[Non-approved antidotes in Japan--Foreign antidotes imported for poisoning countermeasures at the Summit 2000].
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Antidotes↗
Biomedical subjects
M Tamura
Publications and source records attributed to M Tamura.
In vivo transfection of testicular germ cells and transgenesis by using the mitochondrially localized jellyfish fluorescent protein gene.
We aimed to introduce foreign DNA into spermatogenic cells in the testis by injection of the DNA encoding jellyfish fluorescent proteins, green fluorescent protein (GFP) and yellow fluorescent protein (YFP) into the seminiferous tubules and in vivo electroporation. We obtained fluorescent spermatozoa only when using the gene of the YFP protein fused to a mitochondrial localization signal peptide. Intracytoplasmic injection into oocytes of these spermatozoa gave fluorescent fetuses and pups. Almost all of the individuals produced from fluorescent spermatozoa were transgenic. We confirmed integration of the gene into chromosomes and its transmission into offspring. This is the first report of gene transfer into germ cells and subsequent production of transgenic offspring.
Animals↗
Japanese encephalitis virus up-regulates expression of macrophage migration inhibitory factor (MIF) mRNA in the mouse brain.
Macrophage migration inhibitory factor (MIF) is known as a proinflammatory cytokine, glucocorticoid-induced immunomodulator, and pituitary hormone, and contributes to broad-spectrum immune and inflammatory response. To investigate the expression of MIF in the central nervous system in an event of viral infection, we evaluated MIF mRNA expression in the mouse brain infected with Japanese encephalitis virus (JEV). In situ hybridization revealed that MIF mRNA expression was significantly up-regulated in the whole brain by intracranial JEV inoculation at 2 days post-inoculation (d.p.i.). Neurons as well as glial cells expressed MIF transcripts in which some of these cells were co-labeled by double staining for JEV antigens and MIF mRNA. At 4 d.p.i., when typical symptoms of encephalitis were observed, JEV antigen-positive cells were much increased in parallel with enhanced MIF mRNA, consistent with the results of Northern blot analysis. Reverse transcription-polymerase chain reaction showed that MIF mRNA was minimally changed at 1 d.p.i. in comparison with that at 0 d.p.i., but markedly up-regulated after 2 d.p.i. and sustained up to 4 d.p.i. On the other hand, a significant increase of tumor necrosis factor (TNF)-alpha mRNA was observed after only 3 d.p.i. These data suggest the possibility that MIF is involved in virus-induced encephalitis with regard to not only immune responses in the early stage, but also the exacerbation of inflammation in concert with TNF-alpha in the late stages. This is the first evidence demonstrating that MIF is up-regulated in the case of virus-induced encephalitis, which should contribute to the further understanding of the pathological mechanism of JEV-induced encephalitis.
Animals↗
A stable bicyclic compound with two Si=Si double bonds.
In contrast to carbon, silicon does not readily form double bonds, and compounds containing silicon-silicon double bonds can usually be stabilized only by protection with bulky substituents. We have isolated a silicon analog of spiropentadiene 1, a carbon double-ring compound that has not been isolated to date. In the crystal structure of tetrakis[tri(t-butyldimethylsilyl)silyl]spiropentasiladiene 2, a substantial deviation from the perpendicular arrangement of the two rings is observed, and the silicon-silicon double bonds are shown to be distorted. Spectroscopic data indicate pronounced interaction between two remote silicon-silicon double bonds in the molecule. Silicon-silicon bonds may be more accessible to synthesis than previously assumed.
Journal Article↗
Direct interaction of actin with p47(phox) of neutrophil NADPH oxidase.
The cell-free activation of human neutrophil NADPH oxidase is enhanced by actin, and actin filaments formed during activation are suggested to stabilize the oxidase. In an attempt to elucidate the mechanism, we examined the protein-protein interactions between actin and cytosolic components of the oxidase. Far-Western blotting using recombinant phox proteins showed that both alpha- and beta-actin interacted with p47(phox) and rac1, and weakly with rac2. A deletion mutant of p47(phox) proved that its C-terminal region was essential for the interaction. The dissociation constant (K(d)) for interaction between actin and p47(phox) was estimated to be 0.45 microM by surface plasmon resonance, and that between actin and rac1 or rac2 was 1.7 or 4.6 microM, respectively. Far-Western blotting using cytosol as a target showed an interaction between actin and endogenous p47(phox) and rac proteins. These results suggest that actin can directly interact with p47(phox) and possibly with rac in the cells.
Actins↗
Deactivation of neutrophil NADPH oxidase by actin-depolymerizing agents in a cell-free system.
The cell-free activation of human neutrophil NADPH oxidase (O(2)(-)-generating enzyme) is enhanced by actin [Morimatsu, Kawagoshi, Yoshida and Tamura (1997) Biochem. Biophys. Res. Commun. 230, 206--210]. In an attempt to elucidate the mechanism, we examined the effect of actin-depolymerizing agents on the duration of NADPH oxidase in a cell-free system. The addition of DNase I, an F-actin-depolymerizing protein, caused an accelerated deactivation of the oxidase. The deactivation was also facilitated by latrunculin A, a sponge toxin that depolymerizes F-actin. Exogenously added actin prevented the deactivation by DNase I or latrunculin A, whereas EDTA accelerated a dilution-induced deactivation of the oxidase and Mg(2+) ions retarded it. The stability in dilution was found to correlate well with free Mg(2+) concentration. Estimation of F-actin in the system showed that F-actin increased during the oxidase activation and that DNase I or EDTA decreased F-actin content in parallel with the activity. Treatment of the cell-free mixture with a chemical cross-linker prevented the deactivation and F-actin decrease by EDTA. Taken together, these results suggest that actin filaments which grow during the activation of NADPH oxidase prolong the lifetime of the oxidase.
Actins↗
Spontaneous porphyria of the Long-evans cinnamon rat: an animal model of Wilson's disease.
To confirm and extend our previous microspectrophotometric observations of 30-week-old male Long-Evans Cinnamon (LEC) rats, an animal model of human Wilson's disease, we analyzed the porphyrin patterns of the organs, urine, and plasma of LEC rats. Abnormal accumulation of porphyrins, especially highly carboxylated porphyrins (uro- and heptaporphyrin), in the kidneys and liver was seen in male and female LEC rats aged 30 weeks and also in 10-week-old rats, before the onset of spontaneous hepatic dysfunction. Accumulation of copper and iron in the kidneys was not observed in the 10-week-old rats. Massive accumulation of porphyrins was observed only in the kidneys of the 30-week-old male LEC rat, indicating that this symptom is related to sex and age. Renal accumulation of porphyrins was reflected in the rate of urinary porphyrin excretion. Hepatic accumulation of porphyrins appeared to be independent of sex and age. These results indicate that neither renal nor hepatic porphyrin accumulation is the result of renal deposition of metals or of spontaneous hepatic dysfunction and that porphyrinuria in the LEC rat is closely related to the renal accumulation of porphyrins. In contrast to these organs, a reduction in the porphyrin levels was observed in the brain of the LEC rat. This was independent of sex and age. The present work stresses the existence of an abnormal heme metabolism in the LEC rat, and thus, the necessity to study the heme metabolism in human Wilson's disease is strongly suggested.
Aging↗
Clinical features and management of isolated cleft mitral valve in childhood.
OBJECTIVES: We reviewed an institutional experience of isolated cleft mitral valve (ICMV), its clinical features, and management in a pediatric population. BACKGROUND: As ICMV is relatively uncommon, earlier reports highlighted its anatomical and echocardiographic features. Few studies have collated their clinical features with their outcome. METHODS: All patients with ICMV were retrospectively reviewed. Patients who were considered to have an atrioventricular septal defect or variant were excluded. RESULTS: Twenty patients (9 male, 11 female) were diagnosed with ICMV. Seven patients had associated cardiac lesions. The median age of diagnosis was 5.2 years (range 0.4 to 13.6 years). Echocardiography aided by color Doppler demonstrated the ICMV in all patients. However, an incomplete diagnosis was made in 4 of 20 patients before surgery. The severity of the mitral regurgitation (MR) at presentation was mild in 11, moderate in 8, and severe in 1 patient. In the 13 patients without associated cardiac lesions, 5 underwent mitral valve (MV) repair at median age of 5.2 years (range 1.2 to 7.7 years) for moderate to severe MR, 4 being symptomatic. The severity of the MR in seven of the eight unoperated patients has remained unchanged over the follow-up period (median 8.3 years, range 0.7 to 14.4 years). In total, 10 patients underwent MV repair (median 6.4, range 0.4 to 13.8 years). No patient required MV replacement. None of the 10 patients had more than mild MR over the follow-up period (median 0.6, range 0.2 to 11.0 years). CONCLUSIONS: Now readily diagnosable by echocardiography, ICMV is a correctable cause of MR with a good outcome. Surgery is indicated in those patients with moderate to severe MR and probably should be done early following diagnosis.
Adolescent↗
c-fos expression and redox state of cytochrome oxidase of rat brain in hypoxia.
Hypoxic induction of c-fos was studied in rat brains as a function of the cerebral oxygenation state using near-infrared spectroscopy by which the hemoglobin oxygenation state and redox state of mitochondrial cytochrome oxidase could be monitored noninvasively. Following reoxygenation after hypoxia, the expression of c-fos and MAP2 mRNAs was followed by reverse transcription-coupled PCR. The expression of MAP2 remained unchanged throughout all the conditions from 21 to 8% FiO2. Under mildly hypoxia conditions, c-fos mRNA was not induced. Hemoglobin was partially deoxygenated but cytochrome oxidase remained fully oxidized. Severe hypoxia, where cytochrome oxidase was reduced, caused a significant induction of c-fos mRNA At this stage, the oxygen concentration in cerebral tissue fell to < 10(-7) M. These data suggest that the decline in oxidative phosphorylation might be a trigger for the induction of c-fos mRNA.
Animals↗
Structural correlates of an anticarcinoma antibody: identification of specificity-determining residues (SDRs) and development of a minimally immunogenic antibody variant by retention of SDRs only.
Clinical utility of murine mAbs is limited because many elicit Abs to murine Ig constant and variable regions in patients. An Ab humanized by the current procedure of grafting all the complementarity determining regions (CDRs) of a murine Ab onto the human Ab frameworks is likely to be less immunogenic, except that its murine CDRs could still evoke an anti-variable region response. Previous studies with anticarcinoma mAb CC49 showed that light chain LCDR1 and LCDR2 of humanized CC49 could be replaced with the corresponding CDRs of a human Ab with minimal loss of Ag-binding activity. The studies reported in this paper were undertaken to dissect the CC49 Ag-binding site to identify 1) specificity determining residues (SDRs), the residues of the hypervariable region that are most critical in Ag-Ab interaction, and 2) those residues that contribute to the idiotopes that are potential targets of patients' immune responses. A panel of variants generated by genetic manipulation of the murine CC49 hypervariable regions were evaluated for their relative Ag-binding affinity and reactivity to sera from several patients who had been immunized with murine CC49. One variant, designated HuCC49V10, retained only the SDRs of CC49 and does not react with the anti-variable region Abs of the sera from the murine CC49-treated patients. These studies thus demonstrate that the genetic manipulation of Ab variable regions can be accomplished by grafting only the SDRs of a xenogeneic Ab onto human Ab frameworks. This approach may reduce the immunogenicity of Abs to a minimum.
Amino Acid Sequence↗
Effect of electrostatic interactions on the binding of charged substrate to GroEL studied by highly sensitive fluorescence correlation spectroscopy.
The binding processes of GroEL with apo cytochrome c (apo-cyt c) and disulfide-reduced apo alpha-lactalbumin (rLA) in homogeneous solution at low concentration were analyzed by fluorescence correlation spectroscopy (FCS) with extremely high sensitivity. Although apo-cyt c, a positively charged substrate, was tightly bound to GroEL in both the absence and the presence of 200 mM KCl, the strength of the binding was changed with varying salt concentration. Results from experiments when two different salts (KCl or MgCl(2)) were titrated into a sample solution containing GroEL and apo-cyt c clearly showed that the binding strength decreased with increasing salt concentration. On the other hand, the binding affinity of GroEL for rLA, a negatively charged substrate, increased by adding of 200 mM KCl. These results indicate that electrostatic interactions substantially contribute to the binding interactions by manipulating the binding affinity of charged substrates.
Animals↗
Connexin 43-enhanced suicide gene therapy using herpesviral vectors.
Tumor cell transduction with the herpes simplex virus (HSV) thymidine kinase (tk) gene and treatment with ganciclovir (GCV) is a widely studied cancer gene therapy. Connexin (Cx)-dependent gap junctions between cells facilitate the intercellular spread of TK-activated GCV, thereby creating a bystander effect that improves tumor cell killing. However, tumor cells often have reduced connexin expression, thus thwarting bystander killing and the effectiveness of TK/GCV gene therapy. To improve the effectiveness of this therapy, we compared an HSV vector (TOCX) expressing Cx43 in addition to TK with an isogenic tk vector (TOZ.1) for their abilities to induce bystander killing of Cx-positive U-87 MG human glioblastoma cells and Cx-negative L929 fibrosarcoma cells in vitro and in vivo. The results showed that low-multiplicity infection of U-87 MG cells with TOCX only minimally increased GCV-mediated cell death compared with infection by TOZ.1, consistent with the endogenous level of Cx in these cells. In contrast, bystander killing of L929 cells was markedly enhanced by vector-mediated expression of Cx. In vivo experiments in which U-87 MG cells were preinfected at low multiplicity and injected into the flanks of nude mice showed complete cures of all animals in the TOCX group following GCV treatment, whereas untreated animals uniformly formed fatal tumors. TOCX injection into U-87 MG intradermal and intracranial tumors resulted in prolonged survival of the host animals in a GCV-dependent manner. Together, these results suggest that the combination of TK and Cx may be beneficial for the treatment of human glioblastoma.
Animals↗
Effective treatment of experimental glioblastoma by HSV vector-mediated TNF alpha and HSV-tk gene transfer in combination with radiosurgery and ganciclovir administration.
Experiments were carried out in a nude mouse model of human glioblastoma to determine whether gamma-knife radiosurgery combined with herpes simplex virus thymidine kinase (tk) suicide gene therapy and tumor necrosis factor alpha (TNFalpha) gene transfer provided an improved multimodality treatment of this disease. Animals were inoculated intracerebrally with 2 x 10(5) U-87MG human glioblastoma cells to establish brain tumors. At 3 days postinoculation, the tumor region was injected with 2 x 10(6) infectious particles of highly defective herpes simplex viral vectors expressing the viral tk gene with the kinetics of a viral immediate early gene either alone (T.1) or together with TNF alpha (TH:TNF). Subgroups of animals were given daily intraperitoneal injections of ganciclovir (GCV) for 10 days and/or subjected to gamma-knife radiosurgery on the fifth day post tumor-cell implantation. Comparisons of animal survival showed that the TH:TNF vector in combination with radiosurgery and GCV administration provided the most effective therapy; eight of nine animals survived for 75 days compared to four of eight using the next best protocol. These findings suggest that gene therapy in combination with more conventional therapeutic methods may provide an improved strategy for extending the life expectancy of patients afflicted with this ultimately fatal disease.
Animals↗
A case of neonatal-onset carbamoyl-phosphate synthase I deficiency treated by continuous haemodiafiltration.
This is the first case of fulminant neonatal-onset carbamoyl-phosphate synthase I deficiency treated by continuous hemodiafiltration indicating that this is an available and effective procedure for neonates with hyperammonemic coma.
Ammonia↗
X-chromosome inactivation in the human trophoblast of early pregnancy.
To investigate X-chromosome inactivation (XCI) in human trophoblasts during early pregnancy, trophoblast genomic DNA was extracted and analyzed for a Bst XI restriction endonuclease site polymorphism in the X-linked phosphoglycerate kinase gene, after digestion with methylation-sensitive Hpa II (control samples were digested instead with Afa I). Six villous trophoblast DNA samples were informative for the polymorphism (ie, heterozygous) and were derived from women homozygous for the polymorphism. These samples were then evaluated for XCI. In five of the six samples with Hpa II predigestion, the sizes of the two heterozygous band peaks differed; maternal X-chromosome (X(M))-derived alleles showed smaller peak sizes than paternal X-chromosome (X(P))-derived alleles, but the differences varied in degree. In samples obtained by microdissection from formalin-fixed, paraffin-embedded tissues (30 samples from different anchoring villi, and 38 samples from different branch villi), monoclonal band patterns of X(P)-derived alleles were observed more frequently than those of X(M)-derived alleles, but almost half of the samples showed polyclonal patterns. Our results suggest that a skewing of XCI exists in the human trophoblast; however, the degree of nonrandomness due to predominant X(P) inactivation appears to be restricted. It is probable that transcription of the X inactivation center (XIC) begins earlier in mice than in humans.
Clone Cells↗
The attitude of Japanese family caregivers toward the elderly with dementia.
This research examined family caregivers' attitudes toward their elderly relatives living with dementia. A convenience sample of fifty-nine Japanese caregivers was used. Caregiver attitudes were investigated multi-dimensionally (negative attitude, attitude of acceptance, and attitude of active interaction) using a questionnaire developed specifically for this study. The dimensions did not correlate with one another but were associated with different caregiver/elderly characteristics: i.e., negative attitude with short stay use, length of time caregivers and elderly relatives were living together and problem behaviors; attitude of acceptance with sleeping pill use and the type of relationship; and attitude of active interaction with dementia diagnosis. The results have implications for nurses in facilitating constructive caregiver attitudes.
Aged↗
Influence of cetane improvers on the air quality in an urban street canyon.
The concentration distributions of NOx, PM, HC and CO in an urban street canyon have been estimated using a two-dimensional air quality numerical model based on the k-epsilon turbulent model and the atmospheric convection diffusion equation when various cetane improvers were used in diesel fuels. A wind vortex can be found within the street canyon, and the pollutants emitted from the bottom of the street canyon tend to follow the course of the wind field, moving circularly. The addition of cetane improvers can improve the air quality in a street canyon, all of the pollutants were found to decrease with increasing centane number.
Air↗
Exchange transfusion with fluorocarbon for studying synaptically evoked optical signal in rat cortex.
Optical imaging of intrinsic signal is a powerful technique for studying the functional organization of the brain [T. Bonhoeffer, D. S. Kim, D. Malonek, D. Shoham, A. Grinvald, Optical imaging of the layout of functional domains in area 17 and across the area 17/18 border in cat visual cortex, Eur. J. Neurosci. 7 (1995) 1973-1988; M. Hubener, D. Shoham, A. Grinvald, T. Bonhoeffer, Spatial relationships among three columnar systems in cat area 17, J. Neurosci. 17 (1997) 9270-9284; D. Malonek, A. Grinvald, Interactions between electrical activity and cortical microcirculation revealed by imaging spectroscopy: implications for functional brain mapping, Science 272 (1996) 551-554; A. Shmuel, A. Grinvald, Functional organization for direction of motion and its relationship to orientation maps in cat area 18, J. Neurosci. 16 (1996) 6945-6964] [1] [10] [14] [22]. Three components of intrinsic optical signal can be distinguished. Two of these components can be attributed either to changes in blood volume or to changes in oxygen consumption [R.D. Frostig, E.E. Lieke, D.Y. Ts'o, A. Grinvald, Cortical functional architecture and local coupling between neuronal activity and the microcirculation revealed by in vivo high resolution optical imaging of intrinsic signals, Proc. Natl. Acad. Sci. U. S. A. 87 (1990) 6082-6086] [7]. The origin of the third component is not yet clear but the component seems to be based on scattered light [H.U. Dodt, G. D'Arcangelo, E. Pestel, W. Zieglgansberger, The spread of excitation in neocortical columns visualized with infrared-dark field videomicroscopy, NeuroReport 7 (1996) 1553-1558; K. Holthoff, O.W. Witte, Intrinsic optical signals in rat neocortical slices measured with near-infrared dark-field microscopy reveal changes in extracellular space, J. Neurosci. 16 (1996) 2740-2749; B.A. MacVicar, D. Hochman, Imaging of synaptically evoked intrinsic optical signals in hippocampal slices, J. Neurosci. 11 (1991) 1458-1469; L. Trachsel, H.U. Dodt, W. Zieglgansberger, The intrinsic optical signal evoked by chiasm stimulation in the rat suprachiasmatic nuclei exhibits GABAergic day-night variation, Eur. J. Neurosci. 8 (1996) 319-328] [3] [9] [13] [24]. A spectral fitting method with three components is used for the analysis of intrinsic optical signal [M. Nemoto, Y. Nomura, C. Sato, M. Tamura, K. Houkin, I. Koyanagi, H. Abe, Analysis of optical signals evoked by peripheral nerve stimulation in rat somatosensory cortex: dynamic changes in hemoglobin concentration and oxygenation, J. Cereb. Blood Flow Metab. 19 (1999) 246-259] [17]. In order to validate the analysis, we need the knowledge on contribution of signal resulted from hemoglobin to total intrinsic optical signal. The exchange transfusion with fluorocarbon has the advantage that can change the spectral contribution of hemoglobin [M. Ferrari, M.A. Williams, D.A. Wilson, N.V. Thakor, R.J. Traystman, D.F. Hanley, Cat brain cytochrome-c oxidase redox changes induced by hypoxia after blood-fluorocarbon exchange transfusion, Am. J. Physiol. 269 (1995) H417-H424; A.L. Sylvia, C.A. Piantadosi, O(2) dependence of in vivo brain cytochrome redox responses and energy metabolism in bloodless rats, J. Cereb. Blood Flow Metab. 8 (1988) 163-172] [6] [23]. Here we describe a new method of the reduction of hemoglobin signal from somatosensory evoked optical intrinsic signal in rat cortex by the combination of exchange transfusion with fluorocarbon and imaging system of thinned skull cranial window. The method allows for the study of the synaptically evoked changes in light scattering as well as fluorescence of calcium indicator or voltage-sensitive dye without absorption of hemoglobin.
Animals↗