Localization of genes for V+LDL plasma cholesterol levels on two diets in the opossum Monodelphis domestica

Plasma cholesterol levels among individuals vary considerably in response to diet. However, the genes that influence this response are largely unknown. Non-HDL (V+LDL) cholesterol levels vary dramatically among gray, short-tailed opossums fed an atherogenic diet, and we previously reported that two quantitative trait loci (QTLs) influenced V+LDL cholesterol on two diets. We used hypothesis-free, genome-wide linkage analyses on data from 325 pedigreed opossums and located one QTL for V+LDL cholesterol on the basal diet on opossum chromosome 1q [logarithm of the odds (LOD) = 3.11, genomic P = 0.019] and another QTL for V+LDL on the atherogenic diet (i.e., high levels of cholesterol and fat) on chromosome 8 (LOD = 9.88, genomic P = 5 × 10⁻⁹). We then employed a novel strategy involving combined analyses of genomic resources, expression analysis, sequencing, and genotyping to identify candidate genes for the chromosome 8 QTL. A polymorphism in ABCB4 was strongly associated (P = 9 × 10⁻¹⁴) with the plasma V+LDL cholesterol concentrations on the high-cholesterol, high-fat diet. The results of this study indicate that genetic variation in ABCB4, or closely linked genes, is responsible for the dramatic differences among opossums in their V+LDL cholesterol response to an atherogenic diet.     

A quantitative microassay of carbohydrate-mediated cell adhesion to glycoconjugates immobilized on polystyrene plates.

A microadhesion assay that allows the quantitative determination of carbohydrate-mediated cell adhesion to glycoconjugates immobilized on 96-well polystyrene plates has been developed. After dislodging nonadherent cells by centrifugation, specifically bound cells are quantified by colorimetric analysis of a blue formazan product generated from the dye 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide by enzymatic reduction. Carbohydrate specificity of the cell adhesion was demonstrated by inhibition analyses and the general applicability of the assay was proved with indicator cells of three different origins: mouse fibrosarcoma cells, Chang liver cells, and human breast carcinoma cells (MDA-MB 231).     

Design and synthesis of hydroxyethylamine (HEA) BACE-1 inhibitors: Structure–activity relationship of the aryl region

The structure–activity relationship of the prime region of hydroxyethylamine BACE inhibitors is described. Variation in the aryl linker region with 5- and 6-membered heterocycles provided compounds such as 33 with improved permeability and reduced P-gp liability compared to benzyl amine analog 1.     

Discovery of a novel sulfonamide-pyrazolopiperidine series as potent and Efficacious γ-Secretase Inhibitors

Discovery of a series of pyrazolopiperidine sulfonamide based γ-secretase inhibitors and its SAR evolution is described. Significant increases in APP potency on the pyrazolopiperidine scaffold over the original N-bicyclic sulfonamide scaffold were achieved and this potency increase translated in an improved in vivo efficacy.     

Discovery of a novel [3.2.1] benzo fused bicyclic sulfonamide-pyrazoles as potent,selective and efficacious γ-secretase inhibitors

Structure–activity relationship (SAR) of a novel, potent and metabolically stable series of benzo [3.2.1] bicyclic sulfonamide-pyrazoles as γ-secretase inhibitors are described. Compounds that are efficacious in reducing the cortical Aβx-40 levels in FVB mice via oral dose, as well as those with high selectivity over Notch, are highlighted.     

Design, synthesis and structure-activity relationship of novel [3.3.1] bicyclic sulfonamide-pyrazoles as potent γ-secretase inhibitors

The structure-activity relationship (SAR) of a novel, potent and metabolically stable series of sulfonamide-pyrazoles that attenuate β-amyloid peptide synthesis via γ-secretase inhibition is detailed herein. Sulfonamide-pyrazoles that are efficacious in reducing the cortical Aβx-40 levels in FVB mice via a single PO dose, as well as sulfonamide-pyrazoles that exhibit selectivity for inhibition of APP versus Notch processing by γ-secretase, are highlighted.     

Design and synthesis of cell potent BACE-1 inhibitors: Structure–activity relationship of P1′ substituents

Using structure-guided design, hydroxyethylamine BACE-1 inhibitors were optimized to nanomolar Aβ cellular inhibition with selectivity against cathepsin-D. X-ray crystallography illuminated the S1′ residues critical to this effort, which culminated in compounds 56 and 57 that exhibited potency and selectivity but poor permeability and high P-gp efflux.     

Molecular characterization of transketolase (EC 2.2.1.1) active in the Calvin cycle of spinach chloroplasts

A cDNA encoding the Calvin cycle enzyme transketolase (TKL; EC 2.2.1.1) was isolated from Sorghum bicolor via subtractive differential hybridization, and used to isolate several full-length cDNA clones for this enzyme from spinach. Functional identity of the encoded mature subunit was shown by an 8.6-fold increase of TKL activity upon induction of Escherichia coli cells that overexpress the spinach TKL subunit under the control of the bacteriophage T₇ promoter. Chloroplast localization of the cloned enzyme is shown by processing of the in vitro synthesized precursor upon uptake by isolated chloroplasts. Southern blot-analysis suggests that TKL is encoded by a single gene in the spinach genome. TKL proteins of both higher-plant chloroplasts and the cytosol of non-photosynthetic eukaryotes are found to be unexpectedly similar to eubacterial homologues, suggesting a possible eubacterial origin of these nuclear genes. Chloroplast TKL is the last of the demonstrably chloroplast-localized Calvin cycle enzymes to have been cloned and thus completes the isolation of gene probes for all enzymes of the pathway in higher plants.Abbreviations RPE ribulose-5-phosphate 3-epimerase - RPI ribose-5-phosphate isomerase - TKL transketolase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - PGK phosphoglycerate kinase - FBP fructose-1,6-bisphosphatase - SBP sedoheptulose-1,7-bisphosphatase - OPPP oxidative pentose phosphate pathway - Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase - FBA fructose-1,6-bisphosphate aldolase - IPTG isopropyl -d-thiogalactoside - TPI triosephosphate isomerase     

Identification and analysis of four candidate symbiosis genes from 'Chlorochromatium aggregatum', a highly developed bacterial symbiosis

The consortium 'Chlorochromatium aggregatum' currently represents the most highly developed interspecific association between prokaryotes. It consists of green sulfur bacteria, so-called epibionts, which surround a central, motile, chemotrophic bacterium. Four putative symbiosis genes of the epibiont were recovered by suppression subtractive hybridization and bioinformatics approaches. These genes are transcribed constitutively and do not occur in the free-living relatives of the epibiont. The haemagglutinin-like putative gene products of open reading frames (ORFs) Cag0614 and Cag0616 are unusually large and contain repetitive regions and RGD tripeptides. Cag0616 harbours two betagamma-crystalline Greek key motifs. Cag1920 codes for a putative haemolysin whereas the gene product of Cag1919 is a putative RTX-like protein. Based on detailed analyses of Cag1919, the C-terminal amino acid sequence comprises six repetitions of the motif GGXGXD predicted to form a Ca(2+)-binding beta roll. Intact 'C. aggregatum' consortia disaggregated upon the addition of EGTA or pyrophosphate, but stayed intact in the presence of various lectine-binding sugars or proteolytic enzymes. Unlike other RTX toxins, a gene product of Cag1919 could not be detected by (45)Ca(2+) autoradiography, indicating a low abundance of the corresponding protein in the cells. The RTX-type C-terminus coded by Cag1919 exhibited a significant similarity to RTX modules of various proteobacterial proteins, suggesting that this putative symbiosis gene has been acquired via horizontal gene transfer from a proteobacterium.