Cutting edge: stable epigenetic inheritance of regional IFN-gamma promoter demethylation in CD44highCD8+ T lymphocytes

Genomic DNA methylation patterns influence the development and maintenance of function during cellular differentiation. Methylation of regulatory sequences can have long-lasting effects on gene expression if inherited in an epigenetic manner. Recent work suggests that DNA methylation has a regulatory role in differential cytokine gene expression in primary T lymphocytes. Here we show, by clonal lineage analysis, that methylation patterns in the IFN-gamma promoter exhibit long term faithful inheritance in CD44highCD8+ T cells and their progeny, through 16 cell divisions and a clonal expansion of 5 orders of magnitude. Moreover, the demethylated IFN-gamma promoter is faithfully inherited following the withdrawal of T cell stimulation and the loss of detectable IFN-gamma mRNA, consistent with passive rather than active maintenance mechanisms. This represents a form of stable cellular memory, of defined epigenetic characteristics, that may contribute to the maintenance of T cell cytokine expression patterns and T cell memory.     

The duty to recontact: attitudes of genetics service providers

The term "duty to recontact" refers to the possible ethical and/or legal obligation of genetics service providers (GSPs) to recontact former patients about advances in research that might be relevant to them. Although currently this practice is not part of standard care, some argue that such an obligation may be established in the future. Little information is available, however, on the implications of this requirement, from the point of view of GSPs. To explore the opinions of genetics professionals on this issue, we sent a self-administered questionnaire to 1,000 randomly selected U.S. and Canadian members of the American Society of Human Genetics. We received 252 completed questionnaires. The major categories of respondents were physician geneticist (41%), Ph.D. geneticist (30%), and genetic counselor (18%); 72% of the total stated that they see patients. Respondents indicated that responsibility for staying in contact should be shared between health professionals and patients. Respondents were divided about whether recontacting patients should be the standard of care: 46% answered yes, 43% answered no, and 11% did not know. Those answering yes included 44% of physician geneticists, 53% of Ph.D. geneticists, and 31% of genetic counselors; answers were statistically independent of position or country of practice but were dependent on whether the respondent sees patients (43% answered yes) or not (54% answered yes). There also was a lack of consensus about the possible benefits and burdens of recontacting patients and about various alternative methods of informing patients about research advances. Analysis of qualitative data suggested that most respondents consider recontacting patients an ethically desirable, but not feasible, goal. Points to consider in the future development of guidelines for practice are presented.     

Small molecule biaryl FSH receptor agonists. Part 2: Lead optimization via parallel synthesis

Potent small molecule biaryl diketopiperazine FSH receptor agonists such as 10c (EC(50)=13 nM) and 11f (EC(50)=1.2 nM) were discovered through the design, synthesis and evaluation of three biaryl diketopiperazine optimization libraries with over 300 compounds. These libraries were prepared via solid-phase parallel synthesis using a cyclization-release method.     

The small-subunit processome is a ribosome assembly intermediate

The small-subunit (SSU) processome is a large ribonucleoprotein required for the biogenesis of the 18S rRNA and likely corresponds to the terminal knobs visualized by electron microscopy on the 5' end of nascent rRNAs. The original purification of the SSU processome of Saccharomyces cerevisiae resulted in the identification of 28 proteins. Here, we characterize 12 additional protein components, including five small-ribosomal-subunit proteins (Rps4, Rps6, Rps7, Rps9, and Rps14) that had previously been copurified. Our multiple criteria for including a component as a bona fide SSU processome component included coimmunoprecipitation with Mpp10 (an SSU processome component), the U3 snoRNA, and the anticipated pre-rRNAs. Importantly, the association of specific ribosomal proteins with the SSU processome suggests that the SSU processome has roles in both pre-rRNA processing and ribosome assembly. These ribosomal proteins may be analogous to the primary or secondary RNA binding proteins first described in bacterial in vitro ribosome assembly maps. In addition to the ribosomal proteins and based on the same experimental approach, we found seven other proteins (Utp18, Noc4, Utp20, Utp21, Utp22, Emg1, and Krr1) to be bona fide SSU processome proteins.     

Transport of cholesterol across a BeWo cell monolayer: implications for net transport of sterol from maternal to fetal circulation

The placental transport of various compounds, such as glucose and fatty acids, has been well studied. However, the transport of cholesterol, a sterol essential for proper fetal development, remains undefined in the placenta. Therefore, the purpose of these studies was to examine the transport of cholesterol across a placental monolayer and its uptake by various cholesterol acceptors. BeWo cells, which originated from a human choriocarcinoma, were grown on transwells for 3 days to form a confluent monolayer. The apical side of the cells was radiolabeled with either free cholesterol or LDL cholesteryl ester. After 24 h, the radiolabel was removed and cholesterol acceptors were added to the basolateral chamber. Cholesterol was found to be taken up by the apical surface of the placental monolayer, transported to the basolateral surface of the cell, and effluxed to fetal human serum, fetal HDL, or phospholipid vesicles, but not to apolipoprotein A-I. In addition, increasing the cellular cholesterol concentration further increased the amount of cholesterol transported to the basolateral acceptors. These are the first studies to demonstrate the movement of cholesterol across a placental cell from the maternal circulation (apical side) to the fetal circulation (basolateral side).     

Tributyltin and copper effects on encystment and in vitro excystment of Parorchis acanthus larvae

Effects of tributyltin (TBT) and copper (Cu) on cercariae and metacercariae of the trematode Parorchis acanthus (Digenea: Philophthalmidae) were investigated. Cercariae released by the dogwhelk, Nucella lapillus were maintained in natural seawater (SW) or solutions of TBT or Cu ranging from 0.001-100 microg l(-1) and 1-6 mg l(-1) respectively before they encysted. Over 79% of the cercariae encysted in control and test solutions. Low concentrations of TBT reduced encystment success more than low concentrations of Cu. The percentage of cercariae that formed cysts in the highest concentrations of both pollutants was higher than in the controls, perhaps representing an 'emergency response' to the pollutants. Before being induced to excyst in vitro, metacercariae were left in the heavy metal solutions for 3 weeks. Metacercariae exposed as cercariae to TBT and Cu achieved lower percentage excystment success than those that had encysted in SW. Cyst walls provided greater protection against Cu than TBT. It was concluded that TBT and Cu had a detrimental effect on the larval stages of P. acanthus at the higher concentrations used but the cyst wall afforded an element of protection if formed in unpolluted seawater before the larval stages were subjected to the pollutants.     

Genomic effects of IFN-beta in multiple sclerosis patients

The purpose of this report was to characterize the dynamics of the gene expression cascades induced by an IFN-beta-1a treatment regimen in multiple sclerosis patients and to examine the molecular mechanisms potentially capable of causing heterogeneity in response to therapy. In this open-label pharmacodynamic study design, peripheral blood was obtained from eight relapsing-remitting multiple sclerosis patients just before and at 1, 2, 4, 8, 24, 48, 120, and 168 h after i.m. injection of 30 micro g of IFN-beta-1a. The total RNA was isolated from monocyte-depleted PBL and analyzed using cDNA microarrays containing probes for >4000 known genes. IFN-beta-1a treatment resulted in selective, time-dependent effects on multiple genes. The mRNAs for genes implicated in the anti-viral response, e.g., double-stranded RNA-dependent protein kinase, myxovirus resistance proteins 1 and 2, and guanylate binding proteins 1 and 2 were rapidly induced within 1-4 h of IFN-beta treatment. The mRNAs for several genes involved in IFN-beta signaling, such as IFN-alpha/beta receptor-2 and Stat1, were also increased. The mRNAs for lymphocyte activation markers, such as IFN-induced transmembrane protein 1 (9-27), IFN-induced transmembrane protein 2 (1-8D), beta(2)-microglobulin, and CD69, were also increased in a time-dependent manner. The findings demonstrate that IFN-beta treatment induces specific and time-dependent changes in multiple mRNAs in lymphocytes of multiple sclerosis patients that could provide a framework for rapid monitoring of the response to therapy.