Membrane cholesterol in the regulation of aminophospholipid asymmetry and phagocytosis in oxidized erythrocytes

Cholesterol is known to affect several membrane functions, including membrane susceptibility to oxidative stress. In order to gain a better understanding of the relationship between cholesterol contents, structural integrity, and degree of survival in oxidatively stressed erythrocytes, here we analyzed the transbilayer phospholipid distribution, the morphology, and the degree of clearance observed in cholesterol-modified (enriched or depleted) and unmodified (control) erythrocytes exposed to tert-butylhydroperoxide. We report that the modification of cholesterol contents in erythrocytes promotes the externalization of phosphatidylserine (PS) to the membrane surface, which is consistent with a concomitant inhibition of aminophospholipid translocase (APLT) and an increased uptake of modified erythrocytes by macrophages. Moreover, cholesterol depletion modifies the transbilayer aminophospholipid distribution induced by oxidative stress to a great extent, significantly increasing PS externalization, which is associated with the strongest decrease in APLT activity. The loss of normal PS asymmetry is positively correlated with enhanced phagocytosis, and an increase in echinocyte forms is observed in all oxidized erythrocytes. We envisage that PS externalization could be due, at least in part, to the decrease in APLT activity induced by oxidative stress, the activity of which is also dependent on membrane cholesterol contents.     

Silencing of the N family of resistance genes in Nicotiana edwardsonii compromises the hypersensitive response to tombusviruses

The nontarget effects associated with silencing of the N gene in Nicotiana edwardsonii, an amphidiploid species derived from N. glutinosa and N. clevelandii, have been characterized in this study. The N protein confers resistance to Tobacco mosaic virus (TMV), and is representative of a family of nucleotide-binding site leucine-rich repeat proteins present in N. glutinosa. Previous studies have shown that silencing of the N gene or of other plant genes associated with N-mediated defenses abolishes host resistance to TMV, and this effect can be measured through enhancements in movement or replication of TMV in the N-silenced plants. However, the nontarget effects of gene silencing have not been investigated thoroughly. Notably, are the functions of other resistance (R) genes also affected in experiments designed to silence the N gene? To investigate whether heterologous sequences could silence the N gene, we selected an R gene homolog from N. glutinosa that differed from the N gene by approximately 17%, created a hairpin transgene, and developed transgenic N. edwardsonii plants. Expression of this hairpin in the transgenic N. edwardsonii plants compromised the hypersensitive response to TMV, demonstrating that a single hairpin transgene could silence a block of R genes related by sequence similarity. We then investigated whether the response of N-silenced plants to other viruses would be altered, and found that the hypersensitive response triggered against the tombusviruses Tomato bushy stunt virus and Cymbidium ringspot virus also was compromised. This study indicates that a Tombusvirus R gene shares some homology with the N gene, which could facilitate the cloning of this gene.     

Negative regulation of Akt activity by p38alpha MAP kinase in cardiomyocytes involves membrane localization of PP2A through interaction with caveolin-1

Cardiomyocyte-derived cell lines deficient in p38alpha are more resistant to apoptosis owing to lower expression of the pro-apoptotic proteins Bax and Fas and upregulation of the ERK survival pathway. Here, we show that increased Akt activity also contributes to the enhanced survival of p38alpha-deficient cardiomyocytes. We found that the serine/threonine phosphatase PP2A can be targeted to caveolae through interaction with caveolin-1 in a p38alpha-dependent manner. In agreement with this, PP2A activity associated with caveolin-1 was higher in wild type than in p38alpha-deficient cells. Akt was also present in caveolae and incubation of wild-type cells with the PP2A inhibitor okadaic acid increases the levels of Akt activity. Thus, p38alpha-induced re-localization of PP2A to caveolae can lead to dephosphorylation and inhibition of Akt, which in turn would contribute to the decreased survival observed in wild type cells. However, cell detachment impairs the formation of the PP2A/caveolin-1 complex and, as a consequence, phospho-Akt levels and survival are no longer regulated by p38alpha in detached wild type cardiomyocytes. Our results suggest that p38alpha can negatively modulate Akt activity, independently of PI3K, by regulating the interaction between caveolin-1 and PP2A through a mechanism dependent on cell attachment.     

4-Aryl-5-cyano-2-aminopyrimidines as VEGF-R2 inhibitors: synthesis and biological evaluation

A novel series of 4-aryl-5-cyano-2-aminopyrimidines were synthesized and found to have potent VEGF-R2 kinase inhibitory activity. Structure-activity relationships were investigated and compound 14a was shown to be efficacious in a mouse model of corneal neovascularization.     

Specificity of zebrafish retinol saturase: formation of all-trans-13,14-dihydroretinol and all-trans-7,8- dihydroretinol

Metabolism of vitamin A, all-trans-retinol, leads to the formation of 11-cis-retinaldehyde, the visual chromophore, and all-trans-retinoic acid, which is involved in the regulation of gene expression through the retinoic acid receptor. Enzymes and binding proteins involved in retinoid metabolism are highly conserved across species. We previously described a novel mammalian enzyme that saturates the 13-14 double bond of all-trans-retinol to produce all-trans-13,14-dihydroretinol, which then follows the same metabolic fate as that of all-trans-retinol. Specifically, all-trans-13,14-dihydroretinol is transiently oxidized to all-trans-13,14-dihydroretinoic acid before being oxidized further by Cyp26 enzymes. Here, we report the identification of two putative RetSat homologues in zebrafish, one of which, zebrafish RetSat A (zRetSat A), also had retinol saturase activity, whereas zebrafish RetSat B (zRetSat B) was inactive under similar conditions. Unlike mouse RetSat (mRetSat), zRetSat A had an altered bond specificity saturating either the 13-14 or 7-8 double bonds of all-trans-retinol to produce either all-trans-13,14-dihydroretinol or all-trans-7,8-dihydroretinol, respectively. zRetSat A also saturated the 13-14 or 7-8 double bonds of all-trans-3,4-didehydroretinol (vitamin A2), a second endogenous form of vitamin A in zebrafish. The dual enzymatic activity of zRetSat A displays a newly acquired specificity for the 13-14 double bond retained in higher vertebrates and also the evolutionarily preserved activity of bacterial phytoene desaturases and plant carotenoid isomerases. Expression of zRetSat A was restricted to the liver and intestine of hatchlings and adult zebrafish, whereas zRetSat B was expressed in the same tissues but at earlier developmental stages. Exogenous all-trans-retinol, all-trans-13,14-dihydroretinol, or all-trans-7,8-dihydroretinol led to the strong induction of the expression of the retinoic acid-metabolizing enzyme, Cyp26A1, arguing for an active signaling function of dihydroretinoid metabolites in zebrafish. These findings point to a conserved function but altered specificity of RetSat in vertebrates, leading to the generation of various dihydroretinoid compounds, some of which could have signaling functions.     

Effect of transgene concentration, flanking matrix attachment regions, and RecA-coating on the efficiency of mouse transgenesis mediated by intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) of DNA-loaded sperm cells has been shown to be a valuable tool for the production of transgenic animals, especially when DNA constructs with submegabase magnitude are used. In order to optimize and to understand the mechanism of the ICSI-mediated transgenesis, we have evaluated the impact of transgene DNA concentration, transgene flanking with nuclear matrix attachment regions (MARs), and the use of recombinase A (RecA)-coated DNA on the efficiency of mouse transgenesis production by ICSI. Presented data include assays with three DNA constructs; an enhanced green fluorescent protein (EGFP) plasmid of 5.4 kb, this plasmid flanked with two MAR elements (2.3 Kb of the human beta-interferon domain boundaries), and a yeast artificial chromosome (YAC) construct of ~510 kb (the largest transgenic construct introduced by ICSI that we have seen reported). ICSI-mediated transgenesis was done in the B6D2 mouse strain using different concentrations for each construct. Analysis of generated data indicated that ICSI allows the use of higher DNA concentrations than the ones used for pronuclear microinjection, however, when a certain threshold is exceeded, embryo/fetal viability decrease dramatically. In addition, independently of the transgene concentration tested, transgene flanking with MAR sequences did not have a significant impact on the efficiency of this transgenesis method. Finally, we observed that although the overall efficiency of ICSI-mediated transgenesis with fresh spermatozoa and RecA-complexed DNA was similar to the one obtained with the common ICSI-mediated transgenesis approach with frozen-thawed spermatozoa and RecA free DNA, this method was not as efficient in maintaining a low frequency of founder animal mosaicism, suggesting that different mechanisms of transgene integration might result from each procedure.     

Microbiological contamination in stem cell cultures

Cell therapy and regenerative medicine are potentially two of the most exciting aspects of the novel therapeutic methods currently under development. However, these treatments present a number of important biosafety issues, like the possible transmission of microorganisms to the recipients. The most common potential form of contamination in these cell products is by bacteria (including Mycoplasma), yeast and fungi. In our study, 32 stem cell lines and feeder cell lines were analysed. There were 19 contaminated cell passages (12%). The main contaminants were gram positive cocci and Mycoplasma species, followed by gram negative rods and gram positive rods. The Mycoplasma contamination rate was 4%. Stem cell banks and other research centres aim to screen all processed stem cell lines for these microorganisms, and to assure that no contaminants are introduced in the banking procedures. It is a standard part of current good practice in stem cell banks to carry out routine microbiological controls of the stem cell lines and to work in a controlled environment to reduce the probability of contamination in the final product.     

Live attenuated Salmonella as a vector for oral cytokine gene therapy in melanoma

Systemic administration of cytokines has shown therapeutic benefits in cancer patients; however, serious adverse effects associated with direct protein administration prevent the wide use of this approach. We have assessed the capacity of live attenuated Salmonella to act as a vector for oral cytokine-gene therapy. Salmonella orally administered to melanoma-bearing mice was found to accumulate within the tumor, reaching up to 10(5) bacteria per gram of tumor by day 21 after bacterial inoculation. Numbers of bacteria recovered from tumor did not differ from those recovered from liver or spleen at any time point. Recombinant bacteria carrying eukaryotic expression vectors encoding the murine IL-4 or IL-18 genes were administered to groups of mice with established subcutaneous melanoma tumors. We found that a single oral dose of Salmonella carrying any of the cytokine-encoding plasmids resulted in significantly increased survival time, as compared with mice that received Salmonella carrying the parental plasmid or PBS. Increased levels of IFNgamma were found in sera of animals receiving either of the cytokine-encoding bacteria, but not in mice receiving Salmonella alone or PBS. Co-administration of both recombinant bacteria maximized the production of IFNgamma. Overall these results suggest that cytokine-encoding Salmonella can be an effective and safer alternative to systemic administration of cytokines for immunotherapy of cancer.     

The Use of Dye Plants in a Mazahua Community in Central Mexico

Economic Botany - The use of dye plants is part of the cultural inheritance of Santa Rosa de Lima, Mexico. Numerous dye plants are present in the region, but women mainly use indigo and cochineal...