Hepatocyte transplantation (HTx) corrects selected neurometabolic abnormalities in murine intermediate maple syrup urine disease (iMSUD)

Skvorak et al. [1] demonstrated the therapeutic efficacy of HTx in a murine model of iMSUD, confirming significant metabolic improvement and survival. To determine the effect of HTx on extrahepatic organs, we examined the metabolic effects of HTx in brain from iMSUD animals. Amino acid analysis revealed that HTx corrected increased ornithine, partially corrected depleted glutamine, and revealed a trend toward alloisoleucine correction. For amino acid and monoamine neurotransmitters, decreased GABA was partially corrected with HTx, while the l-histidine dipeptide of GABA, homocarnosine, was decreased in iMSUD mice and hypercorrected following HTx. Elevated branched-chain amino acids (BCAA; leucine, isoleucine, and valine) in MSUD can deplete brain tyrosine and tryptophan (the precursors of monoamine neurotransmitters, dopamine (DA) and serotonin (5-hydroxytryptamine; 5-HT)) through competition via the large neutral amino acid transporter. HTx corrected decreased DA levels and the DA metabolite, 3-methoxytyramine, and partially corrected the DA intermediate 3,4-dihydroxyphenylacetate (DOPAC) and 5-HT levels, despite normal tyrosine and tryptophan levels in iMSUD mouse brain. We further observed enhanced intracellular turnover of both DA and 5-HT in iMSUD mouse brain, both of which partially corrected with HTx. Our results suggest new pathomechanisms of neurotransmitter metabolism in this disorder and support the therapeutic relevance of HTx in iMSUD mice, while providing proof-of-principle that HTx has corrective potential in extrahepatic organs.     

Selection of Trichoderma stromaticum isolates for efficient biological control of witches’ broom disease in cacao

Witches’ broom is the most devastating disease of cacao in Brazil, and losses to it entail serious socio-economical and environmental problems. Biological control of the causal agent Moniliophthora perniciosa (Mp) using the antagonistic fungus Trichoderma stromaticum (Ts) is promising, although the identification of superior isolates is necessary. Here, we report a study on the selection of more effective Ts isolates based on field experiments. Sixty-three Ts isolates from a local collection were applied on brooms and placed under typical conditions of shaded-cacao plantations in southeastern Bahia State (Brazil), during two periods of three months each. The percentages of Ts sporulation and incidence and severity of Mp were the parameters used for biocontrol assessments. The results from both experiments were very distinct, indicating a high phenotypic variation in this collection and suggesting a significant effect of the environment in the Ts–Mp interaction. Ts-sporulation rates were negatively correlated with the presence of Mp in the brooms and a number of isolates reduced Mp incidence more efficiently than the reference isolate. Contrasting isolates in their efficiency of reducing Mp incidence were selected and further tested in four subsequent field trials for validation purposes. The results partially confirmed their biocontrol phenotypes but also suggested isolate-specific responses to environmental variations. Inhibition of Mp-basidiospore germination by total protein secreted in culture supernatants of Ts isolates correlated well with field results and revealed a potentially useful procedure for pre-screening of large collections towards selection of better biological control isolates. The characteristics and efficiency of the method as a reliable protocol for identification of superior BCAs in the witches’ broom—cacao pathosystem is discussed.     

Effects of waterlogging on seed germination of three Mediterranean oak species: Ecological implications

Soil water saturation during prolonged periods of time generates a negative impact on nearly all terrestrial plants. In Mediterranean woodlands, precipitation can be very abundant during the wet season, inducing temporary soil waterlogging, coinciding with the seed dispersal and germination time of many species. We investigated the effects of waterlogging on seed germination and early root growth of three coexisting oak species (Quercus canariensis, Q. suber and Q. pyrenaica), by completely flooding of seeds for various periods of time. The three oak species showed a certain level of tolerance to waterlogging, only being affected those seeds subjected for long periods of submersion (over 30 days). Waterlogging during prolonged periods of time decreased the probability of seed germination in the three oak species, lengthened the time to germination, and hampered root development in two of the studied species. The main differences between oak species occurred in terms of root growth (Q. canariensis being the less affected, and Q. suber the most); these differential responses could be related to a species rank of waterlogging tolerance. Thus inter-specific differences in germination responses to waterlogging could contribute to explain, at least partially, species habitat and distribution patterns across landscapes. Seed mass also played an important role on different aspects of germination, though its relative importance varied as function of species and waterlogging treatment. The tolerance to stress induced by waterlogging increased with seed mass, but only in the case of Q. canariensis.     

V H-ATPase along the yeast secretory pathway: Energization of the ER and Golgi membranes

H⁺ transport driven by V H⁺-ATPase was found in membrane fractions enriched with ER/PM and Golgi/Golgi-like membranes of Saccharomyces cerevisiae efficiently purified in sucrose density gradient from the vacuolar membranes according to the determination of the respective markers including vacuolar Ca²⁺-ATPase, Pmc1::HA. Purification of ER from PM by a removal of PM modified with concanavalin A reduced H⁺ transport activity of P H⁺-ATPase by more than 75% while that of V H⁺-ATPase remained unchanged. ER H⁺ ATPase exhibits higher resistance to bafilomycin (I₅₀ = 38.4 nM) than Golgi and vacuole pumps (I₅₀ = 0.18 nM). The ratio between a coupling efficiency of the pumps in ER, membranes heavier than ER, vacuoles and Golgi is 1.0, 2.1, 8.5 and 14 with the highest coupling in the Golgi. The comparative analysis of the initial velocities of H⁺ transport mediated by V H⁺-ATPases in the ER, Golgi and vacuole membrane vesicles, and immunoreactivity of the catalytic subunit A and regulatory subunit B further supported the conclusion that V H⁺-ATPase is the intrinsic enzyme of the yeast ER and Golgi and likely presented by distinct forms and/or selectively regulated.     

Interplay of cysteinyl leukotrienes and TGF-β in the activation of hepatic stellate cells from Schistosoma mansoni granulomas

Hepatic stellate cells (HSCs) have a critical role in liver physiology, and in the pathogenesis of liver inflammation and fibrosis. Here, we investigated the interplay between leukotrienes (LT) and TGF-β in the activation mechanisms of HSCs from schistosomal granulomas (GR-HSCs). First, we demonstrated that GR-HSCs express 5-lipoxygenase (5-LO), as detected by immunolocalization in whole cells and confirmed in cell lysates through western blotting and by mRNA expression through RT-PCR. Moreover, mRNA expression of 5-LO activating protein (FLAP) and LTC₄-synthase was also documented, indicating that GR-HSCs have the molecular machinery required for LT synthesis. Morphological analysis of osmium and Oil-Red O-stained HSC revealed large numbers of small lipid droplets (also known as lipid bodies). We observed co-localization of lipid droplet protein marker (ADRP) and 5-LO by immunofluorescence microscopy. We demonstrated that GR-HSCs were able to spontaneously release cysteinyl-LTs (CysLTs), but not LTB_4, into culture supernatants. CysLT production was highly enhanced after TGF-β-stimulation. Moreover, the 5-LO inhibitor zileuton and 5-LO gene deletion were able to inhibit the TGF-β-stimulated proliferation of GR-HSCs, suggesting a role for LTs in HSC activation. Here, we extend the immunoregulatory function of HSC by demonstrating that HSC from liver granulomas of schistosome-infected mouse are able to release Cys-LTs in a TGF-β-regulated manner, potentially impacting pathogenesis and liver fibrosis in schistosomiasis.     

Nitrogen fixation persists under conditions of salt stress in transgenic Medicago truncatula plants expressing a cyanobacterial flavodoxin

Several recent studies have demonstrated that the expression of a cyanobacterial flavodoxin in plants can provide tolerance to a wide range of environmental stresses. Indeed, this strategy has been proposed as a potentially powerful biotechnological tool to generate multiple‐tolerant crops. To determine whether flavodoxin expression specifically increased tolerance to salt stress and whether it might also preserve legume nitrogen fixation under saline conditions, the flavodoxin gene was introduced into the model legume Medicago truncatula. Expression of flavodoxin did not confer saline tolerance to the whole plant, although the sensitive nitrogen‐fixing activity was maintained under salt stress in flavodoxin‐expressing plants. Our results indicate that flavodoxin induced small but significant changes in the enzymatic activities involved in the nodule redox balance that might be responsible for the positive effect on nitrogen fixation. Expression of flavodoxin can be regarded as a potential tool to improve legume symbiotic performance under salt stress, and possibly other environmental stresses.     

Endoplasmic reticulum stress response in an INS-1 pancreatic β-cell line with inducible expression of a folding-deficient proinsulin

Background  

Cells respond to endoplasmic reticulum stress (ER) stress by activating the unfolded protein response. To study the ER stress response in pancreatic β-cells we developed a model system that allows for pathophysiological ER stress based on the Akita mouse. This mouse strain expresses a mutant insulin 2 gene (C96Y), which prevents normal proinsulin folding causing ER stress and eventual β-cell apoptosis. A double-stable pancreatic β-cell line (pTet-ON INS-1) with inducible expression of insulin 2 (C96Y) fused to EGFP was generated to study the ER stress response.     

Apoptin nucleocytoplasmic shuttling is required for cell type-specific localization, apoptosis, and recruitment of the anaphase-promoting complex/cyclosome to PML bodies

The chicken anemia virus protein Apoptin selectively induces apoptosis in transformed cells while leaving normal cells intact. This selectivity is thought to be largely due to cell type-specific localization: Apoptin is cytoplasmic in primary cells and nuclear in transformed cells. The basis of Apoptin cell type-specific localization and activity remains to be determined. Here we show that Apoptin is a nucleocytoplasmic shuttling protein whose localization is mediated by an N-terminal nuclear export signal (NES) and a C-terminal nuclear localization signal (NLS). Both signals are required for cell type-specific localization, since Apoptin fragments containing either the NES or the NLS fail to differentially localize in transformed and primary cells. Significantly, cell type-specific localization can be conferred in trans by coexpression of the two separate fragments, which interact through an Apoptin multimerization domain. We have previously shown that Apoptin interacts with the APC1 subunit of the anaphase-promoting complex/cyclosome (APC/C), resulting in G(2)/M cell cycle arrest and apoptosis in transformed cells. We found that the nucleocytoplasmic shuttling activity is critical for efficient APC1 association and induction of apoptosis in transformed cells. Interestingly, both Apoptin multimerization and APC1 interaction are mediated by domains that overlap with the NES and NLS sequences, respectively. Apoptin expression in transformed cells induces the formation of PML nuclear bodies and recruits APC/C to these subnuclear structures. Our results reveal a mechanism for the selective killing of transformed cells by Apoptin.     

Population dynamics of Aceodromus convolvuli (Acari: Mesostigmata: Blattisociidae) on spontaneous plants associated with Jatropha curcas in central Brazil

Spontaneously growing plants are commonly considered competitors of cultivated plants. Owing to the lack of specificity of many arthropods, spontaneous plants may be attacked by the same arthropods that attack cultivated plants and they may also harbor natural enemies of organisms harmful to cultivated plants. Aceodromus convolvuli Muma (Blattisociidae) has been reported recently in relatively large numbers in Tocantins state, central Brazil, mostly on Helicteres guazumifolia Kunth (Malvaceae). Very little has been reported about the population dynamics of blattisociid mites under field conditions. The objective of this work was to study the population dynamics of A. convolvuli in Gurupi, Tocantins state, to evaluate its possible interaction with associated mites. Monthly samples were taken from leaves of the 11 most abundant and frequent spontaneous plants in a Jatropha curcas L. (Euphorbiaceae) plantation. About 96.5 % of the specimens of A. convolvuli were collected in the rainy season. The patterns of variation of the population of A. convolvuli and of predators belonging to the family Phytoseiidae were similar, but A. convolvuli was much more numerous than all phytoseiid specimens combined. Highly significant correlations were observed between A. convolvuli densities and relative humidity or diversity of spontaneous plants. When only mites on H. guazumifolia were considered, highly significant correlation was also observed between densities of A. convolvuli and of mites of the family Tetranychidae. The results suggested that A. convolvuli could be a predator of tenuipalpid and/or tetranychid mites. Studies about its biology are needed to determine its preferred food sources and potential as biological control agent.