Progress 10/01/05 to 09/30/11
Outputs
OUTPUTS: Virus infection in humans and animals often lead to damaging inflammation. In these studies we have examined how a number of different viruses trigger the host inflammatory response, how these viruses attempt to subvert this response, and finally we have investigated a number of novel treatments for virus-induced inflammation. Included were the viruses; adenovirus, porcine reproductive and respiratory syndrome virus (PRRSv), and several strains of the influenza A virus. Our studies involved in vitro virus infections, western blots, ELISA's, polymerase chain reactions, immunofluorescence microscopy and various quantitative virus assays. For treatments we examined extracts and purified compounds from several different plants including Larrea tridentata (creosote bush), Echinacea purpurea (purple cone flower), and Hydrastis canadensis (goldenseal). We monitored inflammation using several different parameters including signal transduction, transcription factor activation. The studies were performed using a variety of normal and transformed cell types. The results of our investigations were published in written form in the scientific literature and communicated orally at various scientific conferences. PARTICIPANTS: Culver, C.A., graduate student. Received her Ph.D. degree through this project. Currently employed as a post-doctoral scientist. Eads, D., laboratory technician. Currently working in the veterinary industry. Hansen, R. L., laboratory technician. Returned to school after working on this project and completed her DVM degree at North Carolina State. Currently working as a DVM in North Carolina. Oyegunwa, A.O., graduate student. Completed his Ph.D. working on this project. Currently working as a part-time post doctoral fellow and completing his MBA degree. He intends to work on the business side of the health/biotechnology industry. Cecil, C.E., graduate student. Completed his Ph.D. working on this project. Currently running the Veterinary diagnostics laboratory for the State of North Carolina. Scholle, F., collaborator. A virologist at North Carolina State University with whom we collaborated on this project. Petty, I.T.D., collaborator. A virologist at North Carolina State University with whom we collaborated on this project. Pollara, J., graduate student. A graduate student from Dr. Petty's laboratory with whom we collaborated on this project. Minnis, A. graduate student. Completed her MS degree on this project. Currently working in the biotechnology industry in North Carolina. Cech, N., collaborator. A medicinal chemist at University of North Carolina at Greensboro with whom we collaborated on this project. Kandi, V. graduate student. A graduate student from Dr. Cech's laboratory with whom we collaborated on this project. Davis, J., collaborator. A horticulturalist from North Carolina State University with whom we collaborated on this project. Hamilton, A., research technician. A technician from Dr. Davis's laboratory with whom we collaborated on this project. Sikes, M., collaborator, An immunologist/molecular biologist with whom we collaborated on this project. TARGET AUDIENCES: The target audience for our research is the academic/industrial community developing treatments for inflammation and virus infection. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Each of the viruses we examined was detected by the cells we tested. In response the cells activated an inflammatory response involving activation of both cytosolic processes and de novo transcription. Numerous inflammatory proteins (cytokines and chemokines) and inflammatory lipids (prostaglandins) were produced. We also found that the viruses had the ability to counter the host inflammatory response. Adenovirus, for example, suppressed prostaglandin production soon after it began. Each of the botanical extracts and purified botanical compounds we examined also displayed the ability to suppress inflammation, albeit to different levels. The alkaloid berberine (from goldenseal) and the alkylamide A15 (from Echinacea) were most broadly effective. Berberine also displayed the ability to suppress the growth of influenza A, which activity which should be pursued.
Publications
- No publications reported this period
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: OUTPUTS: Many viruses trigger an inflammatory response which damages host tissues and organs. During this past year we continued our examination of the mechanisms of virus-induced inflammation and methods that can be used to control this response. This year we focused on the underlying molecular mechansims responsible for suppression of inflammation and virus growth. These studies focused on two viruses; H1N1 influenza A strains PR/8/34 and WS/33 and poxvirus strains MVA, cowpox and vaccinia. Several natural plant-derived anti-viral and anti-inflammatory extracts and compounds were examined including those from the Creosote bush (terameprocol), Goldenseal (berberine), and Echinacea(alkylamides). These studies involved a number of different techniques including viral infections, qPCR, ChIP assays, and immunofluorescence. PARTICIPANTS: PARTICIPANTS: Scott Laster, PI, Chad Cecil, Graduate Student, Bola Oyegunwa, Graduate Student, Anna Minnis, Graduate Student, Justin Pollara, Graduate Student, Jason Wilson, Graduate Student, Mike Sikes, NCSU Collaborator, Frank Scholle Ph.D, NCSU Collaborator, Tim Petty, Ph.D., NCSU Collaborator, Jeanine Davis, Ph.D., NCSU Collaborator, Nadja Cech Ph.D, UNC Greensboro Collaborator TARGET AUDIENCES: TARGET AUDIENCES: The target audience for our research is the academic/industrial community developing treatments for inflammation and virus infection. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
OUTCOMES/IMPACTS: Our experiments have revealed that plant-derived compounds have the potential for alleviating virus-induced inflammation. Terameprocol (a poly-phenolic compound in creosote extracts) can inhibit production of both inflammatory cytokines and lipids from virus infected cells. We have found that this compound inhibits the NF-kB-dependent transcription of certain key pro-inflammatory genes. Similarly, alkylamides (from Echinacea) also were shown to strongly inhibit the procuction of inflammatory cytokines and lipids from infected macrophages. Interestingly, extracts of Echinacea plants, with high alkylamide content, did not exert a strong anti-inflammatory effect. Our results suggest that other compounds present in the extracts, perhaps originating with endophytic bacteria, actually stimulate rather than suppress macrophage activity. Similarly, the alkaloid berberine also strongly suppressed production of inflammatory mediators but extracts of goldenseal with high berberine content did not exert this affect. These data indicate that industry compatible extraction processes should be develeped for production of natural products with high anti-viral and anti-inflammatory activity.
Publications
- Cech, N., Kandhi, V. Davis, J. M., Davis, Hamilton, A., Eads, D., and S.M. Laster. 2010. Echinacea and its alkylamides: effects on the influenza A-induced secretion of cytokines and inflammatory lipids from RAW 264.7 macrophage-like cells. Int. Immunopharm. 10:1268-1278.
- Pollara, J.J., Curry, R., Laster, S.M., Eads, D., and I.T.D. Petty, 2010. Inhibiton of poxvirus growth by Terameprocol, a methylated derivative of nordihydroguaiaretic acid. Anti-viral Res. 88:287-295.
- Oyegunwa, A. O., Sikes, M. L., Wilson, J. R., Scholle, F., and S.M. Laster. 2010. Tetra-O-methyl nordihydroguaiaretic acid (Terameprocol) inhibits the NF-kB-dependent transcription of TNF-a and MCP01/CCL2 genes by preventing RelA from binding its cognate sites on DNA. J. Inflamm. 7:59.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Many viruses trigger an inflammatory response which damages host tissues and organs. During this past year we continued our examination of the mechanisms of virus-induced inflammation and methods that can be used to control this response. These studies have involved adenovirus, influenza virus, and several pox viruses. Several natural plant-derived anti-inflammatory extracts and compounds have been examined including those from the Creosote bush, Goldenseal, and Echinacea. The Echinacea and Goldenseal extracts were collected from across the State of North Carolina. These studies involved a number of different techniques including viral infections, ELISA's, Western blots and immunofluorescence. A number of parameters of inflammation were examined, including; production of prostaglandins and leukotrienes, cytokine production, and translocation of NF-KB. The extracts were tested against a number of different cell types including fibroblasts, macrophages and dendritic cells. PARTICIPANTS: Scott Laster, PI, Dawn Eads, Research Specialist, Rebecca Hansen, Research Specialist, Chad Cecil, Graduate Student, Bola Oyegunwa, Graduate Student, Anna Minnis, Graduate Student, Justin Pollara, Graduate Student, Frank Scholle Ph.D, NCSU Collaborator, Tim Petty, Ph.D., NCSU Collaborator, Jeanine Davis, Ph.D., NCSU Collaborator, Nadja Cech Ph.D, UNC Greensboro Collaborator TARGET AUDIENCES: The target audience for our research is the academic/industrial community developing treatments for inflammation and virus infection. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our experiments have revealed that natural products have the potential for alleviating virus-induced inflammation. Terameprocol (a poly-phenolic compound in creosote extracts) can inhibit production of both inflammatory cytokines and lipids. We have found that this compound exterts multiple anti-inflammatory effects including inhibition of NF-kB signaling and direct inhibition of COX-2. Extracts of Goldenseal and its primary alkaloid Berberine also have the ability to block both cytokine and prostaglandin production. Goldenseal and Berberine also can inhibit replication of influenza virus directly. Similar anti-inflammatory effects have been noted with extracts of Echinacea and its alkylamide compounds. On the other hand, Echinacea and its alkylamides did not directly inhibit virus growth. Echinacea extracts also appear to exert their anti-inflammatory effects by mechanisms distict from the other comopunds we have studied. Levels of cytokine mRNA were relatively unaffected suggesting post-transcriptional mechanisms of inhibition. As noted previously, our experiments with Echinacea have also revealed tremendous variability between extracts made from different plants. Some are highly effective while others contain virtually no activity. We have now evaluted levels of several different alkyalamides and levels of caftaric and cichoric acids and LPS and none has proved to be an effective marker for extract activity. Unknown compounds appear to be mediating the variable effects seen with extracts of Echinacea.
Publications
- Eads, D., Hansen, R.L., Oyegunwa, A.O., Cecil, C.E., Culver, C.A., Scholle, F., Petty, I.T.D., and S.M. Laster. 2009. Terameprocol, a methylated derivative of nordihydroguaiaretic acid, inhibits production of prostaglandins and several key inflammatory cytokines and chemokines. J. Inflamm. 6:2.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The human Adenovirus is important both for the disease it causes and for its use as a live virus vaccine vector. In both cases, inflammation has been noted during adenovirus infection. During this past year we continued our examination of the effects of Adenovirus on production of prostaglandins (PGs). These studies involved a number of different techniques including viral infections, ELISA's, Western blots and immunofluorescence. Our lab is also interested in the identification of novel anti-inflammatory and anti-viral compounds and during the past year we continued our examination of extracts of the creosote bush, Larrea tridentata. We also examined extracts of Echinacea and Goldenseal for anti-inflammatory and anti-viral activity. The Echinacea extracts were collected from 19 different growers throughout the State fo North Carolina. A number of parameters of inflammation were examined, including; production of prostaglandins and leukotrienes, cytokine production, and translocation of NF-KB. The extracts were tested against a number of different cell types including fibroblasts, macrophages and dendritic cells. PARTICIPANTS: Scott Laster, PI, Dawn Eads, Research Specialist, Rebecca Hansen, Research Specialist, Chad Cecil, Graduate Student, Bola Oyegunwa, Graduate Student, Anna Minnis, Graduate Student, Justin Pollara, Graduate Student, Frank Scholle Ph.D, NCSU Collaborator, Tim Petty, Ph.D., NCSU Collaborator, Jeanine Davis, Ph.D., NCSU Collaborator, Nadja Cech Ph.D, UNC Greensboro Collaborator TARGET AUDIENCES: The target audience for our research is the academic/industrial community developing treatments for inflammation and virus infection. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our experiments with adenovirus showed that infection is accompanied by the rapid prostaglandin production. Later, however, as the infection progressed, prostaglandin production stopped. Our studies indicate that Adenovirus can inhibit prostaglandin production by inhibiting the activity of cPLA2, a key enzyme in the prostaglandin pathway. Adenovirus actually hijacks cPLA2 away from the prostaglandin pathway and that this occurs via the vimentin intermediate filaments. Adenovirus infection causes the vimentin filaments to reorganize into the perinuclear region of the cell. Taken together, these data reveal dynamic interactions between Ad5 and the lipid mediator pathways of the host and highlight a novel mechanism by which Ad5 evades the host immune response. Our studies of terameprocol revealed several mechanisms by which this drug inhibits inflammation. Our results indicate that terameprocol prevents transcription of several cytokine and chemokine genes. Our results also show that terameprocol acts as a direct inhibitor of COX-2. In vivo, we also found that terameprocol could inhibit production cytokines and chemokines following ip administration of LPS. Our studies of Echinacea and Goldenseal extracts also revealed anti-inflammatory effects. However, with Echinacea extracts, we also measured considerable variabiltiy. Several of the extracts displayed strong anti-inflammatory activity while many did not display this activity. Apparently, genetic and environmental factors influence the chemical contents of the plants. Alkylamides are believed to be the active, anti-inflammatory compounds in Echinacea. However, alkylamide levels did not vary among the samples suggesting that other unknown compounds are mediating the effects we have observed.
Publications
- No publications reported this period
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: The human Adenovirus is important both for the disease it causes and for its use as a live virus vaccine vector. In both cases, inflammation has been noted during adenovirus infection. During this past year we examined how infection of murine and human fibroblasts by Adenovirus (Ad) serotype 5 (Ad5) affects the expression and activity of cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2), and production of prostaglandins (PGs). These studies involved a number of different techniques including viral infections, ELISA's, Western blots and immunofluorescence. Our lab is also interested in the identification of novel anti-inflammatory and anti-viral compounds and during the past year we examined extracts of the creosote bush, Larrea tridentata, which have been used for centuries by natives of the western American and Mexican deserts to treat a variety of diseases and inflammatory disorders. The beneficial activity of this plant has been linked to the compound
nordihydroguaiaretic acid (NDGA), a strong anti-oxidant found in the leaves and stems of Larrea. Recently, tetra-O-methyl NDGA, also known as terameprocol, has been synthesized and shown to inhibit the growth of certain viruses. Terameprocol can also arrest the growth of certain tumor-derived cell lines and is now in clinical trials for the treatment of human cancer. During the past year we tested whether terameprocol displays anti-inflammatory activity. A number of parameters of inflammation were examined, including; production of prostaglandins and leukotrienes, cytokine production, and translocation of NF-KB.
PARTICIPANTS: Scott Laster, PI Dawn Eads, Research Specialist Rebecca Hansen, Research Specialist Carolyn Culver, Graduate Research Assistant
TARGET AUDIENCES: The target audience for our research is the academic/industrial community studying inflammation, whose goal the development of new anti-inflammatory pharmaceuticals.
Impacts
Our experiments with adenovirus showed that infection is accompanied by the rapid activation of cPLA2 and the cPLA2-dependent release of [3H]arachidonic acid ([3H]AA). Increased expression of COX-2 was also observed after Ad infection as was production of PGE2 and PGI2. Later, however, as the infection progressed, release of [3H]AA and production of PGs stopped. Late stage Ad5-infected cells also did not release [3H]AA or PGs following treatment with a panel of biologically diverse agents. Experiments with UV-inactivated virus confirmed that Ad infection is accompanied by the activation of a host-dependent response that is later inhibited by the virus. Investigations of the mechanism of suppression of the PG pathway by Ad5 did not reveal major effects on the expression or activity of cPLA2 or COX-2. We did note a change in the intracellular position of cPLA2 and found that cPLA2 did not translocate normally in infected cells, raising the possibility that Ad5 interferes
with the PG pathway by interfering with the intracellular movement of cPLA2. Taken together, these data reveal dynamic interactions between Ad5 and the lipid mediator pathways of the host and highlight a novel mechanism by which Ad5 evades the host immune response. In addition, our results offer insight into the inflammatory response induced by many Ad vectors lacking early region gene products. Our studies of terameprocol revealed several important effects. Terameprocol inhibited production of both prostaglandins and leukotrienes. Terapmeprocol also blocked production of certain cytokines and chemokines such as TNF and MCP-1. The results of these studies justify testing terameprocol in vivo as and anti-inflammatory agent.
Publications
- Culver, C. A. and S. M. Laster. 2007. Adenovirus exerts multiple effects on the expression and activity of cPLA2, COX-2, and synthesis of prostaglandins. J. Immunol. 179:4170-4179.
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Progress 10/01/04 to 09/30/05
Outputs
During the past year we examined the effects of adenovirus (Ad) infection on the production of PGE2. Our experiments show complete inhibition of PGE2 production by infection with Ad 5. PGE2 production was blocked in both murine and human cell types following stimulation with a variety of ligands including LPS, A23187, and PMA. Surprisingly, the expression of COX-1 or COX-2 was not blocked by infection with Ad 5; in fact Ad 5 infection strongly induced expression of COX-2 protein. Moving upstream we examined the effects of Ad on cPLA2 expression and activity. We found that release of 3H-arachidonic acid was completely blocked by Ad infection. This effect was seen with both Ad 2 and Ad 5 and occurred with E3 deletion mutants indicating that this is a novel, non-E3 encoded activity. Again we did not find an effect on the expression of cPLA2 or its activity in cell lysates. Agonist induced phosphorylation of cPLA2 serine 505 was also not affected. In an effort to
understand this phenomenon we examined the intracellular position of cPLA2. We found that infection with Ad causes cPLA2 to shift from its normal cytosolic location to the nuclear region of the cell. Both perinuclear and intranuclear staining were noted. Subsequently, following agonist stimulation, the cPLA2 in Ad infected cells did not translocate as it does normally. We conclude therefore that Ad inhibits production of PGE2 by causing the intracellular relocation of cPLA2 to a position that prevents its participation in inflammatory responses. During this pasy year we also studied the mechanism by which NDGA inhibits activation of cPLA2 during TNF-induced apoptosis. Our experiments revealed that the necessary calcium response during TNF-induced apoptosis is blocked by NDGA. At present it is not clear how NDGA inhibits this response.
Impacts
Inflammation is a major problem in a number of autoimmune and toxic pathological situations. The liver and the lungs are especially susceptible. We are studying an enzyme whose activity is necessary for inflammation. By studying the mechanisms that regulate this enzyme's activity we will define new targets for the design of anti-inflammatory pharmaceuticals.
Publications
- Culver, C. A., Michalowski, S. M., Maia, R. C. and S. M. Laster. 2005. The anti-apoptotic effects of nordihydroguaiaretic acid: Inhibition of cPLA2 activation during TNF-induced apoptosis arises from inhibition of calcium signaling. Life Sci., 77:2457-2470.
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Progress 10/01/03 to 09/30/04
Outputs
During the past year we continued our research into cPLA2 and the role it plays in apoptosis. Previous studies from our lab have shown that cPLA2 unexpectedly translocates in the nucleus in cells rendered sensitive to TNF by cycloheximide. During the past year we have found that intranuclear cPLA2 can also be detected in cells rendered sensitive to TNF by infection with adenovirus, suggesting that this may be a common feature of apoptotic pathways. Our experiments also reveal that cPLA2-dependent inflammatory responses are suppressed in adenovirus infected cells. The intranuclear movement of cPLA2 may therefore by triggered by adenovirus in attempt by the virus to suppress inflammation and avoid recognition. We have also made progress on our study of the PRRS virus. During the past year we have established in vitro swine lymphocyte proliferation assays. We are using these assays, along with flow cytometry, to identify the effector cells necessary for immunity to PRRS
and determine whether T cell responses to PRRS are cross-reactive among heterologous strains.
Impacts
The apoptosis inducing activity of TNF is a major problem in a number of autoimmune and toxic pathological situations. The liver and the lungs are especially susceptible to TNF-mediated damage. We are studying an enzyme whose activity is necessary for TNF to cause cell death. By studying the mechanisms that regulate this enzyme's activity we will define new targets for the design of anti-apoptotic pharmaceuticals.
Publications
- Draper, D. W., Harris, V. G., Culver, C. A., and Laster, S. M. 2004. Calcium and its role in the nuclear translocation and activation of cytsolic phospholipase A2 in cells rendered senstive to TNF-induced apoptosis by cycloheximide. J. Immunol. 172:2416-2423.
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Progress 10/01/02 to 09/30/03
Outputs
During the past year we continued our research into signal transduction during TNF-inducd apoptosis. We completed our work on the role of calcium in the activation of cPLA2 during apoptosis. This work was submitted and accepted for publication in the Journal of Immunology. We have also determined that adenovirus infection alters the intracellular position of cPLA2. We are characterizing this effect fully, documenting its affect on cellular responsivess and should be submitting the work for publication shortly. Our lab has also been investigating the mechanism of pathogenesis of the porcine reproductive and respiratory syndrome (PRRS)virus. We produced a recombinant vaccine for PRRS. PRRS genes were cloned by rtPCR and expressed in baculovirus. During the past we attempted to establish an arrangement with an animal health company for production of the vaccine. All companies contacted refused due to legal and patent issues.
Impacts
The apoptosis inducing activity of TNF is a major problem in a number of autoimmune and toxic pathological situations. The liver and the lungs are especially susceptible to TNF-mediated damage. We are studying an enzyme whose activity is necessary for TNF to cause cell death. By studying the mechanisms that regulate this enzyme's activity we will define new targets for the design of anti-apoptotic pharmaceuticals.
Publications
- No publications reported this period
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Progress 10/01/01 to 09/30/02
Outputs
During the past year we continued our research into signal transduction during TNF-inducd apoptosis. We further explored the role of calcium in the activation of cPLA2 during apoptosis. We found that levels of calcium rise first in the perinuclear region, followed later by a large rise in the cytosol. Although this pattern mimics the capacitative entry model, we found that verapamil would block both perinuclear and cytosolic increases, suggesting that the capacitative entry model does not apply to the apoptotic cell. Our lab has also been investigating the mechanism of pathogenesis of the porcine reproductive and respiratory syndrome (PRRS)virus. During the past year we produced a recombinant vaccine for PRRS. PRRS genes were cloned by rtPCR and expressed in baculovirus. Recombinant proteins were purified by electroelution from SDS-PAGE gels and used to immunize 4-6 week old swine. Recombinant ORF4 and ORF5 proved most effective in challenge studies, completely
ablating symptoms and reducing the amount of detactable virus.
Impacts
The apoptosis inducing activity of TNF is a major problem in a number of autoimmune and toxic pathological situations. The liver and the lungs are especially susceptible to TNF-mediated damage. We are studying an enzyme whose activity is necessary for TNF to cause cell death. By studying the mechanisms that regulate this enzyme's activity we will define new targets for the design of anti-apoptotic pharmaceuticals.
Publications
- Feng, W.-H., Laster, S.M., Tompkins, M., Brown, T.T., Xu, J.-S., Gomez, W., Benfield, D., and M B. McCaw. Thymocyte and peripheral blood T lymphocyte subpopulation changes in piglets following in utero infection with porcine reproductive and respiratory syndrome virus. Virology 302:363 - 372. 2002.
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Progress 10/01/00 to 09/30/01
Outputs
During the past year we performed a variety of different experiments relating to signal transduction during TNF-inducd apoptosis. We examined the compound nor-dihydroguiaretic acid (NDGA), which strongly inhibits the TNF-induced apoptosis and the activation of cPLA2, however we did not find any inhibition of phosphorylation. We confirmed that TNF-induced apoptosis is indeed accompanied by an increase in intracellular levels of calcium. During this past year we also tested two other tyrosine phosphatase inhibitors, 3,4-dephostatin and DHMV. Neither compound produced the effects associated with sodium orthovanadate, casting some doubt on our hypothesis of phosphatase regulation of apoptosis. Fluorescence microscopy was used to define the intracellular site of cPLA2 translocation during apoptosis. Using a mAb to cPLA2 we found that during TNF-induced apoptosis, cPLA2 translocates only to the nuclear membrane. The pattern cPLA2 translocation seen during TNF-induced
apoptosis is very different from the intranuclear staining seen with PMA. Finally, during our studies of the phosphorylation of cPLA2 we discovered that expression of the adenovirus E1A gene, suppresses the activation of cPLA2 by PMA. We tested whether the mechanism of inhibition was inhibition of phosphorylation and found that phosphorylation of cPLA2 was unaffected. Our lab has also been investigating the mechanism of pathogenesis of the porcine reproductive and respiratory syndrome virus. Our results show a strong destructive impact on immune system of neonatal swine if the neonates are exposed to the virus in utero. The neonates are also born severely immunocompromised.
Impacts
The apoptosis inducing activity of TNF is a major problem in a number of autoimmune and toxic pathological situations. The liver and the lungs are especially susceptible to TNF-mediated damage. We are studying an enzyme whose activity is necessary for TNF to cause cell death. By studying the mechanisms that regulate this enzyme's activity we will define new targets for the design of anti-apoptotic pharmaceuticals. Our discovery of the inhibitory action of adenovirus may also lead to the desing of novel pharmaceutical compounds.
Publications
- Feng, W. H., Laster, S. M., Tompkins, M., Brown, T., Xu, J. S., Altier, C., Gomez, W., Benfield, D. and M. B. McCaw. In utero infection by porcine reproductive and respiratory syndrome virus is sufficient to increase susceptibility of piglets to challenge by streptococcus suis type II. J. Virol. 75:4889-95, 2001.
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Progress 10/01/99 to 09/30/00
Outputs
Our focus during the past year has been the phosphorylation of cPLA2 during TNF-induced apoptosis. Having observed that phosphorylation does occur, our goal is to determine whether it is necessary for enzyme activation. To address this question we have performed site directed mutagenesis. The human cPLA2 gene, in the pCI mammalian expression vector, has been subjected to mutagenesis using the Quick change site directed mutagenesis kit from Stratagene. The four potential sites of phosphorylation have been changed from serine to alanine; including residues 437, 454, 505, and 727. We are now adding a myc tag to each construct (to monitor expression) and we will then be ready to test each mutant gene in transfection experiments. As a positive control we will begin with the phorbol ester PMA, where phosphorylation of serine 505 has been shown to be necessary for enzyme activation. Our prediction is that mutation of serine 505 will inactivate response to PMA, but not to the
calcium ionophore A23187. Once this experiment has been performed sucessfully we will move ahead to treatment with TNF. In addition, during the past year, while performing preliminary dose response experiments with PMA, we discovered that infection with human adenovirus inhibits the PMA-induced release of arachidonic acid. Our studies have failed to reveal an affect on signal transduction suggesting that the mechanism of inhibition is down stream from activation.
Impacts
The apoptosis inducing activity of TNF is a major problem in a number of autoimmune and toxic pathological situations. The liver and the lungs are especially susceptible to TNF-mediated damage. We are studying an enzyme whose activity is necessary for TNF to cause cell death. By studying the mechanisms that regulate this enzyme's activity we will define new targets for the design of anti-apoptotic pharmaceuticals. Our discovery of the inhibitory action of adenovirus may also lead to the desing of novel pharmaceutical compounds.
Publications
- Wolf, L.A. and Laster, S.M. Characterization of arachidonic acid-induced apoptosis. Cell Biochem. Biophys. 30:353-368, 1999.
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Progress 10/01/98 to 09/30/99
Outputs
During the past year, our research focused on the connection between cPLA2 and the sphingomyelinases in TNF-induced apoptosis. In one set of experiments, we tested whether arachidonic acid and other free fatty acids could induce apoptosis. We found that by reducing the serum concentration in our apoptosis assays it was possible to observe fatty acid-induced apoptosis. Based on these results we examined a number of saturated and unsaturated free fatty acids for their ability to induce apoptosis. We defined a set of pro-apoptotic fatty acids and found that these fatty acids are similar to those reported to induce sphingomyelinase activity. In another set of experiments we examined the intracellular distribution of cPLA2 during apoptosis and found that it translocated to a cytosolic rather than nuclear location. Together both sets of evidence support the hypothesis that arachidonic acid is necessary for sphingomyelinase activation during apoptosis, a hypothesis that will
be tested directly during the coming year.
Impacts
(N/A)
Publications
- Wolf, L. A. and Laster, S.M. 1999. Characterization of arachidonic acid-induced apoptosis. Cell Biochem. Biophys. 30: 353-368.
- Laster, S., Stokes, K., Hadley, L. and O'Brien, J. 1999. Cytosolic phospholipase A2: Its role and regulation during TNF-induced apoptosis. Experimental Biology 99. Abstract.
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Progress 01/01/98 to 12/31/98
Outputs
During the past year our research focused on two areas of cPLA2 and its role in TNF-induced apoptosis; the control of cPLA2 activity by phosphorylation and the induction of apoptosis by arachidonic acid and other free fatty acids. Our studies on the phosphorylation of cPLA2 indicate that during apoptosis cPLA2 is activated by a pathway distinct from the MAP kinase pathway. This conclusion is based on the ability of a number of inhibitors to block cPLA2 activation during apoptosis (BHA, NDGA, Y-VAD, and Z-VAD), but not the activation of cPLA2 by PMA. We also observe a distinct pattern of cPLA2 translocation during apoptosis vs. PMA stimulation supporting the concept of distinct pathways. Translocation is to a golgi or endosomal compartment during apoptosis rather than the nuclear membrane as is seen with PMA. Based on these results we have begun to investigate the sites on cPLA2 that are phosphorylated during apoptosis. A finding that distinct sites are phosphorylated
would confirm that different pathways are involved. Our studies on arachidonic acid induced apoptosis indicate that arachidonic acid is the only 20 C fatty acid that can induce apoptosis. These data, coupled with the lack of expression of lipoxygenase pathway enzymes, suggests that the role of arachidonic acid during apoptosis is to activate one of the sphingomyelinase enzymes. This hypothesis will be investigated during the coming year.
Impacts
(N/A)
Publications
- O'Brien, J.B., Piddington, D.L., Voelkel-Johnson, C., Richards, D.J., Hadley, L.A., and Laster, S.M. 1998. Sustained phosphorylation of cytosolic phospholipase A2 accompanies cycloheximide- and adenovirus-induced susceptibility to TNF1. J. Immunol. 161:1525-1532.
- Wolf, L.A. and Laster, S.M. 1998. Characterization of arachidonic acid-induced apoptosis. Cell Biochem. Biophys. In press.
- O'Brien, J.B., Piddington, D.L., Wolf, L.A., Hadley, L.A., Stokes, K.J. and Laster, S.M. 1998. Cytosolic phospholipase A2 (cPLA2) and TNF-induced apoptosis. Seventh Int. Conf. on TNF and Related Cytokines, Hyannis, MA. May 1998. Abstract.
- O'Brien, J.B. 1998. The sustained phosphorylation of cPLA2 accompanies cytoheximide and adenovirus-induced susceptibility to TNF: Adenovirus E1A has diverse effects on the regulation of cPLA2. Ph.D. Thesis. 87 pp.
- Piddington, D. L. 1998. Lithium chloride can induce susceptibility to TNF in a cPLA2-dependent manner. Ph.D. Thesis. 100 pp.
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Progress 01/01/97 to 12/31/97
Outputs
During the past year our research focused on two areas of TNF-induced apoptosis; the signals necessary for the activation of cPLA2 and the role played by cPLA2. Our investigations into signaling have focused on the regulation of cPLA2 by phosphorylation. We have found that under non- toxic conditions, TNF itself causes only a transient pattern of phosphorylation. cPLA2 is phosphorylated for appx. 30 minutes and then dephosphorylated. A different pattern emerged, however, in cells that have been rendered susceptible to TNF by treatment with cycloheximide. In this case cPLA2 was phosphorylated for an extended period of time (up to 2 hrs). The extended phosphorylation of cPLA2 was also accompanied by a change in enzyme activity in vitro, suggesting that cycloheximide is inhibiting the expression of a TNF-induced phosphatase. Our investigations into the role played by cPLA2 have focused on defining the conditions for arachidonic acid-induced apoptosis. We have found that
under low serum conditions we can indeed induce apoptosis with arachidonic acid. Analogues of arachidonic acid also induced apoptosis, except for ETYA (which has triple bonds), indicating that arachidonic acid is being metabolized. These results suggest that a metabolite of arachidonic acid, probably a leukotriene, is acting as a second messenger during TNF-induced apoptosis.
Impacts
(N/A)
Publications
- OBRIEN, J.B and LASTER, S.M. 1997. Sustained phosphorylation of cPLA2 accompanies adenovirus and cycloheximide sensitization to TNF. NCSU Immunology Symposium. Raleigh, NC (Abstract).
- OBRIEN, J.B. and LASTER, S.M. 1997. Adenovirus EIA has multiple effects on the regulation of MAP Kinases. 1st Triangle Apoptois Meeting, Research Triangle Park, NC (Abstract).
- OBRIEN, J.B. and LASTER, S.M. 1997. Adenovirus E1A has multiple effects on the regulation of MAP Kinases. 17th International Congress of Biochemistry and Molecular Biology. San Francisco, California. Vol. 11, #9, p. A1117. (Abstract).
- PIDDINGTON, D.L. and LASTER, S.M. 1997. LiCl can induce susceptibility to TNF-Induced apoptosis. NCSU Immunology symposium, Raleigh, NC. (Abstract).
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Progress 01/01/96 to 12/30/96
Outputs
The focus continues to be the signals that are necessary for the activation of cPLA2 during TNF-induced apoptosis. Two specific signaling pathways are under investigation; a PLC-dependent pathway involving IP3 and calcium, and a MAP- kinase dependent pathway. We have found previously that IP2 (but not IP3) was produced during apoptosis and that verapamil could inhibit lysis following treatment with TNF and cycloheximide but not in E1A sensitized cells. These data suggest that calcium might be involved as a signal to activate cPLA2. However, the failure to find IP3, combined with the inconsistent effects of verapamil made this conclusion somewhat uncertain. We decided, therefore, to examine directly the metabolism of inositol phospholipids during apoptosis. The results of this investigation confirmed a role for a PLC-dependent pathway and lead to the discovery that lithium can induce susceptibility to TNF. The results of this investigation are now being prepared for
publication. Our study of the MAP-kinase pathway has also lead to some interesting results. Both E1A and cycloheximide alter the ability of TNF to phosphorylate the MAP kinases. We are currently studying whether the alterations are influencing the onset of apoptosis via the activation of cPLA2.
Impacts
(N/A)
Publications
- LASTER, S.M. and MACKENZIE, J.M. 1996. Bleb formation and f actin distribution during mitosis and TNF-induced apoptosis. Microscopy Res. and Tech., 34:272-280.
- VOELKEL-JOHNSON, C., THORNE, T.E. and LASTER, S.M. 1996. Susceptibility to TNF.
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Progress 01/01/95 to 12/30/95
Outputs
The focus of research in our laboratory continues to be the signals that are responsible for activating cPLA2 during TNF- mediated cytolysis. In cells that are rendered susceptible to TNF by an inhibitor of transcription or translation, we have determined that calcium is a necessary signal. During the past year we have begun to investigate the machinery that causes the increase in levels of intracellular calcium. The target of our investigation is phospholipase C. To monitor the activity of this enzyme we are measuring levels of inositol phosphates in prelabeled cells. Our results to date reveal a substantial increase in the level of IP2, a product of PLC metabolism, in cells treated with TNF and cycloheximide but not in cells treated with each compound independently. These results indicate that the target of cycloheximide's sensitizing effect is at or above PLC in the lytic pathway. During the coming year we will attempt to identify the form of PLC that is
activated during this response and to determine whether the 14.7 kDa protein inhibits cytolysis by inhibiting the activity of PLC.
Impacts
(N/A)
Publications
- LASTER, S.M., WOLD, W.S., and GOODING, L.R. 1995. Adenovirus proteins that regulate susceptibility to TNF also regulate the activity of PLA2. Seminars in Virology. 5:431-442.
- VOELKEL-JOHNSON, C., ENTINGH, A.J., WOLD, W.S., GOODING, L.R., and LASTER, S.M., 1995. Activation of intracellular proteases is an early event in TNF-induced apoptosis. J. Immunol. 154:1707-1716.
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Progress 10/01/93 to 12/30/94
Outputs
Research in our laboratory during the past year has focused on the signals that are responsible for activating cPLA2 during TNF induced apoptosis. We had found that cPLA2 was fragmented during apoptosis and that TAME, a serine protease inhibitor, could prevent the release of arachidonic acid from dying cells. We failed however, to demonstrate the formation of a common proteolytic fragment in apoptic cells, and when measured in vitro, cPLA2 activity in dying cells was less than in normal cells. We have concluded, therefore, that proteolytic cleavage of cPLA2 is not necessary for its activation during apotosis. We have found that verapamil, a compound that can prevent extracellular Ca++ from entering the cell, can prevent apoptosis and the release of arachidonic acid from dying cells. Verapamil is effective in tumor cells that are susceptible to TNF and in normal cells that are rendered sensitive either by cycloheximide or by infection with adenovirus. From these results,
we have concluded that Ca++ is a necessary signal during the apoptic response and we are now investigating the regulation of Ca++ in transformed cells and in cells infected by human adenovirus.
Impacts
(N/A)
Publications
- JOHNSON, C.V., CROUTHAMEL, H.D., and LASTER, S.M. 1994. TNF and the life and death of melanocytic cells. Fifth International TNF Congress, Monterey, CA, June, Eur. Cyto. Net. 5:261 (abstract).
- VOELKEL-JOHNSON, C., THORNE, T., SCANLON, M., GOODING, L., WOLD, W., and LASTER, S. 1994. Proteolysis may activate cPLA2 during TNF-induced lysis. Fifth International TNF Congress, Monterey, CA, June, Eur. Cyto. Net. 5:226 (abstract).
- KRAJCSI, P. et al. 1994. Adenovirus proteins that prevent both TNF-induced release of arachidonic acid and TNF induced cytolysis. Fifth Int TNF Congress, Monterey, CA, Eur. Cyto. Net. 5:124 (abstract).
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