Active glycation in neurofibrillary pathology of Alzheimer disease: N(epsilon)-(carboxymethyl) lysine and hexitol-lysine.

Advanced glycation end products are a diverse class of posttranslational modifications, stemming from reactive aldehyde reactions, that have been implicated in the pathogenesis of a number of degenerative diseases. Because advanced glycation end products are accelerated by, and result in formation of, oxygen-derived free radicals, they represent an important component of the oxidative stress hypothesis of Alzheimer disease (AD). In this study, we used in situ techniques to assess N(epsilon)-(Carboxymethyl)lysine (CML), the predominant advanced glycation end product that accumulates in vivo, along with its glycation-specific precursor hexitol-lysine, in patients with AD as well as in young and aged-matched control cases. Both CML and hexitol-lysine were increased in neurons, especially those containing intracellular neurofibrillary pathology in cases of AD. The increase in hexitol-lysine and CML in AD suggests that glycation is an early event in disease pathogenesis. In addition, because CML can result from either lipid peroxidation or advanced glycation, while hexitol-lysine is solely a product of glycation, this study, together with studies demonstrating the presence of 4-hydroxy-2-nonenal adducts and pentosidine, provides evidence of two distinct oxidative processes acting in concert in AD neuropathology. Our findings support the notion that aldehyde-mediated modifications, together with oxyradical-mediated modifications, are critical pathogenic factors in AD.     

Reaction between iron(III) and pyrazoic acid

In the reaction of [Fe(H₂O)₆]³⁺ with pyrazoic acid, reduction of iron(III) to iron(II) is observed. When an excess of iron is present, the reaction involves a transfer of four electrons per mole of acid. At room temperature the redox reaction, which is dependent on hydrogen ion, iron(III) and pyrazoic acid concentrations, is rather slow and is the rate-determining step. The kinetic study was carried out at 50.0 ± 0.1 °C. The redox reaction is followed by a fast reaction of the iron(II) with an excess of ligand, resulting in the production of well-known complexes, where the acid acts as a chelating ligand through the nitrogen and oxygen atoms.     

New methods to evaluate colocalization of fluorophores in immunocytochemical preparations as exemplified by a study on A2A and D2 receptors in Chinese hamster ovary cells.

An important aspect of the image analysis of immunocytochemical preparations is the evaluation of colocalization of different molecules. The aim of the present study is to introduce image analysis methods to identify double-labeled locations exhibiting the highest association of two fluorophores and to characterize their pattern of distribution. These methods will be applied to the analysis of the cotrafficking of adenosine A2A and dopamine D2 receptors belonging to the G protein-coupled receptor family and visualized by means of fluorescence immunocytochemistry in Chinese hamster ovary cells after agonist treatment. The present procedures for colocalization have the great advantage that they are, to a large extent, insensitive to the need for a balanced staining with the two fluorophores. Thus, these procedures involve image processing, visualization, and analysis of colocalized events, using a covariance method and a multiply method and the evaluation of the identified colocalization patterns. Moreover, the covariance method offers the possibility of detecting and quantitatively characterizing anticorrelated patterns of intensities, whereas the immediate detection of colocalized clusters with a high concentration of labeling is a possibility offered by the multiply method. The present methods offer a new and sensitive approach to detecting and quantitatively characterizing strongly associated fluorescence events, such as those generated by receptor-receptor interaction, and their distribution patterns in dual-color confocal laser microscopy.     

Demonstration of phosphorylase activity in the rat brain

Summary The phosphorylase reaction in rat brain is strong, but for its correct evaluation in pathological states certain precautions are needed. Control material from animals of the same age should be used identically and simultaneously with the pathological, preferably freezing blocks from each on the same holder. Several sections should be cut and then the holder rotated through 180° and more sections cut, thus avoiding changes in section thickness which can drastically influence the color reactions, i.e., apparent phosphorylase activity. The time the animal is killed until its brain is frozen should be less than 5 min, preferably 11/2 to 3 min, to limit postmortem changes in the enzyme.Preparing the stock incubating medium and storing it at 4°C does not affect the potency of the medium; this procedure is convenient and eliminates more variables.Small amounts of medium can be placed between two slides set at right angles to each other. The height of the chamber, equal to the thickness of a slide, is uniform.Prefixation of the cryostat sections in cold acetone (4°C) for 5 min can be useful for obtaining sharper enzyme localization.AMP is the nucleotide of choice for stimulating full phosphorylase reaction. The inclusion of glycogen in the incubating medium is also necessary.The optimal incubation time for phosphorylase + branching enzyme is about 1/2 hr for 16 sections. For phosphorylase alone (active or total) it is about 2 hr. Dehydrating, clearing, and mounting in iodine-containing solutions is not recommended nor is it necessary. The best preservation of original colors, which is important for the correct interpretation of the results, is accomplished by sealing the preparations mounted in iodine-glycerin with paraffin.     

Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques

Methotrexate (MTX) is one of the most important drugs for osteosarcoma (OS) treatment. To identify genetic aberrations associated with the development of MTX resistance in OS cells, in addition to the previously reported expression changes of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes, comparative genomic hybridization (CGH)-based techniques were used. The direct comparison between MTX-resistant variants of U-2OS or Saos-2 human OS cell lines with their respective parental cell lines by CGH on chromosomes revealed that development of MTX resistance was associated with gain of the chromosomal regions 5q12-q15 and 11q14-qter in U-2OS variants, and with gain of 8q22-qter in Saos-2 variants. Further analyses by CGH on microarrays demonstrated a progressively increasing gain of mixed lineage leukemia (MLL) gene (11q23) in U-2OS MTX-resistant variants, which was also confirmed by fluorescence in situ hybridization (FISH), in addition to gain of FGR (1p36), amplification/overexpression of DHFR, and slight decrease of RFC expression. In Saos-2 MTX-resistant variants, gain of MYC (8q24.12-q24.13) was detected, together with a remarkable decrease of RFC expression. Further analyses of DHFR, MLL, MYC, and RFC gene status in four additional human OS cell lines revealed that only gain of DHFR and MLL were associated with an inherent lower sensitivity to MTX. These data demonstrate that genetic analyses with complementary techniques are helpful for the identification of new candidate genes, which might be considered for an early identification of MTX unresponsive tumors.