Biomedical subjects
Publications and source records attributed to Y Ito.
The x-ray absorption spectra in metallic W (Tungsten) have been measured above MIII edge using the third generation synchrotron radiation. ALS, Berkeley. Several features caused by multi-electron transitions were detected on MIII in solid phase. [3p4f], [3p5s], and [3p5p] double electron transitions were identified by the Z+1 approximation.
Previous reports have suggested that changes in oligosaccharide structures, especially beta1-6 branching in N-glycans, which are biosynthesized by UDP-N-acetylglucosamine:alpha mannoside beta1,6 N-acetylglucosaminyltransferase (GnT-V), are linked to tumor metastasis and invasion. In the present study, we investigated GnT-V expression in human hepatocellular carcinoma (HCC) tissues. High expression of GnT-V mRNA was observed in both HCC and the surrounding tissues but not in normal liver. Immunohistochemical study using a newly established monoclonal antibody against GnT-V revealed that positive staining of GnT-V was observed in 75% of HCC tissues and 60% of surrounding tissues and that liver cirrhosis showed much stronger staining of GnT-V than chronic hepatitis without liver cirrhosis (p = 0.0035). In contrast, all of 12 cases of atypical adenomatous hyperplasia diffusely expressed GnT-V. beta1-6 branching in N-glycans, products of GnT-V, was increased in HCC tissues with high expression of GnT-V, as judged by lectin blotting. Levels of GnT-V expression in HCC tissues were positively correlated with a low Ki-67 labeling index (p = 0.0009), small size (p < 0.0001), poor differentiation (p < 0.0001) and absence of portal invasion (p = 0.018). Furthermore, HCC cases with low or no expression of GnT-V were more likely to show recurrence than cases with high expression (p = 0.0373). These findings strongly suggest that GnT-V expression is concerned mainly with an early phase of hepatocarcinogenesis.
The ability of the sulfonylurea receptor (SUR) 1 to suppress seizures and excitotoxic neuron damage was assessed in mice transgenically overexpressing this receptor. Fertilized eggs from FVB mice were injected with a construct containing SUR cDNA and a calcium-calmodulin kinase IIalpha promoter. The resulting mice showed normal gross anatomy, brain morphology and histology, and locomotor and cognitive behavior. However, they overexpressed the SUR1 transgene, yielding a 9- to 12-fold increase in the density of [(3)H]glibenclamide binding to the cortex, hippocampus, and striatum. These mice resisted kainic acid-induced seizures, showing a 36% decrease in average maximum seizure intensity and a 75% survival rate at a dose that killed 53% of the wild-type mice. Kainic acid-treated transgenic mice showed no significant loss of hippocampal pyramidal neurons or expression of heat shock protein 70, whereas wild-type mice lost 68-79% of pyramidal neurons in the CA1-3 subfields and expressed high levels of heat shock protein 70 after kainate administration. These results indicate that the transgenic overexpression of SUR1 alone in forebrain structures significantly protects mice from seizures and neuronal damage without interfering with locomotor or cognitive function.
In the present study, 92 people were found to have atrophic gastritis (AG) according to depressed serum levels of pepsinogen I and pepsinogen II in a screening involving 208 Japanese people, participating in a group health check. Serum levels of n-3 polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), were higher in AG than in non-AG individuals, while those of gamma-linolenic acid (GLA) were significantly lower in AG individuals. The odds ratios for high serum DHA and GLA levels in AG subjects were 2.20 (95% C.I.: 1.10-4.39) and 0.34 (95% C.I.: 0.17-0.68), respectively. The above results suggested that GLA plays a role in reducing the incidence of AG, whereas DHA may increase a risk of AG.
Pneumolysin (PLY), an important virulence factor of Streptococcus pneumoniae, is one of the members of thiol-activated cytolysins (TACYs) consisting of four domains. TACYs commonly bind to membrane cholesterol and oligomerize to form transmembrane pore. We have constructed full-length and various truncated PLYs to study the role of domains of PLY in the cytolytic activity. Full-length PLY had binding ability to both cell membrane and immobilized cholesterol. A truncated PLY which comprised only domain 4 molecule, the C-terminal domain of PLY, sustained the binding ability to cell membrane and cholesterol, whereas domain 1-3 molecule had no binding ability to them. Furthermore, the domain 4 molecule inhibited both the membrane binding and the hemolytic activity of full-length PLY. Accordingly, the present results provided the direct evidence that domain 4 was essential for the initial binding to membrane cholesterol and the interaction led to the subsequent membrane damage process.
The Drosophila transient receptor potential protein (TRP) and its mammalian homologues are thought to be Ca(2+)-permeable cation channels activated by G protein (G(q/11))-coupled receptors and are regarded as an interesting molecular model for the Ca(2+) entry mechanisms associated with stimulated phosphoinositide turnover and store depletion. However, there is little unequivocal evidence linking mammalian TRPs with particular native functions. In this study, we have found that heterologous expression of murine TRP6 in HEK293 cells reproduces almost exactly the essential biophysical and pharmacological properties of alpha(1)-adrenoceptor-activated nonselective cation channels (alpha(1)-AR-NSCC) previously identified in rabbit portal vein smooth muscle. Such properties include activation by diacylglycerol; S-shaped current-voltage relationship; high divalent cation permeability; unitary conductance of 25 to 30 pS and augmentation by flufenamate and Ca(2+); and blockade by Cd(2+), La(3+), Gd(3+), SK&F96365, and amiloride. Reverse transcriptase-polymerase chain reaction and confocal laser scanning microscopy using TRP6-specific primers and antisera revealed that the level of TRP6 mRNA expression was remarkably high in both murine and rabbit portal vein smooth muscles as compared with other TRP subtypes, and the immunoreactivity to TRP6 protein was localized near the sarcolemmal region of single rabbit portal vein myocytes. Furthermore, treatment of primary cultured portal vein myocytes with TRP6 antisense oligonucleotides resulted in marked inhibition of TRP6 protein immunoreactivity as well as selective suppression of alpha(1)-adrenoceptor-activated, store depletion-independent cation current and Ba(2+) influx. These results strongly indicate that TRP6 is the essential component of the alpha(1)-AR-NSCC, which may serve as a store depletion-independent Ca(2+) entry pathway during increased sympathetic activity.
In general, massive pulmonary embolism induces severe right ventricular overload, but pathological changes in the right ventricle due to pulmonary embolism is rarely seen. In this report, we describe two autopsy cases of massive pulmonary embolism without pre-existing cardiopulmonary disease. Both cases were accompanied by myocarditis-like changes in the right ventricle and infiltration of a number of polymorphonuclear neutrophils and mononuclear cells into the dilated right ventricular wall. Transmural or subendocardial coagulation necrosis was not apparent. Almost all of the mononuclear cells were immunohistochemically revealed to be CD68-positive macrophages. We speculated that these findings resulted from ischemia due to massive pulmonary embolism.
The RUNX family genes are the mammalian homologs of the Drosophila genes runt and lozenge, and members of this family function as master regulators of definitive hematopoiesis and osteogenesis. The RUNX genes encode the alpha subunit of the transcription factor PEBP2/CBF. The beta subunit consists of the non-RUNX protein PEBP2beta. We found that RUNX1/AML1, which is essential for hematopoiesis, is continuously subjected to proteolytic degradation mediated by the ubiquitin-proteasome pathway. When PEBP2beta is present, however, the ubiquitylation of RUNX1 is abrogated and this causes a dramatic inhibition of RUNX1 proteolysis. Heterodimerization between PEBP2beta and RUNX1 thus appears to be an essential step in the generation of transcriptionally competent RUNX1. Consistent with this notion, RUNX1 was barely detected in PEBP2beta(-/-) mouse. CBF(PEBP2)beta- SMMHC, the chimeric protein associated with inv(16) acute myeloid leukemia, was found to protect RUNX1 from proteolytic degradation more efficiently than PEBP2beta. These results reveal a hitherto unknown and major role of PEBP2beta, namely that it regulates RUNX1 by controlling its turnover. This has allowed us to gain new insights into the mechanism of leukemogenesis by CBFbeta-SMMHC.
BACKGROUND: The aim of the present study was to investigate the feasibility and potential value of the computer-controlled, 3D, echocardiographic reconstruction of the color Doppler-imaged vena contracta (CDVC) and the flow convergence (FC) region as a means of accurately and quantitatively estimating the severity of a ventricular septal defect (VSD). METHODS AND RESULTS: We performed a 3D reconstruction of the CDVC and the FC region in 19 patients with an isolated VSD using an ultrasound system interfaced with a Tomtec computer. The variable asymmetric geometry of the CDVC and the FC region could be 3D-visualized in all patients. The 3D-measured areas of CDVC correlated well with volumetric measurements of the severity of VSD (r=0.97, P:<0.001). Regression analysis between the shunt flow rate (calculated from the product of the area of CDVC and the continuous Doppler-derived velocity time integral) and the corresponding reference results (calculated by cardiac catheterization) demonstrated a close correlation (r=0.95, P:<0.001). There was also a good correlation between shunt flow rates calculated using the conventional 2D, 1-axis measurement of the FC isovelocity surface area with the hemispheric assumption (r=0.95, P:<0.001); shunt flow rates calculated using 3D, 3-axis measurements of the FC region (r=0.97, P:<0.01); and reference results by cardiac catheterization. However, the 2D method substantially underestimated the actual shunt flow rate. CONCLUSIONS: The 3D reconstruction of the CDVC and the FC region may aid in quantifying the severity of VSD.
The two pathways to osteoclastogenesis, RANKL-mediated and CD98-mediated osteoclastogenesis, have recently been reported. RANKL, OCIF, and TIMP-3 mRNAs are not found in monocytes freshly isolated or incubated with anti-FRP-1/CD98hc antibody. RANK, TACE, and M-CSF mRNAs can be detected in these cells. Interestingly, the expressed amount of RANK mRNA increases by cultivation of monocytes with anti-CD98hc antibody and maximal expression is observed in osteoclast-like cells. CD98-mediated cell aggregation and multinucleated giant cell formation are blocked by OCIF. OCIF also suppressed the CD98-mediated induction of Sp1 and c-src mRNAs in monocytes. Soluble RANK shows no effect on CD98-mediated cell aggregation and multinucleated giant cell formation. When blood monocytes were incubated with RANKL and M-CSF, c-src and Sp1 mRNAs were first found in blood monocytes incubated with these cytokines for 7 days. On the contrary, c-src mRNA could be detected 3 h after treatment of blood monocytes with anti-CD98hc mAb. LAT-1 mRNA was not found, and the expression levels of Y(+)LAT-1 and Y(+)LAT-2 mRNAs were not changed in monocytes stimulated without or with anti-CD98hc mAb or RANKL and M-CSF. An inhibitory mAb directed against CD98hc, HBJ 127, shows a suppressive effect on RANKL-mediated cell aggregation and cell fusion. Thus, there is cross-talk between these two pathways.
The c-Abl protein tyrosine kinase is activated by certain DNA-damaging agents and regulates induction of the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). The hematopoietic progenitor kinase 1 (HPK1) has also been shown to act upstream to the SAPK/JNK signaling pathway. We report here that exposure of hematopoietic Jurkat T cells to genotoxic agents is associated with activation of HPK1. The results demonstrate that exposure of Jurkat cells to DNA-damaging agents is associated with translocation of active c-Abl from nuclei to cytoplasm and binding of c-Abl to HPK1. Our findings also demonstrate that c-Abl phosphorylates HPK1 in cytoplasm and stimulates HPK1 activity. The functional significance of the c-Abl-HPK1 interaction is supported by the demonstration that this complex regulates SAPK/JNK activation. Overexpression of c-Abl(K-R) inhibits HPK1-induced activation of SAPK/JNK. Conversely, the dominant negative mutant of HPK1 blocks c-Abl-mediated induction of SAPK/JNK. These findings indicate that activation of HPK1 and formation of HPK1/c-Abl complexes are functionally important in the stress response of hematopoietic cells to genotoxic agents.
The hPar14 protein is a peptidyl prolyl cis/trans isomerase and is a human parvulin homologue. The hPar14 protein shows about 30 % sequence identity with the other human parvulin homologue, hPin1. Here, the solution structure of hPar14 was determined by nuclear magnetic resonance spectroscopy. The N-terminal 35 residues preceding the peptidyl prolyl isomerase domain of hPar14 are unstructured, whereas hPin1 possesses the WW domain at its N terminus. The fold of residues 36-131 of hPar14, which comprises a four-stranded beta-sheet and three alpha-helices, is superimposable onto that of the peptidyl prolyl isomerase domain of hPin1. To investigate the interaction of hPar14 with a substrate, the backbone chemical-shift changes of hPar14 were monitored during titration with a tetra peptide. Met90, Val91, and Phe94 around the N terminus of alpha3 showed large chemical-shift changes. These residues form a hydrophobic patch on the molecular surface of hPar14. Two of these residues are conserved and have been shown to interact with the proline residue of the substrate in hPin1. On the other hand, hPar14 lacks the hPin1 positively charged residues (Lys63, Arg68, and Arg69), which determine the substrate specificity of hPin1 by interacting with phosphorylated Ser or Thr preceding the substrate Pro, and exhibits a different structure in the corresponding region. Therefore, the mechanism determining the substrate specificity seems to be different between hPar14 and hPin1.
To characterize seizure-associated increases in cerebral cortical and thalamic cyclic AMP responsive element (CRE)- and activator protein 1 (AP-1) DNA-binding activities in lethargic (lh/lh) mice, a genetic model of absence seizures, we examined the effects of ethosuximide and CGP 46381 on these DNA-binding activities. Repeated administration (twice a day for 5 days) of ethosuximide (200 mg/kg) or CGP 46381 (60 mg/kg) attenuated both seizure behavior and the increased DNA-binding activities, and was more effective than a single administration of these drugs. These treatments did not affect either normal behavior or basal DNA-binding activities in non-epileptic control (+/+) mice. Gel supershift assays revealed that the increased CRE-binding activity was attributable to activation of the binding activity of CREB, and that the c-Fos-c-Jun complex was a component of the increased AP-1 DNA-binding activity.
A chip which allows the detection of various human health markers from a trace amount of blood has been studied. As a goal, a microcapillary with a 30 x 30 microm cross-section was fabricated using all-dry etching technologies on a 2 x 2 cm SiO2 chip. The coating of the biocompatible 2-methacryloyloxyethylphosphorylcholine (MPC) polymer on the inner quartz wall of the microcapillary demonstrated a sufficiently long adsorption suppression of proteins in the serum on the quartz surface, while rapid stopping occurred for serum injected into the microcapillary with a bare quartz surface. The latter rapid stopping corresponded well to fast electroosmosis flow due to the negatively increasing zeta-potential by the adsorption of proteins on the quartz surface. The electroosmosis pump arranged a downstream of the microcapillary was also developed to inject serum into it. As a preliminary application, a given concentration-standard solution was injected into the ion-sensitive field-effect transistor (ISFET) embedded in the chip, employing the electroosmosis pump arranged downstream of the sensor position. Hence, the pH and Na+ and K+ cation concentrations were measured.
A coronary flow reserve (CFR) of 2.0 has been advocated as the endpoint for coronary intervention therapy. Experience shows, however, that CFR does indeed exceed 2.0 in many cases poststenting, while remaining below 2.0 in others. In this study, we assessed the clinical characteristics and IVUS findings of patients whose CFR remained below 2.0 after stent implantation, specifically 16 patients with CFR below 2.0 (22 lesions, 64 +/- 9 years, 4 female), and 102 patients with CFR above 2.0 (112 lesions, mean age 66 +/- 11 years, 22 female). Patient population comprised patients selected for retrospective study, but participants were selected on the basis of matching patient and lesion characteristics. The IVUS findings showed that incidence of calcified lesions and post-PTCA dissection of hard plaque were higher among patients with CFR < 2.0. Further, IVUS-obtained vascular measurements showed post-PTCA area stenosis to be 58.7 +/- 15.2% in the CFR < 2.0 group, and 45.3 +/- 12.5% among CFR > or = 2.0 patients (P < 0.05). These findings indicate that patients with diffuse calcified lesions or high post-PTCA % area stenosis, as determined by IVUS, are more likely to have lower CFR after stenting.
The radial artery approach is becoming more popular for diagnostic cardiac catheterization and interventional procedures because of its lower incidence of access site complications and decreased patient discomfort after the procedure. However, Allen's test reveals inadequate blood supply through the ulnar artery to the hand, and therefore the approach does not seem to be suitable in 10%-30% of patients. Here we demonstrated a new percutaneous ulnar artery approach for coronary angiography in nine patients. We succeeded in obtaining an entry site into the left ulnar artery in seven patients. The average time for cannulation and that for catheterization procedure were comparable with those of the radial approach previously reported from other laboratories. Complications such as bleeding, loss of an ulnar pulse, ulnar nerve injury, and the formation of an aneurysm or fistula were not observed in any patient. The ulnar approach may be another technique that decreases patient discomfort and risk, while preserving the radial artery as a potential coronary bypass graft for surgical myocardial revascularization. Cathet Cardiovasc Intervent 2001;53:410-414.
We produced transgenic rice calli, which constitutively express each of four KNOX family class 1 homeobox genes of rice, OSH1, OSH16, OSH15, and OSH71, and found that constitutive and ectopic expression of such genes inhibits normal regeneration from transformed calli, which showed continuous growth around their shoot-regenerating stages. Transgenic calli transferred onto regeneration medium began to display green spots, a sign of regeneration, but most of the transformants continued to propagate green spots at given stages. In the normal shoot-regeneration process of calli, expression of endogenous OSH1 was restricted in presumptive shoot-regenerating regions of calli and not observed in other areas. This restricted expression pattern should be required for further differentiation of the regenerating shoots. Thus our present results support the proposed function that KNOX family class 1 homeobox genes play a role in the formation and maintenance of the undetermined meristematic state of cells.
With the recent progress in transfusion medicine, separation and isolation of cells in a large quantity is becoming increasingly important. At present, the continuous cell separation method in a preparative scale is limited to apheresis and elutriation: the former is mainly used for collection of platelet and buffy coat from the whole blood, while the latter separates cells virtually according to their size. Here we introduce a continuous flow method that separates cells entirely based on cell density. The method is gentle and capable of processing a large number of cells. The potential capability of the method was demonstrated on separation of lymphocytes and granulocytes from human buffy coat. Lymphocytes were enriched to 90% in the fraction at density = 1.065 and granulocytes are isolated in fractions at density = 1.075-1.080 while red cells were completely retained at the periphery of the channel. CD34 cells were distributed around 1.065 and coeluted with lymphocytes, suggesting that further enrichment requires focusing the density gradient around 1.070. The method could process 10(9) nucleated cells in 2 hours. Our preliminary results suggest that the present method is an effective and efficient means to separate blood cells.