Effects of hydroxylated polychlorinated biphenyls on mouse liver mitochondrial oxidative phosphorylation

The effects of three tetrachlorobiphenylols [2′,3′,4′,5′-tetrachloro-2-biphenylol (1); 2′,3′,4′,5′-tetrachloro-4-biphenylol (2); and 2′,3′,4′,5′-tetrachloro-3-biphenylol (3)]; three monochlorobiphenylols [5-chloro-2-biphenylol (5), 3-chloro-2-biphenylol (6); and 2-chloro-4-biphenylol (7)] and a tetrachlorobiphenyldiol [3,3′,5,5′-tetrachloro-4,4′-biphenyldiol (4) on respiration, adenosine triphosphatase (ATPase)] activity, and swelling in isolated mouse liver mitochondria have been investigated. Tetrachlorobiphenylols (1–3) and the tetrachlorobiphenyldiol (4) inhibited state-3 respiration in a concentration-dependent manner with succinate as substrate (flavin adenine dinucleotide [FAD]-linked) and the tetrachlorobiphenyldiol (4) caused a more pronounced inhibitory effect on state-3 respiration than the other congeners. The monochlorobiphenylols 5–7 were less active as inhibitors of state-3 mitochondrial respiration and significant effects were observed only at higher concentration (≥0.4 μM). However, in the presence of the nicotinamide adenine dinucleotide (NAD)-linked substrates (glutamate plus malate), hydroxylated PCBs (1–7) significantly inhibited mitochondrial state-3 respiration in a concentration-dependent manner. Compounds 5, 6, and 7 uncoupled mitochondrial oxidative phosphorylation only in the presence of FAD-linked substrate as evidenced by increased oxygen consumption during state-4 respiratory transition, stimulating ATPase activity, releasing oligomycin-inhibited respiration, and inducing mitochondrial swelling (5, 6, and 7). Tetrachlorobiphenylols 1, 2, and 3 had no effect on mitochondrial ATPase activity while the tetrachlorobiphenyldiol, 4, decreased the enzyme activity. The possible inhibitory site of electron transport by these compounds and their toxicologic significance is discussed.     

Differential activation of wild-type and variant forms of estrogen receptor alpha by synthetic and natural estrogenic compounds using a promoter containing three estrogen-responsive elements

Structure-dependent estrogen receptor alpha (ER alpha) agonist and antagonist activities of synthetic and natural estrogenic compounds were investigated in human HepG2, MDA-MB-231 and U2 cancer cell lines. Compounds used in this study include 4'-hydroxytamoxifen, ICI 182,780, bisphenol-A (BPA), 2',4',6'-trichloro-4-biphenylol (3Cl-PCB-OH), 2',3',4',5'-tetrachloro-4-biphenylol (4Cl-PCB-OH), p-t-octylphenol, p-nonylphenol, naringenin, kepone, resveratrol, and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE). Cells were transfected with a construct (pERE(3)) containing three tandem estrogen responsive elements (EREs) and either wild-type estrogen receptor alpha (ER-wt) or variants expressing activation function-1 (ER-AF1) or AF-2 (ER-AF2). The ER agonist activities of the synthetic mono and dihydroxy aromatic compounds are comparable in all three-cell lines, whereas the activities of naringenin, kepone and resveratrol are dependent on cell context and expression of wild-type or variant forms of ER alpha. In contrast, the ER antagonist activities for these compounds were highly complex and, with the exception of 3Cl-PCB-OH, all compounds inhibited E2-induced wild-type or variant ER action. Results of this in vitro study suggest that the estrogenic and antiestrogenic activity of structurally diverse synthetic and natural estrogenic compounds is complex, and this is consistent with published data that often give contradictory results for these compounds.     

Activation of kinase pathways in MCF-7 cells by 17beta-estradiol and structurally diverse estrogenic compounds

17beta-Estradiol (E2) activates non-genomic pathways in MCF-7 cells, and this study investigates the effects of structurally-diverse estrogenic compounds on activation of mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3-K), protein kinase C (PKC), PKA, and calcium calmodulin-dependent kinase IV (CaMKIV). Activation of kinases was determined by specific substrate phosphorylation and transactivation assays that were diagnostic for individual kinases. The compounds investigated in this study include E2, diethylstilbestrol (DES), the phytoestrogen resveratrol, and the following synthetic xenoestrogens, bisphenol-A (BPA), nonylphenol, octylphenol, endosulfan, kepone, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), and 2',3',4',5'-tetrachloro-4-biphenylol (HO-PCB-Cl(4)). With the exception of resveratrol, all the compounds activated PI3-K and MAPK. Activation of PKC by the xenoestrogens was structure-dependent since resveratrol, kepone and HO-PCB-Cl(4) were inactive and only minimal activation of PKA was observed. CaMKIV was activated only by E2 and DES, and HO-PCB-Cl(4) was a potent inhibitor of CaMKIV-dependent activity. These results demonstrate that activation of estrogen receptor-alpha-mediated non-genomic pathways by estrogenic compounds in MCF-7 cells is structure-dependent and can result in activation or inhibition of kinase activities.     

Cyclosporin A-induced oxidative stress is not the consequence of an increase in mitochondrial membrane potential

Cyclosporin A induces closure of the mitochondrial permeability transition pore. We aimed to investigate whether this closure results in concomitant increases in mitochondrial membrane potential (DeltaPsim) and the production of reactive oxygen species. Fluorescent probes were used to assess DeltaPsim (JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl-carbocyanine iodide), reactive oxygen species [DCF, 5- (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester] and [Ca2+][Fluo-3, glycine N-[4-[6-[(acetyloxy)methoxy]-2,7-dichloro-3-oxo-3H-xanthen-9-yl]-2-[2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxyethyl]amino]-5-methylphenoxy]ethoxy]phenyl]-N-[2-[(acetyloxy)methoxy]-2-oxyethyl]-(acetyloxy)methyl ester] in human kidney cells (HK-2 cells) and in a line of human small cell carcinoma cells (GLC4 cells), because these do not express cyclosporin A-sensitive P-glycoprotein. We used transfected GLC4 cells expressing P-glycoprotein as control for GLC4 cells. NIM811 (N-methyl-4-isoleucine-cyclosporin) and PSC833 (SDZ-PSC833) were applied as selective mitochondrial permeability transition pore and P-glycoprotein blockers, respectively. To study the effect of cyclosporin A on mitochondrial function, we isolated mitochondria from fresh pig livers. Cyclosporin A and PSC833 induced a more than two-fold increase in JC-1 fluorescence in HK-2 cells, whereas NIM811 had no effect. None of the three substances induced a significant increase in JC-1 fluorescence in GLC4 cells. Despite this, cyclosporin A, NIM811 and PSC833 induced a 1.5-fold increase in DCF fluorescence (P<0.05) and a two-fold increase in Fluo-3 fluorescence (P<0.05). Studies in isolated mitochondria showed that blockage of mitochondrial permeability transition pores by cyclosporin A affected neither DeltaPsim, ATP synthesis, nor respiration rate. The mitochondrial permeability transition pore blockers cyclosporin A and NIM811, but also the non-mitochondrial permeability transition pore blocker PSC833, induced comparable degrees of reactive oxygen species production and cytosolic [Ca2+]. Neither mitochondria, effects on P-glycoprotein nor inhibition of calcineurin therefore play a role in cyclosporin A-induced oxidative stress and disturbed Ca2+ homeostasis.     

The mitochondrial ATPase. Selective modification of a nitrogen residue in the beta subunit.

1. When mitochondrial ATPase, which has been modified on a single tyrosine residue by 4-chloro-7-nitrobenzofurazan, is incubated at pH 9.0, the 7-nitrobenzofurazan group undergoes an intramolecular transfer to a nitrogen residue. The rate of this transfer is sensitive to the binding of adenine nucleotides to the enzyme. The resulting N-nitrobenzofurazan ATPase has little or no activity. 2. The fluorescence of the N-nitrobenzofurazan group in the modified ATPase is quenched on binding of ADP. 3. Electrophoresis of the modified enzyme in sodium dodecyl sulphate on a 10% polyacrylamide gel shows that the fluorescence of the N-nitrobenzofurazan chromophore is exclusively in the beta subunit. 4. The rate of transfer of the nitrobenzofurazan group from tyrosyl oxygen to nitrogen on the enzyme is compared with the rate of transfer between model compounds. 5. The interaction of the N-nitrobenzofurazan ATPase with aurovertin is reported.     

Microbiology of Saturated Salt Solutions and Other Harsh Environments. IV. New Observations of Ribonucleotide-Induced Recovery of KCl-Habituated Penicillium notatum

Salt-tolerant mutant Penicillium notatum sub-cultured in a glucose-peptone broth saturated with KCl shows continued attenuated growth when transferred to salt-free broth. Additional tests have shown E. coli S-RNA to be inferior to yeast RNA preparations, that base-free phosphate sources are inactive, but that nicotinamide adenine dinucleotide and flavine adenine dinucleotide are moderately active. All phosphate derivatives of adenine, cytosine and guanosine and inosine were active including 5'-polyphosphates, 3'(2')-monophosphates 5'-monophosphates, and adenine 3', 5'-cyclic monophosphate. Uracil derivatives were of low activity at best.Among base precursors, orotic acid was moderately active whereas imidazoles were not. The high activity of inosine 5'-phosphate a precursor of other purine nucleotides suggested that one mode of KCl action might involve a block in conversion of 4-amino-5-imidazole carboxamide ribonucleoside to the hypoxanthine nucleotide.     

Substrate specificity of soluble mitochondrial ATPase]

The parameters of the hydrolysis of ATP and several analogs by soluble mitochondrial ATPase were determined. Vmax of the reaction decreases within the range: 2'-desoxy-ATP greater than ATP greater than etheno-ATP greater than GTP greater than 3'-O-methylATP greater than UTP. ATP, 2'-desoxypATP, 3'O-methyl-ATP, GTP, and etheno-ATP are hydrolysed by soluble mitochondrial ATPase with close Km(app) values. CTP is not hydrolysed by the enzyme and does not inhibit the ATPase reaction at a concentration of 10(-2) M. Nucleoside triphosphate derivatives with an "open" ribose cycle 9-[1',5'-dihydroxy-4-(S)-hydroxymethyl-3'-oxapent-2' (R)-yl]adenyl-5'-triphosphate, and 1-[1',5'-dihydroxy-4'-(S)-hydroxymethyl-3'-oxapent-2'(R)-yl[cytosine-5'-triphosphate are effective inhibitors of ATPase (Ki approximately 5.10(-5)M). Mitochondrial ATPase binds the ATP analogs that have hydrocarbon radicals-(CH2)2-, -(CH2)3-, and (CH2)4- instead of the ribose residues: 9-(2'hydroxyethyl)adenyl-2'-triphosphate, 9-(3'-hydroxypropyl)-adenine-3'-triphosphate, and 9-(4'-hydroxybutyl)adenine-4'-triphosphyl)adenine-4'-triphosphate were not hydrolysed by the enzyme, although they inbibit the ATPase reaction (Ki 2.10(-4)M). 9-(2'-hydroxyethyl)adenine-2'-triphosphate is hydrolysed by ATPase eight times more slowly than ATP. It is suggested that the hydrolysis of the substrates of mitochondrial ATPase is- preceded by the binding of the substrates in a tense conformation in the active site of the enzyme.     

2,2',6,6'-Tetrachlorobiphenyl is estrogenic in vitro and in vivo

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants whose effects on biological systems depend on the number of and the positions of the chlorine substitutions. In the present study we examined the estrogenicity of the fully ortho-substituted PCB, 2,2',6,6'-tetrachlorobiphenyl (2,2',6,6'-TeCB). This PCB was chosen as the prototypical ortho-substituted PCB to test the hypothesis that ortho-substitution of a PCB with no para- or meta-chlorine-substitutions results in enhanced estrogenic activity. The results indicate that 2,2',6,6'-TeCB is estrogenic both in vitro, in the MCF-7 cell focus assay, and in vivo, in the rat uterotropic assay. The estrogenic activity elicited by the addition of 5 microM 2,2',6,6'-TeCB to the medium of MCF-7 cultures was inhibited by the estrogen receptor (ER) antagonist, LY156758, suggesting that 2,2',6,6'-TeCB or a metabolite is acting through an ER-dependent mechanism. Results from competitive binding assays using recombinant human (rh) ER indicate that 2,2',6,6'-TeCB does not bind rhERalpha or rhERbeta. A metabolite of 2,2',6,6'-TeCB, 2,2',6,6'-tetrachloro-4-biphenylol (4-OH-2,6,2',6'-TCB), does bind rhERalpha and rhERbeta and is also 10-fold more estrogenic than 2,2',6,6'-TeCB in the MCF-7 focus assay; however, this metabolite is not detected in the medium of MCF-7 cultures exposed to 2,2',6,6'-TeCB. Taken together, the results suggest that the estrogenicity observed in human breast cancer cells and the rat uterus may be due to 1) an undetected metabolite of 2,2',6,6'-TeCB binding to the ER, 2) 2,2',6,6'-TeCB binding directly to a novel form of the ER, or 3) an unknown mechanism involving the ER.     

Effects of ethacrynic acid and furosemide on phosphory-lation reactions of kidney mitochondria. Inhibition of the adenine nucleotide translocase

Previous reports that ethacrynic acid and furosemide diminish mitochondrial P : O ratios and reduce (Na⁺ + K⁺)-ATPase activity suggested that these diuretics may inhibit mitochondrial phosphorylation reactions. This possibility was initially studied by determining the effects of ethacrynic acid and furosemide on [³²P]ATP exchange activity of rat kidney mitochondria. Concentrations of both drugs at 10⁻⁴ M or greater, significantly inhibited [³²P]ATP exchange. To investigate the mechanism of this inhibition, the effects of ethacrynic acid and furosemide on the ATPase activity of intact mitochondria and sonicated submitochondrial particles were determined. Both diuretics inhibited ATPase activity of intact mitochondria at 10⁻⁴ M. In contrast, ATPase of submitochondrial particles was significantly less susceptible to inhibition by the diuretics. These results suggested that ethacrynic acid and furosemide inhibit adenine nucleotide transport across the mitochondrial membrane. This was directly tested by determining the effects of the diuretics on the mitochondrial adenine nucleotide translocase. At 5 · 10⁻⁴ M, both ethacrynic acid and furosemide significantly inhibited adenine nucleotide transport. These findings suggest that ethacrynic acid and furosemide may diminish renal tubular solute reabsorption by direct inhibition of adenine nucleotide transport across the mitochondrial inner membrane.     

Probing the catalytic subunit of the tonoplast H+-ATPase from oat roots. Binding of 7-chloro-4-nitrobenzo-2-oxa-1,3,-diazole to the 72-kilodalton polypeptide

The purified tonoplast H+-ATPase from oat roots (Avena sativa L. var. Lang) consists of at least three different polypeptides with masses 72, 60, and 16 kDa. We have used covalent modifiers (inhibitors) and polyclonal antibodies to identify the catalytic subunit of the H+-pumping ATPase. The inactivation of ATPase activity by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (Nbd-Cl, an adenine analog) was protected by MgATP or MgADP, and showed kinetic properties consistent with active site-directed inhibition. Under similar conditions, [14C]Nbd-Cl preferentially labeled the 72-kDa polypeptide of the purified ATPase. This binding was reduced by MgATP or 2' (3')-)O-(2,4,6-trinitrophenyl) ATP. Nbd-Cl probably modified cysteinyl--SH or tyrosyl--OH groups, as dithiothreitol reversed both ATPase inactivation and [14C]Nbd-Cl binding to the 72-kDa subunit. The finding that N-ethylmaleimide inhibition of ATPase activity was protectable by nucleotides is consistent with the idea of sulfhydryl groups in the ATP-binding site. Polyclonal antibody made to the 72-kDa polypeptide specifically reacted (Western blot) with a 72-kDa polypeptide from both tonoplast-enriched membranes and the purified tonoplast ATPase, but it did not cross-react with the mitochondrial or Escherichia coli F1-ATPase. The antibody inhibited tonoplast ATPase and H+-pumping activities. We conclude from these results that the 72-kDa polypeptide of the tonoplast H+-ATPase contains an ATP- (or nucleotide-) binding site that may constitute the catalytic domain.     

Differential activation of wild-type estrogen receptor alpha and C-terminal deletion mutants by estrogens, antiestrogens and xenoestrogens in breast cancer cells

17beta-Estradiol (E2), diethylstilbestrol (DES) and several synthetic (or xenoestrogenic) compounds induced transactivation in MCF-7 or MDA-MB-231 cells transfected with wild-type estrogen receptor alpha (ERalpha) and a construct (pERE(3)) containing three tandem estrogen responsive elements (EREs) linked to a luciferase gene. In contrast, the antiestrogens ICI 182,780 and 4-hydroxytamoxifen (4-OHT) were inactive in this assay. We have investigated the effects of these compounds and several structurally-diverse estrogenic compounds on transactivation in cells transfected with pERE(3) and wild-type ERalpha, mutant ERalpha (1-553), and ERalpha (1-537) containing deletions of amino acids 595-554 and 595-538, respectively. These constructs were used to develop an in vitro assay to distinguish between different structural classes of estrogenic compounds. The results obtained using these constructs were highly cell context- and structure-dependent. Neither E2- nor diethylstilbestrol-induced transactivation in MCF-7 (or MDA-MB-231) cells transfected with pERE(3)/ERalpha (1-537) due to partial deletion of helix 12; however, octylphenol and nonlylphenol, resveratrol (a phytoestrogen), kepone and 2',3',4',5'-tetrachloro-4-biphenylol were "estrogenic" in MCF-7 cells transfected with pERE(3)/ERalpha (1-537). Moreover, the structure-dependent estrogenic activities of several synthetic estrogens (xenoestrogens) in MDA-MB-231 cells were different than those observed in MCF-7 cells. These results demonstrate that the estrogenic activity of many synthetic compounds do not require activation function 2 (AF-2) of ERalpha and are mechanistically different from E2. These data suggest that xenoestrogens are selective ER modulators (SERMs).     

Respiration and mitochondrial ATPase in energized mitochondria during isoproterenol-induced cell injury of myocardium.

1. Respiration of mitochondria, membrane potential and mitochondrial ATPase under energized conditions were studied in rat myocardium during cell injury induced by treatment with isoproterenol. 2. Increase in the state 4 rate of respiration and ADP:O ratio, as well as decrease in the state 3 rate and Respiratory Control Ratio (RCR) were found. 3. The optimum pH for RCR and for maximum ATPase activity was shifted to lower values. 4. The state 3 respiration was more sensitive to oligomycin inhibition. 5. The mitochondria showed lower ability to generate membrane potential. 6. An increase in the K0.5 values for catalytic sites II and III of mitochondrial ATPase at pH 7.4 and 5.5 was found. 7. These results are consistent with alterations on the integrity of mitochondrial membrane, and corroborate with the hypothesis of changes on the mitochondrial ATPase during isoproterenol-induced cell injury of myocardium.     

Effects of ethacrynic acid and furosemide on phosphorylation reactions of kidney mitochondria. Inhibition of the adenine nucleotide translocase.

Previous reports that ethacrynic acid and furosemide diminish mitochondrial P : O ratios and reduce (Na+ + K+)-ATPase activity suggested that these diuretics may inhibit mitochondrial phosphorylation reactions. This possibility was initially studied by determining the effects of ethacrynic acid and furosemide on [32P]ATP exchange activity of rat kidney mitochondria. Concentrations of both drugs at 10(-4) M or greater, significantly inhibited [32P]ATP exchange. To investigate the mechanism of this inhibition, the effects of ethacrynic acid and furosemide on the ATPase activity of intract mitochondria and sonicated submitochondrial particles were determined. Both diuretics inhibited ATPase activity of intact mitochondria at 10(-4) M. In contrast, ATPase of submitochondrial particles was significantly less susceptible to inhibition by the diuretics. These results suggested that ethacrynic acid anf furosemide inhibit adenine nucleotide transport across the mitochondrial membrane. This was directly tested by determining the effects of the diretics on the mitochondrial adenine nucleotide translocase. At 5-10(-4) M, both ethacrynic acid and furosemide significantly inhibited adenine nucleotide transport. These findings suggest that ethacrynic acid and furosemide may diminish renal tubular solute reabsorption by direct inhibition of adenine nucleotide transport across the mitochondrial inner membrane.     

The mitochondrial ATPase. Evidence for a single essential tyrosine residue.

1. Evidence is presented which indicates that inactivation of the mitochondrial ATPase from bovine heart by the reagent 4-chloro-7-nitrobenzofurazan results from modification of one tyrosine residue per enzyme molecule. Activity can be restored by a variety of sulphydryl reagents. 2. In sodium dodecyl sulphate, the nitrogenzofurazan group on tyrosine is transfered to newly exposed sulphydryl groups on the enzyme. 3. The rate of transfer of the nitrobenzofurazan moiety from theenzyme to sulphydryl compounds is compared with that for transfer from the model compound N-acetyl-tyrosine-0(7-nitrobenzo-furazan) ethyl ester, the synthesis and properties of which are also described. 4. The ligands ATP and ADP exert a protective effect on the rate of reaction between the mitochondrial ATPase and 4-chloro-7-nitrobenzofurazan. The variation in rate of this reaction with change in pH has also been examined and a pKa of 9.5 estimated for the tyrosine residue. 5. The modification does not prevent substrate binding as judged by changes in the fluorescence of aurovertin, an antibiotic with specific affinity for mitochondiral ATPases. 6. When the ATPase activity of submitochondrial particles is inhibited by 4-chloro-7-nitrobenzo-furazan, there is a parallel decrease in the extent of the energy-linked fluorescence enhancement of 1-anilino-naphthalene-8-sulphonate induced by ATP hydrolysis. Both ATPase activity and the fluorescence enhancement are restored by sluphydryl reagents.     

Deterioration of rat liver mitochondria under conditions of metabolite deprivation.

In a previous study [Parce, Cunningham & Waite (1978) Biochemistry 17, 1634-1639] changes in mitochondrial phospholipid metabolism and energy-linked functions were monitored as coupled mitochondria were aged in iso-osmotic sucrose solution at 18 degrees C. The sequence of events that occur in mitochondrial deterioration under the above conditions have been established more completely. Total adenine nucleotides are depleted early in the aging process, and their loss parallels the decline in respiratory control. Related to the loss of total adenine nucleotides is a dramatic decrease in ADP and ATP translocation (uptake). The decline of respiratory control is due primarily to a decrease in State-3 respiration; loss of this respiratory activity can be related to the decline in ADP translocation. Mitochondrial ATPase activity does not increase significantly until State-4 respiration has increased appreciably. At the time of loss of respiratory control the ATPase activity increases to equal the uncoupler-stimulated activity. The H+/O ratio and P/O ratios do not decrease appreciably until respiratory control is lost. Similarly, permeability of the membrane to the passive diffusion of protons increases only after respiratory control is lost. There observations reinforce our earlier conclusion that there are two main phases in mitochondrial aging. The first phase is characterized by loss of the ability to translocate adenine nucleotides. The second phase is characterized by a decline in the ability of the mitochondrion to conserve energy (i.e. maintain a respiration-driven proton gradient) and to synthesize ATP.     

tBid interaction with cardiolipin primarily orchestrates mitochondrial dysfunctions and subsequently activates Bax and Bak

TNFR1/Fas engagement results in the cleavage of cytosolic Bid to truncated Bid (tBid), which translocates to mitochondria. We demonstrate that recombinant tBid induces in vitro immediate destabilization of the mitochondrial bioenergetic homeostasis. These alterations result in mild uncoupling of mitochondrial state-4 respiration, associated with an inhibition the adenosine diphosphate (ADP)-stimulated respiration and phosphorylation rate. tBid disruption of mitochondrial homeostasis was inhibited in mitochondria overexpressing Bcl-2 and Bcl-XL. The inhibition of state-3 respiration is mediated by the reorganization of cardiolipin within the mitochondrial membranes, which indirectly affects the activity of the ADP/ATP translocator. Cardiolipin-deficient yeast mitochondria did not exhibit any respiratory inhibition by tBid, proving the absolute requirement for cardiolipin for tBid binding and activity. In contrast, the wild-type yeast mitochondria underwent a similar inhibition of ADP-stimulated respiration associated with reduced ATP synthesis. These events suggest that mitochondrial lipids rather than proteins are the key determinants of tBid-induced destabilization of mitochondrial bioenergetics.     

Similarities and differences between the tonoplast-type and the mitochondrial H+-ATPases of oat roots

The native tonoplast and the mitochondrial H+-ATPase from oat roots were compared to determine whether the two enzymes have similar mechanisms. H+ pumping in low-density microsomal vesicles reflected activity from the tonoplast-type ATPase, as ATPase activity and ATP-dependent H+ pumping (quinacrine fluorescence quenching) showed similar sensitivities to inhibition by N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, 4,4'-diisothiocyano-2,2'-stilbene disulfonate, nitrate, quercetin, or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. The tonoplast-type ATPase was stimulated by C1-,Br- greater than HCO3- whereas the mitochondrial ATPase was stimulated by HCO3- much greater than C1-,Br-. Both enzymes hydrolyzed ATP preferentially and were inhibited competitively by AMP or ADP. Apart from resistance to azide, the tonoplast-type ATPase was strikingly similar in its inhibitor sensitivities to the mitochondrial ATPase. The insensitivity to vanadate of both enzymes suggests the reaction mechanisms do not involve a covalent phosphoenzyme. Inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide and protection by ATP suggests tyrosine and cysteine residues are in the catalytic site of the tonoplast ATPase. The mitochondrial ATPase was 100 times more sensitive to N,N'-dicyclohexyl-carbodiimide inhibition than the tonoplast H+-ATPase. These results suggest the tonoplast and the mitochondrial H+-ATPases share common steps in their catalytic and vectorial reaction mechanisms, yet sufficient differences exist to indicate they are two distinct ATPases.     

Sulfation of hydroxychlorobiphenyls. Molecular cloning, expression, and functional characterization of zebrafish SULT1 sulfotransferases.

As a first step toward developing a zebrafish model for investigating the role of sulfation in counteracting environmental estrogenic chemicals, we have embarked on the identification and characterization of cytosolic sulfotransferases (STs) in zebrafish. By searching the zebrafish expressed sequence tag database, we have identified two cDNA clones encoding putative cytosolic STs. These two zebrafish ST cDNAs were isolated and subjected to nucleotide sequencing. Sequence data revealed that the two zebrafish STs are highly homologous, being approximately 82% identical in their amino acid sequences. Both of them display approximately 50% amino acid sequence identity to human SULT1A1, rat SULT1A1, and mouse SULT1C1 ST. These two zebrafish STs therefore appear to belong to the SULT1 cytosolic ST gene family. Recombinant zebrafish STs (designated SULT1 STs 1 and 2), expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as approximately 35 kDa proteins on SDS/PAGE. Purified zebrafish SULT1 STs 1 and 2 displayed differential sulfating activities toward a number of endogenous compounds and xenobiotics including hydroxychlorobiphenyls. Kinetic constants of the two enzymes toward two representative hydroxychlorobiphenyls, 3-chloro-4-biphenylol and 3,3',5,5'-tetrachloro-4,4'-biphenyldiol, and 3,3',5-triiodo-l-thyronine were determined. A thermostability experiment revealed the two enzymes to be relatively stable over the range 20-43 degrees C. Among 10 different divalent metal cations tested, Co2+, Zn2+, Cd2+, and Pb2+ exhibited considerable inhibitory effects, while Hg2+ and Cu2+ rendered both enzymes virtually inactive.     

Adaptive thermogenesis in Brandt's vole (Lasiopodomys brandti) during cold and warm acclimation

Brandt's voles (Lasiopodomys brandti) exposed to cold (5±1 °C) or warm (23±1 °C) showed some physiological and biochemical variations which might be important in adaptation to their environments. Cold acclimation induced increases in resting metabolic rate (RMR) and the serum triiodothyronine (T₃) level, the state-4 respiration of liver and muscle mitochondria were activated after 7 days when animals exposed to cold, and the activity of cytochrome c oxidase (COX) of liver and muscle mitochondria tended to rise with cold exposure. RMR and T₃ level decreased during warm acclimation. The state-4 respiration of liver mitochondria declined after 3 days and muscle after 7 days when animals exposed to warm, and the activities of COX of liver and muscle mitochondria tended to decrease with warm acclimation. The cold activation of liver and muscle mitochondrial respiration (regulated by T₃) was one of the cytological mechanisms of elevating RMR. Both state-4 respiration and COX activity of brown adipose tissue (BAT) mitochondria increased significantly during cold acclimation and decreased markedly after acclimated to warm. The uncoupling protein 1 (UCP1) contents in BAT increased after exposure to cold and decreased after warm acclimation. Nonshivering thermogenesis (NST) plays an important role in the process of thermoregulation under cold acclimation for Brandt's voles. Changes in thermogenesis is a important way to cold adaptation for Brandt's voles in natural environments.     

Synthesis of 5-chloroformycin A, 5-chloro-2''-deoxyformycin A and certain related 5,7-disubstituted 3-beta-D-ribofuranosylpyrazolo[4,3-d] pyrimidines from formycin A.

A facile synthesis of 7-amino-5-chloro-3-beta-D-ribofuranosylpyrazolo [4,3-d]pyrimidine (5-chloroformycin A, 6), 7-amino-5-chloro-3-(2-deoxy-beta-D-erythro-pentofuranosyl) pyrazolo [4,3-d]-pyrimidine (5-chloro-2'-deoxyformycin A, 13) and certain related 5,7-disubstituted pyrazolo[4,3-d]pyrimidine ribonucleosides is described starting with formycin A. Thiation of tri-O-acetyloxoformycin B (4b) with phosphorus pentasulfide, followed 3-beta-D-ribofuranosyl-7-thioxopyrazolo[4,3-d] pyrimidin-5(1H,4H,6H)-one (3b) in excellent yield. Chlorination of 4b with either phosphorus oxychloride or phenyl phosphonicdichloride furnished the key intermediate 5,7-dichloro-3-(2,3, 5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo[4,3-d]pyrimidine (5a), which on deacetylation afforded 5,7-dichloro-3-beta-D-ribofuranosylpyrazolo [4,3-d]pyrimidine (5b). Ammonolysis of 5a with liquid ammonia gave 6, whereas with MeOH/NH3, a mixture of 6 and 7-methoxy-5-chloro-3-beta-D-ribofuranosylpyrazolo[4,3-d]pyrimidine (7) was obtained. Reaction of 6 with lithium azide and subsequent hydrogenation afforded 5-aminoformycin A (10). Treatment of 5a with thiourea gave 5-chloro-3-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl) pyrazolo[4,3-d]pyrimidine-7(1H,6H)-thione (8a), which on further reaction with sodium hydrosulfide furnished 3-beta-D-ribofuranosylpyrazolo [4,3-d]pyrimidine-5,7(1H,4H,6H)-dithione (11). The four-step deoxygenation procedure using phenoxythiocarbonylation of the 2'-hydroxy group of the 3', 5'-protected 6 gave 5-chloro-2'-deoxyformycin A (13).