Increased expression of bitter taste receptors in human allergic nasal mucosa and their contribution to the shrinkage of human nasal mucosa.

OBJECTIVE: Bitter taste receptors (TAS2Rs) are expressed in the extraoral tissues, where they possess various physiological functions. This study is to characterize TAS2Rs expression in normal and allergic nasal mucosa and analyse nasal symptom after challenge with bitter tastes to evaluate their pathophysiological function in normal and allergic nasal mucosa. METHODS: The expression levels of TAS2Rs (TAS2R4, 5, 7, 10, 14, 39, and 43) in nasal mucosa were investigated by real-time PCR, Western blot, and immunohistochemistry. The expression levels of TAS2Rs and Ca(2+) imaging in cultured epithelial cells were measured after stimulation with type 2 cytokines (IL-4, IL-5, and IL-13) or bitter tastes. Nasal symptoms in control subjects and allergic rhinitis patients using visual analogue score and acoustic rhinometry were evaluated before and after stimulation with bitter tastes. Vascular diameter of rat nasal septum was measured before and after treatment with bitter tastes. RESULTS: TAS2Rs tested here were expressed in nasal mucosa where they were commonly distributed in superficial epithelium, submucosal glands, and endothelium. Their expression levels are increased in allergic nasal mucosa and up-regulated in cultured epithelial cells simulated with type 2 cytokines. After treatment with bitter tastes, intracellular Ca(2+) signalling was increased in cultured epithelial cells, and vascular constriction was found in rat nasal septum. Increased nasal patency was observed in human nasal mucosa without pain or sneezing. CONCLUSION AND CLINICAL RELEVANCE: TAS2Rs are constitutively expressed in human nasal mucosa and their expression levels are increased in allergic nasal mucosa, where they could potentially contribute to shrinkage of normal and allergic nasal mucosa.

Seroepidemiology of varicella-zoster virus in Korean adolescents and adults using fluorescent antibody to membrane antigen test.

We conducted a cross-sectional seroepidemiological study in 2012-2013 to determine the seroprevalence of varicella-zoster virus (VZV) in adolescents and adults living in Korea, where varicella vaccination has been recommended universally at age 12-15 months since 2005. Residual serum samples were collected from 1196 healthy adults and adolescents aged ⩾10 years between November 2012 and March 2013. The fluorescent antibody to membrane antigen (FAMA) test and enzyme-linked immunosorbent assay (ELISA) were performed to determine the seroprevalence of VZV. The seroprevalences of VZV were compared between six age groups: 10-19, 20-29, 30-39, 40-49, 50-59, and ⩾60 years. The seroprevalence of VZV in the entire study cohort was 99·1% according to the FAMA test and 93·1% as determined by ELISA. The seroprevalences of the six age groups were as follows: 96·0%, 99·5%, 99·5%, 99·5%, 100%, and 100%, respectively, by the FAMA test, and 83·3%, 93·0%, 93·0%, 97·5%, 94·5%, and 97·5%, respectively, by ELISA. Seroprevalence increased significantly with age (P < 0·001); moreover, the seroprevalence in subjects aged 10-19 years was significantly lower than in other age groups (P < 0·001), as measured by both the FAMA test and ELISA. Thus, strategies to increase protective immunity against VZV in teenagers are necessary.

15-Deoxy-Δ(12,14)-prostaglandin J2 prevents oxidative injury by upregulating the expression of aldose reductase in vascular smooth muscle cells.

The omega-6 fatty acid derivative 15-Deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) is believed to play a role in cellular protection against oxidative stress in diverse cell systems. However, the cellular mechanisms by which protection is afforded by 15d-PGJ2 are not fully elucidated in vascular smooth muscle cells (VSMCs). In this study, we report the finding that 15d-PGJ2 elicited a time and concentration- dependent increase in aldose reductase (AR) expression. This induction was independent of the activation of peroxisome proliferator- activated receptor γ. Inhibition of phosphatidylinositol 3-kinase (PI3K) significantly suppressed the increase in expression and promoter activity of AR induced by 15d-PGJ2. Luciferase reporter assays demonstrated that 15d-PGJ2 targets the multiple stress response regions comprising the antioxidant response element in the promoter of the AR gene. 15d-PGJ2-mediated induction of AR promoter activity was potentiated in the presence of nuclear factor-erythroid 2-related factor 2 (Nrf2), but not in cells expressing dominant negative Nrf2. Cells treated with 15d-PGJ2 were resistant to oxidant-induced apoptotic cell death by inhibiting production of reactive oxygen species. These effects were significantly attenuated in the presence of an AR inhibitor or small interfering RNA against AR, indicating that AR plays a protective role against oxidative injury. Taken together, these findings demonstrate that activation of PI3K by 15d-PGJ2 increases the expression of AR through Nrf2, and increased AR activity may function as an important cellular response against oxidative injury.

Posttranslational synthesis of hypusine: evolutionary progression and specificity of the hypusine modification.

A naturally occurring unusual amino acid, hypusine [N (epsilon)-(4-amino-2-hydroxybutyl)-lysine] is a component of a single cellular protein, eukaryotic translation initiation factor 5A (eIF5A). It is a modified lysine with structural contribution from the polyamine spermidine. Hypusine is formed in a novel posttranslational modification that involves two enzymes, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). eIF5A and deoxyhypusine/hypusine modification are essential for growth of eukaryotic cells. The hypusine synthetic pathway has evolved in eukaryotes and eIF5A, DHS and DOHH are highly conserved, suggesting maintenance of a fundamental cellular function of eIF5A through evolution. The unique feature of the hypusine modification is the strict specificity of the enzymes toward its substrate protein, eIF5A. Moreover, DHS exhibits a narrow specificity toward spermidine. In view of the extraordinary specificity and the requirement for hypusine-containing eIF5A for mammalian cell proliferation, eIF5A and the hypusine biosynthetic enzymes present new potential targets for intervention in aberrant cell proliferation.

Dietary palm oil alters the lipid stability of polyunsaturated fatty acid-modified poultry products.

Laying hens were fed a high polyunsaturated fatty acid (PUFA) diet with three levels [low (LPO, 0%), medium (MPO, 1.5%), and high (HPO, 3.5%)] of palm oil (PO) or a PUFA diet with tocopherol mix (control). Flax and fish oils were used as PUFA sources in all of the diets. Inclusion of tocopherols resulted in a lower (P < 0.05) thiobarbituric acid reactive substances (TBARS) in the control eggs in white and dark meats. Among the PO treatments, eggs from HPO diet had the lowest TBARS (P < 0.05). A higher (P < 0.05) TBARS was observed for eggs, meat, and liver from hens on the LPO diet. No difference was observed between the TBARS of LPO and MPO dark meat. The C22:6 n-3 fatty acid was lower (P < 0.05) in MPO and HPO eggs and the HPO white meat.

Effect of serum and hydrogen peroxide on the Ca2+/calmodulin-dependent phosphorylation of eukaryotic elongation factor 2(eEF-2) in Chinese hamster ovary cells.

Eukaryotic elongation factor eEF-2 mediates regulatory steps important for the overall regulation of mRNA translation in mammalian cells and is activated by variety of cellular conditions and factors. In this study, eEF-2 specific, Ca2+/CaM-dependent protein kinase III (CaM PK III), also called eEF-2 kinase, was examined under oxidative stress and cell proliferation state using CHO cells. The eEF-2 kinase activity was determined in the kinase buffer containing Ca2+ and CaM in the presence of eEF-2 and [gamma-32P] ATP. The eEF-2 kinase activity in cell lysates was completely dependent upon Ca2+ and CaM. Phosphorylation of eEF-2 was clearly identified in proliferating cells, but not detectable in CHO cells arrested in their growth by serum deprivation. The content of the eEF-2 protein, however, was equivalent in both cells. Using a phosphorylation state-specific antibody, we show that oxidant such as H2O2, which triggers a large influx of Ca2+, dramatically enhances the phosphorylation of eEF-2. In addition, H2O2-induced eEF-2 phosphorylation is dependent on Ca2+ and CaM, but independent of protein kinase C. In addition, okadaic acid inhibits phosphoprotein phosphatase 2A(PP2A)-mediated eEF-2 dephosphorylation. These results may provide a possible link between the elevation of intracellular Ca2+ and cell division and suggest that phosphorylation of eEF-2 is sensitive cellular reflex on stimuli that induces intracellular Ca2+ flux.

Regulation of chicken protein tyrosine phosphatase 1 and human protein tyrosine phosphatase 1B activity by casein kinase II- and p56lck-mediated phosphorylation.

Protein tyrosine phosphorylation and dephosphorylation are important in the regulation of cell proliferation and signaling cascade. In order to examine whether phosphatase activity of CPTP1 and HPTP1B, typical nontransmembrane protein tyrosine phosphatase, could be controlled by phosphorylation, affinity-purified PTPs were phosphorylated by CKII and p56lck in vitro. Phosphoamino acid analysis revealed that CPTP1 was phosphorylated on both serine and threonine residues by CKII, and tyrosine residue by p56lck. Phosphatase activity of CPTP1 was gradually increased by three-fold concomitant with phosporylation by CKII. Phosphorylation of HPTP1B by CKII resulted in quick two-fold enhancement of its phosphatase activity within 5 min of incubation and remained in that state. In the presence of CKII inhibitor, heparin or poly(Glu.Tyr), both phosphorylation and enhancement of phosphatase activity of CPTP1 and HPTP1B were mostly blocked. p56lck catalyzed tyrosine phosphorylation of CPTP1 and HPTP1B was only observed by inhibiting the intrinsic tyrosine phosphatase activity. Taken together, these results indicate that CPTP1 or HPTP1B possesses a capability to regulate its phosphatase activity through phosphorylation processes and may participate in the cellular signal cascades.

Characterization of yeast deoxyhypusine synthase: PKC-dependent phosphorylation in vitro and functional domain identification.

The biosynthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl)-lysine] occurs in the eIF-5A precursor protein through two step posttranslational modification involving deoxyhypusine synthase which catalyzes transfer of the butylamine moiety of spermidine to the epsilon-amino group of a designated lysine residue and subsequent hydroxylation of this intermediate. This enzyme is exclusively required for cell viability and growth of yeast (Park, M.H. et al., J. Biol. Chem. 273: 1677-1683, 1998). In an effort to understand structure-function relationship of deoxyhypusine synthase, posttranslational modification(s) of the enzyme by protein kinases were carried out for a possible cellular modulation of this enzyme. And also twelve deletion mutants were constructed, expressed in E. coli system, and enzyme activities were examined. The results showed that deoxyhypusine synthase was phosphorylated by PKC in vitro but not by p56lck and p60c-src. Treatment with PMA specifically increased the relative phosphorylation of the enzyme supporting PKC was involved. Phosphoamino acid analysis of this enzyme revealed that deoxyhypusine synthase is mostly phosphorylated on serine residue and weakly on threonine. Removal of Met1-Glu10 (deltaMet1-Glu10) residues from amino terminal showed no effect on the catalytic activity but further deletion (deltaMet1-Ser20) caused loss of enzyme activity. The enzyme with internal deletion, deltaGln197-Asn212 (residues not present in the human enzyme) was found to be inactive. Removal of 5 residues from carboxyl terminal, deltaLys383-Asn387, retained only slight activity. These results suggested that deoxyhypusine synthase is substrate for PKC dependent phosphorylation and requires most of the polypeptide chains for enzyme activity except the first 15 residues of N-terminal despite of N- and C-terminal residues of the enzyme consist of variable regions.

Deoxyhypusine synthase activity is essential for cell viability in the yeast Saccharomyces cerevisiae.

Deoxyhypusine synthase catalyzes the first step in the posttranslational synthesis of an unusual amino acid, hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine), in the eukaryotic translation initiation factor 5A (eIF-5A) precursor protein. The null mutation in the single copy gene, yDHS, encoding deoxyhypusine synthase results in the loss of viability in the yeast Saccharomyces cerevisiae. Upon depletion of deoxyhypusine synthase, and consequently of eIF-5A, cessation of growth was accompanied by a marked enlargement of cells, suggesting a defect in cell cycle progression or in cell division. Two residues of the yeast enzyme, Lys308 and Lys350, corresponding to Lys287 and Lys329, respectively, known to be critical for the activity of the human enzyme, were targeted for site-directed mutagenesis. The chromosomal ydhs null mutation was complemented by the plasmid-borne yDHS wild-type gene, but not by mutated genes encoding inactive proteins, including that with Lys350-->Arg substitution or with substitutions at both Lys308 and Lys350. The mutated gene ydhs (K308R) encoding a protein with diminished activities (< 1% of wild type) could support growth but only to a very limited extent. These findings provide strong evidence that the hypusine modification is indeed essential for the survival of S. cerevisiae and imply a vital function for eIF-5A in all eukaryotes.

The polyamine-derived amino acid hypusine: its post-translational formation in eIF-5A and its role in cell proliferation.

The unusual amino acid hypusine [N (ε) -(4-amino-2-hydroxybutyl)lysine] is a unique component of one cellular protein, eukaryotic translation initiation factor 5A (eIF-5A, old terminology, eIF-4D). It is formed posttranslationally and exclusively in this protein in two consecutive enzymatic reactions, (i) modification of a single lysine residue of the eIF-5A precursor protein by the transfer of the 4-aminobutyl moiety of the polyamine spermidine to itsε-amino group to form the intermediate, deoxyhypusine [N (ε) -(4-aminobutyl)lysine] and (ii) subsequent hydroxylation of this intermediate to form hypusine. The amino acid sequences surrounding the hypusine residue are strictly conserved in all eukaryotic species examined, suggesting the fundamental importance of this amino acid throughout evolution. Hypusine is required for the activity of eIF-5Ain vitro. There is strong evidence that hypusine and eIF-5A are vital for eukaryotic cell proliferation. Inactivation of both of the eIF-5A genes is lethal in yeast and the hypusine modification appears to be a requirement for yeast survival (Schnier et al., 1991 [Mol Cell Biol 11: 3105-3114]; Wöhl et al., 1993 [Mol Gen Genet 241: 305-311]). Furthermore, inhibitors of either of the hypusine biosynthetic enzymes, deoxyhypusine synthase or deoxyhypusine hydroxylase, exert strong anti-proliferative effects in mammalian cells, including many human cancer cell lines. These inhibitors hold potential as a new class of anticancer agents, targeting one specific eukaryotic cellular reaction, hypusine biosynthesis.

Identification of YHR068w in Saccharomyces cerevisiae chromosome VIII as a gene for deoxyhypusine synthase. Expression and characterization of the enzyme.

Deoxyhypusine synthase catalyzes the formation of deoxyhypusine, the first step in hypusine biosynthesis. Amino acid sequences of five tryptic peptides from rat deoxyhypusine synthase were found to match partially the deduced amino acid sequence of the open reading frame of gene YHR068w of Saccharomyces cerevisiae chromosome VIII (AC:U00061). In order to determine whether the product of this gene corresponds to yeast deoxyhypusine synthase,a 1.17-kilobase pair cDNA with an identical nucleotide sequence to that of the YHR068w coding region was obtained from S. cerevisiae cDNA by polymerase chain reaction and was expressed in Escherichia coli B strain BL21 (DE3). The recombinant protein was found mostly in the E. coli cytosol fraction and comprised approximately 20% of the total soluble protein. The purified form of the expressed protein effectively catalyzed the formation of deoxyhypusine in yeast eIF-5A precursors as well as in human precursor and in those from Chinese hamster ovary cells. The molecular mass of the enzyme was estimated to be 172,000 +/- 4,300 Da by equilibrium centrifugation. The mass of its polypeptide subunit was determined to be approximately 43,000 Da, in close agreement with that calculated for the coding region of the YHRO68w gene. These findings show that this gene is a coding sequence for yeast deoxyhypusine synthase and that the product of this gene exists in a tetrameric form.

Elongation factor-2 in chick embryo is phosphorylated on tyrosine as well as serine and threonine.

An endogenous 95 kDa chick embryo cytosolic protein (p95) was phosphorylated in the presence of [gamma-32P]ATP and the kinase activity for p95 was mostly associated with particulate fraction. Phosphorylation of p95 was prominent in embryos of early developmental stage. Hydrolysis of p95 phosphoprotein yielded phosphotyrosine in addition to phosphothreonine and phosphoserine. Native p95 was also tyrosine-phosphorylated. p95 phosphoprotein was purified by DEAE-Sephacel chromatography and immunoprecipitation with anti-phosphotyrosine antibody and the amino acid sequence was determined. The N-terminal sequence, Val-Asn-Phe-Thr-Val-Asp-Gln-Ile-Arg-Ala-Ile-Met-Asp- Lys-Lys-Ala-Asn-Ile-Arg-Asn-Met-, was found to be identical to those of elongation factor-2 (EF-2) of both rat and hamster. Our results suggest the presence of other EF-2 kinase in chick embryo cell than the previously reported Ca2+/calmodulin-dependent protein kinase III.