Staphylococcus aureus-derived membrane vesicles exacerbate skin inflammation in atopic dermatitis.

BACKGROUND: Skin colonization or infection with Staphylococcus aureus is known to trigger aggravation of atopic dermatitis (AD). However, the exact mechanisms by which S. aureus can worsen AD are unknown. OBJECTIVE: We investigated whether and how S. aureus-derived membrane vesicles (MVs) contribute to worsening of AD. METHODS: Immunohistochemical and immunoelectron microscopic analyses were performed to detect staphylococcal protein A (SPA) in the epidermis of AD lesions. HaCaT cells were treated with S. aureus MVs and were analysed for the expression of cytokine genes. Immunopathology and cytokine gene profiles were analysed after topical application of S. aureus MVs to AD-like skin lesions in a mouse model. RESULTS: The MV component SPA was detected in the keratinocytes as well as in the intercellular space of the epidermis of AD lesions colonized with S. aureus. Intact MVs from S. aureus delivered their components to keratinocytes and stimulated pro-inflammatory cytokine gene expression in vitro. A knock-down of Toll-like receptor 2 or nucleotide-binding oligomerization domain 2 using small interfering RNAs suppressed interleukin-8 gene expression. Topical application of intact S. aureus MVs to AD-like skin lesions in the mouse model induced massive infiltration of inflammatory cells and the resulting eczematous dermatitis. This inflammatory reaction was associated with a mixed Th1/Th2 immune response and enhanced expression of chemokine genes in AD-like skin lesions. CONCLUSIONS AND CLINICAL RELEVANCE: This study showed the importance of S. aureus MVs as a potent mediator for worsening of AD among many exogenous worsening factors of AD. Thus, S. aureus MVs may be regarded as one of the therapeutic targets for the management of AD aggravation.

Inhibition of mTOR affects protein stability of OGT.

Autophagy regulates cellular homeostasis through degradation of aged or damaged subcellular organelles and components. Interestingly, autophagy-deficient beta cells, for example Atg7-mutant mice, exhibited hypoinsulinemia and hyperglycemia. Also, autophagy response is diminished in heart of diabetic mice. These results implied that autophagy and diabetes are closely connected and affect each other. Although protein O-GlcNAcylation is up-regulated in hyperglycemia and diabetes, and O-GlcNAcylated proteins play an important role in metabolism and nutrient sensing, little is known whether autophagy affects O-GlcNAc modification and vice versa. In this study, we suppressed the action of mTOR by treatment of mTOR catalytic inhibitors (PP242 and Torin1) to induce autophagic flux. Results showed a decrease in global O-GlcNAcylation, which is due to decreased OGT protein and increased OGA protein. Interestingly, knockdown of ATG genes or blocking of lysosomal degradation enhanced protein stability of OGT. In addition, when proteasomal inhibitor was treated together with mTOR inhibitor, protein level of OGT almost recovered to control level. These data suggest that mTOR inhibition is a more efficient way to reduce protein level of OGT rather than that of CHX treatment. We also showed that not only proteasomal degradation regulated OGT stability but autophagic degradation also affected OGT stability in part. We concluded that mTOR signaling regulates protein O-GlcNAc modification through adjustment of OGT stability.

Multiple displacement amplification for preimplantation genetic diagnosis of fragile X syndrome.

Preimplantation genetic diagnosis (PGD) has become an assisted reproductive technique for couples that have genetic risks. Despite the many advantages provided by PGD, there are several problems, including amplification failure, allele drop-out and amplification inefficiency. We evaluated multiple displacement amplification (MDA) for PGD of the fragile X syndrome. Whole genome amplification was performed using MDA. MDA products were subjected to fluorescent PCR of fragile X mental retardation-1 (FMR1) CGG repeats, amelogenin and two polymorphic markers. In the pre-clinical tests, the amplification rates of the FMR1 CGG repeat, DXS1215 and FRAXAC1 were 84.2, 87.5 and 75.0%, respectively, while the allele dropout rates were 31.3, 57.1 and 50.0%, respectively. In two PGD treatment cycles, 20 embryos among 30 embryos were successfully diagnosed as 10 normal embryos, four mutated embryos and six heterozygous carriers. Three healthy embryos were transferred to the uterus; however, no clinical pregnancy was achieved. Our data indicate that MDA and fluorescent PCR with four loci can be successfully applied to PGD for fragile X syndrome. Advanced methods for amplification of minuscule amounts of DNA could improve the sensitivity and reliability of PGD for complicated single gene disorders.

Time series analysis of delta neutrophil index as the predictor of sepsis in patients with acute poisoning.

Delta neutrophil index (DNI), which reflects the fraction of immature granulocytes, is used to detect infection and sepsis from noninfectious conditions, but few studies have evaluated in the early stage of acute poisoning. This retrospective observational study was performed on acute poisoning patients who visited to the emergency department (ED) and were consecutively admitted in intensive care units over 18-month period. The serial DNI, conventional inflammatory biomarkers, and culture results were obtained in the ED and after admission. The outcomes were the identification of sepsis, bacteremia, and 30-day mortality. Of 166 patients (mean age, 56.0 years) in this cohort, 59 (35.5%) had sepsis and 29 (17.5%) had bacteremia. Initial and peak DNI fractions 24 h after ED admission were strong independent predictors of sepsis development. Analysis of the area under the curve according to multiple receiver operating characteristics showed that DNI had a higher capability to predict sepsis than other parameters (0.815 for DNI, 0.700 for procalcitonin, 0.681 for C-reactive protein, and 0.741 for white blood cell). Using multivariable logistic regression analysis, it was found that DNI was an independent predictor of sepsis (95% confidence interval (CI) of odds: 1.03-1.18) and bacteremia (95% CI: 1.01-1.14). Therefore, initial and serial measurement of DNI may serve as useful risk predictor for development of sepsis or bacteremia in acute poisoning.

Mismatch repair by efficient nick-directed, and less efficient mismatch-specific, mechanisms in homologous recombination intermediates in Chinese hamster ovary cells.

Repair of single-base mismatches formed in recombination intermediates in vivo was investigated in Chinese hamster ovary cells. Extrachromosomal recombination was stimulated by double-strand breaks (DSBs) introduced into regions of shared homology in pairs of plasmid substrates heteroallelic at 11 phenotypically silent mutations. Recombination was expected to occur primarily by single-strand annealing, yielding predicted heteroduplex DNA (hDNA) regions with three to nine mismatches. Product spectra were consistent with hDNA only occurring between DSBs. Nicks were predicted on opposite strands flanking hDNA at positions corresponding to original DSB sites. Most products had continuous marker patterns, and observed conversion gradients closely matched predicted gradients for repair initiated at nicks, consistent with an efficient nick-directed, excision-based mismatch repair system. Discontinuous patterns, seen in approximately 10% of products, and deviations from predicted gradients provided evidence for less efficient mismatch-specific repair, including G-A-->G-C specific repair that may reflect processing by a homologue of Escherichia coli MutY. Mismatch repair was > 80% efficient, which is higher than seen previously with covalently closed, artificial hDNA substrates. Products were found in which all mismatches were repaired in a single tract initiated from one or the other nick. We also observed products resulting from two tracts of intermediate length initiated from two nicks.

Microbial community in biofilm on membrane surface of submerged MBR: effect of in-line cleaning chemical agent.

The objective of this study was to investigate the change in microbial community pattern with the effect of cleaning agent using a quinone profile that is used for membrane in-line chemical cleaning in SMBR. The dominant quinone types of biofilm were ubiquinone (UQs)-8, -10, followed by menaquinone (MKs)-8(H4), -7 and UQ-9, but those of suspended microorganisms were UQ-8, UQ-10 followed by MKs-8(H4), -7 and -11. Both UQ and MK contents decreased with increasing NaCIO dosage and it seems that there is more resistance from UQ compared to MK. In addition, COD and DOC concentrations increased with increasing NaClO dosage up to 0.05 g-NaCIO/g-SS. The organic degradation performance of the microbial community in the presence of NaClO was impaired. The present study suggested that larger added amounts of NaClO caused an inhibition of organic degradation and cell lysis.

A non-cycling mitotic cyclin in the naturally synchronous cell cycle of Physarum polycephalum.

A universal model of the control of the cell cycle in eukaryotic organisms has emerged from the discovery that MPF (maturation or mitosis promoting factor) is a heterodimer consisting of a catalytic subunit (p34cdc2) and a regulatory subunit (mitotic cyclin) encoded by a pair of conserved genes. A prominent feature of the periodic activation of the protein kinase p34cdc2 is the gradual accumulation of cyclin in interphase and its abrupt degradation in mitosis, which is believed to be required for inactivation of MPF and exit from mitosis. Utilizing the precise natural synchrony of mitosis of the plasmodium of the myxomycete Physarum, the high affinity of the p34cdc2/cyclin B complex to p13suc1 Sepharose beads, and immunological reagents including three different anticyclin B antibodies and the anti-PSTAIR antibody, a transient histone H1 kinase activation but not fluctuation in the abundance of cyclin B have been detected during mitosis. It is argued that cyclin degradation may be required for cytokinesis and/or postmitotic controls of cell proliferation in G1 phase and cell-to-cell signaling in development but not for the inactivation of histone H1 kinase in mitosis.

Thirteen UDPglucuronosyltransferase genes are encoded at the human UGT1 gene complex locus.

The original novel UGT1 complex locus previously shown to encode six different UDP-glucuronosyltransferase (transferase) genes has been extended and demonstrated to specify a total of 13 isoforms. The genes are designated UGT1A1 through UGT1A13p with four pseudo ones. UGT1A2p and UGT1A11p through UGT1A13p have either nucleotide deletions or flawed TATA boxes and are therefore pseudo. In the 5' region of the locus, the 13 unique exons 1 are arranged in a tandem array with each having its own proximal TATA box element and, in turn, are linked to four common exons to allow for the independent transcriptional initiation to generate overlapping primary transcripts. Only the lead exon in the nine viable primary transcripts is predicted to undergo splicing to the four common exons generating mRNAs with identical 3' ends and transferase isozymes with an identical carboxyl terminus. The unique amino terminus specifies acceptor-substrate selection, and the common carboxyl terminus apparently specifies the interaction with the common donor substrate, UDP-glucuronic acid. In the extended region, the viable TATA boxes are either A(A)TgA(AA)T or AT14AT; in the original locus the element for UGT1A1 is A(TA)7A and TAATT/CAA(A) for all of the other genes. UGT1A1 specifies the critically important bilirubin transferase isoform. The relationships of the exons 1 to each other are as follows: UGT1A2p through UGT1A5 comprises a cluster A that is 87-92% identical, and UGT1A7 through UGT1A13p comprises a cluster B that is 67-91% identical. For the two not included in a cluster, UGT1A1 is more identical to cluster A at 60-63%, whereas UGT1A6 is identical by between 48% and 56% to all other unique exons. The locus was expanded from 95 kb to 218 kb. Extensive probing of clones beyond 218 kb with coding nucleotides for a highly conserved amino acid sequence present in all transferases was unable to detect other exons 1. The mRNAs are differentially expressed in hepatic and extrahepatic tissues. This locus is indeed novel, indicating the least usage of exon sequences in specifying different transferase isozymes that have an expansive substrate range.

p21(WAF1) is associated with CDK2 and CDK4 protein during HL-60 cell differentiation by TPA treatment.

TPA-treated HL-60 cells are mainly arrested in G1 by p21(WAF1) accumulation. We investigate the downstream changes following such accumulation. Increased p21(WAF1) is associated with CDK2 and CDK4. pRb is dephosphorylated in the presence of p21-CDK2/4 complexes, and the Rb-E2F1 complex increases after TPA treatment, whereas the Rb-HDAC1 complex decreases slightly. Our results suggest that increased p21(WAF1) is associated with CDK2/4, and that these complexes induce pRb dephosphorylation. In turn, hypophosphorylated pRb are mainly complexed with E2F1, but HDAC1 appears not to be a key component in this process.

A single-stranded telomere binding protein in the nematode Caenorhabditis elegans.

We identified and characterized a protein (STB-1) from the nuclear extract of Caenorhabditis elegans that specifically binds single-stranded telomere DNA sequences, but not the corresponding RNA sequences. STB-1 binding activity is specific to the nematode telomere, but not to the human or plant telomere. STB-1 requires the core nucleotides of GCTTAGG and three spacer nucleotides in front of them for binding. While any single nucleotide change in the core sequence abolishes binding, the spacer nucleotides tolerate substitution. STB-1 was determined to be a basic protein of 45 kDa by Southwestern analyses. STB-1 forms a stable complex with DNA once bound to the telomere.

Lack of mutation at p16INK4A gene but expression of aberrant p16INK4A RNA transcripts in human ovarian carcinoma.

Alterations of the p16INK4A gene are frequent in various human cancers. We investigated p16INK4A gene status in 20 ovarian carcinomas by PCR (polymerase chain reaction), PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) and sequencing techniques. None of the primary tumors showed any mutational or deletional events. However, 19 out of 20 tumors displayed both a methylated and an unmethylated p16INK4A promoter. In some of these samples, we detected aberrant p16INK4A transcripts, with partial deletions of both exons 1 and 2, which could not encode a functional p16INK4A protein. The sequences of the aberrant mRNA revealed common 4-7 nucleotide sequences before and after the deleted region, which might cause abnormal splicing of mRNA transcripts. These results suggest that both promoter methylation and aberrant mRNA processing may interfere with p16INK4A expression in ovarian tumors.

5-Aza-2'-deoxycytidine leads to down-regulation of aberrant p16INK4A RNA transcripts and restores the functional retinoblastoma protein pathway in hepatocellular carcinoma cell lines.

The inactivation of the cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor p16INK4A may be caused by gene deletion, mutation or promoter hypermethylation. We have previously reported that p16INK4A in hepatocellular carcinoma (HCC) tissues and cell lines is inactivated predominantly by promoter hypermethylation rather than genomic aberrations. In the present experiments, we have studied the effects of the demethylating agent, 5-aza-2'-deoxycytidine (5-AZA/decitabine), on the expression of aberrant p16INK4A RNA transcripts and the CDK-retinoblastoma gene pathway in HCC cell lines with p16INK4A promoter hypermethylation. The expression of aberrant p16INK4A RNA transcripts was down-regulated and p16INK4A protein was strongly re-expressed in the HCC cell lines, SNU 354, 398, 423 and 475 after 5-AZA/decitabine treatment for 5 days. The re-expressed p16INK4A was functional, because it bound to and inhibited CDK4 kinase activity, and increased the concentrations of the hypophosphorylated form of retinoblastoma protein (pRB) in cells with a wild type RB gene. Moreover, treatment with the demethylating agent led not only to G1 cell cycle arrest, but also to the increased expression of the senescence-associated marker beta-galactosidase. This up-regulation of p16INK4A mRNA and protein correlated with demethylation of the p16INK4A promoter, and with the down-regulation or disappearance of aberrant p16INK4A transcripts. These results suggest that the aberrant p16INK4A RNA transcript can be transcribed from the methylated p16INK4A gene, and endogenous reactivation of functional p16INK4A mRNA by a demethylating agent can restore the pRB pathway in HCC, and foster the terminal differentiation of the malignant cells. Therefore, demethylating agents, such as 5-AZA/decitabine, may have potential in the treatment of HCC.

4 x 10 Gb/s terrestrial optical free space transmission over 1.2 km using an EDFA preamplifier with 100 GHz channel spacing.

We demonstrate a transmission of 4 x 10 Gb/s WDM channels over 1.2 km of free space in 1.55-microm band. The transmitted beam is coupled into a single-mode fiber through a fiber-pigtailed collimator, which enables the use of standard 100-GHz channel spacing and an optical preamplifier at the receiver. All the received channels have Q values higher than 6.

Apoptotic cell death induced by Acinetobacter baumannii in epithelial cells through caspase-3 activation.

Epithelial cell death induced by Acinetobacter baumannii infection was investigated using in vitro assays. Eight hours after live A. baumannii infection, HeLa cells exhibited detachment from the dish, rounding morphologies, high proportions of trypan blue-positive cells and extensive DNA breakdown with faint apoptotic banding, which is indicative of cells undergoing apoptosis. The enzymatic activity of caspase-3 was increased in cells as early as 2 h after infection. In addition, apoptosis of HeLa cells was induced by treatment with bacterial culture filtrates but not with formalin-killed bacteria. These results indicate that A. baumannii infection triggers apoptosis in HeLa cells through caspase-3 activation.

Antimicrobial resistance of Shigella sonnei in Korea during the last two decades.

Eighty-eight strains of Shigella sonnei isolated in Korea during the period 1980 to 1999 were tested for susceptibility to 13 antimicrobial agents. S. sonnei isolates demonstrated high frequencies of resistance to sulfamethoxazole (97.7%), tetracycline (96.6%), and trimethoprim (95.5%). S. sonnei isolates from the 1990s were more resistant to nalidixic acid than isolates from the 1980s (100 vs 7.7%), while isolates from the 1990s were more susceptible to chloramphenicol than isolates from the 1980s (0 vs 100%). Ampicillin-resistant S. sonnei isolates produced the TEM-1 beta-lactamase with a pI of 5.4. The TEM-1 gene was located on conjugally transferable plasmids in the majority of isolates. S. sonnei isolates were all susceptible to cefotaxime, cefoxitin, ceftazidime, ciprofloxacin, and norfloxacin. These results indicate that cephalosporins and quinolones may be alternative antibiotics for the treatment of S. sonnei infections in Korea.

A method of microinjection: delivering monoclonal antibody 1223 into sea urchin embryos.

In this paper, a simpler method of microinjecting sea urchin embryos without using the conventional microinjection chamber designed by Kiehart is reported. A trough was made on a surface of 0.6% agarose gel dissolved in artificial sea water. Approximately fifty hatched embryos could be loaded in the trough and, consequently, swimming embryos were trapped in the trough. Monoclonal antibody (mAB) 1223 which blocks spiculogenesis in vitro was delivered into the blastocoels of sea urchin embryos to test whether this antibody inhibits spiculogenesis in vivo and also, whether this new technique is effective for the microinjection of the sea urchin embryos. The embryos were injected with mAB1223 at the hatched blastula, early mesenchyme blastula and early gastrula stages, and 63%, 90% and 97% of the embryos did not form spicules at the late gastrula stage, respectively. Therefore, mAB1223 was shown to also block spiculogenesis in vivo. From the fact that spiculogenesis occurred at a lower rate when mAB1223 was injected at the hatched blastula stage than at later stages, it may be speculated that endogenous proteases degraded the injected antibodies. Using this technique, extracellular events in the blastocoel or the function of certain molecules expressed in blastocoel can be easily investigated in vivo.

Creatine kinase B is a target molecule of reactive oxygen species in cervical cancer.

Recently, a procedure for detecting ROS-sensitive proteins that contain active cysteine residues was devdoped. The method is based on the fact that biotin-conjugated iodoacetamide (BIAM) and ROS competitively and selectively react with the active cysteine residues in ROS-sensitive proteins. To investigate the role of ROS in cervical cancer, BIAM labeling on cytosolic proteins in normal and cancer tissues was performed, respectively. The BIAM labeling proteins are separated by 2-dimensional electrophoresis, and then identified by MALDI-TOF mass analysis. ROS-sensitive protein is identified as creatine kinase B containing cysteine residue in active center. Activity of creatine kinase B in normal tissue is higher than that of oxidized form in cervical cancer tissues. The result suggests that ROS play an important role in metabolic regulation in cervical cancer cells. However, molecular mechanisms that ROS and creatine kinase B are integrated into a physiological signal leading to the cellular transformation remain to be elucidated.

Squamous cell lung cancer with solitary subungual metastasis.

Subungual metastasis resulting from internal malignancies is an extremely rare event. A few cases of subungual metastasis from lung cancer have been reported. However, subungual metastasis arising from lung cancer without any other form of distant metastases has not been reported. The misdiagnosis of a solitary subungual metastases as a benign inflammatory lesion is an important problem as it may cause the misdiagnosis of a lower stage of lung cancer. We may be reporting the first case of a subungual metastasis from lung cancer without any other distant metastases.

Developmental patterns of GalBeta1,3(4)GlcNAc alpha2,3-sialyltransferase (ST3Gal III) expression in the mouse: in situ hybridization using DIG-labeled RNA probes.

Sialic acids are key determinants for biological processes, such as cell-cell interaction and differentiation. Sialyltransferases contribute to the diversity in carbohydrate structure through their attachment of sialic acid in various terminal positions on glycolipid and glycoprotein (N-linked and O-linked) carbohydrate groups. Galbeta 1,3(4)GlcNAc alpha2,3-sialyltransferase (ST3Gal III) is involved in the biosynthesis of sLe(x)and sLe(a) known as selectin ligands and tumor-associated carbohydrate structures. The appearance and differential distribution of ST3Gal III mRNA during mice embryogenesis [embryonic (E) days; E9, E11, E13, E15] were investigated by in situ hybridization with digoxigenin-labeled RNA probes coupled with alkaline phosphatase detection. On E9, all tissues were positive for ST3Gal III mRNA expression, whereas ST3Gal III mRNA on E11 was not detected throughout all tissues. On E13, ST3Gal III mRNA was expressed in different manner in various tissues. In this stage, ST3Gal III mRNA was positive only in the liver, pancreas and bladder. On E15, specific signal for ST3Gal III was detected in the liver, lung and forebrain. These results indicate that ST3GAI III is differently expressed at developmental stages of mice embryo, and this may be importantly related with regulation of organogenesis in mice.