Heritable epigenetic variation among maize inbreds

Epigenetic variation describes heritable differences that are not attributable to changes in DNA sequence. There is the potential for pure epigenetic variation that occurs in the absence of any genetic change or for more complex situations that involve both genetic and epigenetic differences. Methylation of cytosine residues provides one mechanism for the inheritance of epigenetic information. A genome-wide profiling of DNA methylation in two different genotypes of Zea mays (ssp. mays), an organism with a complex genome of interspersed genes and repetitive elements, allowed the identification and characterization of examples of natural epigenetic variation. The distribution of DNA methylation was profiled using immunoprecipitation of methylated DNA followed by hybridization to a high-density tiling microarray. The comparison of the DNA methylation levels in the two genotypes, B73 and Mo17, allowed for the identification of approximately 700 differentially methylated regions (DMRs). Several of these DMRs occur in genomic regions that are apparently identical by descent in B73 and Mo17 suggesting that they may be examples of pure epigenetic variation. The methylation levels of the DMRs were further studied in a panel of near-isogenic lines to evaluate the stable inheritance of the methylation levels and to assess the contribution of cis- and trans- acting information to natural epigenetic variation. The majority of DMRs that occur in genomic regions without genetic variation are controlled by cis-acting differences and exhibit relatively stable inheritance. This study provides evidence for naturally occurring epigenetic variation in maize, including examples of pure epigenetic variation that is not conditioned by genetic differences. The epigenetic differences are variable within maize populations and exhibit relatively stable trans-generational inheritance. The detected examples of epigenetic variation, including some without tightly linked genetic variation, may contribute to complex trait variation.     

Epistatic interactions in genetic regulation of t-PA and PAI-1 levels in a Ghanaian population

The proteins, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1), act in concert to balance thrombus formation and degradation, thereby modulating the development of arterial thrombosis and excessive bleeding. PAI-1 is upregulated by the renin-angiotensin system (RAS), specifically by angiotensin II, the product of angiotensin converting enzyme (ACE) cleavage of angiotensin I, which is produced by the cleavage of angiotensinogen (AGT) by renin (REN). ACE indirectly stimulates the release of t-PA which, in turn, activates the corresponding fibrinolytic system. Single polymorphisms in these pathways have been shown to significantly impact plasma levels of t-PA and PAI-1 differently in Ghanaian males and females. Here we explore the involvement of epistatic interactions between the same polymorphisms in central genes of the RAS and fibrinolytic systems on plasma t-PA and PAI-1 levels within the same population (n = 992). Statistical modeling of pairwise interactions was done using two-way ANOVA between polymorphisms in the ETNK2, RENIN, ACE, PAI-1, t-PA, and AGT genes. The most significant interactions that associated with t-PA levels were between the ETNK2 A6135G and the REN T9435C polymorphisms in females (p = 0.006) and the REN T9435C and the TPA I/D polymorphisms (p = 0.005) in males. The most significant interactions for PAI-1 levels were with REN T9435C and the TPA I/D polymorphisms (p = 0.001) in females, and the association of REN G6567T with the TPA I/D polymorphisms (p = 0.032) in males. Our results provide evidence for multiple genetic effects that may not be detected using single SNP analysis. Because t-PA and PAI-1 have been implicated in cardiovascular disease these results support the idea that the genetic architecture of cardiovascular disease is complex. Therefore, it is necessary to consider the relationship between interacting polymorphisms of pathway specific genes that predict t-PA and PAI-1 levels.     

Do inhalation general anesthetic drugs induce the neuronal release of endogenous opioid peptides?

The antagonism of some effects of inhalation general anesthetic agents by naloxone suggests that there may be an opioid component to anesthetic action. There is evidence that this opioid action component is due to neuronal release of endogenous opioid peptides. The strongest evidence is provided by studies that monitor changes in the concentration of opioid peptides in the perfused brain following inhalation of the anesthetic. Indirect or circumstantial evidence also comes from studies of anesthetic effects on regional brain levels of opioid peptides, antagonism of selected anesthetic effects by antisera to opioid peptides and anesthetic-induced changes radioligand binding to opioid receptors. It is likely that some inhalation general anesthetics (e.g., nitrous oxide) can induce neuronal release of opioid peptides and that this may contribute to certain components of general anesthesia (e.g., analgesia). More definitive studies utilizing in vivo microdialysis or autoradiography in selected areas of the brain during induction and successive states of general anesthesia have yet to be conducted.     

The Y deletion gr/gr and susceptibility to testicular germ cell tumor

Testicular germ cell tumor (TGCT) is the most common cancer in young men. Despite a considerable familial component to TGCT risk, no genetic change that confers increased risk has been substantiated to date. The human Y chromosome carries a number of genes specifically involved in male germ cell development, and deletion of the AZFc region at Yq11 is the most common known genetic cause of infertility. Recently, a 1.6-Mb deletion of the Y chromosome that removes part of the AZFc region—known as the “gr/gr” deletion—has been associated with infertility. In epidemiological studies, male infertility has shown an association with TGCT that is out of proportion with what can be explained by tumor effects. Thus, we hypothesized that the gr/gr deletion may be associated with TGCT. Using logistic modeling, we analyzed this deletion in a large series of TGCT cases with and without a family history of TGCT. The gr/gr deletion was present in 3.0% (13/431) of TGCT cases with a family history, 2% (28/1,376) of TGCT cases without a family history, and 1.3% (33/2,599) of unaffected males. Presence of the gr/gr deletion was associated with a twofold increased risk of TGCT (adjusted odds ratio [aOR] 2.1; 95% confidence interval [CI] 1.3–3.6; P = .005) and a threefold increased risk of TGCT among patients with a positive family history (aOR 3.2; 95% CI 1.5–6.7; P = .0027). The gr/gr deletion was more strongly associated with seminoma (aOR 3.0; 95% CI 1.6–5.4; P = .0004) than with nonseminoma TGCT (aOR 1.5; 95% CI 0.72–3.0; P = .29). These data indicate that the Y microdeletion gr/gr is a rare, low-penetrance allele that confers susceptibility to TGCT.     

Acetylcholine turnover rates in rat brain regions during cocaine self-administration

The involvement of cholinergic neurons in the brain processes underlying reinforcement has been recently demonstrated. This experiment assessed the potential role of cholinergic neurons in cocaine reinforcement by measuring the turnover rates of acetylcholine in brain regions of rats self-administering cocaine and in yoked cocaine and yoked vehicle-infused controls. The activity of cholinergic innervations of and/or interneurons in the olfactory tubercle, caudate putamen, diagonal band-pre-optic region, ventral pallidum, lateral and medial hypothalamus, hippocampus, ventral tegmental area and visual cortices reflected by the turnover rates of acetylcholine were significantly altered in rats self-administering cocaine compared to yoked cocaine infused controls. These changes implicate the involvement of cholinergic neurons with cell bodies in the diagonal band-pre-optic region, the medial septum and several brainstem nuclei and interneurons in the caudate-putamen and ventral pallidum in the processes underlying cocaine self-administration. The identified cholinergic neuronal systems may have a broader role in the brain processes for natural reinforcers (i.e. food, water, etc.) since drugs of abuse are believed to produce reinforcing effects through these systems.     

Cell detachment model for an antibody-based microfluidic cancer screening system

We consider cells bound to the floor of a microfluidic channel and present a model of their flow-induced detachment. We approximate hydrodynamic force and cell elastic response using static finite-element simulation of a single cell. Detachment is assumed to occur when hydrodynamic and adhesive forces are roughly equal. The result is extended to multiple cells at the device level using a sigmoidal curve fit. The model is applied to a microfluidic cancer-screening device that discriminates between normal epithelial cells and cells infected with human papillomavirus (HPV), on the basis of increased expression of the transmembrane protein alpha6 integrin in the latter. Here, the cells to be tested are bound to a microchannel floor coated with anti alpha6 integrin antibodies. In an appropriate flow rate range, normal cells are washed away while HPV-infected cells remain bound. The model allows interpolation between data points to choose the optimal flow rate and provides insight into interaction of cell mechanical properties and the flow-induced detachment mechanism. Notably, the results suggest a significant influence of cell elastic response on detachment.     

Further differentiation of murine double-positive thymocytes is inhibited in adenosine deaminase-deficient murine fetal thymic organ culture

Murine fetal thymic organ culture (FTOC) was used to investigate the mechanism by which a lack of adenosine deaminase (ADA) leads to a failure of T cell production in the thymus. We previously showed that T cell development was inhibited beginning at the CD4(-)CD8(-)CD25(+)CD44(low) stage in ADA-deficient FTOC initiated at day 15 of gestation when essentially all thymocytes are CD4(-)CD8(-). In the present study, we asked whether thymocytes at later stages of differentiation would also be sensitive to ADA inhibition by initiating FTOC when substantial numbers of CD4(+)CD8(+) thymocytes were already present. dATP was highly elevated in ADA-deficient cultures, and the recovery of alphabeta TCR(+) thymocytes was inhibited by 94%, indicating that the later stages of thymocyte differentiation are also dependent upon ADA. ADA-deficient cultures were partially rescued by the pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or by the use of apoptotic protease-activating factor-1-deficient mice. Rescue was even more dramatic, with 60- to >200-fold increases in the numbers of CD4(+)CD8(+) cells, when FTOC were performed with an inhibitor of adenosine kinase, the major thymic deoxyadenosine phosphorylating enzyme, or with bcl-2 transgenic mice. dATP levels were normalized by treatment with either carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or an adenosine kinase inhibitor, but not in cultures with fetal thymuses from bcl-2 transgenic mice. These data suggest that ADA deficiency leads to the induction of mitochondria-dependent apoptosis as a consequence of the accumulation of dATP derived from thymocytes failing the positive/negative selection checkpoint.     

Structural and immunocytochemical characterization of the Ginkgo biloba L. sperm motility apparatus

Summary. Ginkgo biloba and the cycads are the only extant seed plants with motile sperm cells. However, there has been no immunocytochemical characterization of these gametes to determine if they share characteristics with the flagellated sperm found in bryophytes and pteridophytes or might give clues as to the relationships to nonflagellated sperm in all other seed plants. To determine characteristics of proteins associated with the motility apparatus in these motile sperm, we probed thin sections of developing spermatogenous cells of Ginkgo biloba with antibodies to acetylated and tyrosinated tubulin and monoclonal antibodies that recognize mammalian centrosomes and centrin. The blepharoplast that occurs as a precursor to the motility apparatus consists of an amorphous core, pitted with cavities containing microtubules and a surface studded with probasal bodies. The probasal bodies and microtubules within the blepharoplast cavities are labeled with antibodies specific to acetylated tubulin. Positive but weak reactions of the blepharoplast core occur with the centrosomereactive antibodies MPM-2 and C-9. Reactions to centrin antibodies are negative at this developmental stage. From this pre-motility apparatus structure, an assemblage of about 1000 flagella and associated structures arises as the precursor to the motility apparatus for the sperm. The flagellar apparatus consists of a three-layered multilayered structure that subtends a layer of spline microtubules, a zone of amorphous material similar to that in the blepharoplast, and the flagellar band. Centrin antibodies react strongly with the multilayered structure, the transition zone of the flagella, and fibrillar material near the flagellar base at the surface of the amorphous material. Both the spline microtubules and all of the tubules in the flagella react strongly with the antibodies to acetylated tubulin. These localizations are consistent with the localizations of these components in pteridophyte and bryophyte spermatogenous cells, although the blepharoplast material surrounding and connecting flagellar bases does not occur in the seedless (nonseed) land plants. These data indicate that despite the large size of ginkgo gametes and the taxonomic separation between pteridophytes and Ginkgo biloba, similar proteins in gametes of both groups perform similar functions and are therefore homologous among these plants. Moreover, the presence of acetylated tubulin in bands of microtubules may be a characteristic shared with more derived non-flagellated sperm of other conifers and angiosperms. Correspondence and reprints: Southern Weed Science Research Unit, USDA Agricultural Research Service, P.O. Box 350, Stoneville, MS 38776, U.S.A.     

Lipid headgroup superlattice modulates the activity of surface-acting cholesterol oxidase in ternary phospholipid/cholesterol bilayers

The relationship between the molecular organization of lipid headgroups and the activity of surface-acting enzyme was examined using a bacterial cholesterol oxidase (COD) as a model. The initial rate of cholesterol oxidation by COD in fluid state 1-palmitoyl-2-oleoyl-phosphatidylethanolamine/1-palmitoyl-2-oleoyl-phosphatidylcholine/cholesterol (POPE/POPC/CHOL) bilayers was measured as a function of POPE-to-phospholipid mole ratio (X(PE)) and cholesterol-to-lipid mole ratio (X(CHOL)) at 37 degrees C. At X(PE) = 0, the COD activity changed abruptly at X(CHOL) approximately 0.40, whereas major activity peaks were detected at X(PE) approximately 0.18, 0.32, 0.50, 0.64, and 0.73 when X(CHOL) was fixed to 0.33 or 0.40. At a fixed X(CHOL) of 0.50, the COD activity increased progressively with PE content and exhibited small peaks or kinks at X(PE) approximately 0.40, 0.50, 0.58, 0.69, and 0.81. When X(PE) and X(CHOL) were systematically varied within a narrow 2-D lipid composition window, an onset of COD activity at X(CHOL) approximately 0.40 and the elimination of the activity peak at X(PE) approximately 0.64 for X(CHOL) >0.40 were clearly observed. Except for X(PE) approximately 0.40 and 0.58, the observed critical PE mole ratios agree closely (+/-0.03) with those predicted by a headgroup superlattice model (Virtanen, J.A., et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4964-4969; Cannon, B., et al. (2006) J. Phys. Chem. B 110, 6339-6350), which proposes that lipids with headgroups of different sizes tend to adopt regular, superlattice-like distributions at discrete and predictable compositions in fluid lipid bilayers. Our results indicate that headgroup superlattice domains exist in lipid bilayers and that they may play a crucial role in modulating the activity of enzymes acting on the cell membrane surface.