Observations on the oxidoreduction of the two cytochromes b in cytochrome c-deficient mitochondria and submitochondrial particles.

1. In cytochrome c depleted mitochondria cytochrome bT is reduced rapidly upon addition of ATP or slowly during state 4 respiration, but cytochrome bK is effectively reduced in such mitochondira respiring upon glutamate plus malate in all energy states. In mitochondria or in submitochondrial particles oxidized NADH or succinate, cytochromes bK and bT were always reduced and oxidized independently. 2. Difference spectra for the two b cytochromes were obtained in the presence of respiratory chain inhibitors. Reduced cytochrome bK in the presence of cyanide can be reoxidized by CoQ2. Cytochrome bT reduced in the presence of antimycin can be reoxidized by O2 if rotenone is added to an NADH-reduced sysem or malonate to a succinate-reduced system. There is no evidence for electron transfer between the two b cytochromes. 3. It is suggested that there is no electron transfer from cytochrome bT to cytochrome bK, but that a cytochrome bKbT dimer accepts electrons from the CoQ pool jointly with cytochrome c1 and another acceptor, perhaps the FeS centre. The major steady state species is b2K+b3T+, and a Q-loop occurs with reduction of CoQ by the fully reduced species b2K+b2T+. All proposed interactions between CoQ and Complex III are 2-electron processes and the change from 2-electrons to 1-electron transfer occurs within Complex III itself.

The nature of DT-diaphorase (EC 1.6.99.2) activity in plasma membrane of astrocytes in primary cultures.

This is the confirmation of an earlier indication (Mersel, M., Malviya, A.N., Hindelang, C. and Mandel, P. (1984) Biochim. Biophys. Acta 778, 144-154) that the plasma membrane of astrocytes in primary cultures is endowed with DT-diaphorase (EC 1.6.99.2) activity. It is observed that the NADPH-2,6-dichloroindophenol diaphorase activity found in the isolated plasma membrane is not inhibited by dicoumarol. DT-diaphorase-type activity is also observed on the cell surface employing dichloroindophenol as external electron acceptor and it is found to be a dicoumarol-sensitive NADH dehydrogenase.

Permeability of amino acids into liposomes.

1. A simple and rapid assay for the measurement of permeability of amino acids into liposome membrane was carried out by using the liposomes trapping D-amino acid oxidase (D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3) inside the membrane. 2. Permeability of amino acids into liposomes depended on the lipid composition of the membrane. Permeability of amino acids into phosphatidylcholine-cholesterol liposomes depended critically on temperature. 3. Permeability also depended on the structure of amino acids. The order of permeability was norvaline greater than isoleucine greater than leucine greater than phenylalanine greater than tryptophan greater than methionine greater than tyrosine, valine greater than threonine greater than serine greater than alanine greater than glycine.

Plasma membrane isolated from astrocytes in primary cultures. Its acceptor oxidoreductase properties.

Rat astrocytes in primary cultures were employed to isolate the plasma membrane. The method for the isolation of plasma membrane was based on the capacity of the cytoskeleton to adhere to the substratum entrapping intracellular organelles during freezing-thawing cycle performed on the cell. By washing the 'surface adherent framework', the untrapped plasma membrane were recovered and density equilibrium centrifugation resulted in the isolated membrane. The isolated plasma membrane was characterized on the basis of a variety of marker enzymes positive to the plasma membrane such as (Na+ + K+)-ATPase or 5'-nucleotidase as well as the lack of conventional markers of other endomembranes. Ultrastructurally the membranes, as isolated here, were mainly vesicular in nature. The isolated plasma membrane was devoid of the dehydrogenase responsible for NADH-cytochrome c reductase activity. However, NADH-ferricyanide reductase activity and the dehydrogenase system catalyzing the transfer of reducing equivalents from NADH or NADPH to dichloroindophenol seems plasma membrane redox system. The identical specific activity employing dichloroindophenol as an electron acceptor with NADH or NADPH as donor indicate a DT-diaphorase (EC 1.6.99.2) like activity in the astrocytes plasma membrane.

Regulation of signalling pathways to the nucleus by dopaminergic receptors.

Dopamine regulates postsynaptic gene expression in the central nervous system. The pattern of gene expression is different from chronic vs acute stimulation of dopaminergic receptors. Signalling to the nucleus through dopamine receptors involves different second messenger systems, and each receptor subtype regulates multiple effectors. Long term adaptive changes in neuronal function following administration of dopaminergic drugs such as antipsychotic agent or drugs of abuse is one such example of molecular plasticity triggered by dopaminergic receptors. Role of dopaminergic receptors in the control of transcriptional events and immediate early gene regulation are reviewed.

The nuclear inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate receptors.

IP3R is located to the inner nuclear membrane. Nuclear IP3R is recognized as a 220 kD immunoreactive protein by antisera raised against purified rat brain IP3R. Antisera against C-terminal 95-108 peptide fragment derived from rat brain IP3R does not reveal immunoreactivity in the nucleus. Nuclear IP3R is sensitive to heparin and is phosphorylated by nuclear PKC, enhancing the efficiency of IP3 in nuclear calcium release. There are two IP4 binding sites located to the nuclear envelope. The nuclear IP4R is sensitive to pH and pH 6.5 is found optimum for the ligand binding. The high affinity IP4R is associated with the outer nuclear membrane and mediates nuclear calcium uptake by IP4. Low affinity IP4R is identified with the inner nuclear membrane and is not involved in IP4 mediated calcium entry into the nucleus. The nature of IP4R associated with the outer nuclear membrane as compared with the one identified with the inner nuclear membrane remains to be elucidated.

Modulation of cytosolic protein kinase C activity by ferricyanide: priming event seems transmembrane redox signalling. A study on transformed C3H/10T1/2 cells in culture.

Transformed 3T3/10T1/2 cultured cells incubated with ferricyanide caused a decrease of 2 mM EDTA extractable cytosolic protein kinase C activity in 2 min, whereas 5 or 20 min ferricyanide treatment reverted the enzyme activity to that observed without ferricyanide. The ferricyanide effect in 2 min was abolished by amiloride and sustained by ouabain. Thus, deactivation-activation of cytosolic protein kinase C is attributed to an unknown signal generation during H+ accumulation coupled with the Na+/H+ exchange phase. In this mechanism the priming event concerns the transmembrane redox process shedding H+ into the cell interior while impermeant ferricyanide acts as a unique electron acceptor.

Calcium transported to isolated rat liver nuclei by nicotinamide adenine dinucleotide is insensitive to thapsigargin.

Calcium uptake by isolated nuclei was mediated by nicotinamide adenine dinucleotide. Oxidized nicotinamide nucleotide analogues were more effective mediators of nuclear calcium uptake. Thapsigargin inhibited ATP-mediated nuclear calcium transport without affecting NAD-mediated nuclear calcium uptake. Whilst DBHQ did not influence ATP-induced calcium transport, it did stimulate NAD-mediated nuclear calcium entry. Calcium channel blockers did not influence the action of NAD. This study provides a further mechanism for nuclear calcium transport regulated by changes in the cytosolic NAD(+)/NADH ratio.

Unexpected stimulation of mitochondrial ADP-ribosylation by cyanide.

Cyanide, the classical inhibitor of the mitochondrial respiratory chain at site III, stimulates ADP-ribosylation of a number of mitochondrial proteins, the major protein being the 50-55 kDa band. Sodium azide, sharing the same inhibitory site, does not have the same effect. Rotenone or antimycin A have no influence on mitochondrial ADP-ribosylation. Data suggest that no apparent correlation exists between oxidoreductase function and protein ADP-ribosylation. Purified nuclear poly(ADP-ribose) polymerase activity was not affected by cyanide. The cyanide effect on mitochondrial ADP-ribosylation seems intriguing and may be attributed to NAD+-CN complex formation, since NAD reacts with cyanide at pH greater than 8 with N-substituted nicotinamide which may prevent inhibition of ADP-ribosylation.

Separation from protein kinase C--a calcium-independent TPA-activated phosphorylating system.

A calcium-independent but 12-O-tetradecanoylphorbol-13-acetate (TPA)- or diacylglycerol-activated phospholipid-dependent phosphorylating activity has been separated from protein kinase C. This has been made possible by employing calcium-dependent hydrophobic interaction chromatography. The material bound to phenyl-Sepharose in the presence of calcium at low ionic strength was eluted with EGTA and was protein kinase C. While the unbound material passing through the phenyl-Sepharose column showed no appreciable protein kinase C activity, instead it had a high phosphorylating activity manifested in the absence of calcium and in the presence of TPA plus phospholipid. The identification of this phosphorylating activity, distinct from protein kinase C, leads to important clues to cellular responses monitored by TPA in the absence of calcium.

Interaction of antimycin with cytochrome b-561. A study in secretory granules and in plasma membrane isolated from chromaffin cells of bovine adrenal medulla.

Cytochrome b-561 in chromaffin granules interacts with antimycin and its alpha-peak shifts 1 nm towards red. When chromaffin granules were treated with Triton X-100 antimycin no effect was observed. Cytochrome b-561 is located in the plasma membrane isolated from the chromaffin cells. The plasma membrane b-561 does not seem to interact with antimycin. A number of NADH or NADPH (acceptor) oxidoreductase activity has been observed in isolated plasma membrane providing clues to the origin of plasma membrane dehydrogenase. The possible role of cytochrome b-561 in secretory granules other than its accredited energy conserving electron transport property is projected.

Impaired gene transcription and nuclear protein kinase C activation in the brain and liver of aged rats.

The expression of the hsp70 and c-fos genes and the activation of nuclear protein kinase C (PKC) were studied in young and aged whole rats under heat-shock conditions. The induction of hsp70 and c-fos genes by heat shock were decreased several fold in the brain as well as in the liver of senescent animals. Nuclear run-off transcription assay indicated that this age-related impairment could be attributed to a block at the level of transcription. Nuclear PKC activation by heat shock was not apparent in old animals. Nuclear PKC involvement in the repression of transcription during senescence is postulated.

Pervanadate elicits proliferation and mediates activation of mitogen-activated protein (MAP) kinase in the nucleus.

There is growing evidence for the role of protein tyrosine phosphatases in controlling such fundamental cellular processes as growth and differentiation. Pervanadate is a potent inhibitor of protein tyrosine phosphatase which has been observed here to induce proliferation in C3H10T1/2 mouse fibroblasts. Pervanadate also translocated/activated p42/44 mitogen-activated protein (MAP) kinase to the cell nucleus. An almost similar pattern of nuclear p42/44 MAP kinase stimulation is seen with TPA. On the other hand, TPA treatment results in a rapid activation of cytosolic MAP kinase which declines with time. Thus pervanadate appears as a very useful tool for studying tyrosine phosphorylation.

Pervanadate-triggered MAP kinase activation and cell proliferation are not sensitive to PD 98059. Evidence for stimulus-dependent differential PD 98059 inhibition mechanism.

A tight and stable complex with corresponding protein kinases and phosphatases establishes coupling between activators and inactivators. One such example is emerging from the studies of the Ras-dependent MAP kinase cascade signaling pathway. Pervanadate, a potent inhibitor of protein tyrosine phosphatase, stimulates MAP kinase and elicits cell proliferation in cultured mouse fibroblasts which is insensitive to PD 98059, the major inhibitor of upstream MEK, whereas serum- or TPA-triggered proliferation is sensitive to PD 98059. It is suggested that imbalanced coordination between protein kinase and protein phosphatase determines the cellular responses such as cell proliferation. The PD 98059-insensitive cell proliferation upon protein tyrosine phosphatase inhibition is attributed to a MEK bypass pathway.

Effects of cadmium on nuclear protein kinase C.

Cadmium is a carcinogen whose genotoxicity is only weak. Besides its tumor-initiating capacity, cadmium may be tumor-promoting, since it interferes with several steps of cellular signal transduction. We have investigated effects of cadmium(II) on protein kinase C (PKC), which is a key enzyme in the control of cellular growth and differentiation. Tumor-promoting phorbol esters cause an activation and translocation of PKC from the cytosol to the plasma membrane and to the nucleus of mammalian cells. In mouse 3T3/10 T 1/2 fibroblasts, cadmium(II) potentiated the effect of phorbol ester on nuclear binding and activation of PKC. Furthermore, in a reconstituted system consisting of rat liver nuclei and rat brain PKC, cadmium stimulated the binding of the enzyme to a 105-kDa protein. We propose a model in which cadmium(II) substitutes for zinc(II) in the regulatory domain of PKC, thus rendering the putative protein-protein binding site exposed. Further work is required to elucidate the potential role of the nuclear PKC binding protein(s) in the control of cell proliferation.

Evaluation of four coagulation tests to detect plasma lupus anticoagulants.

Lupus anticoagulant was detected in 205 newly diagnosed, untreated patients with systemic lupus erythematosus by the following tests: kaolin clotting time, activated partial thromboplastin time, plasma prothrombin time, and, in the last 99 patients, by dilute Russell's viper venom time. In 10 patients, lupus anticoagulant was detected by kaolin clotting time prolongation, corrected by inosithin but not by normal plasma; 12 and 6 of them had prolonged activated partial thromboplastin time and partial plasma prothrombin time, respectively. Only 10 patients had a history of recurrent abortions and/or thrombosis, nine of whom had lupus anticoagulant as shown by the kaolin clotting time test. Of the 99 patients studied by all four tests, 9 showed lupus anticoagulant by both kaolin clotting time and dilute Russell's viper venom time; 7 had a history of abortion and/or thrombosis. The dilute Russell's viper venom time test is easy to perform and not affected by inhibitors to factor VIII or IX. It is recommended as a primary screening test for lupus anticoagulant detection in a hospital clinical laboratory.

Inosithin neutralization test to measure lupus anticoagulants.

The inosithin neutralization test was performed in 14 patients in whom lupus anticoagulant was detected. To test its specificity, it was also performed in 10 patients with severe hemophilia and in three patients with Factor VIII inhibitors. Prolonged kaolin clotting time was corrected by adding varying amounts of inosithin (Asolectin, 0.19 to 100 micrograms), a soybean-derived phospholipid, in all patients with lupus anticoagulant but not in patients with hemophilia or in two patients with Factor VIII inhibitors. In one patient, both Factor VIII inhibitors and lupus anticoagulant were present. The concentration of lupus anticoagulant in a patient was expressed as the amount of inosithin (measured in micrograms) required to normalize the prolonged kaolin clotting time. This amount correlated significantly with the occurrence of thrombosis and/or recurrent abortion. The inosithin neutralization test is useful to measure lupus anticoagulant.

Neuroleptics differentially induce jun family genes in the rat striatum.

The effect of neuroleptics in single administration on the expression of genes of the jun family was studied in the rat striatum. jun B, but not jun D was dose-dependently and transiently induced. This effect was blocked by pretreatment with a specific dopamine D2 agonist. The expression of c-jun was also stimulated, although this did not reach statistical significance. Thus dopamine D2 receptors differentially regulate the expression of jun family members in the striatum.

Up-regulation of dopamine D2 receptor mRNA in rat striatum by chronic neuroleptic treatment.

The effect of prolonged administration of antagonists on rat striatal dopamine D2 receptor binding and mRNA content was examined. Both haloperidol (2 and 4 mg/kg) and sulpiride (10 mg/kg) induced a significant rise in total D2 and D2(444) mRNA level and in Bmax. Regulation of messenger RNA accumulation is thus an important determinant of dopamine D2 receptor density.

Phosphatidic acid activation of protein kinase C in LA-N-1 neuroblastoma cells.

Phosphatidic acid (PA), a hydrolytic product of phospholipase D activity, stimulated cytosolic protein kinase C (PKC) activity when LA-N-1 neuroblastoma cells in culture were treated with PA, without translocating the enzyme to the membrane. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) translocated and activated PKC in a dogmatic manner. Partially purified PKC activity derived from LA-N-1 neuroblastoma cells was stimulated by PA alone or in the presence of phosphatidylserine or TPA, without affecting [3H]phorbol dibutyrate binding, indicating that the site of action of PA was different from the phorbol ester or diacylglycerol binding site. These results suggest an unorthodox pattern of PKC stimulation mediated by PA which appears to be yet another mode of PA signal transduction.