Inhibition of Sphingosine Kinase-2 in a Murine Model of Lupus Nephritis

Sphingosine-1-phosphate (S1P), a potent bioactive lipid, is emerging as a central mediator in inflammation and immune responses. We have previously implicated S1P and its synthetic enzyme sphingosine kinase (SK) in inflammatory and autoimmune disorders, including inflammatory bowel disease and rheumatoid arthritis. Generation of S1P requires phosphorylation of sphingosine by SK, of which there are two isoforms. Numerous studies have implicated SK1 in immune cell trafficking, inflammation and autoimmune disorders. In this study, we set out to determine the role of SK and S1P in lupus nephritis (LN). To this end, we examined S1P and dihydro-S1P (dh-S1P) levels in serum and kidney tissues from a mouse model of LN. Interestingly dh-S1P was significantly elevated in serum and kidney tissue from LN mice, which is more readily phosphorylated by SK2. Therefore, we employed the use of the specific SK2 inhibitor, ABC294640 in our murine model of LN. Treatment with ABC294640 did not improve vascular or interstitial pathology associated with LN. However, mice treated with the SK2 inhibitor did demonstrate decreases in glomerular pathology and accumulation of B and T cells in the spleen these were not statistically different from lpr mice treated with vehicle. LN mice treated with ABC294640 did not have improved urine thromboxane levels or urine proteinuria measurements. Both S1P and dh-S1P levels in circulation were significantly reduced with ABC294640 treatment; however, dh-S1P was actually elevated in kidneys from LN mice treated with ABC294640. Together these data demonstrate a role for SKs in LN; however, they suggest that inhibition of SK1 or perhaps both SK isoforms would better prevent elevations in S1P and dh-S1P and potentially better protect against LN.     

Piophila megastigmata McAlpine 1978 (Diptera: Piophilidae): the first record from northern Africa of this forensically relevant fly

Piophila megastigmata McAlpine (Diptera: Piophilidae) is recorded for the first time from Algeria, northern Africa. This is the first Palaearctic record of the species outside the Iberian Peninsula, and the only record from Africa apart from its type locality in eastern South Africa. The current report extends significantly the known distribution in the Palaearctic Region of a species of forensic interest and of potential economic importance as a food pest.     

Inhibition of caspases inhibits the release of apoptotic bodies: Bcl-2 inhibits the initiation of formation of apoptotic bodies in chemotherapeutic agent-induced apoptosis

During apoptosis, the cell actively dismantles itself and reduces cell size by the formation and pinching off of portions of cytoplasm and nucleus as "apoptotic bodies." We have combined our previously established quantitative assay relating the amount of release of [3H]-membrane lipid to the degree of apoptosis with electron microscopy (EM) at a series of timepoints to study apoptosis of lymphoid cells exposed to vincristine or etoposide. We find that the [3H]-membrane lipid release assay correlates well with EM studies showing the formation and release of apoptotic bodies and cell death, and both processes are regulated in parallel by inducers or inhibitors of apoptosis. Overexpression of Bcl-2 or inhibition of caspases by DEVD inhibited equally well the activation of caspases as indicated by PARP cleavage. They also inhibited [3H]-membrane lipid release and release of apoptotic bodies. EM showed that cells overexpressing Bcl-2 displayed near-normal morphology and viability in response to vincristine or etoposide. In contrast, DEVD did not prevent cell death. Although DEVD inhibited the chromatin condensation, PARP cleavage, release of apoptotic bodies, and release of labeled lipid, DEVD-treated cells showed accumulation of heterogeneous vesicles trapped in the condensed cytoplasm. These results suggest that inhibition of caspases arrested the maturation and release of apoptotic bodies. Our results also imply that Bcl-2 regulates processes in addition to caspase activation.     

A Disrupted Homologue of the Human CLN3 or Juvenile Neuronal Ceroid Lipofuscinosis Gene in Saccharomyces cerevisiae: A Model to Study Batten Disease

1.In order to investigate the biological function of the human CLN3 gene that is defective in Batten disease, we created a yeast strain by PCR-targeted disruption of the yeast gene (YHC3), which is a homologue of the human CLN3 gene.2.The phenotypic characterization revealed that the yhc3 mutants are more sensitive to combined heat and alkaline stress than the wild-type strains as determined by inhibition of cell proliferation.3.This suggests that the yhc3 mutant is a good model to investigate the biological function of human CLN3 gene in mammalian cells and to understand the pathophysiology of juvenile Batten disease.     

Bcl-xL interrupts oxidative activation of neutral sphingomyelinase

Recent studies demonstrate a role for intracellular oxidation in the regulation of neutral sphingomyelinase (N-SMase). Glutathione (GSH) has been shown to regulate N-SMase in vitro and in cells. However, it has not been established whether the effects of GSH in cells are due to direct action on N-SMase. In this study, treatment of human mammary carcinoma MCF-7 cells with diamide, a thiol-depleting agent, caused a decrease in intracellular GSH and degradation of sphingomyelin (SM) to ceramide. The SM pool hydrolyzed in response to diamide belonged to the bacterial SMase-resistant pool of SM. Importantly, pretreatment of MCF-7 cells with GSH, N-acetylcysteine, an antioxidant, or GW69A, a specific N-SMase inhibitor, prevented diamide-induced degradation of SM to ceramide, suggesting that intracellular levels of GSH regulate the extent to which SM is degraded to ceramide and that this probably involves a GW69A-sensitive N-SMase. Unexpectedly, expression of Bcl-xL prevented tumor necrosis factor--induced SM hydrolysis and ceramide accumulation but not the decrease in intracellular GSH. Furthermore, Bcl-xL inhibited diamide-induced SM hydrolysis and ceramide accumulation but not the decrease in intracellular GSH. These results suggest that the site of action of Bcl-xL is downstream of GSH depletion and upstream of ceramide accumulation, and that GSH probably does not exert direct physiologic effects on N-SMase.     

Inability of Agrobacterium tumefaciens ribosomes to translate in vivo mRNAs containing non-Shine-Dalgarno translational initiators

Numerous data accumulated during the last decade have shown that the Shine-Dalgarno (SD) sequence is not a unique initiator of translation for Escherichia coli. Several other sequences, mostly of viral origin, have demonstrated their capability of either enhancing or initiating translation in vivo. A phage T7 gene 10 sequence, called "epsilon" (epsilon), has shown its high enhancing activity on translation in both Escherichia coli and Agrobacterium tumefaciens cells. In this study the epsilon, together with three other nucleotide sequences derived from the 5' non-translated regions of tobacco mosaic virus (TMV), papaya mosaic virus (PMV) and clover yellow mosaic virus (CYMV) RNAs are tested for translation initiation activity in A. tumefaciens cells. The obtained results indicate that none of them was capable of initiating translation in vivo of chloramphenicol acetyltransferase (CAT) mRNA. To determine whether their inactivity was related with structural differences in the ribosomal protein S1, the rpsA gene (coding for S1 protein in E. coli) was co-expressed in A. tumefaciens together with the cat gene placed under the translational control of the above sequences. Our results showed that the rpsA gene product did not make any of the four viral enhancers active in A. tumefaciens cells. The inability of A. tumefaciens ribosomes to translate mRNAs devoid of SD sequences indicates for a substantial difference in the ribosome structure of the two Gram negative bacteria E. coli and A. tumefaciens.     

Asymmetrical membranes and surface tension

The (31)P-nuclear magnetic resonance chemical shift of phosphatidic acid in a membrane is sensitive to the lipid head group packing and can report qualitatively on membrane lateral compression near the aqueous interface. We have used high-resolution (31)P-nuclear magnetic resonance to evaluate the lateral compression on each side of asymmetrical lipid vesicles. When monooleoylphosphatidylcholine was added to the external monolayer of sonicated vesicles containing dioleoylphosphatidylcholine and dioleoylphosphatidic acid, the variation of (31)P chemical shift of phosphatidic acid indicated a lateral compression in the external monolayer. Simultaneously, a slight dilation was observed in the inner monolayer. In large unilamellar vesicles on the other hand the lateral pressure increased in both monolayers after asymmetrical insertion of monooleoylphosphatidylcholine. This can be explained by assuming that when monooleoylphosphatidylcholine is added to large unilamellar vesicles, the membrane bends until the strain is the same in both monolayers. In the case of sonicated vesicles, a change of curvature is not possible, and therefore differential packing in the two layers remains. We infer that a variation of lipid asymmetry by generating a lateral strain in the membrane can be a physiological way of modulating the conformation of membrane proteins.     

A close encounter with DNA

Atomic force microscopy, associated with surface science, has the potential to resolve the secondary structure of DNA in liquid form with unusually high resolution and with unprecedented accuracy.     

Functional dichotomy of protein kinase C (PKC) in tumor necrosis factor-alpha (TNF-alpha ) signal transduction in L929 cells. Translocation and inactivation of PKC by TNF-alpha

Tumor necrosis factor-alpha (TNF-alpha) is capable of inducing a variety of biologic responses through multiple signaling pathways. Because of the potential role of protein kinase C (PKC) in apoptosis, we examined the effects and mechanisms of TNF-alpha on PKC regulation, specifically on PKC alpha. In L929 murine fibroblasts, TNF-alpha (0.5- 5 nm) caused potent inhibition of PKC alpha activity and induced translocation of PKC alpha from the cytosol to the membrane. Treatment of cells with TNF-alpha also induced dephosphorylation of PKC alpha as detected by a mobility shift on SDS-polyacrylamide gel and inhibition of PKC phosphorylation as probed by anti-phospho-PKC antibodies. Since PKC is activated directly by diacylglycerol and inactivated indirectly by ceramide, we next examined the roles of these lipid mediators in the regulation of PKC alpha. Addition of TNF-alpha led to accumulation of both ceramide and diacylglycerol. Fumonisin B(1), an inhibitor of ceramide synthase, and glutathione, an inhibitor of neutral sphingomyelinase, both reversed the effect of TNF-alpha on PKC alpha activity, suggesting that ceramide production is necessary for the action of TNF-alpha. The diacylglycerol mimic phorbol 12-myristate 13-acetate was sufficient to cause translocation of PKC alpha, but not the mobility shift. Okadaic acid at 2 nm, a potent protein phosphatase inhibitor, blocked the effects of TNF-alpha on PKC alpha activity, but not on PKC alpha translocation, thus demonstrating that dephosphorylation and translocation are independent processes. These results demonstrate that PKC alpha acts as a downstream target for TNF-alpha and that different lipid-mediated pathways in TNF-alpha signaling lead to opposing signals in the regulation of PKC alpha activity.