Immunoaffinity purification of the lipid transfer protein complex directly from human plasma

The human cholesteryl ester (CE) and triglyceride (TG) exchange protein (denoted LTC or lipid transfer complex) was isolated in a single step from plasma using immunoaffinity batch extraction. Antibodies were raised against two preparations of conventionally purified LTC. LTC-I and LTC-II (purified 20,000-fold and 3500-fold, respectively) were used as immunogens. The antiLTC antibodies were isolated by anion-exchange chromatography and coupled to Affi-Gel 10. Chromatography of plasma on antiLTC Affi-Gel removed all of the CE and TG transfer activity. Moreover, LTC prepared from both antiLTC-I and antiLTC-II-Affi-Gel matrices were identical when analyzed by sodium dodecyl sulfate-polyacrylamide gel LTC electrophoresis. LTC exhibited two protein bands of Mr (apparent) 67,000 and 58,000 and a broad, faintly staining region at greater than 150,000. Analysis of LTC by immunoblotting indicated that both antiLTC-I and antiLTC-II antibodies recognized the same LTC proteins. Isoelectric focussing of LTC gave two pI values, 5.2 and 8.7. These data suggest that LTC is a complex of specific proteins and perhaps lipid. Specific CE and TG exchange activities of immunoaffinity-purified LTC were comparable, although the activities were low with respect to that of the antigen used to generate antiLTC-I. This is not due to contamination of LTC by albumin, lecithin:cholesterol acyltransferase, or apolipoproteins AI, AII, B, CIII, D, or E.     

Possible explanation of the disparity between the in vitro and in vivo measurements of Rubisco activity: a study in loblolly pine grown in elevated pCO2.

Rubisco activity can be measured using gas exchange (in vivo) or using in vitro methods. Commonly in vitro methods yield activities that are less than those obtained in vivo. Rubisco activity was measured both in vivo and in vitro using a spectrophotometric technique in mature Pinus taeda L. (loblolly pine) trees grown using free-air CO2 enrichment in elevated (56 Pa) and current (36 Pa) pCO2. In addition, for studies where both in vivo and in vitro values of Rubisco activity were reported net CO2 uptake rate (A) was modelled based on the in vivo and in vitro values of Rubisco activity reported in the literature. Both the modelling exercise and the experimental data showed that the in vitro values of Rubisco activity were insufficient to account for the observed values of A. A trichloroacetic acid (TCA) precipitation of the protein from samples taken in parallel with those used for activity analysis was co-electrophoresed with the extract used for determining in vitro Rubisco activity. There was significantly more Rubisco present in the TCA precipitated samples, suggesting that the underestimation of Rubisco activity in vitro was attributable to an insufficient extraction of Rubisco protein prior to activity analysis. Correction of in vitro values to account for the under-represented Rubisco yielded mechanistically valid values for Rubisco activity. However, despite the low absolute values for Rubisco activity determined in vitro, the trends reported with CO2 treatment concurred with, and were of equal magnitude to, those observed in Rubisco activity measured in vivo.     

Very low and low density lipoprotein synthesis and secretion by the human hepatoma cell line Hep-G2: effects of free fatty acid

The liver is a major source of the plasma lipoproteins; however, direct studies of the regulation of lipoprotein synthesis and secretion by human liver are lacking. Dense monolayers of Hep-G2 cells incorporated radiolabeled precursors into protein ([35S]methionine), cholesterol ([3H]mevalonate and [14C]acetate), triacylglycerol, and phospholipid ([3H]glycerol), and secreted them as lipoproteins. In the absence of free fatty acid in the media, the principal lipoprotein secretory product that accumulated had a density maximum of 1.039 g/ml, similar to serum low density lipoprotein (LDL). ApoB-100 represented greater than 95% of the radiolabeled apoprotein of these particles, with only traces of apoproteins A and E present. Inclusion of 0.8 mM oleic acid in the media resulted in a 54% reduction in radiolabeled triacylglycerol in the LDL fraction and a 324% increase in triacylglycerol in the very low density lipoprotein (VLDL) fraction. Similar changes occurred in the secretion of newly synthesized apoB-100. The VLDL contained apoB-100 as well as apoE. In the absence of exogenous free fatty acid, the radiolabeled cholesterol was recovered in both the LDL and the high density lipoprotein (HDL) regions. Oleic acid caused a 50% decrease in HDL radiolabeled cholesterol and increases of radiolabeled cholesterol in VLDL and LDL. In general, less than 15% of the radiolabeled cholesterol was esterified, despite the presence of cholesteryl ester in the cell. Incubation with oleic acid did not cause an increase in the total amount of radiolabeled lipid or protein secreted. We conclude that human liver-derived cells can secrete distinct VLDL and LDL-like particles, and the relative amounts of these lipoproteins are determined, at least in part, by the availability of free fatty acid.     

Evidence for Specific Polypeptide Chain Folding in Myelin Basic Protein from Reactions Between Fragments of the Protein and Monoclonal Antibodies

The specificities of two monoclonal IgM antibodies (18.25 and 21.14.2) evoked in mice with guinea pig myelin basic protein were examined and interpreted in terms of a specific folding of the protein's polypeptide chain. Studies with guinea pig and rabbit myelin basic protein fragments showed that a region encompassing the central Phe-Phe (87-88) sequence is obligatory, but not sufficient, for reactivity with antibody 18.25. Appreciable reactivity was observed for rabbit peptides 22-95 and 45-151, and lower, but significant, reactivity was shown by peptide 32-95. Only very weak reactivity was seen with peptide 44-95. No reactivity was observed with peptide 1-95 after its lysine residues were acetylated, acetamidinated, or guanidinated. These results have been interpreted in terms of a polypeptide chain folding that creates an epitope within sequence Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val (84-92). The specific conformation of this epitope, which includes probably the Lys-89 and possibly the Asn-90 and Val-92 side chains, could be formed by the association of sequence 84-92 with either sequence Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-Phe (37-45) or with sequence Val-Leu-Ser-Arg-Phe (108-112) to form beta-sheet structures essentially identical with those that appear to be present in the intact BP [Martenson R.E.J. Neurochem. 46, 1612-1622 (1986)]. The second monoclonal antibody, no. 21.14.2, reacts only with guinea pig myelin basic protein and fragments containing the species-restricted sequence Arg-Ala-Asp-Tyr-Lys-Ser-Lys (129-135).(ABSTRACT TRUNCATED AT 250 WORDS)     

HISTORICAL BIOGEOGRAPHY AND CONTEMPORARY PATTERNS OF MITOCHONDRIAL DNA VARIATION IN WHITE-TAILED DEER FROM THE SOUTHEASTERN UNITED STATES

Mitochondrial DNA (mtDNA) was used to characterize patterns of geographic variation among white-tailed deer (Odocoileus virginianus) populations in the southeastern United States. Fifteen restriction enzymes were employed to survey and map 99 restriction sites in 142 deer from 18 localities in five southeastern states. Phylogenetic analysis revealed three primary groups of haplotypes: (1) southern Florida and the Florida Keys, (2) the remainder of peninsular Florida northward to South Carolina, and (3) the Florida panhandle westward to Mississippi. Geographical heterogeneity in haplotype frequencies suggests that stochastic lineage sorting or isolation by distance are not important determinates of mtDNA differentiation among deer populations. The pattern of mtDNA variation in white-tailed deer is concordant spatially with those observed in unrelated taxa suggesting the common influence of historical biogeographic events. The data (1) support previous hypotheses that relate contemporary patterns of intraspecific phylogeography in northern Florida to the physiogeographic history of the region; and (2) suggest that genetic differentiation in southern Florida may be attributable to episodes of Pleistocene dispersal. Despite potentially high vagility and human intervention, ecological and demographic characteristics of deer have effectively preserved the historical pattern of intraspecific mtDNA differentiation.     

Population genetics and phylogenetics of DNA sequence variation at multiple loci within the Drosophila melanogaster species complex

Two regions of the genome, a 1-kbp portion of the zeste locus and a 1.1- kbp portion of the yolk protein 2 locus, were sequenced in six individuals from each of four species: Drosophila melanogaster, D. simulans, D. mauritiana, and D. sechellia. The species and strains were the same as those of a previous study of a 1.9-kbp region of the period locus. No evidence was found for recent balancing or directional selection or for the accumulation of selected differences between species. Yolk protein 2 has a high level of amino acid replacement variation and a low level of synonymous variation, while zeste has the opposite pattern. This contrast is consistent with information on gene function and patterns of codon bias. Polymorphism levels are consistent with a ranking of effective population sizes, from low to high, in the following order: D. sechellia, D. melanogaster, D.mauritiana, and D. simulans. The apparent species relationships are very similar to those suggested by the period locus study. In particular, D. simulans appears to be a large population that is still segregating variation that arose before the separation of D. mauritiana and D. sechellia. It is estimated that the separation of ancestral D. melanogaster from the other species occurred 2.5-3.4 Mya. The separations of D. sechellia and D. mauritiana from ancestral D. simulans appear to have occurred 0.58- 0.86 Mya, with D. mauritiana having diverged from ancestral D. simulans 0.1 Myr more recently than D. sechellia.      

Interactive direct and plant‐mediated effects of elevated atmospheric [CO2] and temperature on a eucalypt‐feeding insect herbivore

Understanding the direct and indirect effects of elevated [CO₂] and temperature on insect herbivores and how these factors interact are essential to predict ecosystem‐level responses to climate change scenarios. In three concurrent glasshouse experiments, we measured both the individual and interactive effects of elevated [CO₂] and temperature on foliar quality. We also assessed the interactions between their direct and plant‐mediated effects on the development of an insect herbivore of eucalypts. Eucalyptus tereticornis saplings were grown at ambient or elevated [CO₂] (400 and 650 μmol mol^?1 respectively) and ambient or elevated ( + 4 °C) temperature for 10 months. Doratifera quadriguttata (Lepidoptera: Limacodidae) larvae were feeding directly on these trees, on their excised leaves in a separate glasshouse, or on excised field‐grown leaves within the temperature and [CO₂] controlled glasshouse. To allow insect gender to be determined and to ensure that any sex‐specific developmental differences could be distinguished from treatment effects, insect development time and consumption were measured from egg hatch to pupation. No direct [CO₂] effects on insects were observed. Elevated temperature accelerated larval development, but did not affect leaf consumption. Elevated [CO₂] and temperature independently reduced foliar quality, slowing larval development and increasing consumption. Simultaneously increasing both [CO₂] and temperature reduced these shifts in foliar quality, and negative effects on larval performance were subsequently ameliorated. Negative nutritional effects of elevated [CO₂] and temperature were also independently outweighed by the direct positive effect of elevated temperature on larvae. Rising [CO₂] and temperature are thus predicted to have interactive effects on foliar quality that affect eucalypt‐feeding insects. However, the ecological consequences of these interactions will depend on the magnitude of concurrent temperature rise and its direct effects on insect physiology and feeding behaviour.