Henry A. Lardy

[15] Isolation of liver or kidney mitochondria

Publisher Summary This chapter describes the isolation of liver or kidney mitochondria. The selected tissue is disrupted by homogenization in cold isotonic sucrose. Differential centrifugation is then employed to separate the mitochondria from cell debris, red blood cells, nuclei, microsomes, and soluble components. For the isolation of liver mitochondria, the homogenate is distributed into Lusteroid centrifuge cups and centrifuged at 600 g for 10 minutes. The supernatant fraction is decanted and saved. The pellets may be dispersed by using the side of a stirring rod against the wall of the cup or by handoperating the homogenizer. The resuspended material is centrifuged at 600 g for 10 minutes. The supernatant fractions are combined. The pellets are discarded. This washing contributes not only to the yield of the final mitochondrial preparation, but also to its integrity, apparently by permitting the recovery of the larger mitochondria. For the isolation of kidney, the kidney capsule is removed by gently squeezing the kidney through the thumb and forefinger. The kidney is then cut sagittally. The medullary portion is removed and discarded. Mitochondria are then prepared from the cortex following the method described for liver, with the exception that the mitochondrial pellet need be washed only once. Method for testing the quality of mitochondria is also discusses in the chapter.

Antibiotics as tools for metabolic studies. I. A survey of toxic antibiotics in respiratory, phosphorylative and glycolytic systems☆

Abstract Approximately 50 toxic antibiotics were tested as possible inhibitors of respiratory and phosphorylating systems in mitochondria and of glycolysis by Ascites tumor cells. Oligomycin, Nigericin, and Dianemycin inhibited respiration and phosphate uptake in the presence of several substrates. Each of these agents displayed considerable selectivity in these systems as well as in their inhibition of mitochondrial adenosinetriphosphatase (ATPase). Oligomycin inhibits, virtually completely, the ATPase and the ATP-Pi32 exchange reaction in mitochondria and in submitochondrial particles. The effects of Nigericin and Dianemycin are dependent to some extent on the structural integrity of the mitochondria. Two antibiotics which are known to affect electron-carrying systems were found to inhibit, partially, anaerobic glycolysis in Ascites tumor cells. The results affirm the general utility of toxic antibiotics as tools for metabolic investigations.

Generation and characterization of androgen receptor knockout (ARKO) mice: An in vivo model for the study of androgen functions in selective tissues

By using a cre-lox conditional knockout strategy, we report here the generation of androgen receptor knockout (ARKO) mice. Phenotype analysis shows that ARKO male mice have a female-like appearance and body weight. Their testes are 80% smaller and serum testosterone concentrations are lower than in wild-type (wt) mice. Spermatogenesis is arrested at pachytene spermatocytes. The number and size of adipocytes are also different between the wt and ARKO mice. Cancellous bone volumes of ARKO male mice are reduced compared with wt littermates. In addition, we found the average number of pups per litter in homologous and heterozygous ARKO female mice is lower than in wt female mice, suggesting potential defects in female fertility and/or ovulation. The cre-lox ARKO mouse provides a much-needed in vivo animal model to study androgen functions in the selective androgen target tissues in female or male mice.

Effect of surface active agents on the latent ATPASE of mitochondira

In previous work a heat-stable, acetone-soluble fraction from rat liver microsomes was found to stimulate the respiration of mitochondria. By means of an assay system based on the activation of latent mitochondrial ATPase, the active component of the microsomal extract has been identified as free fatty acids. Among the saturated fatty acids, myristic exhibited the greatest activity which diminished progressively as the chain was lengthened or shortened. Introduction of a cis unsaturated bond, but not of a trans unsaturation, enhanced activity. During the interaction, the higher fatty acids or DCA are tightly bound by the mitochondria in contrast to DNP which activates latent ATPase without becoming appreciably bound.

Antioxidant enzyme systems in rat liver and skeletal muscle: Influences of selenium deficiency, chronic training, and acute exercise☆

The influences of selenium deficiency (Se-D), chronic training, and an acute bout of exercise on hepatic and skeletal muscle antioxidant enzymes, i.e., superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX), as well as glutathione S-transferase (GST) and tissue lipid peroxidation, were investigated in post-weaning male Sprague-Dawley rats. Se-D per se depleted GPX in both liver and skeletal muscle but had no effect on SOD or catalase activity. One hour of treadmill running (20 m/min, 0% grade and 27 m/min, 15% grade for untrained and trained rats, respectively) significantly elevated hepatic catalase and cytosolic SOD activity; more prominent activations were found in the Se-D or untrained rats, whereas skeletal muscle antioxidant enzymes were little affected. Ten weeks of training (1 h/day, 5 days/week at 27 m/min, 15% grade) increased hepatic mitochondrial SOD by 23% (P less than 0.05) in Se-D rats. Both hepatic mitochondrial and cytosolic GPX were decreased by training whereas GPX was increased twofold in skeletal muscle mitochondria. Se-independent GPX was elevated by training only in the skeletal muscle mitochondria of Se-D rats. Lipid peroxidation (malondialdehyde formation) was increased by an acute bout of exercise in hepatic mitochondria of the untrained rats and in skeletal muscle mitochondria of the Se-D rats. These data indicate that antioxidant enzymes in liver and skeletal muscle are capable of adapting to selenium deficiency and exercise to minimize oxidative injury caused by free radicals.

Targeting the stromal androgen receptor in primary prostate tumors at earlier stages

To differentiate roles of androgen receptor (AR) in prostate stromal and epithelial cells, we have generated inducible-(ind)ARKO-TRAMP and prostate epithelial-specific ARKO TRAMP (pes-ARKO-TRAMP) mouse models, in which the AR was knocked down in both prostate epithelium and stroma or was knocked out in the prostate epithelium, respectively. We found that loss of AR in both mouse models resulted in poorly differentiated primary tumors with expanded intermediate cell populations. Interestingly, knockdown of both epithelial and stromal AR in ind-ARKO-TRAMP mice at earlier stages resulted in smaller primary prostate tumors with lower proliferation rates, and knockout of AR in pes-ARKO-TRAMP mice resulted in larger primary prostate tumors with higher proliferation rates. The differential proliferation rates, yet with similarly expanded intermediate cell populations, indicated that the prostate stromal AR might play a more dominant role than the epithelial AR to promote primary tumor proliferation at an early stage of tumor. Tissue recombination of human prostate stromal cell lines (WPMY1-v or WPMY1-ARsi) with human prostate cancer epithelial cell lines (PC3-v or PC3-AR9) further demonstrated that the AR might function as a suppressor in epithelial cells and a proliferator in stromal cells in the primary prostate tumors. The dual roles of the AR in prostate epithelium and stroma may require us to reevaluate the target and timing of androgen-deprivation therapy for prostate cancer patients and may suggest a need to develop new drugs to selectively target stromal AR in the primary prostate tumors at earlier stages.

Alteration of membrane permeability to calcium ions during maturation of bovine spermatozoa

Abstract Bovine spermatozoa, maturing in the caudal epididymis, apparently have a low content of mobilizable Ca2+ (6 ± 1 nmoles Ca 2+ 10 8 cells) in situ, but will accumulate added Ca2+ when simply diluted into isotonic media. It is suggested that the low concentrations of Ca2+ (0.5 ± 0.1 mM) and O2 present in epididymal fluids prevent the accumulatin of Ca2+ by sperm prior to ejaculation. Although the seminal plasma that surrounds ejaculated sperm contains 9 ± 1 mM Ca2+, washed ejaculated sperm also have a low Ca2+ content (7 ± 1 nmoles Ca 2+ 10 8 cells). Moreover, in contrast to epididymal sperm, washed ejaculated sperm are incapable of accumulating Ca2+ supplied exogenously. Evidence is presented that bovine seminal fluids contain a component that is added to the surface membranes of the sperm at ejaculation which prevents or delays the active uptake of Ca2+ by these cells.

Inhibition of Phosphofructokinase by Quinone Methide and α-Methylene Lactone Tumor Inhibitors

The plant-derived tumor inhibitors taxodone, taxodione, vernolepin, eupacunin, and euparotin acetate each inhibit the sulfhydryl enzyme, phosphofructokinase. The substrates, fructose-6-phosphate and adenosine triphosphate, protect the enzyme from this, inhibition as does the addition of dithiothreitol to the inhibitors. Incubation of taxodione with phosphofructokinase is associated with the loss of about one sulfhydryl group per inhibitor molecule, and the substrates protect six sulfhydryl groups per protomer of 93,000 daltons.

Ergosteroids II: Biologically Active Metabolites and Synthetic Derivatives of Dehydroepiandrosterone

An improved procedure for the synthesis of 3 beta-hydroxyandrost-5-ene-7,17-dione, a natural metabolite of dehydroepiandrosterone (DHEA) is described. The synthesis and magnetic resonance spectra of several other related steroids are presented. Feeding dehydroepiandrosterone to rats induces enhanced formation of several liver enzymes among which are mitochondrial sn-glycerol 3-phosphate dehydrogenase (GPDH) and cytosolic malic enzyme. The induction of these two enzymes, that complete a thermogenic system in rat liver, was used as an assay to search for derivatives of DHEA that might be more active than the parent steroid. Activity is retained in steroids that are reduced to the corresponding 17 beta-hydroxy derivative, or hydroxylated at 7 alpha or 7 beta, and is considerably enhanced when the 17-hydroxy or 17-carbonyl steroid is converted to the 7-oxo derivative. Several derivatives of DHEA did not induce the thermogenic enzymes whereas the corresponding 7-oxo compounds did. Both short and long chain acyl esters of DHEA and of 7-oxo-DHEA are active inducers of the liver enzymes when fed to rats. 7-Oxo-DHEA-3-sulfate is as active as 7-oxo-DHEA or its 3-acetyl ester, whereas DHEA-3-sulfate is much less active than DHEA. Among many steroids tested, those possessing a carbonyl group at position 3, a methyl group at 7, a hydroxyl group at positions 1, 2, 4, 11, or 19, or a saturated B ring, with or without a 4-5 double bond, were inactive.

Further studies on the potassium requirements of mitochondria

Abstract Added K + is necessary for maximum rates of respiration and phosphorylation by rat liver mitochondria when a phosphate accepting system such as glucose and hexokinase is present. K + is also necessary for maximum rates of respiration and phosphorylation when microsomes, or an acetone-soluble fraction therefrom, are present. When respiration is maximally stimulated by 2,4-dinitrophenol, added K + does not enhance respiration except that in the 3rd and 4th hour of the experiment it prevents deterioration of the mitochondria. It is concluded that K + participates in transphosphorylation reactions but does not directly influence the oxidative reactions.