Gene transfer to pre-hematopoietic and committed hematopoietic precursors in the early mouse Yolk Sac: a comparative study between <Emphasis Type="Italic">in situ</Emphasis> electroporation and retroviral transduction

Background  

Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated. While intra-embryonic precursors harbour the features of hematopoietic stem cells (HSC), precursors formed earlier in the yolk sac (YS) display limited differentiation and self-renewal potentials. The mechanisms leading to the generation of the precursors in both sites are still largely unknown, as are the molecular basis underlying their different potential. A possible approach to assess the role of candidate genes is to transfer or modulate their expression/activity in both sites. We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.     

European society of intensive care medicine study of therapeutic hypothermia (32-35°C) for intracranial pressure reduction after traumatic brain injury (the Eurotherm3235Trial)

Background

Severe malaria remains a major cause of global morbidity and mortality. Despite the use of potent anti-parasitic agents, the mortality rate in severe malaria remains high. Adjunctive therapies that target the underlying pathophysiology of severe malaria may further reduce morbidity and mortality. Endothelial activation plays a central role in the pathogenesis of severe malaria, of which angiopoietin-2 (Ang-2) has recently been shown to function as a key regulator. Nitric oxide (NO) is a major inhibitor of Ang-2 release from endothelium and has been shown to decrease endothelial inflammation and reduce the adhesion of parasitized erythrocytes. Low-flow inhaled nitric oxide (iNO) gas is a US FDA-approved treatment for hypoxic respiratory failure in neonates.

Methods/Design

This prospective, parallel arm, randomized, placebo-controlled, blinded clinical trial compares adjunctive continuous inhaled nitric oxide at 80 ppm to placebo (both arms receiving standard anti-malarial therapy), among Ugandan children aged 1-10 years of age with severe malaria. The primary endpoint is the longitudinal change in Ang-2, an objective and quantitative biomarker of malaria severity, which will be analysed using a mixed-effects linear model. Secondary endpoints include mortality, recovery time, parasite clearance and neurocognitive sequelae.

Discussion

Noteworthy aspects of this trial design include its efficient sample size supported by a computer simulation study to evaluate statistical power, meticulous attention to complex ethical issues in a cross-cultural setting, and innovative strategies for safety monitoring and blinding to treatment allocation in a resource-constrained setting in sub-Saharan Africa.

Trial Registration

ClinicalTrials.gov Identifier: NCT01255215     

Subcellular distribution of chromogranins A and B in bovine adrenal chromaffin cells

The major secretory granule proteins chromogranins A (CGA) and B (CGB) have recently been shown to play critical roles in inositol 1,4,5-trisphosphate-dependent intracellular Ca(2+) mobilizations. We determined here the subcellular distribution of CGA and CGB based on 3D-images of chromaffin cells, and found that approximately 95% of cellular CGA was present in secretory granules while approximately 5% was in the endoplasmic reticulum (ER), whereas approximately 57% of cellular CGB was in secretory granules while approximately 24% and approximately 19% were in the ER and nucleus, respectively. These results suggest that chromogranins are at the center of intracellular Ca(2+) homeostasis in secretory cells.     

Phosphorylation of focal adhesion kinase at tyrosine 861 is crucial for Ras transformation of fibroblasts

Although elevated expression and increased tyrosine phosphorylation of focal adhesion kinase (FAK) are crucial for tumor progression, the mechanism by which FAK promotes oncogenic transformation is unclear. We have therefore determined the role of FAK phosphorylation at tyrosine 861 in the oncogenic transformation of NIH3T3 fibroblasts. FAK phosphorylation at tyrosine 861 was increased in both constitutively H-Ras-transformed and H-Ras-inducible NIH3T3 cells, in parallel with cell transformation. However, H-Ras-inducible cells transfected with the nonphosphorylatable mutant FAK Y861F showed decreased migration/invasion, focus forming activity and anchorage-independent growth, compared with either wild-type or kinase-defective FAK. In contrast to unaltered FAK/Src activity, the association of FAK and p130(CAS) was decreased in FAK Y861F-transfected cells, and FAK phosphorylation at tyrosine 861 enhanced this association in vitro. Consistently, FAK Y861F-transfected cells were defective in activation of c-Jun NH(2)-terminal kinase and in expression of matrix metalloproteinase-9 during transformation. Taken together, these results strongly suggest that FAK phosphorylation at tyrosine 861 is crucial for H-Ras-induced transformation through regulation of the association of FAK with p130(CAS).     

A proteomic approach for dissecting H-Ras signaling networks in NIH/3T3 mouse embryonic fibroblast cells

To elucidate an understanding into H-Ras protein network, we have established various oncogene H-Ras-expressing NIH/3T3 mouse embryonic fibroblast cell clones, which are expressing G12V H-Ras, G12R H-Ras, and G12V/T35S H-Ras proteins under the tight control of expression by an antibiotic doxycycline. Here we provide a catalog of proteome profiles in total cell lysate derived from the oncogenic and partial loss of function H-Ras-expressing NIH/3T3 cells. In this biological context, we compared total proteome changes by the combined methods of 2-DE, quantitative image analysis and MALDI-TOF-MS analysis both commonly in oncogenic and partial loss of function H-Ras expression system. Thus, we tried to dissect H-Ras signaling pathway, especially a downstream effector molecule, Raf in NIH/3T3 cells using proteomics tools. In this study, we centralized upon the proteome profile changes as common targets for oncogenic H-Ras and a partial loss of function H-Ras in the H-Ras-expressing cells. Thirteen protein spots were selected as what the staining intensities on the gels for 2-DE images from both kinds of cells were consistently changed in their protein expression level. Differentially regulated expression was further confirmed for some subsets of candidates by semiquantitative RT-PCR and Western blot analysis using specific antibodies. Taken together, our results obtained and present here show that the comparative analysis of proteome from oncogenic and partial loss of function H-Ras-expressing cells has yielded interpretable data to elucidate the protein network directly and/or indirectly.     

Role of RepB in the replication of plasmid pJB01 isolated from Enterococcus faecium JC1

The plasmid pJB01 (GenBank Accession No. AY425961) isolated from the pathogenic bacterium, Enterococcus faecium JC1, is 2235 base pairs in length and consists of a putative double-strand origin (dso), a single-strand origin, a counter-transcribed RNA, and three open reading frames. A comparison of a few replication factors and motifs, bind and nic regions, for replication initiation on the nucleotide sequence level revealed that it belongs to the pMV158 family among RC-replicating plasmids. A runoff DNA synthesis assay demonstrated that nicking occurred between G525 and A526, which is located on the internal loop of a putative secondary structure in the dso. Unlike all the other plasmids of the pMV158 family having two or three direct repeats, pJB01 has three non-tandem direct repeats of 5'-CAACAAA-3' separated by four nucleotides, as the RepB-binding site in the dso. Moreover, the nick site on the internal loop is located at 77 nucleotides upstream from the RepB-binding region. Irrespective of the structural difference of direct repeats from other members of the pMV158 family, we think, it is still a new member of this plasmid family. The introduction of mutations in conserved regions of RepB confirmed that RepB N-moiety is important for nicking/nick-closing activity. Within N-moiety, especially all of the motif R-III, the Y100 in R-IV and Y116 in R-V residues, played particularly critical roles in this activity, however, for its binding, both of the N- and C-moieties of RepB were needed.     

Phosphorylation and concomitant structural changes in human 2-Cys peroxiredoxin isotype I differentially regulate its peroxidase and molecular chaperone functions

The H2O2-catabolizing peroxidase activity of human peroxiredoxin I (hPrxI) was previously shown to be regulated by phosphorylation of Thr90. Here, we show that hPrxI forms multiple oligomers with distinct secondary structures. HPrxI is a dual function protein, since it can behave either as a peroxidase or as a molecular chaperone. The effects of phosphorylation of hPrxI on its protein structure and dual functions were determined using site-directed mutagenesis, in which the phosphorylation site was substituted with aspartate to mimic the phosphorylated status of the protein (T90D-hPrxI). Phosphorylation of the protein induces significant changes in its protein structure from low molecular weight (MW) protein species to high MW protein complexes as well as its dual functions. In contrast to the wild type (WT)- and T90A-hPrxI, the T90D-hPrxI exhibited a markedly reduced peroxidase activity, but showed about sixfold higher chaperone activity than WT-hPrxI.     

The distribution and evolutionary history of the PRP8 intein

Background  

We recently described a mini-intein in the PRP8 gene of a strain of the basidiomycete Cryptococcus neoformans, an important fungal pathogen of humans. This was the second described intein in the nuclear genome of any eukaryote; the first nuclear encoded intein was found in the VMA gene of several saccharomycete yeasts. The evolution of eukaryote inteins is not well understood. In this report we describe additional PRP8 inteins (bringing the total of these to over 20). We compare and contrast the phylogenetic distribution and evolutionary history of the PRP8 intein and the saccharomycete VMA intein, in order to derive a broader understanding of eukaryote intein evolution. It has been suggested that eukaryote inteins undergo horizontal transfer and the present analysis explores this proposal.