The role of actin isoforms in somatic embryogenesis in Norway spruce

Background  

Somatic embryogenesis in spruce is a process of high importance for biotechnology, yet it comprises of orchestrated series of events whose cellular and molecular details are not well understood. In this study, we examined the role of actin cytoskeleton during somatic embryogenesis in Norway spruce line AFO 541 by means of anti-actin drugs.     

Liver X receptor-mediated activation of reverse cholesterol transport from macrophages to feces in vivo requires ABCG5/G8

Liver X receptor (LXR) agonists increase both total fecal sterol excretion and macrophage-specific reverse cholesterol transport (RCT) in vivo. In this study, we assessed the effects of ABCG5/G8 deficiency as well as those of LXR agonist-induction of RCT from macrophages to feces in vivo. A [(3)H]cholesterol-labeled macrophage cell line was injected intraperitoneally into ABCG5/G8-deficient (G5/G8(-/-)), heterozygous (G5G8(+/-)), and wild-type G5/G8(+/+) mice. G5/G8(-/-)mice presented increased radiolabeled HDL-bound [(3)H]cholesterol 24 h after the label injection. However, the magnitude of macrophage-derived [(3)H]cholesterol in liver and feces did not differ between groups. A separate experiment was conducted in G5G8(+/+) and G5G8(-/-) mice treated with or without the LXR agonist T0901317. Treatment with T0901317 increased liver ABCG5/G8 expression, which was associated with a 2-fold increase in macrophage-derived [(3)H]cholesterol in feces of G5/G8(+/+) mice. However, T0901317 treatment had no effect on fecal [(3)H]cholesterol excretion in G5G8(-/-) mice. Additionally, LXR activation stimulated the fecal excretion of labeled cholesterol after an intravenous injection of HDL-[(3)H]cholesteryl oleate in G5/G8(+/+) mice, but failed to enhance fecal [(3)H]cholesterol in G5/G8(-/-) mice. Our data provide direct in vivo evidence of the crucial role of ABCG5 and ABCG8 in LXR-mediated induction of macrophage-specific RCT.     

Comparison of virulence and activity of some enzymes ofListeria monocytogenes

The virulence of microorganisms and the activity of some enzymes were compared on 17 strains ofListeria monocytogenes, including 5 Patterson's strains, 4 strains maintained for longer periods on nutrient media and 8 strains freshly or recently isolated from animals in our field material. The LD₅₀ and invasivity index were determined as indicators of virulence. The activity of catalase, glucose and lactic acid dehydrogenases and glutamic-oxalo-acetic transaminase were determined quantitatively. Close agreement was found between the values of both indicators of virulence and the activity of catalase. The quantitative assay of catalase and to some extent of some dehydrogenases, as well as glutamic-oxaloacetic transaminase thus represents a simple and satisfactory method of assessing the virulence ofListeria monocytogenes.     

Impaired accumulation of granulocytes in the lung during ozone adaptation

Respiratory alterations induced by an acute exposure to ozone (O(3)) paradoxically resolve during multiday exposure. This adaptation is characteristically accompanied by a gradual attenuation of lung neutrophilia. As maintenance of neutrophilia at the site of inflammation is due to cytokine-mediated delayed neutrophil apoptosis, which is associated with reduced levels of Bax, a proapoptotic protein, we sought to determine whether defects in these mechanisms could account for O(3) adaptation. Lung granulocytes obtained at different time points from calves exposed to 0.75 ppm O(3) for 12 h/d for 7 consecutive days neither showed enhancement of survival nor Bax deficiency, when compared to blood granulocytes. To further investigate the effects of an exogenous oxidative stress on neutrophil survival, human granulocytes were treated with hydrogen peroxide alone, or in combination with granulocyte/macrophage colony-stimulating factor, an antiapoptotic cytokine. Both treatments led to rapid apoptosis associated with downregulation of Bcl-x(L) and Bcl-2, two antiapoptotic proteins. This study shows that O(3) adaptation is associated with a failure in the mechanisms leading to accumulation of neutrophils at the site of inflammation, and suggests that this defect is due to direct proapoptotic effects of exogenous oxidative stress on granulocytes.     

The role of transmembrane chloride current in afterdepolarisations in canine ventricular cardiomyocytes

The physiological role of chloride currents (Icl) in cardiac cells is poorly understood. The aim of the present study was to reveal the role of Icl in the genesis of early and delayed afterdepolarisations (EADs and DADs, respectively). First we identified Icl under action potential voltage clamp conditions as the anthracene-9-carboxylic acid (ANTRA) (0.5 mmol/l)-sensitive current. The ANTRA-sensitive current was large and outwardly directed at the beginning, while it was moderate and inwardly directed at the end of the action potential. Application of ANTRA under current clamp conditions decreased the depth of the incisura, shifted the plateau upwards and lengthened the duration of action potentials. The effect of ANTRA was studied in three models of afterdepolarisations: the ouabain-induced DAD model, the caesium-induced EAD model, and in the presence of subthreshold concentration of isoproterenol. Preincubation of the cells with 0.5 mmol/l ANTRA failed to induce afterdepolarisations. Ouabain (200 nmol/l) alone caused DADs in 62.5% of the cells within 15 min. When ouabain was applied in the presence of ANTRA, 60% of the myocytes transiently displayed EADs before the development of DADs, and all cells developed DADs within 7 min. Isoproterenol (5 nmol/l) alone failed to induce afterdepolarisations. However, 75% of the cells produced DADs within 6 min when superfused with isoproterenol in the presence of ANTRA. Incubation of the myocytes with 3.6 mmol/l CsCl caused EADs in 71.4% of the cells within 30 min. Application of CsCl in the presence of ANTRA resulted in immediate depolarisation of the membrane from -79.6 +/- 0.4 to -54.2 +/- 3.5 mV. Summarizing our results we conclude that the ANTRA-sensitive current is an important mechanism of defence against afterdepolarisations. Suppression of Icl may thus increase the incidence and accelerate the rate of development of both EADs and DADs.     

Activation of PPARdelta alters lipid metabolism in db/db mice

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors, which heterodimerize with the retinoid X receptor and bind to peroxisome proliferator response elements in the promoters of regulated genes. Despite the wealth of information available on the function of PPARalpha and PPARgamma, relatively little is known about the most widely expressed PPAR subtype, PPARdelta. Here we show that treatment of insulin resistant db/db mice with the PPARdelta agonist L-165041, at doses that had no effect on either glucose or triglycerides, raised total plasma cholesterol concentrations. The increased cholesterol was primarily associated with high density lipoprotein (HDL) particles, as shown by fast protein liquid chromatography analysis. These data were corroborated by the chemical analysis of the lipoproteins isolated by ultracentrifugation, demonstrating that treatment with L-165041 produced an increase in circulating HDL without major changes in very low or low density lipoproteins. White adipose tissue lipoprotein lipase activity was reduced following treatment with the PPARdelta ligand, but was increased by a PPARgamma agonist. These data suggest both that PPARdelta is involved in the regulation of cholesterol metabolism in db/db mice and that PPARdelta ligands could potentially have therapeutic value.     

Potential risk of equine herpes virus 1 (EHV-1) transmission by equine embryo transfer

The objective of this study was to determine whether the 10 wash cycles proposed by the International Embryo Transfer Society (IETS) for bovine embryos efficiently decontaminated equine embryos exposed to equine herpes virus 1 (EHV-1) in vitro. Donor mares and stallions were individually screened and shown to be negative for the virus by PCR detection of EHV-1 DNA in blood leukocytes, semen, and uterine lavages in which embryos were recovered. Twenty embryos were recovered and randomly assigned to one of two groups: 10 embryos were exposed for 24h to infectious EHV-1 at 10(6)TCID(50)/ml, and 10 embryos were used as negative controls. Exposed embryos were washed in accordance with IETS recommendations for ruminant and porcine embryos, before being incubated for 24 h with semiconfluent rabbit kidney (RK13) cells to detect any cytopathic effects (CPE), and finally tested for the presence of EHV-1 viral DNA by PCR. The embryo washing media were also assayed for the virus on RK 13 cells and by PCR. Control embryos were neither exposed to the virus nor washed. EHV-1 was not found in the control embryos, or in the last five washes of the exposed embryos. However, the virus was detected in 7/10 of the embryos exposed to EHV-1 for 24h, as well as in the first five washes of the embryos. The gradual disappearance of EHV-1 from the 10 successive wash solutions from the exposed embryos and the detection of viral DNA in 7/10 washed embryos by PCR, demonstrated that the washing procedure was unable to remove EHV-1 and suggested that EHV-1 could be attached to the acellular layer surrounding embryos (zona pellucida or capsule) or had penetrated the embryo.