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Retinoid X receptors (RXRα, β, and γ) are essential members of the nuclear receptor (NR) superfamily of ligand-dependent transcriptional regulators that bind DNA response elements and control the expression of large gene networks. As obligate heterodimerization partners of many NRs, RXRs are involved in a variety of pathophysiological processes. However, despite this central role in NR signaling, there is still no consensus regarding the precise biological functions of RXRs and the putative role of the endogenous ligands (rexinoids) previously proposed for these receptors. Based on available crystal structures, we introduced a series of amino acid substitutions into the ligand-binding pocket of all three RXR subtypes in order to alter their binding properties. Subsequent characterization using a battery of cell-based and in vitro assays led to the identification of a double mutation abolishing the binding of any ligand while keeping the other receptor functions intact and a triple mutation that selectively impairs interaction with natural rexinoids but not with some synthetic ligands. We also report crystal structures that help understand the specific ligand-binding capabilities of both variants. These RXR variants, either fully disabled for ligand binding or retaining the property of being activated by synthetic compounds, represent unique tools that could be used in future studies to probe the presence of active endogenous rexinoids in tissues/organs and to investigate their role in vivo. Last, we provide data suggesting a possible involvement of fatty acids in the weak interaction of RXRs with corepressors.

Background: Triglycerides are lipids in the human body that are produced from the consumption of daily food and drink. However, elevated serum triglycerides, also known as hypertriglyceridemia (HTG), are key biomarkers indicating an unhealthy status and increased risks of cardiovascular diseases (CVDs) and pancreatitis. Different groups of people have different patterns and styles of cooking and different patterns of consumption, such as hill tribe people, who have their own unique culture and cooking practices. This study aimed to estimate the prevalence of and determine the factors associated with HTG among the hill tribe population in Thailand., Method: A cross-sectional study was performed. Data and a-5 mL blood sample were collected from participants who were members of one of the six main hill tribes in Thailand: Akah, Lahu, Hmong, Yao, Karen, and Lisu. People who lived in 30 selected hill tribe villages and aged 30 years over were asked to participate the study. Pearson correlation and logistic regression were used to detect the correlations and determine the associations between variables, respectively, at a significant level of α = 0.05., Results: A total of 2552 participants participated this study; 65.9% were females, 72.35% were aged 40-69 years, 76.7% had no education, 48.7% worked in the agricultural section, and 71.2% had an annual income of less than 50,000 baht/family. Regarding the triglyceride level, 41.7% of participants had elevated levels of serum triglyceride or HTG; 16.4% had a borderline high level, and 25.3% had a high level. After controlling for all potential confounder factors, three variables were found to be associated with elevated serum triglycerides. Those who were members of the Lahu and Hmong tribes were 1.62 times (95%CI = 1.25-2.01) and 1.63 times (95%CI = 1.23-2.16) more likely to have elevated serum triglycerides than those who were members of the Akha tribe, respectively. Those who used a high quantity of cooking oil for daily cooking were 0.73 times less likely to have an abnormal level of triglycerides than those who used a low quantity of cooking oil for daily cooking (95%CI = 0.58-0.91), and those who had a waist circumference indicating obesity were 1.28 times more likely to have an abnormal level of triglycerides than those who had a normal waist circumference (95%CI = 1.08-1.52)., Conclusion: Public health programs that focus on encouraging people to have regular exercise to reduce their body weight, particularly in some tribes, such as Lahu and Hmong, should be implemented.

Background: Low-density lipoprotein cholesterol (LDL-C) is one of the most important types of cholesterol and has an impact on health. Certain lifestyle and dietary habits in different populations may leads to increased levels of LDL-C, particularly among those with poor education and economic statuses, such as hill tribe people in Thailand. This study aimed to estimate the prevalence of and determine the factors associated with high LDL-C levels among hill tribe people in northern Thailand., Methods: A cross-sectional study was performed to gather information from six main hill tribe populations: Akha, Lahu, Hmong, Yao, Karen, and Lisu. Individuals who were aged over 30 years and living in 30 selected hill tribe villages were invited to participate in the study. A validated questionnaire and 5-mL blood specimens were used to obtain data. Correlation analyses, chi-square tests, t-tests, and logistic regression were used to detect correlations and associations., Results: A total of 2552 participants were recruited into the study; 65.9% were females, and 64.1% were aged younger than 60 years old. Approximately 69.6% of participants had abnormal LDL-C levels; 33.6% had above-optimal levels, 24.3% had borderline high levels, 8.0% had high levels, and 3.7% had very high levels. A total of 17.4% of participants had low high-density lipoprotein cholesterol (HDL-C) and high LDL-C levels, while 14.9% had high triglyceride and LDL-C levels. After controlling for sex, age, religion, education, annual family income, and marital status in the multivariate model, three variables were found to be associated with high LDL-C levels: occupation, the amount of lard used in daily cooking, and glycated hemoglobin (HbA1c). Those who were working as agriculturalists had a 1.34-fold greater chance of having abnormal LDL-C than traders and others (95% CI = 1.09-1.34). Those who used moderate and high quantities of lard in their daily cooking had a 1.45-fold (95% CI = 1.15-1.82) and 1.31-fold (95% CI = 1.04-1.68) greater likelihood of having abnormal LDL-C levels than those who used low quantities, respectively. Those who had abnormal HbA1c levels were less likely to develop abnormal LDL-C levels than those who had normal HbA1c levels (AOR = 0.69, 95% CI = 0.51-92)., Conclusions: Effective public health programs that do not conflict with the cultures of hill tribes are urgently needed, particularly programs encouraging the use of small quantities of lard for daily cooking practices.

The histone methyltransferase EZH2 has been the target of numerous small-molecule inhibitor discovery efforts over the last 10+ years. Emerging clinical data have provided early evidence for single agent activity with acceptable safety profiles for first-generation inhibitors. We have developed kinetic methodologies for studying EZH2-inhibitor-binding kinetics that have allowed us to identify a unique structural modification that results in significant increases in the drug-target residence times of all EZH2 inhibitor scaffolds we have studied. The unexpected residence time enhancement bestowed by this modification has enabled us to create a series of second-generation EZH2 inhibitors with sub-pM binding affinities. We provide both biophysical evidence validating this sub-pM potency and biological evidence demonstrating the utility and relevance of such high-affinity interactions with EZH2., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The major population vulnerable to hand, foot and mouth disease (HFMD) is children aged less than 5 years, particularly those who are cared for at day care centers (DCCs). This study aimed to assess the associations of environmental and sanitation factors with high HFMD occurrence rates in DCCs of northern Thailand., Methods: A case-control study was used to gather information from caregivers and local government administrative officers. DCCs in areas with high and low HFMD occurrence rates were the settings for this study. A validated questionnaire was used to collect environmental and sanitation information from the DCCs. In-depth interviews were used to collect information from selected participants who were working at DCCs and from local government administrative officers on the HFMD capacity and prevention and control strategies in DCCs. Logistic regression analysis was used to determine the associations between many environmental factors and HFMD at the α = 0.05 significance level while the content analysis was used to extract information from the interviews., Results: Two variables were found to be associated with a high rate of HFMD occurrence: the number of sinks available in restrooms and the DCC size. Children attending DCCs that did not meet the standard in terms of the number of sinks in restrooms had a greater chance of contracting HFMD than children who were attending DCCs that met the standard (AOR = 4.21; 95% CI = 1.13-15.04). Children who were attending a large-sized DCC had a greater chance of contracting HFMD than those attending a small-sized DCC (AOR = 3.28; 95% CI = 1.21-5.18). The yearly budget allocation and the strategies for HFMD control and prevention, including collaborations among stakeholders for HFMD control and prevention in DCCs, were associated with the effectiveness of HFMD control and prevention., Conclusions: The number of sinks in restrooms and DCC size are major concerns for HFMD outbreaks. Sufficient budget allocation and good collaboration contribute to effective strategies for preventing and controlling HFMD in DCCs.

Background: Depression is a silent health problem that can lead to severe and sometimes fatal outcomes if individuals are not diagnosed and treated properly; this is particularly true in populations with limited education, low economic status and several barriers to accessing health services, such as the hill tribe people in Thailand., Methods: This cross-sectional study aimed to explore the prevalence of and factors associated with depression among hill tribe individuals aged 30 years and over. A validated questionnaire and the Patient Health Questionnaire-9 (PHQ-9) were used for data collection in an interview format in a private and confidential room. Logistic regression was used to detect the associations of variables with depression at a significance level of α = 0.05., Results: A total of 2,552 participants were recruited for the analysis; 65.9% were females, 79.9% were married, 35.8% were aged 50 years and over, and 54.2% were Buddhist. The majority were uneducated (76.7%), were agriculturalists (48.4%), and had a low family income (71.2%). The overall prevalence of depression was 12.0%. In the multivariate analysis, eight variables were found to be significantly associated with depression among hill tribe adults aged 30 years and over in Thailand. Being female, 50 years or older, married, and Christian; living with a relative; smoking; using opium; and having moderate and high stress levels were associated with depression., Conclusion: Effective public health interventions should be considered to reduce the burden of depression in the hill tribe population by focusing on individuals who are female, 50 years or older, married, Christian, and stressed and those who abuse substances., (© 2020 Published by Elsevier Ltd.)

EP300 and CBP (KAT3A/3B) are two highly homologous, multidomain, epigenetic coregulators that play central roles in transcription through the acetylation of lysine residues on histones and other proteins. Both enzymes have been implicated in human diseases, especially cancer. From a high-throughput screen of 191 000 compounds searching for EP300/CBP histone acetyltransferase (HAT) inhibitors, 18 compounds were characterized by a suite of biochemical enzymatic assays and biophysical methods, including X-ray crystallography and native mass spectrometry. This work resulted in the discovery of three distinct mechanistic classes of EP300/CBP HAT inhibitors, including two classes not previously described. The profiles of an example of each class of inhibitor are described in detail. A subsequent medicinal chemistry effort led to the development of a novel class of orally bioavailable AcCoA-competitive EP300/CBP HAT inhibitors with in vivo activity. We believe that this work will prove to be a useful guide for other groups interested in the development of HAT inhibitors., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Hypertension (HT) is a serious noncommunicable disease that can lead to several health problems when it is not detected or is not properly diagnosed and treated in a timely manner, particularly in individuals living in poor economic and education conditions. The hill tribe population in northern Thailand is a vulnerable population with limited information available regarding HT., Methods: The study aimed to estimate the prevalence of HT and to determine the factors associated with HT among individuals from hill tribes aged 35 years and over and living in northern Thailand. A cross-sectional study was conducted to gather essential information from six main hill tribe groups: the Akha, Lahu, Karen, Hmong, Yao and Lisu tribes in Chiang Rai Province. A simple random method was used to select 30 hill tribe villages (5 villages for each tribe). People aged 35 years and over who lived in the selected villages were invited to participate in the study. A validated questionnaire and a 5-mL blood specimen were used as research instruments. A face-to-face interview was conducted to collect data after informed consent was obtained, and 5-mL blood specimens were drawn to determine the lipid profiles of the participants. Logistic regression was performed to determine the factors associated with HT at the significance level of α = 0.05., Results: A total of 1,287 participants were recruited into the study: 60.5% were females, 30.4% were aged 35-44 years, 65.4% were illiterate, and 83.1% were married. The overall prevalence of HT was 24.3%, and the Yao tribe had the highest prevalence at 18.5%. In the multivariable analysis, three variables were found to be associated with HT: marital status, ability to read Thai, and exercise behavior. Those who were single and divorced had a 2.55 (95% CI = 1.23-5.06) and 2.69 times greater chance (95% CI = 1.10-6.59), respectively, of developing HT than those who were married. Those who could not read Thai had a 2.13 times greater chance (95% CI = 1.50-3.01) of developing HT than those who could read, and those who did not exercise and who exercised sometimes had a 1.96 (95% CI = 1.07-3.58) and 2.24 times greater chance (95% CI = 1.21-4.13), respectively, of developing HT than those who regularly exercised., Conclusion: A health screening program for the identification of new HT among the hill tribe population urgently needs to be implemented, followed by the introduction of a proper exercise program to reduce the risk of HT, particularly for those who are illiterate and for single or divorced people., Competing Interests: None., (AJCD Copyright © 2020.)

Hill tribes are a group of people who live in remote areas in northern Thailand. They typically use untreated water for drinking, that can lead several health problems. The six main hill tribes-Akha, Hmong, Karen, Lahu, Lisu, and Yao-were selected for the study. A validated questionnaire was used for data collection. Water samples were collected from the selected villages and tested for the quality at Mae Fah Luang University, Thailand. Results: the major sources of drinking water were mountain water supplies (74.3%), and commercial bottled water (21.4%). Easy access, sufficiency for the whole year, and food-drug administration sign labeled were the criteria used for selecting sources of drinking water. Colorless and safety were also used as a selection criteria for their drinking water in some tribes. Lisu, Karen, and Hmong treated their drinking water by boiling, while Akha and Lahu stored the water in certain containers to allow particle settling before drinking water without treatment. 42.0% of the water samples had a turbidity values <5 NTU, and total coliform and fecal coliform bacteria were detected in 100.0% of the samples. To prevent water-borne diseases among the hill tribe people, appropriate water treatments such as boiling, filtration, and disinfection are recommended.

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) catalyze the dynamic and reversible acetylation of proteins, an epigenetic regulatory mechanism associated with multiple cancers. Indeed, HDAC inhibitors are already approved in the clinic. The HAT paralogs p300 and CREB-binding protein (CBP) have been implicated in human pathological conditions including several hematological malignancies and androgen receptor-positive prostate cancer. Others have reported CoA-competitive inhibitors of p300 and CBP with cell-based activity. Here, we describe 2 compounds, CPI-076 and CPI-090, discovered through p300-HAT high throughput screening screening, which inhibit p300-HAT via binding at an allosteric site. We present the high resolution (1.7 and 2.3 Å) co-crystal structures of these molecules bound to a previously undescribed allosteric site of p300-HAT. Derivatization yielded actionable structure-activity relationships, but the full-length enzymatic assay demonstrated that this allosteric HAT inhibitor series was artifactual, inhibiting only the HAT domain of p300 with no effect on the full-length enzyme., (© 2019 Author(s).)

In its unliganded form, the retinoic acid receptor (RAR) in heterodimer with the retinoid X receptor (RXR) exerts a strong repressive activity facilitated by the recruitment of transcriptional corepressors in the promoter region of target genes. By integrating complementary structural, biophysical, and computational information, we demonstrate that intrinsic disorder is a required feature for the precise regulation of RAR activity. We show that structural dynamics of RAR and RXR H12 regions is an essential mechanism for RAR regulation. Unexpectedly we found that, while mainly disordered, the corepressor N-CoR presents evolutionary conserved structured regions involved in transient intramolecular contacts. In the presence of RXR/RAR, N-CoR exploits its multivalency to form a cooperative multisite complex that displays equilibrium between different conformational states that can be tuned by cognate ligands and receptor mutations. This equilibrium is key to preserving the repressive basal state while allowing the conversion to a transcriptionally active form., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Combination of biophysical and structural techniques allowed characterizing and uncovering the mechanisms underlying increased binding affinity of lactosamine derivatives for galectin 3. In particular, complementing information gathered from X-ray crystallography, native mass spectrometry and isothermal microcalorimetry showed favorable enthalpic contribution of cation-π interaction between lactosamine aryl substitutions and arginine residues from the carbohydrate recognition domain, which resulted in two log increase in compound binding affinity. This incrementing strategy allowed individual contribution of galectin inhibitor moieties to be dissected. Altogether, our results suggest that core and substituents of these saccharide-based inhibitors can be optimized separately, providing valuable tools to study the role of galectins in diseases., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Humans are chronically exposed to multiple exogenous substances, including environmental pollutants, drugs and dietary components. Many of these compounds are suspected to impact human health, and their combination in complex mixtures could exacerbate their harmful effects. Here we demonstrate that a pharmaceutical oestrogen and a persistent organochlorine pesticide, both exhibiting low efficacy when studied separately, cooperatively bind to the pregnane X receptor, leading to synergistic activation. Biophysical analysis shows that each ligand enhances the binding affinity of the other, so the binary mixture induces a substantial biological response at doses at which each chemical individually is inactive. High-resolution crystal structures reveal the structural basis for the observed cooperativity. Our results suggest that the formation of 'supramolecular ligands' within the ligand-binding pocket of nuclear receptors contributes to the synergistic toxic effect of chemical mixtures, which may have broad implications for the fields of endocrine disruption, toxicology and chemical risk assessment.

Retinoic acid is an important regulator of cell differentiation which plays major roles in embryonic development and tissue remodeling. The biological action of retinoic acid is mediated by three nuclear receptors denoted RARα, β and γ. Multiple studies support that RARβ possesses functional characteristics of a tumor suppressor and indeed, its expression is frequently lost in neoplastic tissues. However, it has been recently reported that RARβ could also play a role in mammary gland tumorigenesis, thus demonstrating the important but yet incompletely understood function of this receptor in cancer development. As a consequence, there is a great need for RARβ-selective agonists and antagonists as tools to facilitate the pharmacological analysis of this protein in vitro and in vivo as well as for potential therapeutic interventions. Here we provide experimental evidences that the novel synthetic retinoid BMS948 is an RARβ-selective ligand exhibiting a full transcriptional agonistic activity and activating RARβ as efficiently as the reference agonist TTNPB. In addition, we solved the crystal structures of the RARβ ligand-binding domain in complex with BMS948 and two related compounds, BMS641 and BMS411. These structures provided a rationale to explain how a single retinoid can be at the same time an RARα antagonist and an RARβ full agonist, and revealed the structural basis of partial agonism. Finally, in addition to revealing that a flip by 180° of the amide linker, that usually confers RARα selectivity, accounts for the RARβ selectivity of BMS948, the structural analysis uncovers guidelines for the rational design of RARβ-selective antagonists.

The gastric pathogen Helicobacter pylori is one of the most genetically diverse of bacterial species. Much of its diversity stems from frequent mutation and recombination, preferential transmission within families and local communities, and selection during persistent gastric mucosal infection. MLST of seven housekeeping genes had identified multiple distinct H. pylori populations, including three from Africa: hpNEAfrica, hpAfrica1 and hpAfrica2, which consists of three subpopulations (hspWAfrica, hspCAfrica and hspSAfrica). Most detailed H. pylori population analyses have used strains from non-African countries, despite Africa's high importance in the emergence and evolution of humans and their pathogens. Our concatenated sequences from seven H. pylori housekeeping genes from 44 Gambian patients (MLST) identified 42 distinct sequence types (or haplotypes), and no clustering with age or disease. STRUCTURE analysis of the sequence data indicated that Gambian H. pylori strains belong to the hspWAfrica subpopulation of hpAfrica1, in accord with Gambia's West African location. Despite Gambia's history of invasion and colonisation by Europeans and North Africans during the last millennium, no traces of Ancestral Europe1 (AE1) population carried by those people were found. Instead, admixture of 17% from Ancestral Europe2 (AE2) was detected in Gambian strains; this population predominates in Nilo-Saharan speakers of North-East Africa, and might have been derived from admixture of hpNEAfrica strains these people carried when they migrated across the Sahara during the Holocene humid period 6,000-9,000 years ago. Alternatively, shared AE2 ancestry might have resulted from shared ancestral polymorphisms already present in the common ancestor of sister populations hpAfrica1 and hpNEAfrica.

Keap1 binds to the Nrf2 transcription factor to promote its degradation, resulting in the loss of gene products that protect against oxidative stress. While cell-active small molecules have been identified that modify cysteines in Keap1 and effect the Nrf2 dependent pathway, few act through a non-covalent mechanism. We have identified and characterized several small molecule compounds that specifically bind to the Keap1 Kelch-DC domain as measured by NMR, native mass spectrometry and X-ray crystallography. One compound upregulates Nrf2 response genes measured by a luciferase cell reporter assay. The non-covalent inhibition strategy presents a reasonable course of action to avoid toxic side-effects due to non-specific cysteine modification., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Helicobacter pylori is a globally important and genetically diverse gastric pathogen that infects most people in developing countries. Eradication efforts are complicated by antibiotic resistance, which varies in frequency geographically. There are very few data on resistance in African strains. Sixty-four Gambian H. pylori strains were tested for antibiotic susceptibility. The role of rdxA in metronidazole (Mtz) susceptibility was tested by DNA transformation and sequencing; RdxA protein variants were interpreted in terms of RdxA structure. Forty-four strains (69%) were resistant to at least 8 μg of Mtz/ml. All six strains from infants, but only 24% of strains from adults, were sensitive (P = 0.0031). Representative Mtz-resistant (Mtz(r)) strains were rendered Mtz susceptible (Mtz(s)) by transformation with a functional rdxA gene; conversely, Mtz(s) strains were rendered Mtz(r) by rdxA inactivation. Many mutations were found by Gambian H. pylori rdxA sequencing; mutations that probably inactivated rdxA in Mtz(r) strains were identified and explained using RdxA protein's structure. All of the strains were sensitive to clarithromycin and erythromycin. Amoxicillin and tetracycline resistance was rare. Sequence analysis indicated that most tetracycline resistance, when found, was not due to 16S rRNA gene mutations. These data suggest caution in the use of Mtz-based therapies in The Gambia. The increasing use of macrolides against respiratory infections in The Gambia calls for continued antibiotic susceptibility monitoring. The rich variety of rdxA mutations that we found will be useful in further structure-function studies of RdxA, the enzyme responsible for Mtz susceptibility in this important pathogen.

The human USP7 deubiquitinating enzyme was shown to regulate many proteins involved in the cell cycle, as well as tumor suppressors and oncogenes. Thus, USP7 offers a promising, strategic target for cancer therapy. Using biochemical assays and activity-based protein profiling in living systems, we identified small-molecule antagonists of USP7 and demonstrated USP7 inhibitor occupancy and selectivity in cancer cell lines. These compounds bind USP7 in the active site through a covalent mechanism. In cancer cells, these active-site-targeting inhibitors were shown to regulate the level of several USP7 substrates and thus recapitulated the USP7 knockdown phenotype that leads to G1 arrest in colon cancer cells. The data presented in this report provide proof of principle that USP7 inhibitors may be a valuable therapeutic for cancer. In addition, the discovery of such molecules offers interesting tools for studying deubiquitination., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Helicobacter pylori is an important agent of gastroduodenal disease in Africa and throughout the world. We sought to determine an optimum method for genotyping H. pylori strains from children and adults in The Gambia, West Africa., Results: Virulence genes were amplified in 127 of 190 cases tested (121 adults and 6 children); each of 60 bacterial cultures, and 116 from DNA extracted directly from biopsies. The proportion of biopsies that were cagA+, the ratio of vacAs1/s2, and vacAm1/m2, and the proportion of mixed strain populations in individual subjects changed with age. Strains lacking virulence cagA and vacA genes and with apparently homogeneous (one predominant strain) infections were more common among infants than adults., Conclusions: In order to detect the range of bacterial genotypes harbored by individual patients, direct PCR proved slightly superior to isolation of H. pylori by biopsy culture, but the techniques were complementary, and the combination of both culture and direct PCR produced the most complete picture. The seemingly higher virulence of strains from adult than infant infections in The Gambia merits further analysis.

Background: The prevalence of Helicobacter pylori including strains with putatively virulent genotypes is high, whereas the H. pylori-associated disease burden is low, in Africa compared to developed countries. In this study, we investigated the prevalence of virulence-related H. pylori genotypes and their association with gastroduodenal diseases in The Gambia., Methods and Findings: DNA extracted from biopsies and H. pylori cultures from 169 subjects with abdominal pain, dyspepsia or other gastroduodenal diseases were tested by PCR for H. pylori. The H. pylori positive samples were further tested for the cagA oncogene and vacA toxin gene. One hundred and twenty one subjects (71.6%) were H. pylori positive. The cagA gene and more toxigenic s1 and m1 alleles of the vacA gene were found in 61.2%, 76.9% and 45.5% respectively of Gambian patients harbouring H. pylori. There was a high prevalence of cagA positive strains in patients with overt gastric diseases than those with non-ulcerative dyspepsia (NUD) (p = 0.05); however, mixed infection by cagA positive and cagA negative strains was more common in patients with NUD compared to patients with gastric disease (24.5% versus 0%; p = 0.002)., Conclusion: This study shows that the prevalence of H. pylori is high in dyspeptic patients in The Gambia and that many strains are of the putatively more virulent cagA+, vacAs1 and vacAm1 genotypes. This study has also shown significantly lower disease burden in Gambians infected with a mixture of cag-positive and cag-negative strains, relative to those containing only cag-positive or only cag-negative strains, which suggests that harbouring both cag-positive and cag-negative strains is protective.

Fourteen temporoparietal fascial free flaps were used for correction of first web space atrophy from ulnar nerve palsy in 13 patients. Ten sustained ulnar nerve injuries and three suffered from leprosy. The procedures were performed under general anesthesia except one leprosy patient with bilateral ulnar nerve palsy in which local anesthesia and brachial block were employed to harvest bilateral free flaps and recipient site preparations, respectively. The follow-up time varied from 4 to 64 months. The postoperative results were satisfactory and there was no resorption of the free flaps. The consistency of the augmented first web space was soft and compressible like natural feel. The size of the flap was more than enough for augmentation of first web space and donor site morbidity was minimal and accepted by all patients. We conclude that temporoparietal fascial free flap is an ideal autogenous tissue for correction of first web space atrophy.

Bilateral temporoparietal fascial free flaps were used for reconstruction of bilateral hand defects in two male patients. A 42-year-old man sustained crushed injury to both hands with avulsion defects and exposed bones and tendons. The two separate procedures were performed under general anesthesia. The temporoparietal fascial free flap was skin grafted on the ward on the following day after the operation. The other patient was a 61-year-old leprosy patient who had bilateral high ulnar nerve palsy for 28 years. One simultaneous procedure was performed under local anesthesia for harvesting the temporoparietal fascial free flaps and under brachial block for preparation of the recipient sites. The free flaps were used for augmentation of the atrophic first web spaces. The postoperative results of the two cases were satisfactory. The functions of both hands were restored with normal gliding mechanism of the tendons in the first case, and permanent correction of the atrophic web spaces was demonstrated in the second case. The temporoparietal fascial free flap is an ideal flap for coverage of hand defects as well as augmentation of first web space atrophy.

In eukaryotic cells, the ubiquitin-proteasome pathway is the major mechanism for targeted degradation of proteins. We show that, in F9 cells and in transfected COS-1 cells, the nuclear retinoid receptors, retinoic acid receptor gamma2 (RARgamma2), RARalpha1, and retinoid X receptor alpha1 (RXRalpha1) are degraded in a retinoic acid-dependent manner through the ubiquitin-proteasome pathway. The degradation of RARgamma2 is entirely dependent on its phosphorylation and on its heterodimerization with liganded RXRalpha1. In contrast, RARalpha1 degradation can occur in the absence of heterodimerization, whereas it is inhibited by phosphorylation, and heterodimerization reverses that inhibition. RXRalpha1 degradation is also modulated by heterodimerization. Thus, each partner of RARgamma/RXRalpha and RARalpha/RXRalpha heterodimers modulates the degradation of the other. We conclude that the ligand-dependent degradation of RARs and RXRs by the ubiquitin-proteasome pathway, which is regulated by heterodimerization and by phosphorylation, could be important for the regulation of the magnitude and duration of the effects of retinoid signals.

The crystal structure of a heterodimer between the ligand-binding domains (LBDs) of the human RARalpha bound to a selective antagonist and the constitutively active mouse RXRalphaF318A mutant shows that, pushed by a bulky extension of the ligand, RARalpha helix H12 adopts an antagonist position. The unexpected presence of a fatty acid in the ligand-binding pocket of RXRalpha(F318A is likely to account for its apparent "constitutivity." Specific conformational changes suggest the structural basis of pure and partial antagonism. The RAR-RXR heterodimer interface is similar to that observed in most nuclear receptor (NR) homodimers. A correlative analysis of 3D structures and sequences provides a novel view on dimerization among members of the nuclear receptor superfamily.

Background: Many synthetic retinoids have been generated that exhibit a distinct pattern of agonist/antagonist activities with the three retinoic acid receptors (RARalpha, RARbeta and RARgamma). Because these retinoids are selective tools with which to dissect the pleiotropic functions of the natural pan-agonist, retinoic acid, and might constitute new therapeutic drugs, we have determined the structural basis of their receptor specificity and compared their activities in animal and yeast cells., Results: There are only three divergent amino acid residues in the ligand binding pockets (LBPs) of RARalpha, RARbeta and RARgamma. We demonstrate here that the ability of monospecific (class I) retinoid agonists and antagonists to bind to and induce or inhibit transactivation by a given isotype is directly linked to the nature of these residues. The agonist/antagonist potential of class II retinoids, which bind to all three RARs but depending on the RAR isotype have the potential to act as agonists or antagonists, was also largely determined by the three divergent LBP residues. These mutational studies were complemented by modelling, on the basis of the three-dimensional structures of the RAR ligand-binding domains, and a comparison of the retinoid agonist/antagonist activities in animal and yeast cells., Conclusions: Our results reveal the rational basis of RAR isotype selectivity, explain the existence of class I and II retinoids, and provide a structural concept of ligand-mediated antagonism. Interestingly, the agonist/antagonist characteristics of retinoids are not conserved in yeast cells, suggesting that yeast co-regulators interact with RARs in a different way than the animal cell homologues do.

The nuclear receptor (NR) coactivator TIF2 possesses a single NR interaction domain (NID) and two autonomous activation domains, AD1 and AD2. The TIF2 NID is composed of three NR-interacting modules each containing the NR box motif LxxLL. Mutation of boxes I, II and III abrogates TIF2-NR interaction and stimulation, in transfected cells, of the ligand-induced activation function-2 (AF-2) present in the ligand-binding domains (LBDs) of several NRs. The presence of an intact NR interaction module II in the NID is sufficient for both efficient interaction with NR holo-LBDs and stimulation of AF-2 activity. Modules I and III are poorly efficient on their own, but synergistically can promote interaction with NR holo-LBDs and AF-2 stimulation. TIF2 AD1 activity appears to be mediated through CBP, as AD1 could not be separated mutationally from the CBP interaction domain. In contrast, TIF2 AD2 activity apparently does not involve interaction with CBP. TIF2 exhibited the characteristics expected for a bona fide NR coactivator, in both mammalian and yeast cells. Moreover, in mammalian cells, a peptide encompassing the TIF2 NID inhibited the ligand-induced AF-2 activity of several NRs, indicating that NR AF-2 activity is either mediated by endogenous TIF2 or by coactivators recognizing a similar surface on NR holo-LBDs.

The retinoic acid receptor alpha (Rara) gene, which maps in the same region as the pulmonary adenoma resistance (Par1) locus on mouse chromosome 11 (Manenti G et al., Nature Genet 12:455-457, 1996), was tested as a candidate gene for Par1. We report here the analysis of loss of heterozygosity, nucleotide sequence comparison, gene expression, and biochemical activity of the Rara gene from the Mus spretus(Par1/+) and A/J (Par1/-) mouse strains. The two Rara alleles were distinguished by two amino-acid variations but had similar biochemical activity and expression levels, leading to the exclusion of Rara as a candidate Par1 gene.

A 28-kDa fragment (residues 178-423) of the human retinoic acid receptor gamma, hRARgamma D3E, encompassing the ligand-binding domain (LBD) was overproduced in Escherichia coli and purified as a monomer to more than 95% purity and homogeneity. The Kd for all-trans retinoic acid binding was 0.6 +/- 0.1 nM. Crystals of the LBD complexed with all-trans retinoic acid were grown at pH 7 from sodium acetate in the presence of detergents using the vapor diffusion method. They diffract to 2.0 A using a synchrotron radiation (lambda=0.91 A) and belong to the tetragonal space group P4(1)2(1)2 with unit cell parameters a=b=60.6 A and c=155.3 A, one monomer per asymmetric unit, a solvent content of ca. 33%, and a Vm value of approximately 2 A3/dalton.

The activity of the ligand-inducible activation function 2 (AF-2) contained in the ligand binding domain (LBD) of nuclear receptors (NRs) is thought to be mediated by transcriptional intermediary factors (TIFs). We have recently reported the isolation and characterization of two novel mouse proteins, designated TIF1 and mSUG1, that interact in a ligand-dependent fashion with the LBD (region E) of several NRs in vivo as well as in vitro. Remarkably, these interactions require the conserved core motif of the AF-2 activating domain (AF-2 AD) and can be blocked by AF-2 antagonists. TIF1 and mSUG1 might therefore represent TIFs/mediators for the ligand-dependent AF-2 of NRs. By comparing the interaction properties of these two putative TIFs with different NRs including the oestrogen (ER), thyroid hormone (TR), vitamin D3 (VDR), retinoic acid (RAR alpha) and retinoid X (RXR) receptors, we demonstrate that: (i) RXR alpha efficiently interacts with TIF1, but not with mSUG1, whereas TR alpha interacts much more efficiently with mSUG1 than with TIF1, and RAR alpha, VDR and ER efficiently interact with both TIF1 and mSUG1; (ii) the amphipathic alpha helix core of AF-2 AD is differentially involved in the interactions of RAR alpha with TIF1 and mSUG1; and (iii) the AF-2 AD cores of RAR alpha and ER are similarly involved in their interaction with TIF1, but not with mSUG1. Thus the interaction interfaces between the various NRs and either TIF1 or mSUG1 may vary depending on the nature of both the receptor and the putative mediator of its AF-2 function. We discuss the possible roles of TIF1 and mSUG1 as mediators of the transcriptional activity of the AF-2 of NRs.

The 2.0-A crystal structure of the ligand-binding domain (LBD) of the human retinoic acid receptor (RAR)-gamma bound to all-trans retinoic acid reveals the ligand-binding interactions and suggests an electrostatic guidance mechanism. The overall fold is similar to that of the human RXR-alpha apo-LBD, except for the carboxy-terminal part which folds back towards the LBD core, contributing to the hydrophobic ligand pocket and 'sealing' its entry site. We propose a 'mouse trap' mechanism whereby a ligand-induced conformational transition repositions the amphipathic alpha-helix of the AF-2 activating domain and forms a transcriptionally active receptor.

The effects of pregnancy or progesterone dominance on the beta-adrenergic responsiveness of the uterus were studied in myometrial membranes from mid- and late-pregnant rats (day 15 and on the 16th h of day 22 of pregnancy respectively) or 24 h after administration of progesterone. Levels of the high (RH)- and low (RL)-affinity states of the beta-adrenergic receptor were determined by competition experiments between 125I-labelled cyanopindolol binding and the selective beta-agonist isoproterenol. The ratio KL/KH (respective dissociation constants) was determined since it also reflects the degree of formation of the high-affinity state of the beta-adrenergic receptor. From day 15 to the 10th h of day 22 of pregnancy, two distinct affinity states were apparent: 80-55% RH (KH = 0.31-0.21 microM) and 45-20% RL (KL = 14-5 microM) with a ratio of KL/KH of 55-34. In the last 6 h before birth, beta-adrenergic receptors underwent uncoupling which was paralleled by decreased responsiveness of myometrial adenylate cyclase to isoproterenol (maximum velocity (Vmax) = 17 +/- 3 vs 44 +/- 3 fmol cyclic AMP/10 min per mg protein on day 15). At this stage of pregnancy, previous exposure to progesterone resulted in a 1.8-fold increase in 125I-labelled cyanopindolol-binding sites (Bmax) and the reappearance of the high-affinity state (67% RH, KH = 0.19 +/- 0.04 (S.E.M.) microM, ratio KL/KH = 81.1 +/- 16.9).(ABSTRACT TRUNCATED AT 250 WORDS)