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Biomedical subjects

Y Miyazaki

Publications and source records attributed to Y Miyazaki.

At least 253 records · Page 14Linked to original sources
PubMed1997

Stimulatory effect of gangliosides on phagocytosis, phagosome-lysosome fusion, and intracellular signal transduction system by human polymorphonuclear leukocytes.

Gangliosides are known to be differentiation-inducing molecules in mammalian stem cells. We studied the interaction between the molecular structure of glycosphingolipids (GSLs) and their promoting mechanisms of the phagocytic processes in human polymorphonuclear leukocytes (PMN). The effect of various gangliosides from mammalian tissues on adhesion, phagocytosis, phagosome-lysosome (P-L) fusion and superoxide anion production was examined by human PMN using heat-killed cells of Staphylococcus aureus-coated with GSLs. Gangliosides GM3, GD1a, GD3 and GT1b showed a marked stimulatory effect on the phagocytosis and P-L fusion in a dose-dependent manner, while ganglioside GM1, asialo GM1 and neutral GSLs did not. The relative phagocytic rate of ganglioside GM3-coated S. aureus was the highest among the tested GSLs. Both P-L fusion rate and phagocytosis of S. aureus were elevated significantly when coated with ganglioside GD1a, GD3 or GT1b, and GT1b gave a five times higher rate than that of the non-coated control. These results suggest that the terminal sialic acid moiety is essential for the enhancement of phagocytosis and that the number of sialic acid molecules in the ganglioside is related to the enhancement of the P-L fusion process. On the other hand, the superoxide anion release from PMN was not affected by ganglioside GM2, GM3, GD1a or GT1b. Furthermore, to clarify the trigger or the signal transduction mechanism of phagocytic processes, we examined the effect of protein kinase inhibitors such as H-7, staurosporine (protein kinase C inhibitor), H-89 (protein kinase A inhibitor), genistein (tyrosine kinase inhibitor), ML-7 (myosin light chain kinase inhibitor), and KN-62 (Ca2+/calmodulin-dependent protein kinase II inhibitor) on ganglioside-induced phagocytosis. H-7, staurosporine and KN-62 inhibited ganglioside-induced phagocytosis in the range of concentration without cell damage, while H-89, genistein and ML-7 did not. Moreover, H-7 and KN-62 inhibited ganglioside-induced P-L fusion. These results suggest that protein kinase C and Ca2+/calmodulin-dependent protein kinase II may be involved in the induction of phagocytosis and P-L fusion stimulated by gangliosides.

Bacterial Adhesion↗
PubMed1997

Amplification of BCR protein associated with oncogenesis in human hepatocellular carcinoma.

The BCR gene is located on human chromosome 22. The normal cellular BCR gene encodes a 160,000-dalton phosphoprotein associated with a serine/threonine kinase activity. The BCR protein is involved in signal transduction. We investigated the expression of the BCR protein in hepatocellular carcinoma (HCC), surrounding noncancerous liver tissue, liver cirrhosis (LC), chronic hepatitis (CH), and normal liver with immunohistochemistry and a western blot analysis. BCR immunoreactivity was detected using a monoclonal antibody. In normal liver, and both CH and LC without association of HCC, the immunoreactivity of the BCR protein was minimal. In contrast, 73% (22 of 30) of noncancerous liver tissue adjacent to the HCC and 40% (12 of 30) of HCC expressed BCR protein; this difference was statistically significant (P < 0.01). The expression of the BCR protein expression correlated with the degree of histological differentiation of HCC (P < 0.05). In addition, the amplification of BCR protein in noncancerous cells was supported by the detection of specific protein using a western blot analysis. In two cases, the expression of BCR protein occurred only in overtly malignant HCC cells. As a result, the expression of the BCR protein may be associated with oncogenesis in human HCC.

Aged↗
PubMed1997

Establishment and characterization of a new erythropoietin-dependent acute myeloid leukemia cell line, AS-E2.

We have established an erythropoietin-dependent human leukemia cell line, AS-E2, from a patient with acute myeloid leukemia. These cells have many characteristics of late erythroid progenitor cells, they are positive for CD36, Glycophorin A, and CD71 but negative for CD41, and positive for benzidine and PAS staining. These cells express GATA-1 and have low affinity erythropoietin (EPO) receptor on their surface. Interestingly, AS-E2 cells are strictly dependent on EPO for their growth and survival; other cytokines including GM-CSF, stem cell factor, or IL-3 cannot support the growth of this cell line. These features are similar to late erythroid lineage cells, like normal BFU-E or CFU-E, and we have demonstrated that EPO stimulation induces the tyrosine phosphorylation of several proteins in AS-E2 cells including the EPO receptor and JAK2 kinase. This new cell line is a useful reagent to study biological and molecular events during the late stages of erythropoiesis, and to understand transforming events in human erythroid cells.

Acute Disease↗
PubMed1997

Mucosal interleukin-1 beta production and acid secretion in enlarged fold gastritis.

BACKGROUND: We have previously shown that eradication of Helicobacter pylori increases acid secretion in H. pylori-associated enlarged fold gastritis. AIM: To investigate whether locally produced interleukin-1 beta is possibly involved in the inhibition of acid secretion in H. pylori gastritis. METHODS: IL-1 beta release from the gastric body mucosa was determined by short-term culture of biopsy specimens in 13 patients with enlarged fold gastritis (all H. pylori-positive), five H. pylori-positive and 10 H. pylori-negative patients without enlarged folds. The acid-inhibitory effect of locally produced IL-1 beta was examined by [14C]-aminopyrine uptake assay using isolated rabbit gastric glands. RESULTS: IL-1 beta release was significantly greater in patients with enlarged fold gastritis, significantly correlated with both basal and tetragastrin-stimulated acid outputs in the H. pylori-positive patients (r = -0.591 and r = -0.641, respectively; P < 0.01), and significantly decreased with concomitant increases in acid secretions after eradication of H. pylori. [14C]-aminopyrine uptake was inhibited by IL-1 beta in a dose-dependent manner. CONCLUSIONS: Increased production of IL-1 beta caused by H. pylori infection is possibly involved in the inhibition of acid secretion in enlarged fold gastritis.

Adult↗
PubMed1997

Determination of the DNA-binding sequences of the Zn(II)2Cys6 zinc-cluster-containing PRIB protein, derived from the basidiomycete Lentinus edodes gene.

The 565 amino-acid PRIB protein with a Zn(II)2Cys6 zinc-cluster DNA-binding motif is the expression product of the priB gene, which is most actively transcribed in an early stage of fruiting-body formation by the basidiomycete, Lentinus edodes. PRIB produced in Escherichia coli using the bacteriophage T7 expression system was purified by ion-exchange chromatographies and then subjected to random binding-site selection analysis using a pool of random 24-bp oligonucleotides with 13-bp PCR primer sites at each end. The oligonucleotides (50 bp) selected for PRIB binding were cloned into pUC19. A total of 303 cloned DNA fragments were picked randomly and sequenced. The PRIB binding sites could be grouped into 25 individual sequences, suggesting a consensus sequence of 16 bp, 5' GGGGGGGACAGGANCC 3'. Gel mobility-shift assaying of 10 randomly selected sequences all revealed a reasonable band shift. DNase I footprinting analysis of the 50-bp DNA fragment containing the sequence most similar to the consensus sequence showed that PRIB protects the entire 16-bp sequence from digestion by DNase I.

Base Sequence↗
PubMed1997

The effect of xenon on spinal dorsal horn neurons: a comparison with nitrous oxide.

We compared the effects of xenon (Xe) on the spinal cord dorsal horn neurons with those of nitrous oxide (N2O) in cats anesthetized with chrolarose and urethane. We assessed the potency of both anesthetics by the inhibition of wide dynamic range neuron responses evoked by cutaneous noxious (pinch) stimulation to a hindpaw. During 70% Xe inhalation, the responses of 7 of 11 neurons to pinch stimulation were suppressed. N2O, 70%, suppressed it in 8 of 11 neurons. The potency of Xe and N2O was compared in six neurons that were suppressed by both anesthetics. After 20 min of Xe inhalation, the response to pinch was suppressed to 49.5% +/- 8.2% (mean +/- SE), while N2O, 70% in oxygen, suppressed it to 45.9% +/- 7.9%. The difference between N2O and Xe was not significant. We conclude that Xe and N2O suppress the spinal cord dorsal horn neurons to a similar degree.

Anesthetics, Inhalation↗
PubMed1997

The rapid spread of recombinants during a natural in vitro infection with two human immunodeficiency virus type 1 strains.

We quantified a population of recombinants in a natural in vitro infection, using wild-type viruses without any pressure. It was found that recombinants emerged early after infection and constituted more than 20% of the whole proviral population 15 days after infection. Furthermore, recombinants were isolated as infectious viruses by simple limiting dilution. These results imply that, in addition to the high mutation rate of human immunodeficiency virus type 1 (HIV-1), recombination among HIV-1 strains plays a significant part in the development of the high diversity of HIV-1.

Base Sequence↗
PubMed1997

Higher concentrations of matrix metalloproteinases in bronchoalveolar lavage fluid of patients with adult respiratory distress syndrome.

This study was designed to investigate possible involvement of type IV collagenolytic matrix metalloproteinases (MMPs; 72-kDa type IV collagenase [MMP-2], 92-kDa type IV collagenase [MMP-9]), and the respective specific tissue inhibitors of these MMPs (TIMP-2 and TIMP-1) in the development of adult respiratory distress syndrome (ARDS). We determined the concentrations of these enzymes in the bronchoalveolar lavage fluid (BALF) from patients with ARDS using newly developed sensitive one-step sandwich enzyme immunoassay methods. BALF obtained from the 17 patients and eight healthy volunteer control subjects were also used for the analysis of the number of the cellular component. Concentrations of the 7S portion of type IV collagen and laminin in the BALF were measured as markers of basement membrane disruption. In the BALF from the ARDS patients, the concentrations of MMP-2 (66.7 +/- 57.0 ng/ml versus < 7.0 ng/ml for controls, p < 0.01) and MMP-9 (118.0 +/- 309.3 ng/ml versus 9.0 +/- 9.5 ng/ml for controls, p < 0.05), and the specific inhibitor of MMP-9 (TIMP-1) (161.0 +/- 145.0 ng/ml versus < 50 ng/ml for controls, p < 0.01) were significantly higher compared with those for healthy control subjects. In the ARDS patients, the concentrations of MMP-2 correlated both with those of 7S collagen and laminin; MMP-9 with the concentration of 7S collagen and the number of neutrophils. These findings suggest that the increased concentration of collagenolytic MMPs in lung plays a role in the pathogenesis of ARDS.

Adult↗
PubMed1997

Fumaric acid, anti-thrombin substance from Rhizopus javanicus.

An anti-thrombin substance (M2) was isolated from a culture broth of Rhizopus javanicus. Accumulation of M2 reached a maximum peak after 14 to 15 days of incubation and then decreased. The yield of M2 was 500 mg from 1 L of culture broth. M2 inhibited thrombin activity, and its 50% inhibition concentration in a reaction mixture containing 50 microL of 12.5 NIH unit/mL thrombin and 200 microL of 0.33% bovine fibrinogen was 63 microM. M2 had a specific activity for thrombin, but it was less responsive to plasmin, tissue-type plasminogen activator, urokinase, plasma kallikrein and glandular kallikrein. The structure of M2 was identified as fumaric acid by elementary analysis, FAB/MS, 1H-NMR, 13C-NMR and IR spectra.

Animals↗
PubMed1997

[Brain uptake ratio as an index of cerebral blood flow obtained with 99mTc-ECD].

A new index of cerebral blood flow (brain uptake ratio, BUR) using 99mTc-ECD was developed and evaluated in 66 patients (132 cerebral hemispheres). BUR was calculated from brain count in anterior planar image (60-80 sec after injection of 99mTc-ECD) divided by the summation of the count of aortic arch during first transit of radionuclide. BUR correlated well with brain perfusion index (BPI) obtained with Patlak plot method (r = 0.960, p < 0.001). In conclusion, BUR is useful as a simple and non-invasive index reflecting cerebral blood flow.

Adolescent↗
PubMed1997

[A case of hearing loss caused by overdrainage of cerebrospinal fluid after ventriculo-peritoneal shunting procedure].

We describe a case of intracranial hypotension syndrome due to overdrainage of cerebrospinal fluid presented with hearing loss after ventriculoperitoneal shunting procedure. A 69-year-old man suffering from subarachnoid hemorrhage presented with an angiogram showing two aneurysms, one of the right internal carotid and one of the middle cerebral artery. Neck clipping was performed. One month later, he developed normal pressure hydrocephalus (NPH), which was treated by ventriculoperitoneal (NPH), which was treated by ventriculoperitoneal shunting procedure using low pressure Pudenz system. Trias of NPH were improved by insertion of shunt system. However, he complained of hearing loss which was worsened by upright position and improved by lying down. Such kinds of phenomenon were demonstrated by audiogram showing that the transitory decrease of hearing and electrocochleography showing the elongation of N1 latency at upright position. These data suggested that his hearing loss was caused by inner ear or auditory nerve lesion. After the shunt system was replaced into the antisiphon device, his hearing disturbance improved. Axial computed tomography of bone window at the level of orbitomeatal line demonstrated widely perilymphatic duct on both sides. This finding suggested that the fluctuation of intracranial pressure was easily transmitted into the cochlear through the widened perilymphatic duct, resulting in hearing disturbance.

Aged↗
PubMed1997

[Detection of antigen specific T cell clonality in autoimmune diseases and epitope analysis of autoantigen-reactive T cells].

In order to develop an ultimate therapy to overcome autoimmune diseases, it is very important to identify whether or not autoantigen-reactive T cells accumulate in such diseases. A novel method was established to analyze T cell clonality using a combination of reverse transcriptase-polymerase chain reaction of T cell receptor beta chain transcripts and single-strand conformation polymorphism (RT-PCR/SSCP). Using RT-PCR/SSCP, clonal expansion of T cells were identified in NOD mouse, collagen induced arthritis model, rheumatoid arthritis, Crohn's disease, Sjögren's syndrome, bone marrow transplantation, pregnancy, malignant tumors, HTLV-I associated myopathy, and interstitial pneumonia. By using a tandem mass spectrometry or a lymphocytic proliferation assay, T cell epitopes were mapped in NOD mice, EAE, MCTD, and melanoma.

Animals↗
PubMed1997

Effect of a new monoclonal anti-glycoprotein IX antibody, KMP-9, on high shear-induced platelet aggregation.

Human platelet glycoprotein Ib/IX complex acts as a receptor for von Willebrand factor. It is widely accepted that glycoprotein Ib is the essential receptor component, but the role of glycoprotein IX is still unclear. We produced a new monoclonal anti-glycoprotein IX antibody (KMP-9) by the hybridoma technique using platelets from a patient with Glanzmann's thrombasthenia. The epitope of KMP-9 was localized to the C-terminal 8 kD fragment of glycoprotein IX using ELISA analysis of polyethylene-pin-synthesized peptides, as well as Western blot analysis of platelets after digestion with N-glycosidase and Staphylococcus aureus V8 protease. KMP-9 partially inhibited high shear stress-induced platelet aggregation, but had no effect on aggregation induced by ristocetin or low shear stress. Its inhibitory effect on high shear stress-induced aggregation was weaker than that of anti-glycoprotein Ib or anti-glycoprotein IIb/IIIa monoclonal antibodies. A 21-mer synthetic peptide (glycoprotein IX L110-G130) inhibited the binding of KMP-9 to platelets. It also competively inhibited the suppression of high shear stress-induced platelet aggregation by KMP-9, but had no direct effect on this aggregation. KMP-9 may be useful to clarify the physiological role of GPIX.

Amino Acid Sequence↗
PubMed1997

[Quantification of brain perfusion SPECT with N-isopropyl-p-iodoamphetamine using noninvasive microsphere method: estimation of arterial input by dynamic imaging].

We have developed a noninvasive method to quantify brain perfusion SPECT with 123I-N-isopropyl-p-iodoamphetamine (IMP) using serial dynamic planar imaging of the initial transit phase. The method is based on the microsphere model, but does not require arterial sampling. Serial dynamic planar imaging was performed for 6 min after the bolus injection of IMP (167 MBq in 1.5 ml), followed by additional planar imaging at 20 min and SPECT scan thereafter. The total arterial input to the brain during the initial 5 min after injection was estimated by the injected dose, with the correction of the lung retention, divided by cardiac output (CO). CO was estimated from the initial transit of IMP in the right heart. Cardiac output index (COI), obtained from the integral of the first transit of IMP in the right heart divided by the injected dose, was calibrated by CO measured by Doppler ultrasonography. Regional cerebral blood flow (rCBF) obtained by this method in normal subjects was acceptable. However, the results may be influenced by the injection technique, and careful attention should be considered for clinical application of this method.

Adult↗
PubMed1997

Human erythropoietin receptor increases GATA-2 and Bcl-xL by a protein kinase C-dependent pathway in human erythropoietin-dependent cell line AS-E2.

Erythropoietin (Epo) is a cytokine known to stimulate proliferation and differentiation of erythroid cells. However, recent gene disruption experiments demonstrated that Epo receptor signaling is not an obligatory step in erythroid differentiation. Here, we describe the role of Epo in proliferation, terminal differentiation, and apoptosis in a novel human Epo-dependent cell line, AS-E2. Upon withdrawal of Epo, the cells ceased to proliferate and underwent apoptotic death. Accompanying this cell death, an increase in the number of hemoglobin-positive cells of approximately 2-fold was observed. This was associated with immediate up-regulation of the GATA-1:GATA-2 ratio and down-regulation of Bcl-xL. Treatment with Epo or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) up-regulated expression of GATA-2 and Bcl-xL, and these elevations were inhibited by inhibitors of protein kinase C (PKC), H7 and H8. HA1004, a structural analogue of H7 but a poor inhibitor of PKC, had no inhibitory effect. Therefore, in AS-E2 cells, it is likely that Epo plays a role in (a) proliferation, (b) inhibition of differentiation, and (c) survival, by maintaining GATA-2 and Bcl-xL expression through activation of PKC.

Apoptosis↗
PubMed1996

Isolation of cDNA clones of the rat mRNAs expressed preferentially in the prenatal stages of brain development.

For better understanding of the molecular mechanisms underlying the developmental processes of the mammalian brain, we isolated rat fetal brain-enriched (FBE) cDNA clones, whose corresponding mRNAs were expressed at least 5-fold more in the fetal brain than in the adult brain. Our modified differential screening procedure, which utilized a two-vector (pT7T3D and pBluescript) system and showed low background levels of colony hybridization for screening, efficiently identified 64 candidate FBE clones from a small number (475) of colonies in the fetal brain cDNA library. After subsequent second screening of the candidate FBE clones by Northern blot analysis, we successfully isolated 22 distinct FBE clones. The nucleotide sequence analysis of the 22 FBE clones revealed that 13 of them had no significant matches to the sequences reported in the databases, whereas 9 of them matched previously reported sequences (alpha-tubulin M alpha 1, beta-tubulin M beta 5, thymosin-beta 10, stathmin, beta-tubulin M beta 2, alpha-internexin, ferritin Lg chain, neuronatin and amphoterin), most of which have been shown to be down-regulated during brain development. We also found that the Northern blot analysis in the second screening could be replaced by cDNA library DNA-Southern blot analysis, in most clones corresponding to relatively abundantly expressed mRNAs. Thus, once the cDNA library is constructed, clone selection will be possible in such clones without the use of additional RNA or Northern blot in screening, allowing the analysis of small brain regions of interest.

Animals↗
PubMed1996

High shear stress can initiate both platelet aggregation and shedding of procoagulant containing microparticles.

Previous studies have demonstrated that a high level of shear stress can produce platelet aggregation without the addition of any agonist. We investigated whether high shear stress could cause both platelet aggregation and shedding of microparticles from the platelet plasma membrane. A coneplate viscometer was used to apply shear stress and microparticle formation was measured by flow cytometry. It was found that microparticle formation increased as the duration of shear stress increased. Both microparticles and the remnant platelets showed the exposure of procoagulant activity on their surfaces. Investigation of the mechanisms involved in shear-dependent microparticle generation showed that binding of von Willebrand factor (vWF) to platelet glycoprotein lb, influx of extracellular calcium, and activation of platelet calpain were required to generate microparticles under high shear stress conditions. Activation of protein kinase C (PKC) promoted shear-dependent microparticle formation. Epinephrine did not influence microparticle formation, although it enhanced platelet aggregation by high shear stress. These findings suggest the possibility that local generation of microparticles in atherosclerotic arteries, the site that pathologically high shear stress could occur, may contribute to arterial thrombosis by providing and expanding a catalytic surface for the coagulation cascade.

Blood Platelets↗