• HLA-G DIMER PROLONGS KIDNEY ALLOGRAFT SURVIVAL BY INHIBITING CD8+T CELL ACTIVATION AND GRANZYME B EXPRESSION

      Ajith, Ashwin; Biomedical Sciences (Augusta University, 2019-12)
      Solid organ transplantation is the preferred therapy for many patients diagnosed with end stage organ failure, however allograft rejection is a significant barrier for graft survival. Patient care involves heavy immunosuppressive drug treatment leading to elevated risk for cancer and other opportunistic infections. Hence there is a need to develop effective alternative approaches to minimize graft rejection. We focused on Human leukocyte antigen G (HLA-G), a nonclassical HLA class Ib molecule critically involved in the maintenance of maternal tolerance to semi-allogeneic fetal tissues during pregnancy and has emerged as a potential therapeutic target to control allograft rejection. We demonstrate here that the level of soluble HLA-G dimer was higher in a group of 90 patients with a functioning renal allograft compared with 40 patients who rejected (RJ) their transplants. The HLA-G dimer level was not affected by demographic status. One of the potential mechanisms in tissue organ allograft rejection involves the induction of granzymes and perforin, which are the main effector molecules expressed by CD8+ cytotoxic T lymphocytes and function to destroy allogeneic transplants. Using genomics, molecular and cellular analyses of cells from T-cell–mediated RJ and nonrejected kidney transplant patients, cells from leukocyte Ig-like receptor B1 (LILRB1) transgenic mice, humanized mice, and genetically engineered HLA-G dimer, we demonstrated a novel mechanism by which HLA-G dimer inhibits activation and cytotoxic capabilities of human CD8+ T cells. This mechanism implicated the downregulation of Granzyme B expression and the essential involvement of LILRB1. Thus, HLA-G dimer has the potential to be a specific and effective therapy for prevention of allograft rejection and prolongation of graft survival.
    • HMGB1-TLR4 Signaling Following Traumatic Brain Injury

      Laird, Melissa D; Department of Neurosurgery (2011-05)
      Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Although preventative measures may reduce the incidence of TBI, over 1.7 million Americans suffer a head injury annually1. Brain edema, the abnormal accumulation of fluid within the brain parenchyma, contributes to elevated intracranial pressure (ICP), brain herniation, and a poor prognosis following head injury2"4. Clinically, the degree of swelling on the first computed tomography (CT) scan directly correlates with patient outcome, demonstrating the need to limit brain edema following head injury5. Unfortunately, current medical therapies do not effectively control brain edema and neurosurgical approaches to alleviate increased ICP are invasive and of limited utility. A longrange goal of our laboratory is to elucidate the molecular and cellular mechanisms that promote cerebral edema, which may aid in the development of novel therapeutics for head trauma patients. A central premise of our hypothesis is that activation of Toll-like receptor 4 (TLR4) increases brain edema following TBI. Toll-like receptors (TLR) are membrane proteins within the interleukin-1 receptor superfamily that mediate innate immunity6"8; however, recent evidence suggests TLR are also expressed within the CNS of humans and rodents9"12. Activation of TLR4 exacerbated neuronal injury and neuroinflammation following cerebral ischemia13,14, although the involvement of TLR4 following TBI has only just begun to be examined and comparatively little is known15,16. Given the association between inflammation, neurological injury, and patient outcome17, TLR4 may represent an unexplored therapeutic target following TBI. We hypothesize that High Mobility Group Box Protein B1 (HMGB1), a putative endogenous ligand forTLR4, is released via an NR2B mechanism and promotes cellular edema following TBI.
    • HSP90 Inhibitors in Sepsis and Sepsis-induced Acute Lung Injury

      Chatterjee, Anuran; Vascular Biology Center (2007-06)
      Severe sepsis is the leading cause of death for patients in intensive care units. Patients with severe sepsis develop multiple organ failure, including acute lung injury, resulting from a dysregulated inflammatory response. Inhibitors of the ubiquitous chaperone, heat shock protein 90 (hsp90), block the activity of certain pro-inflammatory mediators, in vitro. We hypothesized that hsp90 inhibitors may ameliorate the inflammation and acute lung injury associated with severe sepsis. Male C57Bl/6 mice received either one of two hsp90 inhibitors, radicicol or 17-allylaminodemethoxygeldanamycin (17-AAG), at 24, 12, 6 and 0 hr before receiving a lethal dose of endotoxin (6.75 x 104 EU / g body weight). Outcomes included survival and parameters of systemic inflammation (plasma cytokine, chemokine and nitrite/nitrate levels), pulmonary inflammation (lung NF-κB and myeloperoxidase activities, inducible nitric oxide or iNOS expression, iNOS-hsp90 complex formation, leukocyte infiltration), and lung injury (pulmonary capillary leak, expression of endothelial specific cell-adhesion or adherens junction proteins, lung function). Mice pre-treated with vehicle and receiving endotoxin exhibited 100% 24-hr lethality, dramatic increase in all parameters of systemic and pulmonary inflammation, reduced lung function and increased capillary leak associated with reduced expression of functional adherens junction proteins. In comparison, mice receiving either radicicol or 17-AAG prior to endotoxin, exhibited prolonged survival, reduced or abolished increases in systemic and pulmonary inflammatory parameters, normal lung function and attenuated capillary leak with restored expression of adherens junction proteins. Additionally, in an in vitro model of endotoxin and activated neutrophil-induced endothelial barrier dysfunction, we show that pre-incubation of neutrophils or endothelial cells or both with hsp90 inhibitors impart a profound barrier protective effect, which is mediated, at-least in part, through reduced activation of pp60Src kinase (an hsp90 client protein) and phosphorylation of the focal adhesion protein paxillin, a pp60Src kinase substrate. Hsp90 inhibitors are drugs already in use clinically as adjunct cancer treatment. Therefore, these findings point to a potential clinical use of these drugs in sepsis and sepsis induced ALI.
    • Human kidney and urinary arylamidase

      Story, Mary Nell Carbo; Department of Cell and Molecular Biology (1969-06)
    • Human Placental Na+-H+ Exchanger: Properties and Function

      Daniel, Balkovets; Department of Cell and Molecular Biology (1988-07)
    • The hydrolysis of diacetylcycloserine: in vitro and in vivo studies

      Chuang, Augustine, Heng-Lung; Department of Cell and Molecular Biology (1972-12)
    • Hypothalamic AgRP and POMC neurons modulate stress-induced depression-related behaviors

      Fang, Xing; Department of Neuroscience and Regenerative Medicine
      Depression is a common and debilitating mental disease. Currently available antidepressants are not effective for many individuals with depression and our understanding of the underlying mechanisms remain limited. Evidence suggests that hypothalamic arcuate nucleus (ARC) is highly responsive to acute stress. The ARC contains two distinct subpopulations of neurons—expressing orexigenic agouti-related peptide (AgRP) and anorexigenic pro-opiomelanocortin (POMC). AgRP and POMC neurons regulate food intake and the food reward system. It is unknown whether AgRP and POMC neurons are recruited by chronic stress and if their dysfunction may contribute to the development of chronic stress-induced depression-related behaviors. To address this, we have developed a mouse model of chronic unpredictable stress (CUS), which can induce anhedonia and despair behavior that mimic symptoms in human depression. Using this animal model, I investigated the roles of AgRP and POMC neurons in stress responses and stress-induced depression-related behaviors. I demonstrated that CUS decreases activity of AgRP neurons but increases activity of POMC neurons. A chemogenetic approach was used to selectively manipulate the activity of POMC and AgRP neurons, leading to opposite effects of stress-induced depression-related behaviors. These results suggest that AgRP and POMC neurons are differentially involved in stress maladaptation and related behaviors. It provides insight into the mechanisms underlying the development of depression and novel strategies for the treatment of this mental illness.
    • Identification and Characterization o f CRIPlb: A Novel CBi Cannabinoid Receptor Interacting Protein

      Niehaus, Jason L.; Department of Biological Sciences (2006-07)
      G protein-coupled receptors (GPCRs) transduce extracellular stimuli to intracellular signals through their interaction with heterotrimeric G proteins. Signaling diversity and specificity is imparted primarily through variations o f G protein subunits. Protein-protein interactions between intracellular accessory proteins and GPCRs also modify signaling by altering receptor activity or signaling pathways. The ability of intracellular proteins to interact with the CBi cannabinoid receptor was investigated to determine whether particular signaling properties of CBi resulted from interaction with specific CBi interacting proteins. A novel protein named C RIPlb was discovered to interact with the C-terminal tail o f CB). The interaction between CRIPlb and CBi was characterized using the yeast two-hybrid assay. Functional consequences of the CRIPlb- CB| interaction were investigated by examining protein localization by confocal microscopy and measuring CBi mediated N-type Ca2+ channel activity in the presence of CRIPlb by whole-cell patch clamp recordings. The yeast two-hybrid assay indicated that the last nine amino acids of the CBi C-terminal tail were required for interaction with CRIPlb. Heterologous expression of C RIPlb and CBi in HEK 293 cells did not reveal evidence of colocalization, nor was CBi able to significantly traffic C RIPlb to the plasma membrane. However, CRIPlb and CBi were found to colocalize in superior cervical ganglion (SCG) neurons. Whole-cell voltage-clamp recordings of N-type Ca2+ channels in SCG neurons indicated that CRIPlb had no effect on agonist- or inverse agonist-induced modulation of Ca2+ current by CBi. Furthermore, the level of CBi constitutive activity was not significantly altered by CRIPlb. The high affinity of CBi for G proteins, as demonstrated by the ability of CBi to sequester G proteins from other Gi/0 coupled receptors, was unaffected by expression of CRIP lb. These results provide evidence that CRIPlb is a novel CBi accessory protein that interacts with the C-terminal tail of CBi. While CRIPlb and CBi can apparently interact in a neuronal expression system, the ability of CRIPlb to modify CBi signaling was not detected in any of the pathways investigated. Thus, the distinctive signaling properties of CB|, such as constitutive activity and G protein sequestration do not originate from nor are modified by CRIPlb.
    • Identification and characterization of CRIP1b: a novel CB₁ cannabinoid receptor interaction protein

      Niehaus, Jason Lance; School of Graduate Studies (2006-07)
      G protein-coupled receptors. (GPCRs) transduce extracellular stimuli to intracellular signals. through their interaction with heterotrimeric G proteins. Signaling diversity and specificity is imparted primarily through variations of G protein subunits. Protein-protein interactions between intracellular accessory proteins and GPCRs also modify signaling by altering receptor activity or signaling pathways. The ability of intracellular proteins to interact with the CB 1 cannabinoid receptor was investigated to determine whether particular signaling properties of CB 1 resulted from interaction with specific CB 1 interacting proteins. A novel protein named CRIP 1 b was discovered· to interact with the C-terminal tail of CB 1 ~- The interaction between CRIP 1 b and CB 1 was characterized using the yeast two-hybrid assay. Functional consequences of the CRIPlbCB 1 interaction were investigated by examining protein localization by confocal microscopy and measuring CB1 mediated N-type Ca2+ channel activity in the presence of CRIP 1 b by whole-cell patch clamp recordings. The ye_ast two-hybrid assay indicated that the last nine amino acids of the. CB 1 C-terminal tail were required for interaction with CRIP 1 b. Heterologous expression of CRIP 1 b and CB 1 in HEK 293 cells did not reveal evidence of co localization, nor was CB 1 able to significantly traffic CRIP 1 b to the plasma membrane. However, CRIP 1 b and CB1 were found to colocalize in superior cervical ganglion (SCG) neurons. Whole-cell voltage-clamp recordings ofN-type Ca2+ channels in SCG neurons indicated that CRIPlb had no.effect on agonist- or inverse agonist-induced modulation of Ca2 + current by CB1. Furthefrr1:ore, the level of CB 1 constitutive activity was· not significantly altered by CRIP 1 b. The high affinity of CB 1 for G proteins, as demonstrated by the ability of CB 1 to sequester G proteins from other Gu0 coupled receptors, was unaffected by expression ofCRIPlb. These results provide evidence that CRIP 1 b is a novel CB 1 accessory protein that interacts with the C-terminal tail of CB1. While CRIPlb and CB1 can apparently interact in a neuronal expression system, the ability of CRIP 1 b to modify CB 1 signaling was not detected in any of the pathways investigated. Thus, the distinctive signaling properties of CB1, such as constitutive activity and G protein seque.stration do not originate from nor are modified by CRIPlb.
    • Identification and Characterization of Two New Players in DNA Double-Strand Break Repair - PSF and p54(nrb)

      Udayakumar, Durga; Department of Molecular Medicine (2005-12)
      The stability and integrity of a genome depends on how accurately the genetic information is passed on to each daughter of a dividing cell. This accuracy is compromised when the genome is exposed to various stressful conditions, including ionizing radiation (IR), radiomimetic agents such as bleomycin, neocarzinostatin, and etoposide, free radicals generated from metabolic processes, and also errors during replication. The effect is DNA damage resulting in potentially lethal double-strand breaks (DSBs). The causes are as follows: DSBs are also created as intermediates during specialized recombination processes, such as V(D)J recombination, immunoglobulin class switching and somatic hypermutation.
    • Identification of Muscarinic Receptor Subtypes and Arachidonic Acid Metabolites Mediating the Effects of Actylcholine in The Pulmonary Circulation of Rabbits and Cats

      El-Kashef, Hassan; Department of Pharmacology (1987-05)
      Acetylch.oline (ACh) is known ·to reduce vascular pressure throughout the systemic. circulation; in. the·-- pulmonary circulatfon: ACh: can·- produce:either vasodi-1ationr or' ·vasoconstri-ction. For example,. ACh', produces: vasoconstrictfon · in· the, pulmonary ·circula~i on· of rabbits .. but dtlati on in· the· -feline. pu·lmof!ary-, vasculature·~-- The·· mechanism(s-) responsible·' for· the· actions of ACh in the pulmona.ry vasculature-- remain unc;lear. This study was designed to i·nve·st.igate poss·ible mechanisms. responsible for the actions. of ACh in the ra~bit ancf cat pulmonari_.circulation·~- Our hypothesi's is tbat . variations in· the· v.as.cular ·-response~ to: ACh may- be due to differe.nces in med-iators. (e.g. ·prostanoids)· released upon. activation .. of the muscarinic receptor~ These· di.ffe-rences cou 1 d be attributed,· either to. differences ... in muscarinic receptor subtypes.-. or to differences. in the·. metabolism of· arachidonic acid- tn rabbit. vs. cat. In anesthetized rabbits,_ ACh-induced pulmonary=- vasoconstriction-··. was,. totatly, inhibited::'·- by:: the: phosphotipasey A'2· inhibitor· qu-inacrine··,. the cycl o~oxygenase"' inhibitors:; indomethacin·,. and,.~ mec lofenamate·,, the- thtomboxane/ A:2:· synthetase=, i nh~i bi:tot· ]-;.;.{1'- Imidazolyl.)-· Heptano.fc. Acid .. (7;..IHA), and'- by,- the··' thromboxane·· A- 2 . receptor·· antagpntst. SQ:. 29-~.548:, .. but not·,. by·- the· 1 .ipoxygenase:-- inhibitor- nordihydrogua-iaret.ic: acid( NDGA}':. AGh·: s:igni-ftcantly; increased> plasma; thromboxane···B2.' (TXB'2J:· levels;. in·-. anesthetized:·,_ rabb_its:. A.Ch:~induced:. decrease'~ in, system.ic ar.teria:l pressure'" was, not· affected: by:·. any o.f'· the·· i nh'ibftors,. mentionedJ above--._ Small>. doses: of· the, select.i ve:· Mi- muscari nfc. receptor· subtype· antagonists:, pi renzepihe and_:. tr.ihexypheni:d~l,.. s:i gni-f.i cantly:- fnhi bjted:-: the· ACh.-i nduced:~- increase:'-·. i m pulmonary; vascuJa·r res:fstance- but. not: the~ ACh-induced=;· decrease Ht systemic. arterfal~: pressur.e··. The:.- s·ame" dose·· o.f secover.i ne:, .. a-~.- se·l ecti ve, M2-- muscar.i.nic: ii recept.or subtype. antagonist, dld ·not· change the · ACh ·effects on the pu 1 mona ry or· sys.temi c · v qSCu latu,re. Larger doses of pirenzepine, trihexyphen-idyl and\· secoverine,· totally. inhibited· the.- effects:: of ACh in· the .. pu:lmonary· and: systemic: circulatjons. Atrop.ine··. equally:- inh.i.bi ted:· the· effects. of ACn· on'" the pulmonary..- and.: systemic vascul atures- at.- any. do$es~ . . used': In .. fso_lated· rabbit·. lungs .. perfused·:·in:· situ~: w:ith · b]ood·-,_'free· medium;< . . . . . . . the ACh-fnduced· increases fn .. ·pulmonary v.ascular resistance, TXB2 level·s: a:nd ·6-Keto~PG·Fra were significa.nt.ly inhibited. by small concentrations.· of· pi.renzepine but .not· secove·rine. Larger concentrations. of· p.irenzepine~ and:'· · . . secover·ine totally ·inhibited· these· effects of ACh. In -anesthetized c~ts, the· ACh~inducec:l decre-ase in pulmonary vascular resfstance was partially~ · inhibited· by quinacrine and·' indomethacin and· significantly:· .potenti~ted by NOGA. The ACh-induced ·decrease in systemic arterial .pressure was not s·ignificantly affected by· any. of these· inhibitors. Small. doses. of secover-ine:'' but not . pfrenzepi ne;~" i nh.ibited·· the: pulmonary: but,. not .. the:· systemic vascular-. response·, tct.. ACh.,,_ i!!.=- v:ivo~ Howev-er,_. large··· doses; .. of·.· ptrenz:epi ne-, · tri·hexyphen:fdyl. and' s.ecoveri'ne' significantly:· inh.i bited~ the:, .. . . effects. of:. ACh~ in·.: the~: pulmonary;· and. systemic vascu:l ature\ In: isola ted:· cat: 1 ung_s:. perfused~- fm: sitw: w.ith; bJ ood.;.free- · medium~. 'ACh'" di-lated... the·· precons.tricted:: pulmonary: artery. bu.t> tt: did·: not ·s,tgnific.antly:· alter- TXB2 ori 6~ Keto~PGF 1 at 1 eveTs:~.. Smal:T: concentrat:i ons:- of:-· secoverfne-': but· not· pJ.renzep.i ne~-- par.t.ially;' i nhi bi'tedr the· ACh;.. induced~· decrease· in: pulinonar,Yvascular ·· resistance· •. The· pros.tacyc;ti n .. · synthetase fnhi bitor· tranylcypromine· si"gn.f$icantly~ i'nhi bited;'; the,· ACh- induced§ decrease:· in:· · pu]monary.:; va-scu..lar· resi.stance·~- . . i fi: ·These· results. ·indi-cate that l) ACh has . opposite actions in. the systemic {dilatory) versus pulmonary· (constrictor) circulation of rabbi-ts, 2r Arachidon-ic· acid.'metabo'lftes·. mediate. the-. pulmonary: . but. :not·· .the" systemic vascular respon.se- to. ACh, in rabbit, 3}. Thromboxane·· A2 mediates,. the:· pulmonary- vasoconstrictor- response·· to, ACh in rabbit·~ 4} The r.abbit pulmonary . v~scular· muscarinic recepto.rs. are· very·,· sensit:ive to·. p_irenzep.i-ne· . and thus be:have m9re · 1 ike Mi recept~rs, _5) · In· the cat·, the· ACh- induced·· decrease in- p~lmonary but not systemic vascular. pressure is partly mediated by prostacycli'n, 6)' In _the cat, the vascula-r· muscarinic receptors both in the· pulmon~ry. and the systemic beds are not selectively sensitive to . .pirenz.ep.ine ·and- thus behave like. non-Mt receptors and,· 7) In the .cat, the· ·muscarinic receptors. in the· pulmonary. and, systemic vasculat~re· represent different or heterogenous populations of M2. receptors since· they exhibit different affinities·. to secoverine.
    • Identification of professional competencies needed for practicing registered nurses in urban and rural hospitals

      Isler, Barbara A.; School of Nursing (1981-05)
      The purpose of this study was to determine if the hospital- location .s i gni fi cantly influenced the regis ter.ed nurses• competency needs .. A. sample of 112 urban and 80 rural .nurses was selected by Nursing Directors in 23 Georgia hospitals. Data were collected by use of a 113 item . competency questionnaire adapted from Clayton'·s (1978) work in thi.s area .. · Findings suggest the urban o~ rural hospital setting significantly . influences the competencies used by the registered nurse. Other findings include the acceptance percentage for ea-ch competency by the urban or. rural nurse indicating thei~ perception of nee~ and demographic characteristics of the two nurse populations.
    • Identification of RAB11-Family Interacting Proteins (RAB11-F1Ps): Integral Components in Plasma Membrane Recycling

      Hales, Chadwick M; Institute of Molecular Medicine and Genetics (2003-05)
      Given the involvement of Rabl la in each of these cellular processes and given the potential impact of Rabl la on human health and disease, we sought to further establish a role for Rabl la in plasma membrane recycling. Since other Rab proteins have numerous characterized interacting proteins and because the repetoire for Rabl la is currently limited to three identified interacting proteins, we hypothesized that other Rabl la binding partners exist as putative downstream effectors for the small GTPase. We therefore proposed the following three aims: Aim 1: Identify R ab lla interacting proteins. Aim 2: Determine the effect of interacting proteins on membrane recycling. Aim 3: Establish an organizational model of a putative Rabl la complex. The progression of studies herein provides insight into the dynamic and complex process of plasma membrane recycling. Yeast two hybrid screening of a parietal cell cDNA library utilizing dominant active Rabl laS20V as the bait identified Rabl 1-Family Interacting Protein 1 (Rabll-FIPl), a novel R ab lla interacting protein. EST database searches with the R abll-FIPl sequence identified three homologous proteins with high carboxyl-terminal identity. Chapter 1 introduces the new family of Rabl la interacting proteins and provides the initial characterization. Interestingly, these studies indicated an interaction between Rabll-Family Interacting Protein 2 (Rabll-FIP2) and myosin Vb tail. Chapter 2 further describes the Rabl l-FIP2/myosin Vb tail binding and provides functional data placing Rabll-FIP2 as an integral component of the plasma membrane recycling system. Finally, recent studies have indicated a recycling system dependence on different kinase activities. Through kinase inhibitor studies and immunofluorescence imaging, evidence presented in Chapter 3 suggests that R ab lla along with multiple Rabll-FIP proteins function as a complex beginning at the process of endocytosis with movement dependent on multiple phosphorylation events. The ultimate goal throughout these studies is to provide a clearer picture of Rabl la function in plasma membrane recycling so that one day a positive impact on human health can be achieved.