Epigenetic mechanisms in neurological disease

The exploration of brain epigenomes, which consist of various types of DNA methylation and covalent histone modifications, is providing new and unprecedented insights into the mechanisms of neural development, neurological disease and aging. Traditionally, chromatin defects in the brain were considered static lesions of early development that occurred in the context of rare genetic syndromes, but it is now clear that mutations and maladaptations of the epigenetic machinery cover a much wider continuum that includes adult-onset neurodegenerative disease. Here, we describe how recent advances in neuroepigenetics have contributed to an improved mechanistic understanding of developmental and degenerative brain disorders, and we discuss how they could influence the development of future therapies for these conditions.     

JAM-A is both essential and inhibitory to development of hepatic polarity in WIF-B cells

Junctional adhesion molecule (JAM) is involved in tight junction (TJ) formation in epithelial cells. Three JAMs (A, B, and C) are expressed in rat hepatocytes, but only rat JAM-A is present in polarized WIF-B cells, a rat-human hepatic line. We used knockdown (KD) and overexpression in WIF-B cells to determine the role of JAM-A in the development of hepatic polarity. Expression of rat JAM-A short hairpin RNA resulted in approximately 50% KD of JAM-A and substantial loss of hepatic polarity, as measured by the absence of apical cysts formed by adjacent cells and sealed by TJ belts. When inhibitory RNA-resistant human JAM-A (huWT) was expressed in KD cells, hepatic polarity was restored. In contrast, expression of JAM-A that either lacked its PDZ-binding motif (huDeltaC-term) or harbored a point mutation (T273A) did not complement, indicating that multiple sites within JAM-A's cytoplasmic tail are required for the development of hepatic polarity. Overexpression of huWT in normal WIF-B cells unexpectedly blocked WIF-B maturation to the hepatic phenotype, as did expression of three huJAM-A constructs with single point mutations in putative phosphorylation sites. In contrast, huDeltaC-term was without effect, and the T273A mutant only partially blocked maturation. Our results show that JAM-A is essential for the development of polarity in cultured hepatic cells via its possible phosphorylation and recruitment of relevant PDZ proteins and that hepatic polarity is achieved within a narrow range of JAM-A expression levels. Importantly, formation/maintenance of TJs and the apical domain in hepatic cells are linked, unlike simple epithelia.     

Membrane environment exerts an important influence on rac-mediated activation of phospholipase Cγ2

We performed analyses of the molecular mechanisms involved in the regulation of phospholipase Cγ2 (PLCγ2). We identified several regions in the PLCγ-specific array, γSA, that contribute to autoinhibition in the basal state by occlusion of the catalytic domain. While the activation of PLCγ2 by Rac2 requires stable translocation to the membrane, the removal of the domains required for membrane translocation in the context of an enzyme with impaired autoinhibition generated constitutive, highly active PLC in cells. We further tested the possibility that the interaction of PLCγ2 with its activator protein Rac2 was sufficient for activation through the release of autoinhibition. However, we found that Rac2 binding in the absence of lipid surfaces was not able to activate PLCγ2. Together with other observations, these data suggest that an important consequence of Rac2 binding and translocation to the membrane is that membrane proximity, on its own or together with Rac2, has a role in the release of autoinhibition, resulting in interfacial activation.     

Changes in angiotensin receptors expression play a pivotal role in the renal damage observed in spontaneously hypertensive rats

The renal renin-angiotensin system plays a central role in the development of hypertension. The aim of this work was to verify the expression of angiotensin II receptors AT(1)R and AT(2)R in the microsomal fraction of renal cortex and correlate this with the development of hypertension and renal damage in spontaneously hypertensive rats (SHR) using Wistar-Kyoto rats (WKY) as controls. AT(1)R expression increased (126%) and AT(2)R expression decreased (66%) in 4-wk-old SHR; AT(2) expression decreased in 14-wk-old SHR (61%) compared with respective age-matched WKY. These modifications were correlated to the increase in protein kinase C activity and decrease in protein kinase A activity. Four-week-old SHR showed large accumulations of macrophages in kidney glomerulus and the tubulointerstitial area, dense cortical collagen deposition, and arterial proliferative changes in the walls of arterioles and medium-sized vessels. Similar modifications were also observed in 14-wk-old SHR. Four-week-old SHR treated with losartan (30 mg·kg(-1)·day(-1)) or hydralazine (15 and 30 mg·kg(-1)·day(-1)) by gavage for 10 wk did not develop hypertension. The decrease in AT(2)R expression and renal damage observed in SHR remained even after treatment with hydralazine. On the other hand, losartan treatment prevented the modifications observed in 14-wk-old SHR, indicating that renal injuries are caused specifically by AT(1) rather than an increase in blood pressure. Our results indicate that the imbalance in AT(1)R and AT(2)R expression is associated with an inflammatory process that contributes to renal injury in adult SHR and to the development of hypertension.     

Tyrosine residues in phospholipase Cgamma 2 essential for the enzyme function in B-cell signaling

Phospholipase Cgamma (PLCgamma) isoforms are regulated through activation of tyrosine kinase-linked receptors. The importance of growth factor-stimulated phosphorylation of specific tyrosine residues has been documented for PLCgamma1; however, despite the critical importance of PLCgamma2 in B-cell signal transduction, neither the tyrosine kinase(s) that directly phosphorylate PLCgamma2 nor the sites in PLCgamma2 that become phosphorylated after stimulation are known. By measuring the ability of human PLCgamma2 to restore calcium responses to the B-cell receptor stimulation or oxidative stress in a B-cell line (DT40) deficient in PLCgamma2, we have demonstrated that two tyrosine residues, Tyr(753) and Tyr(759), were important for the PLCgamma2 signaling function. Furthermore, the double mutation Y753F/Y759F in PLCgamma2 resulted in a loss of tyrosine phosphorylation in stimulated DT40 cells. Of the two kinases that previously have been proposed to phosphorylate PLCgamma2, Btk, and Syk, purified Btk had much greater ability to phosphorylate recombinant PLCgamma2 in vitro, whereas Syk efficiently phosphorylated adapter protein BLNK. Using purified proteins to analyze the formation of complexes, we suggest that function of Syk is to phosphorylate BLNK, providing binding sites for PLCgamma2. Further analysis of PLCgamma2 tyrosine residues phosphorylated by Btk and several kinases from the Src family has suggested multiple sites of phosphorylation and, in the context of a peptide incorporating residues Tyr(753) and Tyr(759), shown preferential phosphorylation of Tyr(753).     

A rapid method for the determination of microbial biomass by dry weight using a moisture analyser with an infrared heating source and an analytical balance

Aims: Microbial biomass is an important biotechnological parameter. The traditional method for its determination involves an oven‐drying step and equilibration to room temperature before weighing, and it is tedious and time consuming. This work studied the utilisation of a moisture analyser consisting of an efficient infrared‐heating module and an analytical balance for the determination of microbial biomass by dry weight. Methods and Results: The method duration depended on the sample volume and was between 7 and 40 min for sample volumes of 1–10 ml. The method precision depended on the total dry weight analysed – 10 mg of total dry weight being sufficient to achieve coefficients of variation of 5% or less. Comparison with the conventional oven method provided a correlation coefficient r² of 0·99. The recovery of an internal standard ranged between 94·2 and 106·4% with a precision of 1·39–4·53%CV. Conclusions: Validation revealed sufficient method accuracy, precision and robustness and was successfully applied to the study of yeast and bacterial growth kinetics. Techniques are discussed that allow for increased method precision at low biomass concentrations, and equations are provided to estimate required drying time and method precision based on sample volume and total sample dry weight, respectively. Significance and Impact of the Study: This work presents a rapid method for the determination of microbial biomass, allowing for the timely implementation of biomass‐based information in biotechnological and laboratory protocols.     

rac regulates its effector phospholipase Cgamma2 through interaction with a split pleckstrin homology domain

Several isoforms of phospholipase C (PLC) are regulated through interactions with Ras superfamily GTPases, including Rac proteins. Interestingly, of two closely related PLCgamma isoforms, only PLCgamma(2) has previously been shown to be activated by Rac. Here, we explore the molecular basis of this interaction as well as the structural properties of PLCgamma(2) required for activation. Based on reconstitution experiments with isolated PLCgamma variants and Rac2, we show that an unusual pleckstrin homology (PH) domain, designated as the split PH domain (spPH), is both necessary and sufficient to effect activation of PLCgamma(2) by Rac2. We also demonstrate that Rac2 directly binds to PLCgamma(2) as well as to the isolated spPH of this isoform. Furthermore, through the use of NMR spectroscopy and mutational analysis, we determine the structure of spPH, define the structural features of spPH required for Rac interaction, and identify critical amino acid residues at the interaction interface. We further discuss parallels and differences between PLCgamma(1) and PLCgamma(2) and the implications of our findings for their respective signaling roles.     

The fission yeast S-phase cyclin Cig2 can drive mitosis

Commitment to mitosis is regulated by cyclin-dependent kinase (CDK) activity. In the fission yeast Schizosaccharomyces pombe, the major B-type cyclin, Cdc13, is necessary and sufficient to drive mitotic entry. Furthermore, Cdc13 is also sufficient to drive S phase, demonstrating that a single cyclin can regulate alternating rounds of replication and mitosis, and providing the foundation of the quantitative model of CDK function. It has been assumed that Cig2, a B-type cyclin expressed only during S phase and incapable of driving mitosis in wild-type cells, was specialized for S-phase regulation. Here, we show that Cig2 is capable of driving mitosis. Cig2/CDK activity drives mitotic catastrophe—lethal mitosis in inviably small cells—in cells that lack CDK inhibition by tyrosine-phosphorylation. Moreover, Cig2/CDK can drive mitosis in the absence of Cdc13/CDK activity and constitutive expression of Cig2 can rescue loss of Cdc13 activity. These results demonstrate that in fission yeast, not only can the presumptive M-phase cyclin drive S phase, but the presumptive S-phase cyclin can drive M phase, further supporting the quantitative model of CDK function. Furthermore, these results provide an explanation, previously proposed on the basis of computational analyses, for the surprising observation that cells expressing a single-chain Cdc13-Cdc2 CDK do not require Y15 phosphorylation for viability. Their viability is due to the fact that in such cells, which lack Cig2/CDK complexes, Cdc13/CDK activity is unable to drive mitotic catastrophe.     

The cytochrome bc) complex of yeast mitochondria. Site of translation of the polypeptides in vivo.

1.Yeast cells were labelled with radioactive amino acids in the presence of cycloheximide and the cytochrome bc1 complex was isolated from them as described in the preceding paper (Katan, M.B.., Pool, L. & Groot, G.S.P. (1976)Eur. J. Biochem, 65, 95-105). After analysis of this preparation by sodium dodecylsulphate polyacrylamide gel electrophoresis only one band, with an apparent Mr of 32000, was found to have incorporated radioactivity. The amount of label in the band was low, but could be increased approximately 5-fold by preincubating the cells in erythromycin before the labelling period. 2. Cells were labelled in the presence of chloramphenicol and the cytochrome bc1 complex was isolated by (NH4)2SO4 fractionation. Upon electrophoresis in the presence of sodium dodecylsulphate only four of the six bands that belong to the complex were found to have incorporated radioactivity; no radioactivity was found in the bands with an Mr of 40000 and 17000. The same result was obtained after labelling in the presence of acriflavin. If, however, the cytochrome bc1 complex was isolated by immunoprecipitation, all bands were found to have incorporated radioactivity in the presence of chloramphenicol. The amount of radioactivity in the Mr 32000 band was now clearly depressed. 3. It is concluded that of the seven polypeptides of the cytochrome bc1 complex of yeast only one is made on mitochondrial ribosomes. This polypeptide has an Mr of 32000 and is probably associated with cytochrome b.