RESUMO
Cerebral beta-amyloidosis is a central part of the neuropathology of Alzheimer's disease (AD). Quantitation of beta-amyloid plaques in the human AD brain, and in animal models of AD, is an important study endpoint in AD research. Methodologic approaches to the measurement of beta-amyloid in the brain vary between investigators, and these differences affect outcome measures. Here, one quantitative approach to the measurement of beta-amyloid plaques in brain sections was analyzed for sources of variability due to sampling. Brain tissue was from homozygous APP(V717F) transgenic male mice. Sampling variables were at the mouse and microscopic slide and field levels. Results indicated that phenotypic variability in the mouse sample population was the largest contributor to the standard error of the analyses. Within each mouse, variability between slides or between fields within slides had smaller effects on the error of the analyses. Therefore, when designing studies of adequate power, in this and in other similar models of cerebral beta-amyloidosis, sufficient numbers of mice per group must be included in order for change in mean plaque burden attributable to an experimental variable to outweigh phenotypic variability.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/análise , Interpretação Estatística de Dados , Hipocampo/patologia , Processamento de Imagem Assistida por Computador/métodos , Placa Amiloide/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Benzotiazóis , Contagem de Células/métodos , Modelos Animais de Doenças , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Processamento de Imagem Assistida por Computador/instrumentação , Masculino , Camundongos , Camundongos Transgênicos/anatomia & histologia , Camundongos Transgênicos/genética , Camundongos Transgênicos/metabolismo , Microscopia de Fluorescência , Placa Amiloide/genética , Placa Amiloide/metabolismo , Reprodutibilidade dos Testes , Distribuições Estatísticas , Tiazóis/farmacocinéticaAssuntos
Ácido Micofenólico/metabolismo , Animais , Bile/análise , Isótopos de Carbono , Células Cultivadas , Resistência a Medicamentos , Fezes/análise , Glucuronatos/metabolismo , Glucuronidase/metabolismo , Nucleotídeos de Guanina/metabolismo , Haplorrinos , Nucleotídeos de Inosina , Cetona Oxirredutases/antagonistas & inibidores , Células L , Camundongos , Camundongos Endogâmicos , Ácido Micofenólico/análise , Ácido Micofenólico/sangue , Ácido Micofenólico/farmacologia , Ácido Micofenólico/urina , Neoplasias Experimentais/enzimologia , Pentosefosfatos , Pentosiltransferases/metabolismo , Peptídeo Sintases/antagonistas & inibidores , Ratos , Ratos EndogâmicosRESUMO
The fatty acid cyclooxygenase (EC 1.14.99.1) that produces the prostaglandin, thromboxane, and prostacyclin precursor (PGH2), was solubilized from human platelet microsomes in 20 sucrose and 1.0% Triton X-100. The enzyme was purified 300-fold by electrofocusing, Sephadex G-200 gel filtration, and hydrophobic chromatography on ethyl agarose. The cyclooxygenase catalyzed the conversion of arachidonic acid to prostaglandin endoperioxide, PGH2, that was trapped at -25 degrees C and separated on TLC at -20 degrees C. PGH2 was hydrolyzed to HHT in acidic pH, or was chemically converted to PGE2 in slightly alkaline pH in the absence of cofactors. The enzyme showed a broad pH optimum in the range of 7-9. Hemin containing substances such as methemoglobin were absolutely required as cofactors, while tryptophan, epinephrine, phenol, and hydroquinone stimulated the PGH2 formation. Metal ions, such as ZN2+ and Cd2+ inhibited the enzyme reaction at 0.1 to 1 mM. The molecular weight of the purified enzyme was estimated at 79,432 by sodium dodecyl sulfate disc gel electrophoresis at pH 8.0. The properties of the human platelet enzyme was generally similar to the sheep vesicular enzyme in the method of solubilization, pH optimum, and molecular weight.
Assuntos
Plaquetas/enzimologia , Prostaglandina-Endoperóxido Sintases/isolamento & purificação , Cromatografia em Agarose , Cromatografia em Gel , Eletroforese Descontínua , Humanos , Focalização Isoelétrica , Peso Molecular , Prostaglandina-Endoperóxido Sintases/sangue , Prostaglandinas H/metabolismo , UltrafiltraçãoRESUMO
This report describes a microprocedure that may be used for direct measurement of proteoglycans and glycosaminoglycans, after chromatographic elution with chaotropic reagents. The assay is based on the ability of the sulfated glycosaminoglycans to bind to the cationic dye, dimethylmethylene blue, in solution. Inclusion of guanidinium chloride (0.24 M) in the assay resulted in a stable dye-proteoglycan interaction, but eliminated the interference of other anionic macromolecules such as DNA. The assay is rapid, sensitive, and reproducible and therefore useful for processing several samples. Finally, the procedure can be used for quantitative determination of several types of proteoglycans and glycosaminoglycans.
Assuntos
Glicosaminoglicanos/análise , Proteoglicanas/análise , Animais , DNA , Guanidina , Guanidinas , Humanos , Azul de Metileno/análogos & derivados , Microquímica/métodosRESUMO
A rapid and sensitive assay method for 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-coA reductase, EC 1.1.1.34) is described. HMG-coA reductase is demonstrated in dog liver microsomes, and the converted reaction product has been identified as mevalonolactone. The enzyme activity undergoes cyclic variation and increases by more than tenfold 5 h after feeding. The properties of dog liver enzyme are generally similar to the rat liver enzyme in the method of solubilization, cold inactivation, pH optimum, and Km values.