ITPKC and CASP3 polymorphisms and risks for IVIG unresponsiveness and coronary artery lesion formation in Kawasaki disease.

Functional single-nucleotide polymorphisms (SNPs) in inositol 1,4,5-trisphosphate 3-kinase C (ITPKC) (rs28493229) and caspase-3 (CASP3) (rs113420705; formerly rs72689236) are associated with susceptibility to Kawasaki's disease (KD). To evaluate the involvement of these 2 SNPs in the risk for intravenous immunoglobulin (IVIG) unresponsiveness, we investigated 204 Japanese KD patients who received a single IVIG dose of 2 g kg(-1) (n=70) or 1 g kg(-1) daily for 2 days (n=134). The susceptibility allele of both SNPs showed a trend of overrepresentation in IVIG non-responders and, in combined analysis of these SNPs, patients with at least 1 susceptible allele at both loci had a higher risk for IVIG unresponsiveness (P=0.0014). In 335 prospectively collected KD patients who were treated with IVIG (2 g kg(-1)), this 2-locus model showed a more significant association with resistance to initial and additional IVIG (P=0.011) compared with individual SNPs. We observed a significant association when all KD patients with coronary artery lesions were analyzed with the 2-locus model (P=0.0031). Our findings strongly suggest the existence of genetic factors affecting patients' responses to treatment and the risk for cardiac complications, and provide clues toward understanding the pathophysiology of KD inflammation.

Altered expression of dermokine in skin disorders.

BACKGROUND: Although dermokine-ß, a glycoprotein expressed in epithelial cells, does not have significant homology to other proteins, its carboxyl-terminal domain shares a high pI value with many cytokines, suggesting similar functions. OBJECTIVE: To better understand the biology of dermokine, we here determined its localization under pathological conditions and examined factors that regulate its expression. METHODS: We generated an anti-human dermokine-ß/γ monoclonal antibody cross-reacting with the mouse protein. Using this antibody, immunohistological staining and Western blotting of dermokine-ß/γ were performed with various tissue samples. RESULTS: Although human dermokine-ß/γ was expressed in almost all granular layers, upper spinous layers of the skin were also stained with anti-dermokine-ß/γ antibody in inflammatory skin disorders. Dermokine-ß/γ was expressed in keratoacanthoma and a part of well-differentiated squamous cell carcinoma (SCC). However, dermokine-ß/γ was not detected in poorly differentiated SCC or tumours derived from non-keratinocytes. In mice, dermokine-ß/γ-expressed keratinocytes were increased in models of contact hypersensitivity, ultraviolet-irradiated skin injury and wound healing. Consistent with expanded distribution in inflammatory skin diseases, proinflammatory cytokines such as interleukin-1ß, interleukin-12, and tumour necrosis factor-α augmented dermokine-ß/γ expression in cultured human keratinocytes. In contrast, growth factors including epidermal growth factor, insulin-like growth factor-I, keratinocyte growth factor and transforming growth factor-α significantly reduced dermokine expression. CONCLUSION: These results provide novel insights into the physiological and pathological significance of dermokine in the epidermis.

A gene encoding a transmembrane protein is mutated in patients with diabetes mellitus and optic atrophy (Wolfram syndrome).

Wolfram syndrome (WFS; OMIM 222300) is an autosomal recessive neurodegenerative disorder defined by young-onset non-immune insulin-dependent diabetes mellitus and progressive optic atrophy. Linkage to markers on chromosome 4p was confirmed in five families. On the basis of meiotic recombinants and disease-associated haplotypes, the WFS gene was localized to a BAC/P1 contig of less than 250 kb. Mutations in a novel gene (WFS1) encoding a putative transmembrane protein were found in all affected individuals in six WFS families, and these mutations were associated with the disease phenotype. WFS1 appears to function in survival of islet beta-cells and neurons.

Role of tyrosine phosphorylation of HS1 in B cell antigen receptor-mediated apoptosis.

The 75-kD HS1 protein is highly tyrosine-phosphorylated during B cell antigen receptor (BCR)-mediated signaling. Owing to low expression of HS1, WEHI-231-derived M1 cells, unlike the parental cells, are insensitive to BCR-mediated apoptosis. Here, we show that BCR-associated tyrosine kinases Lyn and Syk synergistically phosphorylate HS1, and that Tyr-378 and Tyr-397 of HS1 are the critical residues for its BCR-induced phosphorylation. In addition, unlike wild-type HS1, a mutant HS1 carrying the mutations Phe-378 and Phe-397 was unable to render M1 cells sensitive to apoptosis. Wild-type HS1, but not the mutant, localized to the nucleus under the synergy of Lyn and Syk. Thus, tyrosine phosphorylation of HS1 is required for BCR-induced apoptosis and nuclear translocation of HS1 may be a prerequisite for B cell apoptosis.

Microbes Culture System Using Cellulose Tubes and Photocatalyst-Coated Glass Balls.

Currently, microbes are utilized in many fields, such as medicine, food and environment etc. For more application of microbes, we need a new culture system, which can culture target microbes in large quantities at low cost. Thereupon, we propose a culture system using cellulose tubes. Target microbes are encapsulated inside the cellulose tubes, where they acquire nutrients and oxygen through nano pores of the tubes and are protected from competitive microbes even in open environment. To further increase the amount of oxygen and nutritions available for the target microbes, we propose photocatalyst-coated glass balls (PCGB) to sterilize competing microbes outside the tubes. We experimentally verified the effectiveness of the proposed culture system by culturing Coryne gultamicum as the target microbes.

Japanese collaborative study to assess inter-laboratory variation in factor VII activity assays.

BACKGROUND: The clinical phenotype manifest by patients with factor VII (FVII) deficiency correlates poorly with that predicted by laboratory tests. Despite its importance, there are no data on the variability of inter-laboratory determinations of low to very low plasma FVII activity (FVII:C). METHODS: We distributed three FVII-deficient plasma samples, prepared by immunoaffinity chromatography, to 58 laboratories in Japan. All samples were assayed using standardized reference plasma as a calibrator. Recombinant thromboplastin was also supplied as a common reagent. RESULTS: In the case of sample A, which had a very low FVII:C, the use of standardized reference plasma and thromboplastin, lowered the variability of inter-laboratory measurements, when compared with the variability observed when samples were assayed using the respective laboratory's routine method. CONCLUSIONS: The data obtained indicated that results for samples with a very low FVII:C were greatly influenced by the number of plasma dilutions used in constructing a standard activity curve, and also by the type of calibrator and thromboplastin. Such variability was not seen for samples with moderate FVII:C. We conclude that it is necessary to develop a more sensitive and accurate FVII:C measurement system for the diagnosis and treatment of FVII deficiency.

Decreased activation of carcinogens in the liver of carcinogen-resistant rats.

We have developed a new strain of rats (resistant rat) which exhibit resistance against the carcinogenic action of several carcinogenic compounds. In the present study, we compared the ability of resistant rat liver to activate 3'-methyl-4-dimethylaminoazobenzene and 2-acetylaminofluorene to highly reactive metabolites, which can covalently bind to DNA, with that of carcinogen-sensitive parent strain rats (sensitive rat), using in vitro DNA binding assay. The covalent binding of these carcinogens to calf thymus DNA, catalyzed by the hepatic 9000 x g supernatant fraction from resistant rats pretreated with 3-methylcholanthrene, was about one-half of that catalyzed by the 9000 x g supernatant from sensitive rats which had received the same treatment. These results suggest that the ability of resistant rat liver to activate the carcinogens decreases compared to sensitive rat liver. Further experiments revealed that this decreased activation of the carcinogens in resistant rat livers is due to a low inducibility of 3-methylcholanthrene-inducible forms of cytochrome P-450 mRNA. The hepatic cytosolic Ah receptor concentrations in resistant rats were shown to be significantly lower than those of sensitive rats. Scatchard plot analysis demonstrated, however, that there is no significant difference between the affinity of Ah receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin in these two strains. These data implicate that the hepatic Ah receptor level may be an important factor in determining carcinogen sensitivity in rats.

Genetic resistance to chemical carcinogen-induced preneoplastic hepatic lesions in DRH strain rats.

DRH strain rats were established by inbreeding a closed colony of Donryu rats continuously fed the chemical hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzene for over 10 years. They are highly resistant to chemical induction of liver cancer and preneoplastic lesions. We studied the genetic basis of DRH resistance to preneoplastic lesions by analyzing 108 (F344 x DRH)F2 male rats fed 3'-methyl-4-dimethylaminoazobenzene for 7 weeks. Five parameters of preneoplastic liver lesions were selected for quantitative analysis: (a) number of glutathione S-transferase placental form-positive foci per unit area of liver section; (b) percentage area occupied by the foci; (c) average size of foci; (d) glutathione S-transferase placental form mRNA level; and (e) gamma-glutamyltranspeptidase mRNA level. Furthermore, O6-methylguanine DNA methyltransferase and mannose 6-phosphatase/insulin-like growth factor 2 receptor mRNA levels were quantified. Composite interval mapping analysis showed that there were two remarkably significant clusters of quantitative trait loci affecting preneoplastic liver lesions on chromosomes 1 and 4. These clusters were designated collectively as Drh1 and Drh2, respectively. The functions of the recessive DRH allele of Drh1 and the semidominant DRH allele of Drh2 were to suppress the phenotypes of precancerous lesions. Each cluster showed two to three subpeaks in linkage likelihood plots, suggesting the presence of several closely linked quantitative trait loci affecting preneoplastic lesions. Possible candidate genes at each locus will be discussed. Expression of O6-methylguanine DNA methyltransferase and mannose 6-phosphatase/insulin-like growth factor 2 receptor did not affect DRH resistance to hepatocarcinogenesis, although they were polymorphic between DRH and F344 rats.

A simple method for micropatterning nanofibrous hydrogel film.

This paper propose a new fabrication process for micropatterning a nanofibrous thin film made of bacterial cellulose(BC). BC is a hydrogel produced by specific bacteria and composed of pure cellulosic nanoflbers exhibiting 3D network structure. Such nanofibrous structure is found to be appropriate for adhesion of anchorage-dependent cells. Furthermore, BC shows high biocompatibility and mechanical toughness. Thus, the microfabrication technique for BC broadens potentials for applications. In this study, we report a new method for micropatterning BC film with feature resolution comparable with photolithography technology.

Micro-tube mass production device for microbial culture.

This paper describes mass production system of micro-tubes for microbial culture in an open environment. Microbes are used in many fields, such as food, medicine, environmental and energy. We proposed a microbe culture system using hydrogel micro-tubes, which can protect the target microbes inside from competitive microbes outside of the tubes while allow oxygen and nutrition to diffuse through. The hydrogel micro-tubes can be produced by a microfluidic device, which can precisely control the flow and therefore, the tube geometry. For practical applications of the micro-tube-based microbial culture, one of the biggest challenges is the scale-up of the micro-tube-based culture system, or mass production of the tubes. We developed a fluidic system that can produce multiple micro-tubes in parallel. We characterized the mass-produced micro channels and verified the effectiveness of the system.

Daidzein inhibits insulin- or insulin-like growth factor-1-mediated signaling in cell cycle progression of Swiss 3T3 cells.

An isoflavone compound, daidzein, inhibits the cell proliferation of Swiss 3T3 cells. Analysis of entry in S phase of Swiss 3T3 cells reveals that daidzein blocked cell cycle G1 phase progression 4.6 h after stimulation by bombesin plus insulin. After removal of daidzein, insulin or insulin-like growth factors (IGFs) reinitiate cell cycle progression of daidzein-blocked cells without further addition of bombesin. The order in the mitogenic action of insulin or IGFs is as follows: IGF-1 (5 ng/ml) >> IGF-2 (0.5 microgram/ml) congruent to insulin (1 microgram/ml). Studies in vivo of protein kinase activation by mitogenic stimulation reveal that the treatment with daidzein decreased the activation of a MAP2 phosphorylating protein kinase (MAP2 kinase). In vitro kinase assays showed that daidzein inhibits casein kinase II activity, but does not inhibit MAP2 kinase activity. Activation of casein kinase II by polylysine augments the activity of MAP2 kinase in digitonin-permeabilized 3T3 cells. These results suggest that daidzein blocked G1 phase cell cycle progression of Swiss 3T3 by inhibiting the activity of casein kinase II which is required for the commitment of mitogenic signal by insulin or IGF-1 in G1 phase.

Rapid determination of internal volumes of membrane vesicles with electron spin resonance-stopped flow technique.

We have developed an electron spin resonance (ESR)-stopped flow technique and employed it for the simple and rapid determination of internal volumes of biomembrane vesicles and liposomes. A vesicle suspension containing a neutral and membrane-permeable spin label, 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl (TEMPONE), was mixed in the stopped-flow apparatus with an isotonic solution of relatively impermeable line broadening agents, potassium tris(oxalato)chromate(III) or potassium ferricyanide, and an ESR spectrum was recorded. From the relative intensity of the sharp triplet signal due to TEMPONE in the aqueous space within vesicles, the determination of the internal aqueous volume was straightforward. Using this technique, it is possible to measure intravesicular volumes in 0.1 s. The internal volume of sonicated phospholipid vesicles was approximately 0.3 microliter/mg lipid. The light fraction of sarcoplasmic reticulum membrane vesicles isolated from rabbit skeletal muscle was estimated to have an internal volume of 2.2-2.6 microliter/mg protein in its resting state. Activation of Ca2+ pumps in the membrane upon addition of ATP and Ca2+ ions decreased the internal volume by about 10%. This finding supports the hypothesis that the Ca2+ pump is electrogenic and that the efflux of potassium ions compensates for the influx of positive charges. The present technique is widely applicable to the simple and rapid determination of the internal volumes of membrane vesicles.

Higher induction of heat shock protein 72 by heat stress in cisplatin-resistant than in cisplatin-sensitive cancer cells.

Induction of the heat shock proteins (HSPs) is involved in the increased resistance to cancer therapies such as chemotherapy and hyperthermia. We used two human ovarian cancer cell lines; a cisplatin (CDDP)-sensitive line A2780 and its CDDP-resistant derivative, A2780CP. The concentration of intracellular glutathione (GSH) is higher (2.7-fold increase) in A2780CP cells than in A2780 cells. A mild treatment with a heat stress (42 degrees C for 30 min) induced synthesis of both the heat shock protein 72 (Hsp72) mRNA and the HSP72 protein in A2780CP cells, but not in A2780 cells. In contrast, a severe heat stress (45 degrees C for 30 min) increased synthesis of the HSP72 protein in the two cell lines. The induced level of the HSP72 protein by the severe treatment was higher in A2780CP than in A2780 cells. The gel mobility shift assay showed that DNA binding activities of the heat shock factor (HSF) in the two cell lines were induced similarly and significantly by the mild heat stress. Immunocytochemistry using an anti HSF1 antibody also indicated that mild heat stress activated the HSF1 translocation from the cytosol to the nucleus similarly in the both cell lines. Pretreatment of CDDP-sensitive A2780 cells with N-acetyl-L-cysteine, a precursor of GSH, effectively enhanced induction of the Hsp72 mRNA by the mild heat stress. The present findings demonstrate that induction of the Hsp72 mRNA by the mild heat stress was more extensive in CDDP-resistant A2780CP cells. It is likely that the higher GSH concentration in A2780CP cells plays an important role in promoting Hsp72 gene expression induced by the mild heat stress probably through processes downstream of activation of HSF-DNA binding.

Assembly of the alpha-subunit of Torpedo californica Na+/K(+)-ATPase with its pre-existing beta-subunit in Xenopus oocytes.

The alpha- and beta-subunits of Torpedo californica Na+/K(+)-ATPase were expressed in turn in single oocytes by alternately microinjecting the specific mRNAs for the alpha- and beta-subunits. The mRNA first injected was degraded prior to the injection of the second mRNA by injecting the antisense oligonucleotide specific for the first mRNA. The pre-existing beta-subunit, which had been synthesized by injecting mRNA for the beta-subunit, could assemble with the alpha-subunit expressed later in the single oocytes and the resulting alpha beta complex acquired both ouabain-binding and Na+/K(+)-ATPase activities. On the other hand, formation of the alpha beta complex was not detected when the alpha-subunit was expressed first, followed by the beta-subunit. These data suggest that the beta-subunit acts as a receptor or a stabilizer for the alpha-subunit upon the biogenesis of Na+/K(+)-ATPase.

An inhibitor of inositol-phospholipid-specific phospholipase C.

Q12713 substance, a cyclic peptide from Actinomadura spp., strongly inhibited the enzyme activity of phosphatidylinositol-4,5-bisphosphate-specific phospholipase C (PIP2-PLC) but scarcely inhibited other phospholipases. Kinetic analysis demonstrated that the inhibition of PIP2-PLC activity was competitive with respect to phosphatidylinositol 4,5-bisphosphate. Calcium and magnesium ions had no significant effect on the inhibitory activity. On the contrary, potassium or rubidium ion was essential for the inhibitory activity. Furthermore, NaF and AlCl3-stimulated increase of phosphoinositides was decreased by Q12713 in the cultured 3T3 cells.

Purification and characterization of myosin light-chain kinase from porcine myometrium and its phosphorylation and modulation by cyclic AMP-dependent protein kinase.

Myosin light-chain kinase was purified from porcine myometrium to apparent homogeneity at about 262-fold with an Mr of 130 000 as determined by SDS-polyacrylamide gel electrophoresis and a sedimentation coefficient of 4.5 S. The approximate content of the soluble myosin light-chain kinase was estimated to be about 0.85 microM. The purified enzyme exhibited strict substrate specificity only for 20-kDa myosin light chain and Ka values of 0.6 nM and 0.3 microM for calmodulin and Ca2+, respectively. The enzyme was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase, which resulted in a decrease in the affinity for calmodulin of 4-7-fold without effect on the Vmax. The maximal amount of phosphate incorporated into the enzyme was 0.5-0.8 and 1.0-1.4 mol per mol of the enzyme in the presence and absence of Ca2+ and calmodulin, respectively. In the presence of a subsaturating concentration of calmodulin, the enzyme showed a lower sensitivity for Ca2+ by phosphorylation.

Concentration-dependent differential effects of N-acetyl-L-cysteine on the expression of HSP70 and metallothionein genes induced by cadmium in human amniotic cells.

Cadmium induces the expression of the 70 kDa heat shock protein (HSP70) and metallothionein (MT), both of which are considered to be associated with intracellular glutathione (GSH) metabolism in the cellular protection mechanism against cadmium-induced cellular injury. We determined the effects of N-acetyl-L-cysteine (NAC), which increases the intracellular GSH levels, on the induction of HSP70 and MT gene expression in a cultured cell line of human amniotic cells (WISH) exposed to CdCl2. The mRNA level of MT-II, a major isoform of MT genes, was more prominently increased than that of HSP70 when WISH cells were exposed to CdCl2 (5-15 microM, for 6 h). The treatment of WISH cells with 1.5 and 30 mM NAC for 2 h increased the intracellular GSH levels by 1.4- and 3.1-fold, respectively. Pretreatment of cells with 30 mM NAC significantly reduced both HSP70 and MT-II mRNA levels in the cells exposed to 50 microM CdCl2. This concentration of NAC also efficiently suppressed the cadmium-induced lethality. On the contrary, pretreatment with 1.5 mM NAC suppressed only the induction of HSP70 gene expression in the 50 microM CdCl2-treated cells, and did not inhibit the metal toxicity. However, this low concentration of NAC efficiently suppressed lipid peroxidation which was increased by 50 microM CdCl2. Furthermore, this low concentration of NAC also decreased the CdCl2-induced gene expression of HSP32 which represents a general response to oxidative stress. Taken together, NAC seems to have at least two concentration-dependent functions in WISH cells exposed to CdCl2; the low concentration of NAC can suppress the induction of HSP70 gene expression as well as the increase of lipid peroxidation via an antioxidant pathway, while the high concentration of NAC can suppress the induction of MT-II mRNA as well as cadmium-induced cell death. Our present data suggest that changes in intracellular redox status, as reflected by GSH concentration, have more important effects on the induction of HSP70 mRNA rather than that of MT-II mRNA in human amniotic cells exposed to cadmium.

A chromo box gene from carrot (Daucus carota l.): its cDNA structure and expression during somatic and zygotic embryogenesis.

A cDNA clone, designated DcDB1, was isolated from a cDNA library prepared from embryogenic cell clusters of carrot (Daucus carota L.) and characterized. The cDNA (1416 bp) encoded for a protein of 392 amino acid residues that contained a conserved chromo domain. The chromo domain is a 37 aa region found in both the Polycomo gene product, which is a repressor of homeotic genes, and a heterochromatin protein 1 of Drosophila. This domain is postulated to function in the binding of proteins to chromatin. Genomic blot hybridization experiments suggested that the number of DcCB1 genes in the carrot genome is low. The level of DcCB1 mRNAs was high in somatic embryos at globular and heart-shaped stages but low in torpedo-shaped somatic embryos. The level of DcCB1 transcripts decreased during the formation of seeds. The existence of both homeo and chromo box genes in plants suggests that regulatory mechanisms of developmental genes in plants may resemble those in Drosophila.

Induction of heat shock 70 mRNA by cadmium is mediated by glutathione suppressive and non-suppressive triggers.

The induction mechanism of heat shock 70 (Hsp70) gene by cadmium was investigated. In human amniotic WISH cells, Hsp 70 was induced by cadmium in a dose- and time-dependent manner. Cadmium-induced Hsp70 mRNA levels were enhanced 3- to 4-fold after depletion of intracellular glutathione (GSH) by either diethylmaleate or buthionine sulfoximine. Under these conditions, hydrogen peroxide might increase in the absence of substrate for glutathione peroxidase. We found that exogenous hydrogen peroxide alone induced Hsp70 which was further enhanced significantly after GSH-depletion by diethylmaleate. On the other hand, treatment of cells by diethyldithiocarbamate, an inhibitor of superoxide dismutase, induced Hsp70 2-fold over the level of control. This induction was further stimulated by cadmium even in the presence of GSH. Furthermore, a 4-fold increase of intracellular GSH by the treatment of cells with glutathione isopropyl ester did not diminish the cadmium-induced Hsp70. Gel mobility shift assays of nuclear extracts, from these differently treated cells, with oligonucleotide containing a promoter region of Hsp70 gene revealed that the levels of Hsp70 mRNA observed in the present study corresponded to the changes of transcription. These results imply that the induction of Hsp70 mRNA by cadmium is mediated at least partly via reactive oxygen species and attenuated by cellular GSH and that some part of cadmium-induced Hsp70 can not be eliminated by GSH, suggesting that multiple signals are functioning for this induction.