Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes. As N-hydroxysuccinimidyl ester dyes, the Alexa Fluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5 dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction coefficients. However, both Alexa Fluor dyes were significantly more resistant to photobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugates prepared from these dyes showed prominent blue-shifted shoulder peaks for conjugates of the Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. The anomalous peaks, previously observed for protein conjugates of the Cy5 dye, are presumably due to the formation of dye aggregates. Absorption of light by the dye aggregates does not result in fluorescence, thereby diminishing the fluorescence of the conjugates. The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited significantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyes were significantly more fluorescent than those of the Cy dyes, especially at high degrees of labeling. The results from our flow cytometry, immunocytochemistry, and immunohistochemistry experiments demonstrate that protein-conjugated, long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and other long-wavelength dyes in typical fluorescence-based cell labeling applications.

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The effects of dietary cholesterol on brain amyloid precursor protein (APP) processing were examined using an APP gene-targeted mouse, genetically humanized in the amyloid ␤-peptide (A␤) domain and expressing the Swedish familial Alzheimer's disease mutations. These mice express endogenous levels of APP holoprotein and abundant human A␤. Increased dietary cholesterol led to significant reductions in brain levels of secreted APP derivatives, including sAPP␣, sAPP␤, A␤1-40, and A␤1-42, while having little to no effect on cellassociated species, including full-length APP and the COOH-terminal APP processing derivatives. The changes in levels of sAPP and A␤ in brain all were negatively correlated with serum cholesterol levels and levels of serum and brain apoE. These results demonstrate that secreted APP processing derivatives and A␤ can be modulated in the brain of an animal by diet and provide evidence that cholesterol plays a role in the modulation of APP processing in vivo. APP gene-targeted mice lacking apoE, also have high serum cholesterol levels but do not show alterations in APP processing, suggesting that effects of cholesterol on APP processing require the presence of apoE.Alzheimer's disease (AD) 1 pathology includes extracellular amyloid deposits, intracellular neurofibrillary tangles, synaptic loss, and neuronal death (for a review, see Ref. 1). Alterations in the production or processing of APP have been implicated in the etiology of at least some forms of AD (2, 3). Multiple pathways for APP processing have been described, including a nonamyloidogenic pathway in which a putative ␣-secretase cleaves within the A␤ domain (4, 5), resulting in the formation of a secreted NH 2 -terminal fragment, sAPP␣, and a cell-associated 9-kDa COOH-terminal derivative. Another fraction of APP is processed along an amyloidogenic pathway in which cleavage by a putative ␤-secretase at the NH 2 terminus of the A␤ domain results in the formation of a secreted NH 2 -terminal fragment, sAPP␤ (6), and a cell-associated 12-kDa COOH-terminal derivative that may be the immediate precursor of A␤ (7,8). Cleavage of APP by both ␤-secretase and ␥-secretase results in formation of A␤, 40 or 42 amino acids in length (9, 10), that is found deposited in extracellular amyloid plaques in the AD brain (1, 11).In order to elucidate mechanisms of APP processing and A␤ generation in vivo, an animal model was developed by gene targeting that converted the mouse A␤ sequence to human and incorporated the Swedish familial Alzheimer's disease mutations (12). Enhanced amyloidogenic APP processing by the Swedish mutations, resulting in higher level A␤ production has been well documented in cell culture systems (13-15) and in the APP gene-targeted mice (12). These mice are well suited for investigating modulation of APP processing in vivo, because brain A␤ levels are nearly 10-fold above normal endogenous levels, thereby reducing the stringency for assays to detect A␤, particularly for the less abundant but more amyloidogenic 42-residue form. Further...

The role of oxidative damage in neurodegenerative disease was investigated in mice lacking cytoplasmic Cu/Zn superoxide dismutase (SOD) , created by deletion of the SOD1 gene (SOD1 ؊/؊ ). SOD1 ؊/؊ mice developed a chronic peripheral hindlimb axonopathy. Mild denervation of muscle was detected at 2 months, and behavioral and physiological motor deficits were present at 5-7 months of age. Ventral root axons were shrunken but were normal in number. The somatosensory system in SOD1 ؊/؊ mice was mildly affected. SOD1؊/؊ mice expressing Cu/Zn SOD only in brain and spinal cord were generated using transgenic mice expressing mouse SOD1 driven by the neuron-specific synapsin promoter. Neuron-specific expression of Cu/Zn SOD in SOD1 ؊/؊ mice rescued motor neurons from the neuropathy. Therefore , Cu/Zn SOD is not required for normal motor neuron survival , but is necessary for the maintenance of normal neuromuscular junctions by hindlimb motor neurons. (Am J Pathol 1999, 155:663-672)

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