Sphingosine kinase: biochemical and cellular regulation and role in disease

Sphingolipids have emerged as molecules whose metabolism is regulated leading to generation of bioactive products including ceramide, sphingosine, and sphingosine-1-phosphate. The balance between cellular levels of these bioactive products is increasingly recognized to be critical to cell regulation; whereby, ceramide and sphingosine cause apoptosis and growth arrest phenotypes, and sphingosine-1-phosphate mediates proliferative and angiogenic responses. Sphingosine kinase is a key enzyme in modulating the levels of these lipids and is emerging as an important and regulated enzyme. This review is geared at mechanisms of regulation of sphingosine kinase and the coming to light of its role in disease.     

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.     

Recent patents involving virus nucleotide sequences; host defense, RNA silencing and expression vector strategies

Improved knowledge of the molecular biology of viruses, including recent gains in virus sequence data analysis, has greatly contributed to recent innovations in medical diagnostics, therapeutics, drug development and other related areas. Virus sequences have been used for the development of vaccines and antiviral agents to block the spread of viral infections, as well as to target and battle chronic diseases such as cancer. Virus sequences are now routinely employed in a wide array of RNA silencing technologies. Viruses can also be engineered into expression vectors which in turn can be used as protein production platforms as well as delivery vehicles for gene therapies. This review article outlines a number of patents that have been recently issued with respect to virus sequence data and describes some of their biotechnological applications.     

Déjà vu--a study of duplicate citations in Medline

MOTIVATION: Duplicate publication impacts the quality of the scientific corpus, has been difficult to detect, and studies this far have been limited in scope and size. Using text similarity searches, we were able to identify signatures of duplicate citations among a body of abstracts. RESULTS: A sample of 62,213 Medline citations was examined and a database of manually verified duplicate citations was created to study author publication behavior. We found that 0.04% of the citations with no shared authors were highly similar and are thus potential cases of plagiarism. 1.35% with shared authors were sufficiently similar to be considered a duplicate. Extrapolating, this would correspond to 3500 and 117,500 duplicate citations in total, respectively. Availability: eTBLAST, an automated citation matching tool, and Déjà vu, the duplicate citation database, are freely available at http://invention.swmed.edu/ and http://spore.swmed.edu/dejavu     

Sleeping under the Ocean: Despite Total Isolation,Nuclear Submariners Maintain Their Sleep and Wake Patterns throughout Their Under Sea Mission

BackgroundTo assess the effects of isolation, inadequate exposure to light and specific shift work on the subjective and objective measurements of sleep and alertness of submariners.PurposeA strictly controlled randomized crossover study with the polysomnography recorded twice during the mission.MethodsSetting: Shift and night work with prolonged (70 days) social isolation from the real world (with no phone or Internet contact with families or friends during a routine mission aboard the “Téméraire” French Strategic Submarine with Ballistic Nuclear missiles (SSBN). Participants: 19 submariners working on a 24-hour shift for three days in a row schedule. Interventions: The participants attended two polysomnographic (PSG) recordings of night sleep on Day 21 (D21) and Day 51 (D51) of the 70-day patrol; urine cortisol levels were also taken after sleep, and subjective assessments of sleep, sleepiness, mood and anxiety on D21 and D51. The light and temperature on board were also recorded.ResultsPSG analyses showed that sleep did not significantly vary in length (total sleep time) or in quality between D21 and D51. The mariners reported the same subjective sleep, sleepiness, anxiety or mood (except for a slightly worse score for confusion on D51). Blood cortisol levels did not vary significantly.ConclusionsThese results show that humans living in an isolated environment for more than two months with this specific shift schedule do not suffer from any significant effects on sleep, sleepiness and confusion between D21 and D51, when they follow an organized regular shift pattern with controlled light and temperature.     

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.     

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.     

Secreted recombinant P53 protein from Pichia pastoris is a useful antigen for detection of serum p53: autoantibody in patients with advanced colorectal adenocarcinoma

The detection of P53 alteration by serological method is easier to perform, does not require tumor tissues and is of interest for patients monitoring. In this study, we described the development of a home made ELISA test based on recombinant human P53 protein produced in Pichia pastoris and used as antigen for the detection of serum p53-Abs in colorectal carcinoma patients. The human P53 was secreted as a His-tagged protein by recombinant KM71 strain (Kα21) via the peptide signal α of the Saccharomyces cerevisiae mating type gene. The recombinant P53-His was able to detect p53-Abs in 23.4 % of patients. Serum p53-Abs correlated significantly with surgical treatment (P = 0.007), relapse during follow-up (P = 0.036), depth of invasion (P = 0.036) and the level of CA19-9 (P = 0.034). Survival analysis showed that patients negative for serum p53-Abs exhibited a prolonged disease free survival period (P log rank = 0.012). In conclusion, the secreted recombinant human P53-His produced in P. pastoris seems to be a useful antigen for detection of serum p53 Abs in patients with colorectal carcinoma.     

Positively charged ceramide is a potent inducer of mitochondrial permeabilization

Ceramide-induced cell death is thought to be mediated by change in mitochondrial function, although the precise mechanism is unclear. Proposed models suggest that ceramide induces cell death through interaction with latent binding sites on the outer or inner mitochondrial membranes, followed by an increase in membrane permeability, as an intermediate step in ceramide signal propagation. To investigate these models, we developed a new generation of positively charged ceramides that readily accumulate in isolated and in situ mitochondria. Accumulated, positively charged ceramides increased inner membrane permeability and triggered release of mitochondrial cytochrome c. Furthermore, the positively charged ceramide-induced permeability increase was suppressed by cyclosporin A (60%) and 1,3-dicyclohexylcarbodiimide (90%). These observations suggest that the inner membrane permeability increase is due to activation of specific ion transporters, not the generalized loss of lipid bilayer barrier functions. The difference in sensitivity of ceramide-induced ion fluxes to inhibitors of mitochondrial transporters suggests activation of at least two transport systems: the permeability transition pore and the electrogenic H(+) channel. Our results indicate the presence of specific ceramide targets in the mitochondrial matrix, the occupation of which triggers permeability alterations of the inner and outer mitochondrial membranes. These findings also suggest a novel therapeutic role for positively charged ceramides.