The plastidic 2-cysteine peroxiredoxin is a target for a thioredoxin involved in the protection of the photosynthetic apparatus against oxidative damage

The chloroplastic drought-induced stress protein of 32 kD (CDSP32) is composed of two thioredoxin modules and is induced by environmental and oxidative stress conditions. We investigated whether the plastidic protein BAS1, which is related to eubacterial 2-Cys peroxiredoxin, is a target for CDSP32. Using a CDSP32 active-site mutant, we showed that the BAS1 and CDSP32 proteins form a mixed disulfide complex in vitro. Moreover, affinity chromatography indicated that BAS1 is a major target for CDSP32 in chloroplasts. CDSP32 was able to reduce BAS1 in vitro, and BAS1 displayed CDSP32-dependent peroxidase activity. The function of CDSP32 was investigated in transgenic potato lines without detectable levels of the protein as a result of cosuppression. Under conditions of photooxidative stress induced by incubation with either methyl viologen or t-butyl hydroperoxide or by exposure to low temperature under high light, plants lacking CDSP32 exhibited decreased maximal photosystem II photochemical efficiencies compared with the wild type and transgenic controls. In addition, plants without CDSP32 retained much less chlorophyll than controls under stress, indicating increased damage to photosynthetic membranes. We conclude that CDSP32 is a thioredoxin with a critical role in plastid defense against oxidative damage and that this role is related to its function as a physiological electron donor to the BAS1 peroxiredoxin.     

G protein-coupled receptor-mediated phosphorylation of the activation loop of protein kinase D: dependence on plasma membrane translocation and protein kinase Cepsilon

Protein kinase D (PKD) is a serine/threonine protein kinase activated by G protein-coupled receptor (GPCR) agonists through an incompletely characterized mechanism that includes its reversible plasma membrane translocation and activation loop phosphorylation via a protein kinase C (PKC)-dependent pathway. To gain a better understanding of the mechanism regulating the activation of PKD in response to GPCR stimulation, we investigated the role of its rapid plasma membrane translocation on its activation loop phosphorylation and identified the endogenous PKC isozyme that mediates that event in vivo. We had found that the activation loop of a PKD mutant, with reduced affinity for diacylglycerol and phorbol esters, was only phosphorylated upon its plasma membrane association. We also found that the activation loop phosphorylation and rapid plasma membrane dissociation of PKD were inhibited either by preventing the plasma membrane translocation of PKCepsilon, through abolition of its interaction with receptor for activated C kinase, or by suppressing the expression of PKCepsilon via specific small interfering RNAs. Thus, this study demonstrates that the plasma membrane translocation of PKD, in response to GPCR stimulation, is necessary for the PKCepsilon-mediated phosphorylation of the activation loop of PKD and that this event requires the translocation of both kinases to the plasma membrane. Based on these and previous results, we propose a model of GPCR-mediated PKD regulation that integrates its changes in distribution, catalytic activity, and multisite phosphorylation.     

The HIV-1 capsid protein C-terminal domain in complex with a virus assembly inhibitor

Immature HIV particles bud from infected cells after assembly at the cytoplasmic side of cellular membranes. This assembly is driven by interactions between Gag polyproteins. Mature particles, each containing a characteristic conical core, are later generated by proteolytic maturation of Gag in the virion. The C-terminal domain of the HIV-1 capsid protein (C-CA) has been shown to contain oligomerization determinants essential for particle assembly. Here we report the 1.7-A-resolution crystal structure of C-CA in complex with a peptide capable of inhibiting immature- and mature-like particle assembly in vitro. The peptide inserts as an amphipathic alpha-helix into a conserved hydrophobic groove of C-CA, resulting in formation of a compact five-helix bundle with altered dimeric interactions. This structure thus reveals the details of an allosteric site in the HIV capsid protein that can be targeted for antiviral therapy.     

Feeding level in the period previous to the late fattening phase influences fat composition at slaughter in free-ranged Iberian pigs

A group of 11 pigs was fed with 70 g feed per kg of metabolic weight (H pigs) and another group of 11 pigs was fed with 50g feed per kg of metabolic weight (L pigs). In both experimental groups (H and L pigs), it was observed that the higher initial proportion of C16:0, C18:0 and C18:2 (n-6) in backfat at the beginning of the free-range feeding period, the greater decrease rate of these fatty acid proportions regarding weight gain during the free-range fattening period took place. On the other hand, the greater initial proportion of C18:1 (n-9), the smaller increase rate in the concentration of this fatty acid was observed. The intramuscular neutral lipids from L pigs had higher C18:3 (n-3) and lower proportions of monounsaturated fatty acids (MUFA) than those from H pigs, while intramuscular polar lipids from L pigs had significantly higher proportions of C18:0, PUFA, C18:3 (n-3) and (n-3) and significantly lower MUFA and C18:1 (n-9) proportions than those from H pigs. The alpha-tocopherol concentration found in Longissimus dorsi from L pigs was significantly higher (p < 0.012) than those from H pigs.     

Traditional water mite fixatives and their compatibility with later DNA studies

This work compares frozen water, 70% alcohol, Koenike's and Angelier's fluid as preservation media for water mites in terms of their eventual facilitation of DNA extraction and amplification. The time the mites spent in the fixatives ranged between 1 week and 25 years. Two molecular markers were amplified: 16S ribosomal DNA and CoI mitochondrial DNA. DNA was extractable and could be sequenced from specimens fixed in all the above media, although this generally became more difficult as time progressed. In the light of the known characteristics of these media, the results suggest Angelier's fluid to be the most practical, especially on long expeditions.     

Acute regulation of translation initiation by gonadotropin-releasing hormone in the gonadotrope cell line LbetaT2

The hypothalamic neuropeptide hormone GnRH is the central regulator of reproductive function. GnRH stimulates the synthesis and release of the gonadotropins LH and FSH by the gonadotropes of the anterior pituitary through activation of the G-protein-coupled GnRH receptor. In this study, we investigated the role of translational control of hormone synthesis by the GnRH receptor in the novel gonadotrope cell line LbetaT2. Using immunohistochemical and RIA studies with this model, we show that acute GnRH-induced synthesis and secretion of LH are dependent upon new protein synthesis but not new mRNA synthesis. We examined the response to GnRH and found that activation of cap-dependent translation occurs within 4 h. LHbeta promoter activity was also examined, and we found no increases in LHbeta promoter activity after 6 h of GnRH stimulation. Additionally, we show that increased phosphorylation of translation initiation proteins, 4E-binding protein 1, eukaryotic initiation factor 4E, and eukaryotic initiation factor 4G, occur in a dose- and time-dependent manner in response to GnRH stimulation. Quantitative luminescent image analysis of Western blots shows that 10 nm GnRH is sufficient to cause a maximal increase in factor phosphorylation, and maximal responses occur within 30 min of stimulation. Further, we demonstrate that the MAPK kinase inhibitor, PD 98059, abolishes the GnRH-mediated stimulation of a cap-dependent translation reporter. More specifically, we demonstrate that PD 98059 abolishes the GnRH-mediated stimulation of a downstream target of the ERK pathway, MAPK-interacting kinase. Based on these findings, we conclude that acute GnRH stimulation of LbetaT2 cells increases translation initiation through ERK signaling. This may contribute to the acute increases in LHbeta subunit production.     

Cutting edge: human B cell function is regulated by interaction with soluble CD14: opposite effects on IgG1 and IgE production

The mechanism(s) controlling activation of naive B cells, their proliferation, Ag receptor affinity maturation, isotype switching, and their fate as memory or plasma cells is not fully elucidated. Here we show that between 24 and 60% of CD19+ cells in PBMC bind soluble CD14 (sCD14). Tonsillar B cells also bind sCD14, but preferentially the CD38-ve/low cells. Interaction of sCD14 with B cells resulted in higher levels of IgG1 and marked inhibition of IgE production by activated tonsillar B cells and Ag-stimulated PBMC. We found that sCD14 interfered with CD40 signaling in B cells, inhibited IL-6 production by activated B cells, and increased the kinetics and magnitude of CD40 ligand expression on T cells. Together with the previously reported effects on T cells, these findings define sCD14 as a novel soluble regulatory factor capable of modulating cellular and humoral immune responses by interacting directly with T and B cells.     

Comparative ability to detoxify alder leaf litter in field larval mosquito collections

The larvicidal effects of polyphenols from dietary alder leaf litter were investigated in different field collections of three detritivorous Aedes taxa (Ae. detritus, Ae. cataphylla, Ae. rusticus) and compared to the cytochrome P450 monooxygenase, glutathione S-transferase, and esterase activities. Larvae from polyphenol-rich habitats had a higher tolerance for polyphenols and higher midgut cytochrome P450 and esterase activities than larvae from polyphenol-poor habitats. Furthermore, the role of P450 enzymes in the mechanism of resistance to alder polyphenols was suggested by the synergistic effect in vivo of piperonyl butoxide in the resistant Ae. rusticus. This confirms the importance of polyphenols to larval mosquito performance, and provides evidence for the importance of specific detoxification mechanisms for tolerance to dietary polyphenols. Arch.