In vitro immunosuppressive activity of tacrolimus dihydrodiol precursors obtained by chemical oxidation and identification of a new metabolite of SDZ-RAD by electrospray and electrospray-linked scan mass spectrometry.

Different tacrolimus epoxides and dihydrodiol epoxides arising from the chemical oxidation of the parent drug are described. Open-chain tautomeric forms involving the lactone function were identified for the tacrolimus epoxides. Moreover, the identification by electrospray and electrospray linked scan mass spectrometry of an SDZ-RAD C16-C27 O-demethyl 17, 18-19, 20-21, 22 tris-epoxide new metabolite isolated from pig liver microsomes is reported. The in vitro immunosuppressive activity, using mixed lymphocyte reactions of the two macrolide reported oxidation compounds are discussed.

Mitochondrial matrix granules: their behavior during changing metabolic situations and their relationship to contact sites between inner and outer mitochondrial membranes.

Since their discovery in the early fifties mitochondrial granules have been the subject of many researches. Some twenty years ago two hypotheses on their function were introduced. Peachey thought that the granules were a sink of cations and that they would eventually regulate the concentrations of these ions. Alternatively, Barnard thought that the granules were precursors of the mitochondrial inner membrane. There are only a few data on organic constituents of the granules. Phospholipids (e.g., cardiolipin) glycoprotein or lipids, calcium precipitable lipoprotein, cytochrome c oxidase seem to be present in the granules. There has been much debate on whether calcium is present or not. Reports are mostly based on X-ray microanalysis, the result of which depends on preparation techniques. In heart muscle in stimulating situations the NMG (native matrix granules) move towards the inner membrane and are incorporated in it. They appear to create contact sites between inner and outer mitochondrial membranes in which enzymes can function efficiently. It is hypothetized that the system, NMG-contact sites, forms the structural basis of a regulatory mechanism, by which cells can cope with a high and sudden energy demand.

Freeze-fracture study of heart mitochondria in the condensed or orthodox state.

Rat heart mitochondria were isolated and forced in a well-defined metabolic state. After freeze-fracturing, the intramembrane particle dimension and density on both fracture faces of the inner mitochondrial membrane were measured. No significant differences could be calculated between the diameter of the membrane particles in the five different states. However, the particle density on the fracture faces of the inner mitochondrial membrane in the condensed configuration is significantly smaller than in the orthodox configuration on the 99.5% level of confidence. These results are compared with the literature, where conflicting data have been published about these particle densities.

Immunocytochemical investigation of native matrix granules of the rat heart mitochondrion.

We have examined the ultrastructure and the protein content of native matrix granules (NMG) in rat heart mitochondria, by postembedding immunocytochemistry. Cytochrome c oxidase was found to be present in these granules. It is believed that these granules contain incomplete inner mitochondrial membrane fractions, which can be incorporated in the membrane after stimulation of the metabolism.

Cell junctions and locomotion of the blastoderm edge in gastrulating chick and quail embryos.

The blastoderm edge migrates by the active locomotion of a multilayer of epithelial cells, the so-called margin of overgrowth (MO), that uses the vitelline membrane as its substratum. The structural unity formed by the margin of overgrowth cells and their rapid migration suggest coordination of locomotion between individual cells. Using transmission electron microscopy of thin sections and freeze-fracture, we attempted to determine if the pattern of junctions of the migrating margin of overgrowth is related to the suggested cell--cell cooperation between individual cells in this region. In the leading edge there are large areas of closely apposed cell membranes. Incipient desmosomes and small gap junctions were observed. Tight junctions consisted of isolated strands or isolated networks of tight-junctional strands. In the proximal part of the margin of overgrowth the size of the gap junctions increased and the desmosomes were fully developed. Tight-junctional strands were either isolated or arranged into an isolated network. A broad belt of tight junctions was observed at the transition between margin of overgrowth and non-marginal cells. The distribution of the junctional elements in the MO suggests that junctions contribute to the maintenance of the structural and functional organization of the margin of overgrowth. Furthermore, the spatial distribution of the junctions might give information about the mechanism of locomotion of the margin of overgrowth.

Effect of hypnorm, chloralosane and pentobarbital on the ultrastructure of the inner membrane of rat heart mitochondria.

Rat heart mitochondria were isolated from four groups of animals treated in a different way. The animals of the first group were killed after decapitation (D-group) without previous anaesthesia. The three other groups of animals were anaesthetised with different anaesthetics. The second group (N-group) was anaesthetised with nembutal (sodium pentobarbital), the third group with chloralosane (C-group) and the fourth group with hypnorm (H-group). From these three anaesthetics only nembutal is known to interact with mitochondria. After retrograde perfusion and excision of the heart, mitochondria were prepared from the ventricles by standard methods. After freeze-fracturing the mitochondrial suspension, the intramembrane particle dimension and density on both fracture faces of the inner mitochondrial membrane were measured. The intramembrane particle diameter on the P-face of the inner membrane of the N-group mitochondria was significantly different from D-, C- and H-group mitochondria. Also the density and diameter of the intramembrane particles on the mitochondrial inner membrane of D-group mitochondria compared to C- and H-group mitochondria were significantly different at the 95% level of confidence. Between C- and H-group mitochondria no differences of these parameters were observed. From these results it is clear that, depending on the pretreatment of the animals, a different substructure of the inner membrane of heart mitochondria is obtained.

The structure of Artemia sp. haemoglobins-I. Isolation and characterization of oxygen binding domains obtained by limited tryptic digestion.

Limited tryptic digestion of the extracellular haemoglobins of the crustacea Artemia sp. result in series of discrete fragments which are multiples of 16,000 d. 2. These 16,000 d fragments, together with 50,000 d and 80,000 d fragments have similar amino acid composition and tryptic peptide maps as the intact globin chains. 3. The haem content of the 16,000 d fragments is the same as this of the intact pigment and they can bind oxygen in a non cooperative way. 4. These results strongly support that the 16,000 d fragments represent structural and functional units or domains from which the globin chains are built up.