Distribution of cell-derived cholesterol among plasma lipoproteins: a comparison of three techniques

Three fractionation procedures (immunoaffinity chromatography, two-dimensional nondenaturing electrophoresis, and heparin-agarose affinity chromatography) have been compared in determining the kinetics of free and ester cholesterol transfer in normolipemic native plasma. Similar results were obtained in each case. Cell-derived free cholesterol is initially enriched in high density lipoproteins (HDL) (mainly HDL without apoE); at longer time periods (greater than 10 min) greater proportions are observed in very low density lipoproteins (VLDL) and low density lipoproteins (LDL). The major part of cholesteryl ester (about 90%) was retained in HDL, while VLDL and LDL, which contained about 75% of total cholesteryl ester mass, received only about 10% of cell-derived cholesteryl ester. Within HDL, almost all cholesteryl ester was in the apoE-free fraction. These data provide evidence that lipoprotein free and esterified cholesterol are not at chemical equilibrium in normal plasma, and that cell-derived cholesterol is preferentially directed to HDL. The techniques used had a comparable effectiveness for the rapid fractionation of labile lipoprotein lipid radioactivity.     

Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

Modifications of serotonergic and adrenergic receptor concentrations in the brain of aggressive Canis familiaris

The aim of the study was to measure beta-adrenergic (beta-AR) and serotonergic (5-HTR) receptor concentrations in different brain areas (frontal cortex, hippocampus, hypothalamus and thalamus) of normal and aggressive dogs. Eight adult male dogs, 4.2+/-0.6 years old, showing no clinical signs but aggression, were used for the study. Eight healthy male dogs, 4.4+/-0.8 years old, with no history of neurological and/or behavioural disorders and accidental death, were used as controls. The whole frontal cortex, hippocampus, thalamus and hypothalamus were collected after euthanasia and plasma membrane fractions obtained by ultracentrifugation. beta-AR and 5-HTR were measured by binding assays using specific radioligand [(-)[3H]CGP 12177 and 5-hydroxy[3H]-tryptamine trifluoroacetate, respectively]. A significant decrease in beta-AR levels was observed in the frontal cortex (P=0.001), hippocampus (P<0.0001), and thalamus (P<0.0001) of aggressive dogs compared to controls. As far as 5-HTR are concerned, two receptor subtypes were detected. The two subtypes were classified as low-affinity (5-HTR LA) and high-affinity (5-HTR HA) serotonergic receptors for [3H]-hydroxytryptamine, on the basis of their affinity for [3H]-hydroxytryptamine. 5-HTR LA significantly increased in the whole central nervous system (CNS) area of aggressive dogs (frontal cortex P=0.071; hippocampus P=0.0013; thalamus P<0.0001; hypothalamus P=0.0004); 5-HTR HA significantly increased only in the thalamus (P=0.0005) and hypothalamus (P=0.0002). Results suggest the possible role played by the catecholaminergic and serotonergic systems in canine aggressive behaviour. The understanding of the biological basis of canine aggression may enable the development of pharmacological treatments that would target specific neurotransmitter systems.     

Abnormal phospholipid composition impairs HDL biogenesis and maturation in mice lacking Abca1

Recent studies have demonstrated that the ATP-binding cassette transporter A1 (ABCA1) facilitates the efflux of phospholipids and cholesterol to apoprotein acceptors, leading to the synthesis of HDL. The purpose of this study was to determine the changes in the lipoprotein fractions in Abca1-deficient mice and study the mechanisms responsible for the low levels of HDL when ABCA1 is absent. Plasma phospholipid concentration was decreased by more than 75%, mostly due to a reduction of phosphatidylcholine (PC) in HDL. Abca1(-/-) HDL represents less than 2% of wild-type levels and is smaller and enriched in phospholipids (11.2-fold more than HDL from controls). Compared to wild-type littermates, Abca1(-/-) HDL had a 4-fold increase in PC, whereas lysophosphatidylcholine (LPC) (125-fold), sphingomyelin (SPH) (49-fold), and phosphatidylethanolamine (PE) (18-fold) showed even higher increases. As a consequence, the ratios of LPC/PC, SPH/PC, PE/PC, and phosphatidylinositol + phosphatidylserine (PI+PS)/PC were all much higher in HDL from Abca1(-/-), compared to wild-type HDL. Plasma phospholipid transfer protein (PLTP) and lecithin cholesterol acyltransferase (LCAT) activities were decreased by more than 80%, suggesting that the maturation of HDL is affected. To test this hypothesis, plasma from Abca1(-/-) mice was incubated with CHO cells that are known to express high levels of ABCA1 with the intent of restoring the flux of phospholipid and cholesterol onto apoAI. Compared to native plasma, no change in maturation of HDL was observed. In contrast, a 220% increase in the formation of mature HDL was observed when ABCA1 function and LCAT activities were restored. Taken together, these observations suggest that ABCA1 is necessary for the adequate lipidation of apoAI, which enables the interaction with LCAT and subsequent maturation.     

Extramedullary hematopoiesis in a case of benign mixed mammary tumor in a female dog: cytological and histopathological assessment

Backgroud  

Extramedullary hematopoiesis (EMH) is defined as the presence of hematopoietic stem cells such as erythroid and myeloid lineage plus megakaryocytes in extramedullary sites like liver, spleen and lymph nodes and is usually associated with either bone marrow or hematological disorders. Mammary EMH is a rare condition either in human and veterinary medicine and can be associated with benign mixed mammary tumors, similarly to that described in this case.     

Effects of enrichment of fibroblasts with unesterified cholesterol on the efflux of cellular lipids to apolipoprotein A-I.

This study elucidates the factors underlying the enhancement in efflux of human fibroblast unesterified cholesterol and phospholipid (PL) by lipid-free apolipoprotein (apo) A-I that is induced by cholesterol enrichment of the cells. Doubling the unesterified cholesterol content of the plasma membrane by incubation for 24 h with low density lipoprotein and lipid/cholesterol dispersions increases the pools of PL and cholesterol available for removal by apoA-I from about 0.8-5%; the initial rates of mass release of cholesterol and PL are both increased about 6-fold. Expression of the ATP binding cassette transporter A1 (ABCA1) is critical for this increased efflux of lipids, and cholesterol loading of the fibroblasts over 24 h increases ABCA1 mRNA about 12-fold. The presence of more ABCA1 and cholesterol in the plasma membrane results in a 2-fold increase in the level of specific binding of apoA-I to the cells with no change in binding affinity. Characterization of the species released from either control or cholesterol-enriched cells indicates that the plasma membrane domains from which lipids are removed are cholesterol-enriched with respect to the average plasma membrane composition. Cholesterol enrichment of fibroblasts also affects PL synthesis, and this leads to enhanced release of phosphatidylcholine (PC) relative to sphingomyelin (SM); the ratios of PC to SM solubilized from control and cholesterol-enriched fibroblasts are approximately 2/1 and 5/1, respectively. Biosynthesis of PC is critical for this preferential release of PC and the enhanced cholesterol efflux because inhibition of PC synthesis by choline depletion reduces cholesterol efflux from cholesterol-enriched cells. Overall, it is clear that enrichment of fibroblasts with unesterified cholesterol enhances efflux of cholesterol and PL to apoA-I because of three effects, 1) increased PC biosynthesis, 2) increased PC transport via ABCA1, and 3) increased cholesterol in the plasma membrane.     

HOIL-1L Interacting Protein (HOIP) as an NF-κB Regulating Component of the CD40 Signaling Complex

The tumor necrosis factor receptor (TNFR) superfamily mediates signals critical for regulation of the immune system. One family member, CD40, is important for the efficient activation of antibody-producing B cells and other antigen-presenting cells. The molecules and mechanisms that mediate CD40 signaling are only partially characterized. Proteins known to interact with the cytoplasmic domain of CD40 include members of the TNF receptor-associated factor (TRAF) family, which regulate signaling and serve as links to other signaling molecules. To identify additional proteins important for CD40 signaling, we used a combined stimulation/immunoprecipitation procedure to isolate CD40 signaling complexes from B cells and characterized the associated proteins by mass spectrometry. In addition to known CD40-interacting proteins, we detected SMAC/DIABLO, HTRA2/Omi, and HOIP/RNF31/PAUL/ZIBRA. We found that these previously unknown CD40-interacting partners were recruited in a TRAF2-dependent manner. HOIP is a ubiquitin ligase capable of mediating NF-κB activation through the ubiquitin-dependent activation of IKKγ. We found that a mutant HOIP molecule engineered to lack ubiquitin ligase activity inhibited the CD40-mediated activation of NF-κB. Together, our results demonstrate a powerful approach for the identification of signaling molecules associated with cell surface receptors and indicate an important role for the ubiquitin ligase activity of HOIP in proximal CD40 signaling.     

Induction of 8-azaguanine resistant mutants in human cultured cells exposed to 31 MeV protons

We report results on the induction of 8-azaguanine (8-AG)-resistant mutants in cultured human cells (EUE) exposed to 31 MeV protons. The spontaneous frequency of mutants was 5.6 +/- 0.7 x 10(-6) per viable cell. Gamma rays were taken as reference radiation. Expression times giving the highest frequency of mutants after 31 MeV protons and gamma irradiation were found to be about 10 days for both radiations. The dose-response relationship for mutant induction by protons, as determined at the optimal expression time, was compared to that obtained after gamma rays. The relative biological effectiveness (RBE) is 2.4 +/- 0.5, this value being higher than the RBE value determined for cell survival.     

Identification and characterization of rodent ABCA1 in isolated type II pneumocytes

ATP-binding cassette transporter A1 (ABCA1) promotes transfer of cholesterol and phospholipid from cells to lipid-free serum apolipoproteins. ABCA1 mRNA and protein expression in primary cultures of rodent type II cells was sensitive to upregulation with 5 microM 9-cis-retinoic acid (9cRA) and 6.2 microM 22-hydroxycholesterol (22-OH). The increase in ABCA1 protein levels was time dependent and was maximal after 16 h of exposure to 9cRA + 22-OH. Inducible ABCA1 was also found in transformed cell lines of lung origin: WI38/VA13, A549, and NIH-H441 cells. Stimulation of ABCA1 in rat type II cells by 9cRA + 22-OH resulted in a four- or fivefold enhancement of efflux of radioactive phospholipid or cholesterol, respectively, from the pneumocytes to apolipoprotein AI (apo AI), whereas cAMP (0.3 mM) had no effect. ABCA1-mediated lipid efflux to apo AI was independent of the surfactant secretion pathway, inasmuch as upregulation of ABCA1 resulted in a reduction of secretagogue-stimulated surfactant phospholipid release. These studies demonstrate the presence of functional ABCA1 in type II cells from the lung.