Efficient targeting of adenoviral vectors to integrin positive vascular cells utilizing a CAR-cyclic RGD linker protein

Vascular smooth muscle (VSMC) and endothelial cells (EC) are particularly resistant to infection by type 5 adenovirus (Ad) vectors. To overcome this limitation and target Ad vectors to ubiquitously expressed alpha(V)beta(3/5) integrins, we have generated a linker protein consisting of the extracellular domain of the coxsackie adenovirus receptor (CAR) connected via avidin to a biotinylated cyclic (c) RGD peptide. After optimization of CAR to cRGD and to Ad coupling, infection of mouse heart endothelial cells (H5V) could be augmented significantly, as demonstrated by 600-fold increased transgene expression levels. In EOMAs, a hemangioendothelioma-derived cell line, the fraction of infected cells was enhanced 4- to 6-fold. Furthermore, the fraction of infected primary mouse VSMC was increased from virtually 0% to 25%. Finally, in human umbilical vein endothelial cells, the number of GFP positive cells was enhanced from 2% to 75%. In conclusion, CAR-cRGD is a versatile and highly efficient construct to target Ad vectors to both transformed and primary VSMC and EC.     

First report of Phytopythium vexans causing the “Avocado sadness” in Michoacan,Mexico

Mexico is the main producer, consumer and exporter of avocado in the world, being Michoacan the main producer state contributing more than 80% of the national production. There are phytopathogens that decimate the production causing the death of the tree. Root samples were collected in avocado trees that showed the characteristic symptomatology of the disease known as avocado sadness, the sampling was carried out in four of the main avocado producing towns, in the state of Michoacan, Mexico. The isolation consisted in sowing root tissue in Petri dishes with V8^®-PARPH culture medium, subsequently they were identified morphologically and for species level it was determined by molecular biology, with the PCR-ITS technique. Pathogenicity tests were performed in triplicate with avocado seedlings with more than six leaves. After 24 hours, the inoculated plants expressed decay in the apical part, after 120 hours the leaves showed yellowing and after 15 days there was a generalized wilt on the stem and leaves, re-isolating the phytopathogen Phytopythium vexans. This study confirms the first report of the oomycete P. vexans affecting avocado trees in the most important producing region of the Mexican Republic.     

The impact of sleep deprivation on neuronal and glial signaling pathways important for memory and synaptic plasticity

Sleep deprivation is a common feature in modern society, and one of the consequences of sleep loss is the impairment of cognitive function. Although it has been widely accepted that sleep deprivation affects learning and memory, only recently has research begun to address which molecular signaling pathways are altered by sleep loss and, more importantly, which pathways can be targeted to reverse the memory impairments resulting from sleep deprivation. In this review, we discuss the different methods used to sleep deprive animals and the effects of different durations of sleep deprivation on learning and memory with an emphasis on hippocampus-dependent memory. We then review the molecular signaling pathways that are sensitive to sleep loss, with a focus on those thought to play a critical role in the memory and synaptic plasticity deficits observed after sleep deprivation. Finally, we highlight several recent attempts to reverse the effects of sleep deprivation on memory and synaptic plasticity. Future research building on these studies promises to contribute to the development of novel strategies to ameliorate the effects of sleep loss on cognition.     

Apolipoprotein C-I binds free fatty acids and reduces their intracellular esterification

Mice that overexpress human apolipoprotein C-I (apoC-I) homozygously (APOC1(+/+) mice) are protected against obesity and show cutaneous abnormalities. Although these effects can result from our previous observation that apoC-I inhibits FFA generation by LPL, we have also found that apoC-I impairs the uptake of a FFA analog in adipose tissue. In this study, we tested the hypothesis that apoC-I interferes with cellular FFA uptake independent of LPL activity. The cutaneous abnormalities of APOC1(+/+) mice were not affected after transplantation to wild-type mice, indicating that locally produced apoC-I prevents lipid entry into the skin. Subsequent in vitro studies with apoC-I-deficient versus wild-type macrophages revealed that apoC-I reduced the cell association and subsequent esterification of [(3)H]oleic acid by approximately 35% (P < 0.05). We speculated that apoC-I binds FFA extracellularly, thereby preventing cell association of FFA. We showed that apoC-I was indeed able to mediate the binding of oleic acid to otherwise protein-free VLDL-like emulsion particles involving electrostatic interaction. We conclude that apoC-I binds FFA in the circulation, thereby reducing the availability of FFA for uptake by cells. This mechanism can serve as an additional mechanism behind the resistance to obesity and the cutaneous abnormalities of APOC1(+/+) mice.     

The Hyplip2 locus causes hypertriglyceridemia by decreased clearance of triglycerides

The Hyplip2 congenic mouse strain contains part of chromosome 15 from MRL/MpJ on the BALB/cJ background. Hyplip2 mice show increased plasma levels of cholesterol and predominantly triglycerides (TGs) and are susceptible to diet-induced atherosclerosis. This study aimed at elucidation of the mechanism(s) explaining the hypertriglyceridemia. Hypertriglyceridemia can result from increased intestinal or hepatic TG production and/or by decreased LPL-mediated TG clearance. The intestinal TG absorption and chylomicron formation were studied after intravenous injection of Triton WR1339 and an intragastric load of olive oil containing glycerol tri[(3)H]oleate. No difference was found in intestinal TG absorption. Moreover, the hepatic VLDL-TG production rate and VLDL particle production, after injection of Triton WR1339, were also not affected. To investigate the LPL-mediated TG clearance, mice were injected intravenously with glycerol tri[(3)H]oleate-labeled VLDL-like emulsion particles. In Hyplip2 mice, the particles were cleared at a decreased rate (half-life of 25 +/- 6 vs. 11 +/- 2 min; P < 0.05) concomitant with a decreased uptake of emulsion TG-derived (3)H-labeled fatty acids by the liver and white adipose tissue. The increased plasma TG levels in Hyplip2 mice do not result from an enhanced intestinal absorption or increased hepatic VLDL production but are caused by decreased LPL-mediated TG clearance.     

Heparan sulphate proteoglycans are involved in the lipoprotein lipase-mediated enhancement of the cellular binding of very low density and low density lipoproteins.

We found that LPL enhances the binding to HepG2 cells and fibroblasts of both VLDL and apoE free LDL. In the presence of 1.7 micrograms/ml of purified bovine LPL, the binding of LDL and VLDL was up to 60 fold increased as compared to the control binding. In addition, LPL enhances the binding in LDL-receptor negative fibroblasts to the same extent as it does in normal fibroblasts. The presence of 10 mM of EGTA could not prevent the LPL-mediated enhancement of the binding of both LDL and VLDL to fibroblasts, indicating that the binding is calcium independent. Furthermore, up- and down regulation of the LDL receptor did not influence the binding of these lipoproteins in the presence of LPL. Strikingly, we found that the enhancing effect of LPL on the binding of LDL and VLDL to HepG2 cells could be abolished by preincubation of the cells with heparinase, suggesting that heparan sulphate proteoglycans are involved in the LPL-mediated stimulation. We hypothesize that the enhancement of the cellular binding of LDL and VLDL in the presence of LPL is caused by an LPL-bridging between proteoglycans present on the plasma membrane and the lipoproteins, and that the LDL receptor and LRP are not involved.     

Stimulation of the LDL receptor activity in the human hepatoma cell line Hep G2 by high-density serum fractions

The regulation of the LDL receptor activity in the human hepatoma cell line Hep G2 was studied. In Hep G2 cells, in contrast with fibroblasts, the LDL receptor activity was increased 2.5-fold upon increasing the concentration of normal whole serum in the culture medium from 20 to 100% by volume. Incubation of the Hep G2 cells with physiological concentrations of LDL (up to 700 micrograms/ml) instead of incubation under serum-free conditions resulted in a maximum 2-fold decrease in LDL receptor activity (10-fold decrease in fibroblasts). Incubation with physiological concentrations of HDL with a density of between 1.16 and 1.20 g/ml (heavy HDL) resulted in an approximately 7-fold increase in LDL receptor activity (1.5-fold increase in fibroblasts). This increased LDL receptor activity is due to an increase in the number of LDL receptors. Furthermore, simultaneous incubation of Hep G2 cells with LDL and heavy HDL (both 200 micrograms/ml) resulted in a 3-fold stimulation of the LDL receptor activity as compared with incubation in serum-free medium. 3-Hydroxy-3-methylglutaryl-CoA reductase activity was also stimulated after incubation of Hep G2 with heavy HDL (up to 3-fold). The increased LDL receptor activity in Hep G2 cells after incubation with heavy HDL was independent of the action of lecithin:cholesterol acyltransferase during that incubation. However, previous modification of heavy HDL by lecithin:cholesterol acyltransferase resulted in an enhanced ability of heavy HDL to stimulate the LDL receptor activity. Our results indicate that in Hep G2 cells the heavy HDL-mediated stimulation of the LDL receptor activity overrules the LDL-mediated down-regulation and raises the suggestion that in man the presence of heavy HDL and the action of lecithin:cholesterol acyltransferase in plasma may be of importance in receptor-mediated catabolism of LDL by the liver.     

Apolipoprotein A-IV polymorphism and its effect on plasma lipid and apolipoprotein concentrations

Recently, we determined the apolipoprotein E (apoE) phenotype distribution in 2,000 randomly selected 35-year-old male individuals by slab gel isoelectric focusing of delipidated plasma samples, followed by immunoblotting using anti-apoE antiserum. These blots have been successfully re-used for immunovisualization of apoA-IV isoelectric focusing patterns. In a population sample of 1,393 individuals, four distinct apoA-IV isoforms were detected, encoded by the alleles A-IV*0, A-IV*1, A-IV*2, and A-IV*3 with gene frequencies of 0.002, 0.901, 0.079, and 0.018, respectively. The mean of plasma cholesterol, triglyceride, apoB and E levels did not differ significantly among the different apoA-IV phenotype groups. For these lipoprotein parameters, less than 0.1% of the total phenotypic variance could be accounted for by the APOA-IV gene locus. Our results did not show any effect of apoA-IV polymorphism on plasma apoA-I levels nor could we find any correlation between plasma levels of apoA-I and apoA-IV within the different apoA-IV phenotype groups. The plasma level of apoA-IV in subjects bearing the A-IV*3 allele is significantly lower than in subjects without the A-IV*3 allele (5 mg/dl versus 14 mg/dl). We therefore conclude that, in contrast to the apoE polymorphism, the polymorphism at the APOA-IV locus does not influence any of the levels of the lipoprotein parameters considered except apoA-IV.