NOV/CCN3 induces adhesion of muscle skeletal cells and cooperates with FGF2 and IGF-1 to promote proliferation and survival

During mammalian development, expression of the Nephroblastoma overexpressed gene (NOV/CCN3) is tightly regulated in skeletal muscles. Ex vivo, ectopic expression of NOV blocks myogenic differentiation. NOV also supports endothelial cell adhesion and angiogenesis through interactions with integrins. Integrins play fundamental roles during myogenesis. In this study, we show that NOV mediates adhesion and spreading of myoblasts. Myoblasts adhesion to NOV does not require proteoglycans and is dependent on integrin beta1, whereas spreading involves another RGD-sensitive integrin. The C-Terminal part of NOV as well as full-length is able to support adhesion of myoblasts; in addition, both increase focal-adhesion kinase (FAK) phosphorylation. Furthermore, NOV is an adhesive substrate that, combined with FGF2 or IGF-1, promotes cell specific proliferation and survival, respectively, in a better way than fibronectin. Taken together, these results identify NOV as an adhesion substrate for myoblasts which, in concert with growth factors, could play a role in the physiology of muscle cells.     

Effects of chronic paroxetine treatment on dialysate serotonin in 5-HT1B receptor knockout mice

The role of serotonin (5-HT)1B receptors in the mechanism of action of selective serotonin re-uptake inhibitors (SSRI) was studied by using intracerebral in vivo microdialysis in conscious, freely moving wild-type and 5-HT1B receptor knockout (KO 5-HT1B) mice in order to compare the effects of chronic administration of paroxetine via osmotic minipumps (1 mg per kg per day for 14 days) on extracellular 5-HT levels ([5-HT]ext) in the medial prefrontal cortex and ventral hippocampus. Basal [5-HT]ext values in the medial prefrontal cortex and ventral hippocampus, approximately 20 h after removing the minipump, were not altered by chronic paroxetine treatment in both genotypes. On day 15, in the ventral hippocampus, an acute paroxetine challenge (1 mg/kg i.p.) induced a larger increase in [5-HT]ext in saline-pretreated mutant than in wild-type mice. This difference between the two genotypes in the effect of the paroxetine challenge persisted following chronic paroxetine treatment. Conversely, in the medial prefrontal cortex, the paroxetine challenge increased [5-HT]ext similarly in saline-pretreated mice of both genotypes. Such a challenge produced a further increase in cortical [5-HT]ext compared with that in saline-pretreated groups of both genotypes, but no differences were found between genotypes following chronic treatment. To avoid the interaction with raphe 5-HT1A autoreceptors, 1 micro m paroxetine was perfused locally through the dialysis probe implanted in the ventral hippocampus; similar increases in hippocampal [5-HT]ext were found in acutely or chronically treated wild-type mice. Systemic administration of the mixed 5-HT1B/1D receptor antagonist GR 127935 (4 mg/kg) in chronically treated wild-type mice potentiated the effect of a paroxetine challenge dose on [5-HT]ext in the ventral hippocampus, whereas systemic administration of the selective 5-HT1A receptor antagonist WAY 100635 did not. By using the zero net flux method of quantitative microdialysis in the medial prefrontal cortex and ventral hippocampus of wild-type and KO 5-HT1B mice, we found that basal [5-HT]ext and the extraction fraction of 5-HT were similar in the medial prefrontal cortex and ventral hippocampus of both genotypes, suggesting that no compensatory response to the constitutive deletion of the 5-HT1B receptor involving changes in 5-HT uptake capacity occurred in vivo. As steady-state brain concentrations of paroxetine at day 14 were similar in both genotypes, it is unlikely that differences in the effects of a paroxetine challenge on hippocampal [5-HT]ext are due to alterations of the drug's pharmacokinetic properties in mutants. These data suggest that there are differences between the ventral hippocampus and medial prefrontal cortex in activation of terminal 5-HT1B autoreceptors and their role in regulating dialysate 5-HT levels. These presynaptic receptors retain their capacity to limit 5-HT release mainly in the ventral hippocampus following chronic paroxetine treatment in mice.     

Eicosanoid Release Is Increased by Membrane Destabilization and CFTR Inhibition in Calu-3 Cells

The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2α) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis. This could in part account for the abnormal inflammatory status characteristic of this disease. We postulated that CFTR participates in the regulation of eicosanoid release by direct interaction with a complex containing ANXA1, p11 and cPLA2α. We first analyzed by plasmon surface resonance the in vitro binding of CFTR to the three proteins. A significant interaction between p11 and the NBD1 domain of CFTR was found. We observed in Calu-3 cells a rapid and partial redistribution of all four proteins in detergent resistant membranes (DRM) induced by TNF-α. This was concomitant with increased IL-8 synthesis and cPLA2α activation, ultimately resulting in eicosanoid (PGE2 and LTB4) overproduction. DRM destabilizing agent methyl-β-cyclodextrin induced further cPLA2α activation and eicosanoid release, but inhibited IL-8 synthesis. We tested in parallel the effect of short exposure of cells to CFTR inhibitors Inh172 and Gly-101. Both inhibitors induced a rapid increase in eicosanoid production. Longer exposure to Inh172 did not increase further eicosanoid release, but inhibited TNF-α-induced relocalization to DRM. These results show that (i) CFTR may form a complex with cPLA2α and ANXA1 via interaction with p11, (ii) CFTR inhibition and DRM disruption induce eicosanoid synthesis, and (iii) suggest that the putative cPLA2/ANXA1/p11/CFTR complex may participate in the modulation of the TNF-α-induced production of eicosanoids, pointing to the importance of membrane composition and CFTR function in the regulation of inflammation mediator synthesis.     

Expression of a functional C5a receptor in regenerating hepatocytes and its involvement in a proliferative signaling pathway in rat

Activation of the complement system generates the anaphylatoxin C5a whose activities are mediated through its binding to the widely expressed C5aR. C5aR mRNA and protein expressions are known to be induced in rat hepatocytes under inflammatory conditions. However, little is known about the role of the C5a/C5aR complex in liver and its involvement during a proliferative process. We have evaluated the expression of C5aR in regenerating rat hepatocytes following a partial hepatectomy and in hepatocyte cultures. C5aR induction was observed in hepatocytes from regenerating liver, as well as in normal hepatocytes under a culture-induced stress. The effect of a stimulation by a C5a agonist upon the synthesis of a growth factor/receptor pair (hepatocyte growth factor/c-Met) was also evaluated. Our data demonstrated an up-regulated expression of hepatocyte growth factor and c-Met mRNAs, but we failed to observe a direct mitogenic effect of C5a in culture. However, a significantly increased expression of cyclin E and D1mRNA levels, as well as an increased BrdU incorporation, were observed in rats given an i.v. C5a agonist injection following an 80% partial hepatectomy. These studies demonstrate for the first time that: 1) C5aR is up-regulated during liver regeneration, 2) the binding of C5a to C5aR promotes a growth response, and 3) C5aR is involved in a cell cycle signaling pathway. Taken together, these findings point to a novel role for the hepatic C5aR implicating this complement system in the context of normal or abnormal proliferative pathways.     

Activation of C-Jun N-terminal kinase is required for glutathione transferase A4 induction during oxidative stress, not during cell proliferation, in mouse hepatocytes

Expression of the mouse glutathione transferase Alpha 4 (mGSTA4) has been studied during hepatocyte isolation and in cultured hepatocytes. Transient mGSTA4 induction during liver disruption correlated to strong oxidative stress and induction of the Jun N-terminal kinase (JNK) pathway. Similarly, tumor necrosis factor alpha induced both JNK phosphorylation and mGSTA4 expression while specific JNK inhibitor JNKI1 prevented these two events and JNK activator anisomycin strongly induced mGSTA4 expression. We also found that endogenous JNK and mGSTA4 co-immunoprecipitate. A second mGSTA4 induction occurred 2 days after cell seeding concomitantly to DNA replication and was prevented by treatment with mitogen-activated protein kinase (MEK) inhibitor U0126. Our data demonstrate that mGSTA4 is strongly increased during oxidative stress possibly via JNK pathway and during proliferation via MEK/extracellular signal-regulated kinase pathway, and suggest that mGSTA4 might be an endogenous regulator of JNK activity by direct binding.     

Mortality Patterns and Site Heterogeneity of Severe Malaria in African Children

Background

In this study we aimed to assess site heterogeneity of early, intermediate, and late mortality prediction in children with severe Plasmodium falciparum malaria in sub-Saharan Africa.

Methods

Medical records of 26,036 children admitted with severe Plasmodium falciparum malaria in six hospital research centers between December 2000 to May 2005 were analyzed. Demographic, clinical and laboratory data of children who died within 24 hours (early), between 24 and 47 hours (intermediate) and thereafter (48 hours or later, late mortality) were compared between groups and survivors.

Results

Overall mortality was 4·3% (N = 1,129). Median time to death varied across sites (P<0·001), ranging from 8h (3h–52h) in Lambaréné to 40h (10h–100h) in Kilifi. Fifty-eight percent of deaths occurred within 24 hours and intermediate and late mortality rate were 19% and 23%, respectively. Combining all sites, deep breathing, prostration and hypoglycemia were independent predictors for early, intermediate and late mortality (P<0·01). Site specific independent predictors for early death included prostration, coma and deep breathing at all sites (P<0·001). Site specific independent predictors for intermediate and late death largely varied between sites (P<0·001) and included between 1 and 7 different clinical and laboratory variables.

Conclusion

Site heterogeneity for mortality prediction is evident in African children with severe malaria. Prediction for early mortality has the highest consistency between sites.     

The effect of antibiotic treatment on the supercooling ability of the land snail Helix aspersa (Gastropoda: Pulmonata)

The land snail Helix aspersa is a partially freezing tolerant species whose supercooling ability is limited to ca. -3 to -5 degrees C. One hundred adult snails were subjected to the following two experimental conditions: (i) a starved group, provided with water; (ii) an antibiotic-treated group that was provided with a solution containing a mixture of two antibiotics. The antibiotic group exhibited a T(c) significantly lower than the starved group (-3.94 +/- 1.32 degrees C, n = 40 and -3.07 +/- 0.99, n = 30, t test, p < 0.005). This study showed that bacteria of the gut are likely to elevate animal supercooling points. It is also the first report in which a possible ice-nucleating activity of the gut microflora in a land snail has been suggested by the action of antibiotics on the T(c).