205905234
Motor neuronal protection by L-arginine prolongs survival of mutant SOD1 (G93A) ALS mice.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive paralysis due to motor neuron degeneration. Despite the fact that many different therapeutic strategies have been applied to prevent disease progression, no cure or effective therapy is currently available for ALS. We found that L-arginine protects cultured motor neurons from excitotoxic injury. We also found that L-arginine supplementation both prior to and after the onset of motor neuron degeneration in mtSOD1 (G93A) transgenic ALS mice significantly slowed the progression of neuropathology in lumbar spinal cord, delayed onset of motor dysfunction, and prolonged life span. Moreover, L-arginine treatment was associated with preservation of arginase I activity and neuroprotective polyamines in spinal cord motor neurons. Our findings show that L-arginine has potent in vitro and in vivo neuroprotective properties and may be a candidate for therapeutic trials in ALS.
23992310
Differential regulation of neuronal and inducible nitric oxide synthase (NOS) in the spinal cord of mutant SOD1 (G93A) ALS mice.
Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by degeneration of motor neurons throughout the central nervous system. Mutations of the free radical scavenging enzyme superoxide dismutase-1 (SOD1) are a cause of familial ALS but it is not known how mutations lead to cell death. Free radicals such as nitric oxide (NO) are thought to play a key pathogenic role. NO is synthesized by NO synthases (NOSs) from arginine, which is a rate-limiting factor for NO production. We found that neuronal NOS (nNOS)-positive motor neurons were depleted while inducible NOS (iNOS)-positive activated glial cells were increased in transgenic mtSOD1 (G93A) ALS mice. iNOS expression was up regulated consistent with the increases of motor neuron loss and glial activation and citrulline and NO levels while nNOS expression was decreased in G93A ALS mice. Administration of l-arginine to G93A mice reduced the severity of motor neuron depletion and glial activation. In treated animals, nNOS expression was preserved while citrulline and NO were reduced, possibly due to reduced activation of glia expressing iNOS. Our findings show that high concentrations of NO correlate with iNOS expression rather than nNOS expression in G93A ALS mice. This suggests that therapy focused on iNOS inhibition might be a fruitful direction for future ALS therapeutic trials.
24557128
Activation of Ets-2 by oxidative stress induces Bcl-xL expression and accounts for glial survival in amyotrophic lateral sclerosis.
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by selective degeneration of motor neurons and glial activation. Cell-specific transcriptional regulation induced by oxidative stress may contribute to the survival and activation of astrocytes in the face of motor neuron death. In the present study, we demonstrate an age-dependent increase in Bcl-xL and Ets-2 immunoreactivity that correlates with an increase of glial fibrillary acidic protein (GFAP)-positive cells in the ventral horn of the spinal cord in both ALS transgenic mice [mutant SOD1 (G93A)] and affected humans. Chromatin immunoprecipitation (ChIP) analysis verified that Ets-2 preferentially occupies the Ets-2 binding element in the promoter of Bcl-xL in primary astrocytes under oxidative stress conditions as well as in G93A spinal cords. Ets-2 small-interfering RNA down-regulated the transcriptional activity of Bcl-xL. In primary glial cultures, Bcl-xL overexpression and mutant SOD1 (G93A) both conferred resistance to oxidative stress-induced cell death. Our findings suggest that Ets-2 transcription factor activation of Bcl-xL gene may protect glia from constitutive oxidative stress that is thought to be a key mechanism contributing to the pathogenesis of ALS. This survival pathway may contribute to the glial survival and activation seen in the spinal cord of ALS patients.
981469
Development of near-infrared fluorophore (NIRF)-labeled activity-based probes for in vivo imaging of legumain.
Asparaginyl endopeptidase, or legumain, is a lysosomal cysteine protease that was originally identified in plants and later found to be involved in antigen presentation in higher eukaryotes. Legumain is also up-regulated in a number of human cancers, and recent studies suggest that it may play important functional roles in the process of tumorigenesis. However, detailed functional studies in relevant animal models of human disease have been hindered by the lack of suitably selective small molecule inhibitors and imaging reagents. Here we present the design, optimization, and in vivo application of fluorescently labeled activity-based probes (ABPs) for legumain. We demonstrate that optimized aza-peptidyl Asn epoxides are highly selective and potent inhibitors that can be readily converted into near-infrared fluorophore-labeled ABPs for whole body, noninvasive imaging applications. We show that these probes specifically label legumain in various normal tissues as well as in solid tumors when applied in vivo. Interestingly, addition of cell-penetrating peptides to the probes enhanced cellular uptake but resulted in increased cross-reactivity toward other lysosomal proteases as the result of their accumulation in lysosomes. Overall, we find that aza-peptidyl Asn ABPs are valuable new tools for the future study of legumain function in more complex models of human disease.
20668920
Dynamic light scattering optical coherence tomography.
We introduce an integration of dynamic light scattering (DLS) and optical coherence tomography (OCT) for high-resolution 3D imaging of heterogeneous diffusion and flow. DLS analyzes fluctuations in light scattered by particles to measure diffusion or flow of the particles, and OCT uses coherence gating to collect light only scattered from a small volume for high-resolution structural imaging. Therefore, the integration of DLS and OCT enables high-resolution 3D imaging of diffusion and flow. We derived a theory under the assumption that static and moving particles are mixed within the OCT resolution volume and the moving particles can exhibit either diffusive or translational motion. Based on this theory, we developed a fitting algorithm to estimate dynamic parameters including the axial and transverse velocities and the diffusion coefficient. We validated DLS-OCT measurements of diffusion and flow through numerical simulations and phantom experiments. As an example application, we performed DLS-OCT imaging of the living animal brain, resulting in 3D maps of the absolute and axial velocities, the diffusion coefficient, and the coefficient of determination.
18720184
Synthesis and evaluation of aza-peptidyl inhibitors of the lysosomal asparaginyl endopeptidase, legumain.
Legumain or asparaginly endopeptidase (AEP) is a lysosomal cysteine protease with a high level of specificity for cleavage of protein substrates after an asparagine residue. It is also capable of cleaving after aspartic acids sites when in the acidic environment of the lysosome. Legumain expression and activity is linked to a number of pathological conditions including cancer, atherosclerosis and inflammation, yet its biological role in these pathologies is not well-understood. Highly potent and selective inhibitors of legumain would not only be valuable for studying the functional roles of legumain in these conditions, but may have therapeutic potential as well. We describe here the design, synthesis and in vitro evaluation of selective legumain inhibitors based on the aza-asparaginyl scaffold. We synthesized a library of aza-peptidyl inhibitors with various non-natural amino acids and different electrophilic warheads, and characterized the kinetic properties of inactivation of legumain. We also synthesized fluorescently labeled inhibitors to investigate cell permeability and selectivity of the compounds. The inhibitors have second order rate constants of up to 5 × 10(4)M(-1)s(-1) and IC(50) values as low as 4 nM against recombinant mouse legumain. In addition, the inhibitors are highly selective toward legumain and have little or no cross-reactivity with cathepsins. Overall, we have identified several valuable new inhibitors of legumain that can be used to study legumain function in multiple disease models.
4611561
Protein structure prediction based on fragment assembly and parameter optimization.
We propose a novel method for ab-initio prediction of protein tertiary structures based on the fragment assembly and global optimization. Fifteen residue long fragment libraries are constructed using the secondary structure prediction method PREDICT, and fragments in these libraries are assembled to generate full-length chains of a query protein. Tertiary structures of 50 to 100 conformations are obtained by minimizing an energy function for proteins, using the conformational space annealing method that enables one to sample diverse low-lying local minima of the energy. Then in order to enhance the performance of the prediction method, we optimize the linear parameters of the energy function, so that the native-like conformations become energetically more favorable than the non-native ones for proteins with known structures. We test the feasibility of the parameter optimization procedure by applying it to the training set consisting of three proteins: the 10-55 residue fragment of staphylococcal protein A (PDB ID 1bdd), a designed protein betanova, and 1fsd.
35858545
Extended dosing of etanercept 25 mg can be effective in patients with ankylosing spondylitis: a retrospective analysis
As a measure of healthcare cost containment, the total number of vials of entanercept (25 mg) that can be prescribed for patients with inflammatory arthritis is restricted in Korea. Consequently, attempts to extend the dosing interval while maintaining the efficacy have not been an uncommon clinical practice. The aim of this study was to determine if extended doing interval of etanercept can be effective in patients with ankylosing spondylitis (AS). We performed a retrospective analysis using medical records at a single tertiary hospital. One hundred and nine patients with AS and 79 patients with rheumatoid arthritis (RA) started on etanercept between November 2004 and November 2009 were indentified. Etanercept (25 mg) was started with twice-weekly dosing schedule. Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), C-reactive protein (CRP), and etanercept dosing interval for AS patients at 0, 3, 9, 15, 21 months were reviewed. Dosing interval for RA patients was analyzed for comparison. In AS, mean dosing interval was 4.7 ± 2.1 days at 3 months and was increased to 12.1 ± 7.0 days at 21 months. Despite the progressive increase in the dosing interval, the mean BASDAI declined rapidly at 3 months, and continued to decrease over 21 months. Mean CRP declined after 3 months of therapy and remained low thereafter. In RA, mean dosing interval was 4.0 ± 1.2 days at 3 months and 5.1 ± 1.8 days at 21 months. In conclusion, in AS, extended dosing of etanercept can be effective without compromising clinical and laboratory markers of disease activity as measured by BASDAI and CRP, respectively. Tapering of etanercept was less accommodating in RA when compared to AS.