Phosphorylation of perilipin is associated with indicators of lipolysis in Holstein cows.

Perilipin is a regulatory protein that coats the lipid droplet in adipocytes. In the basal state, perilipin inhibits lipolysis by restricting access of hormone sensitive lipase (HSL) to the lipid droplet. In contrast, during stimulated lipolysis, phosphorylated perilipin interacts with HSL such that the catalytic activity of HSL on its acylglycerol substrate is enhanced. However, the regulation and function of perilipin in vivo has not been defined clearly across comparative animal models. Consequently, this study was undertaken to determine if changes in perilipin mRNA, protein, or phosphorylation state are associated with in vivo indicators of lipolysis in the dairy cow as a model of lipolysis induced by the marked metabolic demands of lactation. Semiquantitative western blotting and quantitative PCR were used to quantify total and phosphorylated HSL and perilipin in adipose tissue obtained from cows in early [5-14 days in milk (DIM), n=11] and mid (176-206 DIM, n=9) lactation. As expected, circulating NEFA and glycerol concentrations, and phosphorylated HSL were greater in early versus mid lactation, indicative of greater lipolytic activity in early lactation. Furthermore, phosphorylated, but not total perilipin abundance, was greater in early lactation when the metabolic demand for energy is greater than in mid lactation. Finally, the abundance of phosphorylated perilipin was positively correlated with circulating glycerol and NEFA concentrations during both early and mid lactation. Collectively, these data support the hypothesis that phosphorylated perilipin is a critical determinant of lipolytic activity stemming from the metabolic demands of lactation.

Role of HPC2/ELAC2 in hereditary prostate cancer.

The HPC2/ELAC2 gene on chromosome 17p was recently identified as a candidate gene for hereditary prostate cancer (HPC). To confirm these findings, we screened 300 prostate cancer patients (2 affected members/family) from 150 families with HPC for potential germ-line mutations using conformation-sensitive gel electrophoresis, followed by direct sequence analysis. The minimum criteria for our families with HPC was the presence of 3 affected men with prostate cancer. A total of 23 variants were identified, including 13 intronic and 10 exonic changes. Of the 10 exonic changes, 1 truncating mutation was identified, a Glu216Stop nonsense mutation. This nonsense variant was found in 2 of 3 affected men in a single family. The remaining nine alterations included five missense, three silent, and one variant in the 3' untranslated region. To additionally test for potential associations of polymorphic variants and increased risk for disease, we genotyped two common polymorphisms, Ser217Leu and Ala541Thr, in 446 prostate cancer patients from 164 families with HPC and 502 population-based controls. The frequency of the Leu217 variant was similar for patients (32.3%) and controls (31.8%), as was the frequency of the Thr541 variant (5.4% among patients versus 5.2% among controls). In contrast to previous reports, we found no association of the joint effects of Leu271 and Thr541 (odds ratio, 1.04; 95% confidence interval, 0.57-1.89). Overall, our results did not reveal any association between these two common polymorphisms and the risk for HPC. The finding of a nonsense mutation in the HPC2/ELAC2 gene confirms its potential role in genetic susceptibility to prostate cancer. However, our data also suggest that germ-line mutations of the HPC2/ELAC2 are rare in HPC and that the variants Leu217 and Thr541 do not appear to influence the risk for HPC. Cumulatively, these results suggest that alterations within the HPC2/ELAC2 gene play a limited role in genetic susceptibility to HPC.

Polymerase chain reaction detection of human immunodeficiency virus DNA in human periradicular lesions.

Human immunodeficiency virus (HIV) has been previously reported to be present in the dental pulp of a patient with AIDS. The present report investigated the feasibility of polymerase chain reaction (PCR) amplification to detect the HIV proviral DNA in cells from periradicular lesions from an HIV-positive patient. The standard PCR amplification with 30 cycles and the nested PCR consisting of two 25-cycle amplifications were used. Samples from each reaction were separated by nondenaturing polyacrylamide gel electrophoresis and stained with ethidium bromide for visualization. Gels were electroblotted to nylon membranes, which were then fixed, denatured, and dried. Membranes were hybridized to specific radioactive oligonucleotide probes and placed next to Kodak XAR film for visualization of the HIV-specific bands. No evidence of HIV-specific reaction was observed in cells (negative control) or in two periradicular lesions from two HIV-negative patients. The ethidium bromide strains revealed that PCR amplification of DNA extracts from two lesions from the HIV-positive patient yielded PCR bands (with both primer pairs) which corresponded to HIV-specific bands of the expected size.