The epithelial zinc transporter ZIP10 epigenetically regulates human epidermal homeostasis by modulating histone acetyltransferase activity.

BACKGROUND: The skin is the first organ that manifests changes in response to zinc deficiency. However, the molecular mechanism underlying how zinc is involved in skin homeostasis, especially its epigenetic regulation, is largely unknown. OBJECTIVES: In this study we demonstrate the importance of zinc levels and the zinc transporter ZIP10 in the epigenetic maintenance of human epidermal homeostasis. METHODS: Adult human skin, including skin appendages, were stained with anti-ZIP10 antibody. Histone acetyltransferase (HAT) activity was assessed after treating human keratinocytes with ZIP10 small interfering (si)RNAs or the zinc chelator TPEN. ZIP10- or HAT-regulated genes were analysed based on limma bioinformatics analysis for keratinocytes treated with ZIP10 siRNAs or a HAT inhibitor, or using a public database for transcription factors. A reconstituted human skin model was used to validate the role of ZIP10 in epidermal differentiation and the functional association between ZIP10 and HAT. RESULTS: ZIP10 is predominantly expressed in the interfollicular epidermis, epidermal appendages and hair follicles. ZIP10 depletion resulted in epidermal malformations in a reconstituted human skin model via downregulation of the activity of the epigenetic enzyme HAT. This decreased HAT activity, resulting from either ZIP10 depletion or treatment with the zinc chelator TPEN, was readily restored by zinc supplementation. Through bioinformatics analysis for gene sets regulated by knockdown of SLC39A10 (encoding ZIP10) and HAT inhibition, we demonstrated that ZIP10 and HATs were closely linked with the regulation of genes related to epidermal homeostasis, particularly filaggrin and metallothionein. CONCLUSIONS: Our study suggests that ZIP10-mediated zinc distribution is crucial for epidermal homeostasis via HATs. Therefore, zinc-dependent epigenetic regulation could provide alternatives to maintaining healthy skin or alleviating disorders with skin barrier defects.

Effect of transforming growth factor-beta1 on expression of the connective tissue growth factor (CCN2/CTGF) gene in normal human gingival fibroblasts and periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Connective tissue growth factor (CCN2/CTGF) plays an important role in wound healing and regulation of the extracellular matrix in periodontal tissue. However, the functional relationship between altered transforming growth factor-beta1 levels and CCN2/CTGF has not been extensively investigated in human gingival fibroblasts and periodontal ligament cells. This study investigated the effects of transforming growth factor-beta1 on the expression of the CCN2/CTGF gene in human gingival fibroblasts and periodontal ligament cells in vitro. MATERIAL AND METHODS: Cells were isolated from normal periodontal tissues and cultured in Dulbecco's modified Eagle's minimal essential medium/F12 containing 10% fetal bovine serum. Subconfluent cells were maintained under serum deprivation for 24 h then treated with Dulbecco's modified Eagle's minimal essential medium/F12 containing 0.5% fetal bovine serum (control) and 0.1, 1, 5 or 10 ng/mL of transforming growth factor-beta1 for 24, 48 or 72 h. The effects of transforming growth factor-beta1 on CCN2/CTGF mRNA expression were measured by reverse transcription-polymerase chain reaction. CCN2/CTGF protein was quantitatively analyzed using enzyme-liked immunosorbent assay. Subcellular distribution of CCN2/CTGF protein in both human gingival fibroblasts and periodontal ligament cells was observed using immunofluorescence microscopy. RESULTS: In both human gingival fibroblasts and periodontal ligament cells, the expression of CCN2/CTGF mRNA and CCN2/CTGF protein was significantly increased, in a dose- and time-dependent manner, in the presence of transforming growth factor-beta1. Moreover, immunofluorescence analysis indicated that immunoreactivity to CCN2/CTGF showed a granular pattern of protein localization. CONCLUSION: The expression of CCN2/CTGF mRNA and protein was induced by transforming growth factor-beta1 in human gingival fibroblasts and periodontal ligament cells. These results suggest that CCN2/CTGF plays an important role in wound healing and in the regeneration of periodontal tissue.

Anaesthetists' role in computer keyboard contamination in an operating room.

To store anaesthetic records in computers, anaesthetists usually input data while still wearing dirty wet gloves. No studies have explored computer contamination in the operating room (OR) or anaesthetists' awareness of the importance of handwashing or hand hygiene. We investigated four components of keyboard contamination: (1) degree of contamination, (2) effect of cleaning with ethyl alcohol, (3) bacterial transmission between gloves and keyboards by tapping keys, and (4) frequency of anaesthetists' performing hand hygiene. Most of the bacteria on keyboards were coagulase-negative staphylococci and Bacillus spp.; however, meticillin-resistant Staphylococcus aureus was also found. Cleaning keyboards with ethyl alcohol effectively reduced bacterial counts. Wet contaminated gloves and keyboards transmitted meticillin-susceptible Staphylococcus epidermidis from one to the other more readily than dry contaminated gloves and keyboards. Only 17% of anaesthetists performed hand hygiene before anaesthesia, although 64% or 69% of anaesthetists performed hand hygiene after anaesthesia or before lunch. To prevent cross-contamination, keyboards should be routinely cleaned according to the manufacturer's instructions and disinfected once daily, or, when visibly soiled with blood or secretions. Moreover, anaesthetists should be aware that they could spread microbes that might cause healthcare-associated infection in the OR. Anaesthetists should perform hand hygiene before and after anaesthesia and remove gloves after each procedure and before using the computer.

Microbial growth in propofol formulations with disodium edetate and the influence of venous access system dead space.

Propofol formulated in lipid supports microbial growth. We hypothesised that propofol with disodium edetate would suppress microbial growth more than propofol without disodium edetate. We examined bacterial growth in vitro and bacterial survival in the dead space of different venous access systems. Bacteria in propofol with disodium edetate (Diprivan; AstraZeneca, London, UK) and without disodium edetate (1% Propofol inj. 'Maruishi'; Maruishi Pharmaceutical Co. Ltd, Osaka, Japan) survived and grew in the dead space of the venous access systems, although propofol with disodium edetate suppressed bacterial growth more than propofol without. Disodium edetate is effective in retarding microbial growth. However, for prevention of healthcare-associated infections, medical professionals should maintain strict aseptic precautions when handling propofol, use disodium edetate-containing formulations, and should consider using venous access systems without dead space.

Propofol EDTA and reduced incidence of infection.

Propofol formulated in a lipid vehicle supports the growth of microorganisms. There have been worldwide reports of extrinsic microbial contamination of propofol leading to outbreaks of serious postoperative nosocomial infections. Therefore it is essential that medical professionals follow strict aseptic precautions when handling propofol, as recommended by manufacturers of propofol and the Centers for Disease Control and Prevention. Non-adherence to these recommendations increases the risk of nosocomial postoperative infections, which impose a heavy burden of morbidity and mortality and have serious economic consequences. It has also been recommended that the use of EDTA-containing formulations of propofol be considered. In vitro studies have confirmed that EDTA added to propofol retards microbial growth. Data on the incidence of nosocomial infections before and after the introduction of propofol with EDTA indicates that there have been no further cluster outbreaks and individual nosocomial infections appear to have been reduced. The addition of EDTA is an additional safety precaution to good aseptic practice.

Indirect emissions of nitrous oxide from regional aquifers in the United Kingdom.

Diffuse pollution of groundwater by agriculture has caused elevated concentrations of nitrate (NO3-) and nitrous oxide (N2O) in regional aquifers. N2O is an important "greenhouse" gas, yet there are few estimates of indirect emissions of N2O from regional aquifers. In this study, high concentrations of N2O (mean 602 nM) were measured in the unconfined Chalk aquifer of eastern England, in an area of intensive agriculture. In contrast, pristine groundwaters from upland regions of England and Scotland, with predominantly natural vegetation cover, were found to have much lower concentrations of N2O (mean 27 nM). A positive relationship between N2O and NO3- concentrations and delta18O-NO3 values of between 3.36 and 16.00/1000 suggest that nitrification is the principal source of N2O. A calculated emission factor (EF5-g) of 0.0019 for indirect losses of N2O from Chalk groundwater is an order of magnitude lower than the value of 0.015 currently used in the Intergovernmental Panel on Climate Change (IPCC) methodology for assessing agricultural emissions. A flux of N2O from the major UK aquifers of 0.04 kg N2O-N ha(-1) a(-1) has been calculated using two approaches and suggests that indirect losses of N2O from regional aquifers are much less significant (<1%) than direct emissions from agricultural soils.

The reciprocal role of Egr-1 and Sp family proteins in regulation of the PTP1B promoter in response to the p210 Bcr-Abl oncoprotein-tyrosine kinase.

Protein-tyrosine phosphatase 1B (PTP1B) is an important regulator of protein-tyrosine kinase-dependent signaling pathways. Changes in expression and activity of PTP1B have been associated with various human diseases; however, the mechanisms by which PTP1B expression is regulated have yet to be characterized. Previously, we have shown that the expression of PTP1B is enhanced by p210 Bcr-Abl and that PTP1B is a specific antagonist of transformation induced by this oncoprotein protein-tyrosine kinase. Here we have characterized the PTP1B promoter and demonstrate that a motif with features of a stress-response element acts as a p210 Bcr-Abl-responsive sequence, termed PRS. We have shown that three C(2)H(2) zinc finger proteins, namely Sp1, Sp3, and Egr-1, bind to PRS. Whereas binding of either Sp1 or Sp3 induced promoter function, Egr-1 repressed Sp3-mediated PTP1B promoter activation. The binding of Egr-1 to PRS is suppressed by p210 Bcr-Abl due to the inhibition of Egr-1 expression, resulting in the enhancement of PTP1B promoter activity. Our data indicate that Egr-1 and Sp family proteins play a reciprocal role in the control of expression from the PTP1B promoter.

Analyses of microbial community within a composter operated using household garbage with special reference to the addition of soybean oil.

A commercially available composter was operated using fixed composition of garbage with or without the addition of soybean oil. The composter was operated without adding seed microorganisms or bulking materials. Microflora within the composter were analyzed by denaturing gradient gel electrophoresis (DGGE) in the case of oil addition, or by 16/18 S rRNA gene sequencing of the isolated microorganisms in the case of no oil addition. The results showed that, irrespective of the addition of oil, the bacteria identified were all gram positive, and that lactobacilli seemed to be the key microorganisms. Based on the results, suitable microflora for use in a household composter are discussed.

5-[4-(3,3-Dimethylbutoxycarbonyl)phenyl]-4-pentynoic acid and its derivatives inhibit ionotropic gamma-aminobutyric acid receptors by binding to the 4'-ethynyl-4-n-propylbicycloorthobenzoate site.

Acyclic noncompetitive antagonists of ionotropic gamma-aminobutyric acid (GABA) receptors, bearing an ester or ether linkage, were designed, synthesized, and assayed for their inhibition of the specific binding of [3H]4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a radiolabeled noncompetitive antagonist, to rat brain and housefly head membranes. 5-[4-(3,3-Dimethylbutoxycarbonyl)phenyl]-4-pentynoic acid (DBCPP), a butyl benzoate analogue, was found to competitively inhibit the binding of [3H]EBOB in rat brain membranes, with an IC50 of 88 nM. The potency conferred by the p-substituent decreased in the order C(triple bond)C(CH2)2COOH > C(triple bond)C(CH2)2COOCH3 > C(triple bond) CH > Br. Pentyl phenyl ethers were equally potent compared with butyl benzoates, while phenyl pentanoates and benzyl butyl ethers were less pont. These compounds were generally less active in housefly head membranes than in rat brain membranes. The introduction of an isopropyl group into the 1-position of the 3,3-dimethylbutyl group of a butyl benzoate and two benzyl butyl ethers caused an increase in potency in housefly GABA receptors, whereas this modification at the corresponding position of other compounds led to an unchanged or decreased potency. In the case of rat receptors, this modification resulted in a decrease in potency except for a phenyl pentanoate. To confirm that DBCPP interferes with GABA receptor function, we performed whole-cell patch clamp experiments with rat dorsal root ganglion neurons in the primary culture. Repeated co-applications of GABA and DBCPP suppressed GABA-induced whole-cell currents with an IC50 of 0.54 microM and a Hill coefficient of 0.7. These findings indicate that DBCPP and its derivatives inhibit ionotropic GABA receptors by binding to the EBOB site and that there might be structural difference in the noncompetitive antagonist-binding site between rat and housefly GABA receptors.

Dissection of signaling cascades through gp130 in vivo: reciprocal roles for STAT3- and SHP2-mediated signals in immune responses.

We generated a series of knockin mouse lines, in which the cytokine receptor gp130-dependent STAT3 and/or SHP2 signals were disrupted, by replacing the mouse gp130 gene with human gp130 mutant cDNAs. The SHP2 signal-deficient mice (gp130F759/F759 were born normal but displayed splenomegaly and lymphadenopathy and an enhanced acute phase reaction. In contrast, the STAT3 signal-deficient mice (gp130FXQ/FXXQ) died perinatally, like the gp130-deficient mice (gp130D/D). The gp130F759/F759 mice showed prolonged gp130-induced STAT3 activation, indicating a negative regulatory role for SHP2. Th1-type cytokine production and IgG2a and IgG2b production were increased in the gp130F759/F759 mice, while they were decreased in the gp130FXXQ/FXXQ immune system. These results indicate that the balance of positive and negative signals generated through gp130 regulates the immune responses.

cDNA cloning of the Cry1Aa receptor variants from Bombyx mori and their expression in mammalian cells.

We cloned cDNA of three variants of BtR175, a putative Bombyx mori receptor for Bacillus thuringiensis Cry1Aa delta-endotoxin by PCR. These variants were likely to be allelic to BtR175. cDNA of BtR175b, the most distant variant from BtR175, was introduced into mammalian cells. BtR175b protein was expressed in the plasma membrane of the cells and showed binding activity to Cry1Aa.

Signaling through Gp130: toward a general scenario of cytokine action.

Cytokines play roles in a wide range of responses such as immune response, hematopoiesis and inflammation. A large volume of studies revealed that cytokines show functional pleiotropy and redundancy. Gp130 is a receptor subunit shared by the interleukin-6 family of cytokines. We describe and discuss signaling through gp130 in relation to a general scenario for cytokine signaling regulating cell growth, differentiation and survival.

[The changes of peripheral skin temperature in elderly patients under spinal anesthesia].

The purpose of this study was to evaluate the changes of body temperature in elderly compared with younger patients during spinal anesthesia. Twenty six patients, ASA I-II were divided into two groups, (13; under 30 years of age, 13; above 60 years) who received spinal anesthesia with 0.3% dibucaine at the interspace between L 3 and L 4. All patients were anesthetized below T 8 level. Central temperature was measured at the forehead, and peripheral skin temperature was measured at the finger tip and the toe tip for 25 minutes at every 5 minutes. In both groups, forehead temperature and finger tip skin temperature were unchanged, but toe tip skin temperature increased 5 minutes after spinal anesthesia. The degree of toe tip skin temperature change was significantly lower (P < 0.01) in elderly patients and the speed was also slower. We recognized that the change in peripheral skin temperature during spinal anesthesia is not different between elderly and younger patients, but the speed of temperature change is slower in elderly patients.

Gab-family adapter proteins act downstream of cytokine and growth factor receptors and T- and B-cell antigen receptors.

We previously found that the adapter protein Gab1 (110 kD) is tyrosine-phosphorylated and forms a complex with SHP-2 and PI-3 kinase upon stimulation through either the interleukin-3 receptor (IL-3R) or gp130, the common receptor subunit of IL-6-family cytokines. In this report, we identified another adapter molecule (100 kD) interacting with SHP-2 and PI-3 kinase in response to various stimuli. The molecule displays striking homology to Gab1 at the amino acid level; thus, we named it Gab2. It contains a PH domain, proline-rich sequences, and tyrosine residues that bind to SH2 domains when they are phosphorylated. Gab1 is phosphorylated on tyrosine upon stimulation through the thrombopoietin receptor (TPOR), stem cell factor receptor (SCFR), and T-cell and B-cell antigen receptors (TCR and BCR, respectively), in addition to IL-3R and gp130. Tyrosine phosphorylation of Gab2 was induced by stimulation through gp130, IL-2R, IL-3R, TPOR, SCFR, and TCR. Gab1 and Gab2 were shown to be substrates for SHP-2 in vitro. Overexpression of Gab2 enhanced the gp130 or Src-related kinases-mediated ERK2 activation as that of Gab1 did. These data indicate that Gab-family molecules act as adapters for transmitting various signals.

STAT3 is required for the gp130-mediated full activation of the c-myc gene.

The signal transducers and activators of transcription (STAT) family members have been implicated in regulating the growth, differentiation, and death of normal and transformed cells in response to either extracellular stimuli, including cytokines and growth factors, or intracellular tyrosine kinases. c-myc expression is coordinately regulated by multiple signals in these diverse cellular responses. We show that STAT3 mostly mediates the rapid activation of the c-myc gene upon stimulation of the interleukin (IL)-6 receptor or gp130, a signal transducing subunit of the receptor complexes for the IL-6 cytokine family. STAT3 does so most likely by binding to cis-regulatory region(s) of the c-myc gene. We show that STAT3 binds to a region overlapping with the E2F site in the c-myc promoter and this site is critical for the c-myc gene promoter- driven transcriptional activation by IL-6 or gp130 signals. This is the first identification of the linkage between a member of the STAT family and the c-myc gene activation, and also explains how the IL-6 family of cytokines is capable of inducing the expression of the c-myc gene.

Synergistic roles for Pim-1 and c-Myc in STAT3-mediated cell cycle progression and antiapoptosis.

The activation of STAT3 by the cytokine receptor gp130 is required for both the G1 to S cell cycle transition and antiapoptosis. We found that Pim-1 and Pim-2 are targets for the gp130-mediated STAT3 signal. Expression of a kinase-defective Pim-1 mutant attenuated gp130-mediated cell proliferation. Constitutive expression of Pim-1 together with c-myc, another STAT3 target, fully compensated for loss of the STAT3-mediated cell cycle progression, antiapoptosis, and bcl-2 expression. We also identified valosine-containing protein (VCP) as a target gene for the Pim-1-mediated signal. Expression of a mutant VCP led cells to undergo apoptosis. These results indicate that Pim-family proteins play crucial roles in gp130-mediated cell proliferation and explain the synergy between Pim and c-Myc proteins in cell proliferation and lymphomagenesis.

STAT3 orchestrates contradictory signals in cytokine-induced G1 to S cell-cycle transition.

The signal transducer and activator of transcription molecules (STATs) play key roles in cytokine-induced signal transduction. However, their role in cell growth has not been clear. In the present study, we show that STAT3 plays a key role in the G1 to S phase cell-cycle transition induced by the cytokine receptor subunit gp130, through the upregulation of cyclins D2, D3 and A, and cdc25A, and the concomitant downregulation of p21 and p27. Furthermore, unexpectedly, we found that gp130 could induce the expression of p21 when STAT3 activation was suppressed. Such contradictory signals regulating cell-cycle progression could be simultaneously delivered from distinct cytoplasmic regions of gp130. We propose an 'orchestrating model' for cytokine and growth factor action in which contradictory signals are orchestrated to produce a specific effect in a target cell.

Different response to inflammation of the multiple mRNAs of rat N-acetylglucosaminyltransferase I with variable 5'-untranslated sequences.

We found that there are at least five subclasses of N-acetylglucosaminyltransferase I (GnT-I; EC 2.4.1.101) mRNA with different 5'-untranslated regions in rat brain. These five subclasses were also expressed in many tissues with distinct tissue-specific patterns. Moreover, they were regulated differently in response to acute-phase inflammation. The expression of the most abundant subclass of GnT-I mRNA in rat liver decreased 2.5-fold in response to inflammation, concomitantly with a significant decrease in the total amount of GnT-I mRNA. In contrast, one of the minor subclasses of GnT-I mRNA was induced 10-fold by inflammation.

Gab1 acts as an adapter molecule linking the cytokine receptor gp130 to ERK mitogen-activated protein kinase.

Gab1 has structural similarities with Drosophila DOS (daughter of sevenless), which is a substrate of the protein tyrosine phosphatase Corkscrew. Both Gab1 and DOS have a pleckstrin homology domain and tyrosine residues, potential binding sites for various SH2 domain-containing adapter molecules when they are phosphorylated. We found that Gab1 was tyrosine phosphorylated in response to various cytokines, such as interleukin-6 (IL-6), IL-3, alpha interferon (IFN-alpha), and IFN-gamma. Upon the stimulation of IL-6 or IL-3, Gab1 was found to form a complex with phosphatidylinositol (PI)-3 kinase and SHP-2, a homolog of Corkscrew. Mutational analysis of gp130, the common subunit of IL-6 family cytokine receptors, revealed that neither tyrosine residues of gp130 nor its carboxy terminus was required for tyrosine phosphorylation of Gab1. Expression of Gab1 enhanced gp130-dependent mitogen-activated protein (MAP) kinase ERK2 activation. A mutation of tyrosine 759, the SHP-2 binding site of gp130, abrogated the interactions of Gab1 with SHP-2 and PI-3 kinase as well as ERK2 activation. Furthermore, ERK2 activation was inhibited by a dominant negative p85 PI-3 kinase, wortmannin, or a dominant negative Ras. These observations suggest that Gab1 acts as an adapter molecule in transmitting signals to ERK MAP kinase for the cytokine receptor gp130 and that SHP-2, PI-3 kinase, and Ras are involved in Gab1-mediated ERK activation.

Dual control of neurite outgrowth by STAT3 and MAP kinase in PC12 cells stimulated with interleukin-6.

IL-6 induces differentiation of PC12 cells pretreated with nerve growth factor (NGF). We explored the signals required for neurite outgrowth of PC12 cells by using a series of mutants of a chimeric receptor consisting of the extracellular domain of the granulocyte-colony stimulating factor (G-CSF) receptor and the cytoplasmic domain of gp130, a signal-transducing subunit of the IL-6 receptor. The mutants incapable of activating the MAP kinase cascade failed to induce neurite outgrowth. Consistently, a MEK inhibitor, PD98059, inhibited neurite outgrowth, showing that activation of the MAP kinase cascade is essential for the differentiation of PC12 cells. In contrast, a mutation that abolished the ability to activate STAT3 did not inhibit, but rather stimulated neurite outgrowth. This mutant did not require NGF pretreatment for neurite outgrowth. Dominant-negative STAT3s mimicked NGF pretreatment, and NGF suppressed the IL-6-induced activation of STAT3, supporting the idea that STAT3 might regulate the differentiation of PC12 cells negatively. These results suggest that neurite outgrowth of PC12 cells is regulated by the balance of MAP kinase and STAT3 signal transduction pathways, and that STAT3 activity can be regulated negatively by NGF.