• hElp3 Directly Modulates the Expression of HSP70 Gene in HeLa Cells via HAT Activity

      Li, Fen; Ma, Jixian; Ma, Yu; Hu, Yanyan; Tian, Shujuan; White, Richard E.; Han, Guichun; Department of Pharmacology and Toxicology (2011-12-21)
      Human Elongator complex, which plays a key role in transcript elongation in vitro assay, is incredibly similar in either components or function to its yeast counterpart. However, there are only a few studies focusing on its target gene characterization in vivo. We studied the effect of down-regulation of the human elongation protein 3 (hELP3) on the expression of HSP70 through antisense strategy. Transfecting antisense plasmid p1107 into HeLa cells highly suppressed hELP3 expression, and substantially reduced expression of HSP70 mRNA and protein. Furthermore, chromatin immunoprecipitation assay (ChIP Assay) revealed that hElp3 participates in the transcription elongation of HSPA1A in HeLa cells. Finally, complementation and ChIP Assay in yeast showed that hElp3 can not only complement the growth and slow activation of HSP70 (SSA3) gene transcription, but also directly regulates the transcription of SSA3. On the contrary, these functions are lost when the HAT domain is deleted from hElp3. These data suggest that hElp3 can regulate the transcription of HSP70 gene, and the HAT domain of hElp3 is essential for this function. These findings now provide novel insights and evidence of the functions of hELP3 in human cells.
    • Internalization Dissociates b2-Adrenergic Receptors

      Lan, Tien-Hung; Kuravi, Sudhakiranmayi; Lambert, Nevin A.; Department of Pharmacology and Toxicology (2011-02-22)
      G protein-coupled receptors (GPCRs) self-associate as dimers or higher-order oligomers in living cells. The stability of associated GPCRs has not been extensively studied, but it is generally thought that these receptors move between the plasma membrane and intracellular compartments as intact dimers or oligomers. Here we show that b2-adrenergic receptors (b2ARs) that self-associate at the plasma membrane can dissociate during agonist-induced internalization. We use bioluminescence-resonance energy transfer (BRET) to monitor movement of β2ARs between subcellular compartments. BRET between b2ARs and plasma membrane markers decreases in response to agonist activation, while at the same time BRET between b2ARs and endosome markers increases. Energy transfer between b2ARs is decreased in a similar manner if either the donor- or acceptor-labeled receptor is mutated to impair agonist binding and internalization. These changes take place over the course of 30 minutes, persist after agonist is removed, and are sensitive to several inhibitors of arrestin- and clathrin-mediated endocytosis. The magnitude of the decrease in BRET between donor- and acceptor-labeled b2ARs suggests that at least half of the receptors that contribute to the BRET signal are physically segregated by internalization. These results are consistent with the possibility that b2ARs associate transiently with each other in the plasma membrane, or that b2AR dimers or oligomers are actively disrupted during internalization.
    • Making Structural Sense of Dimerization Interfaces of Delta Opioid Receptor Homodimers

      Johnston, Jennifer M.; Aburi, Mahalaxmi; Provasi, Davide; Bortolato, Andrea; Urizar, Eneko; Lambert, Nevin A.; Javitch, Jonathan A.; Filizola, Marta; Department of Pharmacology and Toxicology (2011-01-24)
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    • Neuroprotective effects and mechanism of cognitive-enhancing choline analogs JWB 1-84-1 and JAY 2-22-33 in neuronal culture and Caenorhabditis elegans

      Keowkase, Roongpetch; Aboukhatwa, Marwa; Adam, Bao-Ling; Beach, J Warren; Terry, Alvin V.; Buccafusco, Jerry J; Luo, Yuan; Department of Pharmacology and Toxicology (2010-12-16)
      Background: Our previous work indicated that novel analogs of choline have cytoprotective effects in vitro that might be useful in neurodegenerative conditions such as Alzheimer's disease (AD). Furthermore, two lead compounds (JWB1-84-1 and JAY2-22-33) from a library of more than 50 improved cognitive performances in a transgenic mouse model of AD. The purpose of these experiments was to more specifically investigate the neuroprotective capabilities of these lead compounds both in vitro and in vivo.
    • Peroxynitrite Mediates Diabetes-Induced Endothelial Dysfunction: Possible Role of Rho Kinase Activation

      El-Remessy, Azza B.; Tawfik, Huda E.; Matragoon, Suraporn; Pillai, Bindu; Caldwell, Ruth B.; Caldwell, Robert William; Department of Pharmacology and Toxicology; Vascular Biology Center (2010-11-1)
      Endothelial dysfunction is characterized by reduced bioavailability of NO due to its inactivation to form peroxynitrite or reduced expression of eNOS. Here, we examine the causal role of peroxynitrite in mediating diabetes-induced endothelial dysfunction. Diabetes was induced by STZ-injection, and rats received the peroxynitrite decomposition catalyst (FeTTPs, 15â mg/Kg/day) for 4 weeks. Vasorelaxation to acetylcholine, oxidative-stress markers, RhoA activity, and eNOS expression were determined. Diabetic coronary arteries showed significant reduction in ACh-mediated maximal relaxation compared to controls. Diabetic vessels showed also significant increases in lipid-peroxides, nitrotyrosine, and active RhoA and 50% reduction in eNOS mRNA expression. Treatment of diabetic animals with FeTTPS blocked these effects. Studies in aortic endothelial cells show that high glucose or peroxynitrite increases the active RhoA kinase levels and decreases eNOS expression and NO levels, which were reversed with blocking peroxynitrite or Rho kinase. Together, peroxynitrite can suppress eNOS expression via activation of RhoA and hence cause vascular dysfunction.
    • RhoA/Rho-kinase signaling: a therapeutic target in pulmonary hypertension.

      Barman, Scott A; Zhu, Shu; White, Richard E.; Department of Pharmacology and Toxicology (2009-08-26)
      Pulmonary arterial hypertension (PAH) is a devastating disease characterized by progressive elevation of pulmonary arterial pressure and vascular resistance due to pulmonary vasoconstriction and vessel remodeling as well as inflammation. Rho-kinases (ROCKs) are one of the best-described effectors of the small G-protein RhoA, and ROCKs are involved in a variety of cellular functions including muscle cell contraction, proliferation and vascular inflammation through inhibition of myosin light chain phosphatase and activation of downstream mediators. A plethora of evidence in animal models suggests that heightened RhoA/ROCK signaling is important in the pathogenesis of pulmonary hypertension by causing enhanced constriction and remodeling of the pulmonary vasculature. Both animal and clinical studies suggest that ROCK inhibitors are effective for treatment of severe PAH with minimal risk, which supports the premise that ROCKs are important therapeutic targets in pulmonary hypertension and that ROCK inhibitors are a promising new class of drugs for this devastating disease.
    • The role of RhoA/Rho kinase pathway in endothelial dysfunction

      Yao, Lin; Romero, Maritza J.; Toque, Haroldo A.; Yang, Guang; Caldwell, Ruth B.; Caldwell, Robert William; Department of Pharmacology and Toxicology; Vascular Biology Center (2010)
      Endothelial dysfunction is a key event in the development of vascular disease, and it precedes clinically obvious vascular pathology. Abnormal activation of the RhoA/Rho kinase (ROCK) pathway has been found to elevate vascular tone through unbalancing the production of vasodilating and vasoconstricting substances. Inhibition of the RhoA/ROCK pathway can prevent endothelial dysfunction in a variety of pathological conditions. This review, based on recent molecular, cellular, and animal studies, focuses on the current understanding of the ROCK pathway and its roles in endothelial dysfunction.
    • Role of the central cholinergic system in the therapeutics of schizophrenia.

      Terry, Alvin V.; Department of Pharmacology and Toxicology (2009-06-09)
      The therapeutic agents currently used to treat schizophrenia effectively improve psychotic symptoms; however, they are limited by adverse effects and poor efficacy when negative symptoms of the illness and cognitive dysfunction are considered. While optimal pharmacotherapy would directly target the neuropathology of schizophrenia neither the underlying neurobiological substrates of the behavioral symptoms nor the cognitive deficits have been clearly established. Abnormalities in the neurotransmitters dopamine, serotonin, glutamate, and GABA are commonly implicated in schizophrenia; however, it is not uncommon for alterations in the brain cholinergic system (e.g., choline acetyltransferase, nicotinic and muscarinic acetylcholine receptors) to also be reported. Further, there is now considerable evidence in the animal literature to suggest that both first and second generation antipsychotics (when administered chronically) can alter the levels of several cholinergic markers in the brain as well as impair memory-related task performance. Given the well-established importance of central cholinergic neurons to information processing and cognition, it is important that cholinergic function in schizophrenia be further elucidated and that the mechanisms of the chronic effects of antipsychotic drugs on this important neurotransmitter system be identified. A better understanding of these mechanisms would be expected to facilitate optimal treatment strategies for schizophrenia as well as the identification of novel therapeutic targets. In this review, the following topics are discussed: 1) the central cholinergic system in schizophrenia 2) effects of antipsychotic drugs on central cholinergic neurons 3) important neurotrophins in schizophrenia, especially those that support central cholinergic neurons; 4) novel strategies to optimize the therapeutics of schizophrenia via the use of cholinergic compounds as primary (i.e., antipsychotic) treatments as well as adjunctive, pro-cognitive agents.