Evaluation of allelic expression of imprinted genes in adult human blood

Background

Imprinted genes are expressed from only one allele in a parent-of-origin dependent manner. Loss of imprinted (LOI) expression can result in a variety of human disorders and is frequently reported in cancer. Biallelic expression of imprinted genes in adult blood has been suggested as a useful biomarker and is currently being investigated in colorectal cancer. In general, the expression profiles of imprinted genes are well characterised during human and mouse fetal development, but not in human adults.

Methodology/Principal Findings

We investigated quantitative expression of 36 imprinted genes in adult human peripheral blood leukocytes obtained from healthy individuals. Allelic expression was also investigated in B and T lymphocytes and myeloid cells. We found that 21 genes were essentially undetectable in adult blood. Only six genes were demonstrably monoallelic, and most importantly, we found that nine genes were either biallelic or showed variable expression in different individuals. Separated leukocyte populations showed the same expression patterns as whole blood. Differential methylation at each of the imprinting control loci analysed was maintained, including regions that contained biallelically expressed genes. This suggests in some cases methylation has become uncoupled from its role in regulating gene expression.

Conclusions/Significance

We conclude that only a limited set of imprinted genes, including IGF2 and SNRPN, may be useful for LOI cancer biomarker studies. In addition, blood is not a good tissue to use for the discovery of new imprinted genes. Finally, lymphocyte DNA methylation status in the adult may not always be a reliable indicator of monoallelic gene expression.     

Cxc chemokine receptor expression on human endothelial cells.

CXC chemokines play a important role in the process of leukocyte recruitment and activation at sites of inflammation. However, recent evidence suggests that these molecules can also regulate endothelial cell functions such as migration, angiogenesis and proliferation. In this study we have investigated CXC chemokine receptor expression in both primary cultures of human umbilical vein endothelial cells (HUVEC) and the spontaneously transformed HUVEC cell line, ECV304. We found that both cell types express mRNA for chemokine receptors CXCR1, CXCR2 and CXCR4, but not CXCR3. Flow cytometric analysis revealed low levels of CXCR1 but higher levels of CXCR4 cell surface expression. HUVECs responded to SDF-1alpha with a rapid and robust calcium flux, however no calcium flux was seen with either IL-8 or Gro-alpha. HUVECs and ECV304 cells did not proliferate in response to CXC chemokines, although ECV304 cells did migrate towards SDF-1alpha and IL-8. These data demonstrate that HUVECs and the endothelial cell line, ECV304 express functional CXC chemokine receptors.     

Development of multiple strain competitive index assays for <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> using pIMC; a new site-specific integrative vector

Background  

The foodborne, gram-positive pathogen, Listeria monocytogenes, is capable of causing lethal infections in compromised individuals. In the post genomic era of L. monocytogenes research, techniques are required to identify and validate genes involved in the pathogenicity and environmental biology of the organism. The aim here was to develop a widely applicable method to tag L. monocytogenes strains, with a particular emphasis on the development of multiple strain competitive index assays.     

Diurnal Variation in Mood and Performance in a Time-Isolated Environment

In order to document circadian rhythmicity in various psychological functions under the chronobiologically 'pure' condition of temporal isolation, a battery of mood and performance tests were administered about 6 times per day to a heterogeneous group of 18 subjects (ages 19-81, 5 female). Each subject spent about 5 days in temporal isolation, entrained to a routine equivalent to his/her own habitual sleep/wake cycle. Average time of day functions were obtained for the mood and performance variables, and compared to rectal temperature data subjected to exactly the same statistical analysis. Significant time of day effects were found in the mood variables of alertness, sleepiness, weariness, effort required, happiness and well-being. Times of 'best' mood were different from the time of peak temperature. Moreover, the minima of sleepiness, weariness and effort occurred earlier in the day than the maximum of alertness. Significant time of day effects were also found in the speed with which search and dexterity tasks were completed. Only the dexterity tasks showed a complete parallelism with the temperature rhythm.     

Recommendations for a nomenclature system for reporting methylation aberrations in imprinted domains

The analysis of DNA methylation has become routine in the pipeline for diagnosis of imprinting disorders, with many publications reporting aberrant methylation associated with imprinted differentially methylated regions (DMRs). However, comparisons between these studies are routinely hampered by the lack of consistency in reporting sites of methylation evaluated. To avoid confusion surrounding nomenclature, special care is needed to communicate results accurately, especially between scientists and other health care professionals. Within the European Network for Human Congenital Imprinting Disorders we have discussed these issues and designed a nomenclature for naming imprinted DMRs as well as for reporting methylation values. We apply these recommendations for imprinted DMRs that are commonly assayed in clinical laboratories and show how they support standardized database submission. The recommendations are in line with existing recommendations, most importantly the Human Genome Variation Society nomenclature, and should facilitate accurate reporting and data exchange among laboratories and thereby help to avoid future confusion.     

cDNA Libraries from Single Human Preimplantation Embryos

In this paper, the construction, evaluation, and application of cDNA libraries from eight unfertilized oocytes and single four-cell-, seven-cell-, and blastocyst-stage embryos are described. Rapid, reproducible, and efficient procedures for the construction of PCR-based cDNA libraries from fewer than 10 cells were first developed in small populations of fibroblast cells. The human embryo libraries display complexities sufficient (between 10⁵and 10⁶clones) to represent the entire active gene population at these early stages of human development. The ubiquitous cytoskeletal elements, β-actin, keratin-18, and α-tubulin, were detected at the expected frequency. Sequencing of consecutively picked random clones, without selection, showed the presence of a variety of sequences, such as the human transposable element, LINE-1 andAlurepeat sequences, housekeeping genes, and tissue-specific genes, such as α-globin and FMR-1. In addition to cDNAs corresponding to known ESTs (expressed sequence tags) in the GenBank and dbEST databases, a high proportion of novel sequences were detected. Applications of the libraries to several areas of interest, such as expression of CpG-island-containing “tissue-specific” genes, developmental genes expressed in a stage-specific manner, and a search for monoallelic expression of imprinted genes, are described. The libraries are a valuable resource for the study of gene expression during human preimplantation development and obviate the need for research on the human embryos themselves.     

A classification of the deciduous forest of eastern North America

Data from 300 forest stands, scattered over 29 states within the eastern North American deciduous forest, were subjected to detrended correspondence analysis (DCA) and two-way indicator species analysis (TWINSPAN) in an effort to identify classifiable units. Most species are widespread which provide a great deal of continuity in the vegetation.The deciduous forest can be divided into three forest regions: (1) northern, (2) central and (3) southern. The northern region corresponds to the hemlock-white pine-northern hardwood forest of Braun (1950). The central region includes the beech-maple and oak-hickory forests. The beech-maple as identified here includes the mixed mesophytic, beech-maple, maple-basswood and about half of the western mesophytic forests of Braun (1950). The oak-hickory includes Braun's oak-hickory, oak-chestnut and about half of the western mesophytic forests. The southern region coincides with the southern mixed hardwood forests.     

Circadian determinants of subjective alertness

Four healthy male subjects each experienced a temporal isolation experiment lasting several months. During part of each experiment (2-5 weeks), the subject's entire imposed daily routine (including light-dark, rest-activity, and meal routines) was either stretched (two subjects: T = 25.8 hr, 26.0 hr) or reduced (two subjects: T = 22.8 hr, 23.1 hr) to beyond the range of entrainment of the endogenous circadian pacemaker (ECP), which then ran at a different period (tau). Subjective alertness was measured approximately three times per hour (during wakefulness), using a computerized visual analogue scale technique. Circadian rhythms in subjective alertness were then plotted both at tau, the period length of the ECP, and at T, the period length of the imposed sleep-wake cycle (SWC) and light-dark cycle. At tau, the alertness rhythm was closely in phase with the temperature rhythm. At T, the alertness rhythm showed an "inverted-U" function with a peak toward the middle of the subjective day, upon which was superimposed a "postlunch dip" for one subject. Thus, subjective alertness would appear to be under the control fo both ECP and SWC mechanisms, which combine to produce the composite time-of-day function normally observed in a diurnal setting.     

Biogenesis of mitochondria 36

Summary The isolation and characterisation of a mutant affecting the assembly of mitochondrial ATPase is reported. The mutation confers resistance to oligomycin and venturicidin and sensitivity of growth on nonfermentable substrates to low temperature (19°). Genetic analysis indicates that the phenotype is due to a single mutation located on the mitochondrial DNA which is probably allelic with the independently isolated oligomycin resistance mutation [oli1-r].Growth of the mutant at the non-restrictive temperature (28°) yields mitochondria in which the ATPase appears more sensitive to oligomycin than that of the sensitive parental strain. However, when the enzyme is isolated free from the influence of the membrane strong resistance to oligomycin is evident. These data suggest that the component responsible for the oligomycin resistance of the ATPase is part of or subject to interaction with the mitochondrial inner membrane.Measurements of the ATPase content of mitochondria indicate that ATPase production is impaired during growth at 19° C. In addition, studies of the maximum inhibition of mitochondrial ATPase activity by high concentrations of oligomycin suggest a selective lesion in ATPase assembly at low temperature. The nett result is that during growth at 19° only about 10% of the normal level of ATPase is produced of which less than half is membrane integrated and thus capable of oxidative energy production.We propose that the mutation affects a mitochondrially synthesised membrane sector peptide of the ATPase which defines the interaction of F₁ ATPase with specific environments on the mitochondrial inner membrane.