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Glycosylphosphatidylinositol-anchored high density lipoprotein–binding protein 1 (GPIHBP1), an endothelial cell protein, binds LPL in the subendothelial spaces and transports it to the capillary lumen. In Gpihbp1−/−mice, LPL remains stranded in the subendothelial spaces, causing hypertriglyceridemia, but how Gpihbp1−/−mice respond to metabolic stress (e.g., cold exposure) has never been studied. In wild-type mice, cold exposure increases LPL-mediated processing of triglyceride-rich lipoproteins (TRLs) in brown adipose tissue (BAT), providing fuel for thermogenesis and leading to lower plasma triglyceride levels. We suspected that defective TRL processing in Gpihbp1−/−mice might impair thermogenesis and blunt the fall in plasma triglyceride levels. Indeed, Gpihbp1−/−mice exhibited cold intolerance, but the effects on plasma triglyceride levels were paradoxical. Rather than falling, the plasma triglyceride levels increased sharply (from ∼4,000 to ∼15,000 mg/dl), likely because fatty acid release by peripheral tissues drives hepatic production of TRLs that cannot be processed. We predicted that the sharp increase in plasma triglyceride levels would not occur in Gpihbp1−/−Angptl4−/−mice, where LPL activity is higher and baseline plasma triglyceride levels are lower. Indeed, the plasma triglyceride levels in Gpihbp1−/−Angptl4−/−mice fell during cold exposure. Metabolic studies revealed increased levels of TRL processing in the BAT of Gpihbp1−/−Angptl4−/−mice.

ApoC-III is often assumed to retard the intravascular processing of triglyceride-rich lipoproteins (TRLs) by inhibiting LPL, but that view is based largely on studies of free LPL. We now recognize that intravascular LPL is neither free nor loosely bound, but instead is tightly bound to glycosylphosphatidylinositol-anchored HDL-binding protein 1 (GPIHBP1) on endothelial cells. Here, we revisited the effects of apoC-III on LPL, focusing on apoC-IIIʼns capacity to affect the activity of GPIHBP1-bound LPL. We found that TRLs from APOC3transgenic mice bound normally to GPIHBP1-bound LPL on cultured cells in vitro and to heart capillaries in vivo. However, the triglycerides in apoC-III–enriched TRLs were hydrolyzed more slowly by free LPL, and the inhibitory effect of apoC-III on triglyceride lipolysis was exaggerated when LPL was bound to GPIHBP1 on the surface of agarose beads. Also, recombinant apoC-III reduced triglyceride hydrolysis by free LPL only modestly, but the inhibitory effect was greater when the LPL was bound to GPIHBP1. A mutant apoC-III associated with low plasma triglyceride levels (p.A23T) displayed a reduced capacity to inhibit free and GPIHBP1-bound LPL. Our results show that apoC-III potently inhibits triglyceride hydrolysis when LPL is bound to GPIHBP1.

Little is known about arable agriculture in the Early Neolithic (4000–3300calBC, Funnel Beaker Culture) of Southern Scandinavia. Archaeobotanical material is rare and few archaeological sites have yielded more than a small number of charred cereal grains. In this short communication, we present single-entity carbon and nitrogen isotope analyses of charred cereals from Stensborg, an early Funnel Beaker Culture site near Stockholm, Sweden. This cereal assemblage is important as it is large, well-preserved and consists of multiple crop species. Our isotopic results indicate that many of the Stensborg cereal crops had been manured and that there is intra- and inter-species variation in manuring. We interpret these data as evidence of an integrated regime of stock-keeping and small-scale agriculture in the early Funnel Beaker Culture near its northernmost limit.

Mutation of conserved cysteines in proteins of the Ly6 family cause human disease—chylomicronemia in the case of glycosylphosphatidylinositol-anchored HDL binding protein 1 (GPIHBP1) and paroxysmal nocturnal hemoglobinuria in the case of CD59. A mutation in a conserved cysteine in CD59 prevented the protein from reaching the surface of blood cells. In contrast, mutation of conserved cysteines in human GPIHBP1 had little effect on GPIHBP1 trafficking to the surface of cultured CHO cells. The latter findings were somewhat surprising and raised questions about whether CHO cell studies accurately model the fate of mutant GPIHBP1 proteins in vivo. To explore this concern, we created mice harboring a GPIHBP1 cysteine mutation (p.C63Y). The p.C63Y mutation abolished the ability of mouse GPIHBP1 to bind LPL, resulting in severe chylomicronemia. The mutant GPIHBP1 was detectable by immunohistochemistry on the surface of endothelial cells, but the level of expression was ∼70% lower than in WT mice. The mutant GPIHBP1 protein in mouse tissues was predominantly monomeric. We conclude that mutation of a conserved cysteine in GPIHBP1 abolishes the ability of GPIHBP1 to bind LPL, resulting in mislocalization of LPL and severe chylomicronemia. The mutation reduced but did not eliminate GPIHBP1 on the surface of endothelial cells in vivo.

Plasma apoC-III levels correlate with triglyceride (TG) levels and are a strong predictor of CVD outcomes. ApoC-III elevates TG in part by inhibiting LPL. ApoC-III likely inhibits LPL by competing for lipid binding. To probe this, we used oil-drop tensiometry to characterize binding of six apoC-III variants to lipid/water interfaces. This technique monitors the dependence of lipid binding on surface pressure, which increases during TG hydrolysis by LPL. ApoC-III adsorption increased surface pressure by upward of 18 mN/m at phospholipid/TG/water interfaces. ApoC-III was retained to high pressures at these interfaces, desorbing at 21–25 mN/m. Point mutants, which substituted alanine for aromatic residues, impaired the lipid binding of apoC-III. Adsorption and retention pressures decreased by 1–6 mN/m in point mutants, with the magnitude determined by the location of alanine substitutions. Trp42 was most critical to mediating lipid binding. These results strongly correlate with our previous results, linking apoC-III point mutants to increased LPL binding and activity at lipid surfaces. We propose that aromatic residues in the C-terminal half of apoC-III mediate binding to TG-rich lipoproteins. Increased apoC-III expression in the hypertriglyceridemic state allows apoC-III to accumulate on lipoproteins and inhibit LPL by preventing binding and/or access to substrate.

GPIHBP1, an endothelial cell protein, binds LPL in the interstitial spaces and shuttles it to its site of action inside blood vessels. For years, studies of human GPIHBP1 have been hampered by an absence of useful antibodies. We reasoned that monoclonal antibodies (mAbs) against human GPIHBP1 would be useful for 1) defining the functional relevance of GPIHBP1's Ly6 and acidic domains to the binding of LPL; 2) ascertaining whether human GPIHBP1 is expressed exclusively in capillary endothelial cells; and 3) testing whether GPIHBP1 is detectable in human plasma. Here, we report the development of a panel of human GPIHBP1-specific mAbs. Two mAbs against GPIHBP1's Ly6 domain, RE3 and RG3, abolished LPL binding, whereas an antibody against the acidic domain, RF4, did not. Also, mAbs RE3 and RG3 bound with reduced affinity to a mutant GPIHBP1 containing an Ly6 domain mutation (W109S) that abolishes LPL binding. Immunohistochemistry studies with the GPIHBP1 mAbs revealed that human GPIHBP1 is expressed only in capillary endothelial cells. Finally, we created an ELISA that detects GPIHBP1 in human plasma. That ELISA should make it possible for clinical lipidologists to determine whether plasma GPIHBP1 levels are a useful biomarker of metabolic or vascular disease

In mice lacking glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1), the LPL secreted by adipocytes and myocytes remains bound to heparan sulfate proteoglycans (HSPGs) on all cells within tissues. That observation raises a perplexing issue: Why isn't the freshly secreted LPL in wild-type mice captured by the same HSPGs, thereby preventing LPL from reaching GPIHBP1 on capillaries? We hypothesized that LPL–HSPG interactions are transient, allowing the LPL to detach and move to GPIHBP1 on capillaries. Indeed, we found that LPL detaches from HSPGs on cultured cells and moves to: 1) soluble GPIHBP1 in the cell culture medium; 2) GPIHBP1-coated agarose beads; and 3) nearby GPIHBP1-expressing cells. Movement of HSPG-bound LPL to GPIHBP1 did not occur when GPIHBP1 contained a Ly6 domain missense mutation (W109S), but was almost normal when GPIHBP1's acidic domain was mutated. To test the mobility of HSPG-bound LPL in vivo, we injected GPIHBP1-coated agarose beads into the brown adipose tissue of GPIHBP1-deficient mice. LPL moved quickly from HSPGs on adipocytes to GPIHBP1-coated beads, thereby depleting LPL stores on the surface of adipocytes. We conclude that HSPG-bound LPL in the interstitial spaces of tissues is mobile, allowing the LPL to move to GPIHBP1 on endothelial cells.

LPL contains two principal domains: an amino-terminal catalytic domain (residues 1–297) and a carboxyl-terminal domain (residues 298–448) that is important for binding lipids and binding glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1) (an endothelial cell protein that shuttles LPL to the capillary lumen). The LPL sequences required for GPIHBP1 binding have not been examined in detail, but one study suggested that sequences near LPL's carboxyl terminus (residues ∼403–438) were crucial. Here, we tested the ability of LPL-specific monoclonal antibodies (mAbs) to block the binding of LPL to GPIHBP1. One antibody, 88B8, abolished LPL binding to GPIHBP1. Consistent with those results, antibody 88B8 could not bind to GPIHBP1-bound LPL on cultured cells. Antibody 88B8 bound poorly to LPL proteins with amino acid substitutions that interfered with GPIHBP1 binding (e.g., C418Y, E421K). However, the sequences near LPL's carboxyl terminus (residues ∼403–438) were not sufficient for 88B8 binding; upstream sequences (residues 298–400) were also required. Additional studies showed that these same sequences are required for LPL binding to GPIHBP1. In conclusion, we identified an LPL mAb that binds to LPL's GPIHBP1-binding domain. The binding of both antibody 88B8 and GPIHBP1 to LPL depends on large segments of LPL's carboxyl-terminal domain.

LPL hydrolyzes triglycerides in triglyceride-rich lipoproteins along the capillaries of heart, skeletal muscle, and adipose tissue. The activity of LPL is repressed by angiopoietin-like 4 (ANGPTL4) but the underlying mechanisms have not been fully elucidated. Our objective was to study the cellular location and mechanism for LPL inhibition by ANGPTL4. We performed studies in transfected cells, ex vivo studies, and in vivo studies with Angptl4−/−mice. Cotransfection of CHO pgsA-745 cells with ANGPTL4 and LPL reduced intracellular LPL protein levels, suggesting that ANGPTL4 promotes LPL degradation. This conclusion was supported by studies of primary adipocytes and adipose tissue explants from wild-type and Angptl4−/−mice. Absence of ANGPTL4 resulted in accumulation of the mature-glycosylated form of LPL and increased secretion of LPL. Blocking endoplasmic reticulum (ER)-Golgi transport abolished differences in LPL abundance between wild-type and Angptl4−/−adipocytes, suggesting that ANGPTL4 acts upon LPL after LPL processing in the ER. Finally, physiological changes in adipose tissue ANGPTL4 expression during fasting and cold resulted in inverse changes in the amount of mature-glycosylated LPL in wild-type mice, but not Angptl4−/−mice. We conclude that ANGPTL4 promotes loss of intracellular LPL by stimulating LPL degradation after LPL processing in the ER.

SLURP1, a member of the lymphocyte antigen 6 protein family, is secreted by suprabasal keratinocytes. Mutations in SLURP1cause a palmoplantar keratoderma (PPK) known as mal de Meleda. SLURP2, another secreted lymphocyte antigen 6 protein, is encoded by a gene located ∼20 kb downstream from SLURP1. SLURP2 is produced by suprabasal keratinocytes. To investigate the importance of SLURP2, we first examined Slurp2knockout mice in which exon 2–3 sequences had been replaced with lacZand neocassettes. Slurp2–/–mice exhibited hyperkeratosis on the volar surface of the paws (i.e., palmoplantar keratoderma), increased keratinocyte proliferation, and an accumulation of lipid droplets in the stratum corneum. They also exhibited reduced body weight and hind limb clasping. These phenotypes are similar to those of Slurp1–/–mice. To solidify a link between Slurp2deficiency and palmoplantar keratoderma and to be confident that the disease phenotypes in Slurp2–/–mice were not secondary to the effects of the lacZand neocassettes on Slurp1expression, we created a new line of Slurp2knockout mice (Slurp2X–/–) in which Slurp2was inactivated with a simple nonsense mutation. Slurp2X–/–mice exhibited the same disease phenotypes. Thus, Slurp2deficiency and Slurp1 deficiencies cause the same disease phenotypes.

Targeting controlled release core–shell nanocarriers with the potential to overcome multidrug resistant (MDR) lung cancer were prepared based on demethoxycurcumin (DMC) loaded amphiphilic chitosan nanoparticles coated with an anti-EGFR antibody layer. The nanocarriers were characterized with regard to size with dynamic light scattering, SEM, and TEM. The characterization confirmed the nanocarriers to have a surface coating of the anti-EGFR antibody and a final size excellently suited for circulating targeting nanocarriers, i.e., <200 nm in diameter. In vitro drug release revealed extended quasi-Fickian release from the nanocarriers, with the anti-EGFR layer further reducing the release rate. Cell culture experiments using normoxic and MDR hypoxic cells overexpressing EGFR confirmed improved DMC delivery for anti-EGFR coated particles and revealed that the DMC was delivered to the cytoplasmic region of the cells, forming nanoprecipitates in lysosomes and endosomes. The effective endocytosis and targeting of the core–shell nanoparticles resulted in the nanocarriers achieving high cytotoxicity also against MDR cells. The therapeutic potential was further confirmed in an A549 xenograft lung tumor mouse model, where DMC loaded core–shell nanocarriers achieved about 8-fold reduction in tumor volume compared with control group over the 8 weeks of the investigation. Both in vitro and in vivo data suggest the anti-EGFR coated core–shell nanocarriers as highly promising for treatment of hypoxic MDR cancers, especially for non-small cell lung cancer.

AbstractMany species have suffered population declines through loss of suitable habitat. In addition, current agricultural land use and human settlements favor generalist predators, which pose an increasing threat to ground-nesting bird species such as shorebirds (waders). During the last 2 decades, nest exclosures have been used to control nest-predation rates and often improved hatching success. We evaluated the effectiveness of protective nest-cages to boost reproductive success in the endangered southern dunlin (Calidris alpina schinzii) and ultimately to halt the population decline. We found that exclosures successfully increased the survival probability of nests and, thereby, the number of hatchlings, without markedly elevating the predation rate on incubating adults. Nest exclosures did not, however, translate into an increased number of fledglings and recruits produced/breeding adult in the population, showing that factors other than nest survival are also important for population development. Our results highlight that conservation efforts aimed only at removing high nest-predation pressure may be insufficient to preserve declining species such as the southern dunlin.

Abstract: Deactivation of palladium and platinum catalysts due to coke formation was studied during hydrogenation of methyl esters of sunflower oil. The supported metal catalysts were prepared by impregnating γ-alumina with either palladium or platinum salts, and by impregnating α-alumina with palladium salt. The catalysts were reused for several batch experiments. The Pd/γ-Al2O3 catalyst lost more than 50% of its initial activity after four batch experiments, while the other catalysts did not deactivate. Samples of used catalysts were cleaned from remaining oil by repeated extractions with methanol, and the amount of coke formed on the catalysts was studied by temperature-programmed oxidation. The deactivation of the catalyst is a function of both the metal and the support. The amount of coke increased on the Pd/γ-Al2O3 catalyst with repeated use, but the amount of coke remained approximately constant for the Pt/γ-Al2O3 catalyst. Virtually no coke was detected on the Pd/α-Al2O3 catalyst. The formation of coke on Pd/α-Al2O3 may be slower than on the Pd/γ-Al2O3 owing to the carrier’s smaller surface area and less acidic character. The absence of deactivation for the Pt/γ-Al2O3 catalyst may be explained by slower formation of coke precursors on platinum compared to palladium.

Deactivation of palladium and platinum catalysts due to coke formation was studied during hydrogenation of methyl esters of sunflower oil. The supported metal catalysts were prepared by impregnating ?-alumina with either palladium or platinum salts, and by impregnating a-alumina with palladium salt. The catalysts were reused for several batch experiments. The Pd/?-Al2O3catalyst lost more than 50% of its initial activity after four batch experiments, while the other catalysts did not deactivate. Samples of used catalysts were cleaned from remaining oil by repeated extractions with methanol, and the amount of coke formed on the catalysts was studied by temperature-programmed oxidation. The deactivation of the catalyst is a function of both the metal and the support. The amount of coke increased on the Pd/?-Al2O3catalyst with repeated use, but the amount of coke remained approximately constant for the Pt/?-Al2O3catalyst. Virtually no coke was detected on the Pd/a-Al2O3catalyst. The formation of coke on Pd/a-Al2O3may be slower than on the Pd/?-Al2O3owing to the carrier's smaller surface area and less acidic character. The absence of deactivation for the Pt/?-Al2O3catalyst may be explained by slower formation of coke precursors on platinum compared to palladium.

A transient approach has been applied to a system deactivating due to coke formation, in order to determine the kinetics for the main reaction. The method, which was used on a PtSn/Al2O3 catalyst deactivated during propane dehydrogenation, consisted of runs in which the gas composition and temperature were changed in steps. By separating the deactivation from the main reaction kinetics, it was possible to obtain rate equations. A simple power-law model was found to fit the data best. Finally, simulations using the model for the main reaction together with a model for the deactivation mechanism were successfully applied to original data. The paper also discusses problems with non-uniform coke profiles in integral reactors, and errors induced in kinetic parameters by using methods that utilize extrapolation to zero process time when finding mechanistic models.

The formation of coke and the influence of the coke on selectivity were investigated during hydrogenation of acetylene on supported palladium catalysts. It was found that the total amount of coke was not directly related to the increase in formation of undesired ethane. Instead, the surface coverage of hydrogen during the deactivation was found to be a crucial parameter. A catalyst deactivated at low hydrogen surface coverage showed a higher ethane selectivity than a sample deactivated at higher surface coverage of hydrogen when compared under the same reaction conditions. In contrast, the coke formation rate was found to increase with increased hydrogen surface coverage. The role of carbon monoxide was also investigated, and the impact on selectivity and coke formation was explained by the reduced surface coverage of hydrogen in the presence of carbon monoxide. The coke was characterized by temperature-programmed oxidation, and deconvolution of the obtained peaks was carried out using a power-law model.

Pt/Al2O3and Pt–Sn/Al2O3catalysts, coked during propane dehydrogenation, have been studied using temperature-programmed oxidation (TPO). Time on stream, temperature, and reaction gas composition have been varied. Three different peaks were identified from the TPO profiles on the Pt–Sn catalyst and attributed to different types of coke; coke on and in the vicinity of the metal, coke on the carrier, and graphitic coke on the carrier. The amounts of these types were related to reaction conditions. The formation of the coke belonging to the first two peaks in the TPO profiles increases with temperature and partial pressure of propene. Hydrogen, on the other hand, suppresses the formation. The amount of coke that can be attributed to the third peak increases with temperature and propane partial pressure. A model is discussed where a minor part of the coke deactivates the catalyst. This coke is formed in parallel with the coke that is seen in the first two peaks in the TPO experiments. The graphitic coke formed on the carrier is not formed through this route. The experiments with different time on stream revealed that the first peak reached a constant level after about 15 h, while the second one still increased. Hydrogen was very efficient in preventing coke formation and deactivation but could not remove coke already formed on the catalyst. The hydrogenolysis and cracking mechanisms during the propane dehydrogenation are also discussed.