Biology
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Item 5-HT2A/2C Receptors and lordosis behavior in the female rat(1998-05) Wolf, Amy; Uphouse, Lynda; Droge, Michael; Hardcastle, James; Kowalski, Jacek; Mills, NathnielThe effects of hypothalamic infusion of serotonin (5-HT)2A/2C receptor compounds on lordosis behavior were examined in ovariectomized, hormone-primed (0.5 $\mu$g estradiol benzoate, subcutaneuosly (s.c.), followed 48 hr later with 500 $\mu$g progesterone, s.c.) rats. This hormone-priming condition resulted in some rats that were sexually receptive and some that showed low or no sexual behavior. Sexually receptive rats were inhibited when the 5-HT$\rm\sb{2A/2C}$ receptor antagonist, 3-(2-) 4-(4-fluorobenzoyl)-1-piperdinlyl(ethyl) -2,4(1H,3H)-quinazo-linedione tartrate, ketanserin, was infused into the ventromedial nucleus of the hypothalamus (VMN). Hormone-primed ovariectomized rats were more affected by the drug than were proestrous rats. The reason for this apparent difference in sensitivity to the drug is unknown. When ovariectomized, hormone-primed rats with low sexual receptivity received VMN infusion with the 5-HT$\rm\sb{2A/2C}$ receptor agonist, ($\pm$)-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl, DOI, sexual behavior increased. Similar increases in lordosis behavior were observed following infusion with the mixed 5-HT receptor compounds, 2-(1-piperazinyl) quinoline dimaleate, quipazine, and N-(3-trifluoromethyl-phenyl) piperazine hydrochloride, TFMPP. Facilitation of the behavior occurred within 15-20 min of agonist infusion, while inhibition following antagonist infusion occurred more rapidly. These findings are consistent with the hypothesis that 5-HT$\rm\sb{2A/2C}$ receptors in the VMN contribute to the facilitation of female rat sexual behavior.Item Alteration of prenylation: Effect on neurite outgrowth and Rho GTPase signaling(2010-05) Samuel, Filsy; Hynds, DiAnna L.; Uphouse, Lynda; Mo, Huanbiao; Beck, Brian; Schwark, HarrisThe action of the 3-hydroxy-3-methyglutaryl coenyzme A (HMG-CoA) reductase inhibitor, lovastatin, promotes neurite outgrowth in some systems and inhibits it in others (Schulz et al., 2004; Holmberg et al., 2006). Anecdotally, lovastatin also contradictorily relieves or exacerbates memory loss in Alzheimer's patients (Wagstaff et al., 2003; Sparks et al., 2008). We postulate that some of lovastatin effects are due to inhibition of prenylation precursors that alter Rho GTPase localization and loading of GTP. We show here that lovastatin decreases neurite initiation and concurrently increases GTP loading of RhoA in the cytosol and decreases GTP loading of Racl associated with the plasma membrane. We also assess how lovastatin affects the activity of cofilin, which is inactivated by phosphorylation by the common Rho GTPase effector, LIM kinase. We correlate cofilin phosphorylation with effects on actin filament content since cofilin is an actin depolymerizing agent. We use western blot analyses and immunocytochemistry to assess phosphorylated and total cofilin and find no significant deviations from control conditions. However, the amount of filamentous actin decreases in growth cones with lovastatin and lovastatin plus geranylgeraniol reversing this effect. Treatment with lovastatin increases GTP loading of RhoA in the cytosol fraction and decreases GTP loading of Rac1 in the membrane fraction. Together, these results suggest that lovastatin may promote actin depolymerization via Rho GTPases signaling leading to a decrease in the neurite initiation aspect of outgrowth. Elucidating the biochemical actions of lovastatin improves our understanding of how this treatment might mediate its effects in Alzheimer's disease and may facilitate the development of effective therapies for other nervous system disorders like traumatic brain or spinal cord injury.Item Alterations in cerebellar protein synthesis in propylthiouracil-induced hypothyroid rats(1991-05) Jaworski, DianeThyroid hormones play an important role in the regulation of gene expression and cerebellar development in the rat. The drug propylthiouracil was used to disrupt thyroid hormone synthesis. This permitted investigation of gene expression under three different states of hormonal deprivation: early in development (PTU 1), late in development (PTU 2), and continual deprivation (PTU 3). The effects of these deprivations on some aspects of the synthesis of two proteins known to be affected by T$\sb3$, apolipoprotein E (apo E) and transferrin (T$\sb{\rm f}$), were investigated. Thyroid hormone's permissive role on growth and development, morphological features as well as body and organ weights were analyzed and found to be significantly reduced in the hypothyroid state. The development of cerebellar cortical strata and cellular morphology was altered by prenatal, as well as postnatal PTU administration. To establish cerebellar synthesis of apo E and T$\sb{\rm f}$, total RNA was extracted, poly A$\sp+$ RNA isolated and translated in an in vitro reticulocyte lysate system. The specific proteins were immunoprecipitated and analyzed by SDS-PAGE. To correlate these results with the amounts of mRNA's for apo E and T$\sb{\rm f}$, northern hybridization was performed. Both apo E and T$\sb{\rm f}$ levels synthesized in vitro with RNA from control animals increased as development proceeded, with apo E produced in greater quantity. While hepatic apo E synthesis increases in the hypothyroid state (presumably as a result of regulation at the transcriptional level), cerebellar apo E translatable mRNA levels were not affected by PTU treatment. Like hepatic T$\sb{\rm f}$, cerebellar T$\sb{\rm f}$ levels were substantially reduced in the hypothyroid state, as compared to control values. Developmentally, apo E mRNA levels declined, while T$\sb{\rm f}$ mRNA levels and T$\sb{\rm f}$ synthesized in vitro were parallel in control animals. PTU 1 treated animals, in which free T$\sb3$ in the circulation increased after PN day 20, demonstrated increases in both apo E and T$\sb{\rm f}$ mRNA levels at the same time point. It appears that the regulation of apo E and T$\sb{\rm f}$ mRNA levels differ from each other within the cerebellum and may be regulated differently from those of the liver. The increase in in vitro protein synthesis and mRNA levels in PTU 1 treated animals may be a result of developmental recovery, rather than specific T$\sb3$ mediated gene regulation.Item An interaction between estrogen and serotonin in sensory neurons as a key regulator of nociception(8/25/2021) Lulla, Sukhbir Kaur; Averitt, Dayna L.Orofacial pain conditions, such as migraine, are at least two times more common in women and have a complex etiology. Orofacial pain, relayed by trigeminal sensory neurons, is linked to gonadal hormone (estrogen; progesterone) fluctuations. The neurotransmitter serotonin (5HT) is a proinflammatory and pronociceptive mediator in the periphery and has been implicated in several female prevalent pain disorders. 5HT can directly activate trigeminal sensory neurons or can sensitize the transient receptor potential vanilloid 1 ion channel (TRPV1), a cation channel activated by capsaicin and heat. TRPV1 activation results in calcium influx and calcitonin gene-related peptide (CGRP) release, leading to peripheral sensitization that heightens pain sensitivity. Previous studies in male rats have shown that 5HT receptors colocalize with and sensitize TRPV1. Furthermore, serotonergic potentiation of CGRP release occurs from the dental pulp of women during the luteal phase of the menstrual cycle (when gonadal hormones are fluctuating). It is unknown whether estradiol (E2; primary estrogen) enhances or attenuates this serotonergic pain mechanism. As >90% of pain research has been conducted in males, and a small subset examines the trigeminal system, gonadal hormone modulation of female trigeminal sensory neurons is grossly understudied and warrants investigation. Our overarching hypothesis is that hormone status alters serotonergic neuromodulation of the TRPV1-expressing subpopulation of trigeminal sensory neurons. Thus, we examined whether (1) naturally fluctuating gonadal hormones can alter 5HT-evoked pain behaviors, (2) varying E2 concentration and exposure time can alter trigeminal pain signaling and transcriptome, and (3) E2 and estrogen receptors (ERs) can modulate the pronociceptive effects of 5HT on TRPV1 function. We report that exogenous 5HT evokes significant pain behaviors in females during proestrus and estrus. 5HT2A receptor antagonism or a steady-state diestrus level E2 attenuates 5HT-evoked pain behaviors. We also report that ERα, ERβ, and G protein coupled ER (GPER) localize to sensory neurons expressing 5HT2A and TRPV1 mRNA. Lastly, E2 modulates specific trigeminal pain genes and enhances ERα-dependent serotonergic potentiation of CGRP release. Together, the data presented in this dissertation provides behavioral, neuroanatomical, and cellular evidence of a mechanism present in female trigeminal sensory neurons that may exacerbate trigeminal pain disorders in women.Item Analysis of a novel HDAC8-H1.3 complex in several human carcinoma cell lines(5/30/2016) Gonzalez, Rhiannon; Bergel, Michael; Conrad-Webb, Heather; Mills, Nathaniel; Maier, Camelia; Vijayagopal, ParakatThe compaction level of chromatin regulates DNA accessibility, gene expression, and cell division. Transcription factors and other proteins cannot access the DNA within compacted chromatin. Two types of proteins that contribute to chromatin compaction are histone deacetylases (HDACs) and linker histones (H1s). H1s are chromatin-binding structural proteins required for the formation of the higher order chromatin structure. H1 subtypes also differentially regulate transcription and apoptosis. HDACs cause chromatin compaction by deacetylating lysine residues on core histone tails, causing core histones to closely interact with DNA. HDACs regulate many other cellular processes including: mitosis, intracellular trafficking, microtubule dynamics, and cell cycle events, through deacetylation of non-histone proteins. This work describes a novel HDAC8-H1.3 protein complex found in several human cancerous cell lines. The goal of this research was to gather insight as to the function of the HDAC8-H1.3 complex using co-immunoprecipitation, chromatin-binding electrophoretic mobility shift assays, deacetylation assays, immunocytochemistry, confocal microscopy, and complex protein mixture identification by LC/MS/MS. The hypothesis was that HDAC8 and H1.3 work synergistically to cause chromatin compaction. Results showed that H1.3, but not HDAC8 binds to nucleosomes. HDAC8 was found to deacetylate H1.3. In MCF-7 cells the complex was found to associate with ER-Golgi associated vesicles and late endosomes during interphase. This is the first report for a non-mitotic cytoplasmic association of a linker histone and HDAC. Also, this is the first reported association of a linker histone with vesicle trafficking.Item Analysis of the role of SLIK complex in Pol II rRNA synthesis in Saccharomyces cerevisiae(2009-12) Pandyaraj, Shenbaga Priya; Conrad-Webb, Heather; Bergel, Michael; Maier, CameliaAlthough rRNA is synthesized by Pol I under normal physiological conditions, stress conditions trigger the synthesis of rRNA by Pol II in Saccharomyces cerevisiae. Mitochondrial dysfunction triggers the polymerase switch; however, basal levels of Pol II rRNA synthesis are present in ρ + cells growing in non-limiting carbon and nitrogen conditions. Both the Retrograde pathway and the Hog pathway are required for basal Pol II rRNA synthesis (Sagar, 2008). Although Rtg1p and Rtg3p are activated by Rtg2p, neither play a role in Pol II rRNA synthesis; instead Rtg2p and other members of the SLIK histone acetylase complex are crucial for Pol II rRNA synthesis. During elongation SLIK is retained in the elongation complex perhaps replacing the elongator complex to allow more efficient elongation of Pol II transcribed rRNA. Thus, chromatin structure plays a crucial role in the selection between Pol I and Pol II for rRNA synthesis.Item Analyzing a calcium-dependent UV response that results in a global chromatin compaction in mammalian cells(May 2023) Sinha Roy, Rituparna; Bergel, Michael; Gumienny, Tina L; Everts, Helen B; Brower, Christopher; Conrad-Webb, HeatherGenomic integrity is challenged every day by both endogenous and exogenous stresses. Once DNA gets damaged, a series of events follow, starting with recognizing the damage by accessing it, then processing and repairing the damaged DNA, and eventually restoring the lesion area to its pre-damage state. However, the structural change in the global chromatin in response to UV radiation and the etiology of these changes are not well known. To visualize a global chromatin structural changes upon UVB radiation, we used a fluorometric detection approach. We observed large-scale chromatin compaction immediately after UVB radiation in mouse embryonic fibroblasts, human cervical cancer cells (HeLa), and primary human epidermal melanocytes (HEMs). Using immunofluorescence we also demonstrated that UVB-induced chromatin compaction protects DNA from further UV damage. Using confocal microscopy, we demonstrated cellular calcium influx after UV radiation without external retinal addition. Our RNA-seq data identified two G-protein coupled receptors (GPCRs), SSTR4 and P2RY6, associated with calcium signaling, whose expression was upregulated after UVB radiation in HEMs. Chemical inhibition of P2RY6 and IP3 receptors resulted in a significant reduction in calcium influx and chromatin compaction after UV irradiation of cells. Inhibition on other candidate calcium channel, TRPA1, which mediates UVR-induced calcium influx and early melanin synthesis, did not lead to significant inhibition of chromatin compaction in HEMs after UVB radiation. Based on these findings, we propose that a non-retinal UVB phototransduction pathway is involved in an increase in intracellular calcium triggered by the opening of IP3 receptors which leads to activation of the P2RY6 receptor and then further Ca2+ influx. This increase in cellular Ca2+ concentration leads to an increase in nuclear chromatin compaction, which has a DNA protective role. We suggest that SSTR4 maintains the steady state of calcium by reducing the calcium level after UVB-induced calcium surge in primary human epidermal melanocytes. RNA-seq data revealed that the highest number of genes that upregulated was at 4 hours, and the lowest number of genes upregulated was at 20 minutes after UVB radiation. We suggest that UV-induced chromatin compaction reduces gene expression, and as time progresses, local unfolding of chromatin happens, which allows more gene to be expressed. However, some genes seem to be overexpressed despite the immediate global chromatin compaction and the general gene repression that follows UV radiation. This finding suggests that there are some mechanisms to keep the activation of essential genes after UV irradiation, despite the general gene repression. A detailed understanding of the molecular mechanisms that allow human skin cells to detect UV radiation and respond by compacting the chromatin and protecting the DNA will aid in the search for novel therapeutics to reduce sun-inflicted carcinogenesis.Item Anti-proliferative activity of novel amidoximes in human and murine malignant cell lines and in mouse mammary carcinoma(10/25/2019) Gekombe, Amon; Bergel, Michael; Mirsaleh-Kohan, NasrinCancer incidence and mortality is on the rise worldwide, which necessitates newer and more potent therapies to combat cancer. In this study, four novel amidoximes, JJMB5, JJMB6, JJMB7 and JJMB9, were analyzed for their ability to inhibit proliferation of human and murine malignant cell lines, and in mouse mammary carcinoma. We established that JJMB5 and JJMB6 induced inhibition of acetylation of core histones H3K9 and H4K5 prior to apoptosis. Moreover, JJMB5, JJMB6 and JJMB9 inhibited acetylation of core histone H3K27, but JJMB7 did not. Additionally, JJMB5, JJMB7 and JJMB9 inhibited the proliferation of murine mammary malignant cell lines in culture but did not inhibit mouse embryonic fibroblasts, as established by the MTS colorimetric assay. When human and murine malignant cell lines were treated individualy, or in combination of various amidoximes or amidoximes with cisplatin, several combination treatments resulted in significantly lower growth inhibitory concentration (GI50) compared to individual treatments. Once the MTDs for the amidoximes were established, tumor studies were carried out. Results on tumor volume reduction were mixed. For example, mice treated with JJMB7 demonstrated significant tumor volume reduction in the first experiment but not in subsequent experiments. On the other hand, mice treated with JJMB9 had the lowest tumor volumes in all experiments. In The second experiment, tumor volumes were significant up to day 21. There were no differences in lung metastasis area between the treatment groups. Acetylation studies of 4T1 cells from tumors revealed that JJMB9 induced significant inhibition of acetylation of H4K5. Also, JJMB9 was able to induce drug resistance in MCF-7 cells, and this resistance was reversed by cisplatin. The amidoximes have potential if certain changes can be made on the protocol such as decreasing 4T1 cells implanted into the mammary fat pad, amidoxime dose escalation, and using alternate routes of administration.Item Arashi in Black and White(May 2024) Lauland, Nicholas; Nicholas Lauland; Nicholas LaulandAfter a great deal of hard work and experimentation, several changes were identified as necessary to enable Arashi to run in Black and White. Additionally, several optimisations were identified to improve the speed on lower performance black and white models.Item Assessing university biology students’ critical thinking skills resulting from team-based learning with case studies in the classroom(12/15/2017) Collier, Jayme E.; Westmoreland, Sandra L.; Maier, Camelia; Gumienny, Tina L.Research was conducted to measure the effectiveness of new course assessments to increase post-semester exam scores and critical thinking abilities of undergraduate students enrolled in a large lecture Principles of Biology course. New complex problem scenarios were designed to reinforce biological topics studied with Team-Based Learning (TBL) and to promote development of higher order thinking skills. The new problem scenarios were introduced in unit exams following a unit of study with TBL. Research results showed significant increases in student post-semester test scores for specific TBL-aligned questions when compared to previous semester student scores. This result validated the hypothesis that student content knowledge scores would increase due to new TBL-aligned problem scenario assessments. Comparisons between students’ successive problem scenario scores showed significant differences. Comparisons of student scores with different science backgrounds were not significant for post-test scores and problem scenario essays.Item Assessment of focal complexes in B35 Neuroblastoma growthcones following Lovastatin treatment(1/1/2013) Deshmukh, Shweta Dilip; Hynds, DiAnna L.; Uphouse, Lynda L.; Beck, Brian W.Proper development of the nervous system requires efficient and precise axon growth and guidance, growths requiring dynamic focal complex adhesion and de-adhesion in the neuronal growth cone. The formation of focal complexes is regulated in part by guanine triphosphatases (GTPases) of the Rho family, which signal bi-directionally to focal complex constituents. Rho GTPases are also regulated through binding of guanosine triphosphate (GTP), a process facilitated by membrane targeting through geranylgeranylation (a specific type of prenylation) at their C terminus. Prior work in our laboratory demonstrated that decreasing geranylgeranylation with the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase inhibitor, lovastatin, decreases neurite initiation and increases neurite branching and cell body rounding. These effects were reversed by co-treatment with geranylgeraniol, a precursor for geranylgeranylation. In the current work, we determined whether manipulating protein prenylation decreases the association of focal complex proteins (paxillin, talin or vinculin) with β1-integrin in B35 growth cones. B35 neuroblastoma cells were grown on uncoated glass coverslips, or coverslips coated with 25 μg/ml laminin or collagen. Cells were maintained in media were either not treated, or treated with lovastatin (LOV, 20 μM) alone or in combination with geranylgeraniol (GGOH, 10 μM). Focal complex formation in growth cones on the different substrata was assessed using co-immunoprecipitation and double-label immunocytochemistry. Culturing on different substrata did not alter the amount of paxillin or talin that associated with 1 integrin. However, vinculin co-localization with 1 integrin was decreased for cells plated on collagen or laminin; and co-localization with talin was increased in cells cultured on laminin, compared to cells plated on uncoated coverslips. For cells plated on collagen, GGOH decreased the amount of paxillin, but not talin or vinculin, that co-localized with β1 integrin compared to control cells maintained in reduced serum medium or treated with LOV. For cells cultured on their native, secreted substratum (undefined) or laminin, manipulating geranylgeranylation did not alter the association of focal complex proteins with 1 integrin in whole cell lysates, growth cones or cell bodies. However, treatment to alter protein geranylgeranylation (LOV, GGOH, LOV+GGOH) decreased the amount of co-localization of paxillin and talin, and GGOH treatment decreased the amount of vinculin co-localized with 1 integrin in growth cones, compared to cells in reduced serum medium (SCM) for cells cultured on collagen. Together, we interpret these data to indicate that inhibiting protein prenylation decreases growth cone focal adhesions, likely leading to decreased neurite initiation. Understanding the molecular mechanisms that regulate Rho GTPase control of neuronal process extension will facilitate our understanding of nervous system development, as well as identify potential therapeutic targets for treating developmental, traumatic and degenerative neural lesions.Item Assessment of the role of testosterone in the rat gonads: Morphometrical and cytochemical analysis(1996-12) Shuttlesworth, GladisRat testes are composed of somatic cells, primarily Sertoli (SC), myoid (MC) and Leydig cells (LC), and germinal epithelium which gives rise to spermatozoa. Sertoli, myoid, and Leydig cells have androgen receptors, of which Sertoli and myoid cells are thought to be primarily responsive to testosterone. Adjacent to Sertoli and myoid cells are Leydig cells which are the primary source of testosterone (T). The aim of this study is to evaluate the effects of T on testicular somatic cell morphology and changes in protein synthesis profiles, using prenatally irradiated male rats (devoid of germ cells) that are, thus, Sertoli-cell enriched (SCE). Mature SCE rats were treated with Leuprolide (Leupr.) a GnRH agonist, for six weeks to prevent release of LH and FSH from the pituitary, thus, indirectly blocking T production from Leydig cells in the absence of LH. Some SCE rats were treated with testosterone propionate (T.P.) or in combination with Leupr. Testis weight (with and without tunica albuginea) of Leupr. treated groups were reduced to 53% of SCE controls. Seminiferous tubule diameter of Leupr. treated animals was reduced to 70% of the control. Neither testicular weight not tubule diameter were maintained by T replacement in Leupr. treated animals. Testosterone levels were reduced to 30% of SCE controls with Leupr. Androgen replacement with or without Leupr. increased T levels 17 and 13 fold respectively. With Leupr., Leupr. + T.P., or T.P. alone there was a two fold decrease of LH concentration as compared to the SCE control. FSH levels of groups treated with Leupr. alone and in combination with T.P. showed 94% and 81% higher and lower values, receptively, when compared to SCE controls. Leydig cells showed a severe morphological change in the Leupr. treated group and interstitial tissue had an abundance of unidentified cells. Myoid and Sertoli nuclear morphology was unaltered and no statistical difference in SC numbers were observed. Protein profiles of the testicular tissue were analyzed using SDS-PAGE. A newly synthesized protein (36kDa) was secreted into the medium with Leupr. treatment. This protein was not observed in control, or in T.P. treated animals, but it was found occasionally in the Leupr. + T.P. treated group. Results obtained in this study appear to indicate a direct effect of Leuprolide in somatic cell morphology and in changes of the protein synthetic profile.Item Behavior of cadmium in a simulated aquatic ecosystem(1974-08) Kinkade, MelindaItem Beta-Adrenergic receptor subtypes in myocytes, arteries, and arterioles of the porcine heart identified with autoradiographic procedures(2000-12) Boliver, Doug B.; Uphouse, Lynda; Mills, Nathaniel; Droge, MichaelBeta (P) adrenergic receptors are localized in several tissue compartments of the heart, but it has been unclear as to the relative numbers contained in each compartment. The goal of the current study was to use receptor autoradiography to analyze p receptor agonist binding characteristics in different tissue compartments of pig heart including cardiac myocytes (predominately P1), coronary arteries (predominately P1) and coronary arterioles (predominately ~2). In addition, the relative numbers of ~1- and 132-adrenergic receptors in these tissue compartments were estimated. Blocks of porcine anterior interventricular sulcus were frozen and tissue sections cut. Sections were incubated in [1251]-(-) iodopindolol {1251-IPIN) in the presence of 10·5 M of the P agonist, DL-propanolol HCI, or 5 x 10·1 M of the ~1 selective antagonist, CGP 20712A. For the validation experiments radioactivity was determined in the gamma counter and the sections demonstrated rapid binding, saturability and stereoselectivity. For autoradiography, emulsion-coated coverslips were attached to the slides. After exposure, the slides were developed and stained, and grain density quantified. There was a greater density of 13-adrenergic receptors, identified by autoradiographic grains, over arterioles as compared to the left anterior descending (LAD) artery. The LAD artery had only one-sixth the density of 13-adrenergic receptors seen in the arterioles, while the myocytes were equivalent to the arterioles.Item Biology of released protein I: an experimental model murine muscular dystrophy(5/31/1980) Krikorian, Debra; Hines, John; Hardcastle, J.; Rudick, MichaelIt has been previously shown that rapidly transported proteins are released from axons in vitro. A model developed to explain the probable function of released proteins states that the proteins released act as signals from the neurons to control Schwann cell activities. To test the validity of this hypothesis I chose to use Rej/129 dystrophic mice which clearly demonstrates that portions of the sciatic nerves display abnormal myelination patterns. For the purpose of this dissertation my objective was to establish if a difference existed between released proteins from nerves of normal mice and those of dystrophic mice, moreover to investigate these potential differencs. Electrophoresis on Tris gels of protein released from nerves of several species including the mouse show consistent patterns of three bands having R(,f) values of 0.84, 0.89 and 0.98. Separation on tris gels of released proteins from dystrophic mice reveal that the 0.89 band is missing. To demonstrate that the 0.89 protein as well as the 0.84 and 0.98 proteins are transported from the cell body down the axon and therefore are neuronal in origin, spinal cords of mice were injected with {('3)H}leucine. Electrophoresis and scintillation counting of these radiolabelled proteins released from normal mice nerves reveals that these three proteins are present and labelled, therefore they are transported down the axon prior to release. Separation of the radiolabeled proteins released from dystrophic nerves indicated little radioactivity at R(,f) 0.89 which is consistent with desitometric scans of the electrophoretic gels. To further substantiate these results it was decided to utilize the more sensitive double label isotope technique. An unexpected result from the use of this procedure was the appearance of a substantial new peak at R(,f) 0.79. Upon re-examination of the densitometric tracings of stained gels containing released proteins from nerves of dystrophic mice a small peak was noted constituting 2-3% of the protein on the gel. Previously we considered this band insignificant and it did not match any protein bands found in normal animals. In line with the original objective of this research these results show: (1) there is a consistent pattern of proteins released from nerves of all species investigated having R(,f) values of 0.84, 0.89 and 0.98, (2) the 0.89 band is missing in dystrophic mice, (3) all three bands are transported, and (4) although the dystrophic model demonstrated the loss of the 0.89 band there is an appearance of a new band at 0.79. From these results it is concluded that a difference exists between proteins released from the nerves of normal dystrophic mice. This difference is the loss of 0.89 protein and the appearance of the 0.79 band. Because both bands constitute the same relative proportions (2-3%) on densitometric scans of released protein it is suspected that the 0.89 protein has been altered such that it has an R(,f) value of 0.79 in the dystrophic model and therefore reflects a biochemical alteration in the mutant.Item Calcium and strontium ionic flux of lipid bilayers(1974-05) Tavasolian, Badri; Hardcastle, J.E.; Fry, Kenneth; Cockerline, Alan; Hines, John; Hamilton, WalterBilayer lipid membranes were formed from lecithin,. cerebroside, ceramide and tripalmitin. The electrical potential across the lecithin and cerebroside membranes were measured in the presence of Na+, Ca++ and Sr++, and from these membrane specific resistance, transference number and ionic flux were calculated for each membrane in the presence of each ion. The electrical properties of the lecithin membrane in the presence of Na+ were comparable with literature values. The measured potential of the lecithin membrane in presence of ·the various ions were in the order of Sr-H->ca:++>Na+, and for the cerebroside membrane the order was' Sr*>Ca++, Na+. No significant differences were found in the transference numbers of these ions in the two membrane systems. The ionic fluxes across the lecithin membrane·were in the order Na+>Ca++>Sr++ and those for the cerebroside membrane were Na+>Ca++, Sr++. The water permeabilities and ionic fluxes were measured for the several membranes. The water permeability coefficient of lecithin membrane was comparable with that of dog red blood cell. Ca++ fluxes across membranes showed the order of cerebroside-lecithin>lecithin> cerebroside>ceramide>tripalmitin, and sr++ fluxes followed the order: lecithin, cerebrosidelecithin> cerebroside>ceramide, tripalmitin. Sr++ flux across cerebroside membranes was higher than'the Cq.;+ flux across this membrane. Ca* fluxes across c.eramide and cerebrosidelecithin membranes were higher than Sr* fluxes across these membranes. Comparing the flux of each ion in either experiment set, indicated that cation permeselectivity of the membrane depends on the field strength of the membrane negative charges, and ion sieving. It is also dependent on cationic site interaction, site spacing, entropy effects, and hydration energy. The isotopically measured flux was about eight orders of magnitude larger than the flux calculated from the electrical potential. A two component mechanism of Ca++ and Sr-++ transport across the membrane was postulated: (1) linear flux through the lipid kinks and (2) a carrier mediated flux as a result of binding of these cations to lipid molecules accompanied by a "flip-flop" mechanism to transport the ions to the other side of the membrane. This hypothesis agreeswith postulated mechanism of Ca++ and Sr++ transport across intestinal membranes.Item Calcium-dependent global chromatin compaction protects DNA from UV inflicted damage(1/13/2020) Abbas, Mohammad M; Bergel, MichaelEukaryotic genomes are packaged into chromatin, which is the physiological substrate for all DNA-mediated functions, including DNA damage repair. At the DNA damage site, chromatin organization undergoes critical rearrangements during the repair process. These rearrangements around the lesion sites accommodate at least three steps: providing access to the repair factors, repair, and restoring the DNA’s pre-lesion. chromatin architecture. However, the global changes to chromatin after UV-irradiation were less explored and understood. To investigate the relationship between chromatin condensation and UV irradiation, HeLa-S3 cells were irradiated and subjected to micrococcal nuclease digestion analysis. The results showed that chromatin globally commenced compaction five minutes after UV-irradiation. Twenty-four hours after irradiation chromatin returned to the pre-UV steady-state. Southwestern blots showed that cells were irradiated twice at 15 J/m2 with a five minutes break had a significantly lower cyclobutane pyrimidine dimers (CPD) and DNA (6-4) photoproduct (6-4PP) rate in comparison to cells subjected to 30 J/m2, and had no significant difference from cells irradiated with a single dose of 15 J/m2. Western blot analysis demonstrated a post-UV core histone deacetylation wave which followed the chromatin condensation. Western blots analysis of caspase-3, which is activated in apoptotic cells both by extrinsic and intrinsic pathways, showed no caspase-3 activation after five and ten minutes post UV-irradiation. Here, we demonstrate that an environmental genotoxic agent, UV radiation, causes immediate and global chromatin compaction in HeLa cells and this compaction results in a robust reduction in the newly formed lesions. Our data suggest an influx of calcium cations after UV irradiation is directly involved in inducing chromatin compaction.Item Cell cycle dependent association and activation of a histone H1.3-HDAC3 complex(2009-12) Patil, Hemangi; Bergel, Michael; Conrad-Webb, Heather; Hynds, DiAnna L.; Mills, Nathaniel; Root, DouglasEukaryotic DNA is organized with nucleoproteins into a dynamic structure named chromatin. Chromatin compaction and decompaction plays an important role in modulation of various DNA-dependent functions in the cell. Histone deacetylases (HDACs), which remove acetyl groups from core histone proteins, regulate the compaction of the chromatin fiber, thus modulating gene transcription levels. Chromatin binding proteins, such as linker histone H1, are also known to cause chromatin compaction. Histone H1 and HDAC3 were independently reported to be involved in the regulation of mitosis. The hypothesis of this study was that histone H1 and HDAC3 could form a complex that will be involved in chromatin condensation and mitosis regulation. Co-immunoprecipitation assay results demonstrated the formation of stable complexes between HDAC3 and histone H1.3 in HeLaS3 human cervical carcinoma epithelial cells. Although the amount of complex increased during the late-G2 and mitosis phases of the cell cycle, the complex was activated for chromatin deacetylation only in mitosis. Studies in vitro demonstrated that HDAC3 was highly phosphorylated on Ser-424 in the mitotic complex but not the G 2 complex. Further studies also showed that HDAC3 from late-G2 complex can be activated in vitro by phosphorylating on Ser-424 with casein kinase II (CK2). Based on the immunocytochemistry and confocal imaging studies, the HDAC3-H1.3 complexes co-localized with mitotic polar microtubules. The complex included also silencing mediator of retinoic acid receptor and thyroid hormone receptor (SMRT), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and annexin I. GAPDH, annexin I and histone H1 are known to bind tubulins and thus increases confidence that they may tether HDAC3 to the polar spindle mitotic microtubules. This report is the first, to the best of our knowledge, in which histone H1 and a class I HDAC are shown to form a complex. This is also the first record of a co-localization of histone H1 with microtubules in an animal kingdom.Item Cellular responses to the accumulation of neurodegeneration associated protein fragments(8/30/2021) Kasu, Yasar Arfat Taufique; Brower, ChristopherCellular protein quality control consists of an integrated network of molecular pathways engaged in protein folding and protein degradation to ensure protein homeostasis. The loss of protein homeostasis, due to excess production of aberrant proteins or defects in protein degradation, contributes to many diseases including neurodegeneration. As part of the etiology of neurodegeneration, specific neuronal proteins are proteolytically digested into fragments that misfold and form aggregates within cells. In this study, we investigated the cellular response to aggregation-prone protein fragments. To examine the influence of N-terminus on fragment behavior, our studies employed two C-terminal fragments of human TDP43 that are associated with amyotrophic lateral sclerosis and frontal temporal dementia. These fragments are largely identical but differ in their N-termini. Using genetic models in cultured cells, yeast, and mice, as well as fluorescence microscopy, biochemistry, and molecular cloning techniques including the CRISPR/Cas9 system, we found that the N-termini of otherwise similar protein fragments have profound effects on fragment behavior and may influence clinical outcomes in neurodegeneration associated with aggregation. In a follow up study, we discovered that the molecular chaperone, BAG6, can recognize hydrophobic regions of neurodegeneration-associated protein fragments and prevent their intracellular aggregation. Our studies also implicate RNF126, an E3‐ubiquitin ligase, in the degradation of these fragments. We also examined how cells respond to the aggregation-prone fragments in the absence of their degradation and we synthesized a cell-based fluorescent reporter to use in screening applications to discover modulators of protein degradation. These studies contribute to our understanding of how cells cope with toxic misfolded proteins and will aid in the search for novel therapeutics for neurodegenerative disease.Item Characterization and prediction of residues forming functional protein-protein interfaces using network analysis(12/21/2017) Mehta, Isha D.; Hynds, DiAnna L.; Beck, Brian W.; Uphouse, Lynda; Hanson, Laura K.; Brower, ChristopherCharacterization of protein-protein interfaces can provide understanding of the fundamental properties of their functionality. Additionally, such understanding aids functional annotation of the interfaces and prediction to help in drug design. In this work, we classified protein interfaces broadly into two functional categories: Functionally Linked Interfaces of Proteins (FLIPs) and Functionally uncorrelated Contacts (FunCs). We used a method, based on network analysis followed by statistical analysis, to classify protein interfaces based on their functionality. Additionally, the method used for classification of protein interfaces characterizes each subunit in the complex individually allowing to expand the use of our method towards prediction of functionally linked interfaces. We assessed two different types of network constructs, as well as a method combining the best features from both the network constructs, for their classification accuracy and ability to predict functional interfaces. In the first network construct, Residue Interaction Networks (RINs), amino acid interactions are represented by spatial distance cut-offs of 8 Å between two Cα atoms of the residues. This simple approach classified FLIPs from FunCs with a consistent accuracy of 72%. It also identified dissimilarities among FLIP and FunC organizations. The positive correlation of FLIPs and network organization features suggest that FLIPs have a surface rich in concavities, while FunCs have smooth surface. The second network construct, Protein Energy Network, identified interactions among the amino acid residues using PyRosetta generated interaction energy scores. This construct was designed to assimilate chemical information in addition to geometrical information based RIN construct to study energy influenced organization of interfaces. A 70% classification accuracy was achieved with this method; however, the compromise in overall accuracy is balanced by accuracies of categorization of a few functional subcategories such as structural interfaces were classified with 100% accuracy. Finally we combined the identified spatial and energetic organization features from both the constructs and analyzed the classification potential of the combined model. It achieved an accuracy of 73% in classifying FLIPs from FunCs. Analysis of organization features from both the constructs, and also, the combination using Receiver Operating Characteristic (ROC) Curves identified that the classification abilities of combined organization features can be translated to a better prediction method among the three. Predictions of functionally linked interfaces of protein showed that 60% of the observed interfaces were correctly predicted, along with identification at least one connected triplet in 87% of the FLIPs. However, a 75% over prediction rate was observed. Analysis of FunCs on the other hand showed that we identified a connected triplet in 70% of the proteins, but rarely predicted functionally uncorrelated interface residues. Overall, our work suggests that network analysis can successfully characterize, classify and predict functionally linked interfaces of proteins.