Salivary cystatins influence ingestion of capsaicin-containing diets in the rat

Dietary capsaicin consumed by rats over several days induces cystatin-like substances in submandibular saliva. Yet the physiological role of these salivary proteins has not been thoroughly investigated. Salivary cystatins in the rat submandibular glands are known to be induced by chronic treatment with the sympathetic beta-agonist, isoproterenol. In the present study, the possible roles of the salivary proteins on food intake were examined by comparing consumption of a capsaicin-adulterated (0.05%) diet in rats with and without isoproterenol pretreatment (0.1 and 5.0 mg/kg, 5 days). Electrophoretic analysis performed prior to feeding trials revealed that the group pretreated with 5 mg/kg isoproterenol had large amounts of cystatin in the saliva compared with the group pretreated with 0.1 mg/kg isoproterenol and control group. The group treated with 5 mg/kg isoproterenol showed greater consumption of the capsaicin-adulterated diet than the other groups until the 3rd day of trials. Bilateral removal of the submandibular and sublingual glands neutralized the effects of isoproterenol. Induction of salivary cystatins by isoproterenol treatment was not mimicked by systemic and intragastric administration of capsaicin. These results suggest that cystatins are included in the salivary proteins induced by capsaicin and that they contribute to enhanced ingestion of the capsaicin diet. Induction of salivary cystatins may be triggered by irritation of the oral mucosa by capsaicin.     

Possible factors responsible for the toxicity of Cochlodinium polykrikoides,a red tide phytoplankton

Cochlodinium polykrikoides, a harmful red tide dinoflagellate, is highly toxic to fish, but the toxic mechanism is still unknown. Recent study has suggested that C. polykrikoides generates reactive oxygen species (ROS) such as superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)), and the ROS-mediated ichthyotoxicity has been proposed. In this study, we found that the levels of O(2)(-) and H(2)O(2) detected in C. polykrikoides were trace levels as compared with those of Chattonella marina which is well-known to produce ROS. Furthermore, no significant increase in O(2)(-) generation by C. polykrikoides was observed in the presence of lectins such as concanavalin A (Con A) and wheat germ agglutinin (WGA) or fish mucus prepared from skin and gill of yellowtail, whereas C. marina generated increased level of O(2)(-) responding to these stimuli. Interestingly, the cell-free aqueous extract prepared from C. polykrikoides showed toxic effect on the HeLa cells, but the extract of C. marina had no significant effect. Furthermore, gradual accumulation of polysaccharides in the medium was observed during the growth of C. polykrikoides, and the medium gradually became viscous, but no such changes were observed in the medium of C. marina. These results suggest that multiple factors may be responsible for the toxic mechanism of C. polykrikoides.     

Dual abnormal effects of mutant MITF encoded by Mi(wh) allele on mouse mast cells: decreased but recognizable transactivation and inhibition of transactivation

Structural localization of a peptide region, KRQPRNPKTDKLVNE, in the catalytic subunit of (Na(+) + K(+))-ATPase was investigated using a specific antibody directed against this peptide in cultured African green monkey kidney CV-1 cells. Immunofluorescence staining of frozen cell sections shows that an anti-KRQPRNPKTDKLVNE antibody (SSA95) interacts with its antigenic site and binds to the extracellular side of the cell membrane. Indirect immunofluorescence and flow cytometric analyses confirmed the presence of this epitope on intact cell surfaces. These results suggest that the KRQPRNPKTDKLVNE region of the (Na(+) + K(+))-ATPase is expressed on the cellular membrane surface.     

TAK1-TAB1 fusion protein: a novel constitutively active mitogen-activated protein kinase kinase kinase that stimulates AP-1 and NF-kappaB signaling pathways

TAK1 mitogen-activated protein kinase kinase kinase (MAP3K) is activated by its specific activator, TAK1-binding protein 1 (TAB1). A constitutively active TAK1 mutant has not yet been generated due to the indispensable requirement of TAB1 for TAK1 kinase activity. In this study, we generated a novel constitutively active TAK1 by fusing its kinase domain to the minimal TAK1-activation domain of TAB1. Co-immunoprecipitation assay demonstrated that these domains interacted intra-molecularly. The TAK1-TAB1 fusion protein showed a significant MAP3K activity in vitro and activated c-Jun N-terminal kinase/p38 MAPKs and IkappaB kinase in vivo, which was followed by increased production of interleukin-6. These results indicate that the fusion protein is useful for characterizing the physiological roles of the TAK1-TAB1 complex.     

Augmentation of receptor-mediated adenylyl cyclase activity by Gi-coupled prostaglandin receptor subtype EP3 in a Gbetagamma subunit-independent manner.

We previously demonstrated that the mouse EP3beta receptor and its C-terminal tail-truncated receptor (abbreviated T-335) expressed in Chinese hamster ovary cells showed agonist-dependent and fully constitutive Gi activity in forskolin-stimulated cAMP accumulation, respectively. Here we examined the effect of the EP3beta receptor or T-335 receptor on adenylyl cyclase activity stimulated by the Gs-coupled EP2 subtype receptor in COS-7 cells. As a result, sulprostone, a selective EP3 agonist, dose dependently augmented butaprost-stimulated adenylyl cyclase activity in EP3beta receptor- or T-335 receptor-expressing COS-7 cells. However, such adenylyl cyclase augmentation was not attenuated by either pertussis toxin treatment or expression of the PH domain of rat betaARK1, which serves as a scavenger of Gbetagamma subunits, but was partially attenuated by treatment with either 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester, an intracellular Ca(2+) chelator, or W-7, a calmodulin inhibitor. These findings suggest that the C-terminal tail of the EP3beta receptor is not essentially involved in activation of EP2 receptor-stimulated adenylyl cyclase in a Ca(2+)/calmodulin-dependent but Gbetagamma subunit-independent manner.     

Phosphorylation-dependent activation of TAK1 mitogen-activated protein kinase kinase kinase by TAB1

TAK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that is involved in the c-Jun N-terminal kinase/p38 MAPKs and NF-kappaB signaling pathways. Here, we characterized the molecular mechanisms of TAK1 activation by its specific activator TAB1. Autophosphorylation of two threonine residues in the activation loop of TAK1 was necessary for TAK1 activation. Association with TAK1 and induction of TAK1 autophosphorylation required the C-terminal 24 amino acids of TAB1, but full TAK1 activation required additional C-terminal Ser/Thr rich sequences. These results demonstrated that the association between the kinase domain of TAK1 and the C-terminal TAB1 triggered the phosphorylation-dependent TAK1 activation mechanism.     

Isolation and characterization of three alpha-glucosidases from the Japanese quail

We have defined one type of acid alpha-glucosidase and two types of neutral alpha-glucosidases from quail skeletal muscle on the basis of differences in the elution patterns on a DEAE-cellulose column. The appearance of the two neutral alpha-glucosidase isoenzymes was age-dependent. A decrease in acid alpha-glucosidase activity was demonstrated in Japanese quails with glycogenosis type II. The characteristics of these three alpha-glucosidase isoenzymes are described.     

A simple PCR-RFLP method for identification and differentiation of 11 Malassezia species

A PCR-RFLP method targeted toward 26S rDNA and with 2 restriction enzymes, CfoI and BstF51, was developed to identify 11 Malassezia species. Not only type and standard strains but also 13 clinical isolates were identified successfully in this study. The results of identifications were confirmed by DNA sequencing.     

Simultaneous determination of phenylbutazone and its metabolites in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the simultaneous determination of phenylbutazone and its metabolites, oxyphenbutazone and γ-hydroxyphenylbutazone, in plasma and urine. Samples were acidified with hydrochloric acid and extracted with benzene—cyclohexane (1:1, v/v). The extract was redissolved in methanol and chromatographed on a μBondapak C₁₅ column using a mobile phase of methanol—0.01 M sodium acetate buffer (pH 4.0) in a linear gradient (50 to 100% methanol at 5%/min; flow-rate 2.0 ml/min) in a high-performance liquid chromatograph equipped with an ultra-violet absorbance detector (254 nm). The detection limit for phenylbutazone, oxyphenbutazone and for γ-hydroxyphenylbutazone was 0.05 μg/ml.A precise and sensitive assay for the determination of phenylbutazone and its metabolites was established.     

Changes in levels of mRNAs of transforming growth factor (TGF)-β1, -β2, -β3, TGF-β type II receptor and sulfated glycoprotein-2 during apoptosis of mouse uterine epithelium

To examine the roles played by transforming growth factors (TGF)-β1, -β2, -β3, and TGF-β type II receptors in the induction of apoptosis in the mouse uterine epithelium after estrogen deprivation, we investigated the expression of their mRNAs and the mRNA of sulfated glycoprotein-2 (SGP-2). Pellets containing 100 μg estradiol-17β (E₂) were implanted into ovariectomized mice and removed four days later. Apoptotic indices (percentage of apoptotic cells) of both luminal and glandular epithelia increased after E₂ pellets were removed, but administration of progesterone (P), 5-dihydrotestosterone (DHT), or continued implantation of E₂ pellets suppressed this increase. Levels of mRNAs of TGF-β1, -β2, and -β3, and SGP-2 did not increase after estrogen deprivation. However, estrogen deprivation caused a gradual increase in the level of TGF-β type II receptor mRNA, and its level increased about six-fold six days later. Moreover, E₂, P, and DHT markedly decreased the level of TGF-β type II receptor mRNA. In situ hybridization demonstrated that mRNAs of TGF-β1, -β2, -β3 and TGF-β type II receptor were localized to the epithelium. Exogenous administration of TGF-β1 into the uterine stroma induced apoptosis in the epithelium, a finding that suggests that signals produced by TGF-βs can induce apoptosis. Therefore, the present results suggest that increased sensitivity of uterine epithelial cells to TGF-βs, as demonstrated by an increase in TGF-β type II receptor mRNA, is involved in the induction of apoptosis after estrogen deprivation, although signals produced by TGF-βs do not appear sufficient to induce apoptosis.