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W Chen

Publications and source records attributed to W Chen.

At least 595 records · Page 33Linked to original sources
PubMed1998

Evidence of heterogeneity and quantitative differences of the type 1 5alpha-reductase expression in cultured human skin cells--evidence of its presence in melanocytes.

Steroid 5alpha-reductase is of crucial importance in androgen physiology because it catalyzes the conversion of testosterone into the more potent 5alpha-dihydrotestosterone in androgen-regulated target tissues. The enzyme occurs in two isoforms, whereby type 1 isozyme exists mainly in the skin and type 2 in the prostate. By using human cell cultures, we examined cutaneous expression and subcellular localization of type 1 5alpha-reductase in vitro. In immunocytochemistry, type 1 5alpha-reductase was detected in the cytoplasm of cultured human sebocytes, keratinocytes, fibroblasts, dermal microvascular endothelial cells, hair dermal papilla cells, and melanocytes. In western blot studies, two closely lying bands of 21-27 kDa were detected, possibly indicating heterogeneity of the type 1 5alpha-reductase in all the cell types tested, with the exception of beard dermal papilla cells. Northern blot studies revealed most abundant type 1 mRNA in neonatal foreskin keratinocytes, followed by adult facial sebocytes. Occipital hair dermal papilla cells presented higher levels of type 1 5alpha-reductase mRNA than those of beard. These findings were confirmed by semiquantitative reverse transcriptase polymerase chain reaction coupled with high performance liquid chromatography analysis. Taken together, it seems likely that in cultured human skin cells there exist (i) heterogeneity of type 1 5alpha-reductase protein and (ii) quantitative differences in its transcriptional and translational expression levels.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1998

Effect of tea on the formation of DNA adducts by azoxymethane.

1. The effect of black tea on the conversion of azoxymethane (AOM) to DNA reactive metabolites was studied in four groups of the male F344 rat. Each received 1.25% solutions of tea for 2 or 6 weeks, and simultaneous controls drank water. All rats were injected s.c. twice with 15 mg/kg AOM after the first or fifth week respectively, on tea or water, and again 1 week later. Groups were killed 6 h after the last dose, or 18 h later. The liver and colon were rapidly removed and rinsed with buffer solution, pH 7.0. DNA was isolated from these tissues, and DNA methylation was examined by the typical fluorescence of 06-methylated and N-7-methylated products, separated by HPLC. 2. Two or 6 weeks of tea intake failed to affect significantly the formation of alkylated DNA from liver and colon compared with controls drinking water. Only in the group of rats on tea for 6 weeks, and killed 6 h after the last dose of AOM, was the O6-methyldG and 7-methyldG decreased in DNA obtained from colon. 3. Thus, solutions of tea affected the formation of alkylated products in DNA of the colon of rats given AOM only at one time point, but did not do so under most other experimental conditions. The underlying mechanism is based on our previous finding that tea does not affect cytochrome P4502E1 that our group established to be the enzyme metabolizing AOM.

Alkylation↗
PubMed1998

Rab11 is required for trans-golgi network-to-plasma membrane transport and a preferential target for GDP dissociation inhibitor.

The rab11 GTPase has been localized to both the Golgi and recycling endosomes; however, its Golgi-associated function has remained obscure. In this study, rab11 function in exocytic transport was analyzed by using two independent means to perturb its activity. First, expression of the dominant interfering rab11S25N mutant protein led to a significant inhibition of the cell surface transport of vesicular stomatitis virus (VSV) G protein and caused VSV G protein to accumulate in the Golgi. On the other hand, the expression of wild-type rab11 or the activating rab11Q70L mutant had no adverse effect on VSV G transport. Next, the membrane association of rab11, which is crucial for its function, was perturbed by modest increases in GDP dissociation inhibitor (GDI) levels. This led to selective inhibition of the trans-Golgi network to cell surface delivery, whereas endoplasmic reticulum-to-Golgi and intra-Golgi transport were largely unaffected. The transport inhibition was reversed specifically by coexpression of wild-type rab11 with GDI. Under the same conditions two other exocytic rab proteins, rab2 and rab8, remained membrane bound, and the transport steps regulated by these rab proteins were unaffected. Neither mutant rab11S25N nor GDI overexpression had any impact on the cell surface delivery of influenza hemagglutinin. These data show that functional rab11 is critical for the export of a basolateral marker but not an apical marker from the trans-Golgi network and pinpoint rab11 as a sensitive target for inhibition by excess GDI.

Animals↗
PubMed1998

Vitamin B-6 requirement and status assessment of young women fed a high-protein diet with various levels of vitamin B-6.

The vitamin B-6 requirement of young women consuming a constant high-protein diet (1.55 g/kg body wt) and the effect of various ratios of vitamin B-6 to protein on this requirement were studied. Eight women were fed a lactoovovegetarian basal diet containing 0.45 mg vitamin B-6 (2.66 micromol as pyridoxine) and 30 micromol carnitine for 92 d. The protocol consisted of successive baseline adjustment (9 d), depletion (27 d), and repletion (two 21-d and then one 14-d) periods. Vitamin B-6 intakes were 1.60, 0.45, 1.26, 1.66, and 2.06 mg, resulting in ratios of vitamin B-6 (in mg) to protein (in g) for the five periods of 0.016, 0.005, 0.013, 0.017, and 0.021, respectively. Direct and indirect as well as short- and long-term vitamin B-6 status measures were assessed weekly. Regression analysis revealed that the amount of dietary vitamin B-6 required to normalize urinary 4-pyridoxic acid, plasma pyridoxal-P, erythrocyte pyridoxal-P and pyridoxal, and erythrocyte alanine and aspartate aminotransferase activity coefficients to predepletion baseline values was 1.94 mg vitamin B-6/d (0.019 mg vitamin B-6/g protein). This study suggests that the current vitamin B-6 recommended dietary allowance of 1.6 mg/d based on 0.016 mg/g protein is not an adequate intake and may require reevaluation.

Adult↗
PubMed1998

Urinary, plasma, and erythrocyte carnitine concentrations during transition to a lactoovovegetarian diet with vitamin B-6 depletion and repletion in young adult women.

The purpose of the present study was to determine whether vitamin B-6 depletion and repletion influence carnitine concentrations in the plasma, erythrocytes, and urine of young adult women consuming a low-carnitine (30 micromol/d), lactoovovegetarian diet. Doses of vitamin B-6 were administered sequentially as follows: 1.60 mg/d during adjustment for 9 d, 0.46 mg/d during depletion for 27 d, 1.26 mg/d during the first repletion for 21 d, 1.66 mg/d during the second repletion for 21 d, and 2.06 mg/d during the third repletion for 14 d. Urinary carnitine tended to decline but was not significantly different throughout the 92-d study period. Plasma short-chain acylcarnitine and total carnitine decreased during vitamin B-6 depletion; however, the ratio of plasma acylcarnitine to total carnitine was not significantly different during changes in vitamin B-6 intake. Erythrocyte long-chain acylcarnitine increased during vitamin B-6 depletion, but the ratio of erythrocyte acylcarnitine to total carnitine did not respond to changes in vitamin B-6 intake. Plasma free and total carnitine concentrations were only weakly correlated with plasma pyridoxal-P concentration (r = 0.28 and r = 0.29, respectively; P < 0.01). No significant correlations were observed between urinary carnitine excretion or erythrocyte carnitine concentrations and plasma or erythrocyte pyridoxal-P concentrations. Thus, a vitamin B-6 intake of 0.5 mg/d does not affect carnitine concentrations in biological fluids and therefore is unlikely to affect endogenous carnitine synthesis over 27 d. The changes in carnitine indexes that we observed were probably due to adaptation to a vegetarian pattern of exogenous carnitine consumption.

Adult↗
PubMed1998

(-)-Epigallocatechin-3-gallate inhibition of ultraviolet B-induced AP-1 activity.

Green tea polyphenols have been shown to inhibit cancer in a variety of tumor models, including ultraviolet B (UVB)-induced non-melanoma skin cancer. In green tea extracts, the major dry mass constituent is the family of catechins, of which (-)-epigallocatechin-(3)-gallate (EGCG) is considered to be important for the chemopreventive activity. EGCG has been shown to have antioxidant properties, but there has been little progress toward identifying the specific targets and mechanisms of its action. Using cultured human keratinocytes, we show that UVB-induced AP-1 activity is inhibited by EGCG in a dose range of 5.45 nM to 54.5 microM. EGCG is effective at inhibiting AP-1 activity when applied before, after or both before and after UVB irradiation. EGCG also inhibits AP-1 activity in the epidermis of a transgenic mouse model. This work begins to define a mechanism by which EGCG could be acting to inhibit UVB-induced tumor formation.

Animals↗
PubMed1998

The effect of age-dependent increase in fat mass on peripheral insulin action is saturable.

Insulin resistance and increased fat mass (FM) are common in human aging. We aimed to investigate the relationship between the age-dependent increase in FM and insulin resistance (by euglycemic hyperinsulinemic clamp technique), in a homogenous rodent model. The decline in insulin responsiveness was linear until late adulthood when body weight, FM, and epididymal fat reached a critical amount (r > .750, for all). Above this critical point, there was no further decline in insulin responsiveness with aging and with increased BW (p < .00001 for all spline curve analyses). This decline in insulin-mediated glucose uptake was accounted for by a decrease in whole body glycolytic rate with no change in the rate of glycogen synthesis. Thus, in this homogenous model, an early increase in FM is associated with impairment in insulin action until a critical FM is achieved, after which there is no additional insulin resistance with aging. We suggest that decreasing insulin responsiveness, in a heterogeneous group such as humans, will only occur within a specific accretion of visceral or total FM.

Adipose Tissue↗
PubMed1998

Induction of Th2 cell differentiation in the primary immune response: dendritic cells isolated from adherent cell culture treated with IL-10 prime naive CD4+ T cells to secrete IL-4.

A number of observations indicate that exposure to IL-4 is essential for the priming of Th2-type effector T cells and that exposure to IL-12 is essential for the priming of Th1-type effector T cells. However, the initial source of IL-4 in the early immune response has not been clearly identified. Dendritic cells (DC) are the most potent antigen-presenting cells (APC) in priming naive T cells. In this report, we show that DC exposed to IL-10 may play an important role in the priming of IL-4-secreting cells in the early immune response. DC isolated from splenic adherent cell cultures treated with rIL-10 (IL-10-DC) primed naive ovalbumin (OVA)-TCR transgenic T cells to secrete IL-4 upon re-stimulation with OVA and splenic APC. By contrast, DC isolated from rIL-12, rIL-4 or control treated cultures induced almost exclusively Th1-type effector T cells. IL-4 secretion was detected in the primary cultures of IL-10-DC plus naive CD4+ T cells and the priming of IL-4-secreting T cells by IL-10-DC was dependent on endogenous IL-4 production in the priming culture since anti-IL-4 neutralizing antibody completely abrogated the priming of IL-4-secreting cells. Anti-B7-2 but not anti-B7-1 inhibited the ability of IL-10-DC to prime T cells to secrete IL-4. Furthermore, the ability of IL-10 DC to prime for IL-4-secreting T cells was closely related to the down-regulation of CD40 ligand-mediated IL-12 p70 production by DC in the primary cultures and was markedly reduced by adding exogenous IL-12 to the priming cultures. Thus, our findings indicate that early immunologic events that drive Th2 differentiation involve the effects of IL-10 on DC.

Animals↗
PubMed1998

CTLs specific for bcr-abl joining region segment peptides fail to lyse leukemia cells expressing p210 bcr-abl protein.

The aim of the current study was to determine whether immunization with synthetic peptides corresponding to the joining region segment of p210 bcr-abl chimeric protein can elicit CD8+ cytotoxic T lymphocytes (CTLs) capable of specifically lysing leukemia cells. BALB/c mice were immunized with peptides identical to the joining region segment of p210 bcr-abl protein. Class I major histocompatibility complex (MHC)-restricted bcr-abl peptide-specific CD8+ CTLs were elicited. The CTL clones were H-2 Kd restricted and specifically recognized a nonamer peptide of the combined sequence of bcr-abl amino acids but neither bcr nor abl amino acid sequence alone. Despite specificity and substantial lytic potential against syngeneic cell line incubated with exogenously supplied peptides, the bcr-abl peptide-specific CTLs failed to lyse syngeneic murine leukemia cells expressing human p210 bcr-abl protein containing the same bcr-abl joining region peptide sequence. Similarly, the bcr-abl peptide-specific CTLs did not lyse human bcr-abl-positive chronic myelogenous leukemia cells expressing murine class I MHC antigen (i.e., K562 cells infected with vaccinia virus expressing H-2 Kd). The appropriateness of the joining region segment of bcr-abl protein to serve as a T cell target depends upon whether that segment is presented by class I MHC in a concentration high enough to stimulate CTLs. The current experiments using murine peptide-specific CTLs could not establish that the joining region of bcr-abl protein is processed and presented by class I MHC antigen-processing pathway, but the possibility was not ruled out. Alternative models and/or strategies are necessary.

Amino Acid Sequence↗
PubMed1998

Regulation of protease expression in Porphyromonas gingivalis.

Although the strong protease activity of Porphyromonas gingivalis appears to be an important virulence property of these organisms, little information is currently available regarding the regulation of expression of the multiple protease genes. Utilizing the lacZ reporter gene strategy, the environmental factors which regulate the expression of the Arg-gingipain gene rgpA and the prtT protease gene were investigated. These two genes are reciprocally regulated since factors which retarded growth (iron depletion and nutrient limitation) appeared to upregulate rgpA expression while down-regulating prtT expression. However, inactivation of the major rgpA gene resulted in increased transcription of the prtT and tpr protease genes while decreasing expression of the Lys-gingipain kgp gene as detected by Northern blot analysis. By contrast, inactivation of the prtT gene did not significantly affect kgp expression but moderately decreased rgpA mRNA levels. These results indicate that the protease genes of P. gingivalis are not coordinately regulated and suggest that some of these enzymes play specific roles in the physiology and/or virulence of these organisms.

Adhesins, Bacterial↗
PubMed1998

Nitric oxide synthase expression in macrophages of Histoplasma capsulatum-infected mice is associated with splenocyte apoptosis and unresponsiveness.

Splenic macrophages from Histoplasma capsulatum-infected mice express inducible nitric oxide synthase (iNOS), and the iNOS expression correlates with severity of the infection. We examined whether production of NO is responsible for apoptosis and the anti-lymphoproliferative response of splenocytes from mice infected with H. capsulatum. In situ terminal deoxynucleotidyl transferase nick end labeling revealed apoptotic nuclei in cryosections of spleen from infected but not normal mice. Splenocytes of infected mice were unresponsive to stimulation by either concanavalin A or heat-killed H. capsulatum yeast cells. Splenocyte responsiveness was restored by addition to the medium of NG-monomethyl-L-arginine, a known inhibitor of NO production. The proliferative response of splenocytes from infected mice was also restored by depletion of macrophages or by replacement with macrophages from normal mice. In addition, expression of iNOS returned to its basal level when the animals had recovered from infection. These results suggest that suppressor cell activity of macrophages is associated with production of NO, which also appears to be an effector molecule for apoptosis of cultured splenocytes from infected mice.

Animals↗
PubMed1998

Mismatch repair proteins regulate heteroduplex formation during mitotic recombination in yeast.

Mismatch repair (MMR) proteins actively inhibit recombination between diverged sequences in both prokaryotes and eukaryotes. Although the molecular basis of the antirecombination activity exerted by MMR proteins is unclear, it presumably involves the recognition of mismatches present in heteroduplex recombination intermediates. This recognition could be exerted during the initial stage of strand exchange, during the extension of heteroduplex DNA, or during the resolution of recombination intermediates. We previously used an assay system based on 350-bp inverted-repeat substrates to demonstrate that MMR proteins strongly inhibit mitotic recombination between diverged sequences in Saccharomyces cerevisiae. The assay system detects only those events that reverse the orientation of the region between the recombination substrates, which can occur as a result of either intrachromatid crossover or sister chromatid conversion. In the present study we sequenced the products of mitotic recombination between 94%-identical substrates in order to map gene conversion tracts in wild-type versus MMR-defective yeast strains. The sequence data indicate that (i) most recombination occurs via sister chromatid conversion and (ii) gene conversion tracts in an MMR-defective strain are significantly longer than those in an isogenic wild-type strain. The shortening of conversion tracts observed in a wild-type strain relative to an MMR-defective strain suggests that at least part of the antirecombination activity of MMR proteins derives from the blockage of heteroduplex extension in the presence of mismatches.

Base Sequence↗
PubMed1998

Tumor promoter arsenite activates extracellular signal-regulated kinase through a signaling pathway mediated by epidermal growth factor receptor and Shc.

Although arsenite is an established carcinogen, the mechanisms underlying its tumor-promoting properties are poorly understood. Previously, we reported that arsenite treatment leads to the activation of the extracellular signal-regulated kinase (ERK) in rat PC12 cells through a Ras-dependent pathway. To identify potential mediators of the upstream signaling cascade, we examined the tyrosine phosphorylation profile in cells exposed to arsenite. Arsenite treatment rapidly stimulated tyrosine phosphorylation of several proteins in a Ras-independent manner, with a pattern similar to that seen in response to epidermal growth factor (EGF) treatment. Among these phosphorylated proteins were three isoforms of the proto-oncoprotein Shc as well as the EGF receptor (EGFR). Tyrosine phosphorylation of Shc allowed for enhanced interactions between Shc and Grb2 as identified by coimmunoprecipitation experiments. The arsenite-induced tyrosine phosphorylation of Shc, enhancement of Shc and Grb2 interactions, and activation of ERK were all drastically reduced by treatment of cells with either the general growth factor receptor poison suramin or the EGFR-selective inhibitor tyrphostin AG1478. Down-regulation of EGFR expression through pretreatment of cells with EGF also attenuated ERK activation and Shc tyrosine phosphorylation in response to arsenite treatment. These results demonstrate that the EGFR and Shc are critical mediators in the activation of the Ras/ERK signaling cascade by arsenite and suggest that arsenite acts as a tumor promoter largely by usurping this growth factor signaling pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1998

The human sebocyte culture model provides new insights into development and management of seborrhoea and acne.

Seborrhoea and acne are exclusively human diseases and sebaceous gland differentiation is species specific. Therefore, fundamental research on human sebaceous cell function and control requires human in vitro models. The human sebocyte culture model, introduced in 1989, has been used in several studies to elucidate sebaceous gland activity and its regulation at the cellular level. Cultured human sebocytes have been shown to preserve important sebocytic characteristics, although they undergo an incomplete terminal differentiation in vitro. In vitro synthesis of free fatty acids without bacterial involvement and marked interleukin 1 alpha expression at the mRNA and protein levels with no further induction by lipopolysaccharides lead to the assumption that human sebocytes may initiate acne lesions by an intrinsic mechanism. Androgens affected sebocyte activity in vitro in a manner dependent on the localization of the sebaceous glands. In vitro stimulation of sebocyte proliferation by androgens could be completely abolished by spironolactone. Cultured sebocytes strongly expressed type 1 5 alpha-reductase and metabolized testosterone to androstenedione, 5 alpha-androstanedione, 5 alpha-dihydrotestosterone, androsterone and 5 alpha-androstanediol, whereas the levels of 5 alpha-reductase activity were probably not feedback regulated. 4,7 beta-Dimethyl-4-aza-5 alpha-cholestan-3-one, a type 1 5 alpha-reductase inhibitor, induced an early, marked down-regulation of 5 alpha-reductase activity in human sebocytes in vitro, while hydrofinasteride, a type 2 inhibitor, required 10(3)-fold higher concentrations to induce similar effects. Stimulation of sebocyte proliferation by insulin, thyroid-stimulating hormone and hydrocortisone indicates that the hormonal control of the sebaceous gland could be a complex mechanism. Retinoids inhibited sebocyte proliferation in a dose-dependent manner and down-regulated lipid synthesis and sebocyte differentiation in vitro. Isotretinoin was the most potent compound. On the other hand, vitamin A was found essential for sebocyte activity and differentiation in vitro and could be partially substituted by synthetic retinoids. The inhibitory effect of isotretinoin on sebocyte proliferation was barely affected by the presence of vitamin A. The low persistent isotretinoin levels or, more likely, the considerably elevated tretinoin concentrations detected in human sebocytes after treatment with isotretinoin in vitro may be responsible for the inhibitory effect of this compound on sebocyte activity.

Acne Vulgaris↗
PubMed1998

Genetic mapping of soluble guanylyl cyclase genes: implications for linkage to blood pressure in the Dahl rat.

The nitric oxide (NO) signaling system, consisting of NO synthases, soluble guanylyl cyclase, and cGMP, plays a prominent role in salt handling and regulation of blood pressure. Soluble guanylyl cyclases are heme-containing heterodimers (alpha/beta). The alpha1/beta1 isoform has greater NO sensitivity than the alpha1/beta2. It has recently been shown that expression of the beta subunits is altered in the kidney of the Dahl salt-sensitive rat, ie, the beta1 subunit is decreased and the beta2 subunit increased. However, whether soluble guanylyl cyclase is linked to salt sensitivity is not known. In the present study, we investigated linkage of guanylyl cyclase genes to blood pressure. Alpha1 and beta1 gene loci for soluble guanylyl cyclase were mapped to rat chromosome 2, and the beta2 gene locus was mapped to rat chromosome 5 using fluorescent in situ metaphase hybridization. By use of a rat radiation hybrid panel, the gene loci were then further mapped with respect to known quantitative trait locus markers of salt-sensitive hypertension in the Dahl rat on chromosomes 2 and 5. Genes for alpha1 and beta1 were closely linked by two-point analysis to Na+,K+-ATPase alpha1 isoform (LOD of 15.1 and 14.0, respectively) and calmodulin-dependent protein kinase II-delta loci (LOD of 14.3 and 12.9, respectively), which have been previously shown to flank a quantitative trait locus for blood pressure in the Dahl rat. The alpha1 and beta1 genes were closely linked (LOD of 11.3; theta, 0.4). The beta2 gene locus was closely linked to the endothelin-2 (ET-2) locus (LOD of 13.0), which has been shown to cosegregate with blood pressure. We conclude that soluble guanylyl cyclase subunit loci, ie, alpha1, beta1, and beta2, are good candidates for genes controlling salt-sensitive hypertension in the Dahl rat.

Animals↗
PubMed1998

Comparative immunochemical analyses of the developmental expression and distribution of ameloblastin and amelogenin in rat incisors.

Mineralized tissues are unique in using proteins to attract and organize calcium and phosphate ions into a structured mineral phase. A precise knowledge of the expression and extracellular distribution of matrix proteins is therefore very important in understanding their function. The purpose of this investigation was to obtain comparative information on the expression, intracellular and extracellular distribution, and dynamics of proteins representative of the two main classes of enamel matrix proteins. Amelogenins were visualized using an antibody and an mRNA probe prepared against the major alternatively spliced isoform in rodents, and nonamelogenins by antibodies and mRNA probes specific to one enamel protein referred to by three names: ameloblastin, amelin, and sheathlin. Qualitative and quantitative immunocytochemistry, in combination with immunoblotting and in situ hybridization, indicated a correlation between mRNA signal and sites of protein secretion for amelogenin, but not for ameloblastin, during the early presecretory and mid- to late maturation stages, during which mRNA signals were detected but no proteins appeared to be secreted. Extracellular amelogenin immunoreactivity was generally weak near secretory surfaces, increasing over a distance of about 1.25 microm to reach a level slightly above an amount expected if the protein were being deposited evenly across the enamel layer. Immunolabeling for ameloblastin showed an inverse pattern, with relatively more gold particles near secretory surfaces and much fewer deeper into the enamel layer. Administration of brefeldin A and cycloheximide to stop protein secretion revealed that the immunoblotting pattern of amelogenin was relatively stable, whereas ameloblastin broke down rapidly into lower molecular weight fragments. The distance from the cell surface at which immunolabeling for amelogenin stabilized generally corresponded to the point at which that for ameloblastin started to show a net reduction. These data suggest a correlation between the distribution of amelogenin and ameloblastin and that intact ameloblastin has a transient role in promoting/stabilizing crystal elongation. (J Histochem Cytochem 46:911-934, 1998)

Amelogenin↗