Resolvin D1 Suppresses H2O2-Induced Senescence in Fibroblasts by Inducing Autophagy through the miR-1299/ARG2/ARL1 Axis.

ARG2 has been reported to inhibit autophagy in vascular endothelial cells and keratinocytes. However, studies of its mechanism of action, its role in skin fibroblasts, and the possibility of promoting autophagy and inhibiting cellular senescence through ARG2 inhibition are lacking. We induced cellular senescence in dermal fibroblasts by using H2O2. H2O2-induced fibroblast senescence was inhibited upon ARG2 knockdown and promoted upon ARG2 overexpression. The microRNA miR-1299 suppressed ARG2 expression, thereby inhibiting fibroblast senescence, and miR-1299 inhibitors promoted dermal fibroblast senescence by upregulating ARG2. Using yeast two-hybrid assay, we found that ARG2 binds to ARL1. ARL1 knockdown inhibited autophagy and ARL1 overexpression promoted it. Resolvin D1 (RvD1) suppressed ARG2 expression and cellular senescence. These data indicate that ARG2 stimulates dermal fibroblast cell senescence by inhibiting autophagy after interacting with ARL1. In addition, RvD1 appears to promote autophagy and inhibit dermal fibroblast senescence by inhibiting ARG2 expression. Taken together, the miR-1299/ARG2/ARL1 axis emerges as a novel mechanism of the ARG2-induced inhibition of autophagy. Furthermore, these results indicate that miR-1299 and pro-resolving lipids, including RvD1, are likely involved in inhibiting cellular senescence by inducing autophagy.

Upregulated Guanine Deaminase Is Involved in Hyperpigmentation of Seborrheic Keratosis via Uric Acid Release.

Seborrheic keratosis, which is a benign tumor composed of epidermal keratinocytes, develops common in the elderly. Uric acid generated by upregulated guanine deaminase (GDA) has been identified to cause UV-induced keratinocyte senescence in seborrheic keratosis. Seborrheic keratosis is also frequently pigmented. Growing evidences indicate that hyperuricemia is a risk factor of acanthosis nigricans, an acquired skin hyperpigmentation. The objective of this study was to investigate role of GDA and its metabolic end product, uric acid, in hyperpigmentation of patients with seborrheic keratosis using their lesional and non-lesional skin specimen sets and cultured primary human epidermal keratinocytes with or without GDA overexpression or uric acid treatment. GDA-overexpressing keratinocytes or their conditioned media containing uric acid increased expression levels of MITF and tyrosinase in melanocytes. Uric acid released from keratinocytes was facilitated by ABCG2 transporter with the help of PDZK1 interaction. Released uric acid was taken by URAT1 transporter in melanocytes, stimulating melanogenesis through p38 MAPK activation. Overall, GDA upregulation in seborrheic keratosis plays a role in melanogenesis via its metabolic end product uric acid, suggesting that seborrheic keratosis as an example of hyperpigmentation associated with photoaging.

Guanine Deaminase Stimulates Ultraviolet-induced Keratinocyte Senescence in Seborrhoeic Keratosis via Guanine Metabolites.

DNA damage and oxidative stress play a critical role in photoageing. Seborrhoeic keratosis (SK) affects sunlight-exposed sites in aged individuals. This study examined the mechanism of photoageing in SK. The guanine deaminase gene, which is involved in purine metabolism, was upregulated with uric acid levels and p21 in SK. Guanine deaminase was detectable in keratinocytes. Repeated exposure to ultraviolet (UV) increased levels of guanine deaminase, together with DNA damage, such as γ-H2AX and cyclobutane pyrimidine dimer formation, generation of reactive oxygen species, and keratinocyte senescence, which were reversed by guanine deaminase knockdown. However, guanine deaminase overexpression and H2O2 formed γ-H2AX, but not cyclobutane pyrimidine dimer. Loss-of-function guanine deaminase mutants reduced the metabolic end-product uric acid, which was increased by exposure to exogenous xanthine. Repeated exposure to UV increased levels of uric acid. Exogenous uric acid increased cellular senescence, reactive oxygen species, and γ-H2AX, similar to guanine deaminase. Overall, guanine deaminase upregulation increased UV-induced keratinocyte senescence in SK, via uric acid mediated by reactive oxygen species followed by DNA damage.

IL-1 Receptor Antagonist Reduced Chemical-Induced Keratinocyte Apoptosis through Antagonism to IL-1α/IL-1ß.

Extracellular interleukin 1 alpha (IL-1α) released from keratinocytes is one of the endpoints for in vitro assessments of skin irritancy. Although cells dying via primary skin irritation undergo apoptosis as well as necrosis, IL-1α is not released in apoptotic cells. On the other hand, active secretion has been identified in interleukin-1 receptor antagonist (IL-1ra), which was discovered to be a common, upregulated, differentially-expressed gene in a microarray analysis performed with keratinocytes treated using cytotoxic doses of chemicals. This study examined whether and how IL-1ra, particularly extracellularly released IL-1ra, was involved in chemically-induced keratinocyte cytotoxicity and skin irritation. Primary cultured normal adult skin keratinocytes were treated with cytotoxic doses of chemicals (hydroquinone, retinoic acid, sodium lauryl sulfate, or urshiol) with or without recombinant IL-1ra treatment. Mouse skin was administered irritant concentrations of hydroquinone or retinoic acid. IL-1ra (mRNA and/or intracellular/extracellularly released protein) levels increased in the chemically treated cultured keratinocytes with IL-1α and IL-1ß mRNAs and in the chemically exposed epidermis of the mouse skin. Recombinant IL-1ra treatment significantly reduced the chemically-induced apoptotic death and intracellular/extracellularly released IL-1α and IL-1ß in keratinocytes. Collectively, extracellular IL-1ra released from keratinocytes could be a compensatory mechanism to reduce the chemically-induced keratinocyte apoptosis by antagonism to IL-1α and IL-1ß, suggesting potential applications to predict skin irritation.

Increased Skin Irritation by Hydroquinone and Rsetinoic Acid Used in Combination.

BACKGROUND: Hydroquinone (HQ) is frequently combined with retinoic acid (RA) to enhance lightening efficacy, which may also affect skin irritancy. Although skin irritation leads to postinflammatory hyperpigmentation, little research has been performed to compare skin irritancy between each component and the combination. OBJECTIVE: This study was done to examine whether HQ-RA combination increased skin irritation induced by HQ or RA alone. METHODS: Patch testing was performed using maximum therapeutic and higher concentrations of HQ and RA in 10 volunteers, and then, it was performed using their popular therapeutic concentrations and combination in the other 20 volunteers. In vitro irritation was also assessed in primary cultured normal human keratinocytes treated with 80% and 50% cell survival doses of HQ, 80% cell survival dose of RA, and their combination. RESULTS: The combination in patch testing induced stronger erythema than the corresponding concentrations of HQ and RA, which was remarkable with use of combination of higher concentrations. In cultured keratinocytes, the RA combination significantly decreased cell viability, but increased cytotoxicity and extracellular interleukin 1 alpha release with corresponding doses of HQ. CONCLUSION: The results of patch tests and in vitro irritation assessment tests suggested that HQ and RA increased skin irritation when used in combination.

Complementary effect of hydroquinone and retinoic acid on corneocyte desquamation with their combination use.

BACKGROUND: Retinoic acid (RA) enhances skin-lightening capabilities of hydroquinone (HQ), at least in part, by facilitating desquamation which leads to increase penetration of HQ. The desquamation also affects skin irritation levels. The mechanism of RA-induced desquamation, however, has not been completely explored and no such data has been available for HQ uses. OBJECTIVE: To examine the role of HQ, RA, and their combination in the desquamation. METHODS: Primary cultured normal human keratinocytes, which were treated with HQ and/or RA in presence or absence of serine-specific inhibitor Kazal type5 (SPINK5)/lympho-epithelial Kazal-type-related inhibitor (LEKTI) knockdown or recombinant human SPINK5/LEKTI, and biopsied skin samples applied with HQ or RA were examined. Expression levels of corneodesmosin (CDSN), desmocollin1 (DSC1), kallikrein5 (KLK5), KLK7, and SPINK5/LEKTI, and proteolysis activity against extracted human skin epidermal protein were determined using time-course real-time PCR, Western blotting, ELISA, and immunofluorescence staining. RESULTS: HQ increased but RA decreased the synthesis of CDSN and DSC1. HQ reduced corneodesmosome degradation by the upregulation of SPINK5/LEKTI, whereas RA showed opposite results without upregulation of SPINK5/LEKTI. The combination of HQ and RA was close to the sum of the individual components. CONCLUSIONS: HQ reduced corneocyte desquamation. However, RA enhanced desquamation. The combination induced more desquamation than HQ but less than RA.

Effects of low-dose light-emitting-diode therapy in combination with water bath for atopic dermatitis in NC/Nga mice.

BACKGROUND: Light-emitting diode (LED) phototherapy and water bath therapy have beneficial effect on atopic dermatitis (AD)-like skin disease. However, not all current treatments work well and alternative therapies are need. The contribution of combination therapy with low-dose 850 nm LED and water bath was investigated on dermatophagoides farina (Df)-induced dermatitis in NC/Nga mice. METHODS: Low-dose LED (10, 15, and 20 J/cm(2) ) irradiation, water bath (36 ± 1°C) were administered separately and together to the Df-induced NC/Nga mice in acrylic jar once a day for 2 weeks. RESULTS: Combined therapy with low-dose LED therapy and water bath therapy significantly ameliorated the development of AD-like skin lesions. These effects were correlated with the suppression of total IgE, NO, histamine, and Th2-mediated immune responses. Furthermore, combination therapy significantly reduced the infiltration of inflammatory cells and the induction of thymic stromal lymphopoietin (TSLP) in the skin lesions. The beneficial therapeutic effects of this combination therapy might regulate by the inhibition of various immunological responses including Th2-mediated immune responses, inflammatory mediators such as IgE, histamine, and NO, as well as inflammatory cells. CONCLUSIONS: The combination therapy of LED and water bath might be used as an efficacious, safe, and steroid-free alternative therapeutic strategy for the treatment of AD.

Combination of glucosamine and low-dose cyclosporine for atopic dermatitis treatment: a randomized, placebo-controlled, double-blind, parallel clinical trial.

Our recent pilot study showed better outcomes using a combination of low-dose cyclosporine and glucosamine than cyclosporine alone in the treatment of atopic dermatitis (AD). Here, a randomized, placebo-controlled, double-blind, parallel-designed study was planned to compare the efficacy and safety of low-dose cyclosporine and glucosamine combination to low-dose cyclosporine alone for the treatment of patients with moderate to severe AD. AD patients with a Severity Scoring of Atopic Dermatitis (SCORAD) index ≥ 30 were randomly assigned in a 1:1 ratio to receive either cyclosporine 2 mg/kg and glucosamine 25 mg/kg (group A) or cyclosporine and placebo (group B) for 8 weeks. SCORAD indices, serum levels of chemokine ligand 17 and interleukin-31, eosinophil counts, and blood cyclosporine levels were examined before and after treatment. The SCORAD indices for group A (n = 19) were significantly reduced after the treatment and a significant correlation between the changes in the SCORAD indices and changes in the serum levels of chemokine ligand 17, but not interleukin-31, was detected. Glucosamine combined with cyclosporine did not increase adverse events and serum cyclosporine levels compared with cyclosporine alone. Therefore, combination of low-dose cyclosporine and glucosamine may be useful to allow the long-term use of cyclosporine in the treatment of patients with moderate to severe AD.

Retinoic acid and hydroquinone induce inverse expression patterns on cornified envelope-associated proteins: implication in skin irritation.

BACKGROUND: Hydroquinone (HQ) with or without retinoic acid (RA) is routinely used for the treatment of hyperpigmented conditions. Skin irritation is a major problem with popular depigmenting agents, resulting in postinflammatory hyperpigmentation. OBJECTIVE: To examine the molecular mechanism associated with skin irritation by RA or HQ. METHODS: A genome-wide transcriptional profiling analysis was performed using monolayer cultures of human keratinocytes treated with or without irritant doses of RA, HQ, or sodium lauryl sulfate (SLS), a representative irritant. Differentially expressed genes (DEGs) were mapped on human chromosomes using a Manhattan plot. For the validation of candidate DEGs, the chemicals with different concentrations of varying irritation intensities were applied in vitro and in vivo and analyzed using real time-PCR and Western blotting. RESULTS: DEGs mapped to the 1q21 locus, which is composed of a cluster of genes encoding the cornified envelope precursors, showed an inverse expression pattern in response to HQ and RA. Concentrations of RA and HQ that induced a broad range of irritant responses in cultured cells or mice skin also induced inverse effects on the expression of cornified envelope-associated proteins. CONCLUSIONS: Genetic modulation on cornified envelope-associated proteins by RA-induced irritation, which may be involved in physiological skin barrier disturbance, could be inverse to that by HQ- or SLS-induced irritation.

S100B as a potential biomarker for the detection of cytotoxicity of melanocytes.

Skin irritation is one of the most common adverse reactions in hydroquinone (HQ) and retinoic acid (RA). Although melanocytes have rarely been considered to be involved in skin irritation, RA and particularly HQ could induce melanocyte toxicity, resulting in depigmentation. We chose S100B as a candidate gene for melanocytotoxicity from a genome-wide transcriptional profiling analysis after applying irritant doses of HQ, RA and sodium lauryl sulphate (SLS) to cultures of keratinocytes and/or melanocytes. In this study, the role of S100B on melanocyte viability and cytotoxicity was examined. S100B was detected in melanocytes, but not in keratinocytes or fibroblasts. Melanocytes after treatment with increasing concentrations of HQ, RA, SLS and urushiol showed significant increases in intracellular and extracellular S100B expression with reduced viable cell number and increased release of lactate dehydrogenase. No RAGE expression and no significant function of CD166/ALCAM in melanocyte survival and cytotoxicity favoured the role of intracellular S100B in chemically irritated melanocytes. S100B knock-down increased apoptosis through inhibition of PI3K/AKT, NF-κB and ERK activation, suggesting the increased intracellular S100B expression by chemical irritation as a compensatory reaction to reduce cytotoxicity. The numerical decrease in S100B/c-kit-double-positive melanocytes was also examined in human skin epidermis irritated by HQ or RA with stronger staining intensities of S100B. Collectively, the decrease in viable cell number by reduced intracellular S100B levels in vitro and by chemical irritation in vivo suggests that S100B could be a potential biomarker for melanocytes cytotoxicity.

850nm light-emitting-diode phototherapy plus low-dose tacrolimus (FK-506) as combination therapy in the treatment of Dermatophagoides farinae-induced atopic dermatitis-like skin lesions in NC/Nga mice.

BACKGROUND: Light emitting diode (LED) phototherapy is an effective alternative for the treatment of inflammatory skin disorders. Tacrolimus (FK-506) is a potent immunomodulating agent, which has been used to treat AD. Combination therapy is often used in the treatment of AD to improve therapeutic efficacy or to reduce the dose of each drug. OBJECTIVE: To investigate the therapeutic efficacy of monotherapy with either 850nm LED phototherapy or low-dose FK-506, and combination therapy in Dermatophagoides farina (Df)-induced AD-like skin lesions in NC/Nga mice. METHODS: The Df-induced NC/Nga mice with a clinical score of 7 were used for treatment with LED (10 and 25J/cm(2)) alone, low-dose FK-506 (1mg/kg) or in combination. The synergistic effects of combined therapy were evaluated by dermatitis scores, skin histology, skin barrier function, and immunological parameters, such as IgE, NO, Th2-mediated cytokines and chemokines. RESULTS: Combination therapy with 850nm (25J/cm(2)) LED and low-dose FK-506 showed a significant reduction in the severity of skin lesions. Combined therapy decreased in the serum level of IgE, NO, and in the splenic level of Th2-mediated cytokines and chemokines. Combination therapy significantly also reduced the inflammatory cellular infiltrate into the skin lesions. Moreover, combination therapy led to recovery of skin barrier function in the skin lesions. CONCLUSIONS: The use of combination of LED phototherapy and low-dose immunosuppressant improved Df-induced AD-like skin lesions in an NC/Nga mouse model by dominantly reducing IgE, NO, suppressing Th2-mediated immune responses, and inhibiting inflammatory cells, as well as improving skin barrier function.

Three new single nucleotide polymorphisms identified by a genome-wide association study in Korean patients with vitiligo.

Genetic susceptibility is involved in the pathogenesis of vitiligo. Association studies with a whole genome-based approach instead of a single or a few candidate genes may be useful for discovering new susceptible genes. Although the etiology of non-segmental and segmental types is different, the association between gene polymorphisms and vitiligo has been reported, without defining types or in non-segmental type. Whole genome-based single nucleotide polymorphisms (SNPs) were examined in patients with non-segmental and segmental types of vitiligo using the Affymetrix GeneChip 500K mapping array, and 10 functional classes of significant SNPs were selected. Genotyping and data analysis of selected 10 SNPs was performed using real-time PCR. Genotype and allele frequencies were significantly different between both types of vitiligo and three of the target SNPs, DNAH5 (rs2277046), STRN3 (rs2273171), and KIAA1005 (rs3213758). A stronger association was suggested between the mutation in KIAA1005 (rs3213758) and the segmental type compared to the non-segmental type of vitiligo. DNAH5 (rs2277046), STRN3 (rs2273171), and KIAA1005 (rs3213758) may be new vitiligo-related SNPs in Korean patients, either non-segmental or segmental type.

Mechanism underlying the effect of combined therapy using glucosamine and low-dose cyclosporine A on the development of atopic dermatitis-like skin lesions in NC/Nga mice.

Combination therapy is often used in the treatment of atopic dermatitis (AD) to improve clinical efficacy or to spare the dose of each drug. Cyclosporine A (CsA) is a calcineurin inhibitor that was developed for the treatment of AD. Glucosamine (Glu) is a potent immunosuppressant that inhibits Th2-mediated immunity. We previously reported that Glu has an ameliorative effect on the development of the pathology in NC/Nga mice. The aims of our study were to investigate the therapeutic efficacy of combination of Glu and low-dose CsA in dermatophagoides farina (Df)-induced AD-like skin lesions in NC/Nga mice and to determine the underlying therapeutic mechanisms. The Df-induced NC/Nga mice with a clinical score of 7 were used for treatment with Glu (500mg/kg) alone, low-dose CsA (2, 5, and 10mg/kg) or in combination. The clinical scores were reduced significantly by the combination treatment with Glu and low-dose CsA. The suppression of dermatitis by combined therapy was accompanied by decrease in the plasma level of IgE and in the splenic level of IL-4, IL-5, IL-13, TARC and eotaxin. Histological analysis of the skin also revealed that combination treatment significantly reduced the inflammatory cellular infiltrate, including mast cells and eosinophils. Particularly, immunological evaluation reveals an increase of CD4(+)CD25(+) Treg cells in the combined treatment. The induction of TSLP, which leads to systemic Th2 response, was reduced in the skin on combination treatment. The protein expression of filaggrin and involucrin was recovered by combination treatment in the skin lesions, whereas the protein expression of keratin-10 and keratin-14 decreased in the combination treatment. Collectively, our findings suggest that combination treatment of Glu and low-dose CsA leads to the therapeutic effects in Df-induced AD-like skin lesion in NC/Nga mice through inhibition of IgE, inflammatory cellular infiltrate, and recovery of skin barrier function via a mechanism that may inhibition of Th2-mediated immune responses, in part, increment of CD4(+)CD25(+) Treg cells. These results suggest that this combined immunosuppressive treatment may provide important implications for the design of therapeutic strategies aimed at AD treatment.

PDZK1 upregulation in estrogen-related hyperpigmentation in melasma.

The pathogenesis of melasma is unknown, although the potential role of estrogen has been considered. Microarray and real-time PCR analyses revealed that upregulation of PDZ domain protein kidney 1 (PDZK1) is clinically correlated with melasma. Although there has been no report that PDZK1 is involved in pigmentation and/or melanogenesis, PDZK1 expression can be induced by estrogen. In this study, the role of PDZK1 upregulation in melasma was examined, particularly in connection with estrogen, using biopsied skin specimens from 15 patients and monocultures and cocultures of melanocytes and keratinocytes with or without overexpression or knockdown of PDZK1. Estrogen upregulated PDZK1. Overexpression of PDZK1 increased tyrosinase expression and melanosome transfer to keratinocytes, whereas PDZK1 knockdown reduced estrogen-induced tyrosinase expression, through regulation of expression of estrogen receptors (ERs) ER-α and ER-ß. The PDZK1-induced tyrosinase expression and melanosome transfer was regulated by ion transporters such as sodium-hydrogen exchanger (NHE), cystic fibrosis transmembrane conductance regulator (CFTR), and SLC26A3, which showed a specific association with each ER subtype. In the melanosome transfer, PDZK1 also increased phosphorylation of ezrin/radixin/moesin (ERM) and ras-related C3 botulinum toxin substrate 1, but not the expression of proteinase-activated receptor-2. Collectively, upregulation of PDZK1 could have an important role in the development of melasma in connection with estrogen through NHE, CFTR, and SLC26A3.

Irradiation of light emitting diode at 850nm inhibits T cell-induced cytokine expression.

BACKGROUND: An anti-inflammatory effect of light obtained from light-emitting diodes (LEDs) has been discovered, however, limited ranges of wavelengths have been used and the action mechanism has been rarely demonstrated. OBJECTIVE: We sought to analyze the immunomodulatory effect of LED on Jurkat T cells and human T cells. METHODS: Jurkat T cells with/without stimulation were irradiated once or five times using seven ranges of LED wavelengths, from 415nm to 940nm. Cytotoxic effects were examined by an MTT assay. Changes in T cell-induced cytokines, including IL-2, IL-4, IL-10, IL-12 and IFN-gamma, and their upstream signaling molecules, ZAP-70 and PKCθ, were examined by real-time PCR, ELISA, and Western blot analysis. The effect of the LED wavelength, whose effect was identified on Jurkat T cells, was also examined in human CD3+ T cells with/without stimulation and in Dermatophagoides farinae-induced atopic dermatitis (AD) NC/Nga mice. RESULTS: Lower doses of LED irradiation at 850nm inhibited T cell-derived cytokines without inducing cell death in both Jurkat T cells and human T cells. Repeated exposure resulted in a greater increase of inhibitory effects than that observed with a single exposure, and these effects were identified in the NC/Nga AD model. CONCLUSIONS: Although more remains to be clarified, these results may support the clinical application of LED for immune regulation.

A novel function of Siglec-9 A391C polymorphism on T cell receptor signaling.

BACKGROUND: Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are the best-characterized immunoglobulin-type lectins. There is a growing amount of data linking Siglec and autoimmune diseases. The recently identified Siglec-9 inhibits T cell receptor (TCR)-mediated signaling which has been demonstrated by site-directed mutagenesis. In human Siglec-9, at least 8 nonsynonymous SNPs have been detected without functional studies. This study examined the SNP(s) related to TCR-mediated signaling. METHODS: Since the functions of Siglecs are modulated by their interaction with sialic-acid-containing carbohydrate groups, a molecular modeling analysis of carbohydrate binding interactions and an RBC binding analysis were performed using the 8 SNPs. The TCR-mediated signaling was analyzed with the downstream signaling molecules ZAP-70 and IL-2. RESULTS: This study revealed that an A391C polymorphism is the only mutant related to the binding. Jurkat T cells transfected with the A391C mutant reduced the inhibition of ZAP-70 phosphorylation and IL-2 production compared to cells transfected with the wild type. CONCLUSIONS: Siglec-9 A391C was the only polymorphism related to TCR-mediated signaling in human Siglec-9, resulting in less inhibition compared to the wild type.

Validation of nested PCR and a selective biochemical method as alternatives for mycoplasma detection.

Direct culture is the most common way to reliably detect mycoplasma, but it is not practical for the qualitative control of cell therapeutics because of the elaborate culture medium, the prolonged incubation time, and the large sample volumes. Here, we chose two alternative methods using commercial detection kits, the PCR mycoplasma detection kit with nested PCR and the selective biochemical method, MycoAlert(®), and validated them with the direct culture method as a reference. We tested eight mycoplasma species and five validation parameters: specificity, detection limit, robustness, repeatability, and ruggedness, based on the regulatory guidelines in the US Pharmacopoeia. All experiments were performed using fibroblasts spiked with mycoplasma. Specificity tests for both methods included all mycoplasma species, except Mycoplasma pneumonia and M. genitalium for the nested PCR and Ureaplasma urealyticum for the MycoAlert(®) assay. Regarding the detection limit, the nested PCR proved to be as sensitive as the direct culture method and more sensitive than the MycoAlert(®) assay. The predicted median for probit = 0.9 was 54 (44-76) CFU/ml for M. hyorhinis and 16 (13-23) CFU/ml for M. hominis by the nested PCR, but 431 (346-593) CFU/ml and 105 (87-142) CFU/ml, respectively, with MycoAlert(®). Changes in the concentration of reagents, reagent lot, or individual analysts did not influence the results of the examined methods. The results of this study support nested PCR as a valuable alternative for mycoplasma detection.

Association of thymic stromal lymphopoietin gene -847C>T polymorphism in generalized vitiligo.

Thymic stromal lymphopoietin (TSLP) induces naïve CD4+ T cells to produce Th2 cytokines. In addition, to low production of Th2 cytokines, strong Th1 response, which plays an important role in vitiligo development, has been induced by blockade of TSLP or TSLP receptor. This study examined whether a functional TSLP polymorphism was associated with vitiligo. One hundred and sixty Korean patients with vitiligo and 568 healthy Korean individuals were examined for the four SNPs of TSLP gene. Luciferase activity was measured for promoter assay. The genotype and allele frequencies of -847C>T polymorphism were lower in vitiligo patients compared with the controls, whereas those of wild type were higher (P = 0.004, P = 0.017 respectively). None the less, the promoter activity of -847C decreased significantly (P = 0.013) compared with -847T, expecting lower TSLP mRNA levels in the polymorphism. Collectively, C allele at the TSLP -847C>T polymorphism may increase susceptibility to generalized vitiligo through decreasing TSLP mRNA expression levels.

Dissociation/association properties of a dodecameric cyclomaltodextrinase. Effects of pH and salt concentration on the oligomeric state.

As an effort to elucidate the quaternary structure of cyclomaltodextrinase I-5 (CDase I-5) as a function of pH and salt concentration, the dissociation/association processes of the enzyme were investigated under various pH and salt conditions. Previous crystallographic analysis of CDase I-5 indicated that it existed exclusively as a dodecamer at pH 7.0, forming an assembly of six 3D domain-swapped dimeric subunits. In the present study, analytical ultracentrifugation analysis suggested that CDase I-5 was present as a dimer in the pH range of 5.0-6.0, while the dodecameric form was predominant at pH values above 6.5. No dissociation of the dodecamer was observed at pH 7.0 and the above. Gel filtration chromatography showed that CDase I-5 dissociated into dimers at a rate of 8.58 x 10(-2) h(-1) at pH 6.0. A mutant enzyme with three histidine residues (H49, H89, and H539) substituted with valines dissociated into dimers faster than the wild-type enzyme at both pH 6.0 and 7.0. The tertiary structure indicated that the effect of pH on dissociation of the oligomer was mainly due to the protonation of H539. Unlike the pH-dependent process, the dissociation of wild-type CDase I-5 proceeded very fast at pH 7.0 in the presence of 0.2-1.0 M of KCl. Stopped-flow spectrophotometric analysis at various concentrations of KCl showed that the rate constants of dissociation (kd) from dodecamers into dimers were 5.96 s(-1) and 7.99 s(-1) in the presence of 0.2 M and 1.0 M of KCl, respectively.