The replacement histone H2A.Z in a hyperacetylated form is a feature of active genes in the chicken

The replacement histone H2A.Z is variously reported as being linked to gene expression and preventing the spread of heterochromatin in yeast, or concentrated at heterochromatin in mammals. To resolve this apparent dichotomy, affinity-purified antibodies against the N-terminal region of H2A.Z, in both a triacetylated and non-acetylated state, are used in native chromatin immmuno-precipitation experiments with mononucleosomes from three chicken cell types. The hyperacetylated species concentrates at the 5′ end of active genes, both tissue specific and housekeeping but is absent from inactive genes, while the unacetylated form is absent from both active and inactive genes. A concentration of H2A.Z is also found at insulators under circumstances implying a link to barrier activity but not to enhancer blocking. Although acetylated H2A.Z is widespread throughout the interphase genome, at mitosis its acetylation is erased, the unmodified form remaining. Thus, although H2A.Z may operate as an epigenetic marker for active genes, its N-terminal acetylation does not.     

Binding of beta-D-glucopyranosyl bismethoxyphosphoramidate to glycogen phosphorylase b: kinetic and crystallographic studies

In an attempt to identify a new lead molecule that would enable the design of inhibitors with enhanced affinity for glycogen phosphorylase (GP), beta-D-glucopyranosyl bismethoxyphosphoramidate (phosphoramidate), a glucosyl phosphate analogue, was tested for inhibition of the enzyme. Kinetic experiments showed that the compound was a weak competitive inhibitor of rabbit muscle GPb (with respect to alpha-D-glucose-1-phosphate (Glc-1-P)) with a Ki value of 5.9 (+/-0.1) mM. In order to elucidate the structural basis of inhibition, we determined the structure of GPb complexed with the phosphoramidate at 1.83 A resolution. The complex structure reveals that the inhibitor binds at the catalytic site and induces significant conformational changes in the vicinity of this site. In particular, the 280s loop (residues 282-287) shifts 0.4-4.3 A (main-chain atoms) to accommodate the phosphoramidate, but these conformational changes do not lead to increased contacts between the inhibitor and the protein that would improve ligand binding.     

Glutathione preconditioning attenuates Ac-LDL-induced macrophage apoptosis via protein kinase C-dependent Ac-LDL trafficking

Oxidized low-density lipoprotein (ox-LDL) incorporation into intimally resident vascular cells via scavenger receptors marks one of the early steps in atherosclerosis. Cellular apoptotic damage results from two major serial intracellular events: the binding and scavenger receptor-mediated uptake of oxidizable lipoproteins and the intracellular oxidative responses of accumulated lipoproteins. Most molecular approaches to prevent apoptotic damage have focused on singular events within the cascade of lipoprotein trafficking. To identify a multifocal strategy against LDL-induced apoptosis, we evaluated the role of cellular preconditioning by glutathione-ethyl ester (GSH-Et), a native redox regulator, in the prevention of the uptake and apoptotic effects of an oxidizable scavenger receptor-specific ligand, acetylated low-density lipoprotein (Ac-LDL). Our results indicate that GSH-Et-mediated protein kinase C (PKC) pathway modulation regulates Ac-LDL binding and incorporation into GSH-Et preconditioned cells and subsequently delays reactive oxygen intermediate generation and apoptotic conversion. The GSH-Et protective effects on apoptosis and Ac-LDL binding were reversed by calphostin C, a PKC inhibitor, and were accompanied by an increase in PKC phosphorylation. However, the rate of reactive oxygen intermediate accumulation was not increased following calphostin C treatment, suggesting that GSH-Et may play an important nonreactive oxygen-intermediate-based protective role in regulating apoptotic dynamics. Overall, we report on the novel role for GSH-Et preconditioning as a molecular strategy to limit lipoprotein entry into the cells, which presents a proactive modality to prevent cellular apoptosis in contrast with the prevalent antioxidant approaches that treat damage retroactively.     

Characterization of heparin affin regulatory peptide signaling in human endothelial cells

Heparin affin regulatory peptide (HARP) is an 18-kDa secreted growth factor that has a high affinity for heparin and a potent role on tumor growth and angiogenesis. We have previously reported that HARP is mitogenic for different types of endothelial cells and also affects cell migration and differentiation (12). In this study we examined the signaling pathways involved in the migration and tube formation on matrigel of human umbilical vein endothelial cells (HUVEC) induced by HARP. We report for the first time that receptor-type protein-tyrosine phosphatase beta/zeta (RPTPbeta/zeta), which is a receptor for HARP in neuronal cell types, is also expressed in HUVEC. We also document that HARP signaling through RPTPbeta/zeta leads to activation of Src kinase, focal adhesion kinase, phosphatidylinositol 3-kinase, and Erk1/2. Sodium orthovanadate, chondroitin sulfate-C, PP1, wortmannin, LY294002, and U0126 inhibit HARP-mediated signaling and HUVEC migration and tube formation. In addition, RPTPbeta/zeta suppression using small interfering RNA technology interrupts intracellular signals and HUVEC migration and tube formation induced by HARP. These results establish the role of RPTPbeta/zeta as a receptor of HARP in HUVEC and elucidate the HARP signaling pathway in endothelial cells.     

Energy intake, diet composition, energy expenditure, and body fatness of adolescents in northern Greece

Objective: The purpose of this study was to examine energy intake, energy expenditure, diet composition, and obesity of adolescents in Northern Greece. Research Methods and Procedures: Anthropometric measurements were taken for all participants. Height, weight, and skinfold thickness at two sites were measured. BMI and percentage body fat were calculated. Energy intake and macronutrient and micronutrient intakes were determined by a 3‐day weighed dietary diary. Energy expenditure was calculated based on calculated resting metabolic rate (RMR) 1 multiplied by an activity factor based on reported physical activity. Results: Thirty‐one percent of boys and 21% of girls had BMI corresponding to ≥25 kg/m² at 18 years and were classified as overweight. Both overweight boys and girls reported a lower energy intake compared with their non‐overweight counterparts when expressed as kilocalories per kilogram body weight. Overweight children had a higher negative energy balance. Both overweight and non‐overweight adolescents had higher than recommended fat intakes. Mean daily carbohydrate, protein, and fat intake, expressed as grams per kilogram body weight, of overweight adolescents were significantly lower compared with the non‐overweight adolescents. Total daily carbohydrate intake, when expressed in grams, was found to be higher for non‐overweight adolescents. Both overweight boys and girls had lower iron intakes than their non‐overweight counterparts. Overweight boys had statistically lower fiber and niacin intakes than non‐overweight boys. Both overweight and non‐overweight adolescents had lower than recommended iron intakes. Furthermore, overweight adolescents consumed more snacks (potato chips, chocolate bars, pizza, cheese pie, and cream pie), more sugar, jam, and honey, and fewer legumes, vegetables, and fruits than their non‐overweight counterparts. Discussion: Reported energy intake of overweight adolescents was lower than their non‐overweight counterparts. Regarding diet composition overweight subjects had significantly lower intakes of carbohydrates compared with non‐overweight subjects. The food consumption pattern of overweight children showed less adherence to the traditional Mediterranean diet.     

Effect of pH on the structure and stability of Bacillus circulans ssp. alkalophilus phosphoserine aminotransferase: thermodynamic and crystallographic studies

pH is one of the key parameters that affect the stability and function of proteins. We have studied the effect of pH on the pyridoxal-5'-phosphate-dependent enzyme phosphoserine aminotransferase produced by the facultative alkaliphile Bacillus circulans ssp. alkalophilus using thermodynamic and crystallographic analysis. Enzymatic activity assay showed that the enzyme has maximum activity at pH 9.0 and relative activity less than 10% at pH 7.0. Differential scanning calorimetry and circular dichroism experiments revealed variations in the stability and denaturation profiles of the enzyme at different pHs. Most importantly, release of pyridoxal-5'-phosphate and protein thermal denaturation were found to occur simultaneously at pH 6.0 in contrast to pH 8.5 where denaturation preceded cofactor's release by approximately 3 degrees C. To correlate the observed differences in thermal denaturation with structural features, the crystal structure of phosphoserine aminotransferase was determined at 1.2 and 1.5 A resolution at two different pHs (8.5 and 4.6, respectively). Analysis of the two structures revealed changes in the vicinity of the active site and in surface residues. A conformational change in a loop involved in substrate binding at the entrance of the active site has been identified upon pH change. Moreover, the number of intramolecular ion pairs was found reduced in the pH 4.6 structure. Taken together, the presented kinetics, thermal denaturation, and crystallographic data demonstrate a potential role of the active site in unfolding and suggest that subtle but structurally significant conformational rearrangements are involved in the stability and integrity of phosphoserine aminotransferase in response to pH changes.     

Leishmania histone H1 overexpression delays parasite cell-cycle progression, parasite differentiation and reduces Leishmania infectivity in vivo

Episomal expression of Leishmania histone H1 sense mRNAs in Leishmania major promastigotes was found previously to result in overexpression of this molecule and to reduce parasite infectivity in vitro. Herein, we evaluated the in vivo infectivity of these transfectants, in BALB/c mice, and showed that it is dramatically reduced. No lesions were observed in this group of mice and this was associated with an extremely low number of parasites both in the footpad and in the draining lymph nodes. Interestingly, the transfectants-reduced infectivity was associated with a delay in their cell-cycle progression and differentiation to axenic amastigotes, assessed in vitro. Therefore, the dramatic reduction in their infectivity may be attributed to the above-mentioned phenotypic modifications. As the metazoan linker histone H1(0) homologue is known to delay cell-cycle progression in mammalian cells we investigated whether its Leishmania counterpart, which possesses homology to its C-terminal region, when expressed in mammalian cells may also affect their cell-cycle progression. It was thus shown that Leishmania histone H1 expressed in COS7 and NIH 3T3 cells, delays cell-cycle progression in these cells too. The latter strengthens the phenotype observed in Leishmania and provides evidence that critical functions of histone H1 molecules are conserved throughout evolution.     

The low-density lipoprotein (LDL)-associated PAF-acetylhydrolase activity and the extent of LDL oxidation are important determinants of the autoantibody titers against oxidized LDL in patients with coronary artery disease

The oxidation of low-density lipoprotein (LDL) induces immunogenic epitopes, many of which are due to oxidatively modified phospholipids (oxPL). Lysophosphatidylcholine (lyso-PC) which is generated during LDL oxidation through the hydrolysis of oxPL by LDL-associated PAF-acetylhydrolase (PAF-AH) is also immunogenic. We investigated whether the LDL-associated PAF-AH and the extent of LDL oxidation influence the autoantibody titers against oxidized LDL (oxLDL) in patients with stable angina as well as in apparently healthy volunteers. Three types of copper-oxidized LDL, were prepared at the end of the lag, propagation or decomposition phase (oxLDL(L), oxLDL(P) and oxLDL(D), respectively). Similar types of oxidized LDL were prepared after previous inactivation of endogenous PAF-AH [oxLDL(-)]. All these types of oxLDL as well as malondialdehyde-modified LDL (MDA-LDL) were used as antigens. Antibody titers against the above antigens were measured with an ELISA method in the serum of 65 patients with stable angina and 47 apparently healthy volunteers. Both groups exhibited higher autoantibody titers against each type of oxLDL(-) compared to the respective type of oxLDL (P<0.00001). In both groups autoantibody titers were higher when the oxLDL(P) and oxLDL(D) or oxLDL(-)(P) and oxLDL(-)(D) were used as antigens compared to oxLDL(L) (P<0.04) or to oxLDL(-)(L), respectively (P<0.0001 for all comparisons). Patients had significantly higher titers against all types of oxLDL (enriched in lyso-PC) and oxLDL(-) (enriched in intact oxPL) compared to controls. Autoantibody titers against MDA-LDL did not differ between patients and controls. Multivariate logistic regression analysis showed that among the autoantibody titers measured only those towards oxLDL(P) are associated with a significantly higher risk for coronary artery disease. LDL-associated PAF-AH activity may play an important role in decreasing the overall immunogenicity of oxLDL, whereas the extent of LDL oxidation seems to modulate the epitopes formed on oxLDL. Lyso-PC, a major component of oxLDL(P), could be mainly responsible for the elevated autoantibody titers against oxLDL in patients with stable angina.     

Regulation of cAMP homeostasis by the efflux protein MRP4 in cardiac myocytes

Recent studies indicate that members of the multidrug-resistance protein (MRP) family belonging to ATP binding cassette type C (ABCC) membrane proteins extrude cyclic nucleotides from various cell types. This study aimed to determine whether MRP proteins regulate cardiac cAMP homeostasis. Here, we demonstrate that MRP4 is the predominant isoform present at the plasma membrane of cardiacmyocytes and that it mediates the efflux of cAMP in these cells. MRP4-deficient mice displayed enhanced cardiac myocyte cAMP formation, contractility, and cardiac hypertrophy at 9 mo of age, an effect that was compensated transiently by increased phosphodiesterase expression at young age. These findings suggest that cAMP extrusion via MRP4 acts together with phosphodiesterases to control cAMP levels in cardiac myocytes.