Gab1, SHP2, and protein kinase A are crucial for the activation of the endothelial NO synthase by fluid shear stress

Fluid shear stress enhances NO production in endothelial cells by a mechanism involving the activation of the phosphatidylinositol 3-kinase and the phosphorylation of the endothelial NO synthase (eNOS). We investigated the role of the scaffolding protein Gab1 and the tyrosine phosphatase SHP2 in this signal transduction cascade in cultured and native endothelial cells. Fluid shear stress elicited the phosphorylation and activation of Akt and eNOS as well as the tyrosine phosphorylation of Gab1 and its association with the p85 subunit of phosphatidylinositol 3-kinase and SHP2. Overexpression of a Gab1 mutant lacking the pleckstrin homology domain abrogated the shear stress-induced phosphorylation of Akt but failed to affect the phosphorylation or activity of eNOS. The latter response, however, was sensitive to a protein kinase A (PKA) inhibitor. Mutation of Gab1 Tyr627 to phenylalanine (YF-Gab1) to prevent the binding of SHP2 completely prevented the shear stress-induced phosphorylation of eNOS, leaving the Akt response intact. A dominant-negative SHP2 mutant prevented the activation of PKA and phosphorylation of eNOS without affecting that of Akt. Moreover, shear stress elicited the formation of a signalosome complex including eNOS, Gab1, SHP2 and the catalytic subunit of PKA. In isolated murine carotid arteries, flow-induced vasodilatation was prevented by a PKA inhibitor as well as by overexpression of either the YF-Gab1 or the dominant-negative SHP2 mutant. Thus, the shear stress-induced activation of eNOS depends on Gab1 and SHP2, which, in turn, regulate the phosphorylation and activity of eNOS by a PKA-dependent but Akt-independent mechanism.     

Specificity and regioselectivity of the conjugation of estradiol, estrone, and their catecholestrogen and methoxyestrogen metabolites by human uridine diphospho-glucuronosyltransferases expressed in endometrium

Uridine diphospho-glucuronosyltransferases (UGTs) inactivate and facilitate the excretion of estrogens to glucuronides (-G), the most abundant circulating estrogen conjugates. The identity of the conjugated estrogens formed by all known overexpressed UGTs (n = 16) was analyzed by comparison with retention time and mass fragmentation of authentic standards by HPLC tandem mass spectrometry methods. Six UGTs, namely 1A1, 1A3, 1A8, 1A9, 1A10, and 2B7, were found to glucuronidate estradiol (E(2)) and estrone (E(1)), their hydroxyls (OH), and their methoxy derivatives (MeO). Addition of glucuronic acid was catalyzed by specific UGTs at positions 2, 3, and 4 of the estrogens, whereas only E(2) was conjugated at position 17 by UGT2B7. Kinetic parameters indicate that the conjugation of E(2) at position 3 was predominantly catalyzed by 1A1, 1A3, and 1A8 and by 1A8 for E(1). Conjugation of 2-OHE(1)/E(2) and 2- and 4-MeOE(1)/E(2) was selective at position 3, mostly catalyzed by 1A1 and 1A8. Of all UGTs, UGT2B7 demonstrated the highest catalytic activities for estrogens and at least 10- to 50-fold higher activity for the conjugation of genotoxic 4-hydroxycatecholestrogens at position 4, compared with the conjugation of E(2), E(1), and 2-hydroxycatecholestrogens. Its presence was further shown in the endometrium by RT-PCR and immunohistochemistry, localizing in the same cells expressing CYP1B1, involved locally in the formation of 4-hydroxycatecholestrogens. Data show that several UGT enzymes detected in the endometrium are involved in the glucuronidation of E(2) and its 2-OH, 4-OH, and 2-MeO metabolites that exert various biological effects in the tissue.     

Constant vs auto-continuous positive airway pressure in patients with sleep apnea hypopnea syndrome and a high variability in pressure requirement

STUDY OBJECTIVES: Auto-continuous positive airway pressure (CPAP) has been reported to have no more efficacy than constant CPAP in unselected patients with sleep apnea hypopnea syndrome (SAHS). The aim of this study was to evaluate patients judged to be good candidates for auto-CPAP because of a high within-night variability in pressure requirement. DESIGN: Single-blind, randomized, cross-over study (2 x 8 weeks) to compare auto-CPAP with constant CPAP. PATIENTS: Outpatients with moderate-to-severe SAHS attending the chest clinic. INTERVENTIONS: Patients were equipped at home in the auto-CPAP mode (model GK418A; Malinckrodt; Nancy, France), using a 4- to 14-cm H(2)O pressure range. Those individuals having a high within-night variability in pressure requirement, assessed at the end of a 14-day run-in period, were included in the cross-over study. Auto-CPAP was compared with constant CPAP (according to a titration night in the sleep laboratory) in terms of compliance, efficacy on apneas (assessed from the pressure monitor), and sleepiness (assessed on the Epworth sleepiness scale). MEASUREMENTS AND RESULTS: Of 90 consecutive patients with SAHS, 27 patients were selected for a within-night variability in pressure requirement exceeding a given threshold. After completion of the cross-over, 24 patients were evaluable. The median percentage of nights the machine was used was 95.5% (range, 45 to 100%) on constant CPAP, and 96.5% (range, 40 to 100%) on auto-CPAP; the median apnea index recorded by the device was 0.40/h (range, 0 to 2.40/h) on constant CPAP, and 0.45/h (range, 0 to 5.80/h) on auto-CPAP (differences not significant). The mean Epworth sleepiness score was significantly (p < 0.01) lower on auto-CPAP (5.1; SD, 2.8) than on constant CPAP (6.1; SD, 2.8). CONCLUSIONS: In patients selected for a high within-night variability in pressure requirement, auto-CPAP administered via a GK418A device was equivalent to constant CPAP based on a titration night in the sleep laboratory. Subjective ratings for sleepiness were slightly lower on auto-CPAP.     

Crystal structure of a eukaryotic zinc-dependent histone deacetylase, human HDAC8, complexed with a hydroxamic acid inhibitor

Histone deacetylases (HDACs) are a family of enzymes involved in the regulation of gene expression, DNA repair, and stress response. These processes often are altered in tumors, and HDAC inhibitors have had pronounced antitumor activity with promising results in clinical trials. Here, we report the crystal structure of human HDAC8 in complex with a hydroxamic acid inhibitor. Such a structure of a eukaryotic zinc-dependent HDAC has not be described previously. Similar to bacterial HDAC-like protein, HDAC8 folds in a single alpha/beta domain. The inhibitor and the zinc-binding sites are similar in both proteins. However, significant differences are observed in the length and structure of the loops surrounding the active site, including the presence of two potassium ions in HDAC8 structure, one of which interacts with key catalytic residues. CD data suggest a direct role of potassium in the fold stabilization of HDAC8. Knockdown of HDAC8 by RNA interference inhibits growth of human lung, colon, and cervical cancer cell lines, highlighting the importance of this HDAC subtype for tumor cell proliferation. Our findings open the way for the design and development of selective inhibitors of HDAC8 as possible antitumor agents.     

Rheumatoid flat foot and deformity of the first ray

OBJECTIVE: To study the relationship between flat foot and forefoot deformities in rheumatoid arthritis (RA) in order to improve understanding of the progression of deformity and thus provide more appropriate treatment. METHODS: Anteroposterior and lateral weight-bearing radiographs were obtained of 308 feet of patients with RA and 202 feet of patients with neck pain (control feet). RESULTS: In women with RA, we observed with disease duration an increased frequency of flat foot that was correlated with first ray deformity (chiefly metatarsus primus adductus) and severe stages of disability. Flat foot increased very markedly after 3-4 years of disease duration. In control women, flat feet were more frequent after the age of 50 years. CONCLUSION: In RA the inflammatory and mechanical factors leading to foot deformity must receive early medical treatment to avoid progressive hindfoot deformities that lead to disability.     

Alcohol intake and diet in France, the prominent role of lifestyle.

AIM: Moderate alcohol intake is related to a decrease of coronary heart disease. This protective effect may be attributed to ethanol but may also depend on the type of alcoholic beverages. However, these differences may be confounded by lifestyle and diet. We investigated the relationships between alcohol consumption, beverage type preference and socio-economic status, diet and lifestyle. METHODS AND RESULTS: A cross-sectional survey on cardiovascular risk factors and nutrition was carried out from 1995 to 1997 by the French MONICA Centres. A sample of 1110 middle-aged men (45-64 years) was randomly recruited; 12.8% of men were abstainers and 16.3% reported a consumption of #10878;60 g/d alcohol. Smoking, waist-to-hip ratio and hypertension increased along with the amount of alcohol intake. Physical activity (from 40.9% in abstainers to 23.8% in heavy drinkers, p=0.0025), educational level (from 11.9+/-4.4 to 11.1+/-3.8 years, p=0.01), socio-economic status and diet quality index (from 7.1+/-2.3 to 6.3+/-2.0, p<0.0001 after multivariate adjustment) decreased along with the increase of alcohol consumption and were higher among wine drinkers than among beer or mixed drinkers. Diet quality index was 7.1+/-1.9, 6.4+/-1.8 and 6.6+/-1.9 among wine, beer and mixed drinkers, respectively (p=0.007 after multivariate adjustment). CONCLUSION: Moderate alcohol drinkers or wine drinkers have healthy diet and behaviours compared to other drinkers or abstainers. The living area plays a significant role in the dieting behaviours.     

Follicular lymphoma international prognostic index

The prognosis of follicular lymphomas (FL) is heterogeneous and numerous treatments may be proposed. A validated prognostic index (PI) would help in evaluating and choosing these treatments. Characteristics at diagnosis were collected from 4167 patients with FL diagnosed between 1985 and 1992. Univariate and multivariate analyses were used to propose a PI. This index was then tested on 919 patients. Five adverse prognostic factors were selected: age (> 60 years vs 60 years), Ann Arbor stage (III-IV vs I-II), hemoglobin level (< 120 g/L vs 120 g/L), number of nodal areas (> 4 vs 4), and serum LDH level (above normal vs normal or below). Three risk groups were defined: low risk (0-1 adverse factor, 36% of patients), intermediate risk (2 factors, 37% of patients, hazard ratio [HR] of 2.3), and poor risk ( 3 adverse factors, 27% of patients, HR = 4.3). This Follicular Lymphoma International Prognostic Index (FLIPI) appeared more discriminant than the International Prognostic Index proposed for aggressive non-Hodgkin lymphomas. Results were very similar in the confirmation group. The FLIPI may be used for improving treatment choices, comparing clinical trials, and designing studies to evaluate new treatments.