In the United States, 2.8 million people suffer a traumatic brain injury (TBI) annually. Between 25%-50% of TBI injuries happen under alcohol intoxication. It is not understood how alcohol impacts patient outcomes via secondary injury pathways. Secondary injury pathways offer a window for therapeutic interventions, but there has been little success finding effective medications. Slice cultures offer a way to study secondary injury mechanisms in a controlled manner. The transection injury can model excitotoxicy seen following TBI. The current studies examined the effect of alcohol intoxication and withdrawal at the time of injury, and the effect of a calpain inhibitor (MDL-28170) on cell death following a transection injury. Intoxication had no effect on cell death compared to the TBI condition. In the ethanol withdrawal (EWD) study, EWD did not increase cell death following the TBI except at 72 hours. There was no effect of MDL on cell death. The severity of the model may have caused a ceiling effect. Additionally, imaging points may not have been sufficient for proper characterization. Future studies should use a different injury mechanism and other imaging times should be considered.

The first goal of this study was to understand the role of u-calpain in skeletal muscle protein degradation in cultured muscle cells. Several strategies were developed to down-regulate endogenous u-calpain activity and m-calpain activity in rat myotubes. These included over-expression of antisense u-calpain (AnsL), dominant negative u-calpain (DN-u-CL), antisense 30K subunit (AnsS) and fused antisense u-calpain/30K (AnsLS, i.e., 80K/30K). The ability to regulate calpain activity was confirmed by fodrin degradation (an index of calpain activity). Our data supported the contention that u-calpain contributes significantly to total protein degradation in myotubes. Specifically, over-expressing DN-u-calpain reduced total protein degradation by 7.9% (P<0.01) at 24 hr time point and by 10.6% (P<0.01) at a 48 hr time point. Similarly, over-expression of antisense u-CL and the 30K subunit reduced total protein degradation significantly at the 24 hr time point (P<0.05). However, over-expression of the fused antisense (80K/30K) did not affect (P>0.05) the total protein degradation. In addition to this we determined that desmin was a calpain substrate and that calpain could not degrade tropomyosin. The second goal of this study was to evaluate the relationships among u- and m-calpain and the 30KD subunit. The rationale for this study was that our earlier work indicated coordinated regulation of the calpain subunits. Our data demonstrated for the first time that the transcription and translation of u-calpain and 30K, and m-calpain and 30K are coordinately regulated, respectively. However, the expression of u-calpain did not affect the expression of m-calpain The third goal of this study was to develop a skeletal muscle-specific inducible expression system that may be used in transgenic animal research. A skeletal muscle a-actin promoter was used to replace the cytomegalovirus immediate-early promoter (pCMV) in the ecdysone inducible mammalian expression system. LacZ was used as a reporter gene. A beta-galactosidase staining assay and high-sensitivity B-gal activity assay indicated that the skeletal muscle-specific expression system functioned in myotubes. After 48 hr of administration of ponasterone A (inducer), the treated cells had 15-fold higher B-gal activity than the control cells. / Graduation date: 2002

Spinal cord injury, acute pain, and morphine tolerance are important issues in the clinical practice. A primary injury to the spinal cord causes both morphological and biochemical changes with initiation of the devastating secondary pathophysiological pathways that ultimately destroy CNS cells and cause degeneration of nerve fibers. Tissue injury is associated with sensitization of nociceptors and subsequent changes in the excitability of central neurons, known as central sensitization. Nociceptor sensitization and central sensitization are believed to underlie the development of primary and secondary hyperalgesia, respectively. The most efficacious drugs used to relieve pain are the opioid analgesics. Chronic administration leads to the development of tolerance. Tolerance is manifested as a decreased potency of the drug, so that progressively larger doses must be administered to achieve a given level of analgesia. The processes underlying opioid tolerance still need to be elucidated. Recently, it is found calpain-Cdk5 (cyclin-dependent kinase-5)-p35 pathway modulation implicated in neuroprotection, acute nociceptive response, and morphine analgesia. In this thesis, we evaluate calpain inhibitor-MDL28170 and Cdk5 inhibitor-roscovitine against rat spinal cord hemisection, formalin-induced acute nociceptive responses, and chronic morphine tolerance. We found calpain-Cdk5-p35 pathway inhibition could protect spinal cord hemisection and subsequent neurodegeneration, inhibit formalin-induced flinch response involving DARPP-32 (dopamine and c-AMP regulated phosphoprotein, MW=32 kDa) phosphorylation, and reverse right shifted morphine dose-response curve with upregulated ED50 (50% of effective dose) reduction. Taken together, calpain-Cdk5-p35 pathway inhibition is useful in the management of spinal cord injury, acute inflammatory pain, and attenuate morphine tolerance development with further clinical application.

Calpains are intracellular enzymes that merge cysteine protease and calcium sensing activities together in one molecule. They respond to Ca2+ signals and modify the activity of their targets by selective proteolysis. Calpains are involved in normal cellular process like cell migration and apoptosis. The over-activation of calpain due to disturbances in Ca2+ homeostasis or inactivation due to mutations, contribute to diseases like ischemic injury and muscular dystrophy. The classical calpains 1 and 2 are heterodimeric enzymes containing a large (80 kDa) subunit and a small subunit (28 kDa). Dimerization occurs through the 5th EF-hand of penta-EF-hand (PEF) domains present in both large and small subunits. In this study, I have used structural genomics approaches to explore the PEF and C2-like regulatory domains of some of the other 12 human calpain isoforms. I have shown that recombinant PEF domain of skeletal muscle-specific calpain 3 exists as a stable homodimer when produced alone. Modelling studies suggest that there would be no barriers for dimerization of the full-length enzyme through the PEF domains which would place the protease cores at opposite ends of the dimer. Co-expression studies using small subunit were performed with PEF domains of calpains 1, 3, 8, 9, 11, 12 and 13. A differential tagging system was devised to differentiate heterodimers from homodimers. The PEF domains of calpains 1, 3, 9 and 13 co-expressed with the small subunit, while the others failed to express. The PEF domains of calpains 1 and 9 formed heterodimers. Conversely, the PEF domain of calpain 3 formed a homodimer and that of calpain 13 predominantly formed a homodimer with a small amount of heterodimer. Homodimerization of calpains implies they are less-likely to be inhibited by the endogenous calpain inhibitor, calpastatin. C2-like regulatory domains of calpains 5-13 were also studied. The structure of the distal C2-like domain of calpain 7 was solved. It is markedly different from canonical C2 domains and may not bind Ca2+. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2009-02-11 12:30:29.18

Calpains are calcium-dependent, intracellular, multi-domain cysteine proteases involved in many physiological functions regulated by calcium signaling, including cell motility. How calpains are activated in the cell is still unknown because the resting intracellular concentration of Ca2+ is orders of magnitude lower than that needed for half-maximal activation of the enzyme in vitro. Several stratagems by which calpains might overcome this Ca2+ concentration differential have been proposed. It is possible that post-translational modifications like phosphorylation, or accessory proteins that bind to calpain, might facilitate the enzyme’s activation at lower than optimal Ca2+ concentrations. Autoproteolysis (autolysis) and subunit dissociation are two other proposed activation mechanisms that could release constraints on the calpain core by breaking the link between the anchor helix and the small subunit to allow the active site to form. By measuring the rate of autolysis at different sites in calpain, it was demonstrated that while the anchor helix is one of the first targets to be cut, several other potentially inactivating autolysis sites, particularly in Domain III, can also be cleaved within the first minute. Thus autolytic activation would go hand in hand with inactivation. By fractionating and identifying calpain 2 autolysis fragments, I show that the small subunit does not dissociate away from the large subunit, but is proteolyzed to a 40-45 k heterodimer of the penta-EF-hand Domains IV and VI. It is likely that this autolysis-generated heterodimer has previously been misidentified as the small subunit domain VI homodimer that would be produced by subunit dissociation. A calpastatin affinity column was constructed and used to capture recombinant calpain 2 from bacterial cell lysate. This affinity column provides a tool to screen for and capture calpain complexed to potential binding partners in the presence of Ca2+. Here I propose a model for calpain 2 activation in vitro that does not involve autolysis, subunit dissociation, or calpain activators. / Thesis (Master, Biochemistry) -- Queen's University, 2010-10-25 16:03:52.364

Molecular modelling has become widely used in chemistry and biology. The aim of this project is to use a range of molecular modelling techniques to study enzymic actions. This thesis consists of two parts. Part A of this thesis describes computational studies conducted for the calpain-calpastatin system. Calpain is a cysteine protease. Over-expression of calpain is associated with many diseases. Calpastatin is the naturally occurring specific regulator of calpain activity. In this part of the thesis, the dynamic conformational preferences of region B of the inhibitory domain in calpastatin were examined in detail by using molecular dynamics simulations and stochastic dynamic simulations with Monte Carlo sampling. Part B of the thesis explores the structure and function of the enzyme 3-dexoy-D-arabino-heptulosonate 7-phosphate synthase from Mycobacterium tuberculosis (MtuDAH7PS). MtuDAH7PS catalyses the first reaction of the shikimate pathway and is a target for the development of anti-tuberculosis drugs. MtuDAH7PS is found to be synergistically inhibited by combinations of aromatic amino acids (Trp+Phe or Trp+Tyr), but not by any single aromatic amino acids. In this part of the thesis, this unique mechanism of allosteric regulation in MtuDAH7PS was investigated by using a range molecular modelling techniques. Firstly protein crystal structure refinements were conducted and those crystal structures of MtuDAH7PS in complex with various ligand molecules are described in Chapter 4. Secondly, the reaction mechanism and roles of active site residues were investigated in Chapter 5, through docking calculations (both rigid docking and induced fit docking) of a series of designed active site inhibitors. Finally, Chapter 6 discusses the molecular basis of the communication mechanism of allosteric regulation in MtuDAH7PS.

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Thesis (Ph. D.)--Ohio State University, 2009. / Title from first page of PDF file. Includes bibliographical references (p. 202-241).

Thesis (Ph. D.)--West Virginia University, 2004 / Title from document title page. Document formatted into pages; contains xiii, 119 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Afin de lutter efficacement contre les malversations du dopage, il apparaît essentiel de comprendre les mécanismes conduisant au remodelage musculaire. Dans ce but nous avons analysé les effets d’un β2-agoniste, le clenbutérol, sur le remodelage musculaire et les différentes voies de signalisation qui y sont associées. Nous nous sommes particulièrement intéressés au système des calpaïnes qui a souvent été associé à des phénomènes de remodelage musculaire, principalement dans des modèles d’atrophie. Nous avons montré une sollicitation précoce du système des calpaïnes lors d’un traitement chronique au clenbutérol chez le rat associé à une conversion phénotypique dans les muscles EDL et Soléaire et à une hypertrophie dans le muscle EDL uniquement. Puis, nous avons inhibé l’activité des calpaïnes en parallèle d’un traitement au clenbutérol. Les muscles ayant une activité des calpaïnes diminuée et soumis à un traitement au clenbutérol n’ont pas développé de remodelage musculaire. Ces premiers résultats renforcent l’idée d’une implication des calpaïnes dans le remodelage musculaire induit par un traitement chronique au clenbutérol. / To fight doping in an effective manner, it is essential to understand the mechanisms leading to muscle remodeling. For this purpose we analyzed the effects of clenbuterol, on muscle remodeling and various associated signaling pathways. We were particularly interested with the calpain system which has often been associated with muscle remodeling phenomena, mainly in models of atrophy. We have shown that an early calpain system solicitation during chronic treatment with clenbuterol in rats was associated with a phenotypic conversion in the Soleus and EDL muscles and hypertrophy in the EDL muscle. We then inhibited the activity of calpains with a parallel clenbuterol treatment. The muscles with a reduced activity of calpain and treated with clenbuterol did not develop muscle remodeling. These initial results reinforce the idea of an involvement of calpain in the muscle remodeling induced by chronic treatment with clenbuterol.