On the effects of the <Emphasis Type=Italic>Fusarium</Emphasis> toxin deoxynivalenol (DON) administered per os or intraperitoneal infusion to sows during days 63 to 70 of gestation

Six pregnant sows of 180.6 ± 5.6 kg were fed either a Fusarium-contaminated (4.42 mg DON and 48.3 μg ZON per kg, DON per os, n = 3) or a control diet (0.15 mg DON and 5 μg ZON/kg) in the period of days 63 and 70 of gestation. On day 63 of gestation, sows fed the control diet were implanted with an intraperitoneal osmotic minipump (delivery rate of 10 μL/h, for 7 days) containing 50 mg pure (98%) DON in 2 ml 50% DMSO (DON ip, n = 3). Frequent plasma samples were taken to estimate the kinetics after oral and ip DON exposure. The intended continuous delivery of DON by the intraperitoneal minipump could not be shown, as there was a plasma peak (C_max) of 4.2–6.4 ng DON/mL either immediately (sow IP-2+3) or 2.5 h (sow IP-1) after implantation of the pump followed by a one-exponential decline with a mean half-time (t_1/2) of 1.75–4.0 h and only negligible DON plasma concentrations after 12 h. Therefore, the DON ip exposure has to be regarded as one single dose 1 week before termination of experiment. The DON per os sows showed a mean basis level (after achieving a steady state) of DON plasma concentration of about 6–8 ng/mL, as also indicated by the plasma DON concentration at the termination of the experiment. On day 70, caesarean section was carried out, the fetuses were killed immediately after birth, and samples of plasma, urine, and bile were taken to analyze the concentration of DON and its metabolite de-epoxy-DON. At necropsy there were no macroscopic lesions observed in any organ of either sows or piglets. Histopathological evaluation of sows liver and spleen revealed no alterations. The proliferation rate of peripheral blood mononuclear cells (PBMC) with or without stimulation was not affected by the kind of DON treatment. The exposure of pregnant sows at mid-gestation (days 63–70, period of organogenesis) to a Fusarium toxin-contaminated diet (4.42 mg DON and 0.048 mg ZON per kg) or pure DON via intraperitoneal osmotic minipump did not cause adverse effects on health, fertility, maintenance of pregnancy, and performance of sows and their fetuses. However, DON was detected in fetus plasma, indicating that this toxin can pass the placental barrier and may cause changes in the proportion of white blood cells (lower monocyte and neutrophil and higher lymphocyte proportion in DON per os fetuses).     

Tumor-infiltrating dendritic cell precursors recruited by a beta-defensin contribute to vasculogenesis under the influence of Vegf-A

The involvement of immune mechanisms in tumor angiogenesis is unclear. Here we describe a new mechanism of tumor vasculogenesis mediated by dendritic cell (DC) precursors through the cooperation of beta-defensins and vascular endothelial growth factor-A (Vegf-A). Expression of mouse beta-defensin-29 recruited DC precursors to tumors and enhanced tumor vascularization and growth in the presence of increased Vegf-A expression. A new leukocyte population expressing DC and endothelial markers was uncovered in mouse and human ovarian carcinomas coexpressing Vegf-A and beta-defensins. Tumor-infiltrating DCs migrated to tumor vessels and independently assembled neovasculature in vivo. Bone marrow-derived DCs underwent endothelial-like differentiation ex vivo, migrated to blood vessels and promoted the growth of tumors expressing high levels of Vegf-A. We show that beta-defensins and Vegf-A cooperate to promote tumor vasculogenesis by carrying out distinct tasks: beta-defensins chemoattract DC precursors through CCR6, whereas Vegf-A primarily induces their endothelial-like specialization and migration to vessels, which is mediated by Vegf receptor-2.     

Species-specific activation of Cu/Zn SOD by its CCS copper chaperone in the pathogenic yeast Candida albicans

Candida albicans is a pathogenic yeast of important public health relevance. Virulence of C. albicans requires a copper and zinc containing superoxide dismutase (SOD1), but the biology of C. albicans SOD1 is poorly understood. To this end, C. albicans SOD1 activation was examined in baker’s yeast (Saccharomyces cerevisiae), a eukaryotic expression system that has proven fruitful for the study of SOD1 enzymes from invertebrates, plants, and mammals. In spite of the 80 % similarity between S. cerevisiae and C. albicans SOD1 molecules, C. albicans SOD1 is not active in S. cerevisiae. The SOD1 appears incapable of productive interactions with the copper chaperone for SOD1 (CCS1) of S. cerevisiae. C. albicans SOD1 contains a proline at position 144 predicted to dictate dependence on CCS1. By mutation of this proline, C. albicans SOD1 gained activity in S. cerevisiae, and this activity was independent of CCS1. We identified a putative CCS1 gene in C. albicans and created heterozygous and homozygous gene deletions at this locus. Loss of CCS1 resulted in loss of SOD1 activity, consistent with its role as a copper chaperone. C. albicans CCS1 also restored activity to C. albicans SOD1 expressed in S. cerevisiae. C. albicans CCS1 is well adapted for activating its partner SOD1 from C. albicans, but not SOD1 from S. cerevisiae. In spite of the high degree of homology between the SOD1 and CCS1 molecules in these two fungal species, there exists a species-specific barrier in CCS–SOD interactions which may reflect the vastly different lifestyles of the pathogenic versus the noninfectious yeast.     

Differential Effects of apoE4 and Activation of ABCA1 on Brain and Plasma Lipoproteins

Apolipoprotein E4 (apoE4), the leading genetic risk factor for Alzheimer''s disease (AD), is less lipidated compared to the most common and AD-benign allele, apoE3. We have recently shown that i.p. injections of the ATP-binding cassette A1 (ABCA1) agonist peptide CS-6253 to apoE mice reverse the hypolipidation of apoE4 and the associated brain pathology and behavioral deficits. While in the brain apoE is the main cholesterol transporter, in the periphery apoE and apoA-I both serve as the major cholesterol transporters. We presently investigated the extent to which apoE genotype and CS-6253 treatment to apoE3 and apoE4-targeted replacement mice affects the plasma levels and lipid particle distribution of apoE, and those of plasma and brain apoA-I and apoJ. This revealed that plasma levels of apoE4 were lower and eluted faster following FPLC than plasma apoE3. Treatment with CS-6253 increased the levels of plasma apoE4 and rendered the elution profile of apoE4 similar to that of apoE3. Similarly, the levels of plasma apoA-I were lower in the apoE4 mice compared to apoE3 mice, and this effect was partially reversed by CS-6253. Conversely, the levels of apoA-I in the brain which were higher in the apoE4 mice, were unaffected by CS-6253. The plasma levels of apoJ were higher in apoE4 mice than apoE3 mice and this effect was abolished by CS-6253. Similar but less pronounced effects were obtained in the brain. In conclusion, these results suggest that apoE4 affects the levels of apoA-I and apoJ and that the anti-apoE4 beneficial effects of CS-6253 may be related to both central and peripheral mechanisms.     

Dependence of purinergic P2X receptor activity on ectodomain structure

Purinergic receptors (P2XRs) activate and desensitize in response to the binding of extracellular nucleotides in a receptor- and ligand-specific manner, but the structural bases of their ligand preferences and channel kinetics have been incompletely characterized. Here we tested the hypothesis that affinity of agonists for binding domain accounts for a ligand-specific desensitization pattern. We generated chimeras using receptors with variable sensitivity to ATP in order: P2X(4)R > P2X(2a)R = P2X(2b)R P2X(7)R. Chimeras having the ectodomain Ile(66)-Tyr(310) sequence of P2X(2)R and Val(61)-Phe(313) sequence of P2X(7)R in the backbone of P2X(4)R were expressed but were non-functioning channels. P2X(2a) + X(4)R and P2X(2b) + X(4)R chimeras having the Val(66)-Tyr(315) ectodomain sequence of P2X(4)R in the backbones of P2X(2a)R and P2X(2b)R were functional and exhibited increased sensitivity to ligands as compared with both parental receptors. These chimeras also desensitized faster than parental receptors and in a ligand-nonspecific manner. However, like parental P2X(2b)R and P2X(2a)R, chimeric P2X(2b) + X(4)R desensitized more rapidly than P2X(2a) + X(4)R, and the rate of desensitization of P2X(2a)+X(4)R increased by substituting its Arg(371)-Pro(376) intracellular C-terminal sequence with the Glu(376)-Gly(381) sequence of P2X(4)R. These results indicate the relevance of interaction between the ectodomain and flanking regions around the transmembrane domains on ligand potency and receptor activation. Furthermore, the ligand potency positively correlates with the rate of receptor desensitization but does not affect the C-terminal-specific pattern of desensitization.     

Glycerophosphate-dependent hydrogen peroxide production by brown adipose tissue mitochondria and its activation by ferricyanide

Oxidation of glycerophosphate (GP) by brown adipose tissue mitochondria in the presence of antimycin A was found to be accompanied by significant production of hydrogen peroxide. GP-dependent hydrogen peroxide production could be detected by p-hydroxyphenylacetate fluorescence changes or as an antimycin A-insensitive oxygen consumption. One-electron acceptor, potassium ferricyanide, highly stimulated the rate of GP-dependent antimycin A-insensitive oxygen uptake, which was prevented by inhibitors of mitochondrial GP dehydrogenase (mGPDH) or by coenzyme Q(CoQ). GP-dependent ferricyanide-induced peroxide production was also determined luminometrically, using mitochondria or partially purified mGPDH. Ferricyanide-induced peroxide production was negligible, when succinate or NADH was used as a substrate. These results indicate that hydrogen peroxide is produced directly by mGPDH and reflect the differences in the transport of reducing equivalents from mGPDH and succinate dehydrogenase to the CoQ pool. The data suggest that more intensive production of reactive oxygen species may be present in mammalian cells with active mGPDH.     

N-terminal truncations of manganese stabilizing protein identify two amino acid sequences required for binding of the eukaryotic protein to photosystem II and reveal the absence of one binding-related sequence in cyanobacteria

Manganese stabilizing protein (MSP) is an intrinsically disordered extrinsic subunit of photosystem II that regulates the stability and kinetic performance of the tetranuclear manganese cluster that oxidizes water to oxygen. An earlier study showed that deletion of the (1)E-(3)G domain of MSP caused no loss of activity reconstitution, whereas deletion of the (4)K-(10)E domain reduced binding of the protein from 2 to 1 mol of MSP/mol of photosystem II and lowered activity reconstitution to about 50% of the control value [Popelkova et al. (2002) Biochemistry 41, 2702-2711]. In this work we present evidence that deletion of 13 or 14 amino acid residues from the MSP N-terminus (mutants DeltaS13M and DeltaK14M) does not interfere either with functional binding of one copy of MSP to photosystem II or with reconstitution of oxygen evolution activity to 50% of the control level. Both of these mutants exhibit nonspecific binding to photosystem II at higher protein concentrations. Truncation of the MSP sequence by 18 amino acids (mutant DeltaE18M), however, causes a loss of protein binding and activity reconstitution. This result demonstrates that the N-terminal domain (15)T-(18)E is required for binding of at least one copy of MSP to photosystem II. Analyses of CD spectra reveal changes in the structure of DeltaE18M (loss of beta-sheet, gain of unordered structure). Use of the information gained from these experiments in analyses of N-terminal sequences of MSP from a number of species indicates that higher plants and algae possess two recognition domains that are required for MSP binding to PSII, whereas cyanobacteria lack the first N-terminal domain found in eukaryotes. This may explain the absence of a second copy of MSP in the crystal structure of PSII from Synechococcus elongatus [Zouni et al. (2001) Nature 409, 739-743].