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Biomedical subjects

T Miyazaki

Publications and source records attributed to T Miyazaki.

At least 199 records · Page 11Linked to original sources
PubMed1999

[Evaluation of molecular weight of hyaluronate preparations by size-exclusion chromatography].

Hyaluronate (HA), a glycosaminoglycan polysaccharide, has been used as a biomedical polymer to treat osteoarthritis by intra-articular injections and in ophthalmic surgery, such as anterior segment surgery. In this study, the molecular weight (MW) of HA preparations was estimated by size-exclusion chromatography (SEC), using HA and pullulan fractions as molecular weight standards, and the MW values obtained were compared to those obtained with a low angle laser light scattering detector (LALLS). The results showed that the universal calibration with pullulan as the standard is useful for HA preparations. The conditions of SEC for HA were also investigated, and the results suggested that a high ionic strength and low flow rate of the eluent were preferable for high molecular weight HA preparations.

Chromatography↗
PubMed1998

Presynaptic inhibition by dopamine of a discrete component of GABA release in rat substantia nigra pars reticulata.

1. Whole-cell patch clamp recordings were made from substantia nigra pars reticulata (SNr) neurones in rat midbrain slices. Monosynaptic IPSCs were evoked by electrical stimulation of the cerebral peduncle in the presence of the glutamate receptor antagonists CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and AP5 (2-amino-5-phosphonopentanoic acid). 2. IPSCs were predominantly outward at -70 mV (in 124/135 cells), with a reversal potential of -83 mV, a time to peak of 2.6 ms and a decay time constant of 6.5 ms. Faster inward IPSCs were also observed in thirty-five cells, with a time to peak of 1.0 ms, a decay time constant of 2.3 ms, and a reversal potential of -61 mV. Both IPSCs were sensitive to the GABAA receptor antagonists picrotoxin or bicuculline. 3. In cells recorded with Cs+-filled pipettes, the outward IPSC reversal potential was shifted to -76 mV, closer to the estimated Cl- equilibrium potential of -56 mV, while that of the inward IPSC was unchanged at -64 mV. 4. The outward IPSC was reversibly depressed by up to 100 % by dopamine in a concentration-dependent manner with an IC50 of 10.5 microM, while the inward IPSC was relatively insensitive. 5. Dopamine was without effect on cell holding current, or on outward IPSC reversal potential, but it increased paired-pulse IPSC facilitation, consistent with a presynaptic site of action. 6. The D1-like dopamine receptor agonist SKF 38393 (10 microM) depressed the outward IPSC by 43 %, while the D2-like dopamine receptor agonist quinpirole (10 microM) was without effect. 7. It is concluded that GABA-ergic synaptic input onto distal rather than proximal regions of SNr neurones is susceptible to presynaptic inhibition via a D1-like receptor. These inputs are probably from striato-nigral fibres, and their inhibition by dopamine is likely to influence the patterning of basal ganglia output.

Animals↗
PubMed1998

Functional dissection of the cytoplasmic subregions of the IL-2 receptor betac chain in primary lymphocyte populations.

The interleukin 2 (IL-2) receptor betac chain (IL-2Rbetac) is known to regulate the development and function of distinct lymphocyte populations. Thus far, the functions of the IL-2Rbetac cytoplasmic subregions have been studied extensively by using cultured cell lines; however, this approach has limitations with respect to their functions in distinct primary lymphocyte populations. In the present study, we generated mice each expressing a mutant form of an IL-2Rbetac transgene, lacking the cytoplasmic A- or H-region, on an IL-2Rbetac null background. We show that lack of the H-region, which mediates activation of the Stat5/Stat3 transcription factors, selectively affects the development of natural killer cells and T cells bearing the gamma delta T cell receptor. This region is also required for the IL-2-induced proliferation of T cells in vitro, by upregulating IL-2Ralpha expression. In contrast, the A-region, which mediates activation of the Src family protein tyrosine kinase (PTK) members, contributes to downregulation of the T cell proliferation function. The IL-2Rbetac null mutant mice develop severe autoimmune symptoms; these are all suppressed following the expression of either of the mutants, suggesting that neither the Stats nor the Src PTK members are required. Thus, our present approach offers new insights into the functions of these cytoplasmic subregions of the IL-2Rbetac chain.

Animals↗
PubMed1998

Radiation-induced long-lived radicals which cause mutation and transformation.

Using electronic spin resonance (ESR), we found a new type of radical with a long life-time in cells (T1/2>20 h) and which may play a more important role in the induction of mutation and transformation than either the active, short-lived, H, or OH radicals. When cells were treated with dimethyl sulfoxide (DMSO) and l-ascorbic acid (AsA) just before irradiation, the short-lived radicals were well-scavenged. On the other hand, if cells were treated with the scavengers 20 min after irradiation, then AsA scavenged the long-lived radicals, but DMSO did not. AsA treatment 20 min after the start of irradiation drastically reduced both the frequencies of mutation at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus in human cells and morphological transformations in mouse m5S cells, but DMSO treatment did not. In addition, AsA treatment 20 h after irradiation also reduced the mutation frequency in human cells. These results suggested that mutations and morphological transformation are probably caused by the presence of long-lived radicals in the cells, rather than by short lived radicals, and that AsA reacts efficiently with long-lived radicals, resulting in a decrease of the mutations and transformations induced.

Animals↗
PubMed1998

Vasculitis-susceptible genes in mice with a deficit in Fas-mediated apoptosis.

Autoimmune diseases show complex pathological manifestations, which frequently involve systemic vasculitis. This complication is understood to be a manifestation of advanced disease, or to represent distinct entities, restricted by genetic and/or environmental factors. An MRL/Mp strain of mice bearing the Fas deletion mutant gene, lpr (MRL/lpr), spontaneously develop systemic vasculitis coincidentally with glomerulonephritis, arthritis and sialoadenitis, but a C3H/HeJ-lpr/lpr (C3H/lpr) strain does not. Thus, this is a suitable model for analyzing the genetic basis of vasculitis in autoimmune diseases. To genetically dissect these complex pathological manifestations, a linkage analysis of each lesion with polymorphic microsatellite markers was performed by using MRL/lpr x (MRL/lpr x C3H/lpr)F1 backcross mice. Vasculitis-susceptible gene loci were mapped on chromosomes 3 and 4, which were not associated with glomerulonephritis, arthritis and sialoadenitis. These results indicate that systemic vasculitis in MRL/lpr mice may be under the control of host genes which are different from those for other autoimmune diseases.

Animals↗
PubMed1998

An improved retroviral gene transfer technique demonstrates inhibition of CD4-CD8- thymocyte development by kinase-inactive ZAP-70.

ZAP-70 is a Syk family tyrosine kinase that plays an essential role in initiating TCR signals. Deficiency in ZAP-70 causes a defect in the development at CD4+CD8+ thymocytes due to defective TCR-mediated positive and negative selection. Using a newly devised retrovirus gene transfer and an efficient green fluorescence protein detection technique in fetal thymus organ cultures, the present study shows that forced expression in developing thymocytes of a catalytically inactive mutant of ZAP-70, but not wild-type ZAP-70, inhibits T cell development at the earlier CD4-CD8- stage. The ZAP-70 mutant blocked the generation of CD4+CD8+ thymocytes even in the absence of endogenous ZAP-70. Thus, the present results demonstrate a novel technique for gene transfer into developing T cells and suggest that ZAP-70/Syk family tyrosine kinases are involved in the signals inducing the generation of CD4+CD8+ thymocytes.

3T3 Cells↗
PubMed1998

Intracellular free calcium concentration in human taste bud cells increases in response to taste stimuli.

We examined changes of intracellular free calcium concentration [Ca2+]i elicited by taste stimuli of sucrose, denatonium and NaCl in the taste buds of seven human fungiform papillae. In one taste bud we observed an increase in [Ca2+]i induced by only NaCl. In another bud an increase of [Ca2+]i in response to both NaCl and sucrose was found. The Ca2+ responses to NaCl and sucrose occurred in differential areas within the one taste bud. In the other five fungiform papillae [Ca2+]i was not changed by the taste stimuli. These results suggest that an increase of [Ca2+]i participates in taste transduction mechanisms for sucrose and NaCl, and that taste cells in one taste bud may respond to differential stimuli.

Adult↗
PubMed1998

Modulation of thymic selection by expression of an immediate-early gene, early growth response 1 (Egr-1).

The potential involvement of early growth response (Egr)-1, a zinc-finger transcription factor belonging to the immediate-early genes, in positive/negative selection of thymocytes has been implicated by its expression in the population of CD4(+)CD8(+) double positive (DP) cells undergoing selection. To further investigate this possibility, transgenic mice overexpressing Egr-1 in thymocytes were bred with a transgenic mouse line expressing a T cell receptor (TCR) recognizing the H-Y male antigen in the context of H-2(b) class I major histocompatibility complex (MHC) molecules. In Egr-1/TCR H-Y double-transgenic mice, efficient positive selection of H-Y CD8(+) T cells occurred, even in mice on either a nonselecting H-2(d) background or a beta2-microglobulin (beta2m)-deficient background in which the expression of class I MHC heavy chains is extremely low; no positive selection was observed on a Kb-/-Db-/-beta2m-/- background where class I MHC expression is entirely absent. Similarly, when the Egr-1 transgene was introduced into a class II MHC-restricted TCR transgenic mouse line, Egr-1/TCR double-transgenic mice revealed increased numbers of CD4(+) T cells selected by class II MHC, as well as significant numbers of CD8(+) T cells selected by class I MHC (for which the transgenic TCR might have weak affinity). Thus, Egr-1 overexpression allows positive selection of thymocytes via TCR-MHC interactions of unusually low avidity, possibly by lowering the threshold of avidity required for positive selection. Supporting this possibility, increased numbers of alloreactive T cells were positively selected in Egr-1 transgenic mice, resulting in a strikingly enhanced response against allo-MHC. These results suggest that expression of Egr-1 and/or its target gene(s) may directly influence the thresholds required for thymocyte selection.

Animals↗
PubMed1998

Simultaneous determination of beta-lactam antibiotics in milk by ion-pair liquid chromatography.

A simple and rapid ion-pairing liquid chromatographic method was developed for the simultaneous determination of five penicillins (PCs), ampicillin (AB-PC), benzylpenicillin (PC-G), Dicloxacillin (MDI-PC) and nafcillin (NF-PC) in milk. These PCs are most frequently used for the treatment of mastitis of cows. These antibiotics were extracted with acetonitrile from milk and cleaned up by solid-phase extraction with a C18 cartridge. PCs were separated on a Kaseisorb LC ODS-300-5 column with a mobile phase (1 ml/min) of acetonitrile-methanol-0.05 M potassium dihydrogenphosphate (20:10:80, v/v/v) mixture containing 5 mM of sodium 1-decanesulfonate adjusted to pH 3.5 and UV detection at 210 nm. The average recoveries of five PCs from milk fortified at 0.5 and 1.0 micrograms/ml (n = 5) were 79.8-89.4% with relative standard deviations ranging from 2.7 to 7.2% The detection limit of PCs in milk were 0.03-0.05 microgram/ml.

Animals↗
PubMed1998

On the influence of postmortem alcohol diffusion from the stomach contents to the heart blood.

Alcohol concentrations in the mixed left and right heart blood, urine and stomach contents of 186 cadavers were analyzed by gas chromatography in order to find the influence of postmortem diffusion of alcohol from the stomach contents to the heart blood. In 39 cases where blood alcohol concentrations (BACs) were less than 0.10 mg/g, alcohol in the stomach contents was suggested to be due to postmortem production, and the postmortem diffusion of alcohol from the stomach contents to the heart blood was less than 10%. In 147 where BACs were 0.10 mg/g and more, ratios of BAC to urine alcohol concentration (UAC) were 1.0 and more in 47 cases (32%), and less than 1.0 in 100 cases (68%). In 17 of these 147 cases, alcohol concentrations in stomach contents (SACs) were more than ten times as high as BACs. Where the highest ratio of SAC/BAC was 60.1, the BAC of 0.14 mg/g was suspected to be due to drinking. In the case where the highest SAC was 50.8 mg/g, the BAC of 5.18 mg/g, the highest in this study, seemed to be little affected by the diffusion. These results suggest that it is important to compare BAC, UAC and SAC to assess the influence of postmortem diffusion of alcohol from the stomach contents to the heart blood.

Alcoholism↗
PubMed1998

Presynaptic inhibition by noradrenaline of the EPSC evoked in neonatal rat sympathetic preganglionic neurons.

Visually identified and electrophysiologically characterized sympathetic preganglionic neurons (SPNs) were recorded using the whole-cell voltage clamp technique in slices of neonatal rat spinal cord. Monosynaptic excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation of the nucleus intercalatus in the presence of strychnine (5 microM) and bicuculline (10 microM). These EPSCs were abolished by the antagonist of AMPA-type glutamate receptors, 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX; 10 microM). Bath applied noradrenaline (NA; 0.5-50 microM) dose-dependently and reversibly decreased by up to around 60% the amplitude of the EPSC, without affecting the holding current. The EPSC depression by NA was not accompanied by a change in EPSC reversal potential (around +5 mV), nor were inward currents generated by pressure application of glutamate affected by NA application. A comparable degree of EPSC depression was also seen with the alpha2-adrenoceptor agonist clonidine (5 microM), and the alpha2A-agonist oxymetazoline (5 microM), while the alpha1-agonist phenylephrine (100 microM) caused only a 22% depression. The EPSC depression caused by NA (10 microM) was completely antagonized by either the alpha-antagonist phentolamine (10 microM) or the alpha2-antagonist idazoxan (2 microM). Conversely, the beta-adrenoceptor antagonist popranolol (5 microM), and the alpha1-, alpha2B- and alpha2C-antagonist prazosin (2 microM) were without effect. These results indicate that activation of presynaptic alpha2A-adrenoceptors on inputs to SPNs decreases glutamate release.

Adrenergic Fibers↗
PubMed1998

Pyk2 is a downstream mediator of the IL-2 receptor-coupled Jak signaling pathway.

Many cytokines transmit signals to the cell interior through activation of receptor-associated, Janus family protein tyrosine kinases (Jak PTKs). The interleukin-2 receptor (IL-2R) is associated with the Jak1 and Jak3 PTKs, and ligand-induced activation of these PTKs is essential for lymphocyte proliferation. Here, the nonreceptor PTK, Pyk2, was found to be activated following IL-2 stimulation in a Jak-dependent manner. Furthermore, physical association was detected between endogenous Pyk2 and Jak3, and a dominant interfering mutant of Pyk2 inhibited IL-2-induced cell proliferation without affecting Stat5 activation. Collectively, these results suggest that Pyk2 is a newly identified component of the Jak-mediated IL-2 signaling pathway.

Binding Sites↗
PubMed1998

The phenotype of H-2M-deficient mice is dependent on the MHC class II molecules expressed.

For a broader view of the role of H-2M as an accessory molecule in antigen presentation, we investigated the degree to which different MHC class II isotypes and alleles depend on H-2M to function in vivo. We generated H-2M-deficient animals expressing Ek/b or Ak molecules in addition to the Ab molecules already present in the mutant strain, and compared the ability of the different MHC class II molecules to present antigen at the cell surface for recognition by T cells, and contribute to positive selection of CD4+ T cells in the thymus. Biochemical analyses were performed to assess MHC class II maturation, and to determine the peptide content of the molecules. In the absence of H-2M, Ek/b molecules contained a more heterogeneous set of class II-associated invariant chain peptides (CLIP) than Ab did, which, unlike Ab-CLIP complexes, were not SDS-stable. Unlike Ab molecules, both Ek/b and Ak efficiently presented exogenously added peptides to T cells in the absence of H-2M. In addition, epitopes from some proteins, especially those known to be invariant chain independent, were presented by Ak molecules in the mutant animals. To our surprise, expression of Ek/b overcame the positive selection defect observed in H-2M-deficient mice expressing Ab alone. In contrast, Ak expression did not augment positive selection of CD4+ T cells in the mutant animals. Some of these findings in vivo contrast significantly with findings from in vitro studies on murine MHC class II molecules in human DM-deficient cell lines.

Amino Acid Sequence↗
PubMed1998

Ultrastructural evaluation following catheterization of the fallopian tube with a hysteroscopic catheter.

PURPOSE: Our purpose was to assess the morphology and ultrastructural changes in the tubal epithelium following catheterization of the fallopian tube. METHODS: Fallopian tubes were obtained from 20 women who had undergone hysterectomies. Catheterization was performed in 20 tubes using a catheter developed for hysteroscopic tubal embryo transfer. The catheter has a 3-French diameter, tapering to 2 French (0.66 mm) at the tip portion. The 20 contralateral tubes served as controls and were not catheterized. Ultrastructural changes were examined by scanning electron microscopy and transmission electron microscopy. RESULTS: Scanning electron microscopy showed no transformation or defects of the tubal epithelium surface in catheterized or control tubes. Transmission electron microscopy showed no significant differences in the percentage of abnormal desmosomes and the percentage of basement membrane in ciliated and nonciliated cells between catheterized and noncatheterized tubes. No transformation or defects were observed in catheterized or noncatheterized tubes. CONCLUSIONS: These findings suggest that catheterization of the tube using a hysteroscopic catheter caused no acute damage to the tubal epithelium.

Basement Membrane↗
PubMed1998

Serous cystadenocarcinoma of the pancreas with liver metastases.

Serous cystadenocarcinoma of the pancreas is a rare entity. We report a primary tumor of the pancreas in a 56-year-old woman that was histologically indistinguishable from microcystic adenoma, but which behaved in a malignant fashion. Metastatic lesions were found in the liver at the time of the initial operation. Nine years after the initial operation, new metastatic liver nodules were found, and the histologic characteristics of these lesions were quite similar to those of the pancreatic neoplasm. This is a very rare case which may support the existence of the entity, serous cystadenocarcinoma of the pancreas.

Angiography↗
PubMed1998

Prenatal ultrasound diagnosis of small-bowel torsion.

BACKGROUND: We report a case of congenital small-bowel torsion detected by prenatal ultrasonographic examination. CASE: A 27-year-old primigravida was found to have a fetus with a small-bowel obstruction by ultrasound examination during the third trimester. At 35 weeks of gestation, she complained of absence of fetal activity. A nonstress test showed a nonreactive pattern with reduced baseline variability. Sonographic examination revealed heterogeneous echogenicity within the dilated bowel, and loss of peristalsis. Also noted was a small amount of fetal ascites. After birth, torsion of the dilated small bowel associated with ileal atresia was found at exploratory laparotomy. CONCLUSION: Serial sonographic examinations with special attention to changes in the dilated bowel are useful in the management of prenatally diagnosed small-bowel obstruction.

Female↗
PubMed1998

Fibroblast-migration in a wound model of ascorbic acid-supplemented three-dimensional culture system: the effects of cytokines and malotilate, a new wound healing stimulant, on cell-migration.

To assess the migratory response of fibroblasts in vitro, normal human dermal fibroblasts (NHDF) were cultured in the presence of L-ascorbic acid 2-phosphate to induce a multilayered structure. Round wounds were made by punching, and the migratory response was evaluated by counting the number of migrating cells in the wounded areas. Collagenase activity in the culture-medium was then measured. When the wound model was treated with bFGF, IL-1 alpha or PDGF, the migratory response was facilitated with increased collagenase secretion. In contrast, treatment with TGF-beta reduced the migratory response and collagenase secretion. Since the multilayered structure is rich in collagenous matrix, degradation of the matrix by secreted collagenase is probably necessary for the cells to migrate into the wounded areas. Furthermore, malotilate, which is now under development as an agent for wound therapy, facilitated the migratory response of NHDF with increased collagenase secretion in this wound model, suggesting that the wound healing effect of malotilate is in part attributable to stimulated migration of fibroblasts to wounded areas subsequent to extracellular matrix-degradation.

Ascorbic Acid↗