Evidence for a quantitative trait locus affecting low levels of apolipoprotein B and low density lipoprotein on chromosome 10 in Caucasian families

High plasma apolipoprotein B (apoB) and LDL cholesterol levels increase cardiovascular disease risk. These highly correlated measures may be partially controlled by common genetic polymorphisms. To identify chromosomal regions that contain genes causing low plasma levels of one or both parameters in Caucasian families ascertained for familial hypobetalipoproteinemia (FHBL), we conducted a whole-genome scan using 443 microsatellite markers typed in nine multigenerational families with at least two members with FHBL. Both variance components and regression-based linkage methods were used to identify regions of interest. Common linkage regions were identified for both measures on chromosomes 10q25.1-10q26.11 [maximum log of the odds (LOD) = 4.2 for LDL and 3.5 for apoB] and 6q24.3 (maximum LOD = 1.46 for LDL and 1.84 for apoB). There was also evidence for linkage to apoB on chromosome 13q13.2 (LOD = 1.97) and to LDL on chromosome 3p14.1 at 94 centimorgan (LOD = 1.52). Bivariate linkage analysis provided further evidence for loci contributing to both traits (6q24.3, LOD = 1.43; 10q25.1, LOD = 1.74). We evaluated single nucleotide polymorphisms (SNPs) in genes within our linkage regions to identify variants associated with apoB or LDL levels. The most significant finding was for rs2277205 in the 5' untranslated region of acyl-coenzyme A dehydrogenase short/branched chain and LDL (P = 10(-7)). Three additional SNPs were associated with apoB and/or LDL (P < 0.01). Although only the linkage signal on chromosome 10 reached genome-wide statistical significance, there are likely multiple chromosomal regions with variants that contribute to low levels of apoB and LDL and that may protect against coronary heart disease.     

Substrate specificity and effect on GLUT4 translocation of the Rab GTPase-activating protein Tbc1d1

Insulin stimulation of the trafficking of the glucose transporter GLUT4 to the plasma membrane is controlled in part by the phosphorylation of the Rab GAP (GTPase-activating protein) AS160 (also known as Tbc1d4). Considerable evidence indicates that the phosphorylation of this protein by Akt (protein kinase B) leads to suppression of its GAP activity and results in the elevation of the GTP form of a critical Rab. The present study examines a similar Rab GAP, Tbc1d1, about which very little is known. We found that the Rab specificity of the Tbc1d1 GAP domain is identical with that of AS160. Ectopic expression of Tbc1d1 in 3T3-L1 adipocytes blocked insulin-stimulated GLUT4 translocation to the plasma membrane, whereas a point mutant with an inactive GAP domain had no effect. Insulin treatment led to the phosphorylation of Tbc1d1 on an Akt site that is conserved between Tbc1d1 and AS160. These results show that Tbc1d1 regulates GLUT4 translocation through its GAP activity, and is a likely Akt substrate. An allele of Tbc1d1 in which Arg(125) is replaced by tryptophan has very recently been implicated in susceptibility to obesity by genetic analysis. We found that this form of Tbc1d1 also inhibited GLUT4 translocation and that this effect also required a functional GAP domain.     

Phenotypic spectrum of the tubulin-related disorders and functional implications of disease-causing mutations

A spectrum of neurological disorders characterized by abnormal neuronal migration, differentiation, and axon guidance and maintenance have recently been attributed to missense and splice-site mutations in the genes that encode α-tubulin and β-tubulin isotypes TUBA1A, TUBA8, TUBB2B, and TUBB3, all of which putatively coassemble into neuronal microtubules. The resulting nervous system malformations can include different types of cortical malformations, defects in commissural fiber tracts, and degeneration of motor and sensory axons. Many clinical phenotypes and brain malformations are shared among the various mutations regardless of structural location and/or isotype, while others segregate with distinct amino acids or functional domains within tubulin. Collectively, these disorders provide novel paradigms for understanding the biological functions of microtubules and their core components in normal health and disease.     

Wine flavor and aroma

The perception of wine flavor and aroma is the result of a multitude of interactions between a large number of chemical compounds and sensory receptors. Compounds interact and combine and show synergistic (i.e., the presence of one compound enhances the perception of another) and antagonistic (a compound suppresses the perception of another) interactions. The chemical profile of a wine is derived from the grape, the fermentation microflora (in particular the yeast Saccharomyces cerevisiae), secondary microbial fermentations that may occur, and the aging and storage conditions. Grape composition depends on the varietal and clonal genotype of the vine and on the interaction of the genotype and its phenotype with many environmental factors which, in wine terms, are usually grouped under the concept of “terroir” (macro, meso and microclimate, soil, topography). The microflora, and in particular the yeast responsible for fermentation, contributes to wine aroma by several mechanisms: firstly by utilizing grape juice constituents and biotransforming them into aroma- or flavor-impacting components, secondly by producing enzymes that transform neutral grape compounds into flavor-active compounds, and lastly by the de novo synthesis of many flavor-active primary (e.g., ethanol, glycerol, acetic acid, and acetaldehyde) and secondary metabolites (e.g., esters, higher alcohols, fatty acids). This review aims to present an overview of the formation of wine flavor and aroma-active components, including the varietal precursor molecules present in grapes and the chemical compounds produced during alcoholic fermentation by yeast, including compounds directly related to ethanol production or secondary metabolites. The contribution of malolactic fermentation, ageing, and maturation on the aroma and flavor of wine is also discussed.     

Transiently redirected T cells for adoptive transfer

Background aimsT cells can be redirected to reject cancer by retroviral transduction with a chimeric antigen receptor (CAR) or by administration of a bispecific T cell engager (BiTE). We demonstrate that transfection of T cells with messenger (m) RNA coding for CAR is an alternative strategy.MethodsWe describe the pre-clinical evaluation of a method based on transient modification of expanded T cells with a CD19 CAR directed against B-cell malignancies. CAR mRNA was generated under cell-free conditions in a scalable process using recombinant RNA polymerase. Efficient and non-toxic square-wave electroporation was used to load the mRNA into the cytoplasm of T cells with no risk of insertional mutagenesis.ResultsAfter transfection > 80% of T cells were viable, with 94% CAR expression. Transfected T cells were cytolytic to CD19⁺ targets and produced interferon (IFN)-γ in response. Killing of CD19⁺ target cells was demonstrated even at day 8 with undetectable CAR expression. Increasing the concentration of mRNA resulted in higher surface CAR expression, better killing and more IFN-γ release but at the expense of increased activation-induced cell death. Finally, we demonstrated that a second transgene could be introduced by co-electroporation of CXCR4 or CCR7 with CAR to also modify chemotactic responses.ConclusionsWe advocate the transient redirection approach as well suited to meet safety aspects for early phase studies, prior to trials using stably transduced cells once CAR has been proven safe. The simplicity of this methodology also facilitates rapid screening of candidate targets and novel receptors in pre-clinical studies.     

Evolution, insular restriction, and extinction of oceanic land crabs, exemplified by the loss of an endemic Geograpsus in the Hawaiian Islands

Most oceanic islands harbor unusual and vulnerable biotas as a result of isolation. As many groups, including dominant competitors and predators, have not naturally reached remote islands, others were less constrained to evolve novel adaptations and invade adaptive zones occupied by other taxa on continents. Land crabs are an excellent example of such ecological release, and some crab lineages made the macro-evolutionary transition from sea to land on islands. Numerous land crabs are restricted to, although widespread among, oceanic islands, where they can be keystone species in coastal forests, occupying guilds filled by vertebrates on continents. In the remote Hawaiian Islands, land crabs are strikingly absent. Here we show that absence of land crabs in the Hawaiian Islands is the result of extinction, rather than dispersal limitation. Analysis of fossil remains from all major islands show that an endemic Geograpsus was abundant before human colonization, grew larger than any congener, and extended further inland and to higher elevation than other land crabs in Oceania. Land crabs are major predators of nesting sea birds, invertebrates and plants, affect seed dispersal, control litter decomposition, and are important in nutrient cycling; their removal can lead to large-scale shifts in ecological communities. Although the importance of land crabs is obvious on remote and relatively undisturbed islands, it is less apparent on others, likely because they are decimated by humans and introduced biota. The loss of Geograpsus and potentially other land crabs likely had profound consequences for Hawaiian ecosystems.     

1H, 13C, 15N resonance assignment of the chitin-binding protein CBP21 from Serratia marcescens

The 18.8 kDa chitin-binding protein CBP21 from Serratia marcescens has been isotopically labeled and recombinantly expressed. In this paper, we report the ¹H, ¹³C, ¹⁵N resonance assignment of CBP21.     

The putative endoglucanase PcGH61D from Phanerochaete chrysosporium is a metal-dependent oxidative enzyme that cleaves cellulose

Many fungi growing on plant biomass produce proteins currently classified as glycoside hydrolase family 61 (GH61), some of which are known to act synergistically with cellulases. In this study we show that PcGH61D, the gene product of an open reading frame in the genome of Phanerochaete chrysosporium, is an enzyme that cleaves cellulose using a metal-dependent oxidative mechanism that leads to generation of aldonic acids. The activity of this enzyme and its beneficial effect on the efficiency of classical cellulases are stimulated by the presence of electron donors. Experiments with reduced cellulose confirmed the oxidative nature of the reaction catalyzed by PcGH61D and indicated that the enzyme may be capable of penetrating into the substrate. Considering the abundance of GH61-encoding genes in fungi and genes encoding their functional bacterial homologues currently classified as carbohydrate binding modules family 33 (CBM33), this enzyme activity is likely to turn out as a major determinant of microbial biomass-degrading efficiency.     

Hepatic triglyceride contents are genetically determined in mice: results of a strain survey

To assess whether genetic factor(s) determine liver triglyceride (TG) levels, a 10-mouse strain survey of liver TG contents was performed. Hepatic TG contents were highest in BALB/cByJ, medium in C57BL/6J, and lowest in SWR/J in both genders. Ninety and seventy-six percent of variance in hepatic TG in males and females, respectively, was due to strain (genetic) effects. To understand the physiological/biochemical basis for differences in hepatic TG among the three strains, studies were performed in males of the BALB/cByJ, C57BL/6J, and SWR/J strains. In vivo hepatic fatty acid (FA) synthesis rates and hepatic TG secretion rates ranked BALB/cByJ approximately C57BL/6J > SWR/J. Hepatic 1-(14)C-labeled palmitate oxidation rates and plasma beta-hydroxybutyrate concentrations ranked in reverse order: SWR/J > BALB/cByJ approximately C57BL/6J. After 14 h of fasting, plasma-free FA and hepatic TG contents rose most in BALB/cByJ and least in SWR/J. beta-Hydroxybutyrate concentrations rose least in BALB/cByJ and most in SWR/J. Adaptation to fasting was most effective in SWR/J and least in BALB/cByJ, perhaps because BALB/cByJ are known to be deficient in SCAD, a short-chain FA oxidizing enzyme. To assess the role of insulin action, glucose tolerance test (GTT) was performed. GTT-glucose levels ranked C57BL/6J > BALB/cByJ approximately SWR/J. Thus strain-dependent (genetic) factors play a major role in setting hepatic TG levels in mice. Processes such as FA production and hepatic export in VLDL on the one hand and FA oxidation on the other, explain some of the strain-related differences in hepatic TG contents. Additional factor(s) in the development of fatty liver in BALB/cByJ remain to be demonstrated.     

Novel insulin-elicited phosphoproteins in adipocytes

Akt is a key insulin-activated protein kinase. We searched for Akt substrates in 3T3-L1 adipocytes by means of immunoprecipitation with an Akt phosphomotif-specific antibody (PAS antibody). Four insulin-elicited phosphoproteins were isolated and identified by mass spectrometry. The identity of each protein was established by isolating the protein from lysates of untreated and insulin-treated adipocytes with an antibody specific for the protein and showing that the PAS antibody reacted only with the protein in the immunoprecipitate from insulin-treated cells. These proteins have sizes of 47, 75, 105, and 250 kDa on SDS PAGE, and have been designated pp47, 75, 105, and 250. The effect of inhibitors on the phosphorylation of the proteins, the identified sites of phosphorylation, and in vitro phosphorylation by recombinant Akt further indicated that pp47, 105, and 250 are likely to be Akt substrates, whereas pp75 may not be. pp47 and 105 are novel proteins with no known or predicted function. pp75 was previously found as a protein that associated with the colony-stimulating factor receptor, designated as Fms-interacting protein. pp250 is a novel protein with a predicted GTPase activating protein (GAP) domain for Rheb and/or Rap at its carboxy terminus. The subcellular and tissue distributions of the four proteins were determined.