RESUMO
BACKGROUND: The effects of ankle osteoarthritis on gait are noticeable in the clinic, but are difficult to quantify and score without detailed kinematic and kinetic analysis. Evaluationof temporal gait parameters and gait variability is a potential alternative. RESEARCH QUESTION: This study aimed to determine associations between limb and gender with temporal gait parameters and gait variability in ankle OA patients to evaluate the utility of these parameters for gait assessment in a clinical setting. METHODS: Following informed consent, 242 end-stage unilateral ankle OA patients walked at self-selected speed across force plates. Means and stride-to-stride standard deviations (SD) of stride, swing, stance, and double support times were determined for each patient. Limb x Gender ANCOVA models co-varying for walking speed were run for swing and stance times, while stride and double support times were only compared between genders. Statistical analysis was performed in SPSS (α = 0.05). RESULTS: Walking speed affected all measures of interest. After adjusting for walking speed, mean stride time, stride time SD, and stance time SD were 3.5%, 67% and 29% higher among women than men (p = 0.002, 0.035 and 0.02 respectively). Swing time was 12% higher and stance time was 6% lower on the affected side compared to the unaffected side (p < 0.001 for both). SIGNIFICANCE: Women have longer stride times and higher variability, which may indicate higher fall risk. Both genders minimized loading on the affected limb by increasing swing time and reducing stance time on the affected side. Simple, easy to record temporal gait patterns can provide useful insight into gait abnormalities in patients with ankle OA.
Assuntos
Articulação do Tornozelo/fisiopatologia , Análise da Marcha/métodos , Marcha/fisiologia , Osteoartrite/fisiopatologia , Idoso , Fenômenos Biomecânicos , Feminino , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Fatores Sexuais , Caminhada/fisiologia , Velocidade de Caminhada/fisiologiaRESUMO
A quantitative reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed to detect the L1 subunit protein from virus-like particles (VLPs) of human papillomavirus (HPV). The method utilizes heat treatment with a buffer consisting of 50 mM Tris, pH 8.0 containing 8 M guanidine-HCl and 10% 2-mercaptoethanol to dissociate the VLPs into monomeric L1. Following dissociation, the sample is injected onto a C4 or C8 column. The L1 protein is eluted as a single, clearly resolved peak. Elution conditions have been optimized to enhance the separation of L1 from other contaminants. Based on spike recovery studies and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis this method is suitable for quantitation of various partially purified in-process samples and can be used to facilitate purification process development.
Assuntos
Papillomaviridae/isolamento & purificação , Vírus/isolamento & purificação , Western Blotting , Calibragem , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Humanos , Indicadores e Reagentes , Solventes , Proteínas Virais/isolamento & purificaçãoRESUMO
Solvent extraction is a very powerful purification step in the preparation of VAQTA, a highly purified, inactivated hepatitis A vaccine. Extraction of an aqueous product-containing protein solution with chloroform through vigorous shaking causes irreversible denaturation of contaminant proteins at the interface. However, the hepatitis A virus (HAV) remains viable and soluble in the aqueous phase. Because three phases (air, aqueous, and organic) are involved, and the mixing is carried out in individual bottles, there is very little theory available to characterize this process, so it must be studied experimentally. This extraction step was characterized by following the removal of a specific impurity from the aqueous phase as a representative marker for the degree of protein precipitation. These experiments led to the identification and optimization of the important variables controlling the extraction step. They were found to be mixing time and size of vessel, with longer mixing times resulting in higher purity and larger bottle size leading to faster kinetics of impurity removal. These parameters are most likely related to solvent/aqueous interfacial area and the resulting shear due to shaking. We conclude that, to scale up this type of mixing, the kinetics of impurity removal need to be determined experimentally for the systems and equipment under consideration.
RESUMO
The development of the purification process for VAQTA, which results in a highly purified inactivated hepatitis A vaccine, was driven by modifications in the cell-culture and harvest methods which permit hepatitis A virus propagation to support large-scale manufacture. The starting material for the purification was initially a concentrated cell pellet scraped from roller bottles. However, when the cell-culture method was scaled up to use high-surface-area Nunc cell factories or Costar cubes, the early steps in the process had to be modified to handle large volumes of dilute lysate. Membrane concentration was used at first, and a highly purified vaccine was prepared, but virus-poly(nucleic acid) complexes were formed, which reduced the yields in later processing steps. The introduction of a nuclease digestion immediately after harvest followed by capture chromatography on an anion-exchange column eliminated the formation of these complexes and resulted in more consistent performance and higher yields of downstream operations.
Assuntos
Desoxirribonucleases/metabolismo , Vírus da Hepatite A Humana/imunologia , Vacinas contra Hepatite Viral/isolamento & purificação , Sítios de Ligação , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Desoxirribonucleases/química , Filtração , Vacinas contra Hepatite A , Humanos , Ácidos Nucleicos/metabolismo , Especificidade por Substrato , Vacinas de Produtos Inativados/isolamento & purificaçãoRESUMO
Poly(ethylene glycol) precipitation has been successfully used to concentrate and purify hepatitis A virus from crude lysate preparations for production of VAQTA, a highly purified, formalin-inactivated hepatitis A vaccine. Initial results showed that nucleic acids present in the starting material were problematic for the performance of the poly(ethylene glycol) precipitation step. Extensive experiments were carried out to identify processing conditions suitable for vaccine manufacture which would enhance product yield and improve purity. Results of these studies indicated that the earlier practice of concentrating crude virus-containing lysate using semipermeable membranes led to aggregation of high molecular weight nucleic acids. This aggregated material coprecipitated with the virus during the subsequent poly(ethylene glycol) precipitation step; variable amounts of nucleic acids led to inconsistent virus recovery and product purity. Nuclease treatment of the crude lysate preparations decreased the molecular size of the nucleic acids and significantly reduced their coprecipitation with the virus. Further experiments demonstrated that optimal placement of the nuclease treatment was at the lysate stage followed by a capture step using anion exchange chromatography. These steps combined with optimization of the virus concentration, ionic strength, and pH of the poly(ethylene glycol) precipitation led to effective and selective concentration of the virus which significantly enhanced process reproducibility and control.
Assuntos
Vírus da Hepatite A Humana/isolamento & purificação , Polietilenoglicóis , Vacinas de Produtos Inativados/isolamento & purificação , Vacinas contra Hepatite Viral/isolamento & purificação , Precipitação Química , DNA Viral/química , Vacinas contra Hepatite A , Vírus da Hepatite A Humana/imunologia , Concentração de Íons de Hidrogênio , Concentração Osmolar , Cloreto de Sódio/química , Temperatura , Fatores de TempoRESUMO
The origin of the microheterogeneity of a highly purified antiinflammatory humanized monoclonal antibody prepared in mammalian cell culture has been investigated. This antibody is an IgG directed toward human CD18 (a subunit of leukocyte integrins). When the IgG preparation is subjected to isoelectric focusing, it is found to contain four major species with pI values ranging from 6 to 7. Although the relative amounts of each form differ and some species are present only in small quantities, each has been isolated by a combination of high-resolution anion-exchange chromatography and isoelectric focusing. Comparative studies reveal no detectable differences in overall secondary (far UV circular dichroism) or tertiary (intrinsic fluorescence) structure, molecular weight (laser-desorption mass spectroscopy), or antigen binding activity. When each of the isolated species is incubated under conditions which favor deamidation, it is converted to forms of lower pI which appear to correspond to naturally observed species. While the isolated light chain is relatively homogeneous, the heavy chain exhibits a pattern of isoelectric focusing bands similar to that of the intact immunoglobulin. These results suggest that in this case, charge microheterogeneity is due to the sequential deamidation of the immunoglobulin heavy chain.
Assuntos
Anticorpos Monoclonais/química , Antígenos CD/imunologia , Antígenos CD18 , Focalização IsoelétricaAssuntos
Envelhecimento/metabolismo , Química Encefálica , Glutamato-Amônia Ligase/metabolismo , Fígado/química , Proteínas/análise , Proteínas/metabolismo , Animais , Repressão Enzimática , Gerbillinae , Humanos , Oxirredução , Proteínas/química , Proteínas/isolamento & purificação , Ratos , Saccharomyces cerevisiae/enzimologiaRESUMO
During aging a number of enzymes accumulate as catalytically inactive or less active forms. The age-related changes in catalytic activity are due in part to reactions of the protein with "active" oxygen species such as ozone, singlet oxygen, or with oxygen free radicals as are produced during exposure to ionizing radiation or to metal ion catalyzed oxidation (MCO) systems. The levels of oxidized proteins in cultured human fibroblasts from individuals of various ages and in liver and brain extracts of rats of different ages increase progressively with age, and in old rats can represent 30-50% of the total cellular protein. The age-related increase in oxidized protein in rat liver and brain tissue is accompanied by a loss of glutamine synthetase (GS) and glucose-6-P dehydrogenase (G-6-PDH) activities, and to a decrease in the level of cytosolic neutral protease activity which is responsible for the degradation of oxidized (denatured) protein. Of particular significance are the results of experiments showing that similar age-related changes occur in the gerbil brain and that these changes are accompanied by a loss of short-term memory as measured by the radial arm maze technique. Chronic treatment (intraperitoneal injections) of old animals with the free radical spin-trap reagent, N-tert-butyl-alpha-phenylnitrone (PBN) resulted in normalization of the several biochemical parameters to those characteristic of the young animals; coincidentally, the short-term memory index was restored to the young animal values. These results provide the first evidence that there is likely a linkage between the age-dependent accumulation of oxidized enzymes and the loss of physiological function.
Assuntos
Envelhecimento/metabolismo , Proteínas/metabolismo , Animais , Encéfalo/enzimologia , Encéfalo/crescimento & desenvolvimento , Radicais Livres/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Glutamato-Amônia Ligase/metabolismo , Fígado/enzimologia , Fígado/crescimento & desenvolvimento , Modelos Biológicos , Oxirredução , RatosRESUMO
The relationship between Alzheimer disease (AD) and aging is not currently known. In this study, postmortem frontal- and occipital-pole brain samples were obtained from 16 subjects with AD, 8 age-matched controls, and 5 young controls. These samples were analyzed both for protein oxidation products (carbonyl) and the activities of two enzymes vulnerable to mixed-function oxidation, glutamine synthetase and creatine kinase. Glutamine synthetase is more sensitive to mixed-function oxidation than creatine kinase. Carbonyl content rises exponentially with age, at double the rate in the frontal pole compared with the occipital pole. Compared with young controls, both aged groups (AD and age-matched controls) have increased carbonyl content and decreased glutamine synthetase and creatine kinase activities, which are more marked in the frontal than occipital pole in all instances. We conclude that protein oxidation products accumulate in the brain and that oxidation-vulnerable enzyme activities decrease with aging in the same regional pattern (frontal more affected than occipital). However, only glutamine synthetase activity distinguishes AD from age-matched controls: Because glutamine synthetase activity is differentially reduced in the frontal pole in AD, we suggest that AD may represent a specific brain vulnerability to age-related oxidation.
Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Creatina Quinase/metabolismo , Glutamato-Amônia Ligase/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Adulto , Idoso , Envelhecimento , Doença de Alzheimer/enzimologia , Encéfalo/enzimologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Oxirredução , Valores de Referência , Análise de RegressãoRESUMO
Oxygen free radicals and oxidative events have been implicated as playing a role in bringing about the changes in cellular function that occur during aging. Brain readily undergoes oxidative damage, so it is important to determine if aging-induced changes in brain may be associated with oxidative events. Previously we demonstrated that brain damage caused by an ischemia/reperfusion insult involved oxidative events. In addition, pretreatment with the spin-trapping compound N-tert-butyl-alpha-phenylnitrone (PBN) diminished the increase in oxidized protein and the loss of glutamine synthetase (GS) activity that accompanied ischemia/reperfusion injury in brain. We report here that aged gerbils had a significantly higher level of oxidized protein as assessed by carbonyl residues and decreased GS and neutral protease activities as compared to young adult gerbils. We also found that chronic treatment with the spin-trapping compound PBN caused a decrease in the level of oxidized protein and an increase in both GS and neutral protease activity in aged Mongolian gerbil brain. In contrast to aged gerbils, PBN treatment of young adult gerbils had no significant effect on brain oxidized protein content or GS activity. Male gerbils, young adults (3 months of age) and retired breeders (15-18 months of age), were treated with PBN for 14 days with twice daily dosages of 32 mg/kg. If PBN administration was ceased after 2 weeks, the significantly decreased level of oxidized protein and increased GS and neutral protease activities in old gerbils changed in a monotonic fashion back to the levels observed in aged gerbils prior to PBN administration. We also report that old gerbils make more errors than young animals and that older gerbils treated with PBN made fewer errors in a radial arm maze test for temporal and spatial memory than the untreated aged controls. These data can be interpreted to indicate that oxidation of cellular proteins may be a critical determinant of brain function. Moreover, it also implies that there is an age-related increase in vulnerability of tissue to oxidation that can be modified by free radical trapping compounds.
Assuntos
Envelhecimento , Encéfalo/metabolismo , Glutamato-Amônia Ligase/metabolismo , Memória/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Óxidos de Nitrogênio/farmacologia , Marcadores de Spin , Animais , Comportamento Animal/efeitos dos fármacos , Óxidos N-Cíclicos , Radicais Livres , Gerbillinae , Masculino , Memória/efeitos dos fármacos , Metaloendopeptidases/metabolismo , Óxidos de Nitrogênio/química , OxirreduçãoAssuntos
Metais/metabolismo , Proteínas/metabolismo , Envelhecimento/metabolismo , Animais , Arteriosclerose/etiologia , Catarata/etiologia , Humanos , Inflamação/etiologia , Oxigenases de Função Mista/metabolismo , Doenças do Sistema Nervoso/etiologia , Oxirredução , Traumatismo por Reperfusão/etiologiaRESUMO
Myelinolysis occurs following rapid correction of hyponatremia in both humans and experimental animals. Although the mechanism of this effect at present is unknown, we have examined the possibility that a rapid rise in serum sodium following hyponatremia potentiates an oxidative stress and results in the oxidation of cellular proteins. In these studies, rats treated with 1 M NaCl following 3 days of vasopressin-induced hyponatremia exhibited myelinolysis in the corpus striatum and thalamus as well as significant increases in soluble oxidized proteins in the brain. These changes did not occur in rats treated with 0.155 M (0.9%) NaCl following 3 days of hyponatremia.
Assuntos
Encéfalo/metabolismo , Hiponatremia/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Cloreto de Sódio/farmacologia , Animais , Arginina Vasopressina , Encéfalo/patologia , Hiponatremia/induzido quimicamente , Hiponatremia/patologia , Masculino , Bainha de Mielina/ultraestrutura , Especificidade de Órgãos , Oxirredução , Potássio/sangue , Ratos , Ratos Endogâmicos , Valores de Referência , Sódio/sangue , VasopressinasRESUMO
Free radical-mediated oxidative damage has been implicated in tissue injury resulting from ischemia/reperfusion events. Global cortical ischemia/reperfusion injury to Mongolian gerbil brains was produced by transient occlusion of both common carotid arteries. Protein oxidation, as measured by protein carbonyl content, increased significantly during the reperfusion phase that followed 10 min of ischemia. The activity of glutamine synthetase, an enzyme known to be inactivated by metal-catalyzed oxidation reactions, decreased to 65% of control levels after 2 hr of reperfusion that followed 10 min of ischemia. We also report that the free radical spin trap N-tert-butyl-alpha-phenylnitrone [300 mg/kg (body weight)] administered 60 min before ischemia/reperfusion is initiated, partially prevents protein oxidation and protects from loss of glutamine synthetase activity. In addition, we report a N-tert-butyl-alpha-phenylnitrone-dependent nitroxide radical obtained in the lipid fraction of the ischemia/reperfusion-lesioned brains, but there was very little radical present in the comparable sham-operated control brains. These data strengthen the previous observation utilizing in vivo-trapping methods, that free radical flux is increased during the reperfusion phase of the ischemia-lesioned gerbil brain. The loss of glutamine synthetase would be expected to increase the levels of brain L-glutamate. Thus, the oxidative inactivation of glutamine synthetase may be a critical factor in the neurotoxicity produced after cerebral ischemia/reperfusion injury.
Assuntos
Córtex Cerebral/metabolismo , Glutamato-Amônia Ligase/metabolismo , Ataque Isquêmico Transitório/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres , Gerbillinae , Masculino , Oxirredução , Valores de ReferênciaAssuntos
Proteínas Sanguíneas/metabolismo , Proteínas de Neoplasias/metabolismo , Neutrófilos/fisiologia , Células Tumorais Cultivadas/fisiologia , Proteínas Sanguíneas/análise , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Leucemia Promielocítica Aguda , Proteínas de Neoplasias/análise , Oxirredução , Fenil-Hidrazinas , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Previous studies in this laboratory have shown that glutamine synthetase (GS) and other key metabolic enzymes are inactivated by metal-catalyzed oxidation reactions in vitro. Oxidative inactivation renders these proteins highly susceptible to proteolysis, especially to a class of newly identified alkaline proteases which exhibit little or no activity against the native enzymes. These studies have suggested that oxidative inactivation may be an important marking step for intracellular protein degradation. Because many of the enzymes which have been shown to accumulate as inactive or less active forms during aging are readily inactivated by metal-catalyzed oxidation reactions in vitro, we have investigated the possible relationship between protein oxidation and proteolysis during aging and oxidative stress in vivo. Oxidized proteins accumulate in hepatocytes of rats exposed to 100% oxygen during the first 48 h of oxygen treatment. In the interval between 48 and 54 h the levels of oxidized proteins decline sharply. The specific activities of at least two liver enzymes, glutamine synthetase and glucose-6-phosphate dehydrogenase (G-6-PDH), decrease during the 54-h experiment. GS and G-6-PDH specific immunological cross-reactivity remains high during the first 48 h of oxygen treatment and then declines in the interval between 48 and 54 h. During this same interval the levels of alkaline proteases which degrade oxidized proteins increase, indicating that these activities are induced or activated in response to oxidative stress and subsequently degrade the proteins which have become oxidized during the initial phase of oxygen treatment. Oxidized proteins accumulate progressively during aging in hepatocytes from rats 3 to 26 months old, with the largest incremental increase between 20 and 26 months. The increase in protein oxidation is correlated with a loss of specific activity of GS and G-6-PDH without a concomitant loss of immunological cross-reactivity. The levels of alkaline proteases which degrade oxidized proteins in hepatocytes from 26-month-old rats is only 20% that of 3-month-old rats, suggesting that oxidized proteins accumulate in hepatocytes from old rats, in part, because the proteases which degrade them are deficient or defective. moreover, when old rats are subjected to treatment with 100% oxygen, the levels of oxidized proteins continue to increase and the alkaline protease activity remains low, indicating that these protease activities are not increased in response to oxidative stress in old rats.
Assuntos
Fígado/crescimento & desenvolvimento , Oxigênio/farmacologia , Peptídeo Hidrolases/metabolismo , Proteínas/metabolismo , Envelhecimento , Animais , Células Cultivadas , Glucosefosfato Desidrogenase/metabolismo , Glutamato-Amônia Ligase/metabolismo , Cinética , Fígado/efeitos dos fármacos , Fígado/metabolismo , NADP/metabolismo , Oxirredução , Peptídeo Hidrolases/isolamento & purificação , Ratos , Valores de ReferênciaAssuntos
Enzimas/metabolismo , Fígado/enzimologia , Oxigenases de Função Mista/metabolismo , Oxigênio/toxicidade , Envelhecimento , Animais , Glucosefosfato Desidrogenase/metabolismo , Glutamato-Amônia Ligase/metabolismo , Cinética , Fígado/efeitos dos fármacos , Fígado/crescimento & desenvolvimento , Oxirredução , RatosRESUMO
It is evident from the results summarized here that a variety of MFO systems catalyze the oxidation inactivation of enzymes. This likely involves site-directed Fenton-chemistry in which Fe(II) bound to metal binding sites on the protein undergoes peroxidation to form active oxygen species that convert proximal amino acid residues to carbonyl derivatives. Such oxidation is likely involved in the accumulation of altered enzymes during aging, in premature aging diseases, in the killing of bacteria by neutrophils and in protein turnover. In view of these results, the possibility that protein oxidation is implicated in various diseases, viz, arthritis, pulmonary dysfunction, and carcinogenesis deserves consideration.