Apolipoprotein A5 and hypertriglyceridemia in Prague hypertriglyceridemic rats

High plasma triglyceride (TG) level is a major independent risk factor of coronary heart disease. A newly identified Apolipoprotein A5 (Apoa5) gene has been shown to play an important role in determining plasma TG concentrations in humans and mice. Prague hereditary hypertriglyceridemic (HTG) rats are a useful model of human hypertriglyceridemia and other symptoms of metabolic syndrome. Thus, the variation of Apoa5 gene and its expression were studied in this strain under normal conditions and after chronic fructose loading. Lewis and Wistar rats served as normotriglyceridemic controls. Plasma TG were significantly higher in HTG rats in comparison with both control strains. Screening of the coding regions and intron-exon boundaries of Apoa5 gene did not reveal any mutation of this gene in HTG rats in comparison with Lewis and Wistar ones. However, rat Apoa5 gene contains only one intron in contrast with two introns of mouse Apoa5 gene. Under the basal conditions the expression of Apoa5 was lower in all age groups of HTG rats compared to Wistar animals. Furthermore, during chronic fructose loading there were no significant changes of Apoa5 expression in HTG rats, although plasma TG levels rose 3-4 times within first two days of fructose loading and were increased during the whole period of fructose treatment. In conclusion, Apoa5 does not seem to be a genetic determinant of hypertriglyceridemia in HTG rats. The absence of significant changes in Apoa5 gene expression during chronic fructose-induced TG elevation excludes its major role in mechanisms compensating severe hypertriglyceridemia.     

Systemic blood pressure response to the inhibition of two hyperpolarizing pathways: a comparison to NO-synthase inhibition

The impact on blood pressure of two vasodilating mechanisms, underlied by vascular smooth muscle hyperpolarization, was studied and compared to that induced by nitric oxide NO mechanism. Systemic blood pressure, after inhibitory intervention in arachidonic acid metabolism cytochrome P-450 inhibition by miconazole 0.5 mg/100 g b.w. , one of the hyperpolarizing pathways, did not change. After the inhibition of the action voltage-dependent K(+) channels operator by 4-aminopyridine 0.1 mg/100 g b.w. , the other hyperpolarizing pathway, blood pressure declined slightly from 132.3+/-3.2 mm Hg to 116.5+/-5.0 mm Hg, P<0.05 . Inhibition of nitric oxide production L-NAME 5 mg/100 g b.w. increased blood pressure considerably 123.5+/-2.7 mm Hg to 155.4+/-3.1 mm Hg, P<0.001 . After inhibition of the hyperpolarizing pathway by miconazole, hypotension induced by acetylcholine (Ach, 10 microg represented 63.0+/-1.9 mm Hg vs control value 78.6+/-5.2 mm Hg P<0.001 , by bradykinin (BK) 100 microg 59.4+/-3.9 mm Hg vs control value 71.2+/-6.1 mm Hg P<0.05 . After inhibition of the hyperpolarizing pathway by 4-aminopyridine, hypotension induced by ACh 10 microg achieved 64.6+/-2.5 mm Hg vs control value 78.4+/-2.8 mm Hg P<0.001 and that induced by BK 100 microg 56.6+/-5.3 mm Hg vs control value 72.3+/-2.5 mm Hg P<0.001 . ACh or BK hypotension after the inhibition of the above hyperpolarizing pathways was significantly attenuated. On the contrary, after NO-synthase inhibition the hypotension to ACh was significantly enhanced. Blood pressure decrease after ACh 10 microg hypotension was 91.8+/-4.1 mm Hg vs control value 79.3+/-3.3 mm Hg P<0.01 , and after BK 100 microg it was 78.4+/-7.1 mm Hg vs control value 68.3+/-5.2 mm Hg. A different basal BP response, but equally attenuated hypotension to Ach and BK, was detected after the inhibition of two selected hyperpolarizing pathways. In cotrast, the inhibition of NO production elicited an increase in systemic BP and augmentation of ACh and BK hypotension. The effectiveness of further hyperpolarizing mechanisms in relation to systemic BP regulation and nitric oxide level remains open.     

Blood phagocyte activation during open heart surgery with cardiopulmonary bypass

Open heart surgery with a cardiopulmonary bypass (CPB) is associated with a systemic inflammatory response which significantly contributes to adverse postoperative complications. The purpose of this study was to characterize the activation of blood phagocytes during open heart surgery with CPB. Blood samples were collected during and up to 24 h after surgery. The production of reactive oxygen species (ROS) in whole blood, the expression of surface molecules by blood phagocytes and complement activity in the plasma were determined. A cDNA microarray analysis of leukocyte RNA profile of genes was performed related to the inflammatory response. Activation of the complement was already observed at the beginning of CPB. This was followed by an increase in the neutrophil number and in both spontaneous and opsonized zymosan-activated ROS production after the onset of reperfusion. The activation of blood phagocytes was affirmed by changes in surface receptors involved in the adhesion and migration of leukocytes (CD11b, CD62L and CD31). Gene arrays also confirmed the activation of leukocytes 4 h after reperfusion. In conclusion, open heart surgery with a cardiopulmonary bypass was found to be associated with a rapid and pronounced activation of blood phagocytes and complement activation which was partly independent at the onset of CPB.     

Baroreflex sensitivity as an individual characteristic feature

The reproducibility of baroreflex sensitivity (BRS in ms/mmHg; BRSf in mHz/mmHg) determined with respect to the coherence between the variability in systolic blood pressure (SBP) and inter-beat intervals (IBI) or heart rate (HR) was tested. SBP and IBI were recorded beat-to-beat for 5 min (Finapres, breathing at 0.33 Hz) in 116 subjects (aged 19-24 years) sitting at rest three times in periods of one week. BRS and BRSf was determined by a cross-spectral method in a frequency range of 0.067-0.133 Hz. Eight indices were evaluated: BRS(0.1 Hz) /BRSf(0.1 Hz) - the value at a frequency of 0.1 Hz; BRS(COHmax)/BRSf(COHmax) - the value at maximum coherence; BRS(Wcoh)/BRSf - weighted value with respect to coherence values in the whole frequency range; BRS(WPcoh)/BRS(WPcoh) - weighted value with respect to coherence for frequencies with coherence above 0.5. All indices revealed a lower intraindividual than interindividual variability (p<0.001). The individual mean values of BRS or BRSf correlated (p<0.001) with standard deviation of their individual values for all indices. Baroreflex sensitivity is an individual characteristic feature with the highest reproducibility at its low values in spite of its resting variation. Reproducibility is not influenced by modification of the spectral method used.     

Construction, database integration, and application of an Oenothera EST library

Coevolution of cellular genetic compartments is a fundamental aspect in eukaryotic genome evolution that becomes apparent in serious developmental disturbances after interspecific organelle exchanges. The genus Oenothera represents a unique, at present the only available, resource to study the role of the compartmentalized plant genome in diversification of populations and speciation processes. An integrated approach involving cDNA cloning, EST sequencing, and bioinformatic data mining was chosen using Oenothera elata with the genetic constitution nuclear genome AA with plastome type I. The Gene Ontology system grouped 1621 unique gene products into 17 different functional categories. Application of arrays generated from a selected fraction of ESTs revealed significantly differing expression profiles among closely related Oenothera species possessing the potential to generate fertile and incompatible plastid/nuclear hybrids (hybrid bleaching). Furthermore, the EST library provides a valuable source of PCR-based polymorphic molecular markers that are instrumental for genotyping and molecular mapping approaches.     

Lipid oxidation and autophagy in yeast

Autophagy, a process involved in the degradation and the recycling of long-lived proteins and organelles to survive nitrogen starvation, is generally non-selective. However, recent data suggest that selective forms of autophagy exist, that are able to specifically target several organelles, including mitochondria. Conversely, mitochondrial alterations could trigger autophagy. Such a selective form of autophagy might be involved in the elimination of damaged mitochondria. We reported previously that, mitochondria were early targets of rapamycin-induced autophagy. Here we report that rapamycin-induced autophagy is accompanied by the early production of reactive oxygen species and by the early oxidation of mitochondrial lipid. Inhibition of these oxidative effects by resveratrol largely impaired autophagy of both cytosolic proteins and mitochondria, and delayed subsequent cell death. These results support a role of mitochondrial oxidation events in the activation of autophagy.     

Analysis of the individual contributions of Igalpha (CD79a)- and Igbeta (CD79b)-mediated tonic signaling for bone marrow B cell development and peripheral B cell maturation

The individual contribution of Igalpha and Igbeta for BCR-triggered fates is unclear. Prior evidence supports conflicting ideas concerning unique as well as redundant functions for these proteins in the context of BCR/pre-BCR signaling. Part of this ambiguity may reflect the recent appreciation that Igalpha and Igbeta participate in both Ag-independent (tonic) and Ag-dependent signaling. The present study undertook defining the individual requirement for Igalpha and Igbeta under conditions where only ligand-independent tonic signaling was operative. In this regard, we have constructed chimeric proteins containing one or two copies of the cytoplasmic domains of either Igalpha or Igbeta and Igalpha/Igbeta heterodimers with targeted Tyr-->Phe modifications. The ability of these proteins to act as surrogate receptors and trigger early bone marrow and peripheral B cell maturation was tested in RAG2(-/-) primary pro-B cell lines and in gene transfer experiments in the muMT mouse model. We considered that the threshold for a functional activity mediated by the pre-BCR/BCR might only be reached when two functional copies of the Igalpha/Igbeta ITAM domain are expressed together, and therefore the specificity conferred by these proteins can only be observed in these conditions. We found that the ligand-independent tonic signal is sufficient to drive development into mature follicular B cells and both Igalpha and Igbeta chains supported formation of this population. In contrast, neither marginal zone nor B1 mature B cell subsets develop from bone marrow precursors under conditions where only tonic signals are generated.     

Regulation of microtubule formation in activated mast cells by complexes of gamma-tubulin with Fyn and Syk kinases

Aggregation of the high-affinity IgE receptors (FcepsilonRIs) on the surface of granulated mast cells initiates a chain of signaling events culminating in the release of allergy mediators. Although microtubules are involved in mast cell degranulation, the molecular mechanism that controls microtubule rearrangement after FcepsilonRI triggering is poorly understood. In this study, we show that the activation of bone marrow-derived mast cells (BMMCs) induced by FcepsilonRI aggregation or treatment with pervanadate leads to a rapid polymerization of microtubules. This polymerization was not dependent on the presence of Lyn kinase as determined by experiments with BMMCs isolated from Lyn-negative mice. One of the key regulators of microtubule polymerization is gamma-tubulin. Immunoprecipitation experiments revealed that gamma-tubulin from activated cells formed complexes with Fyn and Syk protein tyrosine kinases and several tyrosine phosphorylated proteins from both wild-type and Lyn(-/-) BMMCs. Pretreatment of the cells with Src-family or Syk-family selective tyrosine kinase inhibitors, PP2 or piceatannol, respectively, inhibited the formation of microtubules and reduced the amount of tyrosine phosphorylated proteins in gamma-tubulin complexes, suggesting that Src and Syk family kinases are involved in the initial stages of microtubule formation. This notion was corroborated by pull-down experiments in which gamma-tubulin complex bounds to the recombinant Src homology 2 and Src homology 3 domains of Fyn kinase. We propose that Fyn and Syk kinases are involved in the regulation of binding properties of gamma-tubulin and/or its associated proteins, and thus modulate the microtubule nucleation in activated mast cells.