Imaging in five dimensions: time-dependent membrane potentials in individual mitochondria.

Because of its importance in the chemiosmotic theory, mitochondrial membrane potential has been the object of many investigations. Significantly, however, quantitative data on how energy transduction might be regulated or perturbed by the physiological state of the cell has only been gathered via indirect studies on isolated mitochondrial suspensions; quantitative studies on individual mitochondria in situ have not been possible because of their small size, their intrinsic motility, and the absence of appropriate analytical reagents. In this article, we combine techniques for rapid, high resolution, quantitative three-dimensional imaging microscopy and mathematical modeling to determine accurate distributions of a potentiometric fluorescent probe between the cytosol and individual mitochondria inside a living cell. Analysis of this distribution via the Nernst equation permits assignment of potentials to each of the imaged mitochondrial membranes. The mitochondrial membrane potentials are distributed over a narrow range centered at -150 mV within the neurites of differentiated neuroblastoma cells. We find that the membrane potential of a single mitochondrion is generally remarkably stable over times of 40-80 s, but significant fluctuations can occasionally be seen. The motility of individual mitochondria is not directly correlated to membrane potential, but mitochondria do become immobile after prolonged treatment with respiratory inhibitors or uncouplers. Thus, three spatial dimensions, a key physiological parameter, and their changes over time are all quantitated for objects at the resolution limit of light microscopy. The methods described may be readily extended to permit investigations of how mitochondrial function is integrated with other processes in the intact cell.     

Alterations in the hepatic enzymes following experimental lead poisoning

An investigation on the influence of lead toxicity on some of the hepatic enzymes was studied in rats both after a shorter interval of 15 d and after longer intervals of 60 and 90 d. Three different doses of lead as 5, 10, and 50 mg/kg body wt were administered orally on every alternate day. Whereas significant inhibition of succinic dehydrogenase was seen following lead poisoning, the activity acid and alkaline phosphatase increased with lead intoxication. The histoarchitecture of the liver was grossly intact. Liver accumulated less lead compared to kidney at 60 and 90 d.     

Seroepidemiological study of human hydatidosis and trichinosis, with the indirect hemagglutination reaction, in San Juan de la Costa County. Osorno, X Region, Chile. 1990-1991]

San Juan de la Costa County (40 degrees 45' South lat., 73 degrees 19' West long.) is located in the Osorno province, South of Chile. Its population is 8,486 inhabitants. The basic economic activities are agriculture, cattle raising, timber production and manufacture of wood and coal. According to official reports, the incidence of human hydatidosis and trichinosis in this locality in 1989 were 24 and 59 per 100,000 respectively. In order to contribute to a better knowledge of the epidemiology of human hydatidosis and trichinosis in San Juan de la Costa County, an indirect hemagglutination test (IHAT) for these parasitoses was performed to 511 randomized people. Nine (1.8%) individuals resulted positive for hydatidosis and twenty four (4.7%) were positive for trichinosis. Some considerations on the corresponding prophylactic measures are proposed.     

Synthesis and expression of genes encoding tuna, pigeon, and horse cytochromes c in the yeast Saccharomyces cerevisiae.

Genes encoding tuna, pigeon, and horse cytochromes c were constructed with synthetic oligodeoxyribonucleotides having preferred codons and portions of the iso-1-cytochrome c-encoding gene from the yeast Saccharomyces cerevisiae. The genes were ligated into an expression vector, which contains the normal 5'- and 3'-untranslated regions of the yeast iso-1-cytochrome c gene, and were integrated in single copy into the chromosome. Yeast strains were also constructed with multiple integrated copies of the pigeon gene. The heterologous and normal mRNA levels of the single-copy strains were equivalent. Although the N-terminal methionines were completely cleaved in the heterospecific proteins, the levels of trimethylation of Lys72 and acetylation of N-terminal glycines ranged from 39-78% and 10-70%, respectively. Horse cytochrome c was produced at a nearly normal level, whereas the pigeon and tuna cytochromes c were produced at approx. 40% of the normal levels. The levels of the cytochromes c and growth of the mutant yeast strains indicated that the heterospecific cytochromes c had approx. 50% specific activity in vivo.     

The role of the epidermal growth factor-1 and hydrophobic stack domains of human factor IX in binding to endothelial cells.

To determine the function and specificity in factor IX of the first epidermal growth factor (EGF)-like domain and the eight-amino acid hydrophobic stack encoded by exon C (residues 39-46), these domains were replaced by the corresponding polypeptide regions of factor X and chimeric proteins were produced in human embryo kidney cells. Both chimeras were activated by factor XIa at a rate similar to plasma factor IX and exhibited calcium-dependent fluorescence quenching similar to plasma factor IX. Both chimeras competed equally for binding to the endothelial cell receptor. Our findings make it unlikely that the first EGF-like domain or the hydrophobic stack of factor IX are responsible for the specific binding of factor IX to its endothelial cell receptor.