Inhibition of mitochondrial alpha-ketoglutarate dehydrogenase by 1-methyl-4-phenylpyridinium ion

Effects of 1-methyl-4-phenylpyridinium ion (MPP+) on the activities of NAD+- or NADP+-linked dehydrogenases in the TCA cycle were studied using mitochondria prepared from mouse brains. Activities of NAD+- and NADP+-linked isocitrate dehydrogenases, NADH- and NADPH-linked glutamate dehydrogenases, and malate dehydrogenase were little affected by 2 mM of MPP+. However, alpha-ketoglutarate dehydrogenase activity was significantly inhibited by MPP+. Kinetic analysis revealed a competitive type of inhibition. Inhibition of alpha-ketoglutarate dehydrogenase may be one of the important mechanisms of MPP+-induced inhibition of mitochondrial respiration, and of neuronal degeneration.     

Continuous internalization of tumor necrosis factor receptors in a human myosarcoma cell line

The cell dynamics of the receptor for tumor necrosis factor (TNF) were examined in TNF-sensitive KYM cells derived from human myosarcoma. With receptor synthesis inhibited by cycloheximide, the half-life of the surface TNF receptor was 2 h in the absence of TNF and 30 min in its presence, suggesting that the TNF receptor is non-recycling and that its internalization is accelerated by TNF. During cell incubation with TNF receptor degradation suppressed by chloroquine, the number of surface TNF receptors remained approximately constant, but the total number of surface and internal TNF receptors increased gradually, at 3 h reaching 1.5 times the initial number, thus suggesting continuous synthesis, externalization, internalization, and degradation of the TNF receptor in the absence of cycloheximide. On cell incubation with 125I-TNF, the intracellular quantity of the pulse-labeled TNF-receptor complex promptly increased, reaching a maximum at 20 min, and then gradually declined, thus confirming that the TNF receptor is internalized as a TNF-receptor complex in the presence of TNF. During incubations with protein synthesis suppressed by cycloheximide following surface TNF receptor digestion by trypsin, TNF receptors reappeared on the cell surface, increasing in number to a peak at 60 min and gradually decreasing, and cells previously exposed to cycloheximide with or without TNF showed no recurrence of surface TNF receptors, suggesting that the TNF receptor is non-recycling. The results of the study thus suggest that the TNF receptor is continuously internalized and degraded intracellularly by lysosomes without being recycled regardless of the presence or absence of TNF and, further, that its internalization is accelerated when it is part of the TNF-receptor complex.     

13th International Congress of Biochemistry

Cytochrome caa₃ (cytochrome oxidase) from the thermophilic bacterium PS3 can exhibit full catalytic activity in the presence of ascorbate and TMPD or other electron donors and in the absence of added soluble c-type cytochromes. It appears to possess only a low-affinity and not a high-affinity site for the soluble cytochromes. Proteoliposomal cytochrome caa₃ develops an effective membrane potential in the presence of ascorbate and TMPD or PMS, in the absence of added soluble cytochrome c. Reduction of the a₃ centre is blocked in the presence of cyanide. During reductive titrations of the cyanide-inhibited enzyme, electrons initially equilibrate among three centres, the c haem, the a haem and one of the associated Cu atoms. During steady-state turnover, electrons probably enter the complex via the bound c haem; the a haem and perhaps an associated Cu_A atom are reduced next. It is concluded that, despite its size and hydrophobic association with the aa₃ complex, the haem c-containing subunit can behave in an analogous way to that of mammalian cytochrome c, bound at the high-affinity site of the eucaryotic enzyme.     

Different and synergistic actions of human tumor necrosis factor and interferon-gamma in damage of liposome membranes

The effects of human recombinant tumor necrosis factor (TNF) and interferon-gamma (IFN-gamma) in damage of liposome membranes were examined to elucidate the molecular mechanism of their antiproliferative actions on tumor cells. The extent of membrane damage was assayed by measuring the rate of release of the fluorescent dye calcein encapsulated in the liposomes at different pH values in the presence of TNF and/or IFN-gamma. At pH values below about 5, TNF bound to phospholipid liposomes composed of mixtures of phosphatidyl-serine and phosphatidylcholine in molar ratios of 2:1 and 1:2 and caused rapid release of calcein. In contrast, IFN-gamma induced very slow leakage of dye although it bound almost completely to the membranes, suggesting that it causes much less membrane damage than TNF. Small amounts of these two antitumor factors bound to phosphatidylcholine liposomes in the pH range of 4-7, inducing relatively slow leakage of calcein. In the presence of both TNF and IFN-gamma at pH 5, the maximal leakage rate was twice the sum of the rates with the two proteins individually, and the rate depended on the TNF/IFN-gamma ratio, indicating synergistic effects of TNF and IFN-gamma in induction of membrane damage. These different and synergistic actions on liposome membranes may account for the different antitumor properties of the two antitumor cytokines and their synergism.     

Live imaging of <Emphasis Type="Italic">Drosophila</Emphasis>gonad formation reveals roles for Six4 in regulating germline and somatic cell migration

Background  

Movement of cells, either as amoeboid individuals or in organised groups, is a key feature of organ formation. Both modes of migration occur during Drosophila embryonic gonad development, which therefore provides a paradigm for understanding the contribution of these processes to organ morphogenesis. Gonads of Drosophila are formed from three distinct cell types: primordial germ cells (PGCs), somatic gonadal precursors (SGPs), and in males, male-specific somatic gonadal precursors (msSGPs). These originate in distinct locations and migrate to associate in two intermingled clusters which then compact to form the spherical primitive gonads. PGC movements are well studied, but much less is known of the migratory events and other interactions undergone by their somatic partners. These appear to move in organised groups like, for example, lateral line cells in zebra fish or Drosophila ovarian border cells.     

Distribution of retinylester-storing stellate cells in the arrowtooth halibut, Atheresthes evermanni

Hepatic stellate cells play a major role in retinylester storage in mammals, but the retinoid-storing state in nonmammalian vertebrates remains to be elucidated. In this study, we examined retinoids and retinoid-storing cells in the arrowtooth halibut, Atheresthes evermanni. High-performance liquid chromatography analyses revealed the highest concentrations of stored retinoids (retinol and retinylester, 6199 nmol/g) in the pyloric cecum, a teleost-specific organ protruding from the intestine adjacent to the pylorus. Considerable amounts of retinoids were also stored in the intestine (3355 nmol/g) and liver (1891 nmol/g), and small amounts in the kidney (102 nmol/g). Very small amounts or no retinoids were detected in the heart, gill, skeletal muscle, and gonads (less than 2 nmol/g). Use of gold chloride staining and fluorescence microscopy to detect retinoid autofluorescence showed that, in the pyloric cecum and intestine, retinoid-storing cells were localized in the lamina propria mucosae. Under electron microscopy, cells containing well-developed lipid droplets, which are common morphological characteristics of the hepatic stellate cells of mammals, were observed in the lamina propria mucosae of the pyloric cecum. Thus, the distribution of stellate cells with retinoid-storing capacity differs between this halibut and mammals, suggesting that the retinoid-storing site has shifted during vertebrate evolution.