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.

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.

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.

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.

Tec tyrosine kinase links the cytokine receptors to PI-3 kinase probably through JAK.

Tec/Btk tyrosine kinases are members of a subgroup of Src tyrosine kinase family. They are reported to be activated in response to cytokines, such as IL-3 and IL-6. Janus kinases (JAKs) are known to associate with certain cytokine receptors, e.g. gp130, the signal transducing subunit of IL-6 receptor, and the common beta chain of IL-3 receptor, which can be activated upon receptor dimerization in response to cytokines. Here we show the association between Jak1/Jak2 and Tec or Jak1 and Btk. Furthermore, Jak1 but not Jak2 induces tyrosine phosphorylation of Btk, but not Tec. These observations suggest that upon cytokine stimulation JAKs activate Tec/Btk or induce their dimerization resulting in endogenous tyrosine phosphorylation. Furthermore using a yeast two-hybrid system we have identified the target molecules for Tec, the p85 and p55PIK subunits of PI-3 kinase, and Vav. Tec associated with Vav through its SH2 domain independently of its kinase activity. In contrast the p85 and p55PIK subunits of PI-3 kinase associated with the SH2-kinase domain of Tec, dependent on Tec kinase activity. Consistent with these, IL-6 or IL-3 induced the association between Tec and the p85 subunit of PI-3 kinase in mammalian cells. These findings suggest that Tec tyrosine kinase links cytokine receptors to PI-3 kinase probably through JAKs.

An alternative pathway for STAT activation that is mediated by the direct interaction between JAK and STAT.

JAK is believed to be an essential tyrosine kinase that mediates signals from the cytokine receptor to its downstream events. JAK associates with the cytoplasmic domain of the type I cytokine receptor superfamily and upon the ligand stimulation it can be activated, resulting in the receptor phosphorylation. In signaling from gp130, a common signal transducer for the IL-6 family cytokines, STAT3, a transcription factor that contains an SH2 domain, is recruited by phosphotyrosines on gp130 and is subsequently phosphorylated by gp130-associated JAKs. In this study, we attempted to find a new target for JAK that is directly activated by JAK, independent of gp130 tyrosine phosphorylation, by using a yeast two-hybrid system. In the process we found that the JH2 domain of JAK1, JAK2 or JAK3 could specifically associate with the carboxy-terminal portion of STAT5, but not with STAT3 or STAT1. The interaction was confirmed using both a transient expression system in a cell line and a GST-fusion protein binding assay. Furthermore, we showed that the activation of STAT5 via gp130 did not need any phosphotyrosines on gp130 while that of STAT3 strictly depended on phosphotyrosines on gp130. Mutations of STAT5 that eliminated the interaction with JAK1 reduced the activation of STAT5 upon the gp130 stimulation, although such mutants could be still activated through erythropoietin receptor. These results indicate that STATs are activated through cytokine receptors by two distinct mechanisms, one dependent on receptor tyrosine phosphorylation and the other mediated by the JAK-STAT direct interaction.

Properties of the reaction center of the thermophilic purple photosynthetic bacterium Chromatium tepidum.

Reaction centers were purified from the thermophilic purple sulfur photosynthetic bacterium Chromatium tepidum. The reaction center consists of four polypeptides L, M, H and C, whose apparent molecular masses were determined to be 25, 30, 34 and 44 kDa, respectively, by polyacrylamide gel electrophoresis. The heaviest peptide corresponds to tightly bound cytochrome. The tightly bound cytochrome c contains two types of heme, high-potential c-556 and low-potential c-553. The low-potential heme is able to be photooxidized at 77 K. The reaction center exhibits laser-flash-induced absorption changes and circular dichroism spectra similar to those observed in other purple photosynthetic bacteria. Whole cells contain both ubiquinone and menaquinone. Reaction centers contain only a single active quinone; chemical analysis showed this to be menaquinone. Reaction center complexes without the tightly bound cytochrome were also prepared. The near-infrared pigment absorption bands are red-shifted in reaction centers with cytochrome compared to those without cytochrome.

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.

Studies on lectins of amago (Oncorhyncus rhodurus). I. Amago ova lectin and its receptor on homologous macrophages.

A lectin was found in the ova of amago, a Japanese trout (Oncorhyncus rhodurus), which agglutinates rabbit, rat and human B-type erythrocytes. The hemagglutination was specifically inhibited by monosaccharides, L-rhamnose, D-galactose, and their C2 and C4 analogs, and p-nitrophenyl-alpha-D-galactoside and melibiose, indicating a binding specificity for alpha-L-rhamnosyl or alpha-D-galactosyl type sugar moiety. To study its interaction with homologous cells, amago peritoneal macrophages were isolated from corn starch-stimulated peritoneal exudates. The lectin-rabbit erythrocyte complexes were found to adhere onto the macrophages harvested on the 4th day or later after the stimulation, but not to those obtained within 3 days; the latter macrophages acquired the complex-binding capacity when cultured for 3 to 4 days in vitro. These findings indicated that a lectin receptor is expressed on peritoneal macrophages after inflammatory stimulation. Similar lectin receptor-bearing macrophage-like-cells were also detected during in vitro amago head kidney culture. This suggested that the inflammatory induced peritoneal macrophages may be differentiated from the head kidney macrophage precursor cells and during this process the ova lectin receptors also become expressed.

Adult-onset subacute sclerosing panencephalitis: immunocytochemical and electron microscopic demonstration of the viral antigen.

We report a case of subacute sclerosing panencephalitis (SSPE) in a 52 year-old man, who developed rapidly progressive mental deterioration, myoclonic seizures, quadriplegia, and remained incapacitated until his death 4 years after the onset of symptoms. Immunocytochemical and electron microscopic studies are reported. Titers of measles virus antibodies were consistently high in both serum and cerebrospinal fluid, and periodic synchronous discharges were recorded on EEG. Suppressed cellular immunity was noted in skin test with phytohemagglutinin. The brain was extensively destroyed by inflammatory processes. There were either laminar or widespread areas of cortical necrosis associated with neuronophagia, neuronal loss, glial proliferation, and perivascular lymphocytic cuffing. Numerous intranuclear inclusions, in the neurons and glial cells, stained with immunoperoxidase using antiserum to SSPE virus; ultrastructurally, these inclusions were made of tubular nucleocapsids of paramyxovirus. Neurofibrillary changes were occasionally encountered in the pigmented neurons. The white matter showed extensive loss of myelinated fibers and increased numbers of astrocytes with bizarre nuclei. This well-documented case of SSPE in an adult might be related to a condition of impaired cellular immunity.

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.

Platelet abnormalities in a dog suffering from gangrenous mastitis by Staphylococcus aureus infection.

Severe gangrenous mastitis due to Staphylococcus aureus infection was diagnosed in a 7 year-old intact female beagle which was presented with swelling of mammary glands after dystocia. Leukocytosis (25,200-48,600/microliters), decreased platelets (107,000-179,000/microliters), and abnormal platelet pattern continued during the critical condition. Consistent with platelet pattern, large platelets were observed in the blood smear. The number of leukocytes and platelets rapidly returned to normal during treatment, and the platelet pattern was also restored. The number and pattern of platelet may provide a clue for the evaluation of the clinical condition and/or severity of the lesions in the dog with mastitis.

[Four cases of adult Listeria monocytogenes infection in the last 5 years--hepatic necrotic foci in the adult septic case].

We have reported on the clinical courses of 4 cases of adult Listeria monocytogenes (Lm) infection, and the autopsy findings of 2 cases, those we have observed over the past 5 years. They were 2 cases of meningitis, 1 case of meningitis and sepsis and 1 case of sepsis. These 4 cases had CML, neoplastic angioendotheliosis, SLE and post-renal transplant condition, as their underlying diseases, and all were receiving immunosuppressive therapy. One meningitis patient who recovered showed mild liver dysfunction during her clinical course. The other 3 patients who died had jaundice at the time of onset and severe liver dysfunction. The 2 cases those were autopsied were the sepsis cases. The one with an acute course and hepatic failure showed multiple miliary necrotic foci in the liver, where the presence of Lm in the cells could be verified. The other autopsy case, which had received adequate antibiotic therapy and the Lm infection had been cured, showed no necrotic foci in the liver. The case that had necrotic foci in the liver was the first such adult case in Japan. We have discussed the hepatic Lm infection in adult compromised hosts, which conventionally has not been considered a serious problem.

[A patient with Cornelia de Lange syndrome with difficulty in orotracheal intubation].

We experienced a difficult orotracheal intubation in a patient with Cornelia de Lange syndrome. The patient was an eight-year-old girl with Cornelia de Lange syndrome, cleft palate and tetralogy of Fallot who underwent emergency hemicolectomy for strangulation ileus. Orotracheal intubation using a Macintosh laryngoscope was unsuccessful. However, intubation using an endotracheal tube through the laryngeal mask airway with a fiberoptic bronchoscope was successful. The patient's condition was stable during both intubation and operation. In conclusion, we must be careful on endotracheal intubation of patients with congenital anomalies.

[Changes in forehead and sole tissue temperature during cardiopulmonary bypass for acquired cardiac disease].

The forehead tissue temperature (FT-T) and the sole tissue temperature (ST-T) were measured and recorded by a deep body thermometer (Terumo Corp.) during open heart surgery. The changes in FT-T and ST-T after cardiopulmonary bypass (CPB) showed two characteristic patterns; the convergence pattern and the dissociation pattern. It is said that the dissociation pattern can notes a poor peripheral circulation. The deverging point of the two patterns was studied in 65 patients with acquired cardiac disease. We divided the patients into two groups taking 3.5 degrees C of the FT-T and ST-T difference (DT) after 2 hour weaning from CPB. The number of the patients in convergence group (group I) was 42, and that in the dissociation group (group II) was 23. The two groups were compared with the DT and the time required for the rise in each temperature from CPB rewarming to CPB weaning. There was no intergroup difference in the DT when the FT-T began to rise upon CPB rewarming. However, the DT was 3.8 +/- 2.3 (mean +/- SD) degrees C in group I and 7.1 +/- 1.8 degrees C in group II when the ST-T began to rise, and 4.2 +/- 2.5 degrees C in group I and 6.9 +/- 1.4 degrees C in group II when the FT-T reached its peak; the figures were significantly lower in group I (P < 0.01). The time required for the rise in ST-T was significantly shorter in group I (14.8 +/- 17.1 min) than in group II (25.8 +/- 14.7 min).(ABSTRACT TRUNCATED AT 250 WORDS)

[Bacterial contamination of anesthesiologists' hands and the efficacy of handwashing].

The purpose of this study was to examine bacterial contamination, especially by transient skin flora, that were on the hands of trainee and diplomate anesthesiologists during general anesthesia as well as to evaluate the efficacy of washing hands with running water or with three alcohol based antiseptic solutions using a modified glove juice method. The bacterial counts on the anesthesiologists' hands were 3.21 +/- 0.66 [log10 (mean +/- SD)] during induction, 255 +/- 1.15 during maintenance of anesthesia, 2.67 +/- 1.10 during extubation and 3.57 +/- 0.74 at the end of anesthesia. The diplomates' hands were more contaminated than those of the trainees during both intubation and extubation. Washing hands with running water or antiseptic solutions was effective to reduce bacterial contamination, but there was no disinfectant effect of antiseptic solutions against the bacteria that adhered to the hands after drying those solutions. Therefore to prevent nosocomial infection, anesthesiologists should wash their hands with running water or the antiseptic solution after each contact.

[Importance of instructing anesthesiologists in handwashing against bacterial contamination].

The importance of handwashing in preventing the spread of nosocomial infection has been stressed. Using a modified glove juice method, we have already shown the efficacy of washing of anesthesiologists' hands with alcohol based antiseptic solutions against bacterial contamination. The efficacy of handwashing, however, varied because many anesthesiologists washed only the palm and the back. In the present study, we instructed them to wash their hands using rubbing method, from finger tip to the wrist until drying the solutions completely. We compared the efficacy of handwashing, by decrease of bacteria counts after general anesthesia, between the instructed method and the customary way which was done ordinarily in the ward. The instructed method was more effective on the decrease of bacteria counts than the customary way. In the instructed method, the efficacy of handwashing was not different between the antiseptic solutions. In conclusion, doctors not only should practice handwashing with the antiseptic solutions after each contact with the patient, but also wash their hands from the finger tip to the wrist until they become completely dry.

[Difficult adult airway and endotracheal intubation].

In 1984, Cormack and Lehane defined laryngoscopic view in four grades. As the view worsens, the difficulty of intubation may increase but it is not clear. In this study, we examined the endotracheal intubation techniques to the grade III or IV airways. Some 48 patients were determined as grade III and IV. In 26 patients the conventional endotracheal intubation technique (conventional technique) was selected. In 20 patients endotracheal intubation was performed over the gum-elastic bougie (bougie technique). In two patients laryngeal mask airway, fiberoptic bronchoscope and handmade flexible guide tube were used as aids to endotracheal intubation (guide technique). Nineteen patients with conventional technique and 6 patients with bougie technique required the external laryngeal pressure. In conclusion, the grade III or IV airways were not always difficult to intubate. But when the conventional technique failed, the gum-elastic bougie or laryngeal mask airway was a fairly useful aid to endotracheal intubation. Moreover our handmade flexible guide tube made the intubation through the laryngeal mask airway safe and reliable.

[Changes in respiration and hemodynamics during open heart surgery without blood transfusion].

The changes in respiration and hemodynamics during open heart surgery were studied in 25 patients undergoing coronary artery bypass grafting without blood transfusion. The respiratory and circulatory parameters were measured at the time of anesthetic induction and after cardiopulmonary bypass (CPB). The values after CPB were compared with those at the time of anesthetic induction. The values of HR, CI, SVI and mPAP increased, and the values of mAP, SVRI and PVRI decreased after CPB. The PaO2, BE and pH decreased but PaCO2, A-aDO2 and QS/QT increased after CPB. Although VO2I and DO2I increased after CPB, OER (VO2I/DO2I) was unchanged. Arterial lactate, pyruvate and cortisol levels increased after weaning from CPB. Hemodynamics during open heart surgery without blood transfusion showed hyperdynamic state after CPB. Hypoxia was not evident in the peripheral tissue. This suggests that the depression of the oxygen delivery with hemodilution is compensated by hyperdynamic circulation. Coronary artery bypass grafting without blood transfusion seems to offer no clinical problems.