Functional vanilloid receptors in cultured normal human epidermal keratinocytes

Vanilloid receptor subtype 1, VR1, is an ion channel that serves as a polymodal detector of pain-producing chemicals such as capsaicin and protons in primary afferent neurons. Here we showed that both capsaicin and acidification produced elevations in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in cultured human epidermal keratinocytes. The capsaicin- and acidification-evoked increases in [Ca(2+)](i) were inhibited by capsazepine, an antagonist to VR1. VR1-like immunoreactivity was observed in the cells. These findings suggest that functional VR1-like protein is present and functions as a sensor against noxious chemical stimuli, such as capsaicin or acidification, in epidermal keratinocytes.     

Allowable level of lifetime cadmium intake calculated from the individuals in the Jinzu River basin of Japan

We investigated a dose-response relationship between renal dysfunction and liftime cadmium intake in individual subjects using logistic regression analysis and calculated the allowable level of lifetime cadmium intake among the inhabitants of the Jinzu River basin. From the participants of 1967 and 1968 health examinations, target subjects in whose hamlet the cadmium concentration in rice was known and whose history of residence was also known were selected. Cadmium concentrations in rice from data analyzed by the Toyama Prefecture from 1971 to 1976 were used. The urinary examination was done by semiquantitative determination of protein and glucose. All odds ratios for lifetime cadmium intake obtained from logistic regression analysis were more than 1 in both males and females who had resided in their current hamlet since birth with and without subjects who moved from nonpolluted areas and with or without the control group. The allowable levels of lifetime cadmium intake were calculated by substituting the abnormality rates of urinary findings of the controls 40, 50, 60, and 70 yr old into the logistic regression formula. The allowable levels of lifetime cadmium intake were less than 1.58 g for both sexes and each age group using proteinuria with glucosuria measurements.     

Evaluation of a mass screening program for lysinuric protein intolerance in the northern part of Japan

Lysinuric protein intolerance (LPI:MIM 222700) is an autosomal recessive disease characterized by defective transport of the dibasic amino acids. We recently reported a local cluster of LPI in the northern part of Japan (Koizumi et al., 2000). Mutational analysis of the LPI patients in this local cluster revealed they were exclusively homozygous for the R410X mutation. The effectiveness of early intervention with citrulline therapy (200 mg/kg per day) and protein restriction (1.5 g/kg per day) was confirmed in these patients. Mass screening was conducted in 4,568 newborn babies between 1999 and 2002, which was estimated to cover 100% of almost all newborns delivered in the screened area. Forty heterozygous newborns were found (0.88%), leading to an estimated incidence of LPI of 1:51,984. The number of people that required screening to detect one case was 51,984, and the cost for mass screening was 30 cents/person (a total of dollars 15,600). This is comparable to, or even less than, the cost of currently screened diseases in Japan. Therefore, we conclude that a mass screening program for LPI can be introduced effectively and economically into an area where an LPI cluster is located as the result of a founder mutation.     

Beta 3-adrenergic receptor is involved in feeding regulation in chicks

We examined whether the brain beta 3-adrenergic receptor (B3-AR) is involved in the feeding regulation of chicks. Intracerebroventricular (ICV) injection of BRL37344, a B3-AR agonist, reduced food intake of chicks under ad libitum, but not fasting, feeding conditions. The ICV injection of BRL37344 did not affect chick posture or locomotion activity suggesting that BRL37344 inhibited feeding without induction of sleep-like behavior as caused by norepinephrine. Furthermore, the rectal temperature increased following the ICV injection of BRL37344. Intraperitoneal administration of BRL37344 did not reduce food intake under ad libitum feeding condition. The present study demonstrated that the brain B3-AR is involved in the inhibition of feeding in chicks. We also suggested that activation of the brain affects the energy metabolism in chicks.     

Localization of sepiapterin reductase in pigment cells of Oryzias latipes

Body colors of poikilothermal vertebrates are derived from three distinct types of pigment cells, melanophores, erythro/xanthophores and irido/leucophores. It is well known that melanin in melanophores is synthesized by tyrosinase within a specific organelle termed the melanosome. Although sepiapterin reductase (SPR) is an important enzyme involved in metabolizing biopterin and sepiapterin (a conspicuous pteridine as a coloring pigment in xanthophores) the distribution of SPR has not been shown in pigment cells. An antibody raised in rabbits against rat SPR was used to demonstrate the presence of SPR in pigment cells of Oryzias latipes. This study, which used immunohistochemistry with fluorescence or peroxidase/diaminobenzidine as markers, revealed that SPR could be detected readily in xanthophores, but only faintly in melanophores. These results suggest that sepiapterin is metabolized within xanthophores. Moreover, these experiments show that a protein sharing immunological cross-reactivity with rat SPR is located in teleost O. latipes xanthophores, which is significant considering the relationship of pteridine metabolism between poikilothermal vertebrates and mammals. Further progress in investigations of the roles of pteridines in vertebrates will be promoted by using these fish which can be bred in mass rather easily in the laboratory.     

Molecular structure of bacterial endotoxin (Escherichia coli Re lipopolysaccharide): implications for formation of a novel heterogeneous lattice structure

Analyses of crystals of Escherichia coli Re lipopolysaccharide (LPS) formed after storage in 1% triethylamine indicate that the LPS molecules are assembled to form a monolayered structure consisting of a novel heterogeneous lattice structure, the greater part of which is occupied by one kind of lattice (lattice I), corresponding to the acyl chain portion of lipid A, and the remainder is occupied by the other kind of lattice (lattice II), corresponding to the 3-deoxy-Dmanno-octulosonic acid (dOclA) dimer and the N-acetylglucosamine disaccharide of lipid A. X-ray diffraction reveals that the type of cell is monoclinic (a = 5.53 A, b = 27.2 A, c = 6.47 A, alpha = 90 degrees, beta = 125.8 degrees, gamma = 90 degrees ). Atomic force microscopy shows that crystals consist of multiple layers; the thickness of a layer corresponds to the b-axis value, and two types of surface topographies are visualized. One, regarded as the view onto the acyl chain ends, is two-dimensional arrays of oval bodies that constitute the lattice, with the lattice constants corresponding to the a- and c-axes and the angle of beta (lattice I). The other, regarded as the view onto the dOclA dimers, is two-dimensional arrays of dromedary-back-like bodies that constitute the lattice with axes of 9.0 and 10.7 A and the angle of 65 degrees formed by both axes (lattice II). Based on these results, we present the molecular model of E. coli Re LPS.     

Correlated gene expression between beta-1,4-galactosyltransferase V and N-acetylglucosaminyltransferase V in human cancer cell lines

Since our previous study showed that the gene expression level of beta-1,4-galactosyltransferase (beta-1,4-GalT) V is only increased in mouse NIH3T3 transformant and that beta-1,4-GalT V preferentially galactosylates the GlcNAcbeta1 --> 6Man branch of oligosaccharides [Shirane et al. (1999) Biochem. Biophys. Res. Commun. 265, 434-438], whether its gene expression is correlated with malignant transformation was investigated. Northern blot analysis of beta-1, 4-GalTs I, II, III, IV, V, and VI and N-acetylglucosaminyltransferase (GlcNAcT)V in human cancer cell lines showed that the gene expression levels of beta-1,4-GalT V but not other beta-1,4-GalTs are strongly correlated with those of GlcNAcT V whose activity was shown to increase by malignant transformation. These results indicate that beta-1,4-GalT V is involved in the galactosylation of highly branched oligosaccharides characteristic of malignantly transformed cells.     

Rapid and simple detection of PCR product DNA: a comparison between Southern hybridization and fluorescence polarization analysis

The essential aim of this study was to compare two different methods, Southern hybridization and fluorescence polarization (FP) assay. They both detect specific hybridization and were examined using common asymmetric PCR products and probes. FP assay clearly showed the hybridization of probe DNAs with the asymmetric PCR products of their target genes. Southern blot patterns presented excellent consistency with the results of FP assay. In both methods, two types of Shiga toxin (vero toxin) genes held in enterohaemorrhagic Escherichia coli (EHEC) were used as target genes. For detection of the two genes, stx1 and stx2, two respective DNA probes were synthesized. Both in FP assay and in Southern hybridization, the probe for stx1 hybridized only with the product of stx1 and vice versa. The results of the DNA detection using different methods were completely in agreement. Moreover, FP assay makes it possible to detect the hybridization rapidly. In our high NaCl concentration condition, hybridization between the probes and the asymmetric PCR products could be monitored within about 15min.     

Antinociceptive substances from Incarvillea delavayi

Antinociceptive activities of an Incarvillea delavayi extract, as well as its constituents, 8-epideoxyloganic acid and delavayine A, were evaluated in the acetic acid induced writhing test in mice. An oral administration of the delavayi extract weakly decreased the number of writhings and stretchings in this test, in a dose-dependent manner. Furthermore, orally administered 8-epideoxyloganic acid showed weak antinociceptive activity, whereas administration by subcutaneous injection did not. However, subcutaneous injection of delavayine A, a novel monoterpene alkaloid, showed a more significant level of antinociceptive activity.     

Activation and Functional Analysis of Janus Kinase 2 in BA/F3 Cells Using the Coumermycin/Gyrase B System

Janus kinase 2 (Jak2) protein tyrosine kinase plays an important role in interleukin-3– or granulocyte–macrophage colony-stimulating factor–mediated signal transduction pathways leading to cell proliferation, activation of early response genes, and inhibition of apoptosis. However, it is unclear whether Jak2 can activate these signaling pathways directly without the involvement of cytokine receptor phosphorylation. To investigate the specific role of Jak2 in the regulation of signal transduction pathways, we generated gyrase B (GyrB)–Jak2 fusion proteins, dimerized through the addition of coumermycin. Coumermycin induced autophosphorylation of GyrB–Jak2 fusion proteins, thus bypassing receptor activation. Using different types of chimeric Jak2 molecules, we observed that although the kinase domain of Jak2 is sufficient for autophosphorylation, the N-terminal regions are essential for the phosphorylation of Stat5 and for the induction of short-term cell proliferation. Moreover, coumermycin-induced activation of Jak2 can also lead to increased levels of c-myc and CIS mRNAs in BA/F3 cells stably expressing the Jak2 fusion protein with the intact N-terminal region. Conversely, activation of the chimeric Jak2 induced neither phosphorylation of Shc or SHP-2 nor activation of the c-fos promoter. Here, we showed that the GyrB–Jak2 system can serve as an excellent model to dissect signals of receptor-dependent and -independent events. We also obtained evidence indicating a role for the N-terminal region of Jak2 in downstream signaling events.