In vivo transport of folded EGFP by the DeltapH/TAT-dependent pathway in chloroplasts of Arabidopsis thaliana

Among the protein translocation pathways of the thylakoid membrane in chloroplasts, the DeltapH/TAT pathway is unique in several aspects. In vitro transport assays with isolated chloroplasts or thylakoids have defined the trans-thylakoidal proton gradient as the sole requirement for effecting transport. From these studies, evidence has also accumulated indicating that, in contrast to the remaining protein transport pathways present in the thylakoid membrane, the DeltapH/TAT pathway is able to mediate the transport of folded proteins. The present work has established a novel approach to demonstrate the transport of folded proteins by this pathway in vivo. For this purpose, Arabidopsis thaliana plants were stably transformed with gene constructs expressing enhanced green fluorescent protein (EGFP) alone or fused to the transit peptides of different chloroplast proteins under the control of the 35S CAMV promoter. The intracellular and intraorganellar distribution of EGFP in the resulting transformants showed that while all the chloroplast transit peptides efficiently mediated the transport of EGFP into plastids, only those specific for the DeltapH/TAT pathway were able to direct the protein into the thylakoid lumen as well. This could be demonstrated both by fluorescence and immunoelectron microscopy. Analysis of isolated and fractionated chloroplasts using western blot and spectrofluorometric assays confirmed the presence of folded EGFP solely within the thylakoid lumen of these lines. These results strongly suggest that the protein adopts a folded state in the chloroplast stroma and thus, can only be translocated further into the chloroplast lumen by the DeltapH/TAT pathway.     

Aflatoxin M1 effects on Xenopus laevis development

BACKGROUND: The principal Aflatoxin B(1) (AFB(1)) hydroxylated metabolite excreted in milk is Aflatoxin M(1) (AFM(1)) classified in group 2B by the International Agency for Research on Cancer (IARC). Human exposure to AFM(1) is due to the consumption of contaminated dairy products and partly to endogenous production through AFB(1) liver metabolism. METHODS: Since no data are available on AFM(1) embryotoxicity, its lethal and teratogenic potential was investigated using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). Stage-8 blastulae were exposed to AFM(1) at 1, 4, 16, 64, and 256 microg/L concentrations until stage 47, free-swimming larva. RESULTS: A slight increase of mortality and malformed larva percents was found in AFM(1)-exposed groups but these differences were not statistically significant in comparison with the controls. CONCLUSIONS: Therefore, AFM(1) is a non-embryotoxic compound when evaluated with a FETAX model at concentrations under the conditions tested. However, AFM(1) merits further studies using mammals as experimental models to identify a possible risk during human pregnancy.     

Proteome analysis of rat liver mitochondria reveals a possible compensatory response to endotoxic shock

Organ failure induced by endotoxic shock has recently been associated with affected mitochondrial function. In this study, effects of in vivo lipopolysaccharide-challenge on protein patterns of rat liver mitochondria in treated animals versus controls were studied by two-dimensional electrophoresis (differential image gel electrophoresis). Significant upregulation was found for ATP-synthase alpha chain and superoxide dismutase [Mn]. Our data suggest that endotoxic shock mediated changes in the mitochondrial proteome contribute to a compensatory reaction (adaptation to endotoxic shock) rather than to a mechanism of cell damage.     

Regulatory effects of synthetic liver X receptor- and peroxisome-proliferator activated receptor agonists on sterol transport pathways in polarized cerebrovascular endothelial cells

The blood-brain barrier contributes to maintain brain cholesterol metabolism and protects this uniquely balanced system from exchange with plasma lipoprotein cholesterol. Brain capillary endothelial cells, representing a physiological barrier to the central nervous system, express apolipoprotein A-I (apoA-I, the major high-density lipoprotein (HDL)-associated apolipoprotein), ATP-binding cassette transporter A1 (ABCA1), and scavenger receptor, class B, type I (SR-BI), proteins that promote cellular cholesterol mobilization. Liver X receptors (LXRs) and peroxisome-proliferator activated receptors (PPARs) are regulators of cholesterol transport, and activation of LXRs and PPARs has potential therapeutic implications for lipid-related neurodegenerative diseases. To clarify the functional impact of LXR/PPAR activation, sterol transport along the: (i) ABCA1/apoA-I and (ii) SR-BI/HDL pathway was investigated in primary, polarized brain capillary endothelial cells, an in vitro model of the blood-brain barrier. Activation of LXR (24(S)OH-cholesterol, TO901317), PPARalpha (bezafibrate, fenofibrate), and PPARgamma (troglitazone, pioglitazone) modulated expression of apoA-I, ABCA1, and SR-BI on mRNA and/or protein levels without compromising transendothelial electrical resistance or tight junction protein expression. LXR-agonists and troglitazone enhanced basolateral-to-apical cholesterol mobilization in the absence of exogenous sterol acceptors. Along with the induction of cell surface-located ABCA1, several agonists enhanced cholesterol mobilization in the presence of exogenous apoA-I, while efflux of 24(S)OH-cholesterol (the major brain cholesterol metabolite) in the presence of exogenous HDL remained unaffected. Summarizing, in cerebrovascular endothelial cells apoA-I, ABCA1, and SR-BI represent drug targets for LXR and PPAR-agonists to interfere with cholesterol homeostasis at the periphery of the central nervous system.     

The yeast tumor suppressor homologue Sro7p is required for targeting of the sodium pumping ATPase to the cell surface

The SRO7/SOP1 encoded tumor suppressor homologue of Saccharomyces cerevisiae is required for maintenance of ion homeostasis in cells exposed to NaCl stress. Here we show that the NaCl sensitivity of the sro7Delta mutant is due to defective sorting of Ena1p, the main sodium pump in yeast. On exposure of sro7Delta mutants to NaCl stress, Ena1p fails to be targeted to the cell surface, but is instead routed to the vacuole for degradation via the multivesicular endosome pathway. SRO7-deficient mutants accumulate post-Golgi vesicles at high salinity, in agreement with a previously described role for Sro7p in late exocytosis. However, Ena1p is not sorted into these post-Golgi vesicles, in contrast to what is observed for the vesicles that accumulate when exocytosis is blocked in sec6-4 mutants at high salinity. These observations imply that Sro7p has a previously unrecognized role for sorting of specific proteins into the exocytic pathway. Screening for multicopy suppressors identified RSN1, encoding a transmembrane protein of unknown function. Overexpression of RSN1 restores NaCl tolerance of sro7Delta mutants by retargeting Ena1p to the plasma membrane. We propose a model in which blocked exocytic sorting in sro7Delta mutants, gives rise to quality control-mediated routing of Ena1p to the vacuole.     

Assessing the Viability of Bacterial Species in Drinking Water by Combined Cellular and Molecular Analyses

The question which bacterial species are present in water and if they are viable is essential for drinking water safety but also of general relevance in aquatic ecology. To approach this question we combined propidium iodide/SYTO9 staining (“live/dead staining” indicating membrane integrity), fluorescence-activated cell sorting (FACS) and community fingerprinting for the analysis of a set of tap water samples. Live/dead staining revealed that about half of the bacteria in the tap water had intact membranes. Molecular analysis using 16S rRNA and 16S rRNA gene-based single-strand conformation polymorphism (SSCP) fingerprints and sequencing of drinking water bacteria before and after FACS sorting revealed: (1) the DNA- and RNA-based overall community structure differed substantially, (2) the community retrieved from RNA and DNA reflected different bacterial species, classified as 53 phylotypes (with only two common phylotypes), (3) the percentage of phylotpes with intact membranes or damaged cells were comparable for RNA- and DNA-based analyses, and (4) the retrieved species were primarily of aquatic origin. The pronounced difference between phylotypes obtained from DNA extracts (dominated by Betaproteobacteria, Bacteroidetes, and Actinobacteria) and from RNA extracts (dominated by Alpha-, Beta-, Gammaproteobacteria, Bacteroidetes, and Cyanobacteria) demonstrate the relevance of concomitant RNA and DNA analyses for drinking water studies. Unexpected was that a comparable fraction (about 21%) of phylotypes with membrane-injured cells was observed for DNA- and RNA-based analyses, contradicting the current understanding that RNA-based analyses represent the actively growing fraction of the bacterial community. Overall, we think that this combined approach provides an interesting tool for a concomitant phylogenetic and viability analysis of bacterial species of drinking water.     

Authentication of primordial characteristics of the CLBL-1 cell line prove the integrity of a canine B-cell lymphoma in a murine in vivo model

Cell lines are key tools in cancer research allowing the generation of neoplasias in animal models resembling the initial tumours able to mimic the original neoplasias closely in vivo. Canine lymphoma is the major hematopoietic malignancy in dogs and considered as a valuable spontaneous large animal model for human Non-Hodgkin's Lymphoma (NHL). Herein we describe the establishment and characterisation of an in vivo model using the canine B-cell lymphoma cell line CLBL-1 analysing the stability of the induced tumours and the ability to resemble the original material. CLBL-1 was injected into Rag2(-/-)γ(c) (-/-) mice. The generated tumor material was analysed by immunophenotyping and histopathology and used to establish the cell line CLBL-1M. Both cell lines were karyotyped for detection of chromosomal aberrations. Additionally, CLBL-1 was stimulated with IL-2 and DSP30 as described for primary canine B-cell lymphomas and NHL to examine the stimulatory effect on cell proliferation. CLBL-1 in vivo application resulted in lymphoma-like disease and tumor formation. Immunophenotypic analysis of tumorous material showed expression of CD45(+), MHCII(+), CD11a(+) and CD79αcy(+). PARR analysis showed positivity for IgH indicating a monoclonal character. These cytogenetic, molecular, immunophenotypical and histological characterisations of the in vivo model reveal that the induced tumours and thereof generated cell line resemble closely the original material. After DSP30 and IL-2 stimulation, CLBL-1 showed to respond in the same way as primary material. The herein described CLBL-1 in vivo model provides a highly stable tool for B-cell lymphoma research in veterinary and human medicine allowing various further in vivo studies.     

Diarrhea,Stimulation and Growth Predict Neurodevelopment in Young North Indian Children

Background and Objective

Infants and young children in low to middle-income countries are at risk for adverse neurodevelopment due to multiple risk factors. In this study, we sought to identify stimulation and learning opportunities, growth, and burden of respiratory infections and diarrhea as predictors for neurodevelopment.

Methods

We visited 422 North Indian children 6 to 30 months old weekly for six months. Childhood illnesses were assessed biweekly. At end study, we assessed neurodevelopment using the Ages and Stages Questionnaire 3^rd ed. (ASQ-3) and gathered information on stimulation and learning opportunities. We identified predictors for ASQ-3 scores in multiple linear and logistic regression models.

Results

We were able to explain 30.5% of the variation in the total ASQ-3 score by the identified predictors. When adjusting for child characteristics and annual family income, stimulation and learning opportunities explained most of the variation by 25.1%. Height for age (standardized beta: 0.12, p<.05) and weight for height z-scores (std. beta: 0.09, p<.05) were positively associated with the total ASQ-3 score, while number of days with diarrhea was negatively associated with these scores (std. beta: -0.13, p<0.01).

Conclusion

Our results support the importance of early child stimulation and general nutrition for child development. Our study also suggests that diarrhea is an additional risk factor for adverse neurodevelopment in vulnerable children.