Search PubMed⌕ Search

Biomedical subjects

M Harada

Publications and source records attributed to M Harada.

At least 415 records · Page 23Linked to original sources
PubMed1997

Bovine oocytes from early antral follicles grow to meiotic competence in vitro: effect of FSH and hypoxanthine.

A large number of oocytes are contained in the mammalian ovary. A very small number of these oocytes grow to the final size, mature, and are ovulated. In the ovary there are more early antral follicles than late antral or preovulatory follicles, offering a large pool of oocytes for IVM and IVF if appropriate culture conditions could be devised. In the present study, early antral follicles containing oocytes 90 to 99 microm in diameter were isolated from bovine ovaries. Cumulus-oocyte complexes (COC) with pieces of parietal granulosa (COCG) were then dissected from the follicles. The COCGs were embedded in collagen gels and cultured in Medium 199 with 10% fetal calf serum (FCS) for 8 d. In Experiment 1, the effect of hypoxanthine and FSH on the growth of bovine oocytes was examined. When hypoxanthine (2 and 4 mM) and FSH (10 ng/ml) were added to the culture medium, the number of granulosa cell-enclosed oocytes increased significantly (P < 0.05). All of the oocytes surrounded by granulosa cells showed a normal morphology and were at the germinal vesicle stage, while 75 to 94% of the denuded oocytes were degenerated and had resumed meiosis. The mean diameter of the oocytes showing normal morphology was significantly higher than that measured before culture (P < 0.05). In Experiment 2, the maturational competence of in vitro-grown bovine oocytes was examined. Oocytes which were 90 to 99 microm in diameter before culture did not have meiotic competence. After being in a growth culture of 4 mM hypoxanthine- and 10 ng/ml FSH-supplemented medium for 7 or 11 d, granulosa cell-enclosed oocytes were recovered from the COCGs. No significant difference (P < 0.05) in the diameters of the oocytes was observed between 7 and 11 d of culture (7 d: 107.5 +/- 6.1 microm, n = 30; 11 d: 108.0 +/- 5.3 microm, n = 35). After a subsequent 24 h in a maturation free of hypoxanthine and FSH medium, only 17% of the oocytes cultured for 7 d underwent germinal vesicle breakdown. On the other hand, 89% of the oocytes cultured for 11 d underwent germinal vesicle breakdown, and 11% of the oocytes emitted the first polar body and reached metaphase II. These results demonstrate for the first time that bovine oocytes harvested from early antral follicles can grow, and acquire meiotic competence in vitro.

Journal Article↗
PubMed1997

Interactions of an antimicrobial peptide, magainin 2, with outer and inner membranes of Gram-negative bacteria.

Magainin peptides, isolated from Xenopus skin, have broad spectra of antimicrobial activity and low toxicities to normal eukaryotic cells, thus being good candidates for therapeutic agents. The mechanism of action is considered to be the permeabilization of bacterial membranes. A number of studies using lipid vesicles have elucidated its molecular detail. However, their interactions with bacteria are not yet well understood. In this paper, we synthesized several magainin analogs with different charges (0 to +6) and hydrophobicities, and systematically studied their interactions with the outer and inner membranes of three species of Gram-negative bacteria (Escherichia coli, Acinetobacter calcoaceticus, Proteus vulgaris). The treatment of the E. coli cells with native magainin 2 (+4) immediately induced the efflux of the intracellular K+ ions and the cell death. A number of blebs were formed on the bacterial surface and the outer membrane became leaky. An increase in the peptide's positive charge enhanced the outer membrane permeabilization and the bactericidal activity. The cationic peptides also effectively permeabilized the inner membranes rich in acidic phospholipids, indicating the importance of electrostatic interactions. Substitution of Trp for Phe simultaneously increased the bactericidal activity and the hemolytic activity. A strategy to develop potent antimicrobial peptides was discussed on the basis of these results.

Acinetobacter↗
PubMed1997

Immunosuppressive activity of cloned natural killer (NK1.1+) T cells established from murine tumor-infiltrating lymphocytes.

To elucidate the role of NK1.1+ T cells in the antitumor immune response, we established cloned NK1.1+ T cell lines from tumor-infiltrating lymphocytes (TIL) of B16 melanoma, and examined their mode of action in generating antitumor effector T cells both in vitro and in vivo. An NK1.1+ T cell clone (TM4.2) was phenotypically CD3+ TCR-alphabeta+ CD4- CD8- NK1.1+, and CD28+. The TM4.2 cells suppressed the in vitro generation of anti-B16 melanoma CTLs, but not the effector function of CTLs. The results using a transwell membrane suggested that their suppressive activity was mediated by both soluble factors and a direct cell to cell interaction. As for the soluble factors, the suppressive activity of the culture supernatant of TM4.2 cells was neutralized by anti-TGF-beta mAb, and the TM4.2 cells actually produced a considerable amount of TGF-beta. On the other hand, the TM4.2 cells showed a high level of cytolytic activity against B cell blasts and CD80-transfected P815, and such cytolytic activity was reduced by the addition of anti-CD80 mAb. In addition, NK1.1+ T cells in the freshly isolated TIL were revealed to express CD28. Furthermore, the TM4.2 cells suppressed the in vitro generation of anti-allo CTLs irrespective of the MHC haplotype. Finally, the TM4.2 cells suppressed the in vivo antitumor immune response. Collectively, these findings demonstrate that NK1.1+ T cells in TIL show immunosuppressive activity in the antitumor immune response through the production of TGF-beta and the preferential cytolysis of B7-expressing cells.

Animals↗
PubMed1997

Altered retinoblastoma protein expression in nonsmall cell lung cancer: its synergistic effects with altered ras and p53 protein status on prognosis.

BACKGROUND: Inactivation of the retinoblastoma (Rb) gene has been documented in various types of cancer, including lung cancer. Alterations of the p53 and ras genes are also common features in the molecular biology of lung carcinoma, and the authors of this article have reported previously on the prognostic significance of both of them. In the present study, the authors evaluated the prognostic significance of the loss of Rb protein expression alone, then performed a combined analysis of Rb protein and ras p21 status (Rb/ras) as well as an analysis of Rb and p53 protein status (Rb/p53) in patients with nonsmall cell lung cancer (NSCLC). METHODS: Ninety-one patients with NSCLC underwent potentially curative resection between 1977 and 1988, 65 of whom received postoperative combination chemotherapy. Tumor specimens were analyzed for Rb protein expression by immunohistochemistry. Univariate and multivariate analyses were performed to assess the association between Rb protein expression and survival. RESULTS: Nineteen (21%) of the 91 NSCLCs showed negative Rb protein expression. Positive or negative Rb protein expression (Rb+ or Rb-) as an individual factor was not statistically correlated with survival or prognosis in this cohort of NSCLC patients, although a tendency among Rb- patients to do worse was observed. The authors then combined the Rb protein status with previously studied results of ras p21 and p53 protein expression in the same tumor specimens, and compared the prognosis between the individuals with theoretically the best pattern of gene expression in their tumors and those with theoretically the worst pattern of expression, i.e., Rb+/ras- versus Rb-/ras+ and Rb+/p53- versus Rb-/p53+. In patients with adenocarcinoma, those with Rb-/ras+ tumors survived for a significantly shorter period after surgery (13% 5-year survival) than those with Rb+/ras- tumors (82% 5-year survival) (P = 0.01). Similarly, patients with Rb-/p53+ tumors survived for a significantly shorter period (20% 5-year survival) compared with those who had Rb+/p53- tumors (73% 5-year survival) (P = 0.008). Rb/ras status was a significant prognostic factor (P = 0.02 by univariate analysis, P = 0.048 by multivariate analysis), and Rb/p53 status tended to be significant as a prognostic factor (P = 0.04 by univariate analysis, P = 0.08 by multivariate analysis). In patients with squamous cell carcinoma, neither Rb/ras nor Rb/p53 status was a significant prognostic factor in this cohort. CONCLUSIONS: These results suggest that combined immunohistochemical analyses of Rb and ras p21 proteins and of Rb and p53 proteins may indicate their potentially synergistic effects on survival and prognosis. These analyses may also be useful for stratifying patients with adenocarcinoma of the lung into different prognostic groups and identifying populations with different risks of recurrence. Larger prospective studies with Stage I NSCLC patients are necessary to confirm the current findings.

Adenocarcinoma↗
PubMed1997

Local injection of OK432 can augment the TH1-type T-cell response in tumor-draining lymph node cells and increase their immunotherapeutical potential.

The effect of local injections with streptococcal preparation OK432 on the therapeutical potential of tumor-draining lymph node (LN) cells was investigated in mice. Peritumoral injections with OK432 on days 2, 4, 6, 8 and 10 showed no effect on the in vivo growth of s.c. inoculated B16F10 melanoma. The B16F10-draining OK432-treated LN cells, however, showed a high level of anti-B16F10 cytolytic activity after an in vitro culture first with both anti-CD3 monoclonal antibody (MAb) and activated B cell blasts, and subsequently with interleukin (IL)-2 without in vitro restimulation. Such in vitro expanded LN cells showed a remarkable antitumor effect against pulmonary metastasis of B16F10 melanoma, even without the concurrent administration of IL-2. In addition, the therapeutical protocol was also found to be moderately effective against poorly immunogenic MCA fibrosarcoma, and the in vivo antitumor effect was specific to the tumor from which the LNs were harvested. Interestingly, 2 kinds of comparative analyses of the cytokines revealed that the B16F10-bearing state induced the draining LN cells to develop a Th2-type response. However, the OK432 treatment was able to effectively augment their Th1-type response. Collectively, our results suggest that peritumoral injections with OK432 significantly increased the therapeutical potential of the tumor-draining LN cells by augmenting their Th1-type response.

Animals↗
PubMed1997

Measurement of nitric oxide in the rat cerebral cortex during hypercapnoea.

Changes in nitric oxide (NO) concentration and cerebral blood flow (CBF) in the parietal cortex during hypercapnoea were investigated in anaesthetized rats, using a NO-selective electrode and laser Doppler flowmetry. When hypercapnoea was induced by inhalation of 5% CO2 for 10 min, both the NO concentration and CBF increased. After administration of 7-nitroindazole, a neuronal NO synthase (nNOS) inhibitor, both the basal NO and CBF decreased, and responses to hypercapnoea were also significantly suppressed by 70.1% and 73.2%, respectively, compared with the control state. These results suggest that NO derived from nNOS is involved not only in maintaining resting cerebral circulation but also in regulating CBF response during hypercapnoea.

Animals↗
PubMed1997

Oral administration of PSK can improve the impaired anti-tumor CD4+ T-cell response in gut-associated lymphoid tissue (GALT) of specific-pathogen-free mice.

We investigated both the effect and the mechanism of oral (p.o.) administration of PSK, a protein-bound polysaccharide derived from Basidiomycetes, on the anti-tumor T-cell response in gut-associated lymphoid tissue (GALT). The p.o. administration of PSK significantly suppressed the growth of colon 26 carcinoma (C-26) inoculated into the subserosal space of the cecum (i.c.), and augmented the tumor-neutralizing activity of the draining mesenteric lymph node (LN) cells. PSK treatment also significantly decreased the levels of immunosuppressive factors such as plasma transforming growth factor (TGF)-beta in the i.c. C-26-inoculated mice. We also evaluated the improving effect of PSK on the anti-tumor T-cell response in GALT by utilizing B7-transfected P815 mastocytoma (B7/P815). The PSK treatment promoted the rejection of i.c.-inoculated B7/P815 and restored the CD4+ T-cell-dependent proliferative response of the draining mesenteric LN cells against in vitro restimulation. Furthermore, the treatment also decreased the TGF-beta production but increased the IFN-gamma production of these cells. The p.o. administration of PSK, however, showed no effect in the CD8+ T-cell-dependent cytolytic activity of the draining mesenteric LN cells after in vitro restimulation. Overall, these results indicate that the p.o. administration of PSK can improve the impaired anti-tumor CD4+ T-cell response in GALT, mainly through a suppression of TGF-beta production and a restoration of IFN-gamma production.

Administration, Oral↗
PubMed1997

Carcinoma of the uterine cervix with myocardial metastasis.

Cardiac metastasis from pelvic malignancies is rare. We experienced a case of carcinoma of the uterine cervix with myocardial involvement in the right ventricle, and treated this patient with conventional methods until her death. The metastatic tumor was incidentally found in the right ventricle, eroding the myocardium. We used CT, MRI, and gallium scintigraphy to assess the lesion. This aggressive lesion proved fatal for our relatively young patient. We report this case for future reference.

Adult↗
PubMed1997

Ultrasonographic evaluation of gastric emptying in normal children and children after pyloromyotomy.

Gastric emptying in the fundus, body, and antrum of the stomach was evaluated by ultrasonography (US) in 41 control children aged 2 to 18 years and 30 patients aged 1 to 19 years who had undergone pyloromyotomy because of hypertrophic pyloric stenosis. The gastric emptying curve decreased in an exponential manner for both control children and patients, while there was no significant difference in gastric emptying time (GET) between control children and patients within any of the age groups. However, GET was faster for younger children in both groups. An X-ray contrast study of the stomach performed in 2 patients who showed markedly delayed GET showed delayed gastric emptying but no significant deformities of the prepylorus. Our results suggest that US is a reliable method of measuring GET in children.

Adolescent↗
PubMed1997

Anti-granulocyte antibody suppression of active and passive anaphylactic shock in WBB6F1-W/Wv mice.

Our previous study revealed that anaphylactic shock can be produced in WBB6F1-W/Wv (abbreviated as W/Wv) mice. Because they are congenitally deficient in mast cells, we are certain that some other cell types are involved as mediator sources. In the present study, with the aim of examining the role of circulatory granulocytes, the effect of monoclonal antibody to a mouse granulocyte antigen, Gr-1, upon active and passive anaphylactic shock was tested in W/Wv mice using several mast cell-bearing strains as references. An intravenous injection of 40 micrograms of anti-granulocyte antibody one day before the antigen challenge produced a marked decrease in neutrophils and nearly complete abolition of lethal shock in W/Wv mice regardless of the sensitizing method. Both prevention of shock and reduction of neutrophils lasted for three days after the treatment with the antibody but not for six days. From these results, a reasonable conclusion would be that circulating Gr-1+ cells (predominantly composed of neutrophils) are the major mediator source. Reference experiments using mast cell-bearing strains revealed that the suppressive effect of anti-granulocyte antibody was also observed against active anaphylactic shock in C3H/HeN mice but not against active and passive anaphylactic shock in the other mice.

Anaphylaxis↗
PubMed1997

Evaluation of bone marrow iron by magnetic resonance imaging.

Bone marrow iron was estimated by magnetic resonance imaging (MRI) using spin-echo sequences with multiple echoes in 22 patients with varying degrees of tissue storage iron. Levels of bone marrow iron concentration (BMIC) were determined chemically in biopsied specimens concurrently. Concentrations of serum iron, serum ferritin, and transferrin saturation were also measured to evaluate body iron status. Significant correlation was observed between BMIC and T2 relaxation rate (1/T2) (r = 0.77; p < 0.001) in all patients with BMIC levels below 400 micrograms/ml, while BMIC was not correlated with T2 in patients with extremely high BMIC levels. MRI was considered to be inappropriate for quantitation of 1/T2 in patients with extremely high BMIC due to an extreme shortening of T2 relaxation time. These observations suggest that MRI may be a useful and noninvasive method for systemic quantitative determination of bone marrow iron.

Adolescent↗
PubMed1997

A sustained increase of cytosolic Ca2+ in gammadelta T cells triggered by co-stimulation via TCR/CD3 and LFA-1.

We previously reported that co-stimulation with LFA-1 triggered apoptosis in gammadelta T cells but not in alphabeta T cells after TCR engagement. We extended our earlier study on TCR/LFA-1 triggered apoptosis to two autoreactive TCR gammadelta and TCR alphabeta T cell clones, which were derived from syngeneic mixed lymphocyte culture of BALB/c mice. A gammadelta T cell clone, KM1, expressed the Vgamma4 and Vdelta5 genes and CD4-CD8-CD45RB+ phenotype; and an alphabeta T cell clone, BASL1.1, expressed Vbeta6 and CD4+CD8-CD45RB+. Both clones produced Th-1-type cytokines in response to syngeneic BALB/c stimulator cells. KM1 underwent apoptosis upon stimulation with immobilized anti-CD3/LFA-1 mAbs, whereas BASL1.1 could proliferate successfully in response to stimulation with the immobilized mAbs. BASL1.1 was able to down-regulate the increased cytosolic Ca2+ after the simultaneous stimulation, but KM1 exhibited a sustained increase of cytosolic Ca2+ after stimulation via CD3 and LFA-1. Similar results with respect to the kinetics of cytosolic Ca2+ were obtained with normal heterogeneous gammadelta and alphabeta T cell populations after co-stimulation via CD3 and LFA-1. Our results suggested that persistently high levels of cytosolic Ca2+ might be related to apoptosis in gammadelta T cell clone triggered by co-stimulation via CD3 and LFA-1.

Animals↗
PubMed1997

Decentration and tilt of polymethyl methacrylate, silicone, and acrylic soft intraocular lenses.

PURPOSE: The purpose of the study is to investigate the periodic changes regarding the decentration and tilt of the intraocular lens (IOL) and to compare any differences in the decentration and tilt among polymethyl methacrylate (PMMA), silicone, and acrylic soft IOLs. METHODS: A total of 225 cataractous eyes undergoing IOL implant surgery were randomized into 3 groups based on the type of IOL: group A, one-piece PMMA IOL; group B, three-piece silicone IOL; and group C, three-piece acrylic soft IOL. Both the length of the decentration and the degree of the tilt of the IOL were quantitated using the Anterior Eye Segment Analysis System (EAS-1000). All eyes underwent EAS-1000 examinations at 1 week as well as 1, 3, 6, 9, and 12 months after surgery. RESULTS: All IOLs were confirmed to be implanted accurately in the capsular bag after continuous curvilinear capsulorhexis was accomplished. No statistically significant differences were observed regarding the IOL decentration or tilt between the various postoperative periods in any of the three IOL groups. Furthermore, the differences regarding both the IOL decentration and the tilt between the three IOLs were not determined to be statistically significant throughout the observation period. CONCLUSIONS: As long as the IOL was placed properly in the capsular bag after the continuous capsulorhexis, neither the decentration nor the tilt of the IOL showed a significant progression up to 12 months after surgery. Furthermore, both the extent of the decentration and tilt almost were the same among the PMMA, silicone, and acrylic soft IOLs.

Acrylic Resins↗
PubMed1997

Pericentric inversion of chromosome 16 and eosinophilia in chronic myelomonocytic leukemia.

We report a case of myelodysplastic syndrome with bone marrow eosinophilia and the chromosomal abnormality, inv(16)(p13q22). Hematologic findings including monocytosis and trilineage myelodysplasia were consistent with chronic myelomonocytic leukemia, and numerous abnormal eosinophils were present in the bone marrow. Chromosomal analysis of all metaphase cells from peripheral blood and bone marrow revealed in(16)(p13q22), which is well known characteristic of acute myelomonocytic leukemia with eosinophilia (M4Eo). Both monocytes and eosinophils in this case may be derived from common leukemic progenitors affected by inv(16)(p13q22).

Aged↗