A temperature gradient method for lipid phase diagram construction using time-resolved x-ray diffraction.

A method that enables temperature-composition phase diagram construction at unprecedented rates is described and evaluated. The method involves establishing a known temperature gradient along the length of a metal rod. Samples of different compositions contained in long, thin-walled capillaries are positioned lengthwise on the rod and "equilibrated" such that the temperature gradient is communicated into the sample. The sample is then moved through a focused, monochromatic synchroton-derived x-ray beam and the image-intensified diffraction pattern from the sample is recorded on videotape continuously in live-time as a function of position and, thus, temperature. The temperature at which the diffraction pattern changes corresponds to a phase boundary, and the phase(s) existing (coexisting) on either side of the boundary can be identified on the basis of the diffraction pattern. Repeating the measurement on samples covering the entire composition range completes the phase diagram. These additional samples can be conveniently placed at different locations around the perimeter of the cylindrical rod and rotated into position for diffraction measurement. Temperature-composition phase diagrams for the fully hydrated binary mixtures, dimyristoylphosphatidylcholine (DMPC)/dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPE)/DPPC, have been constructed using the new temperature gradient method. They agree well with and extend the results obtained by other techniques. In the DPPE/DPPC system structural parameters as a function of temperature in the various phases including the subgel phase are reported. The potential limitations of this steady-state method are discussed.     

HLA Haplotypes with C4B5; evidence for further allelic heterogeneity

Twenty-three individuals from various disease groups and normal controls were identified by immunofixation with anti-C4, C4-dependent lysis, determination of Rg (Rodgers) and Ch (Chido) phenotypes, and immunoblotting with C4-specific mouse monoclonal antibody. We found that one haplotype predominates with the C4B ^* 5 allele, HLA-A11, B22(55), Cw3, Bf ^* S, C4A ^* 4B ^* 5, which also carries the Ch ^{1,–2, 3} haplotype. The B5 allotype was also found with HLA-1360, HLA-1335 in Caucasoids, and HLA-B18 in non-Caucasoids; these carried the Ch ^{–1, –2, –3} haplotype. Our results are in accord with an earlier report of two B5 subtypes, B5Rg⁺ and B5Rg^– (Roos et al. 1984). The specificity of the mouse monoclonal antibodies IC4 and 21312 had been previously related to C4A and C4B, respectively, but our results suggest that they relate more closely to Rg and Ch determinants.     

VNTR and microsatellite polymorphisms within the subtelomeric region of 7q.

The molecular basis of a highly polymorphic RFLP marker, HTY146c3 (D7S591), within the subtelomeric region of human chromosome 7q was determined by restriction-fragment and DNA sequence analysis. Two polymorphic systems were found--a simple base-substitution polymorphism and a GC-rich VNTR element with a core structure of C3AG2C2. In addition, a compound-imperfect CA dinucleotide-repeat element was identified approximately 10-20 kb from the telomeric sequence repeat (T2AG3), demonstrating that microsatellites can extend essentially to the ends of human chromosomes. The microsatellite marker, sAVH-6 (D7S594), is highly polymorphic, with 10 alleles and an observed heterozygosity of 84% found with the CEPH (Centre d'Etude du Polymorphisme Humain) reference pedigree collection. In combination with the RFLPs, the informativeness of the markers contained within 240 kb at the telomere approaches 100%. A unique genetic and physical STS marker, sAVH-6, defines the endpoint of the long arm of human chromosome 7.     

Moderate Alcohol Use and Cardiovascular Disease from Mendelian Randomization

Background

Observational studies show moderate alcohol use negatively associated with ischemic heart disease (IHD) and cardiovascular disease (CVD). However, healthier attributes among moderate users compared to never users may confound the apparent association. A potentially less biased way to examine the association is Mendelian randomization, using alcohol metabolizing genes which influence alcohol use.

Methods

We used instrumental variable analysis with aldehyde dehydrogenase 2 (ALDH2) genotypes (AA/GA/GG) as instrumental variables for alcohol use to examine the association of alcohol use (10 g ethanol/day) with CVD risk factors (blood pressure, lipids and glucose) and morbidity (self-reported IHD and CVD) among men in the Guangzhou Biobank Cohort Study.

Results

ALDH2 genotypes were a credible instrument for alcohol use (F-statistic 74.6). Alcohol was positively associated with HDL-cholesterol (0.05 mmol/L per alcohol unit, 95% confidence interval (CI) 0.02 to 0.08) and diastolic blood pressure (1.15 mmHg, 95% CI 0.23 to 2.07) but not with systolic blood pressure (1.00 mmHg, 95% CI -0.74 to 2.74), LDL-cholesterol (0.03 mmol/L, 95% CI -0.03 to 0.08), log transformed triglycerides (0.03 mmol/L, 95% CI -0.01 to 0.08) or log transformed fasting glucose (0.01 mmol/L, 95% CI -0.006 to 0.03), self-reported CVD (odds ratio (OR) 0.98, 95% CI 0.76 to 1.27) or self-reported IHD (OR 1.10, 95% CI 0.83 to 1.45).

Conclusion

Low to moderate alcohol use among men had the expected effects on most CVD risk factors but not fasting glucose. Larger studies are needed to confirm the null associations with IHD, CVD and fasting glucose.     

Structure-based design of novel dihydroisoquinoline BACE-1 inhibitors that do not engage the catalytic aspartates

The structure–activity relationship of a series of dihydroisoquinoline BACE-1 inhibitors is described. Application of structure-based design to screening hit 1 yielded sub-micromolar inhibitors. Replacement of the carboxylic acid of 1 was guided by X-ray crystallography, which allowed the replacement of a key water-mediated hydrogen bond. This work culminated in compounds such as 31, which possess good BACE-1 potency, excellent permeability and a low P-gp efflux ratio.     

Evaluation of an in vitro model of androgen ablation and identification of the androgen responsive proteome in LNCaP cells

Proteins responsive to androgen and anti-androgen may be involved in the development and progression of prostate cancer and the ultimate failure of androgen-ablation therapy. These proteins represent potential diagnostic and therapeutic targets for improved management of prostate cancer. We have investigated the effect of androgen (R1881) and anti-androgen (bicalutamide) on the androgen-responsive prostate cancer LNCaP cell line using a quantitative gel-based proteomic approach. Prior to analysis, the in vitro system was evaluated for reproducibility and validated by appropriate molecular responses to treatment. Six replicate samples were independently generated and analysed by 2-D DIGE. According to strict statistical criteria, 197 spots were differentially expressed, of which we have successfully identified 165 spots corresponding to 125 distinct proteins. Following androgen supplementation, 108 spots (68 proteins) were increased and 57 spots (39 proteins) were decreased. Essentially no difference was observed between control and anti-androgen-treated samples, confirming the absence of "off-target" effects of bicalutamide. Identified proteins were involved in diverse processes including the stress response and intracellular signalling. The potential contribution to disease of these processes and identified constituent proteins are discussed. This rigorous, statistically supported study of androgen responses has provided a number of potential candidates for development as diagnostic/prognostic markers and drug targets.     

Mutations in the heatshock cognate 70 protein (hsc4) modulate Notch signaling

In our effort to dissect the Notch signaling mechanism we have conducted a screen for mutations that reduce Notch signaling activity. We recovered nine complementation groups as modifiers of the hypomorphic Notch allele notchoid. Apart from the known Notch signaling modulators Notch, Delta and mastermind we isolated alleles in vestigial, wingless, scalloped and clipped, genes known to affect wing morphogenesis. In addition, we identified mutations in Bag, the gene encoding clathrin heavy chain and a dominant mutation of the cytosolic 70 kDa heatshock cognate protein encoded by the hsc4 gene, as Notch signaling modifier. We focused our attention on the latter mutation because it displays dramatic genetic interactions with mutations of the Notch receptor as well as several additional Notch signaling pathway elements. We discuss how hsc4, a gene thought to be involved in subcellular trafficking, may affect the number of functional Notch receptors on the cell surface.