Upregulation of P2Y2 receptors by retinoids in normal human epidermal keratinocytes

Retinoids, vitamin A derivatives, are important regulators of the growth and differentiation of skin cells. Although retinoids are therapeutically used for several skin ailments, little is known about their effects on P2 receptors, known to be involved in various functions in the skin. DNA array analysis showed that treatment of normal human epidermal keratinocytes (NHEKs) with all-trans-retinoic acid (ATRA), an agonist to RAR (retinoic acid receptor), enhanced the expression of mRNA for the P2Y2 receptor, a metabotropic P2 receptor that is known to be involved in the proliferation of the epidermis. The expression of other P2 receptors in NHEKs was not affected by ATRA. ATRA increased the mRNA for the P2Y2 receptor in a concentration-dependent fashion (1 nM to 1 μM). Am80, a synthesized agonist to RAR, showed a similar enhancement, whereas 9-cis-retinoic acid (9-cisRA), an agonist to RXR (retinoid X receptor), enhanced P2Y2 gene expression to a lesser extent. Ca²⁺ imaging analysis showed that ATRA also increased the function of P2Y2 receptors in NHEKs. Retinoids are known to enhance the turnover of the epidermis by increasing both proliferation and terminal differentiation. The DNA microarray analysis also revealed that ATRA upregulates various genes involved in the differentiation of NHEKs. Our present results suggest that retinoids, at least in part, exert their proliferative effects by upregulating P2Y2 receptors in NHEKs. This effect of retinoids may be closely related to their therapeutic effect against various ailments or aging events in skins such as over-keratinization, pigmentation and re-modeling.     

The Middle Region of an HP1-binding Protein, HP1-BP74, Associates with Linker DNA at the Entry/Exit Site of Nucleosomal DNA

In higher eukaryotic cells, DNA molecules are present as chromatin fibers, complexes of DNA with various types of proteins; chromatin fibers are highly condensed in metaphase chromosomes during mitosis. Although the formation of the metaphase chromosome structure is essential for the equal segregation of replicated chromosomal DNA into the daughter cells, the mechanism involved in the organization of metaphase chromosomes is poorly understood. To identify proteins involved in the formation and/or maintenance of metaphase chromosomes, we examined proteins that dissociated from isolated human metaphase chromosomes by 0.4 m NaCl treatment; this treatment led to significant chromosome decondensation, but the structure retained the core histones. One of the proteins identified, HP1-BP74 (heterochromatin protein 1-binding protein 74), composed of 553 amino acid residues, was further characterized. HP1-BP74 middle region (BP74Md), composed of 178 amino acid residues (Lys⁹⁷–Lys²⁷⁴), formed a chromatosome-like structure with reconstituted mononucleosomes and protected the linker DNA from micrococcal nuclease digestion by ∼25 bp. The solution structure determined by NMR revealed that the globular domain (Met¹⁵³–Thr²³⁷) located within BP74Md possesses a structure similar to that of the globular domain of linker histones, which underlies its nucleosome binding properties. Moreover, we confirmed that BP74Md and full-length HP1-BP74 directly binds to HP1 (heterochromatin protein 1) and identified the exact sites responsible for this interaction. Thus, we discovered that HP1-BP74 directly binds to HP1, and its middle region associates with linker DNA at the entry/exit site of nucleosomal DNA in vitro.     

Phosphorylation of aquaporin-2 regulates its water permeability

Vasopressin-regulated water reabsorption through the water channel aquaporin-2 (AQP2) in renal collecting ducts maintains body water homeostasis. Vasopressin activates PKA, which phosphorylates AQP2, and this phosphorylation event is required to increase the water permeability and water reabsorption of the collecting duct cells. It has been established that the phosphorylation of AQP2 induces its apical membrane insertion, rendering the cell water-permeable. However, whether this phosphorylation regulates the water permeability of this channel still remains unclear. To clarify the role of AQP2 phosphorylation in water permeability, we expressed recombinant human AQP2 in Escherichia coli, purified it, and reconstituted it into proteoliposomes. AQP2 proteins not reconstituted into liposomes were removed by fractionating on density step gradients. AQP2-reconstituted liposomes were then extruded through polycarbonate filters to obtain unilamellar vesicles. PKA phosphorylation significantly increased the osmotic water permeability of AQP2-reconstituted liposomes. We then examined the roles of AQP2 phosphorylation at Ser-256 and Ser-261 in the regulation of water permeability using phosphorylation mutants reconstituted into proteoliposomes. The water permeability of the non-phosphorylation-mimicking mutant S256A-AQP2 and non-phosphorylated WT-AQP2 was similar, and that of the phosphorylation-mimicking mutant S256D-AQP2 and phosphorylated WT-AQP2 was similar. The water permeability of S261A-AQP2 and S261D-AQP2 was similar to that of non-phosphorylated WT-AQP2. This study shows that PKA phosphorylation of AQP2 at Ser-256 enhances its water permeability.     

Calcitonin gene-related peptide activates different signaling pathways in mesenteric lymphatics of guinea pigs

The effects of calcitonin gene-related peptide (CGRP) on constriction frequency, smooth muscle membrane potential (V(m)), and endothelial V(m) of guinea pig mesenteric lymphatics were examined in vitro. CGRP (1-100 nM) caused an endothelium-dependent decrease in the constriction frequency of perfused lymphatic vessels. The endothelium-dependent CGRP response was abolished by the CGRP-1 receptor antagonist CGRP-(8-37) (1 microM) and pertussis toxin (100 ng/ml). This action of CGRP was also blocked by the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine (L-NNA; 10 microM), an action that was reversed by the addition of L-arginine (100 microM). cGMP, adenylate cyclase, cAMP-dependent protein kinase (PKA), and ATP-sensitive K+ (K+(ATP)) channels were all implicated in the endothelium-dependent CGRP response because it was abolished by methylene blue (20 microM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 microM), dideoxyadenosine (10 microM), N-[2-(p-bromociannamylamino)-ethyl]-5-isoquinolinesulfonamide-dichloride (H89; 1 microM) and glibenclamide (10 microM). CGRP (100 nM), unlike acetylcholine, did not alter endothelial intracellular Ca2+ concentration or V(m). CGRP (100 nM) hyperpolarized the smooth muscle V(m), an effect inhibited by L-NNA, H89, or glibenclamide. CGRP (500 nM) also caused a decrease in constriction frequency. However, this was no longer blocked by CGRP-(8-37). CGRP (500 nM) also caused smooth muscle hyperpolarization, an action that was now not blocked by L-NNA (100 microM). It was most likely mediated by the activation of the cAMP/PKA pathway and the opening of K+(ATP) channels because it was abolished by H89 or glibenclamide. We conclude that CGRP, at low to moderate concentrations (i.e., 1-100 nM), decreases lymphatic constriction frequency primarily by the stimulation of CGRP-1 receptors coupled to pertussis toxin-sensitive G proteins and the release of NO from the endothelium or enhancement of the actions of endogenous NO. At high concentrations (i.e., 500 nM), CGRP also directly activates the smooth muscle independent of NO. Both mechanisms of activation ultimately cause the PKA-mediated opening of K+(ATP) channels and resultant hyperpolarization.     

Co-occurrence of mutations in both dystrophin- and androgen-receptor genes is a novel cause of female Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder. Here, we report a novel mechanism for the occurrence of DMD in females. In a Vietnamese DMD girl, conventional PCR amplification analysis disclosed a deletion of exons 12–19 of the dystrophin gene on Xp21.2, with a karyotype of 46, XY. Furthermore, a novel mutation in the androgen-receptor gene on Xq11.2-q12 was identified in this girl, which led to male pseudohermaphroditism. Co-occurrence of mutations of these two genes constitutes a novel mechanism underlying female DMD.     

Aspartic acid 405 contributes to the substrate specificity of aminopeptidase B

Aminopeptidase B (EC 3.4.11.6, ApB) specifically cleaves in vitro the N-terminal Arg or Lys residue from peptides and synthetic derivatives. Ap B was shown to have a consensus sequence found in the metallopeptidase family. We determined the putative zinc binding residues (His324, His328, and Glu347) and the essential Glu325 residue for the enzyme using site-directed mutagenesis (Fukasawa, K. M., et al. (1999) Biochem. J. 339, 497-502). To identify the residues binding to the amino-terminal basic amino acid of the substrate, rat cDNA encoding ApB was cloned into pGEX-4T-3 so that recombinant protein was expressed as a GST fusion protein. Twelve acidic amino acid residues (Glu or Asp) in ApB were replaced with a Gln or Asn using site-directed mutagenesis. These mutants were isolated to characterize the kinetic parameters of enzyme activity toward Arg-NA and compare them to those of the wild-type ApB. The catalytic efficiency (kcat/Km) of the mutant D405N was 1.7 x 10(4) M(-1) s(-1), markedly decreased compared with that of the wild-type ApB (6.2 x 10(5) M(-1) s(-1)). The replacement of Asp405 with an Asn residue resulted in the change of substrate specificity such that the specific activity of the mutant D405N toward Lys-NA was twice that toward Arg-NA (in the case of wild-type ApB; 0.4). Moreover, when Asp405 was replaced with an Ala residue, the kcat/Km ratio was 1000-fold lower than that of the wild-type ApB for hydrolysis of Arg-NA; in contrast, in the hydrolysis of Tyr-NA, the kcat/Km ratios of the wild-type (1.1 x 10(4) M(-1) s(-1)) and the mutated (8.2 x 10(3) M(-1) s(-1)) enzymes were similar. Furthermore, the replacement of Asp-405 with a Glu residue led to the reduction of the kcat/Km ratio for the hydrolysis of Arg-NA by a factor of 6 and an increase of that for the hydrolysis of Lys-NA. Then the kcat/Km ratio of the D405E mutant for the hydrolysis of Lys-NA was higher than that for the hydrolysis of Arg-NA as opposed to that of wild-type ApB. These data strongly suggest that the Asp 405 residue is involved in substrate binding via an interaction with the P1 amino group of the substrate's side chain.     

Daily variation of oral malodour and related factors in community-dwelling elderly Thai

doi: 10.1111/j.1741‐2358.2011.00593.x Daily variation of oral malodour and related factors in community‐dwelling elderly Thai Objectives: The purposes of this study were (i) to estimate the prevalence of oral malodour, (ii) to evaluate the daily variation of oral malodour and (iii) to assess associations of volatile sulphur compound (VSC) concentrations with socio‐demographics, health behaviours and oral health status in community‐dwelling elderly Thai. Methods: The subjects were 428 dentate elderly people (67.6 ± 5.6 years) living in Phitsaulok, Thailand. Information on their socio‐demographics, general health and health behaviours was obtained by a questionnaire. Their dental condition, periodontal status and tongue coating were clinically examined. Their flow rates and the pH of unstimulated saliva were also assessed. Oral malodour was measured at four different times of day using an Oral Chroma?. Results: The proportions of subjects diagnosed with oral malodour using the thresholds of H₂S, CH₃SH and (CH₃)₂S were 60.5%, 62.9% and 80.7%, respectively. Concentrations of H₂S showed significant daily variation. Linear regression analysis demonstrated the following significant associations: (i) oral malodour from H₂S and thickness of the tongue coating, (ii) oral malodour from CH₃SH and periodontal pocket depth of 5 mm or more and the presence of gingival bleeding and (iii) oral malodour from (CH₃)₂S and systemic disease, medications and thickness of the tongue coating. Discussion: Oral malodour was shown to be prevalent among the elderly. Daily variation was observed in the concentration of H₂S. Tongue coating, periodontal disease, systemic diseases and medications were related to oral malodour. Therefore, these factors should be taken into consideration in oral malodour treatment and prevention programmes for the elderly.     

The Erf4 Subunit of the Yeast Ras Palmitoyl Acyltransferase Is Required for Stability of the Acyl-Erf2 Intermediate and Palmitoyl Transfer to a Ras2 Substrate

Protein S-palmitoylation is a posttranslational modification in which a palmitoyl group is added to a protein via a thioester linkage on cysteine. Palmitoylation is a reversible modification involved in protein membrane targeting, receptor trafficking and signaling, vesicular biogenesis and trafficking, protein aggregation, and protein degradation. An example of the dynamic nature of this modification is the palmitoylation-depalmitoylation cycle that regulates the subcellular trafficking of Ras family GTPases. The Ras protein acyltransferase (PAT) consists of a complex of Erf2-Erf4 and DHHC9-GCP16 in yeast and mammalian cells, respectively. Both subunits are required for PAT activity, but the function of the Erf4 and Gcp16 subunits has not been established. This study elucidates the function of Erf4 and shows that one role of Erf4 is to regulate Erf2 stability through an ubiquitin-mediated pathway. In addition, Erf4 is required for the stable formation of the palmitoyl-Erf2 intermediate, the first step of palmitoyl transfer to protein substrates. In the absence of Erf4, the rate of hydrolysis of the active site palmitoyl thioester intermediate is increased, resulting in reduced palmitoyl transfer to a Ras2 substrate. This is the first demonstration of regulation of a DHHC PAT enzyme by an associated protein.     

A change in the sensitivity of elongation factor G to oxidation protects photosystem II from photoinhibition in Synechocystis sp. PCC 6803

The repair of photosystem II (PSII) after photodamage is particularly sensitive to oxidative stress and inhibition of such repair is associated with the oxidation of specific cysteine residues in elongation factor G (EF-G), a key translation factor, in the cyanobacterium Synechocystis sp. PCC 6803. Expression of mutated EF-G with a target cysteine residue replaced by serine in Synechocystis resulted in the protection of PSII from photoinhibition. This protection was attributable to the enhanced repair of PSII via acceleration of the synthesis of the D1 protein, which might have been due to reduced sensitivity of protein synthesis to oxidative stress.     

The structural analysis and the role of calcium binding site for thermal stability in mannanase

Mannanase is an important enzyme involved in the degradation of mannan, production of bioactive oligosaccharides, and biobleaching of kraft pulp. Mannanase must be thermostable for use in industrial applications. In a previous study, we found that the thermal stability of mannanase from Streptomyces thermolilacinus (StMan) and Thermobifida fusca (TfMan) is enhanced by calcium. Here, we investigated the relationship between the three-dimensional structure and primary sequence to identify the putative calcium-binding site. The results of site-directed mutagenesis experiments indicated that Asp-285, Glu-286, and Asp-287 of StMan (StDEDAAAdC) and Asp-264, Glu-265, and Asp-266 of TfMan (TfDEDAAAdC) were the key residues for calcium binding affinity. Isothermal titration calorimetry revealed that the catalytic domain of StMan and TfMan (StMandC and TfMandC, respectively) bound calcium with a K_a of 3.02 × 10⁴ M⁻¹ and 1.52 × 10⁴ M⁻¹, respectively, both with stoichiometry consistent with one calcium-binding site per molecule of enzyme. Non-calcium-binding mutants (StDEDAAAdC and TfDEDAAAdC) did not show any calorimetric change. From the primary structure alignment of several mannanases, the calcium-binding site was found to be highly conserved in GH5 bacterial mannanases. This is the first study indicating enhanced thermal stability of GH5 bacterial mannanases by calcium binding.