Very long chain fatty acids activate NADPH oxidase in human dermal fibroblasts

Very long chain fatty acids (VLCFAs) are exclusively oxidized in peroxisomes and their levels are significantly increased in tissues of patients with peroxisomal disorders. Although the biochemical indicators of peroxisomal dysfunction, such as elevated VLCFAs, are well known, the mechanisms of pathogenesis of peroxisomal diseases are unclear. In this study we have examined the effect of VLCFAs on NADPH oxidase (NOX), a complex enzyme system responsible for the production of superoxide anions, in order to understand the oxidative stress-mediated mechanisms involved in pathology of peroxisomal disorders. Varying concentrations (2.5 to 10 microg ml(-1)) of VLCFAs, lignoceric acid and cerotic acid, significantly (p < 0.001) increased the enzymic activity of NOX in cultures of human dermal fibroblasts. VLCFAs did not affect the expression of gp91phox or p22phox whereas the mRNA and protein levels of p47phox were significantly (two or three-fold) increased following treatment of fibroblasts with lignoceric acid or cerotic acid. VLCFAs also caused a significant (p < 0.01) increase in lipid peroxidation in dermal fibroblasts which could be markedly reversed by treatment with apocyanin (10 mM) or superoxide dismutase (SOD, 25 U ml(-1)). With these results, we report for the first time that VLCFAs enhance NOX activity and superoxide anion-mediated lipid peroxidation in cultured dermal fibroblasts. This study proposes a mechanism that may be taking place in vivo during peroxisomal dysfunction and that leads to oxidative stress-mediated pathogenesis.     

Real-time nucleic acid sequence-based amplification assay for detection of hepatitis A virus

A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5' noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.     

Signalling by PI3K isoforms: insights from gene-targeted mice

Phosphoinositide 3-kinases (PI3Ks) generate lipids that control a wide variety of intracellular signalling pathways. Part of this diversity in PI3K actions stems from the broad range of protein effectors of the PI3K lipids. A further layer of complexity is added by the existence of multiple isoforms of PI3K. Gene-targeting studies in the mouse have recently uncovered key roles for specific PI3K isoforms in immunity, metabolism and cardiac function. Remarkably, some of these actions do not require PI3K catalytic activity. In addition, loss-of-expression of certain PI3K genes leads to increased PI3K signalling following insulin stimulation. PI3K gene targeting has, in many cases, led to altered expression of the non-targeted PI3K subunits, making it difficult to exclude that some of the reported phenotypes result from 'knock-on' effects of PI3K gene deletion. Targeting strategies that take into account the complex interplay between members of the PI3K family will be crucial to gain a full understanding of the physiological roles of the isoforms of PI3K.     

Altering a gene involved in nuclear distribution increases the repeat-induced point mutation process in the fungus Podospora anserina

Repeat-induced point mutation (RIP) is a homology-dependent gene-silencing mechanism that introduces C:G-to-T:A transitions in duplicated DNA segments. Cis-duplicated sequences can also be affected by another mechanism called premeiotic recombination (PR). Both are active over the sexual cycle of some filamentous fungi, e.g., Neurospora crassa and Podospora anserina. During the sexual cycle, several developmental steps require precise nuclear movement and positioning, but connections between RIP, PR, and nuclear distributions have not yet been established. Previous work has led to the isolation of ami1, the P. anserina ortholog of the Aspergillus nidulans apsA gene, which is required for nuclear positioning. We show here that ami1 is involved in nuclear distribution during the sexual cycle and that alteration of ami1 delays the fruiting-body development. We also demonstrate that ami1 alteration affects loss of transgene functions during the sexual cycle. Genetically linked multiple copies of transgenes are affected by RIP and PR much more frequently in an ami1 mutant cross than in a wild-type cross. Our results suggest that the developmental slowdown of the ami1 mutant during the period of RIP and PR increases time exposure to the duplication detection system and thus increases the frequency of RIP and PR.     

Inhibition of phosphatidylinositol 3'-kinase induces preferentially killing of PTEN-null T leukemias through AKT pathway

We examined the functional role of the phosphatidylinositol 3'-kinase pathway in the growth and survival of cell lines of T-cell origin. Pharmacological inhibition of PI3'-kinase using LY294002 resulted in apoptosis of acute lymphoblastic T-cell leukemia (T-ALL) cell lines including CEM, Jurkat, and MOLT-4. On the other hand, the cutaneous T-cell lymphoma cell line HUT-78 was found to be refractory to LY294002- inducible apoptosis. Sensitivity or resistance to pharmacological inhibitors of PI3'-kinase correlated with tumor suppressor PTEN gene expression, as sensitive T-ALL cells do not express PTEN and have high level of activated AKT, in contrast to HUT-78 cells. Our data demonstrate that inhibition of PI3'-kinase results in dephosphorylation of AKT and partial inhibition of Bcl-xL expression in T-ALL cells, but not in HUT-78 cells. Interestingly, HUT-78 cells were also found to express higher levels of Bcl-xL protein as compared to T-ALL cells. Inhibition of PI3'-kinase also induces release of cytochrome c from mitochondria and activation of caspase-3 and PARP in all T-ALL cell lines tested, but not in HUT-78 cells. Taken altogether, our data demonstrate that the PI3'-kinase/AKT pathway plays a major role in the growth and survival of PTEN-null T-ALL cells, and identify this cascade as promising target for therapeutic intervention in acute T-cell leukemias.     

Interleukin-7 inactivates the pro-apoptotic protein Bad promoting T cell survival

Interleukin-7 (IL-7) is a cytokine that is required for T cell development and survival. The anti-apoptotic function of IL-7 is partly through induction of Bcl-2 synthesis and cytosolic retention of Bax. Here we show that the Bcl-2 homology 3 domain-only protein, Bad, is involved in cell death following IL-7 withdrawal from D1 cells, an IL-7-dependent murine thymocyte cell line. IL-7 stimulation resulted in the inactivation of Bad by phosphorylation at Ser-112, -136, and -155. The phosphoinositide 3-kinase (PI3K)/Akt pathway has been implicated previously in Bad phosphorylation. In response to IL-7, the PI3K/Akt pathway induced phosphorylation at Ser-136 and -155, but Ser-112 was partly independent of the PI3K/Akt pathway, indicating an as yet unknown pathway in this response. Following IL-7 withdrawal, dephosphorylated Bad translocated from cytosol to mitochondria, bound to Bcl-2, and accelerated cell death. Thus, the inactivation of Bad contributes to the survival function of IL-7.