[Adaptation of English-written health-related quality of life measure for children].
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Child↗
Biomedical subjects
N Takizawa
Publications and source records attributed to N Takizawa.
Inhibition of nuclear export of ribonucleoprotein complexes of influenza virus by leptomycin B.
We have studied nuclear export of influenza virus components using an in vitro transport system with digitonin-treated infected cells. We first monitored the efficiency of export of the viral ribonucleoprotein (vRNP) complex by analyzing viral components with western blotting. We used leptomycin B (LMB), an inhibitor of nuclear export signal (NES)-and its receptor, CRM1/Exportin1-mediated protein export. LMB efficiently inhibited vRNP export, while it did not affect the subcellular localization and export of matrix protein (M) 1 and nonstructural protein (NS) 2. Second, indirect immunofluorescence assays also revealed that vRNP export is sensitive to LMB. NS2 in NS2-transfected cells was not accumulated in nuclei in the presence of LMB, while NS2 in infected cells was found slightly accumulated in nuclei in the presence of LMB. Finally, we performed in vitro RNA synthesis assays using digitonin-treated infected cells and exported fractions. The exported vRNP was RNA synthesis-competent. Analyses using glycerol density gradients showed that a major fraction of M1 and NS2 was not complexed with the exported vRNP. These results suggest that nuclear export of RNA synthesis-competent vRNP is dependent on a LMB-sensitive pathway and that there would be two types of NS2, i.e. LMB-sensitive and -insensitive NS2. The involvement of viral late proteins in vRNP export during late stages of infection is discussed.
Active Transport, Cell Nucleus↗
Microcoaxial electrode for in vivo nitric oxide measurement.
Nitric oxide (NO) is a gaseous mediator involved in various physiological phenomena, such as vasorelaxation and neurotransmission. Investigation of local cellular responses of NO production in vivo and in vitro requires a measurement method with a high spatial resolution. For selective NO measurement, we therefore developed a microcoaxial electrode whose tip diameter is less than 10 microm. Calibration using various concentrations of NO (0.1-1.0 microM) showed that the electrode has good linearity (r = 0.99) and its detection limit is 0.075 microM (S/N = 3). We verified the applicability of this electrode to in vivo and in vitro local measurement NO released from bovine aortic cultured endothelial cells (BAECs) stimulated by acetylcholine (ACh). After the addition of ACh, a transient increase in NO concentration was detected by the electrode. In the presence of NG-nitro-L-arginine methyl ester (L-NAME), a putative NO synthase inhibitor, NO release (peak NO concentration) from RAECs was significantly less than that in the absence of L-NAME (0.18 +/- 0.04 microM vs 0.47 +/- 0.13; P < 0.01). After removal of L-NAME, NO release partially recovered (0.39 +/- 0.10 microM). In conclusion, the microcoaxial electrode was successfully applied to direct and continuous NO measurement in biological systems.
Calibration↗
Reconstitution of protein kinase C-induced contractile Ca2+ sensitization in triton X-100-demembranated rabbit arterial smooth muscle.
1. Triton X-100-demembranated smooth muscle loses Ca2+-sensitizing responsiveness to protein kinase C (PKC) activators while intact and alpha-toxin-permeabilized smooth muscles remain responsive. We attempted to reconstitute the contractile Ca2+ sensitization by PKC in the demembranated preparations. 2. Western blot analyses showed that the content of the PKC alpha-isoform (PKCalpha) was markedly reduced and that the smooth muscle-specific protein phosphatase-1 inhibitor protein CPI-17 was not detectable, while the amount of calponin and actin still remained similar to those of intact strips. 3. Unphosphorylated recombinant CPI-17 alone induced a small but significant contraction at constant Ca2+. Isoform-selective PKC inhibitors inhibited unphosphorylated but not pre-thiophosphorylated CPI-17-induced contraction, suggesting that in situ conventional PKC isoform(s) can phosphorylate CPI-17. 4. Exogenously replenishing PKCalpha alone did not induce potentiation of contraction and only slowly increased myosin light chain (MLC) phosphorylation at submaximal Ca2+. 5. PKC in the presence of CPI-17, but not the [T38A]-CPI mutant, markedly induced potentiation of both contraction and MLC phosphorylation. CPI-17 itself was phosphorylated. 6. In in vitro experiments, CPI-17 was a much better substrate for PKCalpha than calponin, caldesmon, MLC and myosin. 7. Our results indicate that PKC requires CPI-17 phosphorylation at Thr-38 but not calponin for reconstitution of the contractile Ca2+ sensitization in the demembranated arterial smooth muscle.
Animals↗
Diffuse pachygyria with cerebellar hypoplasia: a milder form of microlissencephaly or a new genetic syndrome?
We report on 2 families with diffuse pachygyria and cerebellar hypoplasia, who presented hypotonia, ataxia, seizures, and developmental delay since infancy. Computed tomography (CT) and magnetic resonance imaging (MRI) revealed decreased gyral formation in the cerebral cortex and marked hypoplasia in the cerebellum. Cerebellar hypoplasia is often associated with type 2 lissencephaly; however, our cases showed no polymicrogyria, and their clinical findings were quite mild compared with those of microlissencephaly. Their characteristic phenotype suggested a new genetic syndrome, which was possibly inherited as an autosomal recessive trait.
Cerebellar Diseases↗
Fast and slow recovery phases of goldfish behavior after transection of the optic nerve revealed by a computer image processing system.
As the goldfish is a common experimental animal for vision research, including psychophysical behavior, it is very important to quantitatively score fish behavior. We have previously developed a computer image processing system which can acquire the positional coordinates of goldfish moving freely in an aquarium and determine turning directions (go straight, right or left turn). In the present study, an algorithm to determine tilting angles of moving goldfish was constructed. We also made histograms for quantifying the interaction between pairs of goldfish (two-point distance). By using these histograms, we estimated the time-course of behavioral regeneration after optic nerve transection in goldfish. Control goldfish showed an equal percentage of right or left turns and maintained an upright position in a dorsoventral axis. When the optic nerve of a goldfish was unilaterally sectioned, the goldfish showed predominant turning and slight tilting toward the intact eye. The abnormal turning and tilting behaviors lasted for 10-14 days and then gradually decreased, returning to control behaviors by one month after the unilateral transection. When the optic nerve of a single goldfish was bilaterally sectioned, it did not show any preferential turning and tilting behavior, which is similar to what was observed in control goldfish. However, the trace maps showed that, after bilateral sectioning, fish preferred to cross the center of the tank, which was unlike control fish. In control pairs, one goldfish chased the other with a fixed small range of two-point distances. However, in pairs of goldfish with bilateral transection of the optic nerve, the blind goldfish behaved independently of each other, with a long two-point distance. The long two-point distance of the blind goldfish lasted for at least two months and then slowly returned to control two-point distance by four months after bilateral transection. Such fast and slow recovery in goldfish behaviors evoked after unilateral and bilateral transection of the optic nerve is discussed with respect to reconnection of regenerating optic nerves in the fish central nervous system. This computer image processing system is a useful tool with which we can quickly and easily quantify fish behavior.
Algorithms↗
Cloning and sequence analysis of hydroxyquinol 1,2-dioxygenase gene in 2,4,6-trichlorophenol-degrading Ralstonia pickettii DTP0602 and characterization of its product.
A gene encoding hydroxyquinol 1,2-dioxygenase was cloned from 2,4,6-trichlorophenol-degrading Ralstonia (Pseudomonas) pickettii strain DTP0602. Cell-free extracts of Escherichia coli containing a cloned 1.4-kb StuI-XhoI DNA fragment of R. pickettii DTP0602 hydroxyquinol 1,2-dioxygenase converted hydroxyquinol into maleylacetate and also degraded 6-chlorohydroxyquinol. The 1.4-kb DNA fragment contained one open reading frame (designated hadC) composed of 948 nucleotides. The molecular mass of 34,591 deduced from the gene product (HadC) was in agreement with the size (35 kDa) of the purified HadC protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid sequence of HadC exhibited high homology to that of the hydroxyquinol 1,2-dioxygenase of 2,4,5-trichlorophenoxyacetic acid-degrading Burkholderia cepacia AC1100 (Daubaras, D. L. et al., Appl. Environ. Microbiol., 61, 1279-1289, 1995). The active enzyme had a molecular mass of 68 kDa, suggesting that it is functional as a homodimer. The enzyme also catalyzed the oxidation of pyrogallol and 3-methylcatechol, possible intermediates in the degradation of 2,4,6-trichlorophenol, in addition to 6-chlorohydroxyquinol and hydroxyquinol. The dioxygenase catalyzed both ortho- and meta-cleavage of 3-methylcatechol.
Journal Article↗
Nucleotide sequences and characterization of genes encoding naphthalene upper pathway of pseudomonas aeruginosa PaK1 and Pseudomonas putida OUS82.
A 12,808-nucleotide containing DNA fragment cloned from naphthalene-utilizing (Nah+) Pseudomonas aeruginosa PaK1 was analyzed and compared with the genes (pah(OUS)) of a 14,462-nucleotide DNA fragment from Pseudomonas putida OUS82. The DNA sequence analyses demonstrated that the naphthalene upper-pathway genes and their deduced enzymes were very similar between the two bacteria: nucleotide similarities, 83-93%; amino acid similarities, 79-95%. These genes were also similar to those of the nah operon of plasmid NAH7; in particular, the OUS82 genes were similar to the nah genes, whereas the PaK1 genes were almost identical to the dox genes of Pseudomonas sp. C18. A region homologous with the 84-bp repeated sequence that Eaton (J. Bacteriol., 176, 7757-7762, 1994) has found at a site upstream of he nah operon was found only in a region downstream of the pah(PaK) gene cluster in PaK1 and on both sides of the pah(OUS) gene cluster in OUS82. A PaK1 gene, corresponding to an unknown gene (nahQ) in the nah operon, is located between the 1,2-dihydroxynaphthalene dioxygenase gene and the trans-o-hydroxybenzylindenepyruvate (tHBP A) hydratase-aldolase gene (nahE), and was suggested to be involved in the conversion of naphthalene to salicylate. Just downstream of the pah(PaK) gene cluster, a portion of a region was identical to one-third of the transposase gene (tnpA) in a phenol-catabolic plasmid pEST1226.
Journal Article↗
Evidence for heteromorphic sex chromosomes in males of Rana tagoi and Rana sakuraii in Nishitama district of Tokyo (Anura: Ranidae).
Karyotypes of the Tago brown frog Rana tagoi and stream Tago brown frog Rana sakuraii from a mountain region in the Nishitama district in Tokyo were examined by conventional Giemsa staining, C-banding and late replication (LR)-banding. Chromosome number was 2n = 26 in all cases. The 26 chromosomes consisted of five (1-5) pairs of large chromosomes and eight (6-13) pairs of small chromosomes. Chromosome 10 had a secondary constriction on the long arm. In all frogs, on chromosome pair 8, the XX/XY type sex chromosome was present. C-banding analysis indicated that, in R. sakuraii, neither the X nor Y chromosome possessed interstitial C-bands but each had centromere staining, while in R. tagoi, an interstitial C-band was present on the long arm of the X chromosome. The Y chromosome had no interstitial C-band. LR-banding analysis demonstrated the X and Y chromosomes to have a LR-band on the short arm and two LR-bands, each on the long arm, and the bands on both species to be essentially the same. Heteromorphic sex chromosomes in males of R. sakuraii and R. tagoi were identified for the first time in this study.
Animals↗
Up-regulation of nuclear PP1alpha and PP1delta in hepatoma cells.
Activities of nuclear type 1 protein phosphatase (PP1) were significantly elevated in human HepG2 and rat AH13 hepatoma cells compared with primary cultured hepatocytes. We examined and compared the nuclear PP1 activities during the cell cycle between synchronized HepG2 cells and HGF-stimulated hepatocytes. Nuclear PP1 activity was significantly and more elevated at the G1/S transition in hepatoma cells compared with hepatocytes, although the amounts of PP1 isoforms remained constant. On the contrary, it was found that the basal levels of nuclear PP1 activity were significantly higher in hepatoma cells and that the amounts of PP1alpha and PP1delta were dramatically increased in the nuclear fraction of hepatoma cells.
Animals↗
Age-related changes in the cerebral distribution of 99mTc-ECD from infancy to adulthood.
UNLABELLED: Although cerebral blood flow in infants differs from that in older individuals, the distribution of 99mTc-ethyl cysteinate dimer (ECD) in infants has not been well studied. This study compared 99mTc-ECD distribution in infants and children with that in young adults. METHODS: 99mTc-ECD SPECT was performed on 37 patients suspected of having epilepsy, ranging in age from 3 mo to 26 y. The patients were divided into two age-matched groups, a drug-free group (n = 19) and a drug-taking group (n = 18), according to their anticonvulsant medication status at the time of examination. 99mTc-ECD (100-740 MBq) was injected interictally, and SPECT data were acquired using a triple-head gamma camera. Mean whole-brain counts were obtained from 10 sequential SPECT images. Regions of interest were set bilaterally on five areas of the cerebral cortex and on the basal ganglia, thalamus and cerebellum. The brain perfusion index (BPI) was obtained as a ratio of the mean counts in each region of interest to the mean whole-brain counts. The relationship between BPI and age in each region in the drug-free and drug-taking groups was analyzed separately and together using linear regression. The relationship between five patient age groups (<1 y, n = 4; 1-4 y, n = 9; 5-9 y, n = 8; 10-15 y, n = 7; >15 y, n = 9) and BPI in each region was also examined using multiple comparison analyses. RESULTS: Significant positive correlations between BPI and age in the frontal cortex and cerebellum were confirmed in the drug-free group. Anticonvulsant drugs did not affect the regression lines of BPI in the frontal cortex and cerebellum. Significant differences in BPI between age groups were seen in the parietal cortex, frontal cortex, occipital cortex, basal ganglia, thalamus and cerebellum in all patients. CONCLUSION: Age-related changes in cerebral 99mTc-ECD distribution were confirmed and found to be unaffected by the administration of anticonvulsant drugs. 99mTc-ECD uptake in children and infants is different from cerebral blood flow glucose metabolism as previously reported, especially in the cerebellum.
Adolescent↗
Structure-function relationships in the four repeats of human interphotoreceptor retinoid-binding protein (IRBP).
PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP) binds hydrophobic ligands in the interphotoreceptor space. Human IRBP consists of 1230 amino acids in four 300 amino acid long repeats. We asked: 1. Whether each of the four repeats can bind retinoids or fatty acids, 2. Whether each repeat can prevent retinol degradation in aqueous solutions, 3. Whether a ligand can stabilize the protein from thermal denaturation, 4. Whether the four repeats can be further classified into two groups. Our rationale was to make each repeat from the human cDNA and then examine structural and functional characteristics. METHODS: Individual repeats were produced in E. coli and the whole protein was expressed in baculovirus. Binding properties with all-trans-retinol were characterized by ligand fluorescence enhancement. The quenching of protein fluorescence by retinol, 9-cis-retinal, all-trans-retinoic acid, beta-ionine, alpha-ionine, trans-parinaric acid, and DHA was also examined. Binding curves were analyzed by nonlinear regression. Prevention of retinol decomposition was measured by absorption spectroscopy. Circular dichroism was examined in the far UV range to study protein secondary structure and the near UV range to study ligand binding effects on the tryptophan environment. RESULTS: Temperature dependent denaturation suggests that EcR1 is the most stable of the four repeats. Each repeat possesses the capability of binding 9-cis-retinal, all-trans-retinol, all-trans retinoic acid, docosahexaenoic acid, alpha- and beta-ionine, and trans-parinaric acid. Protein fluorescence quenching by retinol and retinol fluorescence enhancement assays yielded similar binding parameters for each repeat. Each expressed repeat prevents the degradation of retinol in aqueous solutions. CONCLUSIONS: The data contrast with the idea that two or more repeats are needed to bind one molecule of ligand. Each repeat binds both retinoids and analogs, suggesting that each has multiple ligand binding sites or one binding site with affinity for different ligands. Together, the results suggest that each repeat retains all functions of the whole protein. However, there are distinguishing characteristics among the repeats in their ligand binding properties, though the four repeats cannot be classified into just two distinctive groups. Last, these data fit well with the current model of multiple binding sites in IRBP derived from quadruplication of an ancestral monomeric binding protein.
Circular Dichroism↗
Nitrosyl hemoglobin in blood of normoxic and hypoxic sheep during nitric oxide inhalation.
During nitric oxide (NO) inhalation therapy, NO combines with deoxyhemoglobin to form nitrosyl hemoglobin (HbNO). We used electron spin resonance (ESR) spectroscopy to measure HbNO in arterial and mixed venous blood of normoxic and hypoxic sheep during NO inhalation. Our aim was to quantitatively measure HbNO levels in the blood during NO inhalation, because large amounts of HbNO reduce the oxygen capacity of blood, particularly in hypoxia. Another aim was to investigate the transfer of exogenous NO to the alpha-heme iron of hemoglobin. Thirteen sheep were anesthetized with pentobarbital sodium, and 60 parts per million (ppm) NO were administered for 1 h in the presence of normoxia and hypoxia. Two-way analysis of variance revealed that the HbNO level was dependent on the oxygen level (normoxia vs. hypoxia) and NO inhalation, and there was a significant negative correlation between the HbNO level and arterial O2 saturation (SaO2). Although the HbNO level increased during NO inhalation in hypoxia, the HbNO level at SaO2 > 60% was < 11 mumol/l monomer hemoglobin (0.11% of total 10 mmol/l monomer hemoglobin). The peak of the HbNO ESR spectrum in arterial blood is located in almost the same position in mixed venous blood with an asymmetric HbNO signal, indicating that the NO in beta-heme HbNO molecules had been transferred to alpha-heme molecules. The three-line hyperfine structure of HbNO on ESR spectra was distinct in venous blood in hypoxia during NO inhalation, indicating pentacoordinate alpha-NO heme formation in hypoxic blood. In conclusion, the amount of HbNO during 60 ppm NO inhalation did not considerably reduce the oxygen capacity of the blood even in the presence of hypoxia, and the NO of HbNO was transferred to the alpha-heme iron of hemoglobin, forming pentacoordinate alpha-NO heme in mixed venous blood in hypoxia.
Administration, Inhalation↗
Structure-function relationships in interphotoreceptor retinoid-binding protein (IRBP).
PURPOSE: Interphotoreceptor retinoid binding protein (IRBP) binds hydrophobic ligands in the retina. The polypeptide consists of 1230 amino acids in four 300 amino acid long repeats. We asked whether each of the four repeats can bind one retinoid or fatty acid analog. Our rationale was to make protein variants from the human cDNA bearing one or more of the repeats and examine binding capacities and dissociation constants. METHODS: Proteins were characterized by SDS-PAGE, western blotting, N-terminal sequencing, and CD spectroscopy. Binding properties with all-trans-retinol and 16-anthryloxy-palmitic acid (16-AP) were characterized by ligand fluorescence enhancement and curve fitting. RESULTS: Binding capacities varied according to the length of each protein. Each repeat possesses the capability of binding retinol and 16-AP. CONCLUSIONS: The data contrast with the idea that two or more repeats are needed to bind one molecule of ligand. Each repeat binds a retinoid and fatty acid analog, suggesting that each has multiple ligand binding sites or one binding site with affinity for different ligands. Last, these data fit well with the current model of multiple binding sites in IRBP derived from quadruplication of an ancestral monomeric binding protein.
Animals↗
Chronic infantile neurological cutaneous articular syndrome in a patient from Japan.
A case of chronic infantile neurological cutaneous articular (CINCA) syndrome in a Japanese girl, started at the age of 13 days with episodes of fever, rash followed by swollen joint, hepatosplenomegaly, generalized lymphadenopathy and chronic central nervous system involvement, is reported. Some of the findings suggest that this syndrome may be the result of an intrauterine infection. This is the first case of CINCA syndrome in a Japanese girl.
Age of Onset↗
Relationships of subunits of type-1 serine/threonine protein phosphatase to morphology and aggregation of B cells.
To elucidate the roles of serine/threonine protein phosphatases PP1 and PP2A in the morphological changes of B-lymphocytes during development and in immune responses, we investigated alterations of protein levels of catalytic subunits of PP1 and PP2A and regulatory subunits of PP1 including M130/M133, inhibitor-1 (I-1) and inhibitor-2 (I-2) in B-cell lines at different maturational stages and during their aggregation induced by phorbol myristate acetate (PMA). The protein levels of PP1delta and/or M130/M133 were significantly lower in B-cell lines without pseudopods, WEHI-231, BAL-17, Daudi, and CESS, than in those with pseudopods, Bcl.1, A20, M12, and SKW6.4, whereas the amounts of PP1alpha and PP2A were similar among them. During aggregation of A20 and CESS cells induced by PMA, an activator of PKC, the amount of PP1delta was progressively decreased, and this decrease was blocked by H7, an inhibitor of PKC. The amount of PP1alpha was constant under these conditions. Okadaic acid, an inhibitor of PP1 and PP2A, also induced aggregation of A20 cells at concentrations sufficient to inhibit PP1, but not at lower concentrations that inhibit PP2A alone. These results suggest that myosin light chain phosphatase composed of PP1delta and M130/M133 is involved in the maintenance and regulation of cytoskeletal structures in B-lymphocytes.
Animals↗
Alterations in type-1 serine/threonine protein phosphatase PP1alpha in response to B-cell receptor stimulation.
In response to stimulation of B-cells through cell surface IgM, the activity of the serine/threonine protein phosphatase PP1, but not PP2A, was transiently decreased and reached a minimum 10-20 min after the stimulation. The decrease was more profound in the immature B-cell line WEHI-231, than in the mature B-cell line BAL-17. Under these conditions, PP1alpha, an isoform of PP1, showed unique alterations in the patterns of several spots with distinct isoelectic points in the Western blot after two-dimensional electrophoresis, whereas another isoform, PP1delta, did not show any alteration. PP1gamma1 and PP1gamma2 were not detected in B-cells. Similar alterations in these spots were observed in B-cells stimulated by PMA. When partially purified PP1 consisting of PP1alpha and PP1delta was incubated with [gamma-32P]ATP and PKC, radioactive spots of PP1alpha could be detected, but no spot of PP1delta was detected. Because differences in sequence among PP1 isoforms are mostly restricted to their C-terminals, phosphorylation rates of the C-terminal peptides containing the PKC-phosphorylation motif were compared. The C-terminal peptide of PP1alpha is a better substrate for PKC than those of PP1gamma1 and PP1gamma2, and is phosphorylated at the serine residue corresponding to Ser-325 of PP1alpha. The corresponding C-terminal region of PP1delta does not contain the phosphorylation site. On the other hand, there was a large difference in subcellular distribution of PP1delta, but not PP1alpha, between immature and mature B-cells. From these results, it was strongly suggested that PP1alpha is involved, via phosphorylation by PKC, in the regulation of signal transduction in response to the stimulation of B-cells through cell surface IgM.
Amino Acid Sequence↗
The relation between two molecular species of P-450 in adult testis and 17 alpha-hydroxylase and 17,20-lyase activities.
Two P-450s from adult pig testis were purified to specific contents of 11.2 and 12.0 nmol P-450/mg protein and shown to have minimum molecular weights of 45,000 and 46,000, respectively. The absorption spectra were typical of P-450s. The P-450s were separated from the two fractions by CM-C50 Sephadex column chromatography. One P-450 (M(r) = 45000) exhibited 17,20-lyase activity of 6.78 nmol of androstenedione/min/nmol P-450, on incubation with 17 alpha-hydroxyprogesterone as a substrate. The other P-450 (M(r) = 46,000) exhibited no 17,20-lyase activity. Both P-450s exhibited 17 alpha-hydroxylase activity that amounted to 10 nmol of steroid products. Accordingly, the two molecular species of P-450 are thus markedly different in 17,20-lyase activity toward 17 alpha-hydroxyprogesterone.
Aldehyde-Lyases↗