups of 1 mg/kg/day and more, decrease of creatinine clearance (CCr) value in the groups of 2 mg/kg/day and more, and histopathologically luminal narrowing of the arteriole adjacent the glomerulus in the groups of 1 mg/kg/day and more. These changes were associated with an increase of plasma renin activity (PRA) and urinary thromboxane B2 content and decrease of 6-keto-prostagrandinF1 alpha (6-keto-PGF1 alpha) content. Nilvadipine, which is one of the Ca2+ antagonist and is known to have renal vasodilating activity, prevented both biochemical and histopathological changes due to tacrolimus. The results indicated that the acute nephrotoxicity of tacrolimus was derived from impairment of glomerular function associated with the constriction of the renal arteriole brought about by the drug. All of these renal disorders induced by tacrolimus recovered completely or partially when the drug was withdrawn for 2 or 4 weeks. Consequently, the acute nephrotoxicity of tacrolimus in SHR was considered to be reversible.

6-Ketoprostaglandin F1 alpha↗

PubMed1992

A rapid and simple quantification of human apolipoprotein E-rich high-density lipoproteins in serum.

A rapid and simple method for the quantitative determination of human serum apo E-rich high-density lipoproteins is described. A sample was divided into two parts; one part was mixed with an equal volume of 13% polyethylene glycol 6000, and the other part was mixed with a solution containing dextran sulfate, sodium phosphotungstate, and Mg2+, respectively. The mixed solutions were centrifuged (2000 g; 15 min). The supernate obtained by the former procedure contained both apo E-rich HDL and apo E-poor HDL, but that obtained by the latter procedure contained solely apo E-poor HDL. The serum apo E-rich HDL concentration in terms of apo E (E) and cholesterol (C), was given by the following equations: E = EP x 2, and C = (CP - CD) x 2, where EP and CP were the concentrations of apo E and cholesterol, respectively, in the supernate obtained with 13% polyethylene glycol, and CD was the concentration of cholesterol in the supernate obtained with the mixture solution of dextran sulfate, sodium phosphotungstate, and Mg2+. Normal serum apo E-rich HDL concentrations were 2.6 +/- 1.5 and 6.7 +/- 2.3 mg/dl (means +/- SD, n = 38) in terms of apo E and cholesterol, respectively. Apo E-rich HDL was increased strikingly in the sera from three patients with hepatobiliary diseases.

Adult↗

PubMed1992

The 27-kD diphtheria toxin receptor-associated protein (DRAP27) from vero cells is the monkey homologue of human CD9 antigen: expression of DRAP27 elevates the number of diphtheria toxin receptors on toxin-sensitive cells.

Diphtheria toxin (DT) receptor associates with a 27-kD membrane protein (DRAP27) in monkey Vero cells. A cDNA encoding DRAP27 was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence revealed that DRAP27 is the monkey homologue of human CD9 antigen. DRAP27 is recognized by CD9 antibodies. A human-mouse hybrid cell line (3279-10) possessing human chromosome 5, sensitive to DT, but not expressing CD9 antigen, was used for transfection experiments with DRAP27. When the cloned cDNA encoding DRAP27 was transiently expressed in 3279-10 cells, the total DT binding capacity was three to four times higher than that of untransfected controls. Transfectants stably expressing DRAP27 have an increased number of DT binding sites on the cell surface. Furthermore, the transfectants are 3-25 times more sensitive to DT than untransfected cells, and the sensitivity of these cells to DT is correlated with the number of DRAP27 molecules on the surface. However, when the cloned cDNA was introduced into mouse cell lines that do not express DT receptors, neither an increased DT binding nor enhancement of DT sensitivity was observed. Hence, we conclude that DRAP27 itself does not bind DT, but serves to increase DT binding and consequently enhances DT sensitivity of cells that have DT receptors. 12 proteins related to DRAP27/CD9 antigen were found through homology search analysis. These proteins appear to belong to a new family of transmembrane proteins.

Amino Acid Sequence↗

PubMed1992

Cloning, nucleotide sequences, and enzymatic properties of glucose dehydrogenase isozymes from Bacillus megaterium IAM1030.

Bacillus megaterium is known to have several genes that code for isozymes of glucose dehydrogenase. Two of them, gdhI and gdhII, were cloned from B. megaterium IAM1030 in our previous work (T. Mitamura, R. V. Evora, T. Nakai, Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Ferment. Bioeng. 70:363-369, 1990). In the present study, two new genes, gdhIII and gdhIV, were isolated from the same strain and their nucleotide sequences were identified. Each gene has an open reading frame of 783 bp available to encode a peptide of 261 amino acids. Thus, a total of four glucose dehydrogenase genes have been cloned from B. megaterium IAM1030. In addition, this strain does not seem to have other glucose dehydrogenase genes that can be distinguished from the four cloned genes so far examined by Southern hybridization analysis. The two newly cloned genes were expressed in Escherichia coli cells, and the products, GlcDH-III and GlcDH-IV, were purified and characterized and compared with the other isozymes, GlcDH-I and GlcDH-II, encoded by gdhI and gdhII, respectively. These isozymes showed different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (GlcDH-I greater than GlcDH-III = GlcDH-IV greater than GlcDH-II), although they have the same number of amino acid residues. Double-immunodiffusion tests showed that GlcDH-I is immunologically different from the other isozymes and that GlcDH-III and GlcDH-IV are identical to one another but a little different from GlcDH-II. These glucose dehydrogenases were stabilized in the presence of 2 M NaCl. The effect of NaCl was especially large for GlcDH-III, which is most unstable enzyme. Kinetic studies showed that these isozymes are divided into two groups with respect to coenzyme specificity, although they can utilize both NAD and NADP: GlcDH-III and GlcDH-IV prefer NAD, and GlcDH-I and GlcDH-II prefer NADP. The phylogenic relationship of these glucose dehydrogenase genes is also discussed.

Amino Acid Sequence↗

PubMed1991

Apolipoproteins in rat cerebrospinal fluid: a comparison with plasma lipoprotein metabolism and effect of aging.

Cerebrospinal fluid (CSF) apo E concentrations, determined by a sensitive sandwich ELISA, were 411.3 +/- 76.0 and 454.3 +/- 51.8 micrograms/dl (mean +/- S.D.) for young rats (8-12 weeks old, n = 7) and old rats (36-40 weeks old, n = 10), respectively. Age-related increase, which was conspicuous in serum apo E (21.2 +/- 2.4 vs 60.9 +/- 14.1 mg/dl for young and old rats, respectively), was not observed in CSF apo E. CSF apo A-I concentrations, determined by ELISA, were extremely low in the both groups (less than 10 micrograms/dl). Neither CSF apo A-I nor CSF apo E correlated to any of the plasma lipoprotein components, indicating the presence of largely independent lipoprotein metabolism in the rat central nervous system. Apo E is present in CSF in the form of apo E-rich HDL1 with particle sizes similar to those of plasma E-rich HDL1.

Aging↗

PubMed1991

A sensitive sandwich enzyme-linked immunosorbent assay of rat apolipoprotein A-I: effect of various sample treatments on apolipoprotein A-I immunoreactivity and an application to young and aged rat sera.

A highly sensitive sandwich enzyme-linked immunosorbent assay for rat apo A-I was developed. Samples and standards were added to each well of microtiter plates precoated with immunoaffinity-purified IgG. Bound apo A-I was detected with immunoaffinity-purified Fab'-horseradish peroxidase conjugate by a colorimetric method. The sensitivity reached 2.5 pg/well, and the working range for the measurement of serum apo A-I concentration was 0.1 to 1.0 ng/well. The mean intra- and interassay coefficients of variation were 2.8 and 4.1%, respectively. The epitopes of apo A-I in serum were effectively exposed by the use of 6 mol/liter guanidine.HCl. Serum apo A-I concentrations in 36- to 40-week-old rats (62.3 +/- 8.6 mg/dl, mean +/- SD, n = 16) were significantly higher (P less than 0.05) than those in 8- to 12-week-old rats (55.1 +/- 4.3 mg/dl, n = 9). But the age-related change of serum apo A-I was much smaller than that of serum apo E. Apo A-I was contained in smaller HDL particles (or HDL2) in normal rat serum.

Aging↗

PubMed1991

Torsion of parovarian cyst--report of two cases.

A parovarian cyst oxiginates from the tissue of the broad ligament, predominantly from mesothelium covering the peritoneum but also from paramesonephric and mesonephric remnants. Clinically, torsion of a parovarian cyst is uncommon, and it is difficult to distinguish it from torsion of other adnexal masses, an ovarian accident, appendicitis, etc. Recently, we experienced two cases of torsion of parovarian cysts. In one case, it was associated with 32 weeks' intrauterine gestation. In this case, pelvic sonography during the first and second trimester showed no cystic lesions. In the other case, a lower abdominal pain continued about two weeks. A sonogram revealed a very small cyst like a follicle. These twisted parovarian cysts were removed at laparotomy. The clinical and pathological features of the torsion of parovarian cysts are briefly discussed and the literature is reviewed.

Adult↗

PubMed1990

[Avascular necrosis of the femoral head: chronological change of MR images].

Tl-weighted MR images of thirty-six hips in 25 patients with avascular necrosis of the femoral head were obtained two to five times during the course of 2 to 26 months. We investigated these MR images in the light of the chronological change and compared them with plain radiographs. MR images change in 16 femoral heads; in general, the abnormal low intensity area in the femoral head reduced in extent and the internal high intensity area became smaller of disappeared. Thirteen femoral heads among them became more flattened on plain radiographs in the same period. It is noted that four different zones are defined in the femoral head after bone necrosis takes place: the dead bone marrow, the dead marrow which still contains fat, the reactive interface and the hyperemic bone marrow. In Tl-weighted MR images, the dead bone marrow, the reactive interface and the hyperemic bone marrow are demonstrated as low intensity area, while the dead marrow containing fat may remain high in intensity. On the basis of this knowledge of histopathology and MR images of this disease, we suggest that reduction of the abnormal low intensity area and disappearance of the internal high intensity area on MR images can be regarded as diminution of hyperemia in the living bone marrow and loss of fat in the dead bone marrow, respectively.

Adult↗

PubMed1990

Sandwich enzyme-linked immunosorbent assay of rat apolipoprotein E.

A sandwich ELISA for rat apo E was developed. Blood was drawn from rats that had been administered with triamcinolone diacetate to increase apo E-rich HDL in serum. Apo E was purified from the d less than 1.225 g/ml lipoproteins, and antiserum was raised in rabbits. Diluted samples and standards were added into the wells of polystyrene microtiter plates precoated with immunoaffinity-purified IgG and incubated for 90 min. After washing, immunoaffinity-purified Fab'-horseradish peroxidase conjugate was added to each well and incubated for 90 min. The bound enzyme was assayed by a colorimetric method. Samples and standards were pretreated with 6 M guanidine. HCl to maximize the antigenic response of apo E. The sensitivity lies around 1 pg apo E, and the working range was 0.1 to 1.0 ng. All assay procedures were completed within 4-5 hr. The mean intra- and interassay coefficients of variation were 1.8 and 4.1%, respectively. Serum apo E concentrations were 21.2 +/- 2.1 and 61.3 +/- 17.0 mg/dl (mean +/- SD) for young (8-12 weeks old, n = 9) and old (36-40 weeks old, n = 16) rats, respectively. As determined by gel filtrations, most of the apo E in fasted rat serum was associated with larger HDL particles (or HDL1) and a small portion of apo E was present in a free form.

Aging↗

PubMed1990

Possible involvement of lymphocyte activating factor (LAF) as an endogenous pyrogen in fever induced by the cell wall skeleton of Nocardia rubra (N-CWS).

We investigated whether interleukin-1 (IL-1) acts as an endogenous pyrogen (EP) on the fever caused by the cell wall skeleton of Nocardia rubra (N-CWS) in guinea pigs. IL-1 activity was expressed as potency of lymphocyte activating factor (LAF). When guinea pig peritoneal macrophages were pulse-stimulated with N-CWS (1-100 micrograms/ml), dose-dependent LAF activity was detected in the supernatants after culture for 4 h. Gel filtration of the culture supernatants on Sephadex G-200 showed that the fractions with LAF activity were not the same as those with cytotoxic activity for L-929 cells, which was measured as an index of tumor necrosis factor (TNF) in parallel with LAF activity. Pyretic activity was detected both in the fractions with LAF activity and in those with cytotoxic activity for L-929 cells. Furthermore, when these macrophages were pulse-stimulated again, this time with the supernatant obtained from macrophages previously pulse-stimulated with N-CWS, LAF and cytotoxic activity for L-929 cells continued to be released from the macrophages. We suggest that IL-1 might be a possible EP in the process of fever elicited by N-CWS, and that such an EP stimulates the macrophages to release further IL-1 or TNF. The resultant long-lasting fever would thus be caused by the continuous release of an EP.

Animals↗