The relationship between NADPH-dependent lipid peroxidation and degradation of cytochrome P-450 in adrenal cortex mitochondria

The relationship between NADPH-dependent lipid peroxidation and the degradation of cytochrome P-450 has been studied in bovine adrenal cortex mitochondria. Malondialdehyde formation is accompanied by a corresponding decrease in total cytochrome P-450 content. Inhibitors of lipid peroxidation also prevent the loss of cytochrome P-450, further demonstrating a direct relationship between NADPH-dependent lipid peroxidation and degradation of P-450. To differentiate between cytochrome P-450(11)beta and P-450scc, steroid-induced difference spectra were used to evaluate P-450 degradation. These measurements provide the first evidence that both P-450's are degraded during NADPH-dependent lipid peroxidation with P-450(11)beta being much more susceptible to this process.     

Titration of the binding sites for the oligomycin-sensitivity conferring protein in beef heart submitochondrial particles

The binding parameters of the oligomycin-sensitivity conferring protein (OSCP) in inside-out particles from beef heart mitochondria have been tested by means of two assays, the oligomycin-sensitive ATP-Pi exchange, and the oligomycin-sensitive ATP hydrolysis. The total number of OSCP binding sites in A particles was equal to 220 pmol/mg particle protein. Each mole of ATPase active site was able to bind 1.1 +/- 0.5 mol OSCP with Kd 1.7 nM.     

Possible roles of calcium and ammonium in the development of bitter pit in apple

Apple trees ( Malus pumila Mill . var. domestica Fuji/ Malus prunifolia rootstock) showed a high susceptibility to bitter pit when supplyed with ammonium salt instead of nitrate (control) in the nutrient solution. When apple fruit was affected by bitter pit, a lower calcium as well as a higher nitrogen and ammonium-nitrogen contents was observed in the fruit flesh near the calyx end. The activity of the mitochondrial Ca²⁺-uptake of the fruit flesh near the calyx end was higher when the tree was grown with ammonium salt than when grown with nitrate. Both the activities of succinate: cytochrome c oxidoreductase and the mitochondrial Ca²⁺-uptake per g of tissue were higher in affected fruit than in healthy fruit. Each of chlorpromazine, N-(6-aminohexyl)-5-chloro-l-napthalenesulfonamide (W-7) and N-(6-aminohexyl)-l-naphthalenesulfonamide (W-5), calmodulin antagonists, was infiltrated into the fruit for 20 min under reduced pressure (about 1 × 10⁴ Pa). Few days later, numerous bitter pit-like spots were observed in both fruit treated with W-7 and chlorpromazine, while only a few spots were observed after the infiltration with W-5, a less potent calmodulin antagonist. A possible mechanism for the occurrence of bitter pit is discussed.     

Molecular Diagnosis of Meloidogyne Species

Genetic variation within nuclear and mitochondrial DNA of Meloidogyne species and host races has been evaluated for the development of root-knot nematode molecular diagnostics. This review summarizes the distinctive features of several useful DNA-based assays for plant-parasitic nematodes, focusing upon the direct application of these procedures for Meloidogyne detection, identification, and systematics.     

Restriction Enzyme Analysis of Mitochondrial DNA of Acanthamoeba Strains in Japan

ABSTRACT. Eight isolates, identified as either Acanthamoeba castellanii or A. polyphaga from human eye infections, contact lens containers, and soil in Japan, were characterized by restriction fragment length polymorphisms (RFLP) of mitochondrial DNA (mtDNA). Mitochondrial DNA was digested with either Bgl II, Eco R I, Hind III, Hpa I, Sca I or Xba I, electrophoresed in agarose gels, and stained with ethidium bromide. Four distinct RFLP phenotypes that refer to the collection of six fragment size patterns obtained for a single strain with six enzymes, were discovered among the eight strains used in this study. Three strains morphologically classified as A. polyphaga share a single RFLP phenotype with the Ma strain of A. castellanii. The interspecific sequence differences of 7.06–12.74% in DNA nucleotide were estimated from the proportion of DNA fragments shared by each pair of mtDNA.     

Variation in mitochondrial DNA and post-glacial colonization of north western Europe by brown trout

A purified mitochondrial DNA (mtDNA) probe was used to examine restriction fragment length polymorphisms produced by six restriction enzymes ( Xba I, Eco RV, Ava II, Hinf I, Hae III, Mbo I) in 915 brown trout from western Europe. A total of 20 composite haplotypes were found with one to seven haplotypes in individual populations. Icelandic trout samples from north, south, east, and west coast drainages showed only a single common haplotype in contrast to the high level of polymorphism found in Irish and Scottish populations. The phylogeny of mtDNA haplotypes and the pattern of haplotype distribution suggests that post-glacial colonization of brown trout in NW Europe was more complex than the dual colonization model which has been proposed on the basis of differential LDH-5* allele distribution. For example, Lough Melvin (Ireland) appears to have been independently     

Molecular characterization,relatedness and antifungal susceptibility of the basidiomycetous hormographiella species and Coprinus cinereus from clinical and environmental sources

Hormographiella-like strains, isolated from different natural substrates and producing sclerotia and occasionally basidiomata of Coprinus cinereus, were compared morphologically and using molecular techniques with clinical strains of Hormographiella aspergillata and H. verticillata. Analysis of restriction fragment length polymorphisms of ribosomal and mitochondrial-like DNA confirmed interspecific differences between H. aspergillata and H. verticillata, supporting the morphological data, and helped demonstrate that H. aspergillata is the anamorph of C. cinereus. The latter was confirmed also by crossing tests. The analysis of the mtDNA restriction profiles revealed intraspecific variability in C. cinereus, which allowed differentiation of clinical and environmental strains. Due to the implication of C. cinereus and Hormographiella in human opportunistic infections, the antifungal susceptibility test is included. Results show that all strains were susceptible to miconazole, itraconazole and ketoconazole but not to flucytosine and fluconazol. Susceptibility against amphotericin B was variable; while H. verticillata was susceptible, four out of seven C. cinereus strains tested were resistant.     

Mitochondrial DNA polymorphism and phylogenetic relationships inHevea brasiliensis

Using fourteen random mitochondrial DNA probes, we have examined restriction fragment length polymorphism (RFLP) in wild and cultivatedHevea brasiliensis. A total of 395 accessions, including 345 from various prospectings collected in Brazil, Colombia and Peru and 50 cultivated clones, were analyzed. Two other species (H. benthamiana andH. pauciflora) were also included in the study for comparison. The high level of mitochondrial polymorphism allowed us to divide all the accessions analyzed into 212 distinct genotypes. The genetic variability of cultivated clones was limited to four genotypes forming two clusters. In contrast, considerable genetic variation was found in the wild collections. In almost all cases, accessions displaying the same RFLP profile were restricted to the same geographical area (same or neighbor administrative districts). In addition, accessions whose genetic closeness was predicted by RFLP profiles were also clustered according to geographical origin. In a few cases, however, similar RFLP profiles were found for accessions originating from geographically distant districts. This discrepancy can be explained either by seed dispersion (by river) or possibly by similar genetic events occurring independently in different geographical locations. Chloroplast DNA RFLP was also analyzed in 217 accessions, representative of 126 distinct mitochondrial genotypes. Very few differences were found, indicating that the chloroplast genome is more highly conserved than the mitochondrial genome.     

Inhibition of the mitochondrial Ca2+ uniporter by pure and impure ruthenium red

Commercial ruthenium red is often purified by a single recrystallization as described by Luft, J.H. (1971) Anat Rec 171, 347–368, which yields small amounts of material having an apparent molar extinction coefficient of 67,400 at 533 nm. A simple modification to the procedure dramatically improves the yield, allowing crystallization to be repeated. Three times recrystallized ruthenium red has an apparent extinction coefficient of 85,900, the highest value reported to date. Both crude and highly purified ruthenium red can be shown to inhibit reverse activity of the mitochondrial Ca²⁺ uniporter (uncoupled mitochondria), provided that care is taken to minimize and account for Ca²⁺ release through the permeability transition pore. Crude ruthenium red is 7–10 fold more potent than the highly purified material in this regard, on an actual ruthenium red concentration basis. The same relative potency is seen against forward uniport (coupled mitochondria), however, the I₅₀ values are 10 fold lower for both the crude and purified preparations. These data demonstrate unambiguously that the energy state of mitochondria affects the sensitivity of the Ca²⁺ uniporter to ruthenium red preparations, and that both the forward and reverse reactions are subject to complete inhibition. The data suggest, however, that the active inhibitor may not be ruthenium redper se, but one or more of the other ruthenium complexes which are present in ruthenium red preparations.Abbreviations CCP carbonyl cyanide p-chlorophenylhydrazone - CSA cyclosporin A - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid     

Creatine metabolism and the consequences of creatine depletion in muscle

Currently, considerable research activities are focussing on biochemical, physiological and pathological aspects of the creatine kinase (CK) — phosphorylcreatine (PCr) — creatine (Cr) system (for reviews see [1, 2]), but only little effort is directed towards a thorough investigation of Cr metabolism as a whole. However, a detailed knowledge of Cr metabolism is essential for a deeper understanding of bioenergetics in general and, for example, of the effects of muscular dystrophies, atrophies, CK deficiencies (e.g. in transgenic animals) or Cr analogues on the energy metabolism of the tissues involved. Therefore, the present article provides a short overview on the reactions and enzymes involved in Cr biosynthesis and degradation, on the organization and regulation of Cr metabolism within the body, as well as on the metabolic consequences of 3-guanidinopropionate (GPA) feeding which is known to induce a Cr deficiency in muscle. In addition, the phenotype of muscles depleted of Cr and PCr by GPA feeding is put into context with recent investigations on the muscle phenotype of gene knockout mice deficient in the cytosolic muscle-type M-CK.Abbreviations Cr creatine - Crn creatinine - PCr phosphorylcreatine - CK creatine kinase - M-CK cytosolic muscle type CK isoenzyme - Mi-CK mitochondrial CK isoenzyme - AGAT L-arginine: glycine amidinotransferase - GAMT S-adenosylmethionine: guanidinoacetate methyltransferase - Arg arginine - Met methionine - GPA guanidinopropionate=-guanidinopropionate - PGPA phosphorylated GPA - GBA 3-guanidinobutyrate=-guanidinobutyrate - CPEO chronic progressive external ophthalmoplegia