p23 protects the human aryl hydrocarbon receptor from degradation via a heat shock protein 90-independent mechanism.

The aryl hydrocarbon receptor (AHR) is a ligand-activated signaling molecule which is involved in diverse biological functions ranging from cancer metastasis to immune regulation. This receptor forms a cytoplasmic complex with Hsp90, p23, and XAP2. We have previously reported that down-regulation of p23 triggers degradation of the AHR protein, uncovering a potentially dynamic event which controls the cellular AHR levels without ligand treatment. Here we investigate the underlying mechanisms for this p23 effect using wild-type HeLa and the p23 knockdown HeLa cells. Reduction of the Hsp90 and XAP2 contents, however, did not affect the AHR protein levels, implying that this p23 effect on AHR is more than just alteration of the cytoplasmic complex dynamics. Association of p23 with Hsp90 is not important for the modulation of the AHR levels since exogenous expression of p23 mutants with modest Hsp90-binding affinity effectively restored the AHR message and protein levels. The protein folding property of p23 which resides at the terminal 50-amino acid region is not involved for this p23 effect. Results from our interaction study using the affinity purified thioredoxin fusion proteins and GST fusion proteins showed that p23 directly interacts with AHR and the interaction surface lies within AHR amino acid 1-216 and p23 amino acid 1-110. Down-regulation of the p23 protein content promotes the ubiquitination of AHR, indicating that p23 protects AHR from the ubiquitin-meditated protein degradation.

Beta tubulin affects the aryl hydrocarbon receptor function via an Arnt-mediated mechanism.

We have been studying the requirement for the aryl hydrocarbon receptor nuclear translocator (Arnt)-dependent DNA complex formation, which precedes the activation of gene transcription. Using DEAE chromatography, we have obtained a Sf9 insect fraction F5 that is highly enriched with beta-tubulin. F5 inhibits the formation of the AhR gel shift complex and this inhibition is sensitive to protease, suggesting that proteins that are present in this F5 fraction are responsible for the inhibition. Additional experiments have revealed that this inhibition is less pronounced in the presence of anti-beta-tubulin IgG and beta-tubulin enriched fraction from pig brain also inhibits the AhR gel shift formation. Sf9 beta-tubulin interacts with Arnt and suppresses the binding of the AhR/Arnt heterodimer to its corresponding enhancer. Human beta4-tubulin, which shares high sequence identity with Sf9 beta-tubulin, suppresses the AhR-dependent luciferase expression by reducing the nuclear Arnt content and retaining Arnt in the cytoplasm. Fluorescence studies using the GFP fusion of human beta4-tubulin have revealed that beta4-tubulin prevents the localization of Arnt in Sf9 cells. Here we have provided evidence suggesting that beta-tubulin may regulate the physiological content of Arnt.

Comparison of sebum secretion, skin type, pH in humans with and without acne.

Differences of skin type and pH between subjects with and without acne have not been investigated. In addition, the relationship between sebum secretion and pH in these populations has not been determined. This study assessed the differences in objective and subjective skin types between these two groups. Secondly, this study evaluated the difference in pH on five facial areas (forehead, nose, chin, right and left cheeks) between the two populations. Lastly, the relationship between pH and sebum secretion was analyzed in each population. Sebum casual levels (CL) of the five facial areas in 36 Koreans with acne and 47 Koreans without acne were measured by using a Sebumeter SM 815 and subjects were classified into objective skin types by CL. Subjects reported the type of skin they believed they had, which determined the subjective skin type. The pH levels of the five facial areas were measured by the Skin-pH-Meter PH 905. Data were assessed with adequate statistical tests depending on data type and distribution. Among the five areas, the nose of the subjects with acne showed a significantly higher CL, compared to the subjects without acne. This difference in CL on the nose resulted in the difference in CL on the T-zone and mean facial sebum excretions (MFSE). Although CL differed, objective skin types did not differ between the two groups (P > 0.05), but the subjective skin types differed significantly (P = 0.001). In addition, the objective skin types were significantly different than the subjective skin types in subjects with acne (P = 0.001), whereas the two skin types did not differ in subjects without acne. Subjects with acne actually overestimated their skin types and stated their skin types were "oilier" than they were. In respect to pH, none of the five areas differed significantly between the two groups. Among the five sites in subjects with acne, CL showed a significant negative correlation with pH on the left (r (2 )=0.12) and right (r ( 2 )=0.15) cheeks, which resulted in a significant negative correlation on the U-zone (r ( 2 )=0.14). In contrast, in subjects without acne, there was a significant negative correlation between CL and pH on the forehead (r ( 2 )=0.10) and chin (r ( 2 )=0.16), which led to a significant negative correlation on the T-zone (r ( 2 )=0.14).

Effects of switching pravastatin to cerivastatin on C-reactive protein, butyrylcholinesterase, and lipids.

OBJECTIVE: C-reactive protein (CRP) concentrations, butyrylcholinesterase (BChE) activity, total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglycerides (TG) were evaluated in patients switched from pravastatin to cerivastatin. The purpose of this study was to determine whether a more potent statin (cerivastatin) would further affect CRP, whether a relation ship between CRP and BChE existed, and if there were any relationships between CRP or BChE and lipids. In view of the withdrawal of cerivastatin from the market, studies considering the effects of conversion of patients from one statin to another are warranted. RESEARCH DESIGN AND METHODS: Thirty-seven patients actively taking pravastatin (10 mg-40 mg) were switched to cerivastatin (0.2 mg-0.8 mg) at the initial visit in the Lipid Clinic at David Grant Medical Center, Travis Air Force Base. Samples were collected before the conversion (pravastatin phase) and at 6 weeks and 12 weeks post-conversion. Patients were excluded from the study if they were taking gemfibrozil concomitantly. Patients were counseled on the adverse effects of cerivastatin, including rhabdomyolsis. RESULTS: Median CRP levels at the pravastatin phase, 6 weeks of cerivastatin, and 12 weeks of cerivastatin, were 0.380 mg/dL, 0.403 mg/dL, and 0.364 mg/dL (p = 0.772), respectively. Median BChE activity at the pravastatin phase, 6 weeks of cerivastatin, and 12 weeks of cerivastatin were 0.338 micromol/mL/min, 0.332 micromol/mL/min, 0.33 micromol/mL/min (p = 0.746), respectively. A negative correlation was observed between CRP and BChE at baseline only (r = -0.353, p = 0.032). There was a significant decline in mean TC (p < 0.001) and median LDL (p < 0.001) and a significant increase in mean HDL (p = 0.017) over the three time points. Numerically TG declined, but it was not statistically significant (p = 0.649). No correlations were observed between CRP or BChE and any of the lipids. Gender, aspirin use, and the presence of CHD or diabetes did not affect CRP levels or BChE activity. CONCLUSION: Median CRP remained stable with pravastatin and cerivastatin use, although TC and LDL decreased. The further decline observed with LDL, but not CRP suggests differing effects of statins on LDL and CRP. Limitations include no serum levels prior to statin use and small sample size; thus, future studies are needed to address the relationship between cholesterol and CRP and the mechanism of action of statins on CRP.

TNF-alpha detection using a flow cytometric assay to determine cellular responses to anthrax vaccine.

This study describes a four-color flow cytometric assay that detects CD4+ T cell responses to the anthrax vaccine. Whole blood from seven volunteers who previously obtained the anthrax vaccine was inoculated in vitro with varying concentrations of the anthrax antigen. TNF-alpha and IFN-gamma production from memory CD4+ T cells were measured and compared to a control group who never received the anthrax vaccine. The optimal antigen concentration for TNF-alpha was determined to be around 7.5 microg/ml and IFN-gamma production was not detected. This assay will be used in future larger prospective studies to further evaluate the cellular immune responses induced by the anthrax vaccine.