Studies of expression, signaling, and trafficking of the beta2-adrenergic receptor in response to beta-agonist

Koryakina, Yulia University of Arkansas for Medical Sciences 2009 해외공개박사

The beta2-adrenergic receptor is a prototypical member of a diverse family of G-protein coupled receptors (GPCRs), the largest family of receptors for hormones and drugs. The beta2-adrenergic receptor is an important target for medications in asthma and cardiovascular disease. The intracellular location of the beta2-adrenergic receptor in tissues is likely to have a significant impact on how the tissues respond to beta-agonist medications. For that reason, it is important to understand mechanisms underlying signaling and regulation of the receptor in response to agonists. Thus, method for visualizing beta2-adrenergic receptor would be of utility. In the study, described in Chapter II, I characterized a panel of six beta2-adrenergic receptor antibodies for their ability to recognize human and rat beta2-adrenergic receptor in HEK 293 cells in indirect immunofluorescence assays. We identified one antibody capable of recognizing human but not rat beta2-adrenergic receptor, one antibody capable of recognizing rat but not human receptor, and one antibody recognizing both rat and human beta2-adrenergic receptors. The antibodies were used to localize beta2-adrenergic receptors in primary cultures of rat airway epithelial and smooth muscle cells. In HEK 293 cells, the receptors were localized to the cell surface. In contrast, about half of the receptors were found in intracellular compartment in primary cultures. We anticipate that the antibodies we identified will serve as a valuable tool for studies of expression and trafficking of the beta 2-adrenergic receptor in tissues. N-terminal polymorphisms of the beta2-adrenergic receptor consist of substitution of glycine for arginine at amino acid position 16 and substitution of glutamic acid for glutamine at position 27. Studies have shown that N-terminal polymorphisms of the beta2-adrenergic receptor may influence therapeutic responses to beta-agonists. In my second project, described in Chapter III, I used a recombinant expression system to study the effect of beta-agonist on the expression, trafficking and signaling of the beta2-adrenergic receptor with N-terminal polymorphisms. In ligand binding assays, I found that beta2-adrenergic receptor isoforms were differentially down-regulated as a result of prolonged beta-agonist treatment. Importantly, these differences in down-regulation are translated into changes in functional cyclic AMP response. To explore the mechanism of differential down-regulation, a detailed temporal and spatial analysis of trafficking of the YFP-tagged polymorphic receptors was performed. The results of our experiments are consistent with the notion that enhanced down-regulation of isoforms with Arg16 could be due to enhanced traffic of isoforms with Arg16 in lysosomal compartment for degradation. This may provide the basis for differential response to beta-agonist therapy in patients.

Identification of Cdk1 as the vinblastine activated Bcl-xL kinase

Terrano, David University of Arkansas for Medical Sciences 2009 해외공개박사

Despite detailed knowledge of the components of the spindle-assembly checkpoint, a molecular explanation of how cells die after prolonged spindle checkpoint activation, and thus how microtubule inhibitors and other anti-mitotic drugs ultimately elicit their lethal effects, has yet to emerge. Mitotically arrested cells typically display extensive phosphorylation of two key anti-apoptotic proteins, Bcl-xL and Bcl-2, and evidence suggests that phosphorylation disables their anti-apoptotic activity. However, the responsible kinase has remained elusive. This dissertation presents evidence that Cdk1/cyclin B catalyzes Bcl-xL/Bcl-2 phosphorylation during mitosis and vinblastine-induced mitotic arrest. Preliminary experiments are presented first where two Bcl-xL kinase substrates, a full length protein (HisBclxL DeltaC) and a fusion peptide (GST-BL) were purified and tested as specific Bcl-xL kinase substrates. HisBclxL DeltaC was an ineffective and non-selective substrate because it was equally phosphorylated by size exclusion fractions, whole cell extracts, and cytosolic and mitochondrial fractions from both control and vinblastine treated KB-3 cells. In contrast, GST-BL, where the fused BL peptide contains amino acids 41-70 of Bcl-xL including the vinblastine induced phosphorylation site Ser62, was phosphorylated only in size exclusion fractions from vinblastine treated KB-3 cells. However, the GST-only negative control was also extensively phosphorylated indicating poor specificity of the substrate. Both HisBclxL DeltaC and GST-BL were therefore not suitable as Bcl-xL kinase substrates in partially-purified extracts or subcellular fractions, but the experiments did provide the foundation to develop a small peptide substrate, termed FL62, harboring only the vinblastine induced phosphorylation site, Ser62. Characterization of the kinase in vitro using FL62 as a substrate revealed it corresponded to Cdk1/cyclin B. Furthermore, Cdk inhibitors specifically inhibited Bcl-xL/Bcl-2 phosphorylation induced by vinblastine in cell culture. Furthermore, during normal mitosis, Bcl-xL and Bcl-2 were partially and transiently phosphorylated in a Cdk-dependent. The findings suggest a model whereby a switch in the duration of Cdk1 activation, from transient during mitosis to sustained during mitotic arrest, dramatically increases the extent of Bcl-xL/Bcl-2 phosphorylation, resulting in inactivation of their anti-apoptotic function. Thus, phosphorylation of antiapoptotic Bcl-2 proteins acts as a sensor for Cdk1 signal duration and as a functional link coupling mitotic arrest to apoptosis.

Structure-function studies of the superfamily 1B helicases Dda and Pif1

Blair, Lauren Paige University of Arkansas for Medical Sciences 2008 해외공개박사

Helicases are proteins that unwind double-stranded nucleic acids. Dda helicase from bacteriophage T4 has served as an excellent model for understanding the molecular mechanism of this class of enzymes. Study of the structure of Dda may reveal why some helicases translocate in a 5'-to-3' direction on DNA, while others translocate in a 3'-to-5' direction. Attaining a structure of Dda has proven difficult because the protein fails to readily form crystals and is too large for NMR. Also, Dda has been shown to interact with the T4 single stranded DNA binding protein, gp32. We have developed a homology model of the enzyme that we are using to guide further studies that will examine the structural and functional significance of the interaction between Dda and gp32, and how this interaction impacts translocation on DNA. We tested the structural model by examining the protein surface using two common methods for mapping protein domains and for examining protein surfaces: limited proteolysis and chemical footprinting. We also performed a functional analysis of Dda variants with amino acid alterations at sites known to interact with DNA in other helicases. In the process, we identified the site of Dda interaction on DNA as well as the site of Dda interaction with gp32. The Pif1 helicase from Saccharomyces cerevisiae is in the same superfamily and has the same directionality as Dda. We identified the interactome of Pif1 and found that it interacts with Rim1, a mitochondrial SSB. We also performed a basic analysis of Pif1 post translational modifications. These studies taken together allow us to present a mechanism for SF1B helicases taking into account their interaction with DNA and SSBs.

Neonatal phencyclidine or ketamine treatment modifies preweaning behaviors, but only neonatal PCP treatment alters adult locomotor activity in male and female Sprague-Dawley rats

Boctor, Sherin Yousry University of Arkansas for Medical Sciences 2009 해외공개박사

Treatment with the N-methyl-D-aspartate receptor antagonists ketamine (KET) or phencyclidine (PCP) can trigger apoptotic neurodegeneration in neonatal rodents; however, little is known about the behavioral alterations resulting from such neurodegeneration. Here, rats were sc treated with: saline; 10 mg/kg PCP (1x/day) on postnatal days (PNDs) 7, 9 and 11; 20 mg/kg KET (6 injections every 2 hours on PND 7); or a similar regimen of ketamine and 250 mg/kg L-carnitine (LC) on PND 7, with a single injection of 250 mg/kg LC on PNDs 8-11 (KLC). Post-injection, home cage behavior of each pup was categorized on PNDs 7-11. Slant board (PNDs 8-11), forelimb hang (PNDs 12-16), prepulse inhibition (PPI; PND 25), grip strength and motor coordination (PND 22 or 71), locomotor sensitization (PND 42), spatial alternation (PNDs 22-70) and residential running wheel activity (PNDs 72-77) were also examined. The initial KET or KLC treatments on PND 7 elevated abnormal home cage activity; however, behavior of KLC-treated pups returned to control levels more quickly than that of the KET-treated pups, indicating the protective effects of LC. PCP treatment caused substantial abnormal home cage activity on each treatment day (PND 7, 9, and 11). Latencies to turn on the slant board on PND 8 were significantly longer for KET- and PCP-treated pups and on PND 10 for PCP-treated pups. On PND 12, the forelimb hang time of PCP-treated pups was significantly shorter. Despite the magnitude of PCP-induced preweaning changes, PPI behaviors of PCP-, KET-, and KLC-treated groups were comparable to controls. Developmentally treated KET- or KLC-treated rats responded to a 5 mg/kg KET challenge with activity similar to controls which received the same challenge. However, developmental PCP treatment induced significant sensitization to a 3 mg/kg PCP challenge relative to controls which received the same challenge, causing substantially increased open field activity. Performance on a spatial alternation task was unaffected by neonatal KET, KLC or PCP treatment; however, neonatal PCP treatment elevated light and dark period running wheel activity. These data indicate that developmental PCP treatment causes short- and long-term behavioral alterations and suggest that neonatal KET treatment does not result in lasting behavioral modifications.

Characterization of ade-M26 hotspot RNA and its role in the activation of meiotic homologous recombination in the fission yeast Schizosaccharomyces pombe

Wagnon, Jacy Lee University of Arkansas for Medical Sciences 2009 해외공개박사

Homologous recombination during meiosis is essential to ensure proper chromosome segregation. Improper chromosome segregation can lead to meiotic aneuploidy, which is the leading cause of congenital birth defects, mental retardation, and spontaneous pregnancy loss in humans. Errors in the positioning and frequency of recombination are features of all meiotic aneuploidies studied. Meiotic homologous recombination is induced by double-stranded DNA (dsDNA) breaks and is clustered preferentially at recombination hotspots, such as the ade6-M26 hotspot in the fission yeast Schizosaccharomyces pombe. Evidence that transcription helps activate the ade6-M26 hotspot includes the binding of Atf1, an ATF/CREB transcription factor, to a CRE-like DNA site, M26, that is required for hotspot activity. The ade6 promoter is also essential for hotspot activity. Interestingly, these hotspot-activating factors also trigger the formation of a short, hotspot-specific RNA. In this dissertation, it is reported that no single discrete site in the promoter is required for hotspot activity or hotspot RNA production, as evidenced by a functional dissection of the ade6 promoter region. Hotspot RNA is induced in meiosis and under stress conditions in an Atf1-dependent manner. Furthermore, hotspot RNA is processed post-transcriptionally in a pathway that is distinct from both nonsense-mediated decay and RNA interference. These results provide evidence of a previously unknown mechanism of post-transcriptional gene regulation by ATF/CREB proteins. M26 DNA sites moved along the ade6 open reading frame that have hotspot activity also produce short RNA transcripts whose 5' ends map near the M26 sites as do dsDNA breaks. These data suggest that hotspot RNA may help direct the positioning of recombination-initiating dsDNA breaks. Neither artificially shortened ade6-M26 RNA nor hotspot RNA is sufficient for hotspot activity. However, loss of processed hotspot RNA results in a 50% reduction in hotspot activity. These results indicate that hotspot RNA is required for full hotspot activity at ade6-M26 , although it is not essential for all hotspot activity. This dissertation work shows that a specific processed RNA molecule is required for the full activity of a meiotic recombination hotspot, increasing the number of processes known to be mediated, at least in part, by RNA or RNA metabolism.

Cannabinoid receptor inverse agonists as novel therapeutic agents

Seely, Kathryn A University of Arkansas for Medical Sciences 2009 해외공개박사

The purpose of these studies was to investigate cannabinoid receptor inverse agonists, which decrease the constitutive activity of cannabinoid receptors, as innovative therapies and explore unique interactions of these compounds with mu-opioid receptors (MOR). Amyotrophic lateral sclerosis (ALS) is a neuroinflammatory disease characterized by degeneration of spinal motor neurons. During ALS, CB2 receptors, localized on immune cells, are up-regulated within the spinal cord, indicating a prospective drug target. Since multiple drugs acting by different and distinct mechanisms may be superior treatments, curcumin, a polyphenol in the spice turmeric with anti-inflammatory and anti-oxidant properties, may also represent a novel ALS treatment. The results suggested CB2 inverse agonists prolong survival longer than vehicle treated controls. Also, curcumin lengthens survival but concurrent treatment with curcumin and CB2 inverse agonists antagonize each treatment alone. Therefore, CB2 inverse agonists and curcumin may be novel ALS treatments but concurrent treatment of these compounds should be avoided. Since curcumin and CB2 inverse agonists appeared to antagonize each other, we investigated the binding of curcumin and other polyphenols, such as trans-resveratrol in red wine, to cannabinoid receptors. Surprisingly, these polyphenols bind CB1 receptors, the most abundant G-protein coupled receptor in the central nervous system, selectively with nanomolar affinity and act as antagonists/inverse agonists. Importantly, curcumin and tran-resveratrol produce weight loss similar to a CB1 inverse agonist control suggesting the potential development of polyphenols as anti-obesity treatments. Lastly, a recent study demonstrated a CB1 inverse agonist enhanced MOR function suggesting the constitutive activity of CB1 receptors modulates the function of MOR. Therefore, we investigated the interaction of CB1 and MOR using AM251, a CB1 selective inverse agonist. The results suggest that interactions between AM251 and MOR are not entirely mediated by CB1/MOR interactions because AM251 binds directly to MOR as an antagonist with nanomolar affinity. Therefore, AM251 modulation of MOR may be occurring via AM251 binding directly to MOR as well as interactions between CB1 and MOR. Overall, these studies suggest that cannabinoid receptor inverse agonists may be developed as novel therapeutic options for many diseases, such as ALS and obesity.

Southern rural black women's explanatory models of heart disease

Evans, Laura K. Briant University of Arkansas for Medical Sciences 2009 해외공개박사

Background/significance. Cardiovascular disease (CVD) kills more women than any other cause of death. However, black women are more likely than white women to underestimate their risk for CVD and less likely to participate in leisure-time physical activity (LTPA). Increasing LTPA can potentially decrease the risk for CVD. The purpose of this study was to explore black women's explanatory models (EMs) of the etiology, risk, and prevention of CVD. Methods. An ethnographic design was used to explore EMs of CVD of 20 southern rural black women with at least 1 modifiable risk factor for CVD. Participants were recruited using convenience sampling at monthly staff meetings at a long-term care facility [n=14]. The other 6 participants were recruited through snowball sampling. Individual in-depth semi-structured audiotaped interviews were conducted primarily in women's homes. Descriptive statistics, content analysis, and constant comparison were used to analyze data. Results. Participants were 40 to 58 years, and 19 of 20 were overweight. Other risk factors included: hypertension [n=14] and dyslipidemia [n=7]. Ten reported walking 1 to 2 times a week; only 3 reported LTPA at recommended levels. Although participants were queried about CVD, they focused solely on heart disease. Qualitative data revealed 3 factors of the "Causes of Heart Disease": (1) Lifestyle- "[E]atin' a lot of greasy food." (2) Physiologic Causes- "[P]eople that have diabetes will have heart problems." and (3) Going to Get It- "[A woman] can't prevent it if it's hereditary;" and 4 factors of women's "Thoughts about Physical Activity": (1) Thoughts about How Exercise Affects the Heart - "I would say it [exercise] wouldn't help them not to get it. I feel like it wouldn't be as bad." (2) Barriers to Exercise- "I just don't have the energy to exercise." (3) Reasons to Exercise - "It's a stress reliever for me to be able to walk." and (4) Taking Action- "I don't do it [exercise] as often as I should.". Discussion. Misconceptions regarding the inevitability of CVD, incongruous thoughts about the role of LTPA, and lack of personalization of CVD risk indicate that further education is needed, as is the development of interventions to assist women to increase LTPA.

Molecular regulation of inflammation by the proteasome

Cullen, Sarah University of Arkansas for Medical Sciences 2009 해외공개박사

Proteasome inhibition has become synonymous with inhibition of NF-kappaB activity. However, hyperactive NF-kappaB responses accompany physiological conditions in which proteasomal function is compromised, i.e. advancing age, geriatric diseases, and bortezomib resistance. These paradoxical NF-kappaB responses are likely to be impervious to proteasomal defects because they stem from atypical NF-kappaB signaling induced by proteasome-independent mechanisms. Associated with redox stimuli and various growth factors, this atypical pathway does not require proteasome for NF-kappaB nuclear translocation; however, a potential role for proteasome in nuclear signaling remains unexplored. We now demonstrate that proteasome stringently controls transcription of inflammatory mediators regulated by this atypical NF-kappaB pathway. Proteolytic activity of the proteasome mediates the removal of the NF-kappaB subunit, p65/RelA, from inflammatory genes, thereby terminating atypical NF-kappaB-dependent transcriptional responses. Since proteasome inhibition results in sustained transcription of particular inflammatory genes, this implies that such exaggerated transcription results from prolonged nuclear NF-kappaB activity and persistently remodeled chromatin. In accordance with our hypothesis, proteasome inhibits persistent nucleosome remodeling activity by the SWI/SNF class of ATP-dependent remodeling complex. In summary, proteasome targets promoter-associated subunits of both NF-kappaB and the SWI/SNF complexes to terminate their activities. Thus, an inability to down-regulate NF-kappaB activity induced by proteasome-independent mechanisms may contribute to the hyperactive NF-kappaB responses accompanying physiological conditions characterized by proteasomal defects, i.e. advancing age, geriatric diseases, and bortezomib resistance.

Characterization of copper, zinc superoxide dismutase in vivo: Zinc-deficient SOD1 as a potential neurotoxic species in Amyotrophic Lateral Sclerosis

Bhogaraju, Venkata K University of Arkansas for Medical Sciences 2009 해외공개박사

Amyotrophic Lateral Sclerosis (ALS or MND) is a lethal neurodegenerative disease characterized by the progressive death of motor neurons. Mutations to Cu, Zn Superoxide Dismutase (SOD1) remain the only known cause in humans and transgenic animals, inducing neurotoxicity by an unknown mechanism. The only clues as to how and why the enzyme is selectively toxic to motor neurons come from work with recombinant enzymes. The properties which give rise to the "gain of function" toxicity of SOD1 mutants has yet to be identified, however, mounting evidence suggests that Zn binding and labile cysteine residues play key roles. The goal of this project was to characterize a mutant SOD1 (hS0D1G93A) purified from transgenic rat spinal cords as a function of disease onset and progression, and test these proposed properties in vivo . This required developing new methodology to purify the enzyme in such a way to preserve its native properties. This was accomplished using a novel HPLC purification method that yielded > 99% pure enzyme. In addition, new experimental protocols were designed and optimized to determine the SOD1 specific activity, the content of Cu, Zn, and reduced thiols, all as a function of disease severity. Along with the first report of specific activity, status of the Cu, Zn and thiol content for in vivo expressed SOD1, these results revealed that a substantial fraction of SOD1 was Zn-deficient in vivo which increases with disease progression. This finding has potential implications in ALS as Zn-deficient SOD1 was demonstrated to be both pro-oxidant and pro-aggregatory in vitro. Thus, these results provide both proof of concept for a novel method of measuring the in vivo metallation state of Zn-binding proteins, and support the fundamental hypothesis that Zn-deficient SOD1 correlates with the severity of motor neuron disease. To summarize, these results identified Zn-deficient SOD1 as a primary toxic species or toxic intermediate in causing ALS and thus, could be a novel target for therapeutic intervention.

Promoting personhood among nursing home residents living with dementia

Buron, Bill University of Arkansas for Medical Sciences 2009 해외공개박사

Dementia is one of the leading risk factors for admission into a nursing home (NH) facility. In the traditional medical model, care practices are often systematic and impersonal, and there is little incentive or encouragement for nursing staff to learn about the unique needs of individual residents. In this impersonal environment, residents living with dementia (RLWD), who often lack the capacity to communicate, can be ignored. Unmet needs can lead to disruptive behavior among residents, and frustration, burnout and high turnover among nursing staff. In this study, a pretest-posttest control group quasi-experimental design was used to evaluate the effect of individualized, or person-centered Life History Collages on (1) changes in nursing staff knowledge of individual residents, and (2) nursing staff perceptions of (a) knowing the person, (b) staff to resident communication, and (c) staff to staff communication. Thirty-six nursing staff (18 intervention and 18 control) and 5 RLWD (3 intervention and 2 control) participated. Open and closed-ended questionnaires were used to collect staff (1) knowledge about individual residents, and (2) perceptions of person-centered care (PCC) practices pretest, posttest, and again at 3 weeks post-intervention. The PI utilized multivariate analysis of covariance (MANCOVA) to compare mean (1) levels of knowledge about individual RLWD, and (2) perceptions of PCC practices between the intervention and control nursing staff members posttest, and 3 weeks post-intervention, controlling for pretest levels. Analyses of covariance (ANCOVA) were used to determine the effect of collage exposure, or no collage exposure on the mean nursing staff (1) knowledge of three dependent variables: (a) family, (b) jobs/careers, and (c) past and current likes, dislikes, and interests, and 2) perceptions of (a) knowing the person, (b) staff to resident communication, and (c) staff to staff communication posttest, and 3 weeks post-intervention, controlling for pretest levels. Based on Kitwood's Theory of Personhood, improved knowledge and perceptions of RLWD may lead to improved personhood status. Elevated personhood status has strong implications for caregiving practices in the NH setting. The Personhood Model for Dementia Care is discussed to illustrate the importance of various levels of personhood, and person-centered care practices for RLWD in NH settings.