Specificity of biochemical and biophysical tests in Duchenne and myotonic muscular dystrophy, carrier states, and congenital myotonia.
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Adult↗
Biomedical subjects
D A Butterfield
Publications and source records attributed to D A Butterfield.
A comparative electron spin resonance study of the erythrocyte membrane in myotonic muscular dystrophy.
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Cell Membrane↗
Electron spin resonance studies of erythrocytes from patients with myotonic muscular dystrophy.
Electron magnetic resonance experiments have demonstrated that spin-labeled myotonic erythrocyte membranes have spectra that are recognizably different from those of normal erythrocytes. The spin label incorporated in the erythrocyte membranes of patients having myotonic muscular dystrophy is apparently located in a less polar and somewhat more fluid region than the label in a normal membrane. Although the mechanisms of molecular interaction and their relationship to enzymatic differences is unclear, the results lend confirmation to the suggestion that myotonic muscular dystrophy may be a disease resulting from a basic membrane abnormality.
Cell Membrane↗
Protective effect of carnosine during nitrosative stress in astroglial cell cultures.
Formation of nitric oxide by astrocytes has been suggested to contribute, via impairment of mitochondrial function, to the neurodegenerative process. Mitochondria under oxidative stress are thought to play a key role in various neurodegenerative disorders; therefore protection by antioxidants against oxidative stress to mitochondria may prove to be beneficial in delaying the onset or progression of these diseases. Carnosine has been recently proposed to act as antioxidant in vivo. In the present study, we demonstrate its neuroprotective effect in astrocytes exposed to LPS- and INFgamma-induced nitrosative stress. Carnosine protected against nitric oxide-induced impairment of mitochondrial function. This effect was associated with decreased formation of oxidatively modified proteins and with decreased up-regulation oxidative stress-responsive genes, such as Hsp32, Hsp70 and mt-SOD. Our results sustain the possibility that carnosine might have anti-ageing effects to brain cells under pathophysiological conditions leading to degenerative damage, such as aging and neurodegenerative disorders.
Animals↗
Time-dependence of, and effects of inhibition and cellular aging on, chloride efflux across erythrocyte membranes in Huntington's disease.
Previous studies have demonstrated an increased rate of chloride transport across erythrocyte membranes in Huntington's disease, a process regulated at the external side of the major transmembrane protein Band 3. A marked effect of time was noted in 3 Huntington's disease samples that were studied more than 4 h after obtaining the blood. In order to study anion transport more closely in Huntington's disease and the apparent time-dependence of chloride efflux in this disorder, we have performed several sets of experiments. Chloride transport in Huntington's disease erythrocytes was found to be extremely sensitive to time with the efflux rate constant decreasing by approximately 30% over a 24 h period. Chloride transport in control cells was unaffected by time. Inhibition studies with the specific anion transport blocker 4,4'-diisothiocyanto-2,2'-disulfonic acid stilbene (DIDS) demonstrated that the same degree of inhibition of chloride transport could be achieved at a much lower concentration of DIDS in Huntington's disease than in controls. Comparison of chloride efflux in fractions enriched in young and old erythrocytes, respectively, obtained by density centrifugation of fresh blood, demonstrated that only in the young fraction of cells was chloride efflux diminished with time in Huntington's disease. Chloride transport in in vivo aged Huntington's cells or both young and old control cells was essentially not dependent on time. These results are discussed in terms of proposed molecular mechanisms for neuronal loss in this disorder. The alterations in chloride efflux in extraneural erythrocytes are consistent with a proposed cell-surface membrane defect involving a protein in Huntington's disease.
Adult↗
In vitro and in vivo oxidative stress associated with Alzheimer's amyloid beta-peptide (1-42)
The amyloid beta-peptide (A beta)-associated free radical oxidative stress model for neuronal death in Alzheimer's disease (AD) brain predicts that neuronal protein oxidation is a consequence of A beta-associated free radicals [8]. In this study we have used both in vitro and in vivo models of beta-amyloid (A beta) toxicity to detect free radical induced oxidative stress by the measure of protein carbonyl levels. These model systems employed cultured hippocampal neurons exposed to exogenous synthetic A beta(1-42) and transgenic Caenorhabditis elegans (C. elegans) animals expressing A beta(1-42). We also investigated the importance of the A beta(1-42) Met35 residue for free radical formation in peptide solution and for peptide-induced protein oxidation and neuronal toxicity in these model systems. A beta(1-42) in solution yielded an EPR spectrum, suggesting that free radicals are associated with this peptide; however, neither the reverse [A beta(42-1)] nor methionine-substituted peptide [A beta(1-42)Met35Nlc] gave significant EPR spectra, suggesting the importance of the methionine residue in free radical formation. A beta(1-42) addition to cultured hippocampal neurons led to both neurotoxicity (30.1% cell death, p < 0.001) and increased protein oxidation (158% of controls, p < 0.001). and both of those effects were not observed with reverse or Met35Nle substituted peptides. C. elegans transgenic animals expressing human A beta(1-42) also had significantly increased in vivo protein carbonyls (176% of control animals, p < 0.001), consistent with our model. In contrast, transgenic animals with a Met35cys substitution in A beta(1-42) showed no increased protein carbonyls in vivo, in support of the hypothesis that methionine is important in A beta-associated free radical oxidative stress. These results are discussed with reference to the A beta-associated free radical oxidative stress model of neurotoxicity in AD brain.
Alzheimer Disease↗
Spin labeling and kinetic studies of a membrane-immobilized proteolytic enzyme.
Membrane-based bioreactors can greatly influence the rate and extent of chemical reactions and consequently lower the costs associated with the corresponding engineering processes. However, in order to progress in this area, greater understanding of the relationship of the structure and function of bioreactor systems is required. In this study, a proteolytic enzyme, papain (EC 3.4.22.2), was covalently coupled onto the surface of a vinyl alcohol/vinyl butyral copolymer (PVB) membrane employing either glutaraldehyde (GA) or 1,1'-carbonyldiimidazole (CDI). Various kinetic and performance properties of the immobilized papain were studied. It was found that these characteristics of the membrane-bound papain depended on the immobilization method. The CDI-immobilized papain bioreactor was used, although the apparent Michaelis constant, Km, of the CDI-immobilized papain was larger than that of the GA-immobilized enzyme. In separate experiments, a six-carbon spacer was also used between the membrane support and the covalently-linked enzyme. It was found that the insertion of the spacer reduced the disturbance of the enzyme system, resulting in a decreased Km, which was now closer to the value for the free enzyme. Electron paramagnetic resonance (EPR) techniques of spin labeling were used for the first time to examine the conformational change and the active site structure of an enzyme covalently immobilized to a membrane. The structural changes of the active site of papain upon immobilization with and without a spacer were in agreement with the functional properties of the enzyme.
Binding Sites↗
Kinetics and active fraction determination of a protease enzyme immobilized on functionalized membranes: mathematical modeling and experimental results.
A detailed study on the performance of a membrane bioreactor is presented, considering diffusion reaction models with product adsorption and structure-function correlations. The enzyme papain was utilized for experimental investigation both in the homogeneous state and on a modified polysulfone (MPS) membrane. Variation of enzyme loading on the membrane and enzyme concentration in the solution state depicted decreasing activity with increasing loading. The performance of the bioreactor was simulated using a diffusion reaction model within a recirculation loop. Electron paramagnetic resonance (EPR) spectroscopy was utilized to study the conformational changes of the active site of papain immobilized on the MPS membrane. Two models were applied to correlate the structure and function of the biocatalyst, based on loading (kinetics) and EPR (structure). The active fractions, lambda, determined from the two models were 0.29 and in the range 0.25-0.3, respectively. The intrinsic kinetics (Vmax) for the immobilized enzyme as determined by the correlations were in the range 101-121 micromol/(g.min), compared to 111 micromol/(g.min) for the homogeneous enzyme. This proves that the immobilized enzyme kinetics do approach homogeneous kinetics for papain on the MPS membrane, when corrected for adsorption and conformational changes.
Catalysis↗
Electron paramagnetic resonance studies of the effects of tri-n-butyltin on the physical state of proteins and lipids in erythrocyte membranes.
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Blood Proteins↗
Effects of beta-(N-methylamino)-L-alanine on cytoskeletal proteins of erythrocyte membranes.
The interaction of the amino acid beta-(N-methylamino)-L-alanine, a neurotoxin found in the seed of the false sago palm, with erythrocyte membranes has been monitored by electron paramagnetic resonance techniques of spin labeling. This neurotoxin did not alter the motion or order of bilayer lipids, but a highly-significant and dose-dependent alteration in the physical state of cytoskeletal proteins was observed. These results are discussed in reference to potential mechanisms involved in the neurotoxicity produced by beta-(N-methylamino)-L-alanine and in reference to the unusual neurological disorders among the Chamorro population of Guam and other Marianas Islands.
Amino Acids, Diamino↗
Nitrosative stress, cellular stress response, and thiol homeostasis in patients with Alzheimer's disease.
Alzheimer's disease (AD) is a neurodegenerative disorder with cognitive and memory decline, personality changes, and synapse loss. Increasing evidence indicates that factors such as oxidative and nitrosative stress, glutathione depletion, and impaired protein metabolism can interact in a vicious cycle, which is central to AD pathogenesis. In the present study, we demonstrate that brains of AD patients undergo oxidative changes classically associated with a strong induction of the so-called vitagenes, including the heat shock proteins (HSPs) heme oxygenase-1 (HO-1), HSP60, and HSP72, as well as thioredoxin reductase (TRXr). In inferior parietal brain of AD patients, a significant increase in the expression of HO-1 and TRXr was observed, whereas HO-2 expression was decreased, compared with controls. TRHr was not increased in AD cerebellum. Plasma GSH was decreased in AD patients, compared with the control group, and was associated with a significant increase in oxidative stress markers (i.e., GSSG, hydroxynonenal, protein carbonyl content, and nitrotyrosine). In AD lymphocytes, we observed an increased expression of inducible nitric oxide synthase, HO-1, Hsp72, HSP60, and TRXr. Our data support a role for nitrative stress in the pathogenesis of AD and indicate that the stress-responsive genes, such as HO-1 and TRXr, may represent important targets for novel cytoprotective strategies.
Aged↗
Redox modulation of heat shock protein expression by acetylcarnitine in aging brain: relationship to antioxidant status and mitochondrial function.
There is significant evidence to show that aging is characterized by a stochastic accumulation of molecular damage and by a progressive failure of maintenance and repair processes. Protective mechanisms exist in the brain which are controlled by vitagenes and include members of the heat shock system, heme oxygenase-I, and Hsp70 as critical determinants of brain stress tolerance. Given the broad cytoprotective properties of the heat shock response, molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. Acetyl-L-carnitine is proposed as a therapeutic agent for several neurodegenerative disorders, and the present study reports that treatment for 4 months of senescent rats with acetyl-L-carnitine induces heme oxygenase-1 as well as Hsp70 and SOD-2. This effect was associated with upregulation of GSH levels, prevention of age-related changes in mitochondrial respiratory chain complex expression, and decrease in protein carbonyls and HNE formation. We hypothesize that maintenance or recovery of the activity of vitagenes may delay the aging process and decrease the risk of age-related diseases. Particularly, modulation of endogenous cellular defense mechanisms via acetyl-L-carnitine may represent an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration.
Acetylcarnitine↗
Oxidation of cytosolic proteins and expression of creatine kinase BB in frontal lobe in different neurodegenerative disorders.
The presence of the biomarkers of oxidative damage, protein carbonyl formation and the inactivation of oxidatively sensitive brain creatine kinase (CK BB, cytosolic isoform), were studied in frontal lobe autopsy specimens obtained from patients with different age-related neurodegenerative diseases: Alzheimer's disease (AD), Pick's disease (PkD), diffuse Lewy body disease (DLBD), Parkinson's disease (PD), and age-matched control subjects. The CK activity was significantly reduced in the frontal lobe of AD, PkD and DLBD subjects, and CK BB-specific mRNA was significantly reduced in AD and DLBD. Protein carbonyl content was significantly increased in AD, PkD and DLBD. The results of this study confirm that the presence of biomarkers of oxidative damage is related to the presence of histopathological markers of neurodegeneration. Our data suggest that oxidative damage contributes to the development of the symptoms of frontal dysfunction in AD, PkD and DLBD. The development of frontal dysfunction in idiopathic PD might be secondary to oxidative damage and neuronal loss primarily located in the nigrostriatal system. The results of CK BB expression analysis demonstrate that the loss of the isoenzyme in different neurodegenerative diseases is likely the consequence of its posttranslational modification, possibly oxidative damage. Changes in CK BB expression may be an early indicator of oxidative stress in neurons.
Aged↗
Water transport across red-cell membranes following reductive methylation of the major transmembrane protein, Band 3: implications to increased divalent cation membrane permeability.
1H-T-NMR methods in conjunction with normally membrane-impermeable Mn2+ were used to study the effect of reductive methylation of specific lysine residues of Band 3, the major transmembrane protein, on water permeability. At 21 degrees C, the water apparent transverse relaxation time (T2) was decreased by nearly 16% (p less than .00001) for cells with modified Band 3. Atomic absorption measurements of control and methylated cells showed an increased level of Mn2+ in the erythrocyte cytosol following methylation. This increased level of this paramagnetic relaxation agent is sufficient to relax interior water protein to the values obtained. Thus, following specific methylation of band 3, increased membrane permeability to divalent cations is observed. The results are discussed with reference to possible conformation changes of Band 3 following methylation, and the findings are interpreted be mean that the conformation of Band 3 has influence on cation permeability to erythrocyte membranes.
Anion Exchange Protein 1, Erythrocyte↗