ABSTRACT
We propose a tool for a rapid high-resolution detection of calcium ions which can be used in parallel with other techniques. We have applied a new approach by photo-oxidation of diaminobenzidine in presence of the emission of an excited fluorochrome specific for calcium detection. This method combines the selectivity of available fluorophores to the high spatial resolution offered by transmission electron microscopy to detect even fluorescing molecules even when present in low amounts in membrane-bounded organelles. We show in this paper that Mag-Fura 2 photoconversion via diaminobenzidine oxidation is an efficient way for localizing Ca2+ ions at EM level, is easily carried out and reproducible, and can be obtained on a good amount of cells, since the exposition in our conditions is not limited to the direct irradiation of the sample via an objective but obtained with a germicide lamp. The end product is sufficiently electron dense to be detected clearly when present in sufficient amount within a membrane boundary.
Subject(s)
Calcium Signaling , Calcium , Fluorescent Dyes/chemistry , Fura-2/analogs & derivatives , Calcium/analysis , Calcium/metabolism , Fura-2/chemistry , HeLa Cells , Humans , Microscopy, Fluorescence/methodsSubject(s)
Antineoplastic Agents/therapeutic use , Janus Kinase 2/genetics , Primary Myelofibrosis/genetics , Pyrazoles/therapeutic use , Splenomegaly/drug therapy , Adult , Aged , Aged, 80 and over , Alleles , Female , Genotype , Humans , Male , Middle Aged , Myeloproliferative Disorders/complications , Myeloproliferative Disorders/drug therapy , Myeloproliferative Disorders/genetics , Nitriles , Primary Myelofibrosis/complications , Primary Myelofibrosis/drug therapy , Proportional Hazards Models , Pyrimidines , Splenomegaly/etiology , Treatment OutcomeABSTRACT
Horses, asses and zebras belong to the genus Equus and are the only extant species of the family Equidae in the order Perissodactyla. In a previous work we demonstrated that a key factor in the rapid karyotypic evolution of this genus was evolutionary centromere repositioning, that is, the shift of the centromeric function to a new position without alteration of the order of markers along the chromosome. In search of previously undiscovered evolutionarily new centromeres, we traced the phylogeny of horse chromosome 5, analyzing the order of BAC markers, derived from a horse genomic library, in 7 Equus species (E. caballus, E. hemionus onager, E. kiang, E. asinus, E. grevyi, E. burchelli and E. zebra hartmannae). This analysis showed that repositioned centromeres are present in E. asinus (domestic donkey, EAS) chromosome 16 and in E. burchelli (Burchell's zebra, EBU) chromosome 17, confirming that centromere repositioning is a strikingly frequent phenomenon in this genus. The observation that the neocentromeres in EAS16 and EBU17 are in the same chromosomal position suggests that they may derive from the same event and therefore, E. asinus and E. burchelli may be more closely related than previously proposed; alternatively, 2 centromere repositioning events, involving the same chromosomal region, may have occurred independently in different lineages, pointing to the possible existence of hot spots for neocentromere formation. Our comparative analysis also showed that, while E. caballus chromosome 5 seems to represent the ancestral configuration, centric fission followed by independent fusion events gave rise to 3 different submetacentric chromosomes in other Equus lineages.
