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Biomedical subjects

K Nakata

Publications and source records attributed to K Nakata.

At least 37 records · Page 2Linked to original sources
PubMed2002

BAL findings in a patient with pulmonary alveolar proteinosis successfully treated with GM-CSF.

BACKGROUND: Idiopathic pulmonary alveolar proteinosis (PAP) has recently been recognised as a disease of impaired alveolar macrophage function caused by neutralising anti-granulocyte-macrophage colony-stimulating (anti-GM-CSF) autoantibodies. Subcutaneous recombinant human GM-CSF is a novel treatment for PAP, but its mechanism of action is unclear. METHODS: Clinical, functional, and bronchoalveolar lavage (BAL) findings were prospectively evaluated in a patient with PAP treated with daily subcutaneous GM-CSF 8 microg/kg for 12 weeks. RESULTS: Treatment resulted in improvements in dyspnoea, lung function, and peak cycle ergometry performance. In serum and BAL fluid the titre of anti-GM-CSF autoantibodies was raised at baseline and markedly reduced on treatment. At baseline the BAL fluid cellular profile showed a decrease in the absolute number and the percentage of macrophages (50%) and an increase in lymphocytes (45%), predominantly CD4+. This cellular distribution remained unchanged after 6 and 12 weeks of treatment while macrophages became morphologically normal and functionally improved. Extracellular proteinaceous material completely disappeared. CONCLUSIONS: Clinically successful treatment of PAP with GM-CSF was associated with a profound reduction in GM-CSF neutralising autoantibodies, improvement in alveolar macrophage morphology and function, but persistent BAL lymphocytosis.

Adult↗
PubMed2001

[Analysis of hematological findings affecting the efficiency of the autologous peripheral blood stem cells harvest].

As a predictor of the timing of peripheral blood stem cell harvest (PBSCH) and the yields of PBSC, the efficacy of measurement of CD34+ cell count in PB has been reported. However this measurement is difficult to carry out on the day of PBSCH in most general hospitals because of not having their own flow cytometry. Therefore in order to evaluate the usefulness of several clinical factors which are easily performed in general hospitals as a guide for PBSCH, we analyzed the correlation between these factors and the harvest efficiency (HE) of PBSCH. Eighty-one PBSCHs mobilized by chemotherapy with granulocyte colony-stimulating factor (G-CSF)(n = 63) or chemotherapy alone (n = 18) were carried out in 33 patients with various malignancies. In the group of PBSCHs mobilized by chemotherapy with G-CSF, a strong correlation was observed between HE and the percentage of immature cells in peripheral WBC (IMA-%) on the harvest day (r = 0.724), and between HE and the count of immature cells in PB (IMA-Co) on the same harvest day (r = 0.629). However routine blood counts, except IMA-% and IMA-Co, and the time (days) required for hemopoietic recovery after mobilizing chemotherapy did not correlate with HE. In the group of PBSCHs mobilized by chemotherapy alone, several hematological findings (WBC count, monocyte count and %, IMA-Co, platelet count, reticulocyte-@1000) on the harvest day and recovering velocity of WBC and platelet correlated with HE. These findings suggested that IMA-% and IMA-Co were the most useful factors for successful PBSCH widely mobilized by chemotherapy with G-CSF in general hospitals.

Antigens, CD34↗
PubMed2001

Retrovirus-mediated gene therapy for hepatocellular carcinoma with reversely oriented therapeutic gene expression regulated by alpha-fetoprotein enhancer/promoter.

In the present study, to achieve more selective and efficient therapeutic gene expression in hepatoma cells, we compared the therapeutic efficacies of the retroviral vectors expressing the herpes simplex virus thymidine kinase (HSV-tk) gene by the alpha-fetoprotein (AFP) enhancer/promoter in the forward (LNAFE0.3TK) and reverse (LN[AFE0.3TK]R) orientation to the vector long terminal repeats. By Northern blotting, the level of the HSV-tk mRNA in LN[AFE0.3TK]R-infected HepG2 human hepatoma cells was much higher than that in LNAFE0.3TK-infected cells. Consistent with this, LN[AFE0.3TK]R infection into HepG2 cells caused a greater cytotoxicity by ganciclovir exposure together with a stronger bystander effect than LNAFE0.3TK infection. In an animal model, intratumorous injection of LN[AFE0.3TK]R with ganciclovir treatment resulted in pronounced growth inhibition of HepG2 tumor. Thus, the reversely oriented therapeutic gene expression under the control of AFP enhancer/promoter is a possible candidate for the retrovirus-mediated gene therapy for hepatocellular carcinoma.

Animals↗
PubMed2001

High-performance liquid chromatographic method to analyze picomole levels of glutathione, cysteine and cysteinylglycine and its application to pre-cancerous rat livers.

A HPLC-based method for quantifying glutathione, cysteine and cysteine-containing peptide is described. N-(2,4-Dinitrophenylaminoethyl)maleimide was synthesized from ethylenediamine, 2,4-dinitrochlorobenzene and maleic anhydride. The maleimide was reacted at 40 degrees C and pH 5.8 for 10 min with thiol compounds such as glutathione or cysteine. An aliquot of the reaction mixtures was applied to a reversed-phase column (4.6 x 150 mm) of HPLC. When glutathione and cysteine were simultaneously assayed, the column was eluted with a gradient of acetonitrile in potassium phosphate (pH 7.0). The derivatives were monitored at 350 nm. Good liner relationships existed between peak area and concentration of glutathione or cysteine from 10 pmol to 2 nmol. The recovery tests from rat liver homogenate were 99.7+/-2.2% for glutathione and 104.9+/-3.8% for cysteine. By this method gamma-glutamylcysteine, cysteinylglycine, and homocysteine could be also quantified. The determination limits of glutathione, cysteine and other thiol compounds were 5 pmol. The method is simple: a sample solution is mixed with the labelling reagent and an aliquot of the reaction mixture is applied to a standard HPLC. The hepatic levels of cysteine and glutathione in pre-cancerous rats were determined by this method. The cysteine level in pre-cancerous livers was extremely elevated in comparison to that of the control groups, while no difference was observed in the glutathione contents between the pre-cancerous and control groups.

Animals↗
PubMed2001

Asymmetric epoxidation with a photoactivated [Ru(salen)] complex.

(Nitrosyl)(salen)ruthenium(II) complex 1 was found to serve as an efficient catalyst for the epoxidation of conjugated olefins under photoirradiation, with 2,6-dichloropyridine N-oxide (2) or tetramethylpyrazine N,N'-dioxide as a stoichiometric oxidant. High enantioselectivity was achieved irrespective of the substitution pattern of olefins. The choice of solvent depends on stability of the resulting epoxides: high enantioselectivity is generally observed in the reaction with ethereal solvents, but use of benzene is recommended when the resulting epoxides are acid-sensitive.

Journal Article↗
PubMed2001

X-linked inhibitor of apoptosis protein (XIAP) inhibits caspase-3 and -7 in distinct modes.

The inhibitor of apoptosis proteins (IAP) regulates cell death by inhibiting caspases. The region of X-linked (X) IAP containing the second baculovirus IAP repeat domain (BIR2) is sufficient for inhibiting caspase-3 and -7. In this study, we found that the modes of inhibition of these two caspases were different: caspase-3 is inhibited in a competitive manner whereas caspase-7 inhibition occurs through a mixed competitive and noncompetitive mechanism. Binding assays revealed that the inhibition of caspase-3 by XIAP was totally dependent on the interaction between the active site of caspase-3 and the linker region between the BIR1 and BIR2 domains of XIAP. In contrast, the active site and the NH(2)-terminal region of caspase-7 bound to the linker region and the BIR2, respectively. Moreover the BIR2 with a mutated linker region, which inhibited caspase-3 very weakly, still bound to and inhibited caspase-7. Furthermore, a chimeric caspase-7/3 comprising the NH(2)-terminal portion of caspase-7 and COOH-terminal portion of caspase-3 was inhibited by XIAP by a mixed competitive and noncompetitive mechanism. Our results suggest that the linker region between BIR1 and BIR2 domains is responsible for active site-directed, competitive inhibition of both caspase-3 and -7, whereas the BIR2 itself is involved in noncompetitive inhibition of caspase-7.

Base Sequence↗
PubMed2001

Clinical usefulness of the branched DNA assay version 2 in predicting the efficacy of Interferon treatment in a group of chronic HCV patients based on serotype.

Interferon (IFN) response depends upon pretreatment viral loads and viral genotype/serotype. This study investigated the difference in response to IFN in different viral load groups of 96 chronic hepatitis C patients and compared version 1 (bDNA1.0) and version 2 (bDNA2.0) of the branched DNA assay, according to serotype. On univariate analysis, viral load (P=0.0001, by bDNA 1.0; P=0.0020, by bDNA 2.0,), serotype (P=0.0053) and age (P=0.0073) were significant predictors of IFN response. On multivariate analysis, serotype (odds ratio, 5.44; 95% confidence interval, 1.94-15.24; P<0.01) was a stronger predictor of IFN response than age or viral load (by bDNA2.0). In very high (>6.7 log eq/ml), high (6.0 approximately 6.69 log eq/ml) and low (<6 log eq/ml) viral load groups (by bDNA2.0), complete response was 25, 55 and 92.6% in serotype 2 (sero-2), and 10, 20 and 71.4% in serotype 1 (sero-1), respectively. In sero-2, bDNA2.0 can detect high viral loads underestimated by bDNA1.0. In a low viral load group (by bDNA2.0), IFN response is dependent upon serotype; sero-2 responded better than sero-1. However, in high and very high viral load groups, sensitivities of bDNA1.0 and bDNA2.0 are not effective in clinically distinguishing CR from non-response, and aid in patient selection for IFN therapy.

Journal Article↗
PubMed2001

Skeletal malformations caused by overexpression of Cbfa1 or its dominant negative form in chondrocytes.

During skeletogenesis, cartilage develops to either permanent cartilage that persists through life or transient cartilage that is eventually replaced by bone. However, the mechanism by which cartilage phenotype is specified remains unclarified. Core binding factor alpha1 (Cbfa1) is an essential transcription factor for osteoblast differentiation and bone formation and has the ability to stimulate chondrocyte maturation in vitro. To understand the roles of Cbfa1 in chondrocytes during skeletal development, we generated transgenic mice that overexpress Cbfa1 or a dominant negative (DN)-Cbfa1 in chondrocytes under the control of a type II collagen promoter/enhancer. Both types of transgenic mice displayed dwarfism and skeletal malformations, which, however, resulted from opposite cellular phenotypes. Cbfa1 overexpression caused acceleration of endochondral ossification due to precocious chondrocyte maturation, whereas overexpression of DN-Cbfa1 suppressed maturation and delayed endochondral ossification. In addition, Cbfa1 transgenic mice failed to form most of their joints and permanent cartilage entered the endochondral pathway, whereas most chondrocytes in DN-Cbfa1 transgenic mice retained a marker for permanent cartilage. These data show that temporally and spatially regulated expression of Cbfa1 in chondrocytes is required for skeletogenesis, including formation of joints, permanent cartilages, and endochondral bones.

Animals↗
PubMed2001

NOTCH4 gene polymorphism and susceptibility to schizophrenia and schizoaffective disorder.

The NOTCH4 gene is located at 6p21.3, a site which several studies have shown to have significant linkage with schizophrenia. Recently, an exceptionally strong association was reported between NOTCH4 gene polymorphisms and schizophrenia in British patients. We re-examined their findings using a Japanese population. We genotyped three kinds of polymorphisms, SNP1 in the 5' flanking region, SNP2 in the promoter region and CTG repeats in exon 1 of the NOTCH4 gene of schizophrenics (N=188), patients with schizoaffective disorder (N=39) and controls (N=143). Genotypic distributions and allelic frequencies of SNP1, SNP2 and CTG repeats of the NOTCH4 gene did not show significant associations with schizophrenia or schizoaffective disorder. Neither they showed association with schizophrenia subcategories, hebephrenic and paranoid type schizophrenia, nor with subgroups of schizophrenia with and without positive family history of psychoses. The present study found that the NOTCH4 gene does not confer susceptibility to schizophrenia and schizoaffective disorders, at least in Japanese subjects, in contrast to the findings in British subjects.

Adult↗
PubMed2001

Geranylgeranylacetone induces antiviral gene expression in human hepatoma cells.

Geranylgeranylacetone (GGA), an isoprenoid compound, is used clinically as an anti-ulcer drug. Since some isoprenoids including retinoids have anti-tumor and anti-viral activities in a variety of cell types, we investigated whether GGA could induce anti-viral proteins in human hepatoma cells. The HuH-7 and HepG2 cells were treated with GGA, and expression of anti-viral proteins such as 2'5'-oligoadenylate synthetase (2'5'-OAS) and double-stranded RNA-dependent protein kinase (PKR) in these cells was analyzed. GGA stimulated 2'5'-OAS and PKR gene expression at the transcriptional level through the formation of interferon-stimulated gene factor 3 (ISGF3), which regulates both gene transcription. By Western blotting, GGA induced expression of signal transducers and activators of transcription 1, 2 (STAT1, STAT2) and p48 proteins, components of ISGF3, together with the phosphorylation of STAT1. These results suggest that GGA acts as a potent inducer of anti-viral gene expression by stimulating the ISGF3 formation in human hepatoma cells.

2',5'-Oligoadenylate Synthetase↗
PubMed2001

Effect of carvedilol on atrioventricular conduction in the ischemic heart.

We compared the effects of carvedilol on atrial-His and His-ventricular conduction with those of propranolol in isolated rat hearts. Hearts were perfused retrograde, and atrial-His and His-ventricular intervals were measured. The effective doses that increased conduction times by 25% were 10(-6) M for atrial-His and 3x10(-6) M for His-ventricular for propranolol, and 8x10(-8) M for atrial-His and 10(-8) M for His-ventricular for carvedilol. Prazosin did not affect the atrial-His and His-ventricular intervals. After ischemia-reperfusion, atrial-His and His-ventricular intervals increased to a greater extent with 10(-6) M carvedilol. To determine the direct membrane effect, we examined the transmembrane action potential in guinea pig papillary muscle. Both drugs decreased the maximum upstroke velocity equally. Our data indicate that carvedilol had a greater effect on atrioventricular conduction in the setting of ischemia-reperfusion than did propranolol. This effect of carvedilol was not due to its alpha-adrenoceptor blocking property or to a direct membrane effect.

Action Potentials↗
PubMed2001

Physical and functional interactions between Zic and Gli proteins.

Zic and Gli family proteins are transcription factors that share similar zinc finger domains. Recent studies indicate that Zic and Gli collaborate in neural and skeletal development. We provide evidence that the Zic and Gli proteins physically and functionally interact through their zinc finger domains. Moreover, Gli proteins were translocated to cell nuclei by coexpressed Zic proteins, and both proteins regulated each other's transcriptional activity. Our result suggests that the physical interaction between Zic and Gli is the molecular basis of their antagonistic or synergistic features in developmental contexts and that Zic proteins are potential modulators of the hedgehog-mediated signaling pathway.

3T3 Cells↗
PubMed2001

Recombination and gene conversion-like events may contribute to ABO gene diversity causing various phenotypes.

We identified five different alleles, tentatively named ABO*O301, *0302, *R102, *R103, and *A110, in Japanese individuals possessing the blood group O phenotype. These alleles lack the guanine deletion at nucleotide position 261 which is shared by a majority of O alleles. Nucleotide sequence analysis revealed that *0301 and *0302 had single nonsynonymous substitutions compared with *A101 or *A102 responsible for the A1 phenotype. Analysis of intron 6 at the ABO gene by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing revealed that *R102 and *R103 had chimeric sequences of A-02 and B-02, respectively, from exons 6 to 7. In the analysis of five other chimeric alleles detected in the same manner, we identified a total of four different recombination-breakpoints within or near intron 6. When 510 unrelated Japanese were examined, the frequency of the chimeric alleles generated by recombination in intron 6 or exon 7 was estimated to be 1.7%. In addition, we found that *O301, *A110, *C101, *A111, and 35% of *A102 had a unique A-B-A chimeric sequence at intron 6, presumed to originate from a gene conversion-like event. We had previously established that *A110 also had an A-O2-A chimeric sequence around nucleotide position 646 in exon 7. Thus this allele has an A-B-A-O2-A chimeric sequence from intron 6 to exon 7 probably generated by two different gene conversions. Similar patchwork sequences around nucleotide position 646 in exon 7 were observed in two other new alleles responsible for the Ax and B3 phenotypes. Thus, the site is presumably a hotspot for gene conversion. These results indicate that both recombination and gene conversion-like events play important roles in generating ABO gene diversity.

ABO Blood-Group System↗
PubMed2001

Xenopus Polycomblike 2 (XPcl2) controls anterior to posterior patterning of the neural tissue.

A novel gene, Xenopus Polycomblike 2 (XPcl2), which encodes a protein similar to Drosophila Polycomblike was cloned and characterized. Polycomblike belongs to the Polycomb group proteins, which maintain stable expression patterns for the clustered homeotic genes in the Drosophila embryo by forming multimeric complexes on chromatin. XPcl2 shows greater amino acid sequence homology to human and mouse M96 (hPcl2, mPcl2) than Xenopus Pcl1 (XPcl1), mouse Tctex3 (mPcl1) and human PHF1 (hPcl1), indicating that at least two types of Polycomblike genes are conserved between amphibians and mammals. XPcl2 mRNA is present both maternally and zygotically, and the temporal expression profile is distinct from XPcl1, another member of the Polycomblike family in Xenopus. XPcl2 is highly expressed in the anterior-dorsal region of Xenopus following the neurula stage in a manner similar to XPcl1. Overexpression of XPcl2 disturbs the development of the anterior central nervous system, eye and cement gland. In the XPcl2-overexpressing embryo, a hindbrain marker, Krox20, and a spinal cord marker, HoxB9, are expressed more posteriorly, suggesting an alteration in the anterior-posterior patterning of the neural tissue. In addition, XPcl2 represses Zic3- and noggin-induced anterior neural markers, but not neural crest markers in animal cap explants. These results indicate that XPcl2 regulates anterior neural tissue development and the anterior-posterior patterning of the neural tissue.

Amino Acid Sequence↗
PubMed2001

Overestimated frequency of a possible emphysema-susceptibility allele when microsomal epoxide hydrolase is genotyped by the conventional polymerase chain reaction-based method.

A recent association study suggested that the His113 variant of microsomal epoxide hydrolase (mEPHX) may confer a risk for development of emphysema, presumably by increasing susceptibility to smoking injury. Before considering a possible role of this enzyme in pulmonary disease, we attempted to characterize the genetic polymorphism further. The Tyr/His113 polymorphism within exon 3 of mEPHX was initially examined in 62 healthy individuals by conventional methods involving polymerase chain reaction (PCR)-based determination of a restriction fragment length polymorphism (RFLP). Genomic nucleotide sequences, including the polymorphic site and the downstream primer sequence, were further analyzed in 95 unrelated, healthy Japanese volunteers by single-stranded conformation polymorphism (SSCP) analysis and direct sequencing. Genotyping by the first method (PCR-RFLP) revealed that the allelic distribution in our test population apparently deviated from Hardy-Weinberg equilibrium. Sequence analysis showed that a synonymous nucleotide substitution, AAG to AAA (Lys119), was located just within the published primer site. The AAA at codon 119 was present only in alleles with Tyr113, and its frequency reached 0.31 in our panel of 190 Japanese alleles. This substitution potentially hampered PCR amplification because of the nucleotide mismatch, with the result that the frequency of the Tyr113 variation was underestimated. The frequency of His113, a possible emphysema susceptibility allele of the mEPHX gene, was thus overestimated when human DNA samples were genotyped in the conventional way. Depending on the population(s) tested, this anomaly could represent a pitfall for PCR-based association studies.

Alleles↗
PubMed2001

Comparison of antimycobacterial activity of grepafloxacin against Mycobacterium avium with that of levofloxacin: accumulation of grepafloxacin in human macrophages.

The bactericidal activity of two new quinolones, grepafloxacin and levofloxacin, against five strains of Mycobacterium avium was investigated in vitro. The minimum inhibitory concentrations (MICs) of these two quinolones, determined by the broth microdilution method, were comparable for all strains tested. In contrast, grepafloxacin suppressed the intracellular growth of all the strains in monocyte-derived macrophages more strongly than levofloxacin, when the cells infected with these strains were incubated for 7 days in the presence of various concentrations of the two new quinolones. To find the reason for the strengthened intracellular killing activity of grepafloxacin, we determined the ratio of the concentration of the new quinolones in the cells to that in the medium (C/M concentration ratio). The C/M concentration ratio of grepafloxacin was increased to 34.7 by 7 days, whereas that of levofloxacin at 7 days was only 12.3. These data suggested that a higher level of intraphagocytic accumulation of grepafloxacin endows it with greater mycobactericidal activity.

Anti-Infective Agents↗
PubMed2001

Distribution of the high-affinity choline transporter in the central nervous system of the rat.

In cholinergic nerve terminals, Na(+)- and Cl(-)-dependent, hemicholinium-3-sensitive, high-affinity choline uptake is thought to be the rate-limiting step in acetylcholine synthesis. The high-affinity choline transporter cDNA responsible for the activity was recently cloned. Here we report production of a highly specific antibody to the high-affinity choline transporter and distribution of the protein in the CNS of the rat. The antibody stained almost all known cholinergic neurons and their terminal fields. High-affinity choline transporter-immunoreactive cell bodies were demonstrated in the olfactory tubercle, basal forebrain complex, striatum, mesopontine complex, medial habenula, cranial nerve motor nuclei, and ventral horn and intermediate zone of the spinal cord. Noticeably, high densities of high-affinity choline transporter-positive axonal fibers and puncta were encountered in many brain regions such as cerebral cortex, hippocampus, amygdala, striatum, several thalamic nuclei, and brainstem. Transection of the hypoglossal nerve resulted in a loss of high-affinity choline transporter immunoreactivity in neurons within the ipsilateral hypoglossal motor nucleus, which paralleled a loss of immunoreactivity to choline acetyltransferase. The antibody also stained brain sections from human and mouse, suggesting cross-reactivity. These results confirm that the high-affinity choline transporter is uniquely expressed in cholinergic neurons and is efficiently transported to axon terminals. The antibody will be useful to investigate possible changes in cholinergic cell bodies and axon terminals in human and rodents under various pathological conditions.

Acetylcholine↗