Single-cell investigation by laser scanning confocal microscopy of cytochemical alterations resulting from extracellular oxidant challenge

Confocal laser scanning fluorescence microscopy coupled to image analysis was employed in order to develop and evaluate procedures for the appraisal at the single-cell level of: (1) protein-bound 4-hydroxynonenal, the specific product of membrane peroxidation (by means of immunocytochemistry with biotin-avidin revelation); (2) protein oxidation (by reaction of protein carbonyls with 2,4-dinitrophenyl-hydrazine followed by immunocytochemistry of dinitrophenyl moieties); and (3) cellular protein thiols (by direct alkylation of sulfhydryl groups with thiol-specific fluorescent reagents possessing different cell permeabilities). The procedures proved able to reveal the subcellular distribution of cytochemical parameters useful as indices of oxidative stress conditions, and may allow redox phenotyping of isolated cells, which would provide an efficient tool in selected experimental models.     

Phospholipid vesicle aggregation induced by human myelin basic protein

Human myelin basic protein isolated from the brains of individuals who died with multiple sclerosis was more potent in inducing the aggregation of egg phosphatidylcholine vesicles than was the basic protein isolated from the brains of normal individuals. The portion of myelin basic protein which bound to egg phosphatidylcholine vesicles was separated from the free protein by sucrose density gradient centrifugation. Similar amounts of basic protein from normal or from multiple sclerosis brains are bound to the lipid and no consistent differences in the N^G, N^G dimethyl-arginine content of the protein fractions have been found.     

Localization and synthesis of alpha-fetoprotein in chorioallantoic membrane from chick embryo.

Alpha-fetoprotein (AFP) is a major globulin of the embryonic serum of mammals, birds, and other vertebrates. It is synthesized chiefly by the liver and/or the yolk sac. The aim of this work was to confirm the occurrence of AFP in the chorioallantoic membrane (CAM) from 14-day chick embryo. AFP had previously been detected by immunoelectrophoresis in CAM extracts under the suspicion that it could be a mere artifact resulting from blood contamination. The immunohistochemical study of the CAM carried out for this purpose revealed the protein to be solely located in the mesodermal layer. The joint use of organ culture and immunoperoxidase techniques has enabled us to find evidence for the synthesis of AFP in the cells of this layer. These results confirm the occurrence of such a significant carrier globulin to embryonic development in one more tissue that can be added to the short list of AFP-producing tissues.     

Plasmodium cynomolgi: the hsp 70 gene.

The hsp 70 gene of Plasmodium cynomolgi was isolated and characterized. As expected the gene is highly similar to that of the hsp 70 gene of Plasmodium falciparum (98% at the protein level, 82% at the nucleotide level). Surprisingly, the hsp 70 gene appears to be present in a single copy in all the P. cynomolgi strains tested, a finding that has implications for the parasite's ability to undergo a heat shock response.