Glucocorticoids regulate transendothelial fluid flow resistance and formation of intercellular junctions

The regulation of transendothelial fluid flow by glucocorticoidswas studied in vitro with use of human endothelial cells cultured fromSchlemm's canal (SCE) and the trabecular meshwork (TM) in conjunctionwith computer-linked flowmeters. After 2-7 wk of 500 nMdexamethasone (Dex) treatment, the following physiological, morphometric, and biochemical alterations were observed: a 3- to 5-foldincrease in fluid flow resistance, a 2-fold increase in therepresentation of tight junctions, a 10- to 30-fold reduction in themean area occupied by interendothelial "gaps" or preferential flow channels, and a 3- to 5-fold increase in the expression of thejunction-associated protein ZO-1. The more resistive SCE cells expressed two isoforms of ZO-1; TM cells expressed only one. To investigate the role of ZO-1 in the aforementioned Dex effects, itsexpression was inhibited using antisense phosphorothioate oligonucleotides, and the response was compared with that observed withthe use of sense and nonsense phosphorothioate oligonucleotides. Inhibition of ZO-1 expression abolished the Dex-induced increase inresistance and the accompanying alterations in cell junctions and gaps.These results support the hypothesis that intercellular junctions arenecessary for the development and maintenance of transendothelial flowresistance in cultured SCE and TM cells and are likely involved in themechanism of increased resistance associated with glucocorticoid exposure.

     

Glutamate-cysteine ligase attenuates TNF-induced mitochondrial injury and apoptosis

Glutathione (GSH) is important in free radical scavenging, maintaining cellular redox status, and regulating cell survival in response to a wide variety of toxicants. The rate-limiting enzyme in GSH synthesis is glutamate-cysteine ligase (GCL), which is composed of catalytic (GCLC) and modifier (GCLM) subunits. To determine whether increased GSH biosynthetic capacity enhances cellular resistance to tumor necrosis factor-alpha- (TNF-alpha-) induced apoptotic cell death, we have established several mouse liver hepatoma (Hepa-1) cell lines overexpressing GCLC and/or GCLM. Cells overexpressing GCLC alone exhibit modest increases in GCL activity, while cells overexpressing both subunits have large increases in GCL activity. Importantly, cells overexpressing both GCL subunits exhibit increased resistance to TNF-induced apoptosis as judged by a loss of redox potential; mitochondrial membrane potential; translocation of cytochrome c to the cytoplasm; and activation of caspase-3, caspase-8, and caspase-9. Analysis of the effects of TNF on these parameters indicates that maintaining mitochondrial integrity mediates this protective effect in GCL-overexpressing cells.     

Vesicular stomatitis virus glycoprotein: a transducing coat for SFV-based RNA vectors

BACKGROUND: Semliki Forest virus (SFV) vectors have a great potential for the induction of protective immunity in a large number of clinical conditions including cancer. Such a potential accounts for the huge efforts made to improve the in vivo expression from SFV vectors. It is noteworthy that efficient in vivo expression strongly relies on the ability to deliver high-titre vectors. To achieve this, the generation of recombinant SFV particles, using independent expression systems for structural SFV genes, has been proposed. However, despite several modifications in the production process, a risk of contamination with replication-competent, or partially recombined, virus has remained. METHODS: Here, we exploit the ability of the vesicular stomatitis virus glycoprotein (VSV-G), expressed in trans, to hijack full-length genomic SFV RNA into secreted virus-like particles (VLPs). To allow SFV vector mobilisation, we designed a CMV driven SFV vector in which the internal 26S promoter has been extensively mutated. With this vector, mobilisation events were monitored using the Green Fluorescent Protein (GFP). The production procedure involves a sequential transfection protocol, of plasmids expressing the VSV-G and the SFV vector respectively. RESULTS: We show that the VLPs are effective for cellular delivery of SFV vectors in a broad range of human and non-human cellular targets. Furthermore, production of VLPs is easy and allows, through concentration, the harvest of high-titre vector. CONCLUSIONS: The present paper describes a convenient process aimed at mobilising full length SFV vectors. A major issue to consider, while developing clinically relevant gene transfer vectors, is the risk of undesirable generation of replication competent by-products. Importantly, as the VSV-G gene shares no homology with the SFV genome, our VLPs offer a strong guarantee of biosafety.     

The Na,K-ATPase alpha 2 isoform is expressed in neurons,and its absence disrupts neuronal activity in newborn mice

Na,K-ATPase is an ion transporter that impacts neural and glial physiology by direct electrogenic activity and the modulation of ion gradients. Its three isoforms in brain have cell-type and development-specific expression patterns. Interestingly, our studies demonstrate that in late gestation, the alpha2 isoform is widely expressed in neurons, unlike in the adult brain, in which alpha2 has been shown to be expressed primarily in astrocytes. This unexpected distribution of alpha2 isoform expression in neurons is interesting in light of our examination of mice lacking the alpha2 isoform which fail to survive after birth. These animals showed no movement; however, defects in gross brain development, muscle contractility, neuromuscular transmission, and lung development were ruled out. Akinesia suggests a primary neuronal defect and electrophysiological recordings in the pre-B?tzinger complex, the brainstem breathing center, showed reduction of respiratory rhythm activity, with less regular and smaller population bursts. These data demonstrate that the Na,K-ATPase alpha2 isoform could be important in the modulation of neuronal activity in the neonate.     

New spatially explicit method for detecting extracellular protease activity in biofilms

A novel method of detecting extracellular protease activity at biofilm-substratum interfaces was developed. This method utilizes fluorescent molecules bound to cellulose substrata with a lectin. Extracellular proteases degrade the lectin and release the fluorochrome into solution. This new technique and a standard dissolved-substrate assay detected similar responses of biofilm extracellular protease activity to experimental manipulation of N supply. Combination of this technique with confocal scanning laser microscopy allowed direct visualization of microspatial patterns of bacterial distribution and extracellular protease activity at the biofilm-substratum interface.     

DNA immunisation. New histochemical and morphometric data

Splenic germinal center reactions were measured during primary response to a plasmidic DNA intramuscular injection. Cardiotoxin-pretreated Balb/c mice were immunized with DNA plasmids encodmg or not the SAG1 protein, a membrane antigen of Toxoplasma gondii. Specific anti-SAG1 antibodies were detected on days 16 and 36 after injection of coding plasmids. The results of ELISAs showed that the SAG1-specific antibodies are of the IgG2a class. Morphometric analyses were done on serial immunostained cryosections of spleen and draining or non-draining lymph nodes. This new approach made it possible to evaluate the chronological changes induced by DNA immunisation in the germinal centres (in number and in size). Significant increases in the number of germinal centres were measured in the spleen and only in draining lymph nodes after plasmid injection, the measured changes of the germinal centers appeared to result from the adjuvant stimulatory effect of the plasmidic DNA since both the coding and the noncoding plasmid DNA induced them. No measurable changes were recorded in the T-dependent zone of lymph organs.     

P52rIPK regulates the molecular cochaperone P58IPK to mediate control of the RNA-dependent protein kinase in response to cytoplasmic stress

The 52 kDa protein referred to as P52(rIPK) was first identified as a regulator of P58(IPK), a cellular inhibitor of the RNA-dependent protein kinase (PKR). P52(rIPK) and P58(IPK) each possess structural domains implicated in stress signaling, including the charged domain of P52(rIPK) and the tetratricopeptide repeat (TPR) and DnaJ domains of P58(IPK). The P52(rIPK) charged domain exhibits homology to the charged domains of Hsp90, including the Hsp90 geldanamycin-binding domain. Here we present an in-depth analysis of P52(rIPK) function and expression, which first revealed that the 114 amino acid charged domain was necessary and sufficient for interaction with P58(IPK). This domain bound specifically to P58(IPK) TPR domain 7, the domain adjacent to the TPR motif required for P58(IPK) interaction with PKR, thus providing a mechanism for P52(rIPK) inhibition of P58(IPK) function. Both the charged domain of P52(rIPK) and the TPR 7 domain of P58(IPK) were required for P52(rIPK) to mediate downstream control of PKR activity, eIF2alpha phosphorylation, and cell growth. Furthermore, we found that P52(rIPK) and P58(IPK) formed a stable intracellular complex during the acute response to cytoplasmic stress induced by a variety of stimuli. We propose a model in which the P52(rIPK) charged domain functions as a TPR-specific signaling motif to directly regulate P58(IPK) within a larger cytoplasmic stress signaling cascade culminating in the control of PKR activity and cellular mRNA translation.     

Cercosporin-deficient mutants by plasmid tagging in the asexual fungus<Emphasis Type="Italic"> Cercospora nicotianae</Emphasis>

We have successfully adapted plasmid insertion and restriction enzyme-mediated integration (REMI) to produce cercosporin toxin-deficient mutants in the asexual phytopathogenic fungus Cercospora nicotianae. The use of pre-linearized plasmid or restriction enzymes in the transformation procedure significantly decreased the transformation frequency, but promoted a complicated and undefined mode of plasmid integration that leads to mutations in the C. nicotianae genome. Vector DNA generally integrated in multiple copies, and no increase in single-copy insertion was observed when enzymes were added to the transformation mixture. Out of 1873 transformants tested, 39 putative cercosporin toxin biosynthesis ( ctb) mutants were recovered that showed altered levels of cercosporin production. Seven ctb mutants were recovered using pre-linearized plasmids without the addition of enzymes, and these were considered to be non-REMI mutants. The correlation between a specific insertion and a mutant phenotype was confirmed using rescued plasmids as gene disruption vectors in the wild-type strain. Six out of fifteen rescued plasmids tested yielded cercosporin-deficient transformants when re-introduced into the wild-type strain, suggesting a link between the insertion site and the cercosporin-deficient phenotype. Sequence analysis of a fragment flanking the insert site recovered from one insertion mutant showed it to be disrupted in sequences with high homology to the acyl transferase domain of polyketide synthases from other fungi. Disruption of this polyketide synthase gene ( CTB1) using a rescued plasmid resulted in mutants that were defective in cercosporin production. Thus, we provide the first molecular evidence that cercosporin is synthesized via a polyketide pathway as previously hypothesized.Communicated by E. Cerdá-Olmedo