Statistical aspects of quantitative image analysis of beta-amyloid in the APP(V717F) transgenic mouse model of Alzheimer's disease.

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.

Decreased p38 MAPK activity in end-stage failing human myocardium: p38 MAPK alpha is the predominant isoform expressed in human heart.

Short duration exposure to cellular stresses have been shown to activate p38 mitogen-activated protein kinase (MAPK) in cultured rat ventricular cardiomyocytes and isolated perfused hearts; however, effects of chronic stress on p38 MAPK are not well understood. This study determined whether alterations in the p38 MAPK pathway occurred prior to end-stage human heart failure. The p38 MAPK alpha isoform was detectable in human cardiac tissue. However, carefully controlled analysis of protein and message in this study demonstrated an absence of the p38 MAPK beta -isoform. Low levels of message for the non-SB203580 sensitive p38 MAPK gamma and delta isoforms were also detected in both normal and failing human myocardium. Ischemic and idiopathic end-stage failing human hearts were compared to non-failing hearts for both p38 alpha MAPK protein level and total p38 MAPK activity. Western blotting techniques demonstrated no significant changes in total p38 alpha MAPK content. However, approximately 75% decreases in active/phosphorylated p38 MAPK (P<0.005) were observed in both ischemic and idiopathic failing hearts compared to non-failing hearts. In-gel kinase assays confirmed that activated p38 MAPK, detected by Western blotting, phosphorylated its potential downstream targets. When compared to non-failing hearts, approximately 46% decreases in p38 MAPK phosphorylation of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK-2) were observed in ischemic and idiopathic failing hearts (P=0.03 and P=0.04 respectively). Active p38 MAPK was localized to sarcomeric structures in the cytosol of myocytes by confocal immunofluorescence microscopy. The correlation between decreased MAPKAPK-2 phosphorylation and loss of active p38 MAPK in failing human myocytes suggests that decreases in the activation of p38 MAPK alpha, the predominant cardiac isoform, occur prior to end-stage heart failure.

Protein kinase C-alpha and -epsilon modulate connexin-43 phosphorylation in human heart.

We have previously demonstrated that protein kinase C (PKC)- alpha expression is significantly elevated in failing human left ventricle, with immunostaining showing increased PKC- alpha localization at the intercalated disks of cardiomyocytes. In the present study we sought to determine, in the failing heart, if PKC- alpha interacted with connexin-43 (Cx-43) both spatially and functionally, and to compare the association of PKC- alpha/Cx-43 with that of PKC- epsilon, a PKC isozyme that does not significantly increase in failing hearts. The possibility of a PKC- alpha or PKC- epsilon/Cx-43 association in non-failing hearts was also investigated. Co-immunoprecipitation of PKC- alpha or PKC- epsilon and Cx-43 in non-failing and failing left ventricle was achieved using antibodies to PKC- alpha or Cx-43. Confocal microscopy confirmed that PKC- alpha distribution within the cardiomyocyte included co-localization with connexin-43 in both failing and non-failing myocardium. In a similar manner, confocal imaging of PKC- epsilon showed cardiomyocyte distribution in both cytosol and membrane, and colocalization of PKC- epsilon with Cx-43. Recombinant PKC- alpha or - epsilon increased PKC activity significantly above endogenous levels in the co-immunoprecipitated Cx-43 complexes (P<0.05). However, phosphorylation of purified human Cx-43 (isolated from failing human left ventricle) by recombinant PKC- alpha or PKC- epsilon resulted in only PKC- epsilon mediated Cx-43 phosphorylation. Thus, in the human heart PKC- alpha, PKC- epsilon, and Cx-43 appear to form a closely associated complex. Whereas only PKC- epsilon directly phosphorylates Cx-43, both PKC isoforms result in increased phosphorylation within the Cx-43 co-immunoprecipitated complex.

Preattentive and attentive visual search in individuals with hemispatial neglect.

Preattentive and attentive visual processing was examined in patients with hemispatial neglect, hemispatial neglect with hemianopia, and control participants. In the preattentive search task, targets possessed a unique feature that was not shared by distractors. In the attentive search task, targets lacked a feature that was present in the distractors. Preattentive search was normal in 3 neglect patients with cortical lesions but not in 2 neglect patients with hemianopia. A 4th neglect patient without hemianopia with a subcortical infarct abnormally used serial search mechanisms in the preattentive task. Neglect patients were characteristically impaired in the contralesional field in the attentive search task. This study demonstrates preserved explicit detection of visual features in cases of hemispatial neglect.

Tibenelast, 5,6-diethoxybenzo(B)thiophene-2-carboxylic acid, sodium salt (LY186655), is an orally active anti-asthma compound in the guinea pig.

Anaphylactic shock was induced in actively sensitized guinea pigs by free inhalation of a high dose of ovalbumin (10 mg/ml) aerosol. Tibenelast (LY186655), 5,6-diethoxybenzo(b)-thiophene-2-carboxylic acid, sodium salt, proved to be a potent orally active compound against anaphylactic shock induced by high dose antigen aerosol. When a lower aerosol challenge (0.05 mg/ml) was employed, bronchoconstriction was observed with a concomitant increase in lung resistance (RL) and a fall in dynamic compliance (Cdyn). Tibenelast at 25 mg/kg p.o. prevented these changes. Tibenelast was 10 times more potent than aminophylline by i.v. administration; normalization of pulmonary function was achieved at 1 mg/kg i.v. Tibenelast was synergistic with epinephrine. Combination of no-effect doses of epinephrine (0.025 mg/kg s.c.) and tibenelast (0.1 mg/kg i.v.) normalized pulmonary function. The oral dose response curve of tibenelast was enhanced with the co-administration of epinephrine. These data suggest that tibenelast may act at a site different from that of epinephrine, although the mechanism of action of tibenelast is unclear at present. Tibenelast may be of significant value in the treatment of asthma and other respiratory diseases.

Microdetermination of proteoglycans and glycosaminoglycans in the presence of guanidine hydrochloride.

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.

Purification and characterization of fatty acid cyclooxygenase from human platelets.

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.

Dog liver 3-hydroxy-3-methylglutaryl coenzyme A reductase.

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.