High-resolution neutron and X-ray diffraction room-temperature studies of an H-FABP-oleic acid complex: study of the internal water cluster and ligand binding by a transferred multipolar electron-density distribution.

Crystal diffraction data of heart fatty acid binding protein (H-FABP) in complex with oleic acid were measured at room temperature with high-resolution X-ray and neutron protein crystallography (0.98 and 1.90 Šresolution, respectively). These data provided very detailed information about the cluster of water molecules and the bound oleic acid in the H-FABP large internal cavity. The jointly refined X-ray/neutron structure of H-FABP was complemented by a transferred multipolar electron-density distribution using the parameters of the ELMAMII library. The resulting electron density allowed a precise determination of the electrostatic potential in the fatty acid (FA) binding pocket. Bader's quantum theory of atoms in molecules was then used to study interactions involving the internal water molecules, the FA and the protein. This approach showed H⋯H contacts of the FA with highly conserved hydrophobic residues known to play a role in the stabilization of long-chain FAs in the binding cavity. The determination of water hydrogen (deuterium) positions allowed the analysis of the orientation and electrostatic properties of the water molecules in the very ordered cluster. As a result, a significant alignment of the permanent dipoles of the water molecules with the protein electrostatic field was observed. This can be related to the dielectric properties of hydration layers around proteins, where the shielding of electrostatic interactions depends directly on the rotational degrees of freedom of the water molecules in the interface.

Perdeuteration: improved visualization of solvent structure in neutron macromolecular crystallography.

The 1.8 Šresolution neutron structure of deuterated type III antifreeze protein in which the methyl groups of leucine and valine residues are selectively protonated is presented. Comparison between this and the 1.85 Šresolution neutron structure of perdeuterated type III antifreeze protein indicates that perdeuteration improves the visibility of solvent molecules located in close vicinity to hydrophobic residues, as cancellation effects between H atoms of the methyl groups and nearby heavy-water molecules (D2O) are avoided.

Diagnosis and management of melanoma with regional lymph node metastases: a population-based study in France.

BACKGROUND: Stage III melanoma represents a borderline situation regarding the potential curability of this potentially aggressive cancer and consequently, regional lymph node metastases (RLNM) are a major challenge for melanoma management. OBJECTIVE: To describe the management of melanoma with RLNM as practised in France in 2008 and compare results with previous data from 2004, considering that new French recommendations were published in 2005. METHODS: Retrospective population-based study in five regions of France totalling 8.3 million inhabitants, targeting all incident cases of RLNM diagnosed in 2008. Questionnaires were mailed to physicians to identify cases and collect data, with verification by cancer registries for cases diagnosed concomitantly with the primary tumour using sentinel lymph node biopsies (SLNB). RESULTS: Data were collected for 101 patients in 2008, and compared to 89 cases treated in 2004. Palpation by a dermatologist was the most common circumstance of diagnosis of RLNM in 2008 (36%), followed by SLNB (29%), self-palpation by the patient (16%) and lymph node ultrasonography (6%), without significant modification from 2004. After lymphadenectomy an adjuvant therapy was proposed in 62% of cases, mainly consisting in high-dose interferon (HD-IFN) (80%). Overall, HD-IFN was proposed in 49% of cases, but effectively started in only 40% of cases after being proposed, and prematurely withdrawn in 28%, showing major changes as compared with 2004 (33%, 77% and 67%, respectively, P < 0.05). Adjuvant chemotherapy was not proposed to any patients in 2008, compared to 29% in 2004. Surveillance procedures included medical imaging less often in 2008 (76%) than in 2004 (92%) (P = 0.004), but more often included FDG-PET (23% vs. 12%, P = 0.09). CONCLUSION: Overall, actual practice was in accordance with French recommendations. The main developments from 2004 to 2008 were the disappearance of adjuvant chemotherapies and a more accurate selection of patients for adjuvant interferon.

The role of general practitioners in diagnosis of cutaneous melanoma: a population-based study in France.

BACKGROUND: Little data are available concerning the role of general practitioners (GPs) in the diagnosis of melanoma. OBJECTIVES: To evaluate the actual role of GPs in a population-based study covering five regions of France and 8·2 million inhabitants. MATERIALS AND METHODS: A survey of cancer registries and pathology laboratories, and questionnaires to practitioners were used to identify incident melanomas in 2008, and evaluate characteristics of patients (age, sex, area of residence, social isolation), tumours (Breslow, ulceration, location, histological type), and GPs (training, conditions of practice), and their influence on patterns of diagnosis and Breslow thickness. RESULTS: Among 898 melanomas, 376 (42%) were first diagnosed in a general practice setting (GP group). Breslow thickness was much higher in the GP group than in other melanomas (median: 0·95 vs. 0·61 mm, P < 0·0001). Multivariate analysis identified an older age, lower limb location, nodular subtype and Breslow thickness as factors associated with the GP group. Within this group, 52·5% of melanomas were detected by patients (median Breslow thickness: 1·30 mm) and 47·5% by GPs (median Breslow thickness: 0·80 mm, P = 0·0009), including 8% during a systematic full-body skin examination. Previous GP training on melanoma was associated with active detection by GPs. Male sex and social isolation of patients were associated with thicker melanomas, whereas active detection by GPs was associated with thinner CMs. CONCLUSIONS: GPs play a key role in melanoma diagnosis in France, but still frequently detect thick tumours. Increasing awareness and training of GPs and focusing attention on male and/or socially isolated patients should help to improve early detection of melanoma.

Comparison of hydrogen determination with X-ray and neutron crystallography in a human aldose reductase-inhibitor complex.

Protonation states determination by neutron (2.2 A at room temperature) and X-ray (0.66 A at 100 K) crystallographic studies were compared for a medium size enzyme, human aldose reductase (MW=36 kDa), complexed with its NADP+ coenzyme and a selected inhibitor of therapeutic interest. The neutron resolution could be achieved only with the ab initio fully deuterated protein and the subsequent crystallization in D2O of the complex. We used the largest good-quality crystal (1.00x0.67x0.23 mm, i.e. volume of 0.15 mm3) that we were able to grow so far. Both studies enable the determination of protonation states, with a clear advantage for neutrons in the case of less-ordered atoms (B>5 A2). Hydrogen atoms are best determined by a complementary analysis of the Fourier maps obtained from both methods.

High-resolution neutron protein crystallography with radically small crystal volumes: application of perdeuteration to human aldose reductase.

Neutron diffraction data have been collected to 2.2 Angstrom resolution from a small (0.15 mm(3)) crystal of perdeuterated human aldose reductase (h-AR; MW = 36 kDa) in order to help to determine the protonation state of the enzyme. h-AR belongs to the aldo-keto reductase family and is implicated in diabetic complications. Its ternary complexes (h-AR-coenzyme NADPH-selected inhibitor) provide a good model to study both the enzymatic mechanism and inhibition. Here, the successful production of fully deuterated human aldose reductase [h-AR(D)], subsequent crystallization of the ternary complex h-AR(D)-NADPH-IDD594 and neutron Laue data collection at the LADI instrument at ILL using a crystal volume of just 0.15 mm(3) are reported. Neutron data were recorded to 2 Angstrom resolution, with subsequent data analysis using data to 2.2 Angstrom. This is the first fully deuterated enzyme of this size (36 kDa) to be solved by neutron diffraction and represents a milestone in the field, as the crystal volume is at least one order of magnitude smaller than those usually required for other high-resolution neutron structures determined to date. This illustrates the significant increase in the signal-to-noise ratio of data collected from perdeuterated crystals and demonstrates that good-quality neutron data can now be collected from more typical protein crystal volumes. Indeed, the signal-to-noise ratio is then dominated by other sources of instrument background, the nature of which is under investigation. This is important for the design of future instruments, which should take maximum advantage of the reduction in the intrinsic diffraction pattern background from fully deuterated samples.

[Knowledge and prevention of tick-bite borreliosis: survey of the population in Alsace, an endemic area]. / Connaissance et prévention des borrélioses par piqûres de tiques. Enquête dans la population d'une région endémique européenne, l'Alsace.

INTRODUCTION: European borreliosis, known to the general public as 'Lyme disease' in analogy with the American form, which it differs from in many points, is endemic in the area of Alsace and is a public health problem. The level of knowledge and prevention of the population with regard to the disease has never been assessed in France. MATERIAL AND METHODS: A survey was conducted in all the national health examination centers in Alsace using a self-applied questionnaire. The data collected concerned the socio-demographical characteristics, knowledge on the disease, history of tick bites, fear the disease creates, frequency of visits to the forest, prevention habits when visiting the forest and the attitude adopted in the case of a tick bite. RESULTS: Out of the 600 persons included, 99 (16.5 p.cent) had been bitten by ticks once or more over the past year. The existence of Lyme's disease was known to 74 p.cent of the consultants, 63 p.cent claimed they were worried by the disease and 43 p.cent knew that the first manifestation is redness spreading over the skin. Twenty-seven percent wore trousers and long sleeves when visiting the forest and 19 p.cent inspected their body carefully on their return. The persons least well informed were also those who did little to protect themselves against tick bites. They often were under 30 years old, lived in urban settings and had few diplomas. Those who had frequent spare time in forest and those who had a history of tick bites were the best informed and protected themselves better. The fear of the disease was associated with better knowledge and more appropriate behaviour. DISCUSSION: This study shows that a large percent of the population in Alsace is exposed to tick bites. Tick bite borreliosis is relatively well known, but protection remains insufficient. Better knowledge of the disease is related to better prevention. Information and teaching campaigns for the general public could specifically target the young people, urban dwellers and those with few diplomas. Specific messages of prevention could be delivered to the most exposed at-risk subjects (i.e. those bitten by ticks or having leisure in forest) at the places of their leisure or medical consultations.

Ultrahigh resolution drug design I: details of interactions in human aldose reductase-inhibitor complex at 0.66 A.

The first subatomic resolution structure of a 36 kDa protein [aldose reductase (AR)] is presented. AR was cocrystallized at pH 5.0 with its cofactor NADP+ and inhibitor IDD 594, a therapeutic candidate for the treatment of diabetic complications. X-ray diffraction data were collected up to 0.62 A resolution and treated up to 0.66 A resolution. Anisotropic refinement followed by a blocked matrix inversion produced low standard deviations (<0.005 A). The model was very well ordered overall (CA atoms' mean B factor is 5.5 A2). The model and the electron-density maps revealed fine features, such as H-atoms, bond densities, and significant deviations from standard stereochemistry. Other features, such as networks of hydrogen bonds (H bonds), a large number of multiple conformations, and solvent structure were also better defined. Most of the atoms in the active site region were extremely well ordered (mean B approximately 3 A2), leading to the identification of the protonation states of the residues involved in catalysis. The electrostatic interactions of the inhibitor's charged carboxylate head with the catalytic residues and the charged coenzyme NADP+ explained the inhibitor's noncompetitive character. Furthermore, a short contact involving the IDD 594 bromine atom explained the selectivity profile of the inhibitor, important feature to avoid toxic effects. The presented structure and the details revealed are instrumental for better understanding of the inhibition mechanism of AR by IDD 594, and hence, for the rational drug design of future inhibitors. This work demonstrates the capabilities of subatomic resolution experiments and stimulates further developments of methods allowing the use of the full potential of these experiments.

[Malignant dermohypodermic melanoma of the soft tissues]. / Mélanome des tissus mous dermohypodermique.

INTRODUCTION: Melanoma of the soft tissue is a rare tumor that develops in most cases in the deep structures, only rarely involving the skin surface. We report an exceptional case of melanoma of the superficial soft tissue, exclusively involving the skin surface. OBSERVATION: A 35-year-old man presented with a superficial, pink, soft, mobile, lesion on the thigh. The histological examination revealed a tumoral proliferation of the reticular dermis and hypodermis, composed of pale giant cells grouped in nests or in lobules separated by connective ducts. Immunostaining was positive for HMB45, protein S-100, vimentine and NKIC3. It was negative for EMA, CD34, smooth muscle actin, cytokeratine and desmine. The search for a metastatic localization was negative. Extensive exeresis was performed. Three years later, the patient was still in complete remission. The macroscopic, histological and immunohistochemical examinations concluded in the diagnosis of a strictly dermohypodermic melanoma of the soft tissues. DISCUSSION: Our case report of a melanoma of the soft tissues is original because of the superficial localization of the tumor that, to our knowledge, has never been reported. It underlines the interest of performing systematic biopsies of any fast growing cutaneous lesion of recent discovery.

Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands.

The crystal structures of the ligand-binding domain (LBD) of the vitamin D receptor complexed to 1alpha,25(OH)(2)D(3) and the 20-epi analogs, MC1288 and KH1060, show that the protein conformation is identical, conferring a general character to the observation first made for retinoic acid receptor (RAR) that, for a given LBD, the agonist conformation is unique, the ligands adapting to the binding pocket. In all complexes, the A- to D-ring moieties of the ligands adopt the same conformation and form identical contacts with the protein. Differences are observed only for the 17beta-aliphatic chains that adapt their conformation to anchor the 25-hydroxyl group to His-305 and His-397. The inverted geometry of the C20 methyl group induces different paths of the aliphatic chains. The ligands exhibit a low-energy conformation for MC1288 and a more strained conformation for the two others. KH1060 compensates this energy cost by additional contacts. Based on the present data, the explanation of the superagonist effect is to be found in higher stability and longer half-life of the active complex, thereby excluding different conformations of the ligand binding domain.

[Severe pruriginous acne in dialysed renal failure. Diagnostic difficulties and efficacy of isotretinoin]. / Acné prurigineuse grave chez l'insuffisant rénal dialysé. Difficultés diagnostiques et efficacité de l'isotrétinoïne.

INTRODUCTION: The occurrence of acne in dialysed renal failure patients has rarely been reported and the clinical characteristics and therapeutic issues rarely studied in these patients. CASE REPORTS: Two men and two women, 33 to 56 years-old, with chronic renal failure and no past history of acne, developed severe acne under dialysis. The acne was excoriated in all cases and associated with prurigo-like lesions and intense pruritus, which made diagnosis difficult. Acne was profuse on the face and the trunk, but also on the neck (1 case) and the upper limbs (2 cases). No patient was taking acne-inducing substances. Various to therapies attempting to control pruritus were ineffective. However, anti-acne treatments (cyclines associated with local tretinoin in 1 case and oral isotreninoin in 3 cases) led to complete regression of the acne, pruritus and the prurigo-like lesions without relapse after a follow-up time of 4 months to 2 years. DISCUSSION: Pruritus is frequent during renal failure. However, the occurrence of unexplained acne has only rarely been reported. Our patients' clinical picture was original, characterized by the late development, under dialysis, of severe and pruriginous acne, the pathogenesis of which is unknown. Because of the clinical and therapeutic implications (impaired quality of life, pigmentation or scarring and remarkable efficacy of oral isotretinoin) this clinical picture merits more attention, and the modalities for the prescription of isotretinoin in this context should be defined.

Structural basis for isotype selectivity of the human retinoic acid nuclear receptor.

The human retinoic acid receptor (hRAR) belongs to the family of nuclear receptors that regulate transcription in a ligand-dependent way. The isotypes RARalpha,beta and gamma are distinct pharmacological targets for retinoids that are involved in the treatment of various skin diseases and cancers, in particular breast cancer and acute promyelocytic leukemia. Therefore, synthetic retinoids have been developed aiming at isotype selectivity and reduced side-effects. We report the crystal structures of three complexes of the hRARgamma ligand-binding domain (LBD) bound to agonist retinoids that possess selectivity either for RARgamma (BMS184394) or for RARbeta/gamma (CD564), or that are potent for all RAR-isotypes (panagonist BMS181156). The high resolution data (1.3-1. 5 A) provide a description at the atomic level of the ligand pocket revealing the molecular determinants for the different degrees of ligand selectivity. The comparison of the complexes of the chemically closely related retinoids BMS184394 and CD564 shows that the side-chain of Met272 adopts different conformations depending on the presence of a hydrogen bond between its sulfur atom and the ligand. This accounts for their different isotype selectivity. On the other hand, the difference between the pan- and the RARbeta, gamma-selective agonist is probably due to a steric discrimination at the level of the 2-naphthoic acid moiety of CD564. Based on this study, we propose a model for a complex with the RARgamma-specific agonist CD666 that shows the possible applications for structure-based drug design of RAR isotype-selective retinoids.

Crystal structure of the human RXRalpha ligand-binding domain bound to its natural ligand: 9-cis retinoic acid.

The pleiotropic effects of active retinoids are transduced by their cognate nuclear receptors, retinoid X receptors (RXRs) and retinoic acid receptors (RARs), which act as transcriptional regulators activated by two stereoisomers of retinoic acid (RA): 9-cis RA (9-cRA) and all-trans RA (a-tRA). Among nuclear receptors, RXR occupies a central position and plays a crucial role in many intracellular signalling pathways as a ubiquitous heterodimerization partner with numerous other members of this superfamily. Whereas RARs bind both isomers, RXRs exclusively bind 9-cRA. The crystal structure of the ligand-binding domain (LBD) of human RXRalpha bound to 9-cRA reveals the molecular basis of this ligand selectivity and allows a comparison of both apo and holo forms of the same nuclear receptor. In the crystal, the receptor is monomeric and exhibits a canonical agonist conformation without direct contacts between the ligand and the transactivation helix H12. Comparison with the unliganded RXRalpha LBD structure reveals the molecular mechanisms of ligand-induced conformational changes and allows us to describe at the atomic level how these changes generate the proper protein interface involved in nuclear receptor-coactivator interaction.

Enantiomer discrimination illustrated by high-resolution crystal structures of the human nuclear receptor hRARgamma.

The human retinoic acid receptor (hRAR) is a member of the nuclear receptor superfamily that regulates the transcription of target genes in a ligand-dependent manner. The three hRAR isotypes are targets for retinoids that are used in the treatment of various diseases, including breast cancer and skin diseases. Drug efficiency and safety depend on the pharmacological activity of enantiomers, which can differ because of the chiral environment generated by the target. We report the crystal structures of the hRARgamma ligand-binding domain bound to two enantiomers, the active BMS270394 and the inactive BMS270395, solved at 1.6 A and 1.7 A resolution, respectively. The crystal structures reveal that in both enantiomers, the hydroxyl moiety attached to the chiral center forms a hydrogen bond to the Met-272 sulfur atom, thus imposing a conformation of BMS270395 that differs significantly from that observed for BMS270394 and other known retinoids. BMS270395 adopts an energetically unfavorable conformation, accounting for its inactivity; in contrast, the conformation of BMS270394 is close to an energy minimum. Our high-resolution data allow rationalization of enantiomer discrimination by the receptor and provide a model system for the pharmacological properties of enantiomeric pairs.

The structure of human aldose reductase bound to the inhibitor IDD384.

The crystallographic structure of the complex between human aldose reductase (AR2) and one of its inhibitors, IDD384, has been solved at 1.7 A resolution from crystals obtained at pH 5.0. This structure shows that the binding of the inhibitor's hydrophilic head to the catalytic residues Tyr48 and His110 differs from that found previously with porcine AR2. The difference is attributed to a change in the protonation state of the inhibitor (pK(a) = 4.52) when soaked with crystals of human (at pH 5.0) or pig lens AR2 (at pH 6.2). This work demonstrates how strongly the detailed binding of the inhibitor's polar head depends on its protonation state.

The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand.

The action of 1 alpha, 25-dihydroxyvitamin D3 is mediated by its nuclear receptor (VDR), a ligand-dependent transcription regulator. We report the 1.8 A resolution crystal structure of the complex between a VDR ligand-binding domain (LBD) construct lacking the highly variable VDR-specific insertion domain and vitamin D. The construct exhibits the same binding affinity for vitamin D and transactivation ability as the wild-type protein, showing that the N-terminal part of the LBD is essential for its structural and functional integrity while the large insertion peptide is dispensable. The structure reveals the active conformation of the bound ligand and allows understanding of the different binding properties of some synthetic analogs.

Dimerization of Escherichia coli DNA-gyrase B provides a structural mechanism for activating the ATPase catalytic center.

DNA-gyrase exhibits an unusual ATP-binding site that is formed as a result of gyrase B subunit dimerization, a structural transition that is also essential for DNA capture during the topoisomerization cycle. Previous structural studies on Escherichia coli DNA-gyrase B revealed that dimerization is the result of a polypeptidic exchange involving the N-terminal 14 amino acids. To provide experimental data that dimerization is critical for ATPase activity and enzyme turnover, we generated mutants with reduced dimerization by mutating the two most conserved residues of the GyrB N-terminal arm (Tyr-5 and Ile-10 residues). Our data demonstrate that the hydrophobic Ile-10 residue plays an important role in enzyme dimerization and the nucleotide-protein contact mediated by Tyr-5 side chain residue helps the dimerization process. Analysis of ATPase activities of mutant proteins provides evidence that dimerization enhances the ATP-hydrolysis turnover. The structure of the Y5S mutant of the N-terminal 43-kDa fragment of E. coli DNA GyrB subunit indicates that Tyr-5 residue provides a scaffold for the ATP-hydrolysis center. We describe a channel formed at the dimer interface that provides a structural mechanism to allow reactive water molecules to access the gamma-phosphate group of the bound ATP molecule. Together, these results demonstrate that dimerization strongly contributes to the folding and stability of the catalytic site for ATP hydrolysis. A role for the essential Mg(2+) ion for the orientation of the phosphate groups of the bound nucleotide inside the reactive pocket was also uncovered by superposition of the 5'-adenylyl beta-gamma-imidodiphosphate (ADPNP) wild-type structure to the salt-free ADPNP structure.

Binding of aldose reductase inhibitors: correlation of crystallographic and mass spectrometric studies.

Aldose reductase is a NADP(H)-dependent enzyme, believed to be strongly implicated in the development of degenerative complications of Diabetes Mellitus. The search for specific inhibitors of this enzyme has thus become a major pharmaceutic challenge. In this study, we applied both X-ray crystallography and mass spectrometry to characterize the interactions between aldose reductase and four representative inhibitors: AminoSNM, Imirestat, LCB3071, and IDD384. If crystallography remains obviously the only way to get an extensive description of the contacts between an inhibitor and the enzymatic site, the duration of the crystallographic analysis makes this technique incompatible with high throughput screenings of inhibitors. On the other hand, dissociation experiments monitored by mass spectrometry permitted us to evaluate rapidly the relative gas-phase stabilities of the aldose reductase-inhibitor noncovalent complexes. In our experiments, dissociation in the gas-phase was provoked by increasing the accelerating voltage of the ions (Vc) in the source-analyzer interface region: the Vc value needed to dissociate 50% of the noncovalent complex initially present (Vc50) was taken as a gas-phase stability parameter of the enzyme-inhibitor complex. Interestingly, the Vc50 were found to correlate with the energy of the electrostatic and H-bond interactions involved in the contact aldose reductase/inhibitor (Eel-H), computed from the crystallographic model. This finding may be specially interesting in a context of drug development. Actually, during a drug design optimization phase, the binding of the drug to the target enzyme is often optimized by modifying its interatomic electrostatic and H-bond contacts; because they usually depend on a single atom change on the drug, and are easier to introduce than the hydrophobic interactions. Therefore, the Vc50 may help to monitor the chemical modifications introduced in new inhibitors. X-ray crystallography is clearly needed to get the details of the contacts and to rationalize the design. Nevertheless, once the cycle of chemical modification is engaged, mass spectrometry can be used to select a priori the drug candidates which are worthy of further crystallographic investigation. We thus propose to use the two techniques in a complementary way, to improve the screening of large collections of inhibitors.

Production of crystals of human aldose reductase with very high resolution diffraction.

As the action of human aldose reductase (hAR) is thought to be linked to the pathogenesis of diabetic complications, much effort has been directed towards the analysis of the catalytic mechanism and the development of specific inhibitors. Here, the crystallization of recombinant hAR with its cofactor NADP+ at 277 K in the presence of the precipitating agent PEG 6000 is reported. The crystals diffract to high resolution (1.1 A) and belong to the P21 space group with unit-cell parameters a = 49.97, b = 67.14, c = 48. 02 A, beta = 92.2 degrees with one molecule per asymmetric unit. Seleno-substituted hAR crystals were also produced and diffract to 1. 7 A on a conventional X-ray source.