99 research outputs found
Sphingomyelin synthases regulate production of diacylglycerol at the Golgi
No full textSMS [SM (sphingomyelin) synthase] is a class of enzymes that produces SM by transferring a phosphocholine moiety on to ceramide. PC (phosphatidylcholine) is believed to be the phosphocholine donor of the reaction with consequent production of DAG (diacylglycerol), an important bioactive lipid. In the present study, by modulating SMS1 and SMS2 expression, the role of these enzymes on the elusive regulation of DAG was investigated. Because we found that modulation of SMS1 or SMS2 did not affect total levels of endogenous DAG in resting cells, whereas they produce DAG in vitro, the possibility that SMSs could modulate subcellular pools of DAG, once acute activation of the enzymes is triggered, was investigated. Stimulation of SM synthesis was induced by either treatment with short-chain ceramide analogues or by increasing endogenous ceramide at the plasma membrane, and a fluorescently labelled conventional C1 domain [from PKC (protein kinase C)] enhanced in its DAG binding activity was used to probe subcellular pools of DAG in the cell. With this approach, we found, using confocal microscopy and subcellular fractionation, that modulation of SMS1 and, to a lesser extent, SMS2 affected the formation of DAG at the Golgi apparatus. Similarly, down-regulation of SMS1 and SMS2 reduced the localization of the DAG-binding protein PKD (protein kinase D) to the Golgi. These results provide direct evidence that both enzymes are capable of regulating the formation of DAG in cells, that this pool of DAG is biologically active, and for the first time directly implicate SMS1 and SMS2 as regulators of DAG-binding proteins in the Golgi apparatu
Sphingomyelin synthase 1 activity is regulated by the BCR-ABL oncogene
No full textSphingomyelin synthase (SMS) produces sphingomyelin (SM) while consuming
ceramide (negative regulator of cell proliferation) and forming diacylglycerol (DAG) (a
mitogenic factor). Therefore enhanced SMS activity could favor cell proliferation. To
examine if dysregulated SMS contributes to leukemogenesis, we measured SMS
activity in several leukemic cell lines and found that it is highly elevated in K562 chronic
myelogenous leukemia (CML) cells. The increased SMS in K562 cells was caused by
the presence of Bcr-abl, hallmark of CML, as stable expression of Bcr-abl elevated SMS
activity in HL-60 cells while inhibition of the tyrosine kinase activity of Bcr-abl with
Imatinib mesylate, decreased SMS activity in K562 cells. The increased SMS activity
was the result of up-regulation of the Sms1 isoform. Inhibition of SMS activity with D609
(a pharmacological SMS inhibitor) or down-regulation of SMS1 expression by siRNA,
selectively inhibited the proliferation of Bcr-abl positive cells. The inhibition was
associated with an increased production of ceramide and a decreased production of
DAG, conditions that antagonize cell proliferation. A similar change in lipid profile was
also observed upon pharmacological inhibition of Bcr-abl (K526 cells) and siRNAmediated
down-regulation of BCR-ABL (HL-60/Bcr-abl cells). These findings indicate
that Sms1 is a downstream target of Bcr-abl, involved in sustaining cell proliferation of
Bcr-abl positive cells
VANET Authentication Using Trust Distribution in a Clustered Environment
No full textSecurity is a prime concern in providing communication between vehicles in a Vehicular Ad-Hoc Network (VANET), where they share information among themselves and nearby fixed equipments. Unlike wired networks, the unique characteristics of Vehicular Ad hoc Networks (VANETs) pose a number of non-trivial challenges to security design. This chapter presents a threshold security mechanism with a mobility based clustering for Inter Vehicle Communication (IVC). Nodes that have similar moving pattern are grouped into a cluster. Unlike other clustering algorithms, it takes the moving pattern of the vehicles into consideration along with the driver’s intention. The stability of clusters is estimated based on relative mobility of cluster members. A threshold cryptographic scheme is employed on top of the clusters to protect routing information and data traffic. To ensure distributed trust in the clustered environment, the private key (k) is divided into n pieces in such a way that k is easily reconstruct able from any p number of pieces. Even complete knowledge of (p-1) pieces reveals absolutely no information about k.</jats:p
THE ROLE OF TIS1 AND TIS21 IN TRANSFORMATION OF JB6 CELLS
No full textThe 12-0-tetradecanoylphorbol-13-acetate (TPA) inducible sequence genes are primary response genes induced in Swiss 3T3 cells by TPA (Lim, et al., 1987). Previous studies showed that a subset of TIS genes, TIS21 and TIS1, were
preferentially induced by TPA and also by epidermal growth factor in promotion resistant (P-) murine epidermal JB6 cells. Their protein levels were also greater in TPA-treated promotion resistant (P-) cells than promotion sensitive (P+)
cells (Cmarik, et al., 1994). Based on these observations, it was proposed that the high level of TIS1 and TIS21 proteins in P- cells could be a factor that inhibits tumor promoterinduced transformation. The strategy used to test this
hypothesis was to cause repression of TIS1 or TIS21 protein synthesis by constructing TIS21 or TIS1 antisense RNA expression plasmids. TIS21 cDNA was subcloned in the pcDNA3 vector and TIS1 in the pCEP4 vector. Antisense orientation was confirmed by restriction digestion and sequencing. These plasmids were transfected into P- cells subsequently grown in selective medium containing G418 medium for TIS21 transfectants and hygromycin for TIS1 transfectants. Greater than 25 independent clones of TIS21 or TIS1 were obtained. They were then analyzed for introduced TIS21 or TIS1 antisense RNA expression by northern analysis. The endogenous TIS21 or TIS1 mRNA was expressed, whereas the antisense RNA expression was not observed. Protein extracts from some TIS21 or TIS1 transfectants were subjected to western analysis. There was no decrease in the TPA-induced level of TIS21 or TIS1 proteins
in antisense transfectants compared with that of the vector controls as would be observed with functional antisense. Thus, if any antisense RNAs were expressed, they did not inhibit the synthesis of their respective proteins. Southern
analysis was carried out to determine if TIS1 or TIS21 antisense DNA is present in the cell. Of six TIS21 transfectants tested, none showed the presence of the TI521 antisense DNA, whereas two out of six TIS1 transfectants, pET1j and pET1p appeared to contain the TIS1 antisense DNA. These observations reflect that most of the clones obtained do not have the introduced gene and the few that do have the gene did not express the antisense RNA. Probably during clonal selection the cells lost the antisense DNA and most of the clones obtained are the ones without the antisense DNA. Because no clones with functional antisense that effectively blocked TIS1 or TIS21 protein induction in the P- cells were isolated, the hypothesis that the high level of TIS1 or TIS21 proteins in the P- cells can inhibit tumor promoter-induced transformation could not be validated
Soil Aggregate-Associated Microbial Community Structure and Nitrogen Transformations in Three Different Tillage Systems
No full textSoil management practices such as tillage largely influence soil aggregates and microbial communities that reside on different soil aggregate size fractions. Microbial communities that are central to the N cycle determine plant-available N and N losses via leaching and gaseous emissions. Although some research studies described the role of soil aggregates in C sequestration, very little information is available regarding the link between the soil aggregate size fractions and N cycling processes. The objectives of this research were: 1) to determine the population size of N mineralizers, nitrifiers, and denitrifiers by measuring the potential rates of N mineralization, nitrification, and denitrification, respectively; 2) to assess the activities of enzymes involved in the N mineralization process; 3) to quantify the gross N mineralization, nitrification, and immobilization rates; and 4) to relate the microbial community composition and rates of N processes associated with soil aggregate size fractions of no-till, chisel, and moldboard tillage systems.
Soil and microbial biomass C and N were 1.5 to 2 times greater in no-till than in moldboard systems and 15 to 20% greater in intermediate aggregates (0.5-1 mm) than other aggregates. Aggregate size had significant effects on potential N mineralization, nitrification, and denitrification rates for all three tillage systems. Potential activities of N-acetyl-β-glucosaminidase, L-glutaminase, and arylamidase were also significantly different (p<0.05) among aggregate size fractions. However, L-asparaginase activity did not vary significantly among aggregate sizes but did differ among tillage systems. This shows that different aggregate size fractions accommodate distinct microbial populations and communities associated with N cycling. Therefore, soil microbial community composition and gross N transformation rates associated with soil aggregate size fractions of the three tillage systems were quantified using the 15N pool dilution method. Nitrogen fluxes estimated from the FLUAZ program demonstrated that gross N mineralization, nitrification, and immobilization rates were significantly greater (1.5-2 times) in no-till than in chisel and moldboard systems. Gross N mineralization and nitrification rates were approximately 20 to 25% greater in intermediate aggregates (0.5-1mm) than in other aggregate size fractions. NMS analysis of the microbial community composition analyzed by Phospho Lipid Fatty Acid (PLFA) method both before (A = 0.3205, P <0.005) and after (A = 0.1951, P <0.005) 15N addition illustrated that microbial communities differed with tillage systems but not with aggregate size. Thus, this study demonstrated that higher microbial biomass in long-term no-till soils has resulted in more rapid N turnover via balanced N mineralization, nitrification, and immobilization processes than in tilled soils. However, more detailed studies involving the measurement of NO3 leaching losses are required to formulate best N management practices in different tillage systems
Secure Route Discovery in DSR against Black Hole Attacks in Mobile Ad Hoc Networks
No full textA Mobile Ad hoc Network (MANET) is a collection of wireless nodes communicating over multi-hop paths without any infrastructure. Nodes must cooperate to provide necessary network functionalities. The security in routing protocols like Dynamic Source Routing (DSR) can be compromised by a “Black Hole” attack. Here, a malicious node claims to have the shortest path to the destination and attracts all traffic and drops them, leading to performance degradation. The situation becomes worse when two or more nodes cooperate and perform the “Cooperative black hole” attack. This chapter proposes a solution based on probing to identify and prevent such attacks. The proposed solution discovers a secure route between the source and destination by identifying and isolating the attacking nodes. Simulation results show that the protocol provides better security and performance in terms of detection time, packet delivery ratio, and false negative probability in comparison with trust and probe based schemes.</jats:p
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