60 research outputs found
Analyzing CSF tau concentrations in Alzheimer's disease and control patients
No full textThe purpose of this project is to establish a phage-based biomarker screening assay to recognize AD in human blood and cerebrospinal samples. Specifically, I will be completing ELISA screening of available phage that were identified in prior semesters to identify candidate phage for for further testing.
Phase 1 is to identify phages that recognize PTM changes in tau, and phase 2 is to test the phage in a large array of clinical samples of human blood and cerebrospinal fluid
Redox regulation of cysteine-dependent enzymes
No full textIt is well-established that maintenance of the intracellular redox (i.e., reduction-oxidation) state is critical for cell survival and that prolonged or abnormal perturbations toward oxidation result in cell dysfunction. This is exemplified by the widespread observation of oxidative stress in many pathological conditions, as well as the positive effects of antioxidants in treating certain conditions or extending the life span itself. In addition to the effects of oxidation on the lipid bilayer and modification of DNA in the nucleus, proteins are also modulated by the redox state. One of the primary targets of oxidation within a protein is the AA cysteine, whose thiol side chain is highly sensitive to all types of oxidizing agents. Although this sensitivity is used to prevent oxidation within the cell by potent defense mechanisms, such as glutathione, the use of cysteine in the active site of enzymes leaves them open to oxidant-mediated damage. Whether the damage is due to a pathological condition or to postmortem mediated loss of redox homeostasis, cysteine-dependent enzymes are targets of all forms of reactive oxygen, nitrogen, and sulfur species. A greater understanding of the redox-mediated control of cysteine-dependent enzymes opens the door to the selective use of antioxidants to prevent or reverse the cellular damage their inhibition causes.Journal Articl
-Methyl-d-Aspartate Receptor Subtypes
No full textSince excitotoxicity has been implicated in a variety of neuropathological conditions, understanding the pathways involved in this type of cell death is of critical importance to the future clinical treatment of many diseases. The N-methyl-D-aspartate (NMDA) receptor has become a primary focus of excitotoxic research because early studies demonstrated that antagonism of this receptor subtype was neuroprotective. However, initial pharmacological agents were not clinically useful due to the adverse effects of complete NMDA receptor blockade. Understanding the biochemical properties of the multitude of NMDA receptor subtypes offers the possibility of developing more effective and clinically useful drugs. With the discovery of the basis of heterogeneity of NMDA receptors through molecular biological approaches, many new potential therapeutic targets have been uncovered, and several model systems have been developed for the study of NMDA receptor-mediated cell death. This review discusses these models and the current understanding of the relationship between NMDA receptor subtypes and excitotoxicity.Journal Articl
Thiol-protease oxidation in age-related neuropathology
No full textIncreased oxidative stress is a hallmark of every major neurodegenerative disease that has been studied. Numerous biomarkers of oxidative stress have been found, indicating that waves of oxidation had, at one time or another, overwhelmed antioxidant defenses, leaving behind a host of oxidized DNA, lipids, and proteins in their path. Although some level of oxidation may be beneficial, perhaps mediated by a hormetic response, the extent and types of oxidation detected in neuropathological states would suggest that oxidative stress contributes to a loss of homeostasis and cellular dysfunction. Although there are many targets of oxidants, this review emphasizes protein oxidation with a focus on an important group of redox-sensitive enzymes, the thiol-proteases. Both the direct and the indirect effects of oxidation and their potential importance in neurodegeneration are considered.Journal ArticleFinal article publishe
Calpains: Intact and active?
No full textCalpains are a family of calcium-dependent thiol-proteases which are proposed to be involved in many physiological processes as well as pathological conditions. Calpains are likely to be involved in processing of numerous enzymes and cytoskeletal components, thereby linking their activity to a variety of intracellular events. Although widely studied, the precise mechanism(s) involved in calpain activation and activity in vivo remain poorly understood. Initial studies suggested that calpain exists primarily as an inactive proenzyme that required autolytic cleavage for activation. It was also hypothesized that calpain associated with membrane phospholipids, serving to increase calcium sensitivity, facilitating autolytic conversion and thus activating the enzyme. These hypotheses, however, have not been universally accepted and there is increasing evidence that intact, non-autolyzed calpain is the physiologically active calpain form.Journal Articl
Measurement of calpain activity in vitro and in situ using a fluorescent compound and tau as substrates
No full textCalpains play important roles in numerous physiological and pathological processes (1,2) by catalyzing the limited proteolysis of a wide variety of protein substrates. The activity of calpain toward a specific substrate is regulated not only by calcium, but also by numerous other factors including calpain activator proteins (3), redox state (4,5), and the phosphorylation state of the substrate (6, 7, 8). In order to determine the relative contributions of these and other factors to the activity of calpain, it is necessary to be able to measure calpain activity both in vitro and in situ, and these assays are the subject of this chapter.Book Chapte
NMDA receptor pharmacology: Perspectives from molecular biology
No full textThe NMDA receptor is an important target for drug development, with agents from many different classes acting on this receptor. While the severe side effects associated with complete NMDA receptor blockade have limited clinical usefulness of most antagonists, the understanding of the multiple forms of NMDA receptors provides an opportunity for development of subtype specific agents with potentially fewer side effects. Different NMDA receptor subtypes are assembled from combinations of NR1 and NR2 subunits with each subunit conveying distinct properties. The NRI subunit is the glycine binding subunit and exists as 8 splice variants of a single gene. The glutamate binding subunit is the NR2 subunit, which is generated as the product of four distinct genes, and provides most of the structural basis for heterogeneity in NMDA receptors. Pharmacological heterogeneity results from differences in the structure of ligand binding regions, as well as structural differences between subtypes in a modulatory region called the LIVBP-like domain. This region in NR1 and NR2B controls the action of NR2B-selective drugs like ifenprodil, while this domain in receptors containing the NR2A subunit controls the action of NR2A-selective drugs such as zinc. This suggests that NMDA receptor subtype selective drugs can be created, and further understanding of subtype specific mechanisms ultimately may allow successful use of NMDA receptor antagonists as therapeutic agents.Journal Articl
Protease activity in post-mortem red swamp crayfish (Procambarus clarkii) muscle stored in modified atmosphere packaging
No full textProtease activity during storage is thought to be an important contributor to decreased shelf life of fresh seafood. To examine this, three batches of red swamp crayfish ( Procambarus clarkii) tails, placed on trays, were packed with a polyvinyl chloride film (aerobic packaging or AP), under vacuum (vacuum packaging or VP), or under a modified atmosphere (MAP: 80% CO 2/10% O 2/10% N 2), and proteolytic activity was measured on days 0, 1, 3, 6, and 10 during storage at 2 degrees C. The crude extract from the crayfish digestive system (gut) did not have an apparent role in muscle proteolysis as negligible proteolytic activity was detected. However, the loss of calpastatin (the endogenous calpain inhibitor) was identified in MAP-stored muscle samples on day 10, suggestive of high m-calpain activity. Tail samples stored in AP showed no appreciable proteolysis, but those stored in MAP and VP showed significant decreases in the levels of 53, 66, 71, and 110 kDa polypeptides during storage. The observed proteolytic activity and myofibrillar protein degradation did not correspond to muscle textural properties as the MAP samples had an increased toughness ( P < 0.05) after storage for 10 days. These findings suggest that other physicochemical mechanisms are involved in postmortem alteration in the crayfish muscle structure under the packaging systems investigated.Journal Articl
A region of the rat N-methyl-D-aspartate receptor 2A subunit that is sufficient for potentiation by phorbol esters
No full textN-methyl-D-aspartate (NMDA) receptors are modulated by protein kinase C (PKC) in vivo and in heterologous expression systems. In heterologous expression systems, PKC-mediated modulation is subunit specific with NR2A-containing receptors being potentiated by phorbol 12-myristate 13-acetate (PMA), while NR2C-containing receptors are inhibited or unaffected. In the present study we have produced chimeric receptors containing NR2A and NR2C to define the components of NR2A which are sufficient for potentiation by PMA. Amino acids 1105-1400 of NR2A placed onto the C-terminus of NR2C at amino acid 1102 was the minimum region sufficient for producing a PMA-stimulated receptor. This suggests that this region contains structural determinants for PKC-mediated potentiation of NR2A receptors.Journal Articl
Prediction of Vancomycin Dose for Recommended Trough Concentrations in Pediatric Patients With Cystic Fibrosis
No full textVancomycin is a key antibiotic used in the treatment of multiple conditions including infections associated with cystic fibrosis and methicillin-resistant Staphylococcus aureus. The present study sought to develop a model based on empirical evidence of optimal vancomycin dose as judged by clinical observations that could accelerate the achievement of desired trough level in children with cystic fibrosis. Transformations of dose and trough were used to arrive at regression models with excellent fit for dose based on weight or age for a target trough. Results of this study indicate that the 2 proposed regression models are robust to changes in age or weight, suggesting that the daily dose on a per-kilogram basis is determined primarily by the desired trough level. The results show that to obtain a vancomycin trough level of 20 μg/mL, a dose of 80 mg/kg/day is needed. This analysis should improve the efficiency of vancomycin usage by reducing the number of titration steps, resulting in improved patient outcome and experience.Journal ArticleFinal article publishe
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