Association of breeding conditions with prevalence of osteochondrosis in foals.

Osteochondrosis (OC) is the most common developmental orthopaedic disease in horses and represents a major problem to the horse industry. The complete mechanism of this multifactorial disease is not yet elucidated, but it is accepted that OC lesions are the result of intrinsic genetic and external factors. The aim of the present work was to evaluate the relationship between breeding management and OC. Breeding conditions were recorded, and a radiological examination was performed in 223 foals. Feeding practice and housing management were analysed in a multivariate model to determine risk factors for OC in three periods: gestation, birth to weaning and weaning to one-year-old. The major breakthrough of this study is the significant relationship between OC development and (1) the maternal nutrition during gestation and (2) the type of housing of the foals during their first year. It appears that mares fed with concentrates during gestation are more likely to produce foals that are subsequently affected by OC compared with other mares (P<0.05). Foals housed exclusively at pasture until one year of age are significantly less affected than foals exclusively housed in box or, alternatively, in box and at pasture (P<0.05). These results underline the role of the energy metabolism and the level of exercise in the aetiologic process of the disease, and help to develop preventive strategies during the crucial period of gestation to one year of age of the foal.

Recent advances in the development of selective CB(2) agonists as promising anti-inflammatory agents.

The high distribution of CB(2) receptors in immune cells suggests their important role in the control of inflammation. Growing evidence offers this receptor as an attractive therapeutic target: CB(2) selective agonists are able to modulate inflammation without triggering psychotropic effects. This review will summarize the literature on the implication of CB(2) in inflammation and CB(2) selective agonists with anti-inflammatory activity.

Structural insight into PPARgamma ligands binding.

Peroxisome Proliferator Activated Receptors (PPARs) are a family of three related nuclear receptors first cloned in 1990. Their involvement in glucidic and lipidic homeostasis quickly made them an attractive target for the treatment of metabolic syndrome, the most prevalent mortality factor in developed countries. They therefore attracted much synthetic efforts, more particularly PPARgamma. Supported by a large number of crystallographic studies, data derived from these compounds lead to a fairly clear view of the agonist binding mode into the Ligand Binding Domain (LBD). Nearly all the compounds conform to a three-module structure, with a binder group involved in a series of hydrogen bonds in front of the ligand-dependent Activation Function (AF2), a linker mostly arranged around a phenoxyethyl and an effector end occupying the large cavity of the binding site. Following the marketing of the glitazones and the observation of the hepatotoxicity of troglitazone, variations in the binder led to the glitazars, and then pharmacomodulations have been undertaken on the two other modules, leading to a large family of highly related chemical structures. Some compounds, while still adhering to the three-module structure, diverge from the mainstream, such as the phthalates. Curiously, these plasticizers were known to elicit biological effects that led to the discovery of PPARs but were not actively studied as PPARs agonists. As the biological effects of PPARs became clearer, new compounds were also found to exert at least a part of their actions by the activation of PPARgamma.

Characterisation of novel defective thiopurine S-methyltransferase allelic variants.

Human thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) is a key enzyme in the detoxification of thiopurine drugs widely used in the treatment of various diseases, such as inflammatory bowel diseases, acute lymphoblastic leukaemia and rheumatic diseases. The TPMT gene is genetically polymorphic and the inverse relationship between TPMT activity and the risk of developing severe hematopoietic toxicity is well known. In this study, the entire coding sequence of TPMT, together with its 5'-flanking promoter region, was analysed in patients with an intermediate phenotype for thiopurine drug methylation. Four polymorphisms were identified, two previously described, c.356A>C (p.Lys(119)Thr, TPMT*9) and c.205C>G (p.Leu(69)Val, TPMT*21), and two novel missense mutations, c.537G>T (p.Gln(179)His, TPMT*24) and c.634T>C (p.Cys(212)Arg, TPMT*25). Structural investigations, using molecular modeling, were undertaken in an attempt to explain the potential impact of the amino acid substitutions on the structure and activity of the variant proteins. Additionally, in order to determine kinetic parameters (K(m) and V(max)) of 6-thioguanine (6-TG) methylation, the four variants were expressed in a recombinant yeast expression system. Assays were performed by HPLC and the results were compared with those of wild-type TPMT. The p.Leu(69)Val and the p.Cys(212)Arg substitutions encode recombinant enzymes with a significantly decreased intrinsic clearance compared to that of the wild-type protein, and, consequently, characterise non-functional alleles of TPMT. The p.Lys(119)Thr and the p.Gln(179)His substitutions do not affect significantly the catalytic activity of the corresponding variant proteins, which prevents to unambiguously describe these latter alleles as defective TPMT variants.

PPARgamma as a new therapeutic target in inflammatory bowel diseases.

The peroxisome proliferator activated receptor gamma(PPARgamma) is a nuclear receptor highly expressed in the colon and playing a key role in bacterial induced inflammation. Regulation of colon inflammation by this receptor has been well demonstrated in many experimental models of colitis but also in patients with ulcerative colitis, characterised by impaired expression of PPARgamma confined to their colon epithelial cells. Recent data showing that PPARgamma was the major functional receptor mediating the common aminosalicylate activities in inflammatory bowel diseases (IBD) have also reinforced the roles of this receptor in the control of intestinal inflammation. The aims of this review are to discuss the potential roles of PPARgamma in the physiopathology of IBD, as well as the emerging therapeutic strategies targeting this receptor.

Solid-phase synthesis and pharmacological evaluation of a library of peptidomimetics as potential farnesyltransferase inhibitors: an approach to new lead compounds.

Oncogenic Ras proteins whose activation is farnesylation by farnesyltransferase have been seen as important targets for novel anticancer drugs. Inhibitors of this enzyme have already been developed as potential anti-cancer drugs, particularly by rational design based on the structure of the CA(1)A(2)X carboxyl terminus of Ras. Synthesis of a peptidomimetics library via solid-phase synthesis using the Multipin method is described here. The most active hits on cellular assays were resynthesized and enzymatic activity was measured. Compounds A1, A5 and A7 present significant activity on the isolated enzyme (IC(50)=117, 57.3 and 28.5 nM) and their molecular docking in the active site of the enzyme provides details on key interactions with the protein.

Design, synthesis and in vitro evaluation of novel benzo[b]thiophene derivatives as serotonin N-acetyltransferase (AANAT) inhibitors.

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) is the penultimate enzyme in melatonin (5-methoxy-N-acetyltryptamine) biosynthesis. It is the key-enzyme responsible of the nocturnal rhythm of melatonin production in the pineal gland. Specific AANAT inhibitors could be useful for treatment of different physiopathological disorders encountered in diseases such as seasonal affective disorders or obesity. On the basis of previous works and 3D-QSAR studies carried out in our laboratory, we have synthesized and evaluated four novel benzo[b]thiophene derivatives designed as AANAT inhibitors. Compound 13 exhibited high inhibitory activity (IC50 = 1.4 microM) and low affinities for both MT, (1100 nM) and MT2 (1400 nM) receptors.

Design, synthesis and in vitro evaluation of novel derivatives as serotonin N-acetyltransferase inhibitors.

Serotonin N-acetyltransferase (arylalkylamine N-acetyl-transferase, AANAT) is an enzyme that catalyses the first rate limiting step in the biosynthesis of melatonin (5-methoxy-N-acetyltryptamine). Different physiopathological disorders in human may be due to abnormal secretion of melatonin leading to an inappropriate exposure of melatonin receptors to melatonin. For that reason, we have designed, synthesized and evaluated as inhibitors of human serotonin N-acetyltransferase, a series of compounds that were able to react with coenzyme A to give a bisubstrate analog inhibitor. Compound 12d was found to be a potent AANAT inhibitor (IC50 = 0.18 microM).

Organogenesis of lung and kidney in Thoroughbreds and ponies.

Equine lung and kidney organogenesis has not previously been examined with the use of unbiased stereological techniques. The present study examined healthy (control) pony and Thoroughbred lungs and kidneys to establish baseline data of organ development from before birth until maturity at age 3-18 years. Whole left lungs and kidneys were collected from 45 equine postmortem examinations (34 Thoroughbred, 11 pony). Stereological techniques were used to estimate whole kidney, cortex and medulla volume, total glomerular number and volume-weighted mean glomerular volume, lung volume, total terminal bronchiolar duct ending number and total gas exchange surface area. Lungs were demonstrated to be more developed at birth in ponies compared with Thoroughbreds. Thoroughbreds showed continued lung development after birth, a unique micromorphogenic postnatal development. Kidneys were developed equally in ponies and Thoroughbreds. This study has provided data on the baseline development of the equiune lung and kidney which can be used in further studies to examine whether the development of these organs is affected by specific illnesses.

Three-dimensional quantitative structure-activity relationships of cyclo-oxygenase-2 (COX-2) inhibitors: a comparative molecular field analysis.

The three-dimensional quantitative structure-activity relationship (3D-QSAR) approach using comparative molecular field analysis (CoMFA) was applied to an extensive series of 305 varied diarylheterocyclic derivatives known as COX-2 selective inhibitors. X-ray crystal structure of COX-2 bound with SC-558, a selective COX-2 inhibitor, was used to derive the putative bioactive conformation of these inhibitors. Five statistically significant models were obtained from the randomly constituted training sets (229 compounds) and subsequently validated with the corresponding test sets (76 compounds). The best predictive model (n = 229, q(2) = 0.714, N = 8, r(2) = 0.905, s = 0.291, F = 261.545) was selected for further comparison of the CoMFA contour maps obtained for steric, electrostatic, and lipophilic fields with the enzyme structure. The high level of compatibility with the COX-2 enzyme topology shows the great accuracy of this model that can predict inhibitory activities for a wide range of compounds and offers important structural insight into designing novel antiinflammatory drugs prior to their synthesis.

Conformational analysis of tripeptide Ac-Lys-Pro-Val-NH2, COOH-terminal sequence of alpha-MSH.

Alpha-melanocyte stimulating hormone (alpha-MSH) is an endogenous linear tridecapeptide which interacts with the melanocortin receptors (MC1-R to MC5-R) to mediate its biological effects. Antipyretic and anti-inflammatory activities of alpha-MSH are due to the COOH-terminal peptide sequence, Lys-Pro-Val (alpha-MSH[11-13]). This tripeptide might be useful as a therapeutic agent in the control of fever and inflammatory reactions. With this aim, a theoretical conformational study of the tripeptide has been carried out using molecular dynamics. The obtained conformational space has been classified into families according to the letter-code convention to partition the phi-psi map. The lowest energy conformations of each family were used as templates to design six models of conformationally constrained nonpeptide analogues.

Conformation of the tripeptide Cbz-Pro-Leu-Trp-OBzl(CF3)2 deduced from two-dimensional 1H-NMR and conformational energy calculations is related to its affinity for NK1-receptor.

Chemical modifications of dual NK1/NK2 ligand Cbz-Gly-Leu-Trp-OBzl(CF3)2 (1) enabled us to create a high NK1 selective ligand Cbz-Pro-Leu-Trp-OBzl(CF3)2 (2). A determination of the conformational behavior of tripeptide 2 in solution is described. The 1D and 2D 1H-NMR techniques (COSY and ROESY) were used to assign resonances. Observed interproton distance restraints were considered to characterize conformational behavior. Spectral data indicate that tripeptide 2 presents a rigidified structure in DMSO stabilized by H-bond in two gamma-turns. Agreement with experimental data was obtained by averaging the 1H-NMR parameters over several combinations of low-energy conformations.

Synthesis and pharmacological evaluation of analgesic 6-substituted 2(3H)-benzothiazolones.

A series of novel 6-substituted-2(3H)-benzothiazolones were synthesized and studied as analgesic agents. Among these compounds, two of them were found to exhibit potent analgesic activity in several in vivo tests (acetic acid writhing, Koster, carrageenan and PGE2 hyperalgesia). In these tests the most active compound of this series, i.e. 6-benzoyl-2(3H)-benzothiazolone (4a) was found to be superior to acetylsalicylic acid and equivalent to glafenine. The present study allows to conclude that 4a represents a new type of antinociceptive agent acting in periphery by inhibiting the cyclo-oxygenase pathway and promoting the release of an opioid peptide.

Synthesis and preliminary pharmacological results on new naphthalene derivatives as 5-HT(4) receptor ligands.

The indole derivative GR 113808 is currently used as the reference ligand for labelling the 5-HT(4) serotoninergic receptors. Previous works in our laboratories established the bioisosteric equivalency of the indole heterocycle and naphthalene in a series of melatonin receptor ligands. Based on this knowledge we designed new analogues of GR 113808 by introducing two bioisosteric modifications: firstly, the indole ring was replaced by a naphthalene one and secondly, the ester linkage was replaced by an amide group. Compound 8 emerged within this novel series as it displayed high and selective affinity at 5-HT(4) receptors (Ki 5-HT(4) = 6 nM, Ki 5-HT(3) = 100 nM, Ki values at other 5-HT receptors were higher than 1000 nM). Compound 8 is currently undergoing further pharmacological evaluation.

Scanning electron microscopy of the microcotyledonary placenta of the horse (Equus caballus) in the latter half of gestation.

Scanning electron microscopy was used to examine the microstructure of the maternal and fetal placenta from 25 pregnant ponies (Equus caballus) throughout the second half of gestation. Samples of placenta, 2-6 cm(2)in area, were collected from the antimesometrial region of the pregnant horn at 105 days (n=1), 165-219 days (n=5), 260 days (n=3), 270-277 days (n=3), 313-337 days (n=11) and immediately after spontaneous delivery at term (n=2). The maternal microcaruncle appears to be created from a clustering of about 16 uterine crypts encapsulated in a connective tissue sheath. There is a gestational increase in the depth of the microcaruncle during the second half of pregnancy. The fetal microcotyledon appears to be formed by a clustering of individual fetal villi. The length and branching of the villi increased considerably during the last 2-3 months of gestation. Tufts of from three to six branches were seen arising from close to the base of a villous stem. Branching of the villous stem occurred not only at the base but also along the secondary and tertiary branches. There was evidence of continued branching at the tips of the villi in the few days before birth.

Critical role of tyrosine 277 in the ligand-binding and transactivating properties of retinoic acid receptor alpha.

Retinoic acid receptors specifically bind all-trans-retinoic acid (RA) and function as RA-inducible transcriptional regulatory factors. Binding of RA to RARalpha, beta, and gamma is sensitive to nitration with tetranitromethane, a tyrosine-specific modifying reagent. To identify tyrosine residue(s) that are important for RA binding, we carried out chemical modification experiments with purified RARalpha ligand-binding domain (RARalpha-LBD) subjected to partial acid hydrolysis and selective proteolysis. The chemically modified peptides containing each of the three Tyr residues present in the RARalpha-LBD sequence were then analyzed and identified by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC/ESI-MS). We found that RA binding to RARalpha-LBD protected Tyr(277)-containing peptides from nitration. Protection of Tyr(277) could result either from direct masking by the bound ligand or from ligand-induced changes in receptor conformation and tyrosine accessibility. The role of Tyr residues was further documented by site directed mutagenesis using three site-specific RARalpha mutants: Y208A, Y277A, and Y362A. The affinity for RA of these mutant receptors was in the range of that of the wild-type protein, except for the Y277A receptor mutant, which displays a 15-20-fold reduction in affinity and transactivation activity for RA. Whereas mutation of Tyr(277) into alanine had a variable effect on different agonists and antagonists binding, it caused a dramatic decrease of retinoid-dependent transactivation activity. This later effect was also observed with mutation of Tyr(277) into phenylalanine. It is unlikely that major conformational changes are responsible for the lower affinity of RA binding and RA-dependent transactivation since these mutants displayed wild-type dimerization and DNA-binding activities. Limited proteolysis revealed that upon ligand binding, the Y277A mutant induced a conformational change slightly different from that obtained with the wild-type protein. These data could suggest that Tyr(277) play a critical role in the ligand-induced conformational changes required for the activation of RARalpha.

[Treatment of low rectal cancer by conservative rectal resection after preoperative irradiation. Long term results and prospective study]. / Traitement du cancer du bas rectum par résection rectale conservatrice après irradiation pré-opératoire. Résultats à long terme d'une étude prospective.

AIMS: A prospective study was undertaken to assess the feasibility and long term results of sphincter-preserving rectal excision after preoperative radiation (35 Gy). PATIENTS AND METHODS: From 1986 to 1990, 42 patients were included in the study. Thirty four (81%) could be managed by rectal excision and stapled coloanal anastomosis. They had an adenocarcinoma located at a mean distance of 55 +/- 13 mm (range: 20-80) from the anal verge. RESULTS: Eight specimens were free of tumor. The 26 others were tabulated as follows according to the Astler-Coller staging: A = 2, B1 = 15, B2 = 5, C1 = 1, C2 = 3. The mean distal free margin was 16 +/- 11 mm (range: 1-40). The follow-up period ranged from 5 to 9 years. Six patients (18%) experienced postoperative complications including minor anastomotic leakage (n = 3), bowel obstruction (n = 2), major diarrhea requiring fecal diversion (n = 1). The functional result was good in all but 3 patients (9%) who experienced a supra anastomotic stenosis and underwent a permanent colostomy. A pelvic recurrence was observed in 5 patients (15%) after a postoperative delay ranging from 11 to 50 months. At 5 years, 17 patients (50%) were alive free of cancer, 14 (41%) of them having a good functional result without colostomy. CONCLUSION: This work demonstrates that in most cases low rectal carcinoma can be safely managed by sphincter-preserving rectal excision after preoperative radiation. It strongly suggests that the long-term pelvic recurrence rate is similar to the one observed after abdomino-perineal excision. However both procedures and patients selection must be carefully performed.

Lymphoid hypoplasia and somatic cloning.

BACKGROUND: Adult somatic cloning by nuclear transfer is associated with high rate of perinatal mortality but there is still no evidence that nuclear transfer itself is responsible for these failures. We report on a longlasting defect linked to somatic cloning. METHODS: Skin cells grown from an ear biopsy specimen from a 15-day-old calf were used as a source of nuclei. The donor animal was a clone of three females obtained from embryonic cells. Clinical examination, haematological, and biochemical profiles, and echocardiography of the somatic clone were done from birth to death. FINDINGS: After 6 weeks of normal development, the somatic cloned calf had a sudden and rapid fall in lymphocyte count and a decrease in haemoglobin. The calf died on day 51 from severe anaemia. Necropsy revealed no abnormality except thymic atrophy and lymphoid hypoplasia. INTERPRETATION: Somatic cloning may be the cause of long-lasting deleterious effects. Our observation should be taken into account in debates on reproductive cloning in human beings.

Pharmacophoric search and 3D-QSAR comparative molecular field analysis studies on agonists of melatonin sheep receptors.

Conformational analysis was used to characterize the agonist pharmacophore for melatonin sheep brain receptor recognition and activation. The molecular geometry shared by all conformations of the selected active ligands was determined. Assuming that all the compounds interact at the same binding site at the receptor level, 2-iodomelatonin pharmacophoric conformation served as a template for the superimposition of 64 structurally heterogeneous agonists constituting the training set used to perform a three-dimensional quantitative structure-activity relationship study via the comparative molecular field analysis method. A statistically significant model was obtained for the totality of the compounds (n = 64, q2 = 0.62, N = 6, r2 = 0.96, s = 0.28, F = 249) with steric, electrostatic, and lipophilic relative contributions of 28%, 35%, and 37%, respectively. The predictive power of the proposed model was discerned by successfully testing the 78 agonist ligands constituting the test set. The model so obtained and validated brings important structural insights to aid the design of novel melatoninergic agonist ligands prior to their synthesis.