Efficacy switching SAR of mGluR5 allosteric modulators: highly potent positive and negative modulators from one chemotype

A series of metabotropic glutamate 5 receptor (mGluR5) allosteric ligands with positive, negative or no modulatory efficacy is described. The ability of this series to yield both mGluR5 PAMs and NAMs with single-digit nanomolar potency is unusual, and the underlying SAR is detailed.     

Identification of protein disulfide isomerase as an endothelial hypoxic stress protein

Endothelial cells (EC) exposed to hypoxia upregulate a unique set of five stress proteins. These proteins are upregulated in human and bovine aortic and pulmonary artery EC and are distinct from heat shock or glucose-regulated proteins. We previously identified two of these proteins as the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and enolase and postulated that the remaining proteins were also glycolytic enzymes. Using SDS-PAGE, tryptic digestion, and NH(2)-terminal amino acid sequencing, we report here the identification of the 56-kDa protein as protein disulfide isomerase (PDI). PDI is upregulated by hypoxia at the mRNA level and follows a time course similar to that of the protein, with maximal upregulation detected after exposure to 18 h of 0% O(2). Neither smooth muscle cells nor fibroblasts upregulate PDI to the same extent as EC, which correlates with their decreased hypoxia tolerance. Upregulation of PDI specifically in EC may contribute to their ability to tolerate hypoxia and may occur through PDI's functions as a prolyl hydroxylase subunit, protein folding catalyst, or molecular chaperone.     

Callose deposition and breakdown, followed by phytomelan synthesis in the seed coat ofGasteria verrucosa (Mill.) H. Duval

Summary In the seed coat ofGasteria verrucosa the deposition of phytomelan takes place during seed development in three stages. Phytomelan is a black cell wall material which is chemically very inert. First the radial walls and part of the transverse cell wall of the outer epidermis of the outer integument become thickened by exocytosis of dictyosome vesicles. Callose is deposited at the tangential plasma membrane against those walls. After the callose deposition about two thirds of the original cell volume is filled with callose. During the second stage the callose is broken down, probably into glucose monomers or small polymers. At the same time cellulose is deposited at the outer tangential plasma membrane, forming a wall between the dissolving callose and the plasma membrane. In the third phase small granules appear in the solution of dissolved callose. which grow out and finally fuse to form a block of phytomelan, consisting of spherical 15-nm units. Remarkable is the function of the callose: it determines the size of the phytomelan block, and it probably functions as carbohydrate source for the phytomelan synthesis and/or for the cellulose inner layer. In this study transmission electron microscopy and cryo scanning electron microscopy are used to study the three developmental stages of the formation of the phytomelan layer.     

HIF-2alpha regulates glyceraldehyde-3-phosphate dehydrogenase expression in endothelial cells

Endothelial cells (EC) express both hypoxia inducible factor-1alpha (HIF-1alpha) and -2alpha (HIF-2alpha), yet their roles in the EC hypoxic response are unclear. Hypoxia upregulates the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EC through a 5' hypoxic regulatory element (HRE). We compared the upregulation of GAPDH in human lung microvascular EC to that in hep3B cells, another cell type known to express both HIF-1alpha and HIF-2alpha. GAPDH mRNA increased to a lesser extent in hypoxic hep3B cells than in EC, yet upregulation occurred through the same HRE that was active in EC. HIF-1alpha protein induction in response to hypoxia was similar in both cell types. In contrast, HIF-2alpha protein levels were upregulated to a greater extent and for a longer period of time by hypoxia in EC than in hep3B cells. Correspondingly, electrophoretic mobility supershift assays showed that, in EC, there was preferential binding of HIF-2alpha to the GAPDH HRE while, in hep3B cells, there was binding of both HIF-1alpha and HIF-2alpha. The preferential binding of HIF-2alpha to the GAPDH HRE in EC may account for their higher level of induction of GAPDH. These findings suggest that cell-specific patterns of HIF-1alpha and HIF-2alpha expression lead to cell-specific gene upregulation during hypoxia.     

Intestinal tumorigenesis is not affected by progesterone signaling in rodent models

Clinical data suggest that progestins have chemopreventive properties in the development of colorectal cancer. We set out to examine a potential protective effect of progestins and progesterone signaling on colon cancer development. In normal and neoplastic intestinal tissue, we found that the progesterone receptor (PR) is not expressed. Expression was confined to sporadic mesenchymal cells. To analyze the influence of systemic progesterone receptor signaling, we crossed mice that lacked the progesterone receptor (PRKO) to the Apc(Min/+) mouse, a model for spontaneous intestinal polyposis. PRKO-Apc(Min/+) mice exhibited no change in polyp number, size or localization compared to Apc(Min/+). To examine effects of progestins on the intestinal epithelium that are independent of the PR, we treated mice with MPA. We found no effects of either progesterone or MPA on gross intestinal morphology or epithelial proliferation. Also, in rats treated with MPA, injection with the carcinogen azoxymethane did not result in a difference in the number or size of aberrant crypt foci, a surrogate end-point for adenoma development. We conclude that expression of the progesterone receptor is limited to cells in the intestinal mesenchyme. We did not observe any effect of progesterone receptor signaling or of progestin treatment in rodent models of intestinal tumorigenesis.     

Evolutionary correlation between control region sequence and restriction polymorphisms in the mitochondrial genome of a large Senegalese Mandenka sample

We present here the first comparative analysis at the population level between Restriction Fragment Length Polymorphism (RFLP) and control region sequence polymorphism in a large and homogeneous Senegalese Mandenka sample. Eleven RFLP haplotypes and 60 different sequences are found in 119 individuals, revealing that a very high level of mtDNA diversity can be maintained in a small population. A sequence neighbor- joining tree and an analysis of molecular variance show that sequences associated with a given restriction haplotype are evolutionarily highly correlated: sequencing generally leads to the subtyping of RFLP haplotypes. Evolutionary relationships among RFLP haplotypes inferred from restriction site differences are in good agreement with those inferred from sequence data. A single difference is observed and is likely due to a single restriction homoplasy having occurred in the control region. Selective neutrality tests on both RFLP and sequence data accept the hypotheses of mtDNA neutrality and population equilibrium. The deep coalescence times (exceeding 50,000 yr) of sequences associated with the two most frequent restriction haplotypes confirm that the Niokolo Mandenka population has not passed through a recent bottleneck and that gene flow is maintained among West African populations despite ethnic differences.