Hormonal regulation of expression of the endogenous and transfected human growth hormone gene

Expression of the endogenous human GH (hGH) gene in response to glucocorticoids, thyroid hormone, and insulin was studied in cultures of dispersed GH-secreting human pituitary adenomas. Results were compared to those obtained when the hGH gene was transfected into rat pituitary tumor cells (GC). In the human pituitary cells the glucocorticoid dexamethasone [(Dex) 10(-6) M] increased the release of GH and the levels of GH mRNA by 2 to 4-fold (P less than 0.05). T3 (10(-8) M) had no effect on GH mRNA but increased hGH release by 2- to 6-fold (P less than 0.01). Insulin (5 x 10(-9) M) alone had no significant effect on either hGH mRNA or protein, but blunted the effect of Dex. Among 11 of 18 GC cell clones transfected with the hGH gene with detectable hGH mRNA expression, Dex increased hGH mRNA levels in seven and T3 treatment reduced hGH mRNA levels in eight. Conversely, rat GH mRNA levels from the endogenous rat gene were increased by either Dex or T3 in all 18 clones. Insulin alone or in combination with T3 or Dex was found to increase hGH mRNA levels in some cell lines and to decrease hGH mRNA levels in others; these effects were correlated strongly (r = 0.88; P less than 0.001) with the influence of insulin on the endogenous rat GH gene, implying that individual cellular differences can simultaneously affect the insulin responsiveness of both genes.(ABSTRACT TRUNCATED AT 250 WORDS)     

CYP3A4-V and prostate cancer in African Americans: causal or confounding association because of population stratification?

CYP3A4-V, an A to G promoter variant associated with prostate cancer in African Americans, exhibits large differences in allele frequency between populations. Given that the African American population is genetically heterogeneous because of its African ancestry and subsequent admixture with European Americans, case-control studies with African Americans are highly susceptible to spurious associations. To test for association with prostate cancer, we genotyped CYP3A4-V in 1376 (2 N) chromosomes from prostate cancer patients and age- and ethnicity-matched controls representing African Americans, Nigerians, and European Americans. To detect population stratification among the African American samples, 10 unlinked genetic markers were genotyped. To correct for the stratification, the uncorrected association statistic was divided by the average of association statistics across the 10 unlinked markers. Sharp differences in CYP3A4-V frequencies were observed between Nigerian and European American controls (0.87 and 0.10, respectively; P<0.0001). African Americans were intermediate (0.66). An association uncorrected for stratification was observed between CYP3A4-V and prostate cancer in African Americans (P=0.007). A nominal association was also observed among European Americans (P=0.02) but not Nigerians. In addition, the unlinked genetic marker test provided strong evidence of population stratification among African Americans. Because of the high level of stratification, the corrected P-value was not significant (P=0.25). Follow-up studies on a larger dataset will be needed to confirm whether the association is indeed spurious; however, these results reveal the potential for confounding of association studies by using African Americans and the need for study designs that take into account substructure caused by differences in ancestral proportions between cases and controls.     

Linkage of prostate cancer susceptibility loci to chromosome 1

Three prostate cancer susceptibility genes have been reported to be linked to different regions on chromosome 1: HPC1 at 1q24-25, PCAP at 1q42-43, and CAPB at 1p36. Replication studies analyzing each of these regions have yielded inconsistent results. To evaluate linkage across this chromosome systematically, we performed multipoint linkage analyses with 50 microsatellite markers spanning chromosome 1 in 159 hereditary prostate cancer families (HPC), including 79 families analyzed in the original report describing HPC1 linkage. The highest lod scores for the complete dataset of 159 families were observed at 1q24-25 at which the parametric lod score assuming heterogeneity (hlod) was 2.54 (P=0.0006) with an allele sharing lod of 2.34 (P=0.001) at marker D1S413, although only weak evidence was observed in the 80 families not previously analyzed for this region (hlod=0.44, P=0.14, and allele sharing lod=0.67, P=0.08). In the complete data set, the evidence for linkage across this region was very broad, with allele sharing lod scores greater than 0.5 extending approximately 100 cM from 1p13 to 1q32, possibly indicating the presence of multiple susceptibility genes. Elsewhere on chromosome 1, some evidence of linkage was observed at 1q42-43, with a peak allele sharing lod of 0.56 (P=0.11) and hlod of 0.24 (P=0.25) at D1S235. For analysis of the CAPB locus at 1p36, we focused on six HPC families in our collection with a history of primary brain cancer; four of these families had positive linkage results at 1p36, with a peak allele sharing lod of 0.61 (P=0.09) and hlod of 0.39 (P=0.16) at D1S407 in all six families. These results are consistent with the heterogeneous nature of hereditary prostate cancer, and the existence of multiple loci on chromosome 1 for this disease.     

Using the longest significance run to estimate region-specific p-values in genetic association mapping studies

Background  

Association testing is a powerful tool for identifying disease susceptibility genes underlying complex diseases. Technological advances have yielded a dramatic increase in the density of available genetic markers, necessitating an increase in the number of association tests required for the analysis of disease susceptibility genes. As such, multiple-tests corrections have become a critical issue. However the conventional statistical corrections on locus-specific multiple tests usually result in lower power as the number of markers increases. Alternatively, we propose here the application of the longest significant run (LSR) method to estimate a region-specific p-value to provide an index for the most likely candidate region.     

On the mechanism of ATP-induced shape changes in the human erythrocyte membranes: the role of ATP

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.     

Synthetic biology evolves

Synthetic biology is advancing rapidly as biologists, physicists and engineers are combining their efforts to understand and program cell function. By characterizing isolated genetic components or modules, experimentalists have paved the way for more quantitative analyses of genetic networks. A recent paper presents a method of computational, or in silico, evolution in which a set of components can evolve into networks that display desired behaviors. An integrated approach that includes a strategy of in silico design by evolution, together with efforts exploiting directed evolution in vivo, is likely to be the next step in the evolution of synthetic biology.     

Genome-wide association study identifies novel loci associated with circulating phospho- and sphingolipid concentrations

Phospho- and sphingolipids are crucial cellular and intracellular compounds. These lipids are required for active transport, a number of enzymatic processes, membrane formation, and cell signalling. Disruption of their metabolism leads to several diseases, with diverse neurological, psychiatric, and metabolic consequences. A large number of phospholipid and sphingolipid species can be detected and measured in human plasma. We conducted a meta-analysis of five European family-based genome-wide association studies (N = 4034) on plasma levels of 24 sphingomyelins (SPM), 9 ceramides (CER), 57 phosphatidylcholines (PC), 20 lysophosphatidylcholines (LPC), 27 phosphatidylethanolamines (PE), and 16 PE-based plasmalogens (PLPE), as well as their proportions in each major class. This effort yielded 25 genome-wide significant loci for phospholipids (smallest P-value = 9.88×10⁻²⁰⁴) and 10 loci for sphingolipids (smallest P-value = 3.10×10⁻⁵⁷). After a correction for multiple comparisons (P-value<2.2×10⁻⁹), we observed four novel loci significantly associated with phospholipids (PAQR9, AGPAT1, PKD2L1, PDXDC1) and two with sphingolipids (PLD2 and APOE) explaining up to 3.1% of the variance. Further analysis of the top findings with respect to within class molar proportions uncovered three additional loci for phospholipids (PNLIPRP2, PCDH20, and ABDH3) suggesting their involvement in either fatty acid elongation/saturation processes or fatty acid specific turnover mechanisms. Among those, 14 loci (KCNH7, AGPAT1, PNLIPRP2, SYT9, FADS1-2-3, DLG2, APOA1, ELOVL2, CDK17, LIPC, PDXDC1, PLD2, LASS4, and APOE) mapped into the glycerophospholipid and 12 loci (ILKAP, ITGA9, AGPAT1, FADS1-2-3, APOA1, PCDH20, LIPC, PDXDC1, SGPP1, APOE, LASS4, and PLD2) to the sphingolipid pathways. In large meta-analyses, associations between FADS1-2-3 and carotid intima media thickness, AGPAT1 and type 2 diabetes, and APOA1 and coronary artery disease were observed. In conclusion, our study identified nine novel phospho- and sphingolipid loci, substantially increasing our knowledge of the genetic basis for these traits.     

Microbial Diversity and Resistance to Copper in Metal-Contaminated Lake Sediment

Contamination of habitats with heavy metals has become a worldwide problem. We describe herein the analysis of lake sediment contaminated with high concentrations of copper as a consequence of mine milling disposal over a 100-year period. Copper concentrations in the sediment were found to vary with depth and ranged from 200 to 5500 ppm. Analysis of the microbial community with T-RFLP identified a minimum of 20 operational taxonomic units (OTU). T-RFLP analysis along a depth profile detected as many as nine shared OTUs across 15 centimeters, suggesting a conservation of community structure over this range. Only two genera, Arthrobacter and Ralstonia, were detected among 50 aerobic copper-resistant isolates cultivated on R2A, one of which (Ralstonia sp.) was characterized by the sequestration of copper, identified by electron diffraction scanning, in growing colonies. Scanning electron microscopy showed changes to the outer envelope of the cells when grown in the presence of copper. The copper-resistant Ralstonia isolates were also resistant to Ni, Cd, and Zn, showing two patterns of phenotypic resistant to these three metals in which either resistance to Zn or Ni was expressed in an isolate but never both.     

Synthesis, characterization and photophysical properties of mixed ligand tris(polypyridyl)chromium(III) complexes, [Cr(phen)2L]

A series of new heteroleptic, tris(polypyridyl)chromium(III) complexes, [Cr(phen)₂L]³⁺ (L = substituted phenanthrolines or bipyridines), has been prepared and characterized, and their photophyical properties in a number of solvents have been investigated. X-ray crystallography measurements confirmed that the cationic (3+) units contain only one ligand L plus two phenanthroline ligands. Electrochemical and photophysical data showed that both ground state potentials and lifetime decays are sensitive to ligand structure and the nature of the solvent with the exception of compounds containing L = 5-amino-1,10-phenanthroline (aphen) and 2,2′-bipyrimidine (bpm). Addition of electron-donating groups in the ligand structure shifts redox potentials to more negative values than those observed for the parent compound, [Cr(phen)₃]³⁺. Emission decays show a complex dependence with the solvent. The longest lifetime was observed for [Cr(phen)₂(dip)]³⁺ (dip = 4,7-diphenylphenanthroline) in air-free aqueous solutions, τ = 273 μs. Solvent effects are explained in terms of the affinity of hydrophobic complexes for non-polar solvent molecules and the solvent microstructure surrounding chromium units.