Postmitochondrial Supernatant Research Articles

Modulation of phosphatidylcholine biosynthesis in celery by exogenous fatty acids.

The effects of C16 and C18 fatty acids on the synthesis of phosphatidylcholine were studied in Apium graveolens cell suspension cultures and postmitochondrial supernatants. When cells were exposed to exogenous oleic acid, the rate of phosphatidylcholine biosynthesis increased 1.4-fold within 5 min of the addition of the fatty acid to the culture medium. The sensitivity of microsomal CTP:cholinephosphate cytidylyltransferase (EC 2.7.7.15) to saturated and unsaturated fatty acids was monitored through the addition of unesterified fatty acids to postmitochondrial supernatants. The saturated fatty acids, palmitic and stearic, appeared to have little effect on CTP:cholinephosphate cytidylyltransferase activity, whereas exposure to oleic, linoleic and cis-vaccenic acids resulted in significant increases in enzyme activity. Optimal microsomal CTP:cholinephosphate cytidylyltransferase activities were achieved by the incubation of postmitochondrial supernatants with 500 microM oleate. The exogenous fatty acids were found to be incorporated into microsomal membranes in their unesterified form. Removal of unesterified fatty acids by incubation of microsomal membranes with defatted bovine serum albumin resulted in the reduction of microsomal CTP:cholinephosphate cytidylyltransferase activity; demonstrating that the enzyme requires unesterified unsaturated fatty acids.

The activities of six exo-and endopeptidases in the substantia nigra, neostriatum, and cortex of the rat brain.

We determined the enzymatic activity and crude subcellular distribution of four exopeptidases: Dipeptidylaminopeptidase IV (DAP-IV), Alanyl aminopeptidase (AAP), Prolyl aminopeptidase (PAP) and gamma-Glutamyl transpeptidase (gammaGTP), and two endopeptidases: Postproline endopeptidase (PEP) and "Trypsin-like" peptidase ("T-L" P) in pars compacta (SNPC) and pars reticulata (SNPR) of substantia nigra, caudate-putamen (CAU) and cerebral cortex (CC) of the rat brain. We found: 1) DAP-IV activity is comparatively higher in SNPC and it is equally distributed in the postmitochondrial precipitate (PR) and supernatant (SN) fractions of SNPC, CAU and CC but higher in the SN from SNPR. 2) CC shows the highest activity of AAP and its activity is mainly located in the SN from all areas. 3) The activity of PAP is comparatively higher in SNPC and it is exclusively located in the SN from all areas. 4) gammaGTP activity is similar in all areas but its predominance is in the SN for SNPC and SNPR, and in the PR for CAU and CC. 5) CAU has higher PEP activity (higher in the PR) than CC (higher in the SN); no activity is detected in the substantia nigra. 6) The activity of a "Trypsin-like" peptidase is the highest in SNPC and SNPR; this activity have some predominance in the SN and higher predominance in the same fraction from CAU and CC.

Calpain mediates eukaryotic initiation factor 4G degradation during global brain ischemia.

Global brain ischemia and reperfusion result in the degradation of the eukaryotic initiation factor (eIF) 4G, which plays a critical role in the attachment of the mRNA to the ribosome. Because eIF-4G is a substrate of calpain, these studies were undertaken to examine whether calpain I activation during global brain ischemia contributes to the degradation of eIF-4G in vivo. Immunoblots with antibodies against calpain I and eIF-4G were prepared from rat brain postmitochondrial supernatant incubated at 37 degrees C with and without the addition of calcium and the calpain inhibitors calpastatin or MDL-28,170. Addition of calcium alone resulted in calpain I activation (as measured by autolysis of the 80-kDa subunit) and degradation of eIF-4G; this effect was blocked by either 1 micromol/L calpastatin or 10 micromol/L MDL-28,170. In rabbits subjected to 20 minutes of cardiac arrest, immunoblots of brain postmitochondrial supernatants showed that the percentage of autolyzed calpain I increased from 1.9% +/- 1.1% to 15.8% +/- 5.0% and that this was accompanied by a 68% loss of eIF-4G. MDL-28,170 pretreatment (30 mg/kg) decreased ischemia-induced calpain I autolysis 40% and almost completely blocked eIF-4G degradation. We conclude that calpain I degrades eIF-4G during global brain ischemia.

Selenium-dependent glutathione peroxidase-GI is a major glutathione peroxidase activity in the mucosal epithelium of rodent intestine

Gpx2 mRNA, encoding a selenium-dependent glutathione peroxidase (GPX-GI), has been found to be highly expressed in the gastrointestinal tract (GI) mucosal epithelium. In this study, we show that GPX-GI is produced in the mucosal epithelium of the adult rat GI tract and that the activity levels are comparable to that from GPX-1. Post-mitochondrial supernatant GPX activity from the mucosal epithelium of the complete length of the small intestine was partially purified. A sample enriched for putative GPX-GI was fractionated by SDS-polyacrylamide gel electrophoresis. Polypeptides of 21 kDa and 22 kDa were digested with trypsin. After resolving the tryptic peptides by high pressure liquid chromatography (HPLC), the major peaks were analyzed for their amino acid sequence by Microflow-HPLC-Tandem Mass Spectrometry and automated Edman degradation sequencing. Both methods revealed that the 21-kDa sample contained rat GPX-GI determined by the sequence homology with the deduced mouse GPX-GI polypeptide sequence. Rat GPX-1 was also detected in the samples. AntiGPX-GI and antiGPX-1 antibodies were used to determine the distribution of the respective isoenzyme activities along the length of the intestine and with respect to the crypt to villus axis in rats. GPX-GI and GPX-1 activities were uniformly distributed in the middle and lower GI tract and with respect to the crypt to villus axis. GPX-GI activity accounted nearly the same percentage of the total GPX activity as GPX-1 in all of the these compartments. Studies on the distal ileum segment of wildtype and Gpx1 gene knockout mice showed that GPX-GI activity was also at parity with GPX-1 in the mucosal epithelium of this segment.

Endosome‐lysosome transfer of insulin and glucagon in a liver cell‐free system

The endosome-lysosome transfer of in vivo internalized insulin and glucagon has been studied in a rat liver cell-free system and compared to that of galactosylated bovine serum albumin (GalBSA), a ligand of the asialoglycoprotein receptor. Density-gradient analysis of a postmitochondrial supernatant isolated 8 min after injection of [125I]iodoinsulin showed that the membrane-associated radioactivity (55% of the total) migrated as a single peak at the position of galactosyltransferase, a Golgi marker (1.08-1.10 g/ml). After incubation at 37 degrees C in the presence of ATP, an additional peak of radioactivity (12%) was detected at the position of acid phosphatase, a lysosomal marker (1.12-1.14 g/ml). No such peak was observed in a lysosome-depleted fraction. An ATP-dependent conversion of [125I]iodoinsulin to trichloroacetic-acid-soluble products occurred during incubation (20%) but this was unaffected by lysosome depletion. Gel-filtration and HPLC analysis of acid extracts of gradient fractions isolated after injection of [125I]iodoinsulins selectively labeled at tyrosine residues A14 or B26 revealed the presence of components which differed from intact iodoinsulins by size and/or hydrophobicity. Low molecular-mass components were less abundant and, conversely, intact iodoinsulin and/or high molecular-mass components more abundant in lysosomal fractions than in endosomal fractions. In vivo internalized [125I]iodoglucagon and [125I]iodogalBSA underwent a greater lysosomal transfer (17-21%) and lesser degradation (8-11%) than [125I]iodoinsulin. Glycyl-L-phenylalanine 2-naphtylamide and methionine O-methyl ester, two lysosome-disrupting enzyme substrates, partially released the radioactivity associated with lysosomal fractions (GalBSA > insulin = glucagon) but caused little or no release of that associated with endosomal fractions. Analysis of the alpha and beta subunits of the insulin receptor by cross-linking to [125I]iodoinsulin and Western immunoblotting, respectively, revealed a partial lysosomal transfer of these subunits during endosome-lysosome interaction. We conclude that an endosome-lysosome transfer of insulin and glucagon occurs in a liver cell-free system and suggest that the low recovery of these peptides in lysosomal fractions in vivo results from their rapid degradation within endosomes.

Expression of recombinant human cytochrome P450 1A2 in Escherichia coli bacterial mutagenicity tester strain.

Human cytochrome P450 1A2 is one of the major cytochrome P450s in human liver. It is known to be capable of activating a number of carcinogens such as arylamines and heterocyclic amines. In order to develop the new bacterial mutagenicity test system with human P450, a full length of human P450 1A2 cDNA inserted into pCW bacterial expression vector was introduced to Escherichia coli WP2 uvrA strain which is a well-known E. coli strain for bacterial reverse mutagenicity assay. Expressed human P450 1A2 showed typical P450 hemoprotein spectra. Maximum expression was achieved at 48 hrs after incubating at 30 degrees C in terrific broth containing ampicillin, IPTG and other supplements. High level expression of P 450 1A2 in E. coli WP2 uvrA membranes was determined in SDS-PAGE. The well-known mutagens 2-aminoanthracene and MelQ increased the revertant colonies of E. coli WP2 uvrA expressing human P450 1A2 without an exogenous rat hepatic post-mitochondrial supernatant (S9 fraction) in a dose-dependent manner. The results show that the functional expression of human P450 in bacterial mutagenicity tester strain will provide a useful tool for studying the mechanism of the mutagenesis and carcinogenesis of new drugs and environmental chemicals.

Purification, Characterization and Differentiation-Dependent Expression of a Perchloric Acid Soluble Protein from Rat Kidney

We have recently reported the presence of a novel perchloric acid soluble protein in rat liver (PSP1) that inhibits cell-free protein synthesis in a rabbit reticulocyte system. While studying the perchloric acid soluble proteins from different tissues of rats, we found that the kidney protein cross-reacted with antibody against the PSP1. In this investigation, we have purified a perchloric acid soluble protein from the rat kidney and studied its characterization and expression. The protein extracted from the postmitochondrial supernatant fraction with 5% perchloric acid was purified by ammonium sulfate fractionation and CM-Sephadex chromatography. By immunoscreening with the rabbit antisera against the PSP1, we detected a cDNA that contained an open reading frame of 411 bp, encoding a 137 amino-acid protein with a molecular mass of 14,149 daltons. The deduced amino acid sequence was completely identical with that of PSP1 from rat liver. The perchloric acid soluble protein from rat kidney (K-PSP1) also inhibited cell-free protein synthesis in the rabbit reticulocyte lysate system in a different manner than RNase A. Immunohistochemistry showed that the expression of K-PSP1 increased from fetal 17th day to postnatal 4th week, and it remained almost the same until the 7th week of postnatal age. Furthermore, the expression of K-PSP1 in the kidney of the nephrotic rat model was shown to be differentiation dependent. On the other hand, the expression of K-PSP1 in renal tumor cells was downregulated as compared with intact tissue. These results suggest that the expression of K-PSP1 is regulated in a differentiation-dependent manner in the kidney.

Phosphatidylinositol Transfer Protein (PI-TP) is Required for Regulated Translocation/Activation of Cholinephosphate Cytidylyltransferase (CYTase) in Mle12 Cells • 1770

A major mechanism regulating CYTase, the rate-limiting enzyme in phosphatidylcholine (PC) synthesis, involves lipid-dependent translocation of inactive soluble CYTase (L-form) to membranes where the enzyme is activated. In yeast, the phosphatidylinositol transfer protein SEC14p regulates golgi CYTase activity, but the mechanisms that regulate the translocation/activation of CYTase in mammalian cells have not been completely elucidated. The purpose of this study was to determine whether mammalian PI-TP regulates CYTase in the lung. In cytosol from the MLE12 rat respiratory epithelial cell line, PI-TP co-localized with CYTase L-form fractions. We studied the effect of immunodepleting PI-TP (using anti-PI-TP antisera from GM Helmkanp, U. Kansas) on L-form CYTase activity. PI-TP depletion did not co-precipitate CYTase, but reduced lipid-independent CYTase activity in L-form fractions 3-4.5-fold. To determine the effect of immunodepleting PI-TP on oleic acid (OA)-induced translocation and activation of CYTase in post-mitochondrial supernatants(PMS), PI-TP-depleted and sham-depleted PMS were incubated with 250 μM oleic acid (OA) at 37°C for 20 min and were fractionated on 7.2-36% glycerol gradients. CYTase was analyzed in each fraction by functional assay and immunoblotting. In the absence of OA treatment, 17% of the total CYTase activity was present in the microsomal pellet. OA treatment increased the microsomal CYTase activity to 72.9±18.4% of total CYTase activity in the sham-depleted PMS, but the microsomal CYTase activity increased to only 41.9±17.6% of total activity in the PI-TP-depleted PMS. By contrast, immunoblotting demonstrated 2 to 23-fold more CYTase protein in PI-TP-depleted vs. sham-depleted PMS. Thus, while OA-stimulated translocation of CYTase protein from cytosol to microsomes was higher in the PI-TP-depleted PMS, the specific activity of the translocated enzyme was reduced over 15-fold compared with PI-TP-containing PMS. These data suggest an essential function for PI-TP in the coordinated translocation and activation of CYTase.

Purification and Characterization of 1-O-Acylceramide Synthase, a Novel Phospholipase A2 with Transacylase Activity

A novel pathway for ceramide metabolism, 1-O-acylceramide formation, was previously reported (Abe, A., Shayman, J. A., and Radin, N. S. (1996) J. Biol. Chem. 271, 14383-14389). In this pathway a fatty acid in the sn-2 position of phosphatidylethanolamine or phosphatidylcholine is transferred to the 1-hydroxyl position of ceramide. An enzyme that catalyzes the esterification of N-acetylsphingosine was purified from the postmitochondrial supernatant of calf brain through consecutive steps, including ammonium sulfate fractionation, DEAE-Sephacel, phenyl-Sepharose, S-Sepharose, Sephadex G-75, concanavalin A-agarose, and heparin-Sepharose chromatography. The molecular mass of the enzyme was determined to be 40 kDa by gel filtration on Sephadex G-75. The enzyme bound to concanavalin A-agarose column was eluted with the buffer containing 500 mM alpha-methyl-D-mannopyranoside. Further purification by heparin-Sepharose chromatography resulted in separation of two peaks of enzyme activity. Coincidence between the transacylase activity and a stained protein of a molecular mass of 40 kDa was observed, as determined by SDS-polyacrylamide gel electrophoresis and recovery after separation over an acidic native gel. The second peak of activity from the heparin-Sepharose chromatography represented a purification of 193,000-fold. These results are consistent with the enzyme being a glycoprotein of a molecular mass of about 40 kDa with a single polypeptide chain. The purified enzyme had a pH optimum at pH 4.5. The divalent cations Ca2+ and Mg2+ enhanced but were not essential for the transacylase activity. Neither activation nor inactivation of the enzyme activity was observed in the presence of 2 mM ATP or 2 mM dithiothreitol. Preincubation of the enzyme with 1 mM N-ethylmaleimide, 1 mM phenylmethylsulfonyl fluoride, or 3.1 microM bromoenol lactone, a potent inhibitor of cytosolic Ca2+-independent phospholipase A2, had no significant effect on the enzyme activity. The enzyme activity was completely abolished in the presence of greater than 773 microM Triton X-100. Partial inhibition of the enzyme activity was observed in the presence of 10-100 microg/ml heparin. In the absence of N-acetylsphingosine, the enzyme acted as a phospholipase A2. These results strongly suggest that 1-O-acylceramide synthase is both a transacylase and a novel phospholipase A2.

S-oxygenation of thiourea results in the formation of genotoxic products

Thiourea (TU) is a thyroid carcinogen which has previously been shown to cause genotoxicity in various test systems in vitro and in vivo. The mechanism underlying these effects has not yet been elucidated. The present study addressed the question of whether the formation of oxidized products of TU might be involved in genotoxicity. Chemical oxidation of [14C]TU with hydrogen peroxide in the presence of calf thymus DNA resulted in the formation of [14C]formamidine sulfinate ([14C]FASA), [14C]cyanomide, and [14C]urea and in covalent binding of radioactivity to the DNA. Incubation of V79 Chinese hamster cells with 10-20 mM TU for 18 hr but not for 3 hr, increased the frequency of micronuclei to a slight extent. In cells depleted of glutathione, which can prevent the oxidation of TU, micronucleus induction by TU was more pronounced and detectable both after 3 and 18 hr of incubation. Exposure of the cells to 1.25 to 10 mM FASA for 3-5 hr induced micronuclei, DNA repair synthesis, and gene mutations in the cells. Flavin-containing monooxygenase (FMO], an enzyme known to catalyze the S-oxygenation of TU in liver, could not be detected in the postmitochondrial supernatant (S-9) of the V79 cells. There is evidence, however, that TU can easily autoxidize to S-oxygenated products. Both FASA and TU caused a slight induction of DNA repair synthesis in cultured rat hepatocytes, but FASA was active at lower concentrations than TU. Cyanamide did not elicit repair. The finding that FASA, a product of both the nonenzymatic and the enzymatic S-oxygenation of TU, is genotoxic in cultured mammalian cells provides for the first time a hypothesis to explain the genotoxicity of TU.

Differential activation of mRNA during maturation of bovine oocytes is correlated with changes in poly(A)-tail length

Some aspects of the state of aggregation and functioning of polysomes have been investigated in preparations obtained from rat liver lobes subjected to ischemia. The incroporation of amino acid into protein is reduced in postmitochondrial supernatant fractions, microsomes, and C-ribosomes from ischemic livers. In the presence of cell sap from ischemic livers, normal C-ribosomes, but not normal microsomes, show a reduced incorporating capacity, which is not affected by a fivefold increase of the amount of cell sap in the incubation mixture. The analysis of the polysomal patterns shows that ischemia causes a progressive disaggregation of polyribosomes, with a reduction in the number of heavy aggregates and an increase of dimers and monomers. This disaggregation is best seen in the ribosomes recovered from the fraction bound to the membranes of the endoplasmic reticulum. The determination of RNA -used as a reference basis of the various subcellular fractions—shows that ribosomes are also reduced in number in preparations from ischemic livers. Bound ribosomes are more affected than the free forms. On the whole, ribosomes obtained from ischemic livers seem to be reduced in number, less aggregated, and less efficient in carrying out protein synthesis.

The suitability of gastropods as biomarkers

The measurement and induction of mixed function oxyigenases (MFOs) of limnetic gastropods were investigated to estimate their suitability as biomarkers. A determination of MFO activities was performed through the measurement of ethoxyresorufin-O-dealkylase (EROD), pentoxy-(PROD) and benzoxyresorufin-O-dealkylase-activity (BROD). Optimal measuring conditions of these activities were investigated in preparations of the digestive gland of 4 species of limnetic gastropods. Results indicate that MFO-activity inhibiting substances are accumulating in the microsomal pellet, the fraction commonly used for the measurement of MFO-activities. Therefore, the fraction used for induction studies was the postmitochondrial supernatant (PMS). EROD, PROD, and BROD activity of Planorbis planorbis and Planorbis carinatus were measured after a treatment with Aroclor 1254 for 1-17 days. Maximal induction of EROD and PROD were 6 and 10 times the value in the control group of P. carinatus, respectively induction in P. planorbis was lower. BROD-activity could not been measured in P. planorbis. In P. carinatus, BROD-activity increased to a maximum of 2 times after treatment with Aroclor 1254. The level of induction of EROD-activity is comparable to results described in analog studies with fish.

Effect of brain ischemia and reperfusion on the localization of phosphorylated eukaryotic initiation factor 2 alpha.

Postischemic brain reperfusion is associated with a substantial and long-lasting reduction of protein synthesis in selectively vulnerable neurons. Because the overall translation initiation rate is typically regulated by altering the phosphorylation of serine 51 on the alpha-subunit of eukaryotic initiation factor 2 (eIF-2 alpha), we used an antibody specific to phosphorylated eIF-2 alpha [eIF-2(alpha P)] to study the regional and cellular distribution of eIF-2(alpha P) in normal, ischemic, and reperfused rat brains. Western blots of brain postmitochondrial supernatants revealed that approximately 1% of all eIF-2 alpha is phosphorylated in controls, eIF-2(alpha P) is not reduced by up to 30 minutes of ischemia, and eIF-2(alpha P) is increased approximately 20-fold after 10 and 90 minutes of reperfusion. Immunohistochemistry shows localization of eIF-2(alpha P) to astrocytes in normal brains, a massive increase in eIF-2(alpha P) in the cytoplasm of neurons within the first 10 minutes of reperfusion, accumulation of eIF-2(alpha P) in the nuclei of selectively vulnerable neurons after 1 hour of reperfusion, and morphology suggesting pyknosis or apoptosis in neuronal nuclei that continue to display eIF-2(alpha P) after 4 hours of reperfusion. These observations, together with the fact that eIF-2(alpha P) inhibits translation initiation, make a compelling case that eIF-2(alpha P) is responsible for reperfusion-induced inhibition of protein synthesis in vulnerable neurons.

Oleic acid stimulates rapid translocation of cholinephosphate cytidylyltransferase in type II cells

Activity of cholinephosphate cytidylyltransferase, the rate-limiting enzyme in phosphatidylcholine synthesis, increases rapidly in the lung after birth predominantly due to an increase in membrane-associated activity. Although there is strong evidence that enzyme translocation is a major regulatory mechanism in other cells, this mechanism has not been conclusively demonstrated in intact alveolar type II cells. In this study, we show that oleic acid stimulates rapid translocation of cytidylyltransferase activity and protein from cytosol to microsomes in both primary cultured fetal and adult type II cells and MLE12 cells, a cell line derived from murine distal respiratory epithelial cells. Shifts in subcellular distribution occurred within 5 min of exposure to 200 μM oleic acid. The magnitude of the increases in microsomal enzyme activity and immunoreactive protein levels was several-fold greater in d21 fetal cells than adult type II cells. Oleic acid-induced translocation was confirmed in in vitro translocation experiments. After incubating MLE12 cell postmitochondrial supernatants at 37°C with oleic acid and separation of enzyme isoforms on glycerol density gradients, enzyme activity was decreased in gradient fractions corresponding to both cytosolic isoforms and microsomal activity increased 7.9-fold compared to the distribution of enzyme activity in postmitochondrial supernatants incubated at 4°C without oleic acid. The increase in microsomal activity was associated with an increased incorporation of [ 14C]oleic acid in the membrane free fatty acid fraction. Developmental changes in type II cell membrane lipid composition may induce the rapid translocation/activation of cytidylyltransferase in the lung after birth.

Coincubation of rat renal proximal tubules with hepatic subcellular fractions potentiates the effects of para-aminophenol.

Treatment of rats with para-aminophenol (PAP) (300 mg/kg ip) produced decreases in renal nonprotein sulfhydryl (NPSH) content, oxygen consumption, and adenine nucleotide concentrations 2-4 hr following administration. In contrast, incubation of rat renal tubules with up to 1 mm PAP for 4 hr produced inconsistent changes in renal tubules. This discrepancy suggested that extrarenal metabolism of PAP may be involved in PAP bioactivation and nephrotoxicity. We designed the present studies to test the hypothesis that hepatic metabolism of PAP potentiates the effects of PAP on renal tubules. Incubation of renal tubules with 0.5 mm PAP and 10 mg protein from hepatic postmitochondrial supernatant (S9 fraction) in the absence of glutathione (GSH) for 4 hr did not alter renal oxygen consumption or adenine nucleotide metabolite concentrations. We observed no changes when we incubated tubules with 0.5 mm PAP and 1 mm GSH in the absence of hepatic S9 fraction. However, incubation of renal tubules with 0.5 mm PAP, 1 mm GSH, and 10 mg hepatic S9 protein for 4 hr significantly decreased renal oxygen consumption and adenosine triphosphate and total nucleotide concentrations. These data suggest that the effects of PAP in renal tubules may be potentiated by enzymatic metabolism of PAP, possibly involving oxidation and GSH conjugation. From experiments using hepatic microsomes or cytosol instead of S9 fraction, we found that changes were produced when we incubated tubules with PAP in the presence of hepatic microsomes, but not cytosol. These data suggest that hepatic microsomal metabolism of PAP may contribute to the production of changes in renal tubules in vitro. PAP-induced changes in renal proximal tubules were prevented when we included a beta-nicotinamide adenine dinucleotide phosphate (NADPH) generating system in the incubation medium. The protective effect of NADPH persisted when microsomes were inactivated by incubation with 1-aminobenzotriazole, a cytochrome P450 inhibitor. These data suggest that cytochrome P450-dependent oxidation is not involved in the production or prevention of PAP-induced changes in renal tubules. The enzyme(s) responsible for PAP bioactivation and the mechanism(s) by which NADPH protects renal tubules from PAP-induced decrements in oxygen consumption and adenine nucleotide concentrations are currently unclear.

Studies on the mechanism of antimalarial action of a novel arylene bis(methylketone)

2-Amino-4-(3,5-diacetylphenyl)amino-l,6-dimethylpyrimidinium chloride (CNI-H0294) is a novel arylene bis(methylketone) compound that displays antimalarial activity against chloroquine- and pyrimethamine-resistant Plasmodium falciparum clones. The compound has been found to be concentrated into infected erythrocytes, with 80–179 μM accumulated when parasites were cultured in the presence of 1.0 μM CNI-H0294-Uninfected erythrocytes, in contrast, only accumulated 2.5–3.4 μM CNI-H0294 under identical conditions. Using postmitochondrial supernatants from a number of parasite clones, the compound was found to inhibit dihydrofolate reductase (EC 1.5.1.3) activity with an IC 50 of 243–483 μM. Thus, while CNI-H0294 is not a powerful inhibitor of plasmodial dihydrofolate reductase, the accumulation of the compound into infected erythrocytes, when correlated to the external ED 50 concentration against parasite growth in vitro, reaches concentrations sufficient to inhibit the malarial enzyme.

Mutagenicity of cyclopenta-fused polynuclear aromatic hydrocarbons and a non-polar fraction from a fuel combustion sample in a Salmonella forward mutation assay without exogenous metabolic activation

A series of cyclopenta-fused polynuclear aromatic hydrocarbons (PAH) were tested for mutagenicity in a bacterial forward mutation assay based on resistance to 8-azaguanine (8-AG) in Salmonella typhimurium TM677 in the absence of Aroclor-induced rat liver postmitochondrial supernatant (PMS). All of the aceanthrylenes tested were mutagenic in the absence of PMS, whereas none of the acephenanthrylenes were active. The following mutagenic potency series expressed as the minimum detectable mutagen concentration (MDMC) in nmol/ml was obtained: aceanthrylene (AA) (5.5); cyclopent[ h,i]aceanthrylene (CPAA) (18.2); 6-methylaceanthrylene (6-MeAA) (112); 1,2,6,7-tetrahydrocyclopent[ h,i]aceanthrylene (THCPAA) (166); 1,2-dihydroaceanthrylene (DHAA) (298). Saturation of the cyclopenta rings or methylation at the 6-position of AA reduced, but did not eliminate, mutagenicity measured in the absence of PMS. AA was unusual because it was approximately 4-fold more mutagenic in the absence of PMS than in its presence. The other aceanthrylenes tested were 1.3–10.7 times more mutagenic in the presence of PMS than in its absence to give an MDMC potency series of: CPAA (3.8); 6-MeAA (10.5); AA (19.9); THCPAA (52.9); DHAA (229). Approximately 20% of the PMS-independent mutagenicity in a combustion sample from ethylene burned under fuel-rich conditions was found in a fraction containing only non-polar, 4–7 ring PAHs, widely attributed to be mutagenic only in the presence of PMS. None of this mutagenicity could be attributed to aceanthrylenes, thus other non-polar PAHs appear to possess significant PMS-independent mutagenicity as well.

Subcellular structure containing mRNA for beta subunit of mitochondrial H+-ATP synthase in rat hepatocytes is translationally active.

We have recently reported that the nuclear-encoded mRNA for the beta subunit of mitochondrial H+-ATP synthase (beta-mRNA) is localized in rounded, electron-dense clusters in the cytoplasm of rat hepatocytes. Clusters of beta-mRNA are often found in close proximity to mitochondria. These findings suggested a role for these structures in controlling the cytoplasmic expression and sorting of the encoded mitochondrial precursor. Here we have addressed the question of whether the structures containing beta-mRNA are translationally active. For this purpose a combination of high-resolution in situ hybridization and immunocytochemical procedures was used. Three different co-localization criteria showed that beta-mRNA-containing structures always revealed positive immunoreactive signals for mitochondrial H+-ATP synthase (F1-ATPase), ribosomal and hsc70 proteins. Furthermore, clusters show evidence in situ of developmental changes in the translational efficiency of the beta-mRNA. These findings suggest that structures containing beta-mRNA are translationally active irrespective of their cytoplasmic location. The immunocytochemical quantification of the cytoplasmic presentation of hsc70 in the hepatocyte reveals that approx. 86% of the protein has a dispersed distribution pattern. However, the remaining hsc70 is presented in clusters of which only half reveal positive hybridization for beta-mRNA. The interaction of hsc70 with the beta-F1-ATPase precursor protein is documented by the co-localization of F1-ATPase immunoreactive material within cytoplasmic clusters of hsc70 and by the co-immunoprecipitation of hsc70 with the beta-subunit precursor from liver post-mitochondrial supernatants. Taken together, these results suggest a role for hsc70 in the translation/sorting pathway of the mammalian precursor of the beta-F1-ATPase protein.

Fe2+-CATALYZED PRODUCTION OF CABON MONOXIDE (CO) BY RAT BRAIN TISSUE HOMOGENATES. † 1096

Most CO excreted by mammals results from the degradation of heme. However, some CO is derived from other sources. We studied the Fe2+-initiated peroxidation of tissue homogenate fractions that yield a number of products, including thiobarbituric acid reactive substances (TBARS) and CO. Post-mitochondrial rat tissue supernatants were incubated in the dark with 100μM ascorbate (Asc) and 6 μM Fe2+ for 30 min at 37°C in septum-sealed vials. Control reactions contained 100 μM butylated hydroxytoluene (BHT). CO in the headspace was determined by GC and TBARS in the liquid phase was measured spectrophotometrically. Reaction mixture composition and product formation (n=3, pmoles CO/4 mg fresh weight) are as follows (mean ± SD): Table When brain homogenate was fractionated, CO production occurred with mitochondria (86±27) and microsomes(100±26), but not with the soluble fraction (1±1). Supernatants from brain (242±18), kidney (198±20), spinal cord(159±23), lung (22±5), and spleen (21±4) produced CO, but not those from blood (8±5), intestine (3±2), liver (2±2), nor heart (1±1). Bilirubin inhibited brain peroxidation completely at 3 mg/dL. Iron chelators, such as desferrioxamine, completely suppress CO and TBARS production. We conclude that: CO is a product of Fe2+-initiated peroxidation in vitro, in parallel with TBARS; CO production occurs in membranes only. We speculate that tissue supernatants that do not produce CO are protected from oxidation by endogenous antioxidants, such as bilirubin.

Mutagenicity of the atmospheric transformation products 2-nitrofluoranthene and 2-nitrodibenzopyranone in Salmonella and human cell forward mutation assays

The mutagenicity of the atmospheric transformation products 2-nitrofluoranthene (2-NF) and 2-nitrodibenzopyranone (2-NDBP), as well as a related isomer 3-nitrodibenzopyranone (3-NDBP), was measured in quantitative forward mutation assays with bacteria ( Salmonella typhimurium TM677) and in two metabolically competent human cell lines (MCL-5 and h1A1v2) that differ in their complement of cytochrome P450s and microsomal epoxide hydrolase. 2-NF was a potent mutagen in Salmonella TM677 both in the absence and presence of rat liver postmitochondrial supernatant (PMS). 2-NDBP was non-mutagenic in the absence of PMS, but was mutagenic in its presence. The converse result was obtained for 3-NDBP. The mutagenic potency series in Salmonella in the absence of PMS, expressed as the minimum detectable mutagen concentration (MDMC) in nmol/ml, was: 2-NF, 2.5; 3-NDBP, 16.9; and 2-NDBP, >415. With PMS, the potency series was: 2-NF, 1.2; 2-NDBP, 15.1; and 3-NDBP, 208. Neither 2-NDBP nor 3-NDBP were mutagenic at the tk locus in MCL-5 or h1A1v2 cells at up to 200 nmol/ml. 2-NF was also inactive in MCL-5 cells, but was a potent mutagen in h1A1v2 cells with an MDMC of 0.02 nmol/ml. Cytochrome P450 CYP1A1, present constitutively only in h1A1v2 cells, was implicated in 2-NF activation because mutagenicity was reduced by 55–80% when α-naphthoflavone (ANF) was present during incubation. The lack of mutagenicity in MCL-5 cells was attributed to the inability of 2-NF to induce CYP1A1 activity in this cell line. These data indicate a primary role for ring oxidation in 2-NF activation. Previous emphasis placed upon 2-NDBP as a major mutagen in ambient air may need to be modified in view of the negative results for this compound in the human cell assays and in the absence of PMS in Salmonella TM677. However, these findings support the concern that 2-NF may be a risk to human health.