Bruce Mackler

Studies of mitochondrial development during embryogenesis in the rat

Abstract Marked and progressive increases in the activities of DPNH oxidase, cytochrome c oxidase, succinic dehydrogenase, and mitochondrial ATP-ase were shown to occur between days 10 and 14 of gestation in the rat embryo-fetus. The rise in enzyme activity was paralleled by striking changes in the number and structure of the cristae of the heart mitochondria of the embryo-fetus as demonstrated by electron microscopy.

Biochemical correlates of respiratory deficiency VI. Mitochondrial DNA

The isolation, purification, physical and chemical properties of a mitochondrial DNA of Saccharomyces cerevisiae are described and compared with the nuclear DNA of the same organism. The two entities are clearly distinct in absorption spectra at various pH values, thermal transition temperature in two different solvent systems, buoyant density in CsCl and base composition, all of which are consistent with a GC content of 35 % for nuclear and 21 % for mitochondrial DNA. The latter, a normal double helical molecule, occurs integrated into respiratory particles which renders it DNase resistant. It is present to the extent of 0.71 μg per mg of particle protein in highly aerobic cells of the wild type, decreases during glucose repression and is greatly reduced (⩽ 0.06 μg/mg protein) in a cytoplasmic respiratory-deficient mutant (vegetative petite, ϱ−). The So20,w of the isolated DNA equals 33 s, hence its molecular weight is approximately 2 × 107. Evidence is also presented for its preferential interaction with acridines. The implications of all these findings in the problems of mitochondrial autonomy and self-duplication are discussed.

Studies of DPNH oxidase: properties of a soluble DPNH dehydrogenase

Abstract A soluble highly purified DPNH dehydrogenase has been prepared from beef heart DPNH oxidase. The enzyme catalyzed the oxidation of DPNH by molecular oxygen, cytochrome c , indophenol, flavin-adenine dinucleotide and flavin mononucleotide. Hydrogen peroxide was found to be a product of the reaction when oxygen served as electron acceptor. The flavin component of the enzyme was easily removed from the preparation by dialysis or repeated treatment with ammonium sulfate, and may be bound to the enzyme through thiol linkages.

Reactive oxygen species and DNA oxidation in fetal rat tissues.

It is well recognized that reactive oxygen species (ROS) are formed during the reperfusion of ischemic tissues and ROS may be pathogenic in adult tissues. Although there is little information on the formation and toxicity of ROS during prenatal life, a strong association has been made between limb and possibly brain malformations and uteroplacental ischemia during fetal stages of gestation. It has been proposed that these malformations result from attack by ROS formed during the resumption of placental perfusion. Studies reported here examined formation of ROS in teratogenically sensitive limb and brain and insensitive heart before and during the period of teratogenic sensitivity. Also examined was the formation of ROS following hypoxia and reoxygenation in fetal culture and DNA hydroxylation in sensitive and insensitive fetal tissues during uteroplacental ischemia and reperfusion in vivo. Rates of formation of superoxide anion radical and hydrogen peroxide declined with increasing gestational age whereas those for hydroxyl radical increased. Hydrogen peroxide generation was greatest in insensitive heart whereas hydroxyl radical formation was significantly lower in brain than in limb or heart, which had comparable rates. Hydrogen peroxide generation, which declined significantly in fetuses, but not in membranes with gestation, failed to respond to reoxygenation in vitro. Finally, there were significant increases in DNA hydroxylation in fetal hearts and limbs, but not in brains during uteroplacental ischemia but no further significant change could be detected after reperfusion.

Studies of mitochondrial energy systems during embryogenesis in the rat

Abstract Mitochondria isolated from rat embryos of Day 11 to Day 14 of gestation showed an equal capacity to carry out oxidative phosphorylation with P:O ratios of approximately 3 for pyruvate-malate and 2 for succinate as substrates. Activities of succinic dehydrogenase, cytochrome oxidase, and mitochondrial ATPase remained at nearly constant levels in the preparations during this period of gestation, but DPNH oxidase activity increased over 2-fold. The increase in DPNH oxidase activity could be prematurely induced at Day 11 of gestation of the embryos by prior treatment of the pregnant rats with an atmosphere containing 85% oxygen and 0.4% CO2, but no changes in the growth and development of the treated embryos were found by gross examination.

Studies of oxidative energy deficiency: I. Achondroplasia in the rabbit☆

Abstract Defective oxidative energy formation with the absence of phosphorylation at the cytochrome oxidase region (site III) of the terminal respiratory system has been demonstrated to be present in isolated mitochondrial preparations from the livers of newborn achondroplastic rabbits. Normal-appearing littermates did not exhibit the defect. Terminal electron transport and oxidative activities of the terminal respiratory system as well as mitochondrial ATPase activity (coupling factor I) were similar in both normal and achondroplastic tissue preparations. No significant differences were detectable in the morphology of liver mitochondria from normal and achondroplastic littermates by electron microscopy examination.

Relation of barbiturate structure to DPNH oxidase inhibition

Abstract A series of barbiturates, carbamides, and miscellaneous compounds were examined in order to elucidate the structural requirements for an Amytal-like inhibition of DPNH oxidase. It was found that the essential molecular structure consisted of a nonspecific hydrocarbon group attached to an amide, carbamide, or barbituric acid, these compounds having in common the CONH group. None of the compounds inhibited the oxidation of succinate by succinic oxidase. Similarity was noted between the structural requirements for DPNH oxidase inhibition and for experimental porphyria induction.

Iron deficiency in the rat: effects on oxidative metabolism in distinct types of skeletal muscle.

Summary: Studies performed on iron-deficient and control rats demonstrated that oxidative energy production (phosphorylation) by mitochondria from iron-deficient red and intermediate skeletal muscles was greatly reduced with pyruvate-malate, succinate, and a-glycerophosphate as substrates. Although phosphorylation was also decreased in iron-deficient white skeletal muscle with succinate and pyruvate-malate as substrates, no change was found with a-glycerophosphate as substrate.

Studies of oxidative phosphorylation in Saccharomyces cerevisiae and Saccharomyces carlsbergensis

Abstract Mitochondria isolated from cultures of Saccharomyces cerevisiae and Saccharomyces carlsbergensis , which were harvested late in the stationary phase of growth at a time when full development of the electron transport and associated phosphorylation systems had occurred, carried out oxidative phosphorylation with P:O ratios of approximately 3 and 2 when pyruvate-malate and succinate, respectively, were used as substrates.

Oxidative energy deficiency. II. Human achondroplasia

Abstract Defective oxidative energy formation (with decreased phosphorylation possibly occurring at Site I of phosphorylation, the NADH dehydrogenase region of the terminal respiratory system) has been demonstrated in mitochondrial preparations isolated from muscle biopsy specimens from human subjects with classical achondroplasia.