Genome-wide Screen for Modulation of Hepatic Apolipoprotein A-I (ApoA-I) Secretion

Control of plasma cholesterol levels is a major therapeutic strategy for management of coronary artery disease (CAD). Although reducing LDL cholesterol (LDL-c) levels decreases morbidity and mortality, this therapeutic intervention only translates into a 25–40% reduction in cardiovascular events. Epidemiological studies have shown that a high LDL-c level is not the only risk factor for CAD; low HDL cholesterol (HDL-c) is an independent risk factor for CAD. Apolipoprotein A-I (ApoA-I) is the major protein component of HDL-c that mediates reverse cholesterol transport from tissues to the liver for excretion. Therefore, increasing ApoA-I levels is an attractive strategy for HDL-c elevation. Using genome-wide siRNA screening, targets that regulate hepatocyte ApoA-I secretion were identified through transfection of 21,789 siRNAs into hepatocytes whereby cell supernatants were assayed for ApoA-I. Approximately 800 genes were identified and triaged using a convergence of information, including genetic associations with HDL-c levels, tissue-specific gene expression, druggability assessments, and pathway analysis. Fifty-nine genes were selected for reconfirmation; 40 genes were confirmed. Here we describe the siRNA screening strategy, assay implementation and validation, data triaging, and example genes of interest. The genes of interest include known and novel genes encoding secreted enzymes, proteases, G-protein-coupled receptors, metabolic enzymes, ion transporters, and proteins of unknown function. Repression of farnesyltransferase (FNTA) by siRNA and the enzyme inhibitor manumycin A caused elevation of ApoA-I secretion from hepatocytes and from transgenic mice expressing hApoA-I and cholesterol ester transfer protein transgenes. In total, this work underscores the power of functional genetic assessment to identify new therapeutic targets.     

Anion conductance of frog muscle membranes: one channel, two kinds of pH dependence

Anion conductance and permeability sequences were obtained for frog skeletal muscle membranes from the changes in characteristic resistance and transmembrane potential after the replacement of one anion by another in the bathing solution. Permeability and conductance sequences are the same. The conductance sequence at pH = 7.4 is Cl^- Br^- > NO₃ ^- > I^- > trichloroacetate ≥ benzoate > valerate > butyrate > proprionate > formate > acetate ≥ lactate > benzenesulfonate ≥ isethionate > methylsulfonate > glutamate ≥ cysteate. The anions are divided into two classes: (a) Chloride-like anions (Cl^- through trichloroacetate) have membrane conductances that decrease as pH decreases. The last six members of the complete sequence are also chloride like. (b) Benzoate-like anions (benzoate through acetate) have conductances that increase as pH decreases. At pH = 6.7 zinc ions block Cl^- and benzoate conductances with inhibitory dissociation constants of 0.12 and 0.16 mM, respectively. Chloride-like and benzoate-like anions probably use the same channels. The minimum size of the channel aperture is estimated as 5.5 x 6.5 Å from the dimensions of the largest permeating anions. A simple model of the channel qualitatively explains chloride-like and benzoate-like conductance sequences and their dependence on pH.     

Reproductive burden, locomotor performance, and the cost of reproduction in free ranging lizards

Abstract. A reduction in the locomotor capacity of gravid females is considered to be a cost of reproduction if it leads to an increased risk of mortality. In this study, we measured the change in endurance between gravid and postgravid female side-blotched lizards ( Uta stansburiana ) as a test of the cost of reproduction. We also altered reproductive investment in some females by direct ovarian manipulation (yolkectomy), which decreased reproductive burden by 30%. Regardless of experimental treatment, all females had lower endurance when gravid. Endurance was 28% lower in gravid females from the yolkectomy treatment and 31% lower in the unmanipulated females relative to postoviposition females. The experimental reduction in clutch mass resulted in a 21% increase in endurance of gravid yolkectomy females relative to control females. Postovipositional endurance was significantly higher in the yolkectomized females than unmanipulated females, which suggests that the cost of reproduction carries over to postoviposition performance. Unmanipulated females exhibited a significant negative association between endurance and size-specific burden. Endurance was not correlated with clutch size or size-specific burden in the yolkectomy females. Survivorship to the second clutch was higher in the yolkectomy females. The results from a logistic regression showed the probability of survival to the second clutch was significantly and positively associated with endurance after controlling for the effects of treatment. Our analyses demonstrated that the decrement in performance associated with current reproductive investment represents a cost of reproduction expressed as diminished locomotor performance and lowered survivorship to the next clutch.     

Survival and Nodulating Ability of Indigenous and Inoculated Rhizobium leguminosarum biovar trifolii in Sterilized and Unsterilized Soil Treated with Sewage Sludge

Rhizobium leguminosarum biovar trifolii was detected in soil from 41 of 47 plots, within nine sewage sludge-treated sites with different soil characteristics and heavy metal contents. However, although population size varied widely, there was no consistent correlation with soil heavy metal concentration. Indigenous populations in 20 plots within four selected sites retained their ability to induce effective nodule formation after incubation of soil in the dark for 165 days. In sterilized (γ-irradiated) soil, Rhizobium survival varied from 0.01% to 95% depending on the soil sample and strain used. Metal-resistant strains with non-mucoid colonies survived less well than mucoid metal-sensitive strains. Received: 26 June 2000/Accepted: 31 July 2000     

Density distribution and cation composition of red blood cells in newborn puppies

The density distribution and cation composition of red blood cells from newborn puppies have been studied. The density distribution of red cells from a newborn puppy in a bovine serum albumin density gradient resembles a normal distribution with a peak density at a region less than that found for adult dog red cells. In two weeks the whole distribution shifts toward a more dense region, and a second cell peak appears so that the distribution becomes bimodal. This second cell peak is smaller than the original peak, and it appears at a region of lower density. In nine weeks the distribution becomes a normal one again, but the peak density corresponds to the peak density of the second cell peak which first appeared at two weeks. Evidence has been obtained to show that fetal red cells are located in the more dense cell peak and neonatal cells are in the less dense second peak. These results were obtained by labeling fetal cells with Cr⁵¹ and neonatal cells with Fe⁵⁹. The analysis of the cation content of these cells shows that fetal cells contain more K and Na and have a higher K/Na ratio than adult red cells. Furthermore, neonatal cells contain considerably less cation and hemoglobin than do fetal cells. From a study of the cation and hemoglobin content of red cells appearing in various density fractions it is concluded that fetal cells lose K and Na during the first two weeks after birth. Thus, the change in the density disribution of the erythrocytes is thought to be due to two factors: (1) An increase in the density of fetal cells due to the loss of K and Na and, hence, water during the first two weeks after birth, and (2) the entry of less dense neonatal cells into the circulation.     

In addition to the SH3 binding region, multiple regions within the N-terminal noncatalytic portion of the cAMP-specific phosphodiesterase, PDE4A5, contribute to its intracellular targeting

The long cyclic AMP (cAMP)-specific phosphodiesterase isoform, PDE4A5 (PDE4A subfamily isoform variant 5), when transiently expressed in COS-7 cells, was shown in subcellular fractionation studies to be associated with both membrane and cytosol fractions, with immunofluorescence analyses identifying PDE4A5 as associated both with ruffles at the cell margin and also at a distinct perinuclear localisation. Deletion of the first nine amino acids of PDE4A5 (1) ablated its ability to interact with the SH3 domain of the tyrosyl kinase, LYN; (2) reduced, but did not ablate, membrane association; and (3) disrupted the focus of PDE4A5 localisation within ruffles at the cell margin. This deleted region contained a Class I SH3 binding motif of similar sequence to those identified by screening a phage display library with the LYN-SH3 domain. Truncation to remove the PDE4A5 isoform-specific N-terminal region caused a further reduction in membrane association and ablated localisation at the cell margin. Progressive truncation to delete the PDE4A long isoform common region and then the long isoform-specific UCR1 did not cause any further change in membrane association or intracellular distribution. However, deletion up to the super-short form splice junction generated an entirely soluble 'core' PDE4A species. We propose that multiple sites in the N-terminal noncatalytic portion of PDE4A5 have the potential to associate with intracellular structures and thus define its intracellular localisation. At least two such sites lie within the PDE4A5 isoform-specific N-terminal region and these appear to be primarily responsible for targeting PDE4A5 to, and organising it within, the cell margin; one is an SH3 binding motif able to interact with LYN kinase and the other lies within the C-terminal portion of the PDE4A5 unique region. A third membrane association region is located within the N-terminal portion of UCR2 and appears to be primarily responsible for targeting to the perinuclear region. Progressive N-terminal truncation, to delete defined regions of PDE4A5, identified activity changes occurring upon deletion of the SH3 binding site region and then upon deletion of the membrane association site region located within UCR2. This suggests that certain of these anchor sites may not only determine intracellular targeting but may also transduce regulatory effects on PDE4A5 activity.     

Study of the interaction of Saccharomyces cerevisiae with glucose by particle microelectrophoresis

Saccharomyces cerevisiae NCYC 239 in the presence of glucose at temperatures under 303 K shows a time-dependent lowering of electrophoreric mobility v. At temperatures above 303 K, this time-dependent change in v is in the direction of increased mobilities. Cells suspended in buffer indicate a surface pKa of less than 4, whereas for cells suspended in buffered glucose it is impossible to derive a surface pKa. A kinetic study of the interaction of S. cerevisiae with glucose as a function of temperature allows calculation of an activation energy of 140 kJ X mol-1 for the combined processes of (i) uptake of glucose onto the cell wall, (ii) transfer through the cell wall and membrane, and (iii) the establishment of a steady glucose flux through the wall and membrane.