[Budd-Chiari syndrome associated with hemophagocytic syndrome caused by tuberculosis of bone marrow].
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Adult↗
Biomedical subjects
T Saito
Publications and source records attributed to T Saito.
Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.
Mouse genetic manipulation has provided an excellent system to characterize gene function in numerous contexts. A number of mutants have been produced by using transgenic, gene knockout, and mutagenesis techniques. Nevertheless, one limitation is that it is difficult to express a gene in vivo in a restricted manner (i.e., spatially and temporally), because the number of available enhancers and promoters which can confine gene expression is limited. We have developed a novel method to introduce DNA into in/exo utero embryonic mouse brains at various stages by using electroporation. More than 90% of operated embryos survived, and more than 65% of these expressed the introduced genes in restricted regions of the brain. Expression was maintained even after birth, 6 weeks after electroporation. The use of fluorescent protein genes clearly visualized neuronal morphologies in the brain. Moreover, it was possible to transfect three different DNA vectors into the same cells. Thus, this method will be a powerful tool to characterize gene function in various settings due to its high efficiency and localized gene expression.
Animals↗
Notch1 inhibits differentiation of hematopoietic cells by sustaining GATA-2 expression.
Notch signaling is involved in cell fate decisions in many systems including hematopoiesis. It has been shown that expression of an activated form of Notch1 (aNotch1) in 32D mouse myeloid progenitor cells inhibits the granulocytic differentiation induced by granulocyte colony-stimulating factor (G-CSF). Results of the current study show that aNotch1, when expressed in F5-5 mouse erythroleukemia cells, also inhibits erythroid differentiation. Comparison of the expression levels of several transcription factors after stimulation for myeloid and erythroid differentiation, in the presence or absence of aNotch1, revealed that aNotch1 did not change its regulation pattern with any of the transcription factors examined, except for GATA-2, despite its inhibitory effect on differentiation. GATA-2 was down-regulated when the parental 32D and F5-5 were induced to differentiate into granulocytic and erythroid lineages, respectively. In these induction procedures, however, the level of GATA-2 expression was sustained when aNotch1 was expressed. To ascertain whether maintenance of GATA-2 is required for the Notch-induced inhibition of differentiation, the dominant-negative form of GATA-3 (DN-GATA), which acted also against GATA-2, or transcription factor PU.1, which was recently shown to be the repressor of GATA-2, was introduced into aNotch1-expressing 32D (32D/aNotch1) cells that do not express GATA family proteins other than GATA2. Both DN-GATA and PU.1 reversed the phenotype of 32D/aNotch1 inducing its differentiation when G-CSF was added. Furthermore, enforced expression of HES-1, which is involved in Notch signaling, delayed differentiation of 32D, and again this phenotype was neutralized by DN-GATA. These results indicate that GATA-2 activity is necessary for the Notch signaling in hematopoietic cells.
Animals↗
Real-time spectroscopy of transition states in bacteriorhodopsin during retinal isomerization.
Real-time investigations of the rearrangement of bonds during chemical transformations require femtosecond temporal resolution, so that the atomic vibrations within the reacting molecules can be observed. Following the development of lasers capable of emitting ultrashort laser flashes on this timescale, chemical reactions involving relatively simple molecules have been monitored in detail, revealing the transient existence of intermediate species as reactants are transformed into products. Here we report the direct observation of nuclear motion in a complex biological system, the retinal chromophore of bacteriorhodopsin (bR568), as it undergoes the trans-cis photoisomerization that is fundamental to the vision process. By using visible-light pulses of less than 5 femtosecond in duration, we are able to monitor changes in the vibrational spectra of the transition state and thus show that despite photoexcitation of the anti-bonding molecular orbital involved, isomerization does not occur instantly, but involves transient formation of a so-called 'tumbling state'. Our observations thus agree with growing experimental and ab initio evidence for a three-state photoisomerization model and firmly discount the initially suggested two-state model for this process.
Bacteriorhodopsins↗
Vasopressin differentially modulates noradrenaline release in the rat supraoptic nucleus.
Vasopressin is released not only from axon terminals in the neurohypophysis but also from soma/dendrite regions in the supraoptic nucleus. In order to investigate presynaptic roles of dendritically released vasopressin, we examined effects of local application of vasopressin upon noradrenaline release within the supraoptic nucleus by a microdialysis method. Noradrenaline release within the supraoptic nucleus was facilitated by local perfusion with high K+ or an NMDA receptor antagonist. Vasopressin augmented noradrenaline increase after high K+ but reduced it after an NMDA receptor antagonist, AP-5. The results suggest that dendritically released vasopressin modulates noradrenaline release within the supraoptic nucleus in a bimodal fashion.
Animals↗
Clinical features of patients with reperfused inferior wall acute myocardial infarction complicated by early complete atrioventricular block.
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Aged↗
Heterogeneous transforming growth factor (TGF)-beta unresponsiveness and loss of TGF-beta receptor type II expression caused by histone deacetylation in lung cancer cell lines.
Transforming growth factor (TGF)-beta strongly inhibits epithelial cell proliferation. Alterations of TGF-beta signaling are thought to play a role in tumorigenesis. We show in the present study that most lung cancer cell lines have lost the growth-inhibitory response to TGF-beta signal, and that those with TGF-beta unresponsiveness can be divided into two major groups, TGF-beta type II receptor (TGFbetaRII)(+)/Smad7(+) and TGFbetaRII(-)/Smad7(-), suggesting the heterogeneous mechanisms underlying the TGF-beta responsiveness. The mechanism of the loss of TGFbetaRII expression of the latter group was further studied, identifying aberrant DNA methylation of the promoter region in a limited fraction of cell lines. Interestingly, we found that the alteration of chromatin structure because of histone deacetylation may also be involved, showing a good correlation with loss of TGFbetaRII expression. This notion was supported by the findings of a restriction enzyme accessibility assay, of a chromatin immunoprecipitation assay with anti-acetyl histone antibodies, and of an in vivo induction of TGFbetaRII expression by histone deacetylase inhibitors including trichostatin A (TSA) and sodium butyrate. In vitro induction of TGFbetaRII promoter reporter activity by TSA was also detected and found to require the CCAAT box within the -127/-75 region. A positive regulatory mechanism for TGFbetaRII expression in a TGF-beta-expressing cell line was also investigated, and a TPA-responsive element (TRE)-like motif, TRE2, was detected in addition to the previously reported TRE-like motif Y element in the positive regulatory region. Alterations in two discrete proteins interacting with these two TRE-like motifs were also suspected of being involved in the loss of TGFbetaRII expression. This is the first study to demonstrate that, in addition to the TSA-responsive region and TRE2 motif in the TGFbetaRII promoter, the alteration of histone deacetylation may be involved in the loss of TGFbetaRII expression in lung cancer cell lines.
Acylation↗
Inhibitory effect of bepridil on hKv1.5 channel current: comparison with amiodarone and E-4031.
Effects of bepridil on the depolarization-activated outward K(+) currents (I(out)) in rat atrial myocytes and the human cardiac K(+) (hKv1.5) channel current stably expressed in human embryonic kidney (HEK) 293 cells were examined, and compared with those of amiodarone and N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl] methanesulphonamide dihydrochloride dihydrate (E-4031). Membrane currents were recorded using patch-clamp techniques in enzymatically isolated rat atrial myocytes and HEK 293 cells expressing hKv1.5 channels. Bepridil potently inhibited I(out) elicited by depolarization pulses and prolonged the action potential in rat atrial cells. Bepridil also inhibited the hKv1.5 channel current with the IC(50) value of 6.6 microM. The inhibitory effects of bepridil on the currents in HEK 293 cells were voltage-dependent. Amiodarone weakly inhibited rat atrial I(out) and hKv1.5 channel current. In contrast, E-4031 at a concentration of 10 microM had little influence on these currents. Thus, bepridil inhibits hKv1.5 channel current and the inhibitory effect may be useful for the treatment of atrial fibrillation.
4-Aminopyridine↗
Efficient antigen gene transduction using Arg-Gly-Asp fiber-mutant adenovirus vectors can potentiate antitumor vaccine efficacy and maturation of murine dendritic cells.
Dendritic cells (DCs), the most effective antigen-presenting cells, are being studied as adjuvants or antigen delivery vehicles for eliciting T-cell-mediated antitumor immunity. Gene delivery to DCs provides an intracellular source of antigen for efficient and persistent loading to MHC class I molecules capable of activating CD8(+) CTLs, which play a central role in antitumor immunity. We previously reported that the fiber-mutant adenovirus vector (Ad) harboring the Arg-Gly-Asp (RGD) sequence in the HI loop of its fiber knob could more efficiently transduce the LacZ gene into both murine DC lines and normal human DCs than conventional Ad. In the present study, we compared immunological properties and vaccine efficacy of DC2.4 cells, an immature murine DC line, transduced with an ovalbumin (OVA) gene by fiber-mutant Ad (Ad-RGD-OVA) or conventional Ad (Ad-OVA). Ad-RGD-OVA-infected DC2.4 cells could more efficiently present OVA peptides via MHC class I molecules in a vector particle-dependent manner and induce OVA-specific CTL response by vaccination than Ad-OVA-infected DC2.4 cells. This result was correlated with the efficiency of gene transduction into DC2.4 cells by both types of Ad. Moreover, vaccination with Ad-RGD-OVA-infected DC2.4 cells could achieve an equal or greater antitumor effect against challenge with E.G7-OVA tumor cells with lower doses of Ad on infection or fewer cells for immunization than the vaccination procedure using Ad-OVA-infected DC2.4 cells. In addition, the maturation of DC2.4 cells was promoted by efficient expression of the antigen gene by the Arg-Gly-Asp fiber-mutant Ad. Flow cytometric analysis indicated enhanced expression of MHC class I and II molecules as well as CD80, CD86, CD40, and CD54, and reverse transcription-PCR analysis revealed increased levels of interleukin 12 p40 mRNA. However, infection by Ad-OVA or Ad that did not contain the cDNA of interest (Ad-Null and Ad-RGD-Null) contributed little to phenotypical changes in DC2.4 cells. On the basis of these results, we propose that DC manipulation using the Arg-Gly-Asp fiber-mutant Ad system could advance the development of more effective vaccines and allow for more convenient administration of DC-based gene immunotherapy.
Adenoviridae↗
Significance of serum amyloid A on the prognosis in patients with renal cell carcinoma.
BACKGROUND: Evidence of systemic inflammation, i.e., elevation of serum C-reactive protein, interleukin-6, and/or the erythrocyte sedimentation rate, is correlated to poorer prognosis of patients with renal cell carcinoma (RCC). Serum amyloid A (SAA) has been recognized mainly as acute-phase reactant. METHODS: Serum SAA from 72 patients with RCC were examined. Thirty-eight of 72 patients with RCC had elevated SAA compared with 17 healthy donors. RESULTS: The disease specific survival rate was significantly lower in the elevated SAA group, and SAA level was shown to be an independent prognostic factor by univariate and multivariate analysis. CONCLUSIONS: Evaluation of serum SM level in RCC patients may be a useful prognostic indicator.
Adult↗
The neuronal form of adaptor protein-3 is required for synaptic vesicle formation from endosomes.
Heterotetrameric adaptor complexes vesiculate donor membranes. One of the adaptor protein complexes, AP-3, is present in two forms; one form is expressed in all tissues of the body, whereas the other is restricted to brain. Mice lacking both the ubiquitous and neuronal forms of AP-3 exhibit neurological disorders that are not observed in mice that are mutant only in the ubiquitous form. To begin to understand the role of neuronal AP-3 in neurological disease, we investigated its function in in vitro assays as well as its localization in neural tissue. In the presence of GTPgammaS both ubiquitous and neuronal forms of AP-3 can bind to purified synaptic vesicles. However, only the neuronal form of AP-3 can produce synaptic vesicles from endosomes in vitro. We also identified that the expression of neuronal AP-3 is limited to varicosities of neuronal-like processes and is expressed in most axons of the brain. Although the AP-2/clathrin pathway is the major route of vesicle production and the relatively minor neuronal AP-3 pathway is not necessary for viability, the absence of the latter could lead to the neurological abnormalities seen in mice lacking the expression of AP-3 in brain. In this study we have identified the first brain-specific function for a neuronal adaptor complex.
Adaptor Proteins, Vesicular Transport↗
Functional characterization of cysteinyl leukotriene CysLT(2) receptor on human coronary artery smooth muscle cells.
Cysteinyl leukotrienes (LTC(4), LTD(4), and LTE(4)) are a class of biologically active lipids that exert potent effects on the heart. To assess their roles, we investigated the distribution of their receptors, CysLT(1) and CysLT(2), in the cardiovascular system. CysLT(2) mRNA was detected at high levels in the human atrium and ventricle and at intermediate levels in the coronary artery, whereas CysLT(1) mRNA was barely detected. Further analysis by in situ hybridization revealed that CysLT(2) mRNA was expressed in myocytes, fibroblasts, and vascular smooth muscle cells, but not in endothelial cells. When human coronary smooth muscle cells were stimulated with LTC(4), the intracellular calcium concentration increased in a dose-dependent manner, and this action was partially inhibited by nicardipine. Additionally, these cells showed chemotactic responses to LTC(4). This is the first report on the physiological role of CysLT(2), and the findings suggest that CysLT(2) has biological significance in the cardiovascular system.
Arteries↗
Selective concentration of gold in water to a polystyrene-embedded fiber disk with polyoxyethylene(10)-p-isononylphenyl ether.
A new surfactant-mediated separation method was developed for concentrating traces of gold ion in water. The methodology is based on the combination of selective complexation of gold(III) with polyoxyethylene(10)-p-isononylphenyl ether, PONPE-10, and strong binding of surfactant complex to hydrophobic polystyrene resins embedded in a PTFE fiber disk (Empore disk). A 400-fold concentration of gold(III) was achieved by 400 ml load of the sample containing 0.01% (w/v) PONPE-10 and 0.10 M nitric acid and by the subsequent elution with 1.0 ml of aqueous buffer solution of 0.01 M N-(dithiocarboxyl)sarcosine diammonium. Traces of gold (0.40 ng/l) in river water samples were successfully determined with inductively coupled plasma MS.
Ethers↗
Characterization of a human H9N2 influenza virus isolated in Hong Kong.
Two H9N2 viruses were isolated, for the first time, from humans in Hong Kong in 1999. Isolation of influenza viruses with a novel subtype of the hemagglutinin (HA) drew attention of health care authorities worldwide from the view of pandemic preparedness. Sequence analysis of the HA genes reveals that HA of A/Hong Kong/1073/99 (H9N2) is most closely related to that of A/quail/HK/G1/97 (H9N2) that contains the internal genes similar to those of Hong Kong/97 (H5N1) viruses. Phylogenetic and antigenic analyses demonstrated the diversity among H9 HA. A/Hong Kong/1073/99 was shown to cause a respiratory infection in Syrian hamsters, suggesting that the virus can replicate efficiently in mammalian hosts. We developed a whole virion test vaccine with a formalin-inactivated egg-grown HK1073. Intraperitoneal administration of the vaccine twice to hamsters conferred a complete protection against challenge infection by the MDCK cell-grown homologous virus. Receptor specificity of HK1073 appeared different from that of other avian influenza viruses of H9 subtype which recognize preferentially alpha-2,3 linked sialic acid. Hemagglutination of HK1073 with guinea pig erythrocytes was inhibited by both alpha-2,3 and alpha-2,6 linked sialic acid containing polymers. These data suggested that HK1073 had acquired a broader host range, including humans. Together with data so far available, the present study suggested that isolation of the H9 influenza viruses from humans requires precaution against the emergence of a novel human influenza.
Animals↗
Docking protein Gab2 is phosphorylated by ZAP-70 and negatively regulates T cell receptor signaling by recruitment of inhibitory molecules.
To maintain various T cell responses and immune equilibrium, activation signals triggered by T cell antigen receptor (TCR) must be regulated by inhibitory signals. Gab2, an adaptor protein of the insulin receptor substrate-1 family, has been shown to be involved in the downstream signaling from cytokine receptors. We investigated the functional role of Gab2 in TCR-mediated signal transduction. Gab2 was phosphorylated by ZAP-70 and co-precipitated with phosphoproteins, such as ZAP-70, LAT, and CD3zeta, upon TCR stimulation. Overexpression of Gab2 in Jurkat cells or antigen-specific T cell hybridomas resulted in the inhibition of NF-AT activation, interleukin-2 production, and tyrosine phosphorylation. The structure-function relationship of Gab2 was analyzed by mutants of Gab2. The Gab2 mutants lacking SHP-2-binding sites mostly abrogated the inhibitory activity of Gab2, but its inhibitory function was restored by fusing to active SHP-2 as a chimeric protein. A mutant with defective phosphatidylinositol 3-kinase binding capacity also impaired the inhibitory activity, and the pleckstrin homology domain-deletion mutant revealed a crucial function of the pleckstrin homology domain for localization to the plasma membrane. These results suggest that Gab2 is a substrate of ZAP-70 and functions as a switch molecule toward inhibition of TCR signal transduction by mediating the recruitment of inhibitory molecules to the TCR signaling complex.
Adaptor Proteins, Signal Transducing↗
Acceleration of lung metastasis by up-regulation of CD44 expression in osteosarcoma-derived cell transplanted mice.
The effect of CD44-phenotypic expression on metastasis to the lung was studied using a spontaneous murine osteosarcoma-derived cell line, POS-1, stimulated with lipopolysaccharide (LPS). POS-1 cells were inoculated into the hind paws of 20 C3H/HeJ mice and produced a visible mass in all mice in 5 weeks, and these transplanted tumors resulted in lung metastasis in all mice. The number of metastatic foci in the lungs was 12.0+/-2.1 (mean+/-SD) with LPS-stimulated cells, which was significantly higher than that of unstimulated cells (5.8+/-1.4; N=10 for each; P<0.05). Hyaluronate (HA), a ligand of CD44, inhibited a number of lung metastases in a dose-dependent manner (0.5% HA, 3.0+/-1.1; 0.005% HA, 5.1+/-1.5; without HA, 8.6+/-1.7; N=10 for each; P<0.05, each group with HA versus the group without HA). Adhesion assay by coculturing POS-1 cells and lung microvascular endothelial cells on culture plate showed that the adhesion was significantly lower in HA treated POS-1 than those without HA (1.18+/-0.12 and 2.74+/-0.17, respectively, P<0.05). These results suggest that lung metastasis was accelerated by up-regulation of CD44.
Animals↗
Solid-phase microextraction and gas chromatography-mass spectrometry analysis of p,p' -DDE in biological samples.
A simple and rapid extraction method for the analysis of p,p'-DDE from rat whole blood and tissues was developed using headspace solid-phase microextraction (SPME). A vial containing a sample of p,p'-DDE, sodium hydroxide, and benzophenone as internal standard was heated at 120 degrees C. A polydimethylsiloxane-coated SPME fiber was exposed for 15 min in the headspace of the vial. The fiber needle was then injected and desorbed by exposing the fiber in the injection port of a capillary gas chromatography-mass spectrometry system. The calibration curve demonstrated good linearity throughout the concentration range from 0.02 to 50 microg/g for rat whole blood and liver samples. The limit of detection for p,p'-DDE was 0.020 microg/g using 0.5 g rat whole blood and liver samples. Coefficients of variation ranged from 7.0 to 7.9%. This method was used to analyze a rat whole blood sample after administration of p,p'-DDE.
Animals↗
Alpha2,6-sialylation of cell-surface N-glycans inhibits glioma formation in vivo.
Human gliomas express very high levels of cell-surface alpha2,3-linked terminal sialic acids on glycoproteins bearing N-linked oligosaccharides, most notably on alpha3beta1 integrin, which is the predominant integrin found in these tumors. Alpha2,6-linked terminal sialic acids, however, are not expressed. Two stable transfectants were made using a tumorigenic human glioma cell line, U-373 MG. Galbeta1,4GlcNAc alpha2,6-sialyltransferase (ST6Gal I) transfectants were made to replace the endogenous alpha2,3-linked sialic acids with alpha2,6-linked sialic acids. And Galbeta1,3(4)GlcNAc alpha2,3-sialyltransferase (ST3Gal III) transfectants were made to increase further the expression of cell-surface, N-glycan, alpha2,3-linked sialic acids. Although ST3Gal III transfection resulted in increased invasivity when compared with parental U-373 MG and vector-transfected control cells in vitro, ST6Gal I transfection abolished invasion in vitro and induced alterations in both cell morphology, cell-spreading, and adhesion-mediated protein tyrosine phosphorylation. Furthermore, the ST6Gal I transfectants produced no intracranial tumors in severe combined immunodeficient mice, whereas parental U-373 MG cells, the vector-transfected control cells, and ST3Gal III-transfected U-373 MG cells did. These results suggest that both the linkage and expression levels of the terminal sialic acids of alpha3beta1 integrin N-glycans play an important role in glioma cell-extracellular matrix interactions. Thus, manipulating ST6Gal I gene expression may have therapeutic potential for the treatment of malignant gliomas.
Animals↗