Investigation of factor Xa inhibitors containing non-amidine S1 elements

Several non-amidino S1 derivatives of the 1,2-diaminobenzene-based scaffold (4) were synthesized and evaluated for their ability to bind to the active site and inhibit the human protease factor Xa. A subset of these compounds were also evaluated for their anticoagulant effects in human plasma as measured by prothrombin time (PT).     

1-Pentyl-3-phenylacetylindoles, a new class of cannabimimetic indoles

A new class of cannabimimetic indoles, with 3-phenylacetyl or substituted 3-phenylacetyl substituents, has been prepared and their affinities for the cannabinoid CB1 and CB2 receptors have been determined. In general those compounds with a 2-substituted phenylacetyl group have good affinity for both receptors. The 4-substituted analogs have little affinity for either receptor, while the 3-substituted compounds are intermediate in their affinities. Two of these compounds, 1-pentyl-3-(2-methylphenylacetyl)indole (JWH-251) and 1-pentyl-3-(3-methoxyphenylacetyl)indole (JWH-302), have 5-fold selectivity for the CB1 receptor with modest affinity for the CB2 receptor. GTPgammaS determinations indicate that both compounds are highly efficacious agonists at the CB1 receptor and partial agonists at the CB2 receptor.     

Potential use of loss of heterozygosity in pleural effusions of breast cancer metastases using the microsatellite marker of the 16q22.1 region of the CDH1 gene

OBJECTIVE: To evaluate the presence of allelic loss in 16q22.1, including the locus of E-cadherin, in pleural effusions in breast cancer patients. STUDY DESIGN: Molecular analysis of DNA was performed using a DNA extraction kit (NucleoSpin, Macherey-Nagel, Germany). Loss of heterozygosity (LOH) in primary tumors and pleural effusions was analyzed using a microsatellite marker of the CDH1 gene, D16S265, described in previous studies. LOH was evaluated by radioactive polymerase chain reaction assay in 17 samples of pleural effusions and breast tissues (primary tumors and nonneoplastic adjacent tissue) from breast cancer patients: 7 positive for neoplastic cells, 6 suspected and 4 cases without evidence of neoplastic cells in the effusions. RESULTS: Thirteen cases (76%) were informative. LOH was detected in 5 cases (38.5%). In 3 of them LOH was detected only in the cytologic sample, and in 2 of them LOH was detected in the primary tumor and cytologic sample. CONCLUSION: Results show that LOH in the CDH1 gene can identify tumor cells in pleural effusions when morphologic analysis is difficult.     

Cholesterol dictates the freedom of EGF receptors and HER2 in the plane of the membrane

The flow of information through the epidermal growth factor receptor (EGFR) is shaped by molecular interactions in the plasma membrane. The EGFR is associated with lipid rafts, but their role in modulating receptor mobility and subsequent interactions is unclear. To investigate the role of nanoscale rafts in EGFR dynamics, we used single-molecule fluorescence imaging to track individual receptors and their dimerization partner, human epidermal growth factor receptor 2 (HER2), in the membrane of human mammary epithelial cells. We found that the motion of both receptors was interrupted by dwellings within nanodomains. EGFR was significantly less mobile than HER2. This difference was likely due to F-actin because its depolymerization led to similar diffusion patterns between the EGFR and HER2. Manipulations of membrane cholesterol content dramatically altered the diffusion pattern of both receptors. Cholesterol depletion led to almost complete confinement of the receptors, whereas cholesterol enrichment extended the boundaries of the restricted areas. Interestingly, F-actin depolymerization partially restored receptor mobility in cholesterol-depleted membranes. Our observations suggest that membrane cholesterol provides a dynamic environment that facilitates the free motion of EGFR and HER2, possibly by modulating the dynamic state of F-actin. The association of the receptors with lipid rafts could therefore promote their rapid interactions only upon ligand stimulation.     

Ligand accumulation in autocrine cell cultures

Cell-culture assays are routinely used to analyze autocrine signaling systems, but quantitative experiments are rarely possible. To enable the quantitative design and analysis of experiments with autocrine cells, we develop a biophysical theory of ligand accumulation in cell-culture assays. Our theory predicts the ligand concentration as a function of time and measurable parameters of autocrine cells and cell-culture experiments. The key step of our analysis is the derivation of the survival probability of a single ligand released from the surface of an autocrine cell. An expression for this probability is derived using the boundary homogenization approach and tested by stochastic simulations. We use this expression in the integral balance equations, from which we find the Laplace transform of the ligand concentration. We demonstrate how the theory works by analyzing the autocrine epidermal growth factor receptor system and discuss the extension of our methods to other experiments with cultured autocrine cells.     

Neonatal androgenization of hypogonadal (hpg) male mice does not abolish estradiol-induced FSH production and spermatogenesis

Background  

Testicular development is arrested in the hypogonadal (hpg) mouse due to a congenital deficiency in hypothalamic gonadotropin-releasing hormone (GnRH) synthesis. Chronic treatment of male hpg mice with estradiol induces FSH synthesis and secretion, and causes testicular maturation and qualitatively normal spermatogenesis. As estradiol negative feedback normally inhibits FSH production in the male, this study tested whether this paradoxical response to estradiol in the male hpg mouse might be due to inadequate masculinisation or incomplete defeminization in the neonatal period. Previous studies have demonstrated that treatment of hpg mice with testosterone propionate in the immediate neonatal period is necessary to allow full reproductive behaviors to be expressed following suitable endocrine stimulation at adult ages.     

Genetic evidence for a maternal effect locus controlling genomic imprinting and growth

Crosses between two species of deer mouse (Peromyscus) yield dramatic parent-of-origin effects. Female P. maniculatus (BW) crossed with male P. polionotus (PO) produce animals smaller than either parent. PO females crossed with BW males yield lethal overgrowth that has been associated with loss-of-imprinting (LOI). Previously, we mapped two loci influencing fetal growth. These two loci, however, do not account for the LOI, nor for the dysmorphic phenotypes. Here we report that maternal genetic background strongly influences the LOI. Analyses of crosses wherein maternal genetic background is varied suggest that this effect is likely due to the action of a small number of loci. We have termed these putative loci Meil. Estimation of Meil loci number was confounded by skewed allelic ratios in the intercross line employed. We show that the Meil loci are not identical to any of the DNA methyltransferases shown to be involved in regulation of genomic imprinting.     

Sorghum genome sequencing by methylation filtration

Sorghum bicolor is a close relative of maize and is a staple crop in Africa and much of the developing world because of its superior tolerance of arid growth conditions. We have generated sequence from the hypomethylated portion of the sorghum genome by applying methylation filtration (MF) technology. The evidence suggests that 96% of the genes have been sequence tagged, with an average coverage of 65% across their length. Remarkably, this level of gene discovery was accomplished after generating a raw coverage of less than 300 megabases of the 735-megabase genome. MF preferentially captures exons and introns, promoters, microRNAs, and simple sequence repeats, and minimizes interspersed repeats, thus providing a robust view of the functional parts of the genome. The sorghum MF sequence set is beneficial to research on sorghum and is also a powerful resource for comparative genomics among the grasses and across the entire plant kingdom. Thousands of hypothetical gene predictions in rice and Arabidopsis are supported by the sorghum dataset, and genomic similarities highlight evolutionarily conserved regions that will lead to a better understanding of rice and Arabidopsis.