Reconstitution of Oxidative Phosphorylation and of Oligomycin-Sensitive ATPase by Five- and Six-Subunit Forms of Pea Mitochondrial F(1)-ATPase

Five- and six-subunit forms of F₁-ATPase were purified from pea (Pisum sativum L. cv Homesteader) cotyledon submitochondrial particles. Apart from the usual complement of five subunits, the six-subunit enzyme contained an additional 26,500-dalton protein. Both forms of the F₁-ATPase were used to reconstitute oxidative phosphorylation in F₁-depleted (ASU) as well as in F₁ and oligomycin-sensitivity conferring protein (OSCP)-depleted (ASUA) bovine mitochondrial membranes. The six-subunit enzyme was considerably more efficient in reconstituting the ATP synthesis than the five-subunit enzyme. Both forms of the enzyme were also able to reconstitute the ATPase activity in ASU- as well as in ASUA-particles. There were substantial differences, however, in the oligomycin sensitivity of the ATPase bound to the ASUA-particles: 20 and 60% inhibition by oligomycin was obtained in the case of the five-subunit and six-subunit enzyme, respectively. We conclude, that the 26,500-dalton protein present in the six-subunit F₁-ATPase is responsible for the increase in oligomycin sensitivity of the bound enzyme and functions, therefore, as the plant OSCP.     

Studies on photophosphorylation utilizing methylene diphosphonate analogs of ADP and ATP

Spinach chloroplasts were able to photophosphorylate the ADP analog α,β-methylene adenosine 5′-diphosphate (AOPCP). Phosphorylation of AOPCP was catalyzed by chloroplasts that were washed or dialyzed to remove free endogenous nucleotides. In the presence of glucose, hexokinase, AOPCP and ³²P_i, the ³²P label was incorporated into α,β-methylene adenosine 5′-triphosphate (AOPCPOP).In contrast to photophosphorylation of AOPCP, the ATP analog AOPCPOP was a poor substrate for the ATP-P_i exchange reaction and its hydrolysis was neither stimulated by light and dithiothreitol nor inhibited by Dio-9.Photophosphorylation of AOPCP was inhibited by the α,β- and β,γ-substituted methylene analogs of ATP, while phosphorylation of ADP was unaffected by them. The ATP-P_i exchange was also unaffected by both ATP analogs, while the weak AOPCPOP-P_i exchange was inhibited by the β,γ-methylene analog of ATP.Direct interaction of methylene analogs with the chloroplast coupling factor ATPase was indicated by the enzymatic hydrolysis of AOPCPOP on polyacrylamide gels.     

Altered gene and protein expression by Nm23-H1 in metastasis suppression

The aim of our work was to study (1) the antioxidant properties of lipoic acid (LA) and its reduced metabolite dihydrolipoic acid (DHLA) formed by reduction of LA and (2) the effects of treatment with LA and DHLA on (a) K(+) efflux from human red blood cells and (b) post-ischemic recovery and oxidative stress in isolated perfused rat hearts challenged with an ischemia-reperfusion (IR) sequence. In vitro, we used xanthine and xanthine oxidase to generate superoxide anion, which is not directly measurable by electron paramagnetic resonance (EPR), but specifically oxidizes the spin probe CPH into an EPR-detectable long lasting CP(*) nitroxide radical. While 5 mM of LA was ineffective in reducing the kinetics of CP(*) nitroxide formation, DHLA was shown to lessen this rate in a dose-dependent manner and at 30 mM was even more efficient than 300 UI/ml SOD. These results are in agreement with the fact that DHLA is able to directly scavenge superoxide anion. Red cells are a good model to investigate oxidative damage in biological membranes; hence, we used a suspension of erythrocytes incubated with 2,2(')-azobis(2-amidinopropane) hydrochloride (AAPH) which generates in vitro free radicals. DHLA provided more effective protection of red cells membranes than LA; DHLA was comparable to Trolox for its antioxidant potency. In vivo, treatment of rats (50 mg/kg/day i.p. for 7 days) with LA induced a slight increase in coronary flow (CF) in isolated perfused hearts, after 30 min of global total ischemia. This effect was not associated with an improvement in contractile function and reduction of myocardial oxidative stress. In conclusion, because of their ability to scavenge free radicals, LA and to an even greater degree DHLA were able to protect the membranes of red blood cells. This finding suggests that LA and DHLA might be useful in the treatment of diseases associated with oxidative stress such as diabetes.     

The genus Hyalomma Koch, 1844. IX. Redescription of all parasitic stages of H. (Euhyalomma) impeltatum Schulze & Schlottke, 1930 and H. (E.) somalicum Tonelli Rondelli, 1935 (Acari: Ixodidae)

The ticks Hyalomma (Euhyalomma) impeltatum Schulze &; Schlottke, 1930 and H. (E.) somalicum Tonelli Rondelli, 1935 [a species resurrected for “Hyalomma ? species” of Hoogstraal (1956) and H. erythraeum of Kaiser &; Hoogstraal (1968)] are tentatively considered to belong to the H. (E.) asiaticum group of closely related species. Amongst other features that are fairly similar, males of H. impeltatum can be distinguished from those of H. somalicum by the oval posterior margin of the conscutum, a narrow, subtriangular parma, the lack of ventral sclerotised plaques on median, paramedian and 4th festoons, and an incomplete to complete ivory-coloured stripe on the dorsal aspect of the leg segments; whereas males of H. somalicum have a broad but only slightly convex posterior conscutal margin, in most cases no parma, well-developed sclerotised ventral plaques on all festoons, and only a small ivory-coloured spot on the dorsal aspect of the leg segments. Females of H. impeltatum can be distinguished from those of H. somalicum by the bulging rather than flat preatrial fold of the genital aperture. All parasitic stages of both ticks are illustrated and redescribed, and the characteristics that distinguish the adults from those of other closely related species are detailed. Larger domestic and wild ungulates are the principal hosts of the adults of both ticks. Nymphs and larvae of H. impeltatum parasitise rodents, leporids, birds and lizards, whereas the hosts of the immature stages of H. somalicum are unknown. H. impeltatum is widely distributed in Africa north of the equator, Arabia, the Near East and south-western part of Central Asia; in contrast, H. somalicum has a more limited distribution in East Africa and possibly the Arabian Peninsular. Data on their possible disease relationships are also provided.     

Phylogeny of Rozites, Cuphocybe and Rapacea inferred from ITS and LSU rDNA sequences

Phylogenetic relationships of Rozites, Cuphocybe, and Rapacea were assessed using molecular phylogenetic approaches. These three genera are placed in Cortinariaceae and have been regarded as closely related to Cortinarius. Rozites includes more than 20 species, which are characterized by having both a membranaceous partial veil in the form of a persistent annulus and a membranaceous universal veil. Cuphocye (4 species) lacks an annulus or cortina, but has pigmented veil fibrils or scales. The monotypic genus Rapacea accommodates a distinct taxon with pale, nearly smooth and thick-walled basidiospores. We analyzed 56 sequences of the internal transcribed spacer region (ITS1, ITS2, and the intervening 5.8S rRNA gene) for nine species of Rozites, three species of Cuphocybe, 28 species of Cortinarius, Rapacea mariae and Protoglossum luteum. Two species of Hebeloma were used as outgroup. Large subunit (LSU) rDNA sequences from selected taxa were also analyzed. The results clearly demonstrate that Rozites species are nested within the clade/Cortinarius, and that Rozites is polyphyletic, suggesting that membranaceous veils have evolved several times in the genus Cortinarius. Also Rapacea and Cuphocybe are nested within Cortinarius, making the latter genus paraphyletic. Based on phylogenetic studies, Rozites, Cuphocybe and Rapacea are artificial genera and do not reflect natural relationships.     

The life cycle of Ixodes rubicundus (Acari: Ixodidae) and its adaptation to a hot,dry environment

The life cycle of Ixodes rubicundus, the Karoo paralysis tick, was studied under field conditions in the south-western Orange Free State, South Africa, by placing freshly engorged ticks in small containers. The life cycle extends over 2 years. The two regulating phases in the life cycle, which undergo a morphogenetic diapause during the hot and dry summer months, are the egg and engorged nymph. Possible behavioural diapause in adults which suppresses questing activity before the end of March. can also serve as a third regulating phase. Temperature affects the duration of the pre-oviposition period of engorged females and the period between detachment of engorged larvae from hosts and ecdysis. Commencement of larval hatch is reasonably synchronized, irrespective of the month during which oviposition occurred. Peak activity periods of larvae (April or May) occur during a period of high rainfall and decreasing daily maximum temperatures. The period between detachment of engorged nymphs from hosts and ecdysis is highly variable (8–36 weeks). All developmental stages of the tick occur mainly during autumn and winter and no ticks are active during the hot summer months of December to February. Larvae, nymphs and adults each survive for only one season.     

Factors significant in the diagnostic accuracy of lung cytology in bronchial washing and sputum samples. I. Bronchial washings

Some factors influencing the diagnostic accuracy for primary lung cancer in bronchial washings were studied in 276 consecutive cases seen between 1959 and 1974. Diagnostic accuracy increased during the years under study; the reasons included increasing expertise of the laboratory staff, better documentation of cytologic criteria and improved collection techniques. The overall accuracy was 74%. Detection of malignant cells was highest for squamous-cell and adenosquamous carcinomas (81%), small-cell carcinoma, adenocarcinoma and large-cell carcinoma (70%) and lowest for bronchioloalveolar-cell carcinoma (47%). Accuracy was 84% for central tumors as compared to 30% for peripheral lesions. Tumors of less than 2 cm in diameter yielded very poor results (15%) while those greater than 2 cm yielded 82% accuracy. The specificity of diagnosis of cell type in those specimens with malignant cells was over 93% for squamous-cell carcinoma, small-cell carcinoma and adenocarcinoma, 77% for large-cell carcinoma and below 50% for adenosquamous carcinoma, bronchioloalveolar carcinoma and the uncommon tumors. Two bronchial washings per case gave an appreciably better result (92%) than one per case (68%). The percentage of unsatisfactory specimens from those with cancer was 13.5 and from a control group was 29.9. Reasons for unsatisfactory specimens included limited cellular material, excessive blood and/or leukocytes and drying artifacts.