Intracellular trafficking of pigeon beta-very low density lipoprotein and low density lipoprotein at low and high concentrations in pigeon macrophages

Foam cell formation occurs in vitro at lipoprotein concentrations above 50 microgram/ml in pigeon macrophages. Hypothetically, intracellular trafficking of lipoproteins at higher concentrations may differ from uptake of lipoproteins associated with low concentrations, revealing a separate atherogenic endocytic pathway. Macrophage intracellular trafficking of pigeon beta-very low density lipoprotein (beta-VLDL) and low density lipoprotein (LDL) at low concentrations (12 microgram/ml) near the saturation of high affinity binding sites and high lipoprotein concentrations (50-150 microgram/ml) used to induce foam cell formation were examined. Pigeon beta-VLDL and LDL, differentially labeled with colloidal gold, were added simultaneously to contrast trafficking of beta-VLDL, which causes in vitro foam cell formation, with LDL, which does not. The binding of lipoproteins to cell surface structures, distribution of lipoproteins in endocytic organelles, and the extent of colabeling in the endocytic organelles were determined by thin-section transmission electron microscopy.At low concentrations, the intracellular trafficking of pigeon LDL and beta-VLDL was identical. At high concentrations, LDL was removed more rapidly from the plasma membrane and reached lysosomes more quickly than beta-VLDL. No separate endocytic route was present at high concentrations of beta-VLDL; rather, an increased residence on the plasma membrane, association with nonmicrovillar portions of the plasma membrane, and slower trafficking in organelles of coated-pit endocytosis reflected a more atherogenic trafficking pattern.     

Thermogenic responses of high-altitude adapted rats on low temperatures and low pressures

The male rats were raised in two groups, one at Mt. Yatsugatake (2,100 m above sea level, the average ambient temperature 12.5 degrees C) for 30 days, and the other at a laboratory of Matsumoto (610 m above sea level, the average temperature 20 degrees C). The steady-state oxygen consumptions (VO2) and the rectal temperatures (TR) were measured under exposure conditions of various temperatures combined with different simulated altitudes. The values of VO2 and TR for a control group at 610 m-20 degrees C were regarded as 100% and the relative changes to the control values were obtained at various temperatures in the respective low-pressure condition. When measured at a simulated altitude of 2,000 m on the 2nd day after the rats raised at Mt. Yatsugatake were translocated to Matsumoto, the values at 0 degrees C and 10 degrees C room temperatures, VO2 and TR, were still significantly increased as compared with those of rats raised at Matsumoto. On the 40th day after the translocation from Mt. Yatsugatake, however, the values turned out to exhibit no significant difference in both groups. These results indicated that the greater thermogenesis of high-altitude adapted rats had been established by combined stimuli of low temperatures and low pressures as compared with those of Matsumoto-level adapted rats, but the responses returned to the control level by deadaptation process at 40 days after the translocation.     

Comparison of plasma clearance of low density lipoprotein with beta-very low density lipoprotein or acetoacetylated low density lipoprotein in cholesterol-fed rabbits

The plasma clearance and tissue distribution of radioiodinated low-density lipoprotein (LDL), beta-very low density lipoprotein (beta-VLDL), and acetoacetylated LDL were studied in cholesterol-fed rabbits. Radioiodinated LDL ([125I]LDL) was cleared more slowly than either [125I]beta-VLDL or acetoacetylated-[125I]LDL and its fractional catabolic rate was one-half that of [125I]beta-VLDL and one-ninth that of acetoacetylated-[125I]LDL. Forty-eight hours after the injection of the labeled lipoproteins, the hepatic uptake was the greatest among the organs evaluated with the uptake of [125I]LDL being one-third that of either [125I]beta-VLDL or acetoacetylated-[125I]LDL. The reduction in the hepatic uptake of LDL due to a down-regulation of the receptors would account for this retarded plasma clearance.     

Characterization of the chicken oocyte receptor for low and very low density lipoproteins

The chicken oocyte receptor for low and very low density lipoproteins has been identified and characterized. Receptor activity present in octyl-beta-D-glucoside extracts of oocyte membranes was measured by a solid phase filtration assay, and the receptor was visualized by ligand blotting. The protein had an apparent Mr of 95,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions and exhibited high affinity for apolipoprotein B-containing lipoproteins, but not for high density lipoproteins or lipoproteins in which lysine residues had been reductively methylated. Binding of lipoproteins was sensitive to EDTA, suramin, and treatment with Pronase. In these aspects, the avian oocyte system was analogous to the mammalian low density lipoprotein receptor in somatic cells. Furthermore, a structural relationship between the mammalian and avian receptors was revealed by immunoblotting: polyclonal antibodies directed against the purified bovine low density lipoprotein receptor reacted selectively with the 95-kDa chicken receptor present in crude oocyte membrane extracts.     

Effects of electromagnetic fields of low frequency and low intensity on rat metabolism

A series of experiments on rats have been performed, to study the effects of long time (50 days) exposure to electromagnetic fields of extremely low frequency (ELF, i.e. less than 100 Hz) and amplitude (non thermal), testing whether the metabolic processes would be affected. The background lies on recent observations on the behaviour of isolated enzymes in vitro exposed to EFL fields. In these experiments, the cyclotron (or Larmor) frequency of the metallic ion has been used to "stimulate" the metalloproteins redox-active site, thus obtaining a clear variation of the enzyme functionality. In this paper we have extended for the first time the check to more complex animal metabolism. The novelty of this approach implies that a large amount of data had to be analyzed since it was not possible, in principle, to select only a few parameters among all the potential effects. Several biochemical parameters have been evaluated by comparing their values during the periods of exposure (field ON) and non exposure (field OFF). The evidence that long term exposure to electromagnetic fields with a well defined frequency may have relevant effects on parameters such as body weight, blood glucose and fatty acid metabolism has been obtained.     

Sclerophylly in two contrasting tropical environments: low nutrients vs. low rainfall

The defining characteristics of sclerophylly are mechanical (e.g., hardness, toughness, stiffness), but little is known about how they vary in contrasting environments and contribute to the adaptiveness of sclerophylly. Here we investigate how the degree and nature of sclerophylly in terms of leaf mechanics differ between vegetation of two contrasting stressful environments, maquis on nutrient-deficient, moist sites and dry forest on moderate-nutrient, drier sites. We measured toughness, strength, and stiffness at the level of the whole leaf ("structural") and per unit thickness ("material"). Leaves of maquis plants were on average structurally stiffer, stronger, and tougher than those in dry forest. There was little difference in material properties between habitats, and leaf thickness was the main contributor to differences in structural mechanical properties between habitats. Flexural stiffness varied most among species and habitats, correlating strongly with leaf mass per area and thickness. We suggest that having thicker leaves allows efficient packaging of biomass to reduce branching costs in sunny but stressful environments, with subsequent impacts on structural mechanical properties. Sclerophylly is probably a complex phenomenon, however, with its mechanical constitution arising from both evolved mechanical properties that confer protection or resistance to stress and nonadaptive mechanical consequences of adaptation to stressful environments.     

Enhancement of virus-induced gene silencing in tomato by low temperature and low humidity

Virus-induced gene silencing (VIGS) is an attractive reverse-genetics tool for studying gene function in plants. We showed that silencing of a phytoene desaturase (PDS) gene is maintained throughout TRV-PDS-inoculated tomato plants as well as in their flowers and fruit and is enhanced by low temperature (15 degrees C) and low humidity (30%). RT-PCR analysis of the PDS gene revealed a dramatic reduction in the level of PDS mRNA in leaves, flowers and fruits. Silencing of PDS results in the accumulation of phytoene, the desaturase substrate. In addition, the content of chlorophyll a, chlorophyll b and total chlorophyll in the leaves of PDS-silenced plants was reduced by more than 90%. We also silenced the LeEIN2 gene by infecting seedlings, and this suppressed fruit ripenning. We conclude that this VIGS approach should facilitate large-scale functional analysis of genes involved in the development and ripening of tomato.     

Dodecyl sulphate/polyacrylamide-gel electrophoresis at low pH values and low temperatures.

A simple method is described for dodecyl sulphate/polyacrylamide-gel electrophoresis of pH- and temperature-labile biological intermediates. The method is based on a catalyst system that works at temperatures of 2--4 degrees C and pH values of 2--4 and an appropriate buffer system containing Li+ or Tris [CH2OH--C(CH2OH)2--NH3+] instead of Na+. This system does not lead to the precipitation of 1% dodecyl sulphate.     

Interactions of high density lipoproteins with very low and low density lipoproteins during lipolysis

Interactions of high density lipoproteins (HDL) with very low (VLDL) and low (LDL) density lipoproteins were investigated during in vitro lipolysis in the presence of limited free fatty acid acceptor. Previous studies had shown that lipid products accumulating on lipoproteins under these conditions promote the formation of physical complexes between apolipoprotein B-containing particles (Biochim. Biophys. Acta, 1987. 919: 97-110). The presence of increasing concentrations of HDL or delipidated HDL progressively diminished VLDL-LDL complex formation. At the same time, association of HDL-derived apolipoprotein (apo) A-I with both VLDL and LDL could be demonstrated by autoradiography of gradient gel electrophoretic blots, immunoblotting, and apolipoprotein analyses of reisolated lipoproteins. The LDL increased in buoyancy and particle diameter, and became enriched in glycerides relative to cholesterol. Both HDL2 and HDL3 increased in particle diameter, buoyancy, and relative glyceride content, and small amounts of apoA-I appeared in newly formed particles of less than 75 A diameter. Association of apoA-I with VLDL or LDL could be reproduced by addition of lipid extracts of lipolyzed VLDL or purified free fatty acids in the absence of lipolysis, and was progressively inhibited by the presence of increasing amounts of albumin. We conclude that lipolysis products promote multiple interactions at the surface of triglyceride-rich lipoproteins undergoing lipolysis, including physical complex formation with other lipoprotein particles and transfers of lipids and apolipoproteins. These processes may facilitate remodeling of lipoproteins in the course of their intravascular metabolism.