N-(2-Benzoylphenyl)-L-tyrosine PPARgamma agonists. 3. Structure-activity relationship and optimization of the N-aryl substituent

3-?4-[2-(Benzoxazol-2-ylmethylamino)ethoxy]phenyl?-(2S)-((2- benzoylph enyl)amino)propionic acid (1) and (2S)-((2-benzoylphenyl)amino)-3-?4-[2-(5-methyl-2-phenyloxazol-4-y l)e thoxy]phenyl?propionic acid (2) are peroxisome proliferator-activated receptor gamma (PPARgamma) agonists and have antidiabetic activity in rodent models of type 2 diabetes. As part of an effort to develop the SAR of the N-2-benzoylphenyl moiety of 1 and 2, a series of novel carboxylic acid analogues, 23-66, modified only in the N-2-benzoylphenyl moiety were synthesized from L-tyrosine and evaluated as PPARgamma agonists. In general, only modest changes in the N-2-benzoylphenyl moiety of 1 and 2 are tolerated. More specifically, the best changes involve bioisosteric replacement of one of the two phenyl rings of this moiety. Addition of substituents to this moiety generally produced compounds that are less active in the cell-based functional assays of PPARgamma activity although binding affinity to PPARgamma may be maintained. A particularly promising set of analogues is the anthranilic acid esters 63-66 in which the phenyl ring in the 2-benzoyl group of 1 and 2 has been replaced by an alkoxy group. In particular, (S)-2-(1-carboxy-2-?4-[2-(5-methyl-2-phenyloxazol-4-yl)ethoxy]phen yl? ethylamino)benzoic acid methyl ester (63) has a pKi of 8.43 in the binding assay using human PPARgamma ligand binding domain and a pEC50 of 9.21 in the in vitro murine lipogenesis functional assay of PPARgamma activity. Finally, 63 was found to normalize glycemia when dosed at 3 mg/kg bid po in the Zucker diabetic fatty rat model of type 2 diabetes.     

Distribution of genetic diversity in relation to chromosomal inversions in the malaria mosquito Anopheles gambiae

The epidemiology of malaria in Africa is complicated by the fact that its principal vector, the mosquito Anopheles gambiae, constitutes a complex of six sibling species. Each species is characterized by a unique array of paracentric inversions, as deduced by karyotypic analysis. In addition, most of the species carry a number of polymorphic inversions. In order to develop an understanding of the evolutionary histories of different parts of the genome, we compared the genetic variation of areas inside and outside inversions in two distinct inversion karyotypes of A. gambiae. Thirty-five cDNA clones were mapped on the five arms of the A. gambiae chromosomes with divisional probes. Sixteen of these clones, localized both inside and outside inversions of chromosome 2, were used as probes in order to determine the nucleotide diversity of different parts of the genome in the two inversion karyotypes. We observed that the sequence diversity inside the inversion is more than three-fold lower than in areas outside the inversion and that the degree of divergence increases gradually at loci at increasing distance from the inversion. To interpret the data we present a selectionist and a stochastic model, both of which point to a relatively recent origin of the studied inversion and may suggest differences between the evolutionary history of inversions in Anopheles and Drosophila species.     

Perforation of the gastro-intestinal tract by a foreign body. A case report

According to the research that there were some immune and inflammatory substances in the synovial fluid of temporomandibular joint disturbance syndrome (TMJDS), we improved the traditional intraarticular injection and developed an intraarticular irrigation-injection therapy to treat 43 cases of TMJDS and got a good result. The controls include two groups, the simple rinsing and the traditional injection. Statistical analysis showed a highly significant difference in the effects between test and control groups. In order to study the mechanism of the therapy, the tumor necrosis factor (TNF) in synovial fluid of 8 cases were assessed. It was found that the concentration of TNF was reduced markedly after treatment. We think that the new therapy is effectable for TMJDS and could be used widely.     

Evaluation of bovine-derived bone protein with a natural coral carrier as a bone-graft substitute in a canine segmental defect model

The efficacy of a bone-graft substitute (bovine-derived bone protein in a carrier of natural coral) in the healing of a segmental defect of a weight-bearing long bone was evaluated. Twenty dogs, divided into two groups, underwent bilateral radial osteotomies with creation of a 2.5 cm defect. On one side of each dog, the defect was filled with autogenous cancellous bone graft. Contralateral defects received, in a blinded randomized fashion, cylindrical implants consisting of natural coral (calcium carbonate) or calcium carbonate enhanced with a standard dose of bovine-derived bone protein (3.0 mg/implant; 0.68 mg bone protein/cm3). The limbs were stabilized with external fixators, and all animals underwent monthly radiographs. They were killed at 12 (group 1) or 24 (group 2) weeks, and regenerated bone was studied by biomechanical testing and histology. Radiographic union developed in all 20 radii with autogenous cancellous bone grafts and in all 10 of the radii with the composite implants. None of the radii with implants of calcium carbonate alone showed radiographic evidence of union. This represented a statistically significant difference between implant types. In addition, calcium carbonate implants both with and without bone protein demonstrated radiographic evidence of near total resorption of the radiodense carrier by 12 weeks. This resorption facilitated radiographic evaluation of healing. Mean values for biomechanical parameters of radii with the composite implants exceeded those for the contralateral controls at 12 and 24 weeks; the difference was statistically significant at 12 weeks. Histology revealed scant residual calcium carbonate carrier at either time in the defects with calcium carbonate implants; however, a moderate amount was present in defects with the composite implants. In these specimens, the residual carrier was completely surrounded by newly formed bone that may have insulated the calcium carbonate from further degradation. The present study used a carrier of granular calcium carbonate reconstituted with bovine type-I collagen to deliver an osteoinductive protein to the defect site. This carrier is of nonhuman origin (eliminating the risk of disease transmission or antigenicity) and resorbs rapidly. In this model, bovine-derived bone protein in a natural coral carrier performed consistently better than the gold standard autogenous cancellous bone graft in terms of the amount of bone formation and strength of the healed defect. This may have implications for removal of hardware or resumption of weight-bearing in certain clinical situations. These data also indicate that coralline calcium carbonate alone represents a poor option as a bone-graft substitute in this critical-sized segmental defect model.     

New early tertiary primates and a reappraisal of some Plesiadapiformes

A new study of specimens of paromomyid and microsyopid primates clarifies evolution in these primates and reuslts in new concepts of each family. The paromomyid Phenacolemur and its former synonym, Ignacius, are distinct genera, each with new Early Wasatchian species, Plesiolestes, Palaechthon, Torrejonia, Palenochtha, Navajovius, Micromomys, and Tinimomys are removed from the Paromomyidae and transferred to the Microsyopidae. The Plesiadapiformes are classified in three groups which may be differentiated on incisor and molor structure: Plesiadapoidea (Plesiadapidae, Carpolestidae, and Saxonellidae), Microsyopidae, and Paromomyidae. The Paromomyidae, Microsyopidae (and its two subfamilies), and several genera are rediagnosed in the light of new comparative evidence.     

Structural damage to lactate dehydrogenase during copper iminodiacetic acid metal affinity chromatography

The stability of the enzyme lactate dehydrogenase (LDH) was evaluated by measuring structural damage and activity loss after exposure to copper-iminodiacetic acid (IDA) immobilized metal affinity chromatography (IMAC) under oxidizing conditions at pH 7.0. Oxidizing conditions were produced by adding reductants commonly employed in bioprocessing and biomedical applications (glutathione, beta-mercaptoethanol, dithiothreitol, cysteine, or ascorbate) and/or hydrogen peroxide to the mobile phase. Most of these additives have been shown recently to give rise to metal-catalyzed oxidation (MCO) reactions on copper-iminodicaetic acid IMAC columns. Structural damage in the form of increased susceptibility to proteolytic degradation, fragmentation, and cross-linking were measured. Increased sensitivity to proteolysis was significant in virtually all cases tested, even when activity remained high (>95% specific activity recovered). In contrast fragmentation and cross-linking were minimal in all cases, even when activity was low (<50%). As the damage was believed to have been caused primarily by MCO reactions, preventative measures consistent with this reaction pathway were tested. The most successful measure for all of the conditions studied was addition of the Cu+ chelating agent bicinchoninic acid (BCA) to the mobile phase. Decreased contact time with the column decreased damage in the case where glutathione was added. Removal of dissolved oxygen by nitrogen sparging and use of Tris-acetate buffer in place of phosphate had no measurable effect. The success of BCA addition in reducing structural damage and activity loss strengthens the conclusion that MCO reactions can occur on copper-iminodiacetic acid IMAC columns. However, the addition of BCA and the other protective measures described were not successful in eliminating the increased proteolytic susceptibility observed when LDH in buffer was exposed to the copper-charged column with no oxidizing additives. This suggests that at least one other pathway for damage exists. This damage is difficult to detect as it did not cause statistically significant losses in enzymatic activity, fragmentation, or cross-linking.