The Respiratory Chain of Plant Mitochondria. II. Oxidative Phosphorylation in Skunk Cabbage Mitochondria

Mitochondria were prepared from the spadices of skunk cabbage (Symplocarpus foetidus) whose respiratory rate with succinate and malate showed 15% to 30% sensitivity to cyanide inhibition, and which showed respiratory control by added ADP. The observed respiratory control ratios ranged from 1.1 to 1.4. The change in pH of the mitochondrial suspension was recorded simultaneously with oxygen uptake: alkalinization of the medium, expected for phosphorylation of ADP, coincided with the period of acceleration in oxygen uptake caused by addition of an ADP aliquot. The ADP/O ratios obtained were 1.3 for succinate and 1.9 for malate. In the presence of 0.3 mm cyanide, the ADP/O ratio for succinate was zero, while that for malate was 0.7. These results are consistent with the existence of an alternate oxidase which interacts with the flavoprotein and pyridine nucleotide components of the respiratory chain and which, in the presence of cyanide, allows the first phosphorylation site to function with an efficiency of about 70%. In the absence of respiratory inhibitors, the efficiency of each phosphorylation site is also about 70%. This result implies that diversion of reducing equivalents through the alternate oxidase, thereby bypassing the 2 phosphorylation sites associated with the cytochrome components of these mitochondria, occurs to a negligible extent during the oxidative phosphorylation of ADP or State 3.Addition of ADP or uncoupler to skunk cabbage mitochondria respiring in the controlled state or State 4, results in reduction of cytochrome c and the oxidation of the cytochromes b, ubiquinone and pyridine nucleotide. A site of interaction of ADP with the respiratory chain between cytochromes b and cytochrome c is thereby identified by means of the crossover theorem. Flavoprotein measured by fluorescence is also oxidized upon addition of ADP or uncoupler, but flavoprotein measured by optical absorbance changes becomes more reduced under these conditions. Depletion of the mitochondria by pretreatment with ADP and uncoupler prevents reduction of most of the fluorescent flavoprotein by succinate. These results indicate that skunk cabbage mitochondria contain both high and low potential flavo-proteins characterized by different fluorescence/absorbance ratios similar to those demonstrated to be part of the respiratory chain in mitochondria from animal tissues.     

STUDY OF MITOCHONDRIA IN RAT LIVER : Quantitative Electron Microscopy

The electron microscope has been used to determine the weight distribution of isolated subcellular particles from normal rat liver. The following results are reported: (1) There exist at least two well defined weight populations of subcellular particles; their respective median weights are 1.3 x 10^-14 and 11 x 10^-14 gm. The lighter fraction is considered to consist of lysosomes, the heavier of mitochondria. (2) The mitochondrial fraction shows a log-normal distribution of the particle weight. (3) By the introduction of morphologic criteria, the mitochondrial fraction is divided into two groups, one consisting of a spherical, the other of an oblong type of particle. The data found support the following concepts: (a) Mitochondria increase their weight from a certain size up by linear growth. (b) Mitochondria divide. The division is not necessarily symmetric; in all cases, however, one part of the division product is a spherical particle. It is felt that these results constitute a valuable demonstration of the general capabilities of quantitative electron microscopy and may stimulate many other useful applications of this technique in cytology, bacteriology, and virology.     

Linkage of the acetylcholine transporter-vesamicol receptor to proteoglycan in synaptic vesicles.

The relationship of the acetylcholine transporter-vesamicol receptor (AcChT-VR) to proteoglycan in Torpedo electric organ synaptic vesicles was investigated. The cholate-solubilized VR was immunoprecipitated by a monoclonal antibody directed against the SV1 epitope located in the glycosaminoglycan portion of the proteoglycan. AcChT that was photoaffinity-labeled with a tritiated high-affinity analogue of AcCh [cyclohexylmethyl cis-N-(4-azidophenacyl)-N-methylisonipecotate] and then denatured in sodium dodecyl sulfate also immunoprecipitated. The labeled AcChT exhibited a M(r) range of 100,000-200,000. Proteoglycan did not engage in detectable nonspecific reversible aggregation that might mask the presence of another subunit during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In vesicles permeabilized with cholate, the enzymes keratanase and testicular hyaluronidase inactivated binding of vesamicol and destroyed the SV1 epitope without detectable proteolysis. Other glycosaminoglycan-degrading enzymes were without effect. The results demonstrate that the AcChT-VR and proteoglycan are very strongly linked and that glycosaminoglycan-like polysaccharide controls the conformation of the VR. The unexpected linkage to proteoglycan suggests that AcChT-VR in intact terminals might communicate with extracellular matrix and participate in stabilization and operation of the synapse.     

Activation of ribulose bisphosphate carboxylase in intact chloroplasts by CO2 and light

The activity of ribulose 1,5-bisphosphate (RuBP) car?ylase in intact spinach chloroplasts is shown to depend on light and CO₂. This activity was measured upon lysis of chloroplasts and assay of the initial activity using nonlimiting substrate concentrations. Incubation of chloroplasts at 25 °C in the absence of CO₂ results in a gradual inactivation of the RuBP car?ylase. In the presence of CO₂ the initial activity is preserved or increased. CO₂ is also able to reactivate the chloroplast car?ylase previously inactivated in the absence of CO₂. Upon illumination of the chloroplasts, additional activation was observed. This light activation results from an increased affinity for CO₂ of the chloroplast car?ylase. At pH 7.8, the enzyme in dark-adapted chloroplasts required 112 μ m CO₂ for half activation, while in the light it required 24 μ m CO₂. The light activation was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, carbonylcyanide 3-chlorophenylhydrazone, or dl-glyceraldehyde. Part of the light activation is most likely due to increased Mg²⁺ in the stroma. dl-Glyceraldehyde inhibition also suggests that some intermediate of the photosynthetic carbon cycle is involved. These results suggest that photosynthetic CO₂ assimilation in the chloroplast depends upon the amount of activation of the RuBP car?ylase. This activation is regulated by CO₂ and light-induced changes in the chloroplast stroma such as pH, Mg²⁺, and intermediates of the photosynthetic carbon cycle.     

中国西北沙漠夜间活动的东鳖甲属昆虫二新种(鞘翅目:拟步甲科:漠甲亚科)

记述中国西北地区东鳖甲属2新种:巴丹东鳖甲A.badainica Ba,Ren&Liu sp.nov.和古尔班东鳖甲A.gurbantunggutica Ba&Ren sp.nov.,提供了主要鉴别特征和形态图,并简要讨论了其昼夜活动规律.     

Haemosporidian Parasites of Antelopes and Other Vertebrates from Gabon,Central Africa

Re-examination, using molecular tools, of the diversity of haemosporidian parasites (among which the agents of human malaria are the best known) has generally led to rearrangements of traditional classifications. In this study, we explored the diversity of haemosporidian parasites infecting vertebrate species (particularly mammals, birds and reptiles) living in the forests of Gabon (Central Africa), by analyzing a collection of 492 bushmeat samples. We found that samples from five mammalian species (four duiker and one pangolin species), one bird and one turtle species were infected by haemosporidian parasites. In duikers (from which most of the infected specimens were obtained), we demonstrated the existence of at least two distinct parasite lineages related to Polychromophilus species (i.e., bat haemosporidian parasites) and to sauropsid Plasmodium (from birds and lizards). Molecular screening of sylvatic mosquitoes captured during a longitudinal survey revealed the presence of these haemosporidian parasite lineages also in several Anopheles species, suggesting a potential role in their transmission. Our results show that, differently from what was previously thought, several independent clades of haemosporidian parasites (family Plasmodiidae) infect mammals and are transmitted by anopheline mosquitoes.     

Molecular basis of ion permeability in a voltage‐gated sodium channel

Voltage‐gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na⁺ ≈ Li⁺ ≫ K⁺, Ca²⁺, Mg²⁺) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones.     

Oxysterols in the circulation of patients with the Smith-Lemli-Opitz syndrome: abnormal levels of 24S- and 27-hydroxycholesterol

Infants with the cholesterol synthesis defect Smith- Lemli-Opitz syndrome (SLO) have reduced activity of the enzyme 7-dehydrocholesterol-7-reductase and accumulate 7-dehydrocholesterol, with the highest concentration in the brain. As a result of the generally reduced content of cholesterol, plasma levels of oxysterols would be expected to be reduced. 24S-hydroxycholesterol is almost exclusively formed in the brain, whereas 27-hydroxycholesterol is mainly formed from extrahepatic and extracerebral cholesterol. In accordance with the expectations, sterol-correlated plasma levels of 24S-hydroxycholesterol were reduced in infants with SLO (by about 50%). In contrast, the sterol-correlated levels of 27-hydroxycholesterol in the circulation were markedly increased. No side-chain oxidized metabolites of 7-dehydrocholesterol were detected in the circulation. Recombinant human CYP27 had markedly lower 27-hydroxylase activity toward 7-dehydrocholesterol than towards cholesterol. HEK293 cells expressing 24S-hydroxylase active toward cholesterol had no significant activity towards 7-dehydrocholesterol. The plasma levels of 3 beta,7 alpha-dihydroxy-5-cholestenoic in the patients acid were reduced, suggesting a generally reduced metabolism of 27-oxygenated steroids. It is concluded that the accumulation of 7-dehydrocholesterol in the brains of patients with SLO reduces formation of 24S-hydroxycholesterol. The condition is associated with markedly increased circulating levels of 27-hydroxycholesterol, most probably due to reduced metabolism of this oxysterol. We discuss the possibility that the circulating levels of 24S-hydroxycholesterol may be used as a marker for the severity of the disease.--Bj?rkhem, I., L. Starck, U. Andersson, D. Lütjohann, S. von Bahr, I. Pikuleva, A. Babiker, and U. Diczfaulsy. Oxysterols in the circulation of patients with the Smith-Lemli-Opitz syndrome: abnormal levels of 24S- and 27-hydroxycholesterol. J. Lipid Res. 2001. 42: 366--371.