Postmitochondrial Supernatant Fraction Research Articles

A multi-year study of hepatic biomarkers in coastal fishes from the Gulf of Mexico after the Deepwater Horizon Oil Spill

Following the 2010 Gulf of Mexico oil spill, concerns were raised regarding exposure of fish to crude oil components, particularly polycyclic aromatic hydrocarbons (PAHs). This three year study examined hepatic enzymes in post-mitochondrial supernatant fractions from red snapper (Lutjanus campechanus) and gray triggerfish (Balistes capriscus) collected in the north central Gulf of Mexico between 2011 and 2014. Biomarker activities evaluated included benzo(a)pyrene hydroxylase (AHH), ethoxyresorufin O-deethylase (EROD), glutathione transferase (GST), and glutathione peroxidase (GPx). Mean EROD activity was higher in gray triggerfish (12.97 ± 7.15 pmol/min/mg protein [mean ± SD], n = 115) than red snapper (2.75 ± 1.92 pmol/min/mg protein, n = 194), p < 0.0001. In both species, EROD declined over time between 2011 and 2014. Declines in GST and GPx activities were also noted over this time period for both species. Gray triggerfish liver was fatty, and heptane extracts of the liver fat contained fluorescent substances with properties similar to known PAHs, however the origin of these PAHs is unknown.

Lycopene abrogates Aβ(1–42)-mediated neuroinflammatory cascade in an experimental model of Alzheimer’s disease

BackgroundNeuroinflammation characterized by glial activation and release of proinflammatory mediators is considered to play a critical role in the pathogenesis of Alzheimer's disease (AD). β-Amyloid1–42 (Aβ1–42)-induced learning and memory impairment in rats is believed to be associated with neuronal inflammation. ObjectivesThe present study was designed to investigate the effect of lycopene, a potent antioxidant and anti-inflammatory carotenoid, in intracerebroventricular (ICV) Aβ1–42-induced neuroinflammatory cascade along with learning and memory impairment in rats. Material and methodsICV Aβ1–42 was injected bilaterally followed by treatment with lycopene or rivastigmine for 14 days. Morris water maze and elevated plus maze tests were used to assess the memory function. Rats were sacrificed and brains harvested to evaluate various biochemical parameters and mitochondrial complex activities in postmitochondrial supernatant fractions of cerebral cortex and hippocampus of rat brains. The levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), tumor growth factor β (TGF-β), nuclear factor-κB (NF-κB) and caspase-3 were assessed by enzyme-linked immunosorbent assay analysis. ResultsLycopene remediated Aβ-induced learning and memory deficits in a dose-dependent manner. Aβ1–42-induced mitochondrial dysfunction along with surge of proinflammatory cytokines TNF-α, TGF-β and IL-1β as well as NF-κB and caspase-3 activity in rat brain was significantly reduced with lycopene treatment. ConclusionThe amelioration of Aβ1–42-induced spatial learning and memory impairment by lycopene could be linked, at least in part, to the inhibition of NF-κB activity and the down-regulation of expression of neuroinflammatory cytokines, suggesting that lycopene may be a potential candidate for AD treatment.

Renal and Hepatic Antioxidant Status of Hyperglycemic Rats Treated with Single and Combinatorial Herbal Formulations.

Background/Aim: The present study ascertained the comparative capacities of single and combinatorial herbal formulations of Acanthus montanus, Emilia coccinea, Hibiscus rosasinensis and Asystasia gangetica to exert glycemic control and ameliorate oxidative stress in renal and hepatic tissues of alloxan-induced hyperglycemic rats (HyGR) following 30 days treatment. Materials and Methods: Phytochemical composition of the herbal extracts and fasting plasma glucose concentration (FPGC) of the rats were measured by standard methods. The post mitochondrial supernatant fractions (PMSF) of renal and hepatic tissues homogenates were measured for activity levels of glutathione peroxidase (GPOx), superoxide dismutase (SOD) and catalase (CAT) as well as glutathione (GSH) concentration. Results: Comparatively, flavonoids were the most abundant phytochemical present in the four herbal extracts. FPGC of treated HyGR was with in the range of 66.30±0.71-133.00±0.63 mg/dL compared to the untreated hyperglycemic rat group=368.20±0.72 mg/dL (p<0.05). At the end of the experimental time, treated HyGR liver SOD activities were lower than that of normal group (p<0.05). Liver and kidney GPOx activities of HyGR were 2.36 folds and 3.93 folds lower than the normal rats. Furthermore, liver and kidney CAT activities of the treated HyGR were significantly (p<0.05) lower than the normal rats, whereas liver and kidney GSH/GSSG ratios were significantly (p<0.05) increased when compared with treated HyGR. Conclusion: Combinatorial herbal formulations exerted greater glycemic control than the single herbal formulations and caused a sparing effect on renal and hepatic antioxidants against reactive oxygen species.

Antioxidant Status of Liver and Kidney Homogenates from Hyperglycemic Rats Administered with Single and Combinatorial Herbal Formulations

Aims: The present study ascertained the comparative capacities of single and combinatorial herbal formulations of Acanthus montanus, Emilia coccinea, Hibiscus rosasinensis and Asystasia gangetica to exert glycemic control and ameliorate oxidative stress in renal and hepatic tissues of alloxan-induced hyperglycemic rats (HyGR) following 30 days treatment. Materials and Methods: Phytochemical composition of the herbal extracts and fasting plasma glucose concentration (FPGC) of the rats were measured by standard methods. The post mitochondrial supernatant fractions (PMSF) of renal and hepatic tissues homogenates were measured for activity levels of glutathione peroxidase (GPOx), superoxide dismutase (SOD) and catalase (CAT) as well as glutathione (GSH) concentration. Results: Comparatively, flavonoids were the most abundant phytochemical present in the four herbal extracts. FPGC of treated HyGR was within the range of 66.30 ± 0.71133.00 ± 0.63 mg/dL as against the untreated hyperglycemic rat group=368.20 ± 0.72 mg/dL (p<0.05). At the end of the experimental time, treated HyGR liver SOD activities were lower than that of normal group (p<0.05). Liver and kidney GPOx activities of HyGR were 2.36 folds and 3.93 folds lower than the normal rats. Furthermore, liver and kidney CAT activities of the treated HyGR were significantly (p<0.05) lower than the normal rats, whereas liver and kidney GSH/ GSSG ratios were significantly ( p<0.05) increased when compared with treated HyGR. Conclusion: Combinatorial herbal formulations exerted greater glycemic control than the single herbal formulations and caused a sparing effect on renal and hepatic antioxidants against reactive oxygen species.

Glycemic Indices/Renal and Hepatic Antioxidant Status of Hyperglycemic Rats Treated with Single and Combinatorial Herbal Formulations

The present study ascertained the comparative capacities of single and combinatorial herbal formulations of Acanthus montanus, Emilia coccinea, Hibiscus rosasinensis and Asystasia gangetica to exert glycemic control and ameliorate oxidative stress in renal and hepatic tissues of alloxan-induced hyperglycemic rats (HyGR) following 30 days treatment. Phytochemical composition of the herbal extracts and fasting plasma glucose concentration (FPGC) of the rats were measured by standard methods. The post mitochondrial supernatant fractions (PMSF) of renal and hepatic tissues homogenates were measured for activity levels of glutathione peroxidase (GPOx), superoxide dismutase (SOD) and catalase (CAT) as well as glutathione (GSH) concentration. Comparatively, flavonoids were the most abundant phytochemical present in the four herbal extracts. FPGC of treated HyGR was within the range of 66.30 ± 0.71 - 133.00 ± 0.63 mg/dL as against the untreated hyperglycemic rat group=368.20 ± 0.72 mg/dL (p<0.05). At the end of the experimental time, treated HyGR liver SOD activities were lower than that of normal group (p<0.05). Liver and kidney GPOx activities of HyGR were 2.36 folds and 3.93 folds lower than the normal rats. Furthermore, liver and kidney CAT activities of the treated HyGR were significantly (p<0.05) lower than the normal rats, whereas liver and kidney GSH/GSSG ratios were significantly (p<0.05) increased when compared with treated HyGR. Combinatorial herbal formulations exerted greater glycemic control than the single herbal formulations and caused a sparing effect on renal and hepatic antioxidants against reactive oxygen species.

Use of Enzyme Inhibitors to Evaluate the Conversion Pathways of Ester and Amide Prodrugs: A Case Study Example with the Prodrug Ceftobiprole Medocaril

An approach was developed that uses enzyme inhibitors to support the assessment of the pathways that are responsible for the conversion of intravenously administered ester and amide prodrugs in different biological matrices. The methodology was applied to ceftobiprole medocaril (BAL5788), the prodrug of the cephalosporin antibiotic, ceftobiprole. The prodrug was incubated in plasma, postmitochondrial supernatant fractions from human liver (impaired and nonimpaired), kidney, and intestine as well as erythrocytes, in the presence and absence of different enzyme inhibitors (acetylcholinesterase, pseudocholinesterase, retinyl palmitoyl hydrolase, serine esterases, amidases, and cholinesterase). Hydrolysis was rapid, extensive, and not dependent on the presence of β-nicotinamide-adenine dinucleotide phosphate (reduced form) in all matrices tested, suggesting the involvement of carboxylesterases but not P450 enzymes. Hydrolysis in healthy human plasma was rapid and complete and only partially inhibited in the presence of paraoxonase inhibitors or in liver from hepatic impaired patients, suggesting involvement of nonparaoxonase pathways. The results demonstrate the utility of this approach in confirming the presence of multiple conversion pathways of intravenously administered prodrugs and in the case of BAL5788 demonstrated that this prodrug is unlikely to be affected by genetic polymorphisms, drug interactions, or other environmental factors that might inhibit or induce the enzymes involved in its conversion.

In Vitro Metabolism of Oxymetazoline: Evidence for Bioactivation to a Reactive Metabolite

Oxymetazoline (6-tert-butyl-3-(2-imidazolin-2-ylmethyl)-2,4-dimethylphenol) has been widely used as a nonprescription nasal vasoconstrictor for >40 years; however, its metabolic pathway has not been investigated. This study describes the in vitro metabolism of oxymetazoline in human, rat, and rabbit liver postmitochondrial supernatant fraction from homogenized tissue (S9) fractions and their microsomes supplemented with NADPH. The metabolites of oxymetazoline identified by liquid chromatography (LC)/UV/tandem mass spectrometry (MS/MS), included M1 (monohydroxylation of the t-butyl group), M2 (oxidative dehydrogenation of the imidazoline to an imidazole moiety), M3 (monohydroxylation of M2), M4 (dihydroxylation of oxymetazoline), and M5 (dihydroxylation of M2). Screening with nine human expressed cytochromes P450 (P450s) identified CYP2C19 as the single P450 isoform catalyzing the formation of M1, M2, and M3. Glutathione conjugates of oxymetazoline (M6) and M2 (M7) were identified in the liver S9 fractions, indicating the capability of oxymetazoline to undergo bioactivation to reactive intermediate species. M6 and M7 were not detected in those liver S9 incubations without NADPH. Cysteine conjugates (M8 and M9) derived from glutathione conjugates and hydroxylated glutathione conjugates (M10 and M11) were also identified. The reactive intermediate of oxymetazoline was trapped with glutathione and N-acetyl cysteine and identified by LC/MS/MS. M6 was isolated and identified by one-dimensional or two-dimensional NMR as the glutathione conjugate of a p-quinone methide. We have shown the tendency of oxymetazoline to form p-quinone methide species via a bioactivation mechanism involving a CYP2C19-catalyzed two-electron oxidation. Nevertheless, we conclude that the formation of this reactive species might not be a safety concern for oxymetazoline nasal products because of the typical low-dose and brief dosage regimen limited to nasal delivery.

In Vitro and In Vivo Metabolism of the Radiolytic Compound 2‐Dodecylcyclobutanone

Our knowledge about the metabolism of alkylcyclobutanones (2-ACBs) is limited, and the lack of literature on the metabolism of 2-ACBs causes consumers to doubt the safety of irradiated foods. The objectives of this study were to evaluate the metabolism of 2-dodecylcyclobutanone (2-DCB) and identify any possible metabolite. The 2-DCB was mixed with rat S9 (postmitochondrial supernatant fraction) and beta-nicotinamide adenine dinucleotide phosphate (NADPH) in phosphate buffer (pH 7.4) and incubated for 2 h at 37 degrees C. Then, the incubation mixture was mixed with sodium sulfate and extracted with n-hexane by using a Soxhlet apparatus. The hexane extract was concentrated under nitrogen and injected into the gas chromatography-mass spectrometry (GC-MS) machine running in selective ion monitoring mode (SIM) to measure 2-DCB concentration. The hexane extract from the in vitro and in vivo studies was also derivatized with a silylation reagent and injected into a GC-MS running in full scan mode. The average percentage of 2-DCB recovered from the test incubations was 23%, compared with 50% from the controls. The GC-MS chromatograms of the derivatized samples showed a unique peak in the in vitro test incubations and in the hexane extract of the rat feces that were given 2-DCB. This peak was later identified as 2-doecylcyclobutanol.

Oxidative Stress in the Progression of Alzheimer Disease in the Frontal Cortex

We investigated oxidative stress in human postmortem frontal cortexfrom individuals characterized as mild cognitive impairment (n= 8), mild/moderate Alzheimer disease (n = 4), and late-stage Alzheimer disease (n = 9). Samples from subjects with no cognitive impairment (n = 10) that were age- and postmortem interval-matched with these cases were used as controls. The short postmortem intervalbrain samples were processed for postmitochondrial supernatant, nonsynaptic mitochondria, and synaptosome fractions. Samples were analyzed for several antioxidants (glutathione, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, glucose-6-phosphate dehydrogenase, superoxide dismutase, catalase) and the oxidative marker, thiobarbituric acid reactive substances. The tissue was also analyzed for possible changes in protein damage using neurochemical markers for protein carbonyls, 3-nitrotyrosine, 4-hydroxynonenal, andacrolein. All 3 neuropil fractions (postmitochondrial supernatant, mitochondrial, and synaptosomal) demonstrated significant disease-dependent increases in oxidative markers. The highest changes were observed in the synaptosomal fraction. Both mitochondrial and synaptosomal fractions had significant declines in antioxidants (glutathione, glutathione peroxidase, glutathione-S-transferase, and superoxide dismutase). Levels of oxidative markers significantly correlated with Mini-Mental Status Examination scores. Oxidative stress was more localized to the synapses, with levels increasing in a disease-dependent fashion. These correlations implicate an involvement of oxidative stress in Alzheimer disease-related synaptic loss.

Reduced endoplasmic reticulum luminal calcium links saturated fatty acid-mediated endoplasmic reticulum stress and cell death in liver cells

Chronic exposure to elevated free fatty acids, in particular long chain saturated fatty acids, provokes endoplasmic reticulum (ER) stress and cell death in a number of cell types. The perturbations to the ER that instigate ER stress and activation of the unfolded protein in response to fatty acids in hepatocytes have not been identified. The present study employed H4IIE liver cells and primary rat hepatocytes to examine the hypothesis that saturated fatty acids induce ER stress via effects on ER luminal calcium stores. Exposure of H4IIE liver cells and primary hepatocytes to palmitate and stearate reduced thapsigargin-sensitive calcium stores and increased biochemical markers of ER stress over similar time courses (6 h). These changes preceded cell death, which was only observed at later time points (16 h). Co-incubation with oleate prevented the reduction in calcium stores, induction of ER stress markers and cell death observed in response to palmitate. Inclusion of calcium chelators, BAPTA-AM or EGTA, reduced palmitate- and stearate-mediated enrichment of cytochrome c in post-mitochondrial supernatant fractions and cell death. These data suggest that redistribution of ER luminal calcium contributes to long chain saturated fatty acid-mediated ER stress and cell death.

Topogenesis of Mammalian Oxa1, a Component of the Mitochondrial Inner Membrane Protein Export Machinery

Oxa1 is a mitochondrial inner membrane protein with a predicted five-transmembrane segment (TM1 approximately 5) topology in which the N terminus and a hydrophilic loop, L2, are exposed to the intermembrane space and the C-terminal region and two loops, L1 and L3, are exposed to the matrix. Oxa1 mediates the insertion of mitochondrial DNA-encoded subunits of respiratory complexes and several nuclear DNA-encoded proteins into the inner membrane from the matrix. Compared with yeast Oxa1, little is known about the import and function of mammalian Oxa1. Here, we investigated the topogenesis of Oxa1 in HeLa cells using systematic deletion or mutation constructs and found that (i) the N-terminal 64-residue segment formed a presequence, and its deletion directed the mature protein to the endoplasmic reticulum, indicating that the presequence arrests cotranslational activation of the potential endoplasmic reticulum-targeting signal within mature Oxa1, (ii) systematic deletion of Oxa1 TM segments revealed that the presence of all five TMs is essential for efficient membrane integration, (iii) the species-conserved hexapeptide (GLPWWG) located near the N terminus of TM1 was essential for export of the N-terminal segment and L2 into the intermembrane space from the matrix, i.e. for correct topogenesis of Oxa1, and (iv) GLPWWG placed near the N terminus of TM2 or TM3 in the reporter construct also supported its membrane integration in the Nout-Cin orientation. Together, these results demonstrated that topogenesis of Oxa1 is a cooperative event of all five TMs, and GLPWWG followed immediately by TM1 is essential for correct Oxa1 topogenesis.

Metabolism of Flufenpyr-ethyl in Rats and Mice

The metabolism of flufenpyr-ethyl [ethyl 2-chloro-5-[1,6-dihydro-5-methyl-6-oxo-4-(trifluoromethyl)pyridazin-1-yl]-4-fluorophenoxyacetate] was examined in rats and mice. [Phenyl-(14)C]flufenpyr-ethyl was administered to rats and mice as a single oral dose at a level of 500 mg/kg, and (14)C-excretion was examined. Total (14)C-recoveries within 7 days after administration were 93.2 to 97.5% (feces, 42.0 to 46.0%; and urine, 47.2 to 55.5%) in rats and 92.6 to 96.4% (feces, 26.7 to 32.7%; and urine, 59.9 to 69.7%) in mice. (14)C-Excretion into expired air was not detected in rats (expired air of mice was not analyzed). No marked species- or sex-related differences were observed in the rate of (14)C-elimination, but a relatively higher excretion into the urine of mice was observed compared to that in rats. (14)C-residues in tissue 7 days after administration were relatively high for liver, hair, skin, and kidney, but total (14)C-residues were low, below 0.2% of the dose. An ester cleaved metabolite (S-3153acid) was the major metabolite in feces and urine. Hydroxylation of the methyl group on the C5 of the pyridazine ring and ether cleavage were also observed. No sex-related differences were observed in (14)C-elimination, (14)C-distribution, and metabolite profiles, and metabolism of flufenpyr-ethyl in rats and mice was similar. In vitro metabolism of flufenpyr-ethyl was examined using stomach and intestinal contents and blood and liver S9 fractions (postmitochondrial supernatant fractions) in rats. S-3153acid was detected as a major metabolite in the presence of intestinal contents and blood and liver S9 fractions, and a small amount was also formed in the presence of stomach contents, indicating that the parent compound is rapidly metabolized by intestinal contents and blood and liver S9 fractions through ester cleavage.

Hypolipidemic Effect ofTelfairia occidentalis(Fluted Pumpkin) in Rats Fed a Cholesterol-Rich Diet

Telfairia occidentalis (fluted pumpkin) is one of the commonly consumed leafy vegetables in Nigeria. In order to justify its inclusion in herbal preparations in African traditional medicine, the possible hypolipidemic effect of this vegetable was investigated in rats fed a cholesterol-rich diet. The ability of Questran, a hypolipidemic drug, to attenuate hypercholesterolemia was also examined. Rats were fed with either a basal diet containing cholic acid (0.2%) or a supplemented diet with T. occidentalis at the 3% and 6% levels. Oral administration of cholesterol for 9 consecutive weeks resulted in a significant increase (P < .001) in the relative weight of the heart of cholesterol-fed rats. However, supplemented diets significantly (P < .001) ameliorated the cholesterol-induced enlargement of the heart. Rats fed on supplemented diets had a dose-dependent reduction in plasma and postmitochondrial supernatant fraction (PMF) cholesterol levels. In particular, supplemented diets containing 3% and 6% T. occidentalis decreased plasma and PMF cholesterol levels by 20% and 30% and by 30% and 45%, respectively. A similar decrease in plasma and PMF cholesterol levels was obtained in Questran-treated hypercholesterolemic rats. Furthermore, 3% and 6% T. occidentalis-supplemented diets significantly (P < .05) decreased the cholesterol-induced increase in plasma and PMF low-density lipoprotein (LDL) cholesterol levels by 24% and 48% and by 28% and 52%, respectively. In contrast, there was no significant difference (P > .05) in plasma and PMF triglyceride levels of rats fed on supplemented diets when compared with cholesterol-fed rats. There were significant decreases (P < .05) in lipid peroxidation levels in rats fed on the supplemented diets. Specifically, 3% and 6% T. occidentalis-supplemented diets decreased plasma and PMF lipid peroxidation by 24% and 20% and by 42% and 21%, respectively. This study demonstrates that T. occidentalis may be a useful therapy for hypercholesterolemia through reducing oxidative stress and cholesterol levels.

Tissue Kallikrein mK13 Is a Candidate Processing Enzyme for the Precursor of Interleukin-1β in the Submandibular Gland of Mice

By using Western blot analysis, high levels of 17.5- and 20-kDa interleukin-1beta (IL-1beta) proteins were detected in the submandibular gland (SMG) of mice. Despite this fact, the amount of pro-IL-1beta protein, a precursor of IL-1beta, with a molecular size of 35 kDa in this tissue was below the detectable level, although strong expression of pro-IL-1beta mRNA was observed. A large amount of 17.5-kDa IL-1beta also appeared in the saliva of mice injected with lipopolysaccharide, suggesting that this IL-1beta is a secretory form produced by the SMG. The protein for IL-1beta-converting enzyme, a processing enzyme for pro-IL-1beta, was expressed only at a low level in the SMG as compared with its level in various epithelial tissues or lipopolysaccharide-stimulated macrophages. On the other hand, mK1, mK9, mK13, and mK22, members of the kallikrein family, were detected strongly in the SMG but not in other tissues. By incubation with mK13, but not with mK1, mK9, or mK22, the 35-kDa pro-IL-1beta was cleaved into two major products with molecular masses of 17.5 and 22 kDa, and production was inhibited by phenylmethylsulfonyl fluoride, a serine protease inhibitor, but not by IL-1beta-converting enzyme inhibitors. A peptide segment corresponding to amino acid residues 107-121 of mouse pro-IL-1beta (107WDDDDNLLVCDVPIR) was cleaved by incubation with mK13, generating two peptides, 107WDDDDNL and 114LVCDVPIR. Therefore, kallikrein mK13 would appear to hydrolyze pro-IL-1beta between its Leu113 and Leu114 residues. The results of immunohistochemistry and an autonomic therapy experiment showed that IL-1beta and kallikrein mK13 were co-localized in the secretory granules of granular convoluted tubular cells. Our present results thus suggest kallikrein mK13 is a plausible candidate for the processing enzyme for pro-IL-1beta in the SMG of mice.

Antioxidant enzyme activity and lipid peroxidation in liver of female rats co-exposed to lead and cadmium: Effects of vitamin E and Mn2+

The oxidative status of liver of female rats exposed to lead acetate and cadmium acetate either alone or in combination at a dose of 0.05 mg/kg body wt intraperitoneally for 15 days was studied. After the administration of lead alone, the activity of superoxide dismutase (SOD) decreased in liver, whereas no changes were observed in catalase (CAT) activity, and glutathione (GSH) and thiobarbituric acid (TBARS) levels. Cadmium exposure and combined exposure to lead and cadmium led to decrease in GSH content and increased TBARS levels. Moreover, animals exposed to either cadmium alone or in combination with lead showed a decrease in SOD activity and an increase in CAT activity. The in vitro experiments showed that vitamin E failed to restore the antioxidant enzyme activities in metal treated postmitochondrial supernatant fraction of liver. But Mn2+ ions protected the mitochondria from lipid peroxidation and could completely restore Mn-superoxide dismutase (Mn-SOD) activity following metal intoxication. The results of this study indicate that despite the ability of lead and cadmium to induce oxidative stress the effect in liver is not intensified by combined exposure to both lead and cadmium. The observed changes in various oxidative stress parameters in the liver of rats co-exposed to lead and cadmium may result from an independent effect of lead and /cadmium and also from their interaction such as changes in metal accumulation and content of essential elements like Cu, Zn and Fe. These results suggest that when lead and cadmium are present together in similar concentrations, cadmium mediates major effects due to its more reactive nature.

COX23, a Homologue of COX17, Is Required for Cytochrome Oxidase Assembly

Deletion of reading frame YHR116W of the Saccharomyces cerevisiae nuclear genome elicits a respiratory deficiency. The encoded product, here named Cox23p, is shown to be required for the expression of cytochrome oxidase. Cox23p is homologous to Cox17p, a water-soluble copper protein previously implicated in the maturation of the Cu(A) center of cytochrome oxidase. The respiratory defect of a cox23 null mutant is rescued by high concentrations of copper in the medium but only when the mutant harbors COX17 on a high copy plasmid. Overexpression of Cox17p by itself is not a sufficient condition to rescue the mutant phenotype. Cox23p, like Cox17p, is detected in the intermembrane space of mitochondria and in the postmitochondrial supernatant fraction, the latter consisting predominantly of cytosolic proteins. Because Cox23p and Cox17p are not part of a complex, the requirement of both for cytochrome oxidase assembly suggests that they function in a common pathway with Cox17p acting downstream of Cox23p.

The hydrogenase-like Nar1p is essential for maturation of cytosolic and nuclear iron-sulphur proteins.

The genome of the yeast Saccharomyces cerevisiae encodes the essential protein Nar1p that is conserved in virtually all eukaryotes and exhibits striking sequence similarity to bacterial iron-only hydrogenases. A human homologue of Nar1p was shown previously to bind prenylated prelamin A in the nucleus. However, yeast neither exhibits hydrogenase activity nor contains nuclear lamins. Here, we demonstrate that Nar1p is predominantly located in the cytosol and contains two adjacent iron-sulphur (Fe/S) clusters. Assembly of its Fe/S clusters crucially depends on components of the mitochondrial Fe/S cluster biosynthesis apparatus such as the cysteine desulphurase Nfs1p, the ferredoxin Yah1p and the ABC transporter Atm1p. Using functional studies in vivo, we show that Nar1p is required for maturation of cytosolic and nuclear, but not of mitochondrial, Fe/S proteins. Nar1p-depleted cells do not accumulate iron in mitochondria, distinguishing these cells from mutants in components of the mitochondrial Fe/S cluster biosynthesis apparatus. In conclusion, Nar1p represents a crucial, novel component of the emerging cytosolic Fe/S protein assembly machinery that catalyses an essential and ancient process in eukaryotes.

Aep3p Stabilizes the Mitochondrial Bicistronic mRNA Encoding Subunits 6 and 8 of the H+-translocating ATP Synthase of Saccharomyces cerevisiae

Subunits 6 and 8 of the mitochondrial ATPase in Saccharomyces cerevisiae are encoded by the mitochondrial genome and translated from bicistronic mRNAs containing both reading frames. The stability of the two major species of ATP8/6 mRNA, which differ in the length of the 5'-untranslated region, depends on the expression of several nuclear-encoded factors. In the present study, the product of the gene designated AEP3 (open reading frame YPL005W) is shown to be required for stabilization and/or processing of both ATP8/6 mRNA species. In an aep3-disruptant strain, the shorter ATP8/6 mRNA was undetectable, and the level of the longer mRNA was reduced to approximately 35% that of wild type. Localization of a hemagglutinin-tagged version of Aep3p showed that the protein is an extrinsic constituent of the mitochondrial inner membrane facing the matrix.

Stable expression of human cytochrome P450 2D6*10 in HepG2 cells.

Over 90% of drugs are metabolized by the cytochrome P-450 (CYP) family of liver isoenzymes. The most important enzymes are CYP1A2, 3A4, 2C9/19, 2D6 and 2E1. Although CYP2D6 accounts for <2% of the total CYP liver enzyme content, it mediates metabolism in almost 25% of drugs. In order to study its enzymatic activity for drug metabolism, its cDNA was cloned and a HepG2 cell line stably expressing CYP2D6 was established. Human CYP2D6 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR) from total RNA extracted from human liver tissue and cloned into pGEM-T vector. cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A cell line was established by transfecting the recombinant plasmid of pREP9-CYP2D6 to hepatoma HepG2 cells. Expression of mRNA was validated by RT-PCR. Enzyme activity of catalyzing dextromethorphan O-demethylation in postmitochondrial supernatant (S9) fraction of the cells was determined by high performance liquid chromatography (HPLC). The cloned cDNA had 4 base differences, e.g. 100 C-T, 336 T-C, 408 C-G and 1 457 G-C, which resulted in P34S, and S486T amino acid substitutions, and two samesense mutations were 112 F and 136 V compared with that reported by Kimura et al (GenBank accession number: M33388). P34S and S486T amino acid substitutions were the characteristics of CYP2D6*10 allele. The relative activity of S9 fraction of HepG2-CYP2D6*10 metabolized detromethorphan O-demethylation was found to be 2.31 +/- 0.19 nmol/min(-1)/mg(-1) S9 protein (n=3), but was undetectable in parental HepG2 cells. cDNA of human CYP2D6*10 can be successfully cloned. A cell line, HepG2-CYP2D6*10, expressing CYP2D6*10 mRNA and having metabolic activity, has been established.

The role of CYP 1A1 in the in vitro metabolism of pregnenolone by the liver of rainbow trout embryos

The in vitro metabolism of pregnenolone (P 5) was investigated using whole liver preparations taken from rainbow trout ( Oncorhynchus mykiss) embryos sampled between 55 and 61 days post-fertilization. The intent of the study was to use HPLC techniques to separate and identify the metabolites of hepatic P 5 metabolism and identify the enzyme(s) involved. The major metabolite of [ 3H]P 5 catabolism was [ 3H]7α-hydroxypregnenolone ([ 3H]7αOHP 5), and the enzyme involved was hypothesized to be a cytochrome P450 (CYP) isozyme. To test that hypothesis, whole liver preparations from embryos were pre-treated with selected CYP inhibitors prior to incubation with [ 3H]P 5 and post-mitochondrial supernatant (PMS) fractions of embryo livers were pre-treated with specific antibodies raised against rainbow trout CYP 1A1 prior to incubation with radiolabelled steroid precursor. Three of the four inhibitors used (Miconazole, Clotimazole, Ketokonazole) and the CYP 1A1 antibodies totally blocked the conversion of [ 3H]P 5 to [ 3H]7αOHP 5, and the fourth, Metyrapone, partially blocked the conversion. These results suggest that CYP 1A1 is the major enzyme involved in hepatic catabolism of P 5 by rainbow trout embryos.