We have reported that in vitro Manzamine A potently inhibited LPS-activated rat microglia thromboxane B2 (TXB2) (IC50
Ram P. Naikawadi1, Ni Cheng1, Dianqing Wu2 and Richard D Ye1
1Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612; 2Department of Pharmacology, Yale
Maintenance of vascular endothelial integrity is of great importance to homeostasis of vital organ functions. The small GTPase Rac is one of the key signaling mediators for vascular endothelial functions, but how Rac activation is regulated under various pathophysiological conditions remains incompletely understood. Using gene deletion and knockdown approaches, we investigated the potential role of the phosphoinositide- and G protein b subunitsregulated guanine nucleotide exchange factor P-Rex1 in TNF-a stimulated endothelial cells. P-Rex1, previously found in neutrophils and neurons, is also expressed in endothelial cells. In cultured human lung microvascular endothelial cells (HLMVECs), TNFa stimulation resulted in a decline in transendothelial electrical resistance (TER). Small interference (si) RNA-mediated knockdown of P-Rex1 markedly reversed the loss of TER and reduced intercellular gap formation. HLMVECs receiving PRex1-specific siRNA also displayed significantly reduced Rac activation, reactive oxygen species (ROS) production and phosphotyrosine dependent VE-cadherin phosphorylation upon stimulation with TNF-a, compared to HLMVECs receiving scrambled siRNA. TNF-a stimulated P-Rex1 membrane translocation and the resulting Rac activation is dependent on PI3K. Removal of P-Rex1 from endothelial cells produced a more significant reduction in neutrophil transendothelial migration when compared to removal of P-Rex1 from neutrophils. Moreover, significantly less neutrophils was found in the bronchoalveolar fluid of P-Rex1 deficient mice compared to WT mice after intratracheal instillation of TNF-a. Mice lacking the P-Rex1 gene are also resistant to TNF--induced lung edema, suggesting that PRex1 is a critical mediator of vascular permeability. These results demonstrate for the first time a pivotal role of endothelial P-Rex1 in the modulation of vascular endothelial integrity, suggesting the possibility for targeting endothelial P-Rex1 in the control of lung edema and neutrophil infiltration to inflammatory tissues.
Anlys Olivera, Andrew P Brown, Terry W Moore, Aiming Sun, Fang Hu, Dennis C Liotta, James P Snyder, Hyunsuk Shim,
The inflammatory transcription factor, nuclear factor kappa B (NF-κB), is a critical link between chronic inflammation and cancer. Therefore, we have endeavored to develop novel anti-inflammatory compounds that target NF-κB. Data indicate that curcumin, an ingredient of the curry spice turmeric, acts as a NF-κB inhibitor. EF31 and EF24 are structurally related to curcumin but exhibit increased potency and bioavailability. To examine the relative activity of these compounds in-vitro, mouse RAW264.7 macrophages were treated with EF31, EF24, curcumin (1–100μM) or vehicle (DMSO 1%) for 1 hour. NF-κB pathway activity was assessed following treatment with lipopolysaccharide (LPS) (1μg). LPS induced NF-κB activity in nuclear extracts peaked at 15 minutes, whereas expression of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α peaked at 3 hours. Potent inhibition of NF-κB DNA-binding was observed at concentrations of 5-10μM for EF31, 30-50μM for EF24, and 50-100μM for curcumin. In addition, EF31 exhibited significantly greater inhibition of NF-κB nuclear translocation as well as the induction of downstream inflammatory mediators including pro-inflammatory cytokine mRNA and protein. Moreover, EF31 exhibited significantly greater inhibition of IκB kinase β compared to EF24. Finally, EF31 demonstrated potent toxicity in NF-κB-dependent cancer cell lines while having minimal and reversible toxicity in RAW264.7 macrophages. These data indicate that EF31 is a more potent inhibitor of NF-κB activity than either EF24 or curcumin while exhibiting both anti-inflammatory and anticancer activities. Curcumin analogs including EF31 may represent a novel approach to treat cancer by hindering the effects of inflammation on the initiation and progression of tumors. Thus, EF31 represents a promising curcumin analogue for further therapeutic development.
Inflammation associated with various human diseases and the changes in endothelial cells (ECs) phenotype to support various phases of the inflammatory process are key patho-biological players in lung vascular remodeling and subsequent vascular disorders. In inflammatory states, like rheumatoid arthritis, pulmonary hypertension, connective tissue disease, the number of inflammatory cells, including T lymphocytes, is increased. The CD8+ cytotoxic T cells contain perforins and granzyme B (GrB) which are released from the cytotoxic granules to induce target cell death. The cytotoxic protease GrB can initiate ECs apoptosis by cleaving caspases and other proteins important for proliferation and cell survival. Using in vitro and in vivo approaches we now show that intersectin-1s (ITSN-1s), an essential protein for lung ECs survival, is cleaved by GrB at the sequence IDQD271K and generates an N-terminal cleavage product (GrBEHITSN), of 28 kDa. Significantly, expression of myc-GrB-EHITSN in ECs causes marked increase in cell proliferation, as indicated by bromodeoxiuridine (BrdU) incorporation detected with anti BrdU Ab. Additional Western blot analyses and Enzyme-Linked Immunosorbent Assay applied on lysates or nuclear extracts respectively, prepared from control and myc-GrB-EHITSN-transfected cells, indicate that GrB-EHITSN may be implicated in controlling cell proliferation via persistent activation of p38 MAPK and Elk-1 transcription factor. While the C-terminal GrB cleavage fragment has dominant negative effects on Ras/Erk1/2 survival signaling, expression of GrB-EHITSN and downstream p38 activation further inhibits Erk1/2 phosphorylation. Altogether, our studies provide evidence that loss of full-length ITSN-1s, the proliferative properties of GrB-EHITSN, selective activation of p38 MAPK and downstream Elk1, as well as the modulatory effect of ITSN-1s GrB cleavage products on p38/Erk1/2 MAPK signaling may provide an advantage for p38 MAPK signaling cascade favoring the selection of a hyper-proliferative EC phenotype.
Bethany E. Perez White*, Huiping Zhao and Debra A. Tonetti
Several factors have been identified that influence epithelial-mesenchymal transition (EMT) in breast cancer cells. We previously reported that overexpression of PKCα in breast cancer predicts tamoxifen resistant secondary tumors (Tonetti, 2003). In breast cancer cells, stable overexpression of PKCα (T47D/PKCα) leads to hormone-independent growth as well as tamoxifen resistance (Chisamore, 2001). In our current study we report that PKCα overexpression increases the metastatic potential of breast cancer cells through induction of a nonclasssical EMT. T47D/PKCα cells are morphologically distinct from T47D/neo cells and do not retain the cuboidal structure of epithelial breast cancer cells. Overexpression of PKCα resulted in significantly enhanced migratory capabilities in the Boyden chamber assay. PKC activation by phorbol 12-myristate 13 acetate further enhanced migration in T47D/PKCα cells. Pharmacological inhibition with the classical PKC inhibitor Gö6976 reduced the migratory capacity of T47D/PKCα cells. Transient siRNAmediated knockdown of PKCα (120 h) significantly reduced both basal and NIH3T3 fibroblast conditioned mediainduced migration in T47D/PKCα cells. Levels of adherens junction proteins E-cadherin, α-E-catenin, β-catenin and p120-catenin were significantly downregulated in T47D/PKCα cells compared to T47D/neo cells as determined by western blot. Levels of p120-catenin protein were restored after 120 h of PKCα knockdown, while levels of E-cadherin protein did not increase until after 144 h of PKCα knockdown in T47D/PKCα cells. E-cadherin transcripts analyzed by SYBR green RT-qPCR were significantly higher in T47D/PKCα cells compared to T47D/neo control cells while there was no change in expression of β-catenin or α-E-catenin transcripts. Only p120-catenin transcript levels were significantly lower. Treatment with proteasomal inhibitor MG132 induces accumulation of E-cadherin but not p120-catenin. Taken together, these data suggest that PKCα may be responsible for a nonclassical EMT in breast cancer cells through upstream signaling that leads to transcriptional inhibition of p120-catenin and subsequent degradation of E-cadherin. Further, PKCα expression may not only be predictive of tamoxifen-resistance but of increased potential for metastasis, two of the biggest clinical hurdles that preclude the effective management of breast cancer. Further studies are required to define PKCα as a potential therapeutic target or biomarker.
1Biomedical Sciences Program, College of Health Sciences and 2Department of Pharmaceutical Sciences, Chicago College
Stroke is a common life-threatening neurological disorder that requires rapid workup and treatment. GLP-1 has been found to protect neurons against oxidative insults. Liraglutide is a GLP-1 analogue approved to treat type 2 diabetes mellitus that crosses the blood brain barrier and may potentially treat cerebral ischemia. The aim of this study was to assess the effect of liraglutide on neurological outcome, oxidative stress and infarct volume in rats subjected to cerebral ischemia by middle cerebral artery occlusion (MCAO) in normal as well as in diabetes. Male Sp.Dawley rats were subjected to cerebral ischemia by MCAO. Animals were pretreated with either vehicle or liraglutide (50 μg/kg, s.c.) for 14 days. At 24 h after occlusion, rats were assessed for neurological and motor function. Then, animals were sacrificed for estimation of oxidative stress markers and infarct volume. In the diabetic group, streptozotocin (45 mg/kg, i.p.) was administered 3 days prior to vehicle/liraglutide injection. Non-diabetic and diabetic rats treated with vehicle showed significant (P
Effects of L-DOPA on risk-taking behaviors in rats
Stephanie Tedford1, Sandra Rokosik1,2 and T Celeste Napier1
1Dept. of Pharmacology, and Center for Compulsive Behavior and Addiction, RUSH University Medical Center, 60607;
2Neuroscience Program, Loyola University Medical Center, Maywood, IL 60153
A subset of Parkinson’s disease (PD) patients treated with dopamine (DA) agonists demonstrates impulse control disorders, such as pathological gambling. These side effects are associated with DA D3 receptor (D3R) preferring agonists including pramipexole (PPX) and are rarely seen with monotherapy using L-DOPA, an indirect DA agonist. To better understand the underlying neurobiology of these disorders, we developed a preclinical model of risk-taking using a probability discounting task in laboratory rats. In this task, a subject chooses between a small positive reinforcer that is always delivered and a large reinforcer that is delivered with varying probabilities. Risky behavior is reflected in a preference for the large, uncertain reinforcer. Our lab has implemented this task using intracranial selfstimulation of the medial forebrain bundle (MFB) as the positive reinforcer and determined that PPX increases risktaking in a rat model of PD and sham controls. For the current study, we aimed to assess the propensity of L-DOPA to induce risk-taking. To model PD, rats were bilaterally injected with 6-OHDA or vehicle into the dorsalateral striatum. The rats were also implanted with an electrode into the MFB. A forelimb step task validated motor deficits reflecting successful DAergic lesions of the striatum, and reversal of these deficits established the L-DOPA dose to be tested in the probability discounting task. Dose-response evaluations verified that Emax for improvements in stepping was reached for 3, 6 and 12mg/kg ip L-DOPA. For the second phase of this study, unlesioned rats were trained on the discounting task, and after a stable risk-taking baseline was established, they were treated with 12mg/kg ip L-DOPA 2X a day for 15 days. Risk-taking was assessed 30 min and 4 hr post injection every other day. Data collected thus far reveal that 12mg/kg L-DOPA does not alter risk-taking in control rats. Ongoing studies are determining the effects of LDOPA in PD-like rats. This work was supported by the Michael J. Fox Foundation, the Parkinson’s Disease Foundation Center Grant, Novel Projects Program Rush University and the Rice Foundation.
NMDA receptor blockade during adolescence disrupts the normative development of prefrontal cortical inhibitory
Thomases D, Cass DK, Caballero A, Tseng KY
Department of Cellular & Molecular Pharmacology, Rosalind Franklin University of Medicine and Science, The Chicago
Medical School, North Chicago, IL 60064
Despite the fact that a developmental disruption of cortical inhibitory circuits is thought to contribute to the adolescent onset of cognitive deficits observed in many major psychiatric disorders (e.g., schizophrenia), the neuronal basis and pathophysiological consequences underlying such changes remains unclear. Among the possible players, the N-methyl D-aspartate (NMDA) receptor is thought to play a crucial role in the maturation of parvalbumin (PV)-positive/fastspiking GABA interneurons in the prefrontal cortex (PFC). We hypothesized that if during development, such interneuronal activity does not become enhanced, prefrontal inhibitory control will be altered at maturity. Here we examine how repeated administration of the NMDA antagonist MK-801 during the periadolescent transition (postnatal day -PD- 35 to 40) changes the normal development of PV-positive interneurons in the PFC. We found that the level of PV-positive interneurons is diminished in the adult (PD>65) PFC when animals were treated with MK-801 during adolescence. No apparent changes in PV-immunoreactivity were observed in rats treated during adulthood (PD 75-80). In addition, electrophysiological analyses of cortical network activity indicate that MK-801-induced developmental dysregulation of interneuronal circuits can elicit a sustained, disinhibited PFC state. Such PFC disinhibition is associated with increased responsiveness as well as abnormal synaptic integration of synchronous inputs from the ventral hippocampus. We next examined the behavioral consequences of the decreased prefrontal interneuronal function by using the discrete paired delayed alternation paradigm to measure deficits in prefrontal-dependent working memory and acquired learning. Adolescent-treated rats displayed marked deficits in acquired learning capabilities, relative to controls when tested in adulthood. Together, these results indicate that glutamatergic activation of PV-positive GABAergic interneurons by NMDA receptors is critical for interneuronal development in the PFC. Impairment of such processes in the PFC could contribute to the periadolescent onset of cognitive deficits observed in schizophrenia and related psychiatric disorders. Supported by Rosalind Franklin University (KYT) and National Institute of Health Grant R01-MH086507 (KYT).
Enduring Alterations in the Prefrontal Cortex After a Single In Vivo Exposure of the HIV-1 Protein Tat
WN WAYMAN1,3, HB DODIYA2, AL MICKIEWICKZ2,3, F KASHANCHI4, JH KORDOWER2,3, XT HU1,3 and TC NAPIER1,3
1Department of Pharmacology and 2Neuroscience, and 3Center For Compulsive Behavior and Addiction, Rush University,
Chicago, IL; 4Microbiology, George Mason University, Manassas, VA
HIV-1-infected patients, even with highly active antiretroviral therapy, can develop HIV-associated neurocognitive disorders (HAND). While the neuropathogenesis of HAND is unknown, neurotoxicity of HIV proteins, including HIV-1 Tat (transactivator of transcription), is thought to play a critical role. The prefrontal cortex (PFC), a regulator for cognition and motivated-behaviors, is altered in those with HAND. Our recent studies revealed that Tat enhanced responses of pyramidal cells in the medial PFC to excitatory stimuli; this Tat effect was mediated by over-activated L-type Ca2+ channels with abnormally enhanced Ca2+ influx. For the current study, we hypothesized that a short-term in vivo exposure of Tat at pathophysiological levels will (1) increase the expression of the L-channels, (2) induce astrocytosis, but will not (3) cause neuronal death in the PFC. For these studies, adult rats were given a single unilateral intracerebroventricular (icv) injection of recombinant Tat (80μg/20μl), or its vehicle (n=3/each). Fourteen days after the icv injection, mPFC tissue was harvested and immunohistochemistry (IHC) was conducted. Stereological estimates and optical density were used to quantify Tat-induced increases in (1) L-channels, (2) glial fibrillary acidic protein (GFAP) a marker for astrocytosis, and (3) neuronal nuclear protein (NeuN) a marker for cell death. Our results indicated that the number of cells immunoreactive (ir) for the Cav1.2 Ca2+ L-channels and GFAP were significantly increased within the PFC of Tat-exposed rats compared to vehicle-injected controls. There was no change in the number of cells immunoreactive for NeuN. In summary, neither the astrocytosis nor the upregulation of Ca2+ channels was sufficient to cause the neuronal death that is commonly seen in later time points of HIV infection. However, as Tat can induce hyper-excitability of these pyramidal neurons via L-type Ca2+ channels and these channels are abnormally upregulated, channel blockers may be an efficacious therapeutic target to reduce the impact of HIV-1 proteins on the brain. Supported by the Rice Fdn., McManus Fdn and the Ctr Compul Beh & Addiction, RUMC.
Adolescent rats are more vulnerable to cocaine self-administration than adults
Vivian WC WONG, TUCCI NE, FORD KA, MCCUTCHEON JE, MARINELLI M.
Department of Cellular and Molecular Pharmacology, Rosalind Franklin University of Medicine and Science, North
Chicago, IL 60064
Adolescent rats take cocaine more readily than adults, are sensitive to lower doses, and work harder for access to the drug. Our study shows, for the first time, that adolescents are more sensitive to lower doses of cocaine, and they will work harder to obtain it. Our research is the first to offer scientific evidence that when all opportunities to take drugs are equal, biology alone makes adolescents more likely to use cocaine compared to adults. Adolescence is a period of heightened propensity to develop cocaine addiction in humans. We have previously shown that midbrain dopamine neurons are more active in adolescent rats compared with adults. Given that elevated activity of dopamine neurons is associated with elevated propensity to self-administer cocaine in rats, we tested whether the period of adolescence is associated with higher liability to self-administer cocaine relative to adulthood. Adolescent (PND 42) and adult (~PND 88) rats were compared for their sensitivity to, and motivation for, cocaine using self-administration. Different cohorts of rats were tested either (1) for acquisition of saline or very low to moderate doses of cocaine (75, 150, 600 μg/kg), (2) on a within-session dose-response paradigm (18.75-1200 μg/kg), or (3) on a between-session progressive ratio paradigm (600 μg/kg) in which the ratio was increased every other day (1, 3, 6, 12, 24, 48). In the acquisition tests, adolescents showed higher intake of cocaine than adults at low to moderate doses (150 μg/kg and 600 μg/kg), but did not differ in intake of a very low dose of cocaine (75 μg/kg) or saline. Adolescents and adults did not differ in the within session dose-response paradigm. In the between-session progressive ratio test, adolescents worked more to obtain cocaine than adults, suggesting that adolescents have greater motivation to self-administer cocaine than adults. In conclusion, we show that adolescent rats are more likely to acquire self-administration of cocaine than adults at low to moderate doses of cocaine, and show greater motivation to self-administer this drug. These experiments could help explain the greater addiction liability observed in human adolescents relative to adults.
GLC-ASPET Abstracts 2011: Undergraduate Students
Periadolescent activation of the CB1 cannabinoid receptor prevents the normative developmental facilitation of
parvalbumin-positive interneurons in the prefrontal cortex
Accoh P, Simak N, Tseng KY
Department of Cellular and Molecular Pharmacology, RFUMS / The Chicago Medical School, North Chicago, IL 60064
Recent epidemiological findings indicate that cannabis use during adolescence is associated with an increased risk for schizophrenia and other psychiatric disorders. However, the neuronal basis underlying the association of cannabis use and psychiatric disorders remains unclear. Decreased levels of interneuron’s markers have been repeatedly observed in the prefrontal cortex (PFC) of subjects with schizophrenia. Among cortical interneurons, the subset expressing the protein parvalbumin (PV) are known to inhibit pyramidal neuron firing in the PFC. In addition, PV immunoreactivity is known to correlate to interneuron activity. Here we assessed the effect of repeated administration of the CB1 agonist WIN on interneuron function by measuring PV immunoreactivity in the PFC. We observed that PV immunoreactivity in the normal PFC follows a distinctive developmental trajectory: lowest in juveniles and maximal in adulthood. Repeated administration of WIN during adolescence prevented the developmental enhancement of PV immunoreactivity in the PFC. In contrast, PV staining in the adult PFC was unaffected by WIN exposure. These results indicate that excessive activation of CB1 receptors during adolescence prevents the normal developmental maturation of PV interneuron function in the PFC. We therefore hypothesized that activation of the CB1 cannabinoid receptor during adolescence could alter the developmental maturation of PFC interneurons, a developmental that trigger a disinhibited PFC state and contribute to an increased risk for psychiatric disorders. Supported by NIMH R01 MH086507 (KYT).
BIOCHEMICAL CHARACTERIZATION OF GTP BINDING AND HYDROLYSIS IN AN INTRAGENIC SUPPRESSOR OF A
CONSTITUTIVELY ACTIVE ALLELE OF GSα
Jennifer Haick, Marla Hady, Robin Pals-Rylaarsdam
Benedictine University, Lisle, IL 60532
Heterotrimeric G-proteins cycle between an active/GTP-bound conformation and an inactive/GDP-bound conformation. Reduction in GTP hydrolysis by the G-protein alpha subunit causes constitutive activity of the protein. McCune-Albright Syndrome (MAS) is a disorder caused by a mutation that inhibits GTP hydrolysis in Gsα. Previous work from our laboratory identified an intragenic suppressor of the MAS mutation, which substituted two residues in the GTP-binding site. We have expressed this suppressor allele of Gsα, as well as wild-type (WT) and the MAS alleles in E. coli, partially purified them with affinity chromatography, and measured the ability of the isoforms of Gsα to bind and hydrolyze GTP. WT Gsα both binds and hydrolyzes GTP. The MAS allele can bind GTP well, but hydrolyzes it at a significantly lower rate than the WT protein. The suppressor allele is able to bind GTP, and exhibits an intermediate rate of GTP hydrolysis, suggesting that its suppression of the MAS mutation is due to partial restoration of GTPase function in the GTP binding site.
FINDING NOVEL INTRAGENIC SUPPRESSORS OF A CONSTITUTIVELY ACTIVE ALLELE OF GSα
Daniela Janevska, Eraj Din, Robin Pals-Rylaarsdam
Benedictine University, Lisle, IL 60532
Heterotrimeric G-proteins cycle between an active/GTP-bound conformation and an inactive/GDP-bound conformation. McCune-Albright Syndrome (MAS) is a genetic disorder caused by a mutation (R201H) that inhibits GTP hydrolysis in Gsα, permanently activating the protein. We have developed a yeast model system for MAS in which mutations in the yeast G subunit that mimic MAS (R297H) preven t colony formation. Previous students in the laboratory have used this system to identify an intragenic suppressor of the MAS mutation (L319P/D320V). In this presentation, we will describe our construction of a library of mutations in the yeast G subunit gene that will allow us to identify other suppressors of the MAS mutation using this yeast model. A library of 32,000 unique mutations in the Gα gene was constructed, the DNA purified, and preliminary screening the library for new intragenic suppressors of the MAS mutation has begun. Two new suppressor alleles have been identified. This work will allow future studies to thoroughly map the surface of Gα for sites which can inactivate the MAS allele, driving rational drug discovery of better treatments for MAS patients.
THE EFFECTS OF CADMIUM ON OSTEOCLAST APOPTOSIS & THE NONGENOMIC ROLE OF G-PROTEIN COUPLED
ESTROGEN RECEPTOR (GPER) IN CADMIUM-EXPOSED OSTEOCLAST APOPTOSIS
Garrick Moll, Joy Holowicki, Allison Wilson
Cell Biology/Toxicology, Benedictine University, Lisle, IL 60532
Cadmium has been identified as xenoestrogenic. Cadmium is a metal, yet it has the ability to bind to estrogen receptors. Estrogen is known to induce apoptosis within osteoclasts, which are the bone resorptive cells. Previous studies have shown that cadmium binds to estrogen receptor alpha (ER-α); it is postulated that the binding of cadmium to ER-α located on cellular surfaces inhibits the effect of estrogen on osteoclasts through a nongenomic pathway. Thus, cadmium could lead to premature bone loss and may exacerbate osteoporosis. Osteoclasts differentiated from RAW 264.7 cells and MitoCapture™ assay were used to confirm the findings of previous research that used DAPI/Phallodin staining. The analysis should inform that cadmium acts antagonistically with respect to estrogen, and that cadmium extends the viability of osteoclasts in the presence of estrogen. The second experiment intends to investigate the role of G-protein coupled estrogen receptor (GPER), a third estrogen receptor that also mediates nongenomic pathways in cells. Apoptosis after 1 hour was quantified with MitoCapture™. These tests will help clarify the mechanism of cadmium action in suppressing apoptosis in osteoclasts.
Cigarette smoke induces a pulmonary inflammatory response which leads to an increase activity of Mixed Lineage
Kinase-3 and possibly cell death
Arnav Rana, Velusamy Rangasamy, Gautam Sondarva, Joanna Bakowska and Suneet Mehrotra
Department of Molecular pharmacology and Therapeutics, Loyola University Medical Center, Maywood, IL 60153
Smoking is a central factor in many pathological conditions. Its role in neoplasm, lung and cardiovascular diseases has been well established for years. The mechanism of cigarette smoke toxicity is due to nicotine-enriched aerosol which is inhaled by smokers. The particle size of this aerosol is in micron-range, permitting efficient alveolar deposition and rapid absorption in the systemic blood. However, it is less acknowledged that cigarette smoking affects peripheral blood cell signaling. Cigarette smoke was shown to augment the production of numerous pro-inflammatory cytokines such as TNF-α, IL-1, IL-6, IL-8, GM-CSF and decreases the levels of anti-inflammatory cytokines such as IL-10. In our laboratory we have previously reported that increase in pro-inflammatory cytokine TNF-α, leads to an increase in kinase activity of Mixed Lineage Kinase-3 (MLK-3) a MAP3K member. We and others have reported that increase in MLK-3 kinase activity leads to activation of Jun N-terminal kinase (JNK) pathway, leading to cell death. Thus, we had carried out this study to elucidate the activity of MLK-3 in peripheral blood of smokers and non-smokers. Our results clearly demonstrate that smokers have augmentation in the kinase activity for MLK3, when compared to non-smokers. These results collectively suggest that smokers’ peripheral blood cells are more prone to cell death via activation of MLK3/JNK pathway.
EVALUATING THE ROLE OF TWO POST-TRANSLATIONAL MODIFICATIONS OF PARKINSON’S DISEASE PROTEN α-
SYNUCLEIN IN YEAST MODELS
Keith Solvang and Shubhik DebBurman
Biology Department, Lake Forest College, Illinois 60045
Parkinson’s disease (PD) is an incurable neurodegenerative disorder that affects four million people worldwide. Pathogenesis of PD includes aggregation of the protein α-synuclein into structures called Lewy bodies within dying midbrain dopaminergic neurons. α-Synuclein is also post-translationally modified through several mechanisms. Many neurodegenerative diseases, such as Alzheimer’s disease and Huntington disease, are linked to the imbalance of posttranslational modifications. Thus, a major hypothesis in PD is that the disproportionate levels of specific posttranslational modifications may contribute to those α-synuclein properties that make it toxic. Increasing evidence suggests phosphorylation and nitration are the most significant pathology-related modifications on α-synuclein. Three α-synuclein characteristics commonly linked to its toxicity include its ability to accumulate, to aggregate and to bind membrane phospholipids. However, the link between phosphorylation, nitration and these three properties are still not well understood in organismal models. Our lab has recently developed two yeast models that each recapitulates distinct aspects of these α-synuclein properties. In particular, budding yeast (Saccharomyces cerevisiae) models membrane association and fission yeast (Schizosaccharomyces pombe) models aggregation. Therefore, using both these yeasts, we evaluated the relationship between the pathological characteristics of α-synuclein and serine phosphorylation (S87, S129) and tyrosine nitration (Y39, 125, Y133, Y133). Specifically, we evaluated α-synuclein concentration (via Western blotting), aggregation and membrane association (via live cell e-GFP imaging) and cellular toxicity (via serial dilution colony growth in solid plates), and asked if these properties were altered when we blocked or constitutively activated the phosphorylation or nitration at each relevant site. We report three significant findings here. First, α-synuclein is phosphorylated in budding yeast. Secondly, constitutive phosphorylation decreased the ability to bind membrane phospholipids, however, blocking phosphorylation decreased aggregation and increased toxicity. Finally, nitration status affected membrane association in budding yeast and was toxic in fission yeast. This report provides multiple lines of evidence that post-translational modifications affect several PD-associated properties of α-synuclein. Supported by NIH R15 048508-02, NSF MRI 0115919, NSF CCLI 0310627.
INTRAGENIC SUPPRESSORS OF A CONSTITUTIVELY ACTIVE ALLELE OF GSα
Dahlia Sultan, Raquel Tobar-Rubin*, Robin Pals-Rylaarsdam
Benedictine University, Lisle, IL 60532
Heterotrimeric G-proteins cycle between an active/GTP-bound conformation and an inactive/GDP-bound conformation. Reduction in GTP hydrolysis by the G-protein alpha subunit causes constitutive activity of the protein. McCune-Albright Syndrome (MAS) is a genetic disorder caused by a mutation that inhibits GTP hydrolysis in Gsα. Biopsies from MAS patients show R201H, R201S, and R201C mutations. Previous work modeled the MAS mutation in a yeast system, and identified an intragenic suppressor of the MAS mutation, which substituted two residues in the GTPbinding site. We characterized the ability of mutations at F222 and D223 in human Gsα to suppress the constitutive activity of the protein caused by mutations at R201 found in MAS patients. Site-directed mutagenesis introduced mutations into Gs at residues F222 and D223. Following expression in mammalian cells; basal and receptor-mediated cAMP production was measured by ELISA. The original suppressor, F222P/D223V, reduced basal cAMP levels compared with the R201H allele and signaled through G-protein mediated pathways. F222P and D223V alone each suppressed the constitutive activity of the R201H allele. The F222P/D223V mutation was able to suppress the constitutive activity of a serine substitution at R201. Finally, Substitution of D223 with nonpolar (A), but not acidic (E) or polar (N) residues was able to suppress the R201H constitutive activity similarly to the D223V substitution.