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Jun-Mo Yang

Publications and source records attributed to Jun-Mo Yang.

At least 37 records · Page 2Linked to original sources
PubMed2005

Analysis of calcium-inducible genes in keratinocytes using suppression subtractive hybridization and cDNA microarray.

Terminal differentiation of skin keratinocytes is a vertically directed multistep process that is tightly controlled by the sequential expression of a variety of genes. To gain further insight into the molecular events involved in this process, we used suppression subtraction hybridization (SSH) and cDNA microarray analysis. Messenger RNAs were isolated from primary skin keratinocytes cultured in vitro after treatment with calcium and then SSH was performed. A total of 840 cDNA clones were obtained from subtracted libraries, and these cDNA clones were used to make the microarray slides. Time-course cDNA microarray analysis (1, 3, 7, and 14 days after calcium treatment) revealed the global gene expression profile during keratinocyte differentiation. Of the 840 genes tested, 290 showed a greater than twofold change in expression level at least once over four time points. The genes were clustered into six groups according to their expression pattern using self-organizing map analysis and showed the global feature of function-related regulation. The genes related to keratinocyte differentiation were markedly up-regulated by calcium treatment. In addition, a unique pattern of increase was seen in the expression of genes related to ribosomal proteins. On the other hand, transcripts involved in metabolism, DNA repair, transcription, and translation were generally down-regulated. These results demonstrate the complexity of the gene expression profile that contributes to the spatiotemporal regulation of keratinocyte differentiation.

Calcium↗
PubMed2005

Inhibition kinetics of mushroom tyrosinase by copper-chelating ammonium tetrathiomolybdate.

With a strategy of chelating coppers at tyrosinase active site to detect an effective inhibitor, several copper-specific chelators were applied in this study. Ammonium tetrathiomolybdate (ATTM) among them, known as a drug for treating Wilson's disease, turned out to be a significant tyrosinase inhibitor. Treatment with ATTM on mushroom tyrosinase completely inactivated enzyme activity in a dose-dependent manner. Progress-of-substrate reaction kinetics using the two-step kinetic pathway and dilution of the ATTM revealed that ATTM is a tight-binding inhibitor and high dose of ATTM irreversibly inactivated tyrosinase. Progress-of-substrate reaction kinetics and activity restoration with a dilution of the ATTM indicated that the copper-chelating ATTM may bind slowly but reversibly to the active site without competition with substrate, and the enzyme-ATTM complex subsequently undergoes reversible conformational change, leading to complete inactivation of the tyrosinase activity. Thus, inhibition by ATTM on tyrosinase could be categorized as complexing type of inhibition with a slow and reversible binding. Detailed analysis of inhibition kinetics provided IC50 at the steady-state and inhibitor binding constant (K(I)) for ATTM as 1.0+/-0.2 microM and 10.65 microM, respectively. Our results may provide useful information regarding effective inhibitor of tyrosinase as whitening agents in the cosmetic industry.

Agaricales↗
PubMed2005

Immunoaffinity purification of SRT-tagged human creatine kinase by peptide elution.

The mouse monoclonal antibody (Mab), SRT10, recognizes a linear epitope of 10 amino acids (ThrPheIleGlyAlaIleAlaThrAspThr). When these epitope-tagged fusion proteins are expressed in mammalian cells, the Mab can detect the tagged proteins by immunoblotting, immunocytochemistry and immunoprecipitation. Here, we describe an efficient method for the purification of SRT-tagged recombinant human creatine kinase (CK) transiently expressed in mammalian cells. This method utilizes the expression of the N-terminal- or C-terminal-tagged CK in transiently transfected HEK293 cells followed by binding to anti-SRT-agarose affinity resin and competitive elution with SRT peptide. Recombinant CK was purified near homogeneity as judged by SDS-PAGE.

Amino Acid Sequence↗
PubMed2005

A new type of uncompetitive inhibition of tyrosinase induced by Cl- binding.

A new type of Cl- induced inhibition of mushroom derived tyrosinase has been detected in this study, and it is defined as the reversible partial hyperbolic uncompetitive inhibition. The Cl- binding site was only induced at the state of the enzyme-substrate complex, and this was confirmed with the intrinsic fluorescence changes. As the oxygen bridge is broken by L-DOPA binding, Cl- simultaneously binds to the ES state to form the ESI complex. It is worth noticing that tyrosinase reacts sensitively to Cl- in the manner of a complex interaction, and this indicates that Cl- might be physiologically involved in the regulation of tyrosinase activity.

Ammonium Chloride↗
PubMed2005

Association of human papillomavirus infection with palmoplantar epidermal cysts in Korean patients.

Human papillomavirus (HPV) infection can develop into many different kinds of diseases ranging from simple warts to carcinoma in situ. Some investigators have recently suggested that HPV infection could be closely related to palmoplantar epidermal cysts. The aim of this study was to determine the existence of HPV and its subtype in palmoplantar epidermal cysts of Korean patients. We investigated 13 cases of palmoplantar epidermal cysts by employing clinical, histopathological and immunohistochemical examinations. In addition, PCR was performed using HPV 57 and 60 type primers. Four of the 13 cases (30.8%) showed the distinct histopathological features of palmoplantar epidermal cysts such as parakeratosis, vacuolar structures and eosinophilic inclusion bodies and these 4 cases showed positive immunohistochemical staining reactions for HPV. PCR for HPV detected type 60-positive bands in 5 of the 13 cases (38.5%), which included the 4 cases that were positive for immunohistochemical staining. These results show that HPV is associated with some cases of palmoplantar epidermal cysts in Korean patients, suggesting that HPV may play a role in the pathogenesis of these cysts.

Adolescent↗
PubMed2005

Molecular cloning and expression of human keratinocyte proline-rich protein (hKPRP), an epidermal marker isolated from calcium-induced differentiating keratinocytes.

We isolated a human gene encoding keratinocyte proline-rich protein (hKPRP). hKPRP gene is located in the region of epidermal differentiation complex on chromosome 1q21, and its approximately 2.5 kb mRNA encodes 579 amino acid protein with high proline content (18%). The mRNA level of hKPRP was markedly increased at both 7 and 14 d after treatment with 1.2 mM calcium in cultured normal human epidermal keratinocytes. In situ hybridization demonstrated that hKPRP was expressed in upper granular layer of normal epidermis with characteristic intermittent pattern. In psoriatic lesion, hKPRP expression was increased as compared with normal skin and showed continuous pattern. Immunohistochemical analysis also confirmed the expression of hKPRP at the protein level. Western blot analysis showed that hKPRP protein of approximately 70 kDa size was significantly increased by calcium in a time-dependent manner. In mouse tissue blot assays, the expression of KPRP was detected in stomach and skin tissues, and began at 17.5 embryonic days. Additionally, hKPRP expression was detected in the periderm of human fetal skin from 16 wk estimated gestational age. Together, these results suggest that hKPRP is an epidermal marker expressed in stratified squamous epithelia and has a potential role in keratinocytes differentiation.

Amino Acid Sequence↗
PubMed2005

Early osteolysis following second-generation metal-on-metal hip replacement.

BACKGROUND: Total hip arthroplasty with use of metal-on-metal bearings has been reintroduced as an alternative to the use of metal-on-polyethylene bearings because of theoretical advantages such as reduced wear and a lower prevalence of osteolysis. However, we observed early osteolysis in a cohort of patients who had been managed with second-generation metal-on-metal hip replacements and investigated the possible etiologic role of metal hypersensitivity. METHODS: We retrospectively analyzed 165 patients (169 hips) who had undergone primary cementless total hip replacement with a contemporary metal-on-metal total hip design between 2000 and 2002. After a minimum duration of follow-up of twenty-four months, nine patients (ten hips) had an osteolytic lesion localized to the greater trochanter. Skin-patch tests for hypersensitivity to metals were performed on the nine patients and on nine randomly selected patients with total hip replacements who did not have osteolytic changes and who were matched to the study cohort for age and gender. Microbiological cultures, histopathologic examinations, and immunohistochemical analysis were performed on samples of periprosthetic tissue that were collected during revision arthroplasty on two hips with early osteolysis. RESULTS: The patients with early osteolysis had a significantly higher rate of hypersensitivity reaction to cobalt compared with controls (p = 0.031). The retrieved periprosthetic tissues showed no evidence of metallic staining, but histologic analysis revealed a perivascular accumulation of CD3-positive T-cells and CD68-positive macrophages and an absence of both particle-laden macrophages and polymorphonuclear cells. Immunohistochemical analysis demonstrated that bone-resorbing cytokines such as IL-1beta and TNF-alpha were produced mainly by infiltrating lymphocytes and activated macrophages. CONCLUSIONS: These findings raise the possibility that early osteolysis in patients with this second-generation metal-on-metal hip replacement is associated with abnormalities consistent with delayed-type hypersensitivity to metal. A prospective study in which a large group of patients is evaluated with multiple diagnostic methods is needed in order to establish whether there is a causal relationship between metal hypersensitivity and osteolysis.

Adult↗
PubMed2004

Towards a proteomic analysis of atopic dermatitis: a two-dimensional-polyacrylamide gel electrophoresis/mass spectrometric analysis of cultured patient-derived fibroblasts.

Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disease typically characterized by a distribution of eczematous skin lesions with lichenification, pruritic excoriations, and dry skin with wide varieties of pathophysiologic aspects. Recently, AD was divided into extrinsic and intrinsic forms according to the presence or absence of an allergy. We investigated alterations in protein expression in primary cultured AD cells from the patient biopsy samples by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight. In the primary cultured fibroblasts, we obtained 31 candidate proteins from the two-dimensional gel image analysis in which 18 proteins were up-regulated, eight proteins were down-regulated and five proteins were post-translationally modified. From these 2-DE results, we found several candidate genes matched proteomic expression patterns by semiquantitative reverse transcription PCR. Since the exact mechanism of atopic alterations in fibroblasts remains unknown, our results may provide new clues to aid in understanding AD.

DNA, Complementary↗
PubMed2004

Analytical electron microscopy and electron holography on microstructures and magnetic domain structures of Sm-Co 2:17 magnets.

Microstructures and magnetic domain structures of precipitation-hardened Sm-Co permanent magnets were systematically investigated by analytical electron microscopy and electron holography. By an elemental mapping method with energy-dispersive X-ray spectroscopy, the change in the local distribution of additive elements, i.e. Cu, Fe and Zr, in Sm-Co magnets with various heat treatments was visualized and the enrichment of Zr in the Z-phase with a width of approximately 1 nm was clarified directly. Detailed analysis with electron holography revealed that considerable fluctuation in the distribution of lines of magnetic flux in the step-aged magnet was due to the chemical partitioning of additives and resulted in magnetic hardening during the magnetization process.

Holography↗
PubMed2004

Identification of calcium-inducible genes in primary keratinocytes using suppression-subtractive hybridization.

Terminal differentiation in epidermal keratinocytes involves major biochemical changes including the expression of many new differentiation-specific genes. To further understand this process, we performed suppression-subtractive hybridization of keratinocytes cultured under high-calcium condition, known to induce differentiation in vitro. We randomly isolated 300 clones representing 90 different genes. By reverse Northern blot analyses, 20 different genes were found to be overexpressed, of which 13 were confirmed as differentially expressed genes during keratinocyte differentiation by Northern blot analysis. Of those, five genes, transglutaminase 1, keratin 6, interleukin-1 receptor antagonist, kallikrein 7, and heat shock protein 27, are known to be up-regulated during epidermal differentiation. Six genes, ferritin-L chain, ribosomal protein S6, tumor-associated calcium signal transducer 2, neuroendocrine secretory protein 55, phosphoserine aminotransferase, and neutrophil gelatinase-associated lipocalin, heretofore were not known to be up-regulated during keratinocyte differentiation. We also identified two novel genes. One of these maps to chromosome 1q21 of the epidermal differentiation complex, and its expression level was strongly increased in differentiating keratinocytes. These differentially expressed genes may provide significant opportunities for further understanding of the epidermal keratinocyte differentiation.

Blotting, Northern↗
PubMed2003

Kinetic inactivation study of mushroom tyrosinase: intermediate detection by denaturants.

The unfolding and inhibition study of mushroom tyrosinase have been studied in the presence of different denaturants such as sodium dodecyl sulfate (SDS), guanidine hydrochloride (GdnHCl), and urea. The kinetic two-phase rate constants were commonly measured from semilogarithmic plots of the activity versus time, which resolved into two straight lines, indicating that the inactivation process consisted of fast and slow phases as a first-order reaction. This result also implied that transient partially folded intermediate existed during tyrosinase unfolding pathway. Mushroom tyrosinase had different behaviors to denaturants in regard with: noncooperative binding manner by SDS while cooperative interactions by GdnHCl and urea; in equilibrium state, SDS-micelle never completely inactivated enzyme activity while GdnHCl has single step denaturation and urea induced a typical transition-like process. Various kinetic parameters for each denaturant were calculated and the possible unfolding pathway scheme was discussed.

Agaricus↗
PubMed2003

A new continuous spectrophotometric assay method for DOPA oxidase activity of tyrosinase.

Sensitive assay methods for tyrosinase are essential not only for the understanding the process of pigment production but also for the development of effective inhibitors of tyrosinase. To develop an efficient assay method, we applied thymol blue to reaction mixtures. The enzyme kinetic study revealed that DOPA oxidase activity of tyrosinase in thymol blue-applied reaction system was more sensitively measured, even under lower enzyme units compared with the previous report with significant enhancement of Vmax while affinity change on substrate was not observed. To test whether this method could be applicable to the inhibition and the inactivation kinetic study of tyrosinase, the effect of kojic acid, a well-known tyrosinase inhibitor, and sodium chloride respectively, have been studied. Conclusively, thymol blue method can assay tyrosinase activity with sensitivity and is applicable to the inhibition and the inactivation study of tyrosinase.

Agaricales↗
PubMed2003

Aggregation and folding of recombinant human creatine kinase.

The processes of aggregation and refolding of recombinant human creatine kinase (rHCK) were studied. Most of the rHCK expressed in E. coli was present in the insoluble traction and it could be solubilized in 6 M urea solution. Unfolding of rHCK in 6 M urea showed biphasic kinetic courses (kappa1 = 6.5 x 10(-3) s(-1); kappa2 = 0.54 x 10(-3) s(-1)) as observed by maximum fluorescence wavelength change. During refolding of the rHCK dissolved in urea, significant aggregation was noticed following first-order kinetics. Aggregation rate constants were influenced by the concentration of NaCl, which increased the difference in transition-free energy (deltadeltaG), showing that stabilization of folding intermediates by NaCl could efficiently reduce the formation of insoluble aggregates. Formations of aggregate were also reduced by adjusting temperature, pH, and concentration of rHCK. Refolding of rHCK under the optimized condition which prevented the aggregation also showed multi-kinetic phases (kappa1 = 3.0 x 10(-3) s(-1); kappa2 = 0.64 x 10(-3) s(-1)). Under optimized conditions applied in this study, rHCK could correctly refold retrieving the high specific enzymatic activity.

Creatine Kinase↗
PubMed2003

Effect of thiohydroxyl compounds on tyrosinase: inactivation and reactivation study.

An unusual thioether bridge (Cys-His) has been detected at the active site of mushroom tyrosinase, and the effects of thiohydroxyl compounds such as dithiothreitol (DTT) and beta-mercaptoethanol (beta-ME) on Cu2+ at the active site have been elucidated. Treatment with DTT and beta-ME on mushroom tyrosinase completely inactivated 3,4-dihydroxyphenylalanine oxidase activity in a dose-dependent manner. Sequential kinetic studies revealed that DTT and beta-ME caused different mixed-type inhibition mechanisms: the slope-parabolic competitive inhibition (Ki = 0.143 mM) by DTT and slope-hyperbolic noncompetitive inhibition (Ki = 0.0128 mM) by beta-ME, respectively. Kinetic Scatchard analysis consistently showed that mushroom tyrosinase had multiple binding sites for DTT and beta-ME with different affinities. Reactivation study of inactivated enzyme by addition of Cu2+ confirmed that DTT and beta-ME directly bound with Cu2+ at the active site. Our results may provide useful information regarding interactions of tyrosinase inhibitor for designing an effective whitening agent targeted to the tyrosinase active site.

Agaricales↗