Cellular & Molecular Science

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/31253


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Now showing 1 - 11 of 11
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    (2013-04-12) Liu, Ying
    Purpose: We hypothesize that TIP110 expression is modulated through C-MYC and herein demonstrate that the oncogenic transcription factor C-MYC up-regulates transcription of the RNA binding protein TIP110 through interaction with the TIP110 E-box in the TIP110 promoter, thus ensuring high-level Tip110 expression in proliferating hESCs. TIP110, therefor, regulates OCT4 expression through alternative splicing in hESCs. Methods: To examine direct involvement of C-MYC over TIP110 expression, we over-expressed C-MYC, or silenced C-MYC expression in stem cells to test whether C-MYC regulates Tip110 expression. To identify the elements by which C-MYC could regulate Tip110 expression, both CHIP assay and EMSA were performed. Splicing assay was used to study the alternative splicing of OCT4 by TIP110 protein. Results: There was a clear increase of Tip110 expression with overexpression C-MYC, and a reduction of TIP110 expression with knockdown of C-MYC expression at both mRNA and protein level. The results reveal the binding between TIP110 promoter E-box region and C-MYC protein. The splicing assay further shows that TIP110 alternatively splices OCT4 to produce OCT4A in stem cells. Conclusions: In this study, we demonstrate the interaction between TIP110 promoter E-box region and C-MYC protein. TIP110 expression induces OCT4 exon 1a inclusion and 1b skipping, an event required for the pluripotency of human embryonic stem cells.
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    (2013-04-12) Midde, Krishna
    Purpose: Muscle contracts by cyclic, ATP-driven, reorientations of myosin cross-bridges while they are interacting with actin. It is widely recognized that during force generating step of this interaction, referred to as a power-stroke, myosin head forms a firm attachment to actin, while the torque is provided by the rotation of myosin neck (a cross-bridge consists of a head plus a neck). During contraction, the Regulatory Light Chain of myosin (RLC), which resides on the neck, gets phosphorylated. An important goal of muscle research is to elucidate the role of phosphorylation during active contraction. We set out to measure how the rate of power stroke is affected by phosphorylation in muscle ex vivo. Methods: The rate can only be established when small number of molecules is investigated. This is accomplished by Confocal Fluorescence Correlation Spectroscopy (CFCS). Results: Phosphorylation of RLC speeds up power stroke from ~0.64/s when RLC is de-phosphorylated to ~1.5/s when it is phosphorylated. Conclusions: Using this technique, we show that the state of phosphorylation of the Regulatory Light Chain (RLC) of myosin has a profound effect on kinetics of ex vivo rabbit psoas cross-bridges during contraction.
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    (2013-04-12) Mamik, Manmeet K.
    Purpose: Chemokine CXCL8 is an important neutrophil chemoattractant implicated in various neurodegenerative disorders. We previously reported that HIV-1 infection is linked to upregulation of CXCL8 in brain tissue. We also reported that SHP2 and MAPK pathways regulate the expression of CXCL8 in human astrocytes. In the post-ART era, low level of productive replication of HIV-1 in brain is a critical component of neuropathogenesis regulation. The present study investigated the effect of CXCL8 on productive infection of HIV-1 in human monocytes-derived macrophages (MDM). Methods: Human MDM were infected with blood and brain-derived HIV-1 isolates, HIVADA and HIVJR-FL. HIV p24 levels were assayed from culture supernatants with ELISA. DNA was isolated from infected cells treated with/without CXCL8 (10-100ng/ml) and amplified for two-LTR circles, as a measure of integration We employed human promonocytic cell line U937 as an efficient transfection system. U937 cells were transfected with pHIV-LTR and promoter activity was measured by luciferase reporter assay. Results: Treatment with CXCL8 led to significant upregulation (p<0.01) in HIV-1 p24 levels in supernatants of HIV-infected MDM, as determined by ELISA. Reverse transcriptase (RT) activity was significantly increased (p<0.01) in HIV-infected MDM treated with CXCL8. We compared the formation of 2-LTR circles in CXCL8 treated vs untreated HIV-infected MDM by RT-PCR, as a measure of viral genome integration. Transient transfection of U937 cells with HIV-LTR construct containing luciferase reporter gene resulted in increased luciferase activity when treated with CXCL8. Conclusions: The results show that CXCL8 mediates productive infection of HIV-1 in MDM and induces HIV-1 LTR promoter activity in U937 cells. Detailed understanding of the mechanisms involved could aid in therapeutic intervention strategies by modulating levels of this chemokine in the brain.
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    (2013-04-12) Whitmill, Amanda
    Purpose: HIV Tat-interacting protein of 110 kDa (Tip110) plays important roles in various cellular processes including pre-mRNA splicing, antigen presentation, regulation of transcription, and even embryonic development. Previous studies pinpointing Tip110 knockout in the perinatal death of zebrafish led to the current study concerning the effect of Tip110 knockout in a transgenic mouse model. Taking from the zebrafish study we predicted that Tip110 knockout might primarily affect endoderm differentiation and thus mainly disrupt thymic development, lung development, and the functions of the exocrine pancreas. Thus far our data indicate that embryonic lethality of Tip110-/- mice occurs early after uterine implantation, suggesting a key role for this protein in mammalian development. We have also began investigating differences in the human and mouse versions of this protein as it has been discovered that human Tip110 is processed at the cellular level in a different fashion than mouse Tip110. Methods: A floxed/cre transgenic mouse model was used to determine the time point at which embryonic death occurs. Tip110 floxed mice were bred with eIIa-Cre transgenic mice, the embryos were dissected at various time points post coitum to estimate time of death. In the future tissue fixation and immunohistochemistry, RT-PCR, and in vitro culture of embryos will be performed. To characterize the differences between human and mouse tip110 we have developed recombinant constructs and expressed them in both human and mouse cell lines followed by Western blot analysis, immunoprecipitation, and use of various enzymes and inhibitors to determine their differences. Results: Tip110-/- mice do not survive long enough after uterine implantation for key developmental milestones such as establishment of the placenta, organogenesis, and limb formation to occur. Human and mouse Tip110 have different protein expression patterns depending on whether vector expression occurs in a human or mouse cell line. They also differ in the degree at which they are (de)ubiquitinated and degraded by the 26S proteasomal system. Conclusions: Tip110-/- must disturb a crucial step of early embryogenesis. Future studies with other tissue-specific conditional knockouts may provide insight into the effects of Tip110-/-. There is some subtle change occurring in the human and/or mouse Tip110 protein that will likely explain some of the differences we have noted in terms of the size, protein expression, and rate of degradation in Tip110.
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    (2013-04-12) Smedley, Crystal
    Purpose: Canonical transient receptor potential (TRPC) 6 affects glomerular filtration of proteins. Whether this protein also regulates other glomerular functions is not known. The present study was conducted to test a hypothesis that TRPC6 regulated glomerular filtration rate (GFR) by regulating the contractile function of glomerular mesangial cells (MCs). Methods: GFR in mice was estimated using a two-compartment clearance model. 5% FITC-inulin (3.74 µl/g body weight) was retroorbitally injected and blood samples were periodically collected from the tail vein under the conscious state. GFR in rats was evaluated based on the plasma inulin clearance during intravenous infusion of 1% FITC-inulin at a rate of 1 ml/h/100 g body weight. Blood samples were collected from the left common carotid artery and urine samples were collected from either the bladder or the urether under the anesthetized state (sodium pentobarbital, 50 mg/kg body weight, i.p). In vivo knockdown of TRPC6 in rat kidneys was achieved by injection of shRNA constructs against rat TRPC6 via the left renal artery. Ca2+ imaging in combination of molecular and pharmacological tools were employed to determine a role of TRPC6 in Ca2+ signaling in MCs. Furthermore, MC contractile response was assessed by measuring a change in the planar surface area of cultured MCs in response to Ang II stimulation. Results: We found that GFR in TRPC6 knockout mice was remarkably elevated compared to wild type mice. In rats, acute infusion of Ang II significantly reduced GFR. However, the Ang II-induced GFR decrease was abolished by local knockdown of TRPC6 in kidneys. Immunohistochemistry showed that MCs were the predominantly transfected glomerular cells. Consistently, the GFR of the left kidney (the transfected kidney) significantly decreased by Ang II when transfected with control vectors, but did not have a significant change when transfected with shRNA against TRPC6. There were no significant differences in arterial blood pressure and renal blood flow between TRPC6-knocked down and their control rats. Ca2+ imaging experiments showed that the Ang II-stimulated Ca2+ entry was significantly inhibited in MCs isolated from TRPC6 knockout mice and in human MCs with TRPC6 knocked down. Moreover, the Ang II-stimulated contraction was significantly suppressed in TRPC6 deficient mouse MCs. Conclusions: These results suggest that TRPC6 channel is an important component regulating GFR by modulating MC Ca2+ signaling and contraction.
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    (2013-04-12) Shaygan, Lida
    Purpose: Irritable bowel diseases (IBD), including Crohn's disease and ulcerative colitis, are idiopathic inflammatory disorders of the gastrointestinal tract that lead to chronically inappropriate immune responses against normal flora in the gut. Among other factors, oxidative stress and increased intestinal tissue injury have been indicated in patients with IBD. Hyperbaric oxygen treatment (HBOT) has been successfully used in humans to treat different conditions such as decompression sickness in scuba diving (Gill et al., 2004), carbon monoxide poisoning, and poor wound healing in diabeteic patients (Tiaka et al., 2011). The purpose of this experiment was to determine the effect of hyperbaric oxygen treatment on colon inflammation in irritable bowel disease. Methods: Colitis was induced in mice using dextran sodium sulfate (DSS), according to a previous described model (Wirtz et al., 2007), and one of the groups was treated with hyperbaric oxygen. 24 eight-week old male C57-BL/6 mice (Jackson Laboratories) were divided into 3 groups: a control group, which was only given distilled water; a second group in which IBD was induced through a 14 day DSS (3%) in drinking water, and a third group also induced with a 14 day DSS (3%) in drinking water and simultaneously treated with HBO daily for two hours. Daily weight loss for all mice was recorded, colons were measured, and colon tissue was histologically analyzed. Results: Hyperbaric oxygen treatment of DSS induced mice led to a significant (P <.05) increase in body weight and colon weight/length ratio measurements compared to DSS induced mice that did not undergo hyperbaric oxygen treatment. Conclusions: Hyperbaric oxygen treatment (HBOT) of colitis induced mice does not appear to decrease colon weight/length ratio, a reliable marker of inflammatory responses in IBD (Galvez et al., 2000). However, HBOT does improve survival and maintains body weight at better levels than DSS-induced mice without treatment. Histological analysis of colon tissues is pending and will provide a better indication of the degree of inflammation in DSS induced mice treated with HBOT.
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    (2013-04-12) Nagwekar, Janhavi
    Purpose: Muscle is organized into regular periodic thick myosin and thin actin filaments. Myosin tails interact with each other to form a tight coiled coil rod, and the heads protrude out to interact with actin. Myosin head referred to as cross-bridge has ATPase activity and actin binding domain. The tail has site (Regulatory Light Chain (RLC) domain) which gets phosphorylated during muscle contraction. The degree to which RLC phosphorylation affects the organization of myosin cross-bridges is the question investigated in this project. Methods: Rabbit psoas muscle is the source of sample for experiments in this project. Glycerinated muscle bundles were homogenized and myofibrils were extracted. Myofibrils were phosphorylated with skeletal myosin light chain kinase/calmodulin and dephosphorylated with phosphatase and 1mM EGTA. Essential light chain (LC1) protein site specifically labeled with SeTau dye was exchanged with native LC1 at a minimal concentration in myofibrils. Labeled myofibrils were analyzed for error of the mean of polarized fluorescence (which is a measure of orientation) emanating from myosin cross-bridges. Results: Histograms were plotted from the polarized fluorescence data and the Full Width at Half Maximum (FWHM) of the mean was calculated. This is an index of cross-bridge order. FWHM values in relaxed de-phosphorylated and phosphorylated muscle were 0.213 ± 0.015, and 0.23 ± 0.022, respectively. During active contraction FWHM values in de-phosphorylated and phosphorylated muscle were 0.23 ± 0.039 and 0.387 ± 0.050, respectively. In 20 experiments, statistically significant difference was seen between the groups. Conclusions: Phosphorylation of the RLC disturbs myosin head-to-head and head-to-rod interactions. Low FWHM in de-phosphorylated muscle suggests well organized cross-bridges. Phosphorylation destroys this organization, as indicated by high FWHM.
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    (2013-04-12) Namuduri, Anuradha
    Purpose: Despite a multitude of endogenous mechanisms to compensate for transient decreases in glucose and oxygen delivery to the brain, ischemic stroke remains the third leading cause of death and the leading cause of long-term disability in the United States. Studies in animal models of cerebral ischemia have shown that both endogenous and exogenous estrogens improve histological and behavioral stroke outcomes. Similarly, phytoestrogens derived from soy and other plant products can reduce stroke injury in the lab. These compounds are structurally similar to estrogen and bind to and activate estrogen receptors. Our overall hypothesis is that soy phytoestrogens can induce adaptive responses in the brain to favor increased survival and cell repair. One early adaptive response to ischemia is activation of the oxygen homeostasis mediator hypoxia-inducible factor 1 alpha (HIF-1 alpha), a normally labile protein that is stable under conditions of low oxygen. Despite the importance of HIF-1 alpha in the adaptive response to hypoxia and ischemia, acute overactivation of this protein increases cerebral edema and cell death. The purpose of this research is to determine if soy phytoestrogens (genistein, daidzein, equol) inhibit acute ischemic injury by attenuating the detrimental actions of HIF-1 alpha. Methods: In vitro, HT22 hippocampal cells were treated with 0.1 or 1 micromolar genistein and exposed to oxygen and glucose deprivation to mimic conditions of ischemia within the brain. In vivo, ovariectomized female Sprague Dawley rats were fed a soy-free or high soy diet for 4 weeks followed by 90 minutes experimental stroke and 4-24 hours of recovery. HIF-1 alpha levels were determined by Western blotting for the HIF-1 alpha targets vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), and BCL2/adenovirus E1B 19kDa interacting protein 3 (BNIP3). Results: In HT22 cells, genistein attenuated the HIF-1 alpha response to ischemia and increased degradation of HIF-1 alpha. Rats fed a high soy diet had significantly decreased levels of HIF-1 alpha and it's targets VEGF, eNOS, and BNIP3 in the ischemic hemisphere compared to rats on a soy-free diet. Conclusions: These results suggest that one mechanism of phytoestrogen neuroprotection is the attenuation of acute detrimental effects of HIF-1 alpha. These results further support the potential for dietary phytoestrogens as an effective prophylactic step in the prevention and management of ischemic strokes.
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    (2013-04-12) Duggal, Divya
    Purpose: Myosin consists of two heavy chains and four light chains. Two of the light chains, so called Regulatory Light Chains (RLC), get phosphorylated during muscle contraction. The role of this phosphorylation is currently unknown. In this work we describe our attempts to clarify this matter. Methods: A transgenic mouse was produced in which myosin RLC was devoid of serines that are normally necessary for phosphorylation. Glycerinated muscle bundles were dissected from the tibialis muscle of the mouse, and homogenized to produce myofibrils. Phosphorylation status in W.T muscle was preserved by adding phosphatase inhibitors. Essential light chain (the other two light chains of myosin, ELC1) was specifically labeled with SeTau fluorescent dye and exchanged with native ELC1. Cross-bridges of contracting myofibrils were analyzed for an error of the mean orientation. Results: Number of occurrences (histograms) of a given orientation were plotted vs. the mean orientation. The Full Width at Half Maximum (FWHM) of the mean was calculated. FWHM is an index of cross-bridge order/chaos. Small FWHM means order, large FWHM means chaos. During active contraction FWHM values in de-phosphorylated and phosphorylated muscle were 0.363 ± 0.049 and 0.456 ± 0.083, respectively. Two-tailed t test revealed statistically extremely significant difference (P<<0.001, t=4.83 with 63 degrees of freedom). Confidence intervals were from 0.0545 to 0.131. Conclusions: Phosphorylation of RLC causes considerable increase in the cone of angles within which myosin head interacts with thin filaments to produce force.
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    (2013-04-12) Cisneros, Irma
    Purpose: Methamphetamine (METH) is a highly abused and addictive psychostimulant. METH heightens sexual arousal and decreases inhibition increasing the probability for acquiring human immunodeficiency virus-1 (HIV-1). HIV-1 results in cognitive effects, such as HIV-associated dementia (HAD) characterized by similar neurotoxic mechanisms as METH. Astrogliosis and hyperthermia are key pathological features of METH exposure and HAD. In context of our studies, METH abuse increases brain temperature by approximately 2° C, mimicking a fever common during early HIV-1 infection. A moderate increase in brain temperature exacerbates neuroinflammatory processes synergistically effecting METH/HIV-1-associated neurodegeneration. Methods: Astrocytes sensitivity to METH led to the investigation of astrocyte TAAR1 as a receptor mechanism for METH-induced effects in astrocytes. Previously we showed localization and function of astrocyte TAAR1. Documented TAAR1 thermoregulatory responses led the expansion of our studies to investigate METH-associated hyperthermia in METH/HIV-1-induced astrocyte activation. Results: Furthermore, preliminary data suggest TAAR1 regulation in the presence of thermal stress. Elevated temperatures increased GFAP expression and cytokine secretion. We propose activation of astrocyte TAAR1 mediates METH/HIV-1-induced neurodegeneration, further modulated by METH-associated hyperthermia. Conclusions: The results will lead to understanding of the mechanisms and pathological features associated with METH and HIV-1 neurodegeneration and potential therapeutic targets in the CNS.
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    (2013-04-12) Fraser, Patrick
    Purpose: Folic acid, a member of the family of B vitamins, is provided in elevated concentrations to pregnant women to prevent congenital birth deformities such as cleft palate and neural tube defects. Previous studies have shown in-utero exposure to high concentrations of folic acid can negatively regulate gene expression via hypermethylation of DNA and/or histones in promoter regions of developmentally regulated genes. Our study aims to show a similar effect on the lung-specific, developmentally regulated gene, surfactant protein-A (SP-A) in the fetal lungs of ICR mice exposed in-utero to high concentrations of dietary folic acid. Appropriate SP-A expression is necessary for the normal development and regulation of multiple aspects of the innate and acquired arms of the immune system. Methods: Outbred ICR mice (n=8) were placed on a diet supplemented with high levels of folic acid (HMD) along with the necessary cofactors for methyl metabolism. A control group (n=8) was placed on a standard diet. Multiple rounds of breeding were conducted until four distinct timepoints were collected. The fetal-lung tissues were harvested at the last four days of gestation: 15, 16, 17, and 18 days post-coitum (dpc), respectively. A significant suppression of SP-A, SP-B, and SP-D at term was revealed in the HMD group when quantitative real-time PCR (qRT-PCR) data were analyzed via the comparative CT method. Results: Increased expression of cyclooxygenase 1 and 2 (COX-1, COX-2), markers of inflammation, was observed in these same mice at 17 dpc. Conclusions: These findings suggest a possible predisposition for pulmonary dysfunction in HMD mice as a result of decreased expression of the anti-inflammatory, SP-A.