Instruct: Given a scientific claim, retrieve relevant documents that support or refute the claim.
Query: Improvements in OER catalysts show stable activity over several hundred hours.
Earth-abundant first-row (3d) transition metal–based catalysts have been developed for the oxygen-evolution reaction (OER); however, they operate at overpotentials substantially above thermodynamic requirements. Density functional theory suggested that non-3d high-valency metals such as tungsten can modulate 3d metal oxides, providing near-optimal adsorption energies for OER intermediates. We developed a room-temperature synthesis to produce gelled oxyhydroxides materials with an atomically homogeneous metal distribution. These gelled FeCoW oxyhydroxides exhibit the lowest overpotential (191 millivolts) reported at 10 milliamperes per square centimeter in alkaline electrolyte. The catalyst shows no evidence of degradation after more than 500 hours of operation. X-ray absorption and computational studies reveal a synergistic interplay between tungsten, iron, and cobalt in producing a favorable local coordination environment and electronic structure that enhance the energetics for OER.
Cristae, the organized invaginations of the mitochondrial inner membrane, respond structurally to the energetic demands of the cell. The mechanism by which these dynamic changes are regulated and the consequences thereof are largely unknown. Optic atrophy 1 (OPA1) is the mitochondrial GTPase responsible for inner membrane fusion and maintenance of cristae structure. Here, we report that OPA1 responds dynamically to changes in energetic conditions to regulate cristae structure. This cristae regulation is independent of OPA1's role in mitochondrial fusion, since an OPA1 mutant that can still oligomerize but has no fusion activity was able to maintain cristae structure. Importantly, OPA1 was required for resistance to starvation-induced cell death, for mitochondrial respiration, for growth in galactose media and for maintenance of ATP synthase assembly, independently of its fusion activity. We identified mitochondrial solute carriers (SLC25A) as OPA1 interactors and show that their pharmacological and genetic blockade inhibited OPA1 oligomerization and function. Thus, we propose a novel way in which OPA1 senses energy substrate availability, which modulates its function in the regulation of mitochondrial architecture in a SLC25A protein-dependent manner.
The intracellular Ca(2+) concentration of many nonexcitable cells is regulated by calcium store release and store-operated calcium entry (SOCE). In platelets, STIM1 was recently identified as the main calcium sensor expressed in the endoplasmic reticulum. To evaluate the role of the SOC channel moiety, Orai1, in platelet SOCE, we generated mice expressing a mutated, inactive form of Orai1 in blood cells only (Orai1(R93W)). Platelets expressing Orai1(R93W) were characterized by markedly reduced SOCE and impaired agonist-induced increases in [Ca(2+)](i). Orai1(R93W) platelets showed reduced integrin activation and impaired degranulation when stimulated with low agonist concentrations under static conditions. This defect, however, did not significantly affect the ability of Orai1(R93W) platelets to aggregate or to adhere to collagen under arterial flow conditions ex vivo. In contrast, these adherent Orai1(R93W) platelets were defective in surface phosphatidylserine exposure, suggesting that Orai1 is crucial for the platelets' procoagulant response rather than for other Ca(2+)-dependent cellular responses.
We have investigated HO endonuclease-induced double-strand break (DSB) recombination and repair in a LACZ duplication plasmid in yeast. A 117-bp MATa fragment, embedded in one copy of LACZ, served as a site for initiation of a DSB when HO endonuclease was expressed. The DSB could be repaired using wild-type sequences located on a second, promoterless, copy of LACZ on the same plasmid. In contrast to normal mating-type switching, crossing-over associated with gene conversion occurred at least 50% of the time. The proportion of conversion events accompanied by exchange was greater when the two copies of LACZ were in direct orientation (80%), than when inverted (50%). In addition, the fraction of plasmids lost was significantly greater in the inverted orientation. The kinetics of appearance of intermediates and final products were also monitored. The repair of the DSB is slow, requiring at least an hour from the detection of the HO-cut fragments to completion of repair. Surprisingly, the appearance of the two reciprocal products of crossing over did not occur with the same kinetics. For example, when the two LACZ sequences were in the direct orientation, the HO-induced formation of a large circular deletion product was not accompanied by the appearance of a small circular reciprocal product. We suggest that these differences may reflect two kinetically separable processes, one involving only one cut end and the other resulting from the concerted participation of both ends of the DSB.
Oxysterols are oxygenated cholesterol derivates that are emerging as a physiologically important group of molecules. Although they regulate a range of cellular processes, only few oxysterol-binding effector proteins have been identified, and the knowledge of their binding mode is limited. Recently, the family of G protein-coupled seven transmembrane-spanning receptors (7TM receptors) was added to this group. Specifically, the Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) was shown to be activated by several oxysterols, most potently by 7α,25-dihydroxycholesterol (7α,25-OHC). Nothing is known about the binding mode, however. Using mutational analysis, we identify here four key residues for 7α,25-OHC binding: Arg-87 in TM-II (position II:20/2.60), Tyr-112 and Tyr-116 (positions III:09/3.33 and III:13/3.37) in TM-III, and Tyr-260 in TM-VI (position VI:16/6.51). Substituting these residues with Ala and/or Phe results in a severe decrease in agonist binding and receptor activation. Docking simulations suggest that Tyr-116 interacts with the 3β-OH group in the agonist, Tyr-260 with the 7α-OH group, and Arg-87, either directly or indirectly, with the 25-OH group, although nearby residues likely also contribute. In addition, Tyr-112 is involved in 7α,25-OHC binding but via hydrophobic interactions. Finally, we show that II:20/2.60 constitutes an important residue for ligand binding in receptors carrying a positively charged residue at this position. This group is dominated by lipid- and nucleotide-activated receptors, here exemplified by the CysLTs, P2Y12, and P2Y14. In conclusion, we present the first molecular characterization of oxysterol binding to a 7TM receptor and identify position II:20/2.60 as a generally important residue for ligand binding in certain 7TM receptors.
Tyrosine hydroxylase (TH, EC 1.14.16.2) from beef brain striata was purified 23-fold from an extract of an acetone powder. If this enzyme preparation is treated with a cyclic AMP[-dependent protein phosphorylation system, there is a change in the pH dependence of the enzyme activity. The pH optimum at saturating tetrahydrobiopterin (BH4) concentration is shifted from below pH 6 to about pH 6.7. At pH 7, activation is expressed mainly as an increase in Vmax, whereas at pH 6, activation is expressed mainly as a decrease in Km for the pterin cofactor. Further, even with the control enzyme the Km for pterin cofactor declines precipitously as the pH is increased from 6 toward neutrality. Similar data were obtained with G-25 Sephadex-treated rat striatal TH. Experiments in which rat striatal synaptosomes were used demonstrated that the in situ activation of TH by phosphorylating conditions is expressed primarily as an increase in the maximum rate of dopamine synthesis. These results indicate that changes in TH activity caused by cyclic AMP-dependent protein phosphorylation will depend to a large extent on the pH of the TH environment.
BACKGROUND Orai1/CRACM1 is a principal component of the store-operated calcium channels. Store-operated calcium influx is highly correlated with inflammatory reactions, immunological regulation, and cell proliferation. Epidermal growth factor (EGF), which plays an important role in the regulation of cell proliferation, can activate store-operated calcium channels. However, the consequences of Orai1/CRACM1 overexpression in EGF-mediated lung cancer cells growth are not known. METHODS To investigate the role of Orai1/CRACM1 in EGF-mediated lung cancer cell proliferation, Orai1/CRACM1 plasmids were transfected into cells by lipofection. A cell proliferation assay, immunofluorescence staining, flow cytometry, and real-time polymerase chain reaction were employed to monitor cell proliferation. The calcium influx signals were investigated using a fluorescent-based calcium assay. RESULTS Transfection of Orai1/CRACM1 plasmids resulted in the inhibition of EGF-mediated cell proliferation. ERK1/2 and Akt phosphorylation were inhibited by Orai1/CRACM1 overexpression. Expression of the cell cycle modulator p21 was induced in the Orai1/CRACM1-overexpressing cells, whereas the expression of cyclin D3 was reduced. Flow cytometry revealed that overexpression of Orai1/CRACM1 resulted in G0/G1 cell cycle arrest. Importantly, Orai1/CRACM1 overexpression significantly attenuated EGF-mediated store-operated calcium influx. In addition, application of 2-APB, a store-operated calcium channel inhibitor, resulted in the inhibition of EGF-mediated cancer cell proliferation. CONCLUSIONS We conclude that Orai1/CRACM1 overexpression attenuates store-operated Ca(2+) influx that in turn blocks EGF-mediated proliferative signaling and drives cell cycle arrest.
We isolated a mutant allele of dnaX, encoding the tau and gamma subunits of the DNA polymerase III holoenzyme, that causes extreme cell filamentation but does not affect either cell growth or DNA replication. This phenotype results from a defect in daughter chromosome decatenation during rapid growth. In these cells, ParC, one subunit of topoisomerase IV, no longer associated with the replication factory, as occurs in wild-type cells, and was instead distributed uniformly on the nucleoid; the distribution of ParE, the other subunit of topoisomerase IV, was unaffected. In addition, the majority of topoisomerase IV activity in synchronized cell populations was restricted to late in the cell cycle, when replication was essentially complete. These observations suggest that topoisomerase IV activity in vivo might be dependent on release of ParC from the replication factory.
In vivo activation of Klebsiella aerogenes urease, a nickel-containing enzyme, requires the presence of functional UreD, UreF, and UreG accessory proteins and is further facilitated by UreE. These accessory proteins are proposed to be involved in metallocenter assembly (M. H. Lee, S. B. Mulrooney, M. J. Renner, Y. Markowicz, and R. P. Hausinger, J. Bacteriol. 174:4324-4330, 1992). A series of three UreD-urease apoprotein complexes are present in cells that express ureD at high levels, and these complexes are thought to be essential for in vivo activation of the enzyme (I.-S. Park, M. B. Carr, and R. P. Hausinger, Proc. Natl. Acad. Sci. USA 91:3233-3237, 1994). In this study, we describe the effect of accessory gene deletions on urease complex formation. The ureE, ureF, and ureG gene products were found not to be required for formation of the UreD-urease complexes; however, the complexes from the ureF deletion mutant exhibited delayed elution during size exclusion chromatography. Because these last complexes were of typical UreD-urease sizes according to native gel electrophoretic analysis, we propose that UreF alters the conformation of the UreD-urease complexes. The same studies revealed the presence of an additional series of urease apoprotein complexes present only in cells containing ureD, ureF, and ureG, along with the urease subunit genes. These new complexes were shown to contain urease, UreD, UreF, and UreG. We propose that the UreD-UreF-UreG-urease apoprotein complexes represent the activation-competent form of urease apoprotein in the cell.
Mitochondrial morphological and ultrastructural changes occur during apoptosis and autophagy, but whether they are relevant in vivo for tissue response to damage is unclear. Here we investigate the role of the optic atrophy 1 (OPA1)-dependent cristae remodeling pathway in vivo and provide evidence that it regulates the response of multiple tissues to apoptotic, necrotic, and atrophic stimuli. Genetic inhibition of the cristae remodeling pathway in vivo does not affect development, but protects mice from denervation-induced muscular atrophy, ischemic heart and brain damage, as well as hepatocellular apoptosis. Mechanistically, OPA1-dependent mitochondrial cristae stabilization increases mitochondrial respiratory efficiency and blunts mitochondrial dysfunction, cytochrome c release, and reactive oxygen species production. Our results indicate that the OPA1-dependent cristae remodeling pathway is a fundamental, targetable determinant of tissue damage in vivo.
CaMKII is activated by oxidation of methionine residues residing in the regulatory domain. Oxidized CaMKII (ox-CaMKII) is now thought to participate in cardiovascular and pulmonary diseases and cancer. This invited review summarizes current evidence for the role of ox-CaMKII in disease, considers critical knowledge gaps and suggests new areas for inquiry.
HTRF (Homogeneous Time Resolved Fluorescence) is the most frequently used generic assay technology to measure analytes in a homogenous format, which is the ideal platform used for drug target studies in high-throughput screening (HTS). This technology combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR). In TR-FRET assays, a signal is generated through fluorescent resonance energy transfer between a donor and an acceptor molecule when in close proximity to each other. Buffer and media interference is dramatically reduced by dual-wavelength detection, and the final signal is proportional to the extent of product formation. The HTRF assay is usually sensitive and robust that can be miniaturized into the 384 and 1536-well plate formats. This assay technology has been applied to many antibody-based assays including GPCR signaling (cAMP and IP-One), kinases, cytokines and biomarkers, bioprocess (antibody and protein production), as well as the assays for protein-protein, proteinpeptide, and protein-DNA/RNA interactions. Since its introduction to the drug-screening world over ten years ago, researchers have used HTRF to expedite the study of GPCRs, kinases, new biomarkers, protein-protein interactions, and other targets of interest. HTRF has also been utilized as an alternative method for bioprocess monitoring. The first-generation HTRF technology, which uses Europium cryptate as a fluorescence donor to monitor reactions between biomolecules, was extended in 2008 through the introduction of a second-generation donor, Terbium cryptate (Tb), enhancing screening performance. Terbium cryptate possesses different photophysical properties compared to Europium, including increased quantum yield and a higher molar extinction coefficient. In addition to being compatible with the same acceptor fluorophors used with Europium, it can serve as a donor fluorophore to green-emitting fluors because it has multiple emission peaks including one at 490 nm. Moreover, all Terbium HTRF assays can be read on the same HTRF-compatible instruments as Europium HTRF assays. Overall, HTRF is a highly sensitive, robust technology for the detection of molecular interactions in vitro and is widely used for primary and secondary screening phases of drug development. This review addresses the general principles of HTRF and its current applications in drug discovery.
Mu transposons carrying the chloramphenicol resistance marker have been inserted into the cloned Escherichia coli genes sodA and sodB coding for manganese superoxide dismutase (MnSOD) and iron superoxide dismutase (FeSOD) respectively, creating mutations and gene fusions. The mutated sodA or sodB genes were introduced into the bacterial chromosome by allelic exchange. The resulting mutants were shown to lack the corresponding SOD by activity measurements and immunoblot analysis. Aerobically, in rich medium, the absence of FeSOD or MnSOD had no major effect on growth or sensitivity to the superoxide generator, paraquat. In minimal medium aerobic growth was not affected, but the sensitivity to paraquat was increased, especially in the sodA mutant. A sodA sodB double mutant completely devoid of SOD was also obtained. It was able to grow aerobically in rich medium, its catalase level was unaffected and it was highly sensitive to paraquat and hydrogen peroxide; the double mutant was unable to grow aerobically on minimal glucose medium. Growth could be restored by removing oxygen, by providing an SOD-overproducing plasmid or by supplementing the medium with the 20 amino acids. It is concluded that the total absence of SOD in E. coli creates a conditional sensitivity to oxygen.
Cells deficient in a major DNA double-strand break repair pathway (nonhomologous DNA end joining [NHEJ]) have increased spontaneous chromosome breaks; however, the source of these chromosome breaks has remained undefined. Here, we show that the observed spontaneous chromosome breaks are partially suppressed by reducing the cellular oxygen tension. Conversely, elevating the level of reactive oxygen species by overexpressing the antioxidant enzyme superoxide dismutase 1 (SOD1), in a transgenic mouse, increases chromosome breakage. The effect of SOD1 can also be modulated by cellular oxygen tension. The elevated chromosome breakage correlates histologically with a significant increase in the amount of neuronal cell death in Ku86(-/-) SOD1 transgenic embryos over that seen in Ku86(-/-) embryos. Therefore, oxygen metabolism is a major source of the genomic instability observed in NHEJ-deficient cells and, presumably, in all cells.
Escherichia coli responds to the redox stress imposed by superoxide-generating agents such as paraquat by activating the synthesis of as many as 80 polypeptides. Expression of a key group of these inducible proteins is controlled at the transcriptional level by the soxRS locus (the soxRS regulon). A two-stage control system was hypothesized for soxRS, in which an intracellular redox signal would trigger the SoxR protein as a transcriptional activator of the soxS gene and the resulting increased levels of SoxS protein would activate transcription of the various soxRS regulon genes (B. Demple and C.F. Amábile Cuevas, Cell 67:837-839, 1990). We have constructed operon fusions of the E. coli lac genes to the soxS promoter to monitor soxS transcription. Expression from the soxS promoter is strongly inducible by paraquat in a manner strictly dependent on a functional soxR gene. Several other superoxide-generating agents also trigger soxR(+)-dependent soxS expression, and the inductions by paraquat and phenazine methosulfate were dependent on the presence of oxygen. Numerous other oxidative stress agents (H2O2, gamma rays, heat shock, etc.) failed to induce soxS, while aerobic growth of superoxide dismutase-deficient bacteria triggered soxR-dependent soxS expression. These results indicate a specific redox signal for soxS induction. A direct role for SoxR protein in the activation of the soxS gene is indicated by band-shift and DNase I footprinting experiments that demonstrate specific binding of the SoxR protein in cell extracts to the soxS promoter. The mode of SoxR binding to DNA appears to be similar to that of its homolog MerR in that the SoxR footprint spans the -10 to -35 region of the soxS promoter.
A recent study revealed a mechanism of delaying aging in yeast by a natural compound which specifically impacts mitochondrial redox processes. In this mechanism, exogenously added lithocholic bile acid enters yeast cells, accumulates mainly in the inner mitochondrial membrane, and elicits an age-related remodeling of phospholipid synthesis and movement within both mitochondrial membranes. Such remodeling of mitochondrial phospholipid dynamics progresses with the chronological age of a yeast cell and ultimately causes significant changes in mitochondrial membrane lipidome. These changes in the composition of membrane phospholipids alter mitochondrial abundance and morphology, thereby triggering changes in the age-related chronology of such longevity-defining redox processes as mitochondrial respiration, the maintenance of mitochondrial membrane potential, the preservation of cellular homeostasis of mitochondrially produced reactive oxygen species, and the coupling of electron transport to ATP synthesis.
The region located immediately upstream from the Klebsiella aerogenes urease structural genes was sequenced and shown to possess an open reading frame capable of encoding a 29.8-kDa peptide. Deletions were generated in this gene, denoted ureD, and in each of the genes (ureE, ureF, and ureG) located immediately downstream of the three structural genes. Transformation of the mutated plasmids into Escherichia coli resulted in high levels of urease expression, but the enzyme was inactive (deletions in ureD, ureF, or ureG) or only partially active (deletions in ureE). Ureases were purified from the recombinant cells and shown to be identical to control enzyme when analyzed by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis; however, in every case the activity levels correlated to nickel contents as analyzed by atomic absorption analysis. UreD, UreE, UreF, and UreG peptides were tentatively identified by gel electrophoretic comparison of mutant and control cell extracts, by in vivo expression of separately cloned genes, or by in vitro transcription-translation analyses; the assignments were confirmed for UreE and UreG by amino-terminal sequencing. The latter peptides (apparent M(r)s, 23,900 and 28,500) were present at high levels comparable to those of the urease subunits, whereas the amounts of UreF (apparent M(r), 27,000) and UreD (apparent M(r), 29,300) were greatly reduced, perhaps because of the lack of good ribosome binding sites in the regions upstream of these open reading frames. These results demonstrate that all four accessory genes are necessary for the functional incorporation of the urease metallocenter.
Reactive oxygen species have been linked with neuropathological changes in the central nervous system. Epidemiological studies supported the beneficial effect of supplementation of antioxidants. Superoxide dismutase (SOD) is an endogenous enzyme which can scavenge reactive oxygen species. This study investigated the effect of supplementation with ascorbic acid (vitamin C) on the changes of SOD in cultured neurological cells. Rat brain astrocytes (RBA-1 cells) were incubated with vitamin C and divided into four groups: a control group (without vitamin C) and three treatment groups with vitamin C at 40, 80, and 160 µmol/l. After short-term (2 days) and long-term (7 days) incubation, SOD activity, SOD mRNA level by Northern blotting, and SOD protein amounts by Western blotting were measured. After 2 days of incubation, vitamin C resulted in a decrease in the activity of SOD in a concentration-dependent manner (Mn-SOD from 14.8 ± 1.2 to 13.2 ± 0.5 U/mg protein and Cu/Zn-SOD from 64.8 ± 1.2 to 51.7 ± 0.9 U/mg protein; p < 0.05), and vitamin C also attenuated the Cu/Zn-SOD mRNA level from 100 to 86.3 ± 6.7%; p < 0.01), whereas the protein amounts of these two SODs remained unchanged. After 7 days of incubation with vitamin C, the SOD activity of RBA-1 cells decreased significantly (Mn-SOD from 14.9 ± 0.3 to 11.8 ± 0.3 U/mg protein and Cu/Zn SOD from 61.8 ± 1.8 to 54.6 ± 0.9 U/mg protein; p < 0.01), and the mRNA level was also attenuated (Mn-SOD from 100 to 86.8 ± 8.7% and Cu/Zn-SOD from 100 to 84.7 ± 4.8%; p < 0.01). These results suggest that 2 and 7 days of incubation with relatively high concentrations of vitamin C may downregulate activity and gene expression of SOD in cultured RBA-1 cells.
The bulk of ATP consumed by various cellular processes in higher eukaryotes is normally produced by five multimeric protein complexes (I-V) embedded within the inner mitochondrial membrane, in a process known as oxidative phosphorylation (OXPHOS). Maintenance of energy homeostasis under most physiological conditions is therefore contingent upon the ability of OXPHOS to meet cellular changes in bioenergetic demand, with a chronic failure to do so being a frequent cause of human disease. With the exception of Complex II, the structural subunits of OXPHOS complexes are encoded by both the nuclear and the mitochondrial genomes. The physical separation of the two genomes necessitates that the expression of the 13 mitochondrially encoded polypeptides be co-ordinated with that of relevant nuclear-encoded partners in order to assemble functional holoenzyme complexes. Complex biogenesis is a highly ordered process, and several nuclear-encoded factors that function at distinct stages in the assembly of individual OXPHOS complexes have been identified.
The forkhead O (FoxO) family of transcription factors participates in diverse physiologic processes, including induction of cell-cycle arrest, stress resistance, differentiation, apoptosis, and metabolism. Several recent studies indicate that FoxO-dependent signaling is required for long-term regenerative potential of the hematopoietic stem cell (HSC) compartment through regulation of HSC response to physiologic oxidative stress, quiescence, and survival. These observations link FoxO function in mammalian systems with the evolutionarily conserved role of FoxO in promotion of stress resistance and longevity in lower phylogenetic systems. Furthermore, these findings have implications for aging in higher organisms and in malignant stem cell biology, and suggest that FoxOs may play an important role in the maintenance and integrity of stem cell compartments in a broad spectrum of tissues.
Oxidative phosphorylation (OXPHOS) is the major pathway for ATP production in humans. Deficiencies in OXPHOS can arise from mutations in either mitochondrial or nuclear genomes and comprise the largest collection of inborn errors of metabolism. At present we lack a complete catalog of human genes and pathways essential for OXPHOS. Here we introduce a genome-wide CRISPR "death screen" that actively selects dying cells to reveal human genes required for OXPHOS, inspired by the classic observation that human cells deficient in OXPHOS survive in glucose but die in galactose. We report 191 high-confidence hits essential for OXPHOS, including 72 underlying known OXPHOS diseases. Our screen reveals a functional module consisting of NGRN, WBSCR16, RPUSD3, RPUSD4, TRUB2, and FASTKD2 that regulates the mitochondrial 16S rRNA and intra-mitochondrial translation. Our work yields a rich catalog of genes required for OXPHOS and, more generally, demonstrates the power of death screening for functional genomic analysis.
Exonic splicing enhancers (ESEs) facilitate exon definition by assisting in the recruitment of splicing factors to the adjacent intron. Here we demonstrate that suboptimal 5' and 3' splice sites are activated independently by ESEs when they are located on different exons. However, when they are situated within a single exon, the same weak 5' and 3' splice sites are activated simultaneously by a single ESE. These findings demonstrate that a single ESE promotes the recognition of both exon/intron junctions within the same step during exon definition. Our results suggest that ESEs recruit a multicomponent complex that minimally contains components of the splicing machinery required for 5' and 3' splice site selection.
ErmC' is a methyltransferase that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics by catalyzing the methylation of 23S rRNA at a specific adenine residue (A-2085 in Bacillus subtilis; A-2058 in Escherichia coli). The gene for ErmC' was cloned and expressed to a high level in E. coli, and the protein was purified to virtual homogeneity. Studies of substrate requirements of ErmC' have shown that a 262-nucleotide RNA fragment within domain V of B. subtilis 23S rRNA can be utilized efficiently as a substrate for methylation at A-2085. Kinetic studies of the monomethylation reaction showed that the apparent Km of this 262-nucleotide RNA oligonucleotide was 26-fold greater than the value determined for full-size and domain V 23S rRNA. In addition, the Vmax for this fragment also rose sevenfold. A model of RNA-ErmC' interaction involving multiple binding sites is proposed from the kinetic data presented.
We have characterized the membrane topology of a 60-kDa inner membrane protein from Escherichia coli that is homologous to the recently identified Oxa1p protein in Saccharomyces cerevisiae mitochondria implicated in the assembly of mitochondrial inner membrane proteins. Hydrophobicity and alkaline phosphatase fusion analyses suggest a membrane topology with six transmembrane segments, including an N-terminal signal-anchor sequence not present in mitochondrial Oxa1p. In contrast to partial N-terminal fusion protein constructs, the full-length protein folds into a protease-resistant conformation, suggesting that important folding determinants are present in the C-terminal part of the molecule.
Eicosapentaenoic acid (EPA) has beneficial effects in many inflammatory disorders. In this study, dietary EPA was converted to 17,18-epoxyeicosatetraenoic acid (17,18-EpETE) by ω-3 epoxygenation in the mouse peritoneal cavity. Mediator lipidomics revealed a series of novel oxygenated metabolites of 17,18-EpETE, and one of the major metabolites, 12-hydroxy-17,18-epoxyeicosatetraenoic acid (12-OH-17,18-EpETE), displayed a potent anti-inflammatory action by limiting neutrophil infiltration in murine zymosan-induced peritonitis. 12-OH-17,18-EpETE inhibited leukotriene B4-induced neutrophil chemotaxis and polarization in vitro in a low nanomolar range (EC50 0.6 nM). The complete structures of two natural isomers were assigned as 12S-OH-17R,18S-EpETE and 12S-OH-17S,18R-EpETE, using chemically synthesized stereoisomers. These natural isomers displayed potent anti-inflammatory action, whereas the unnatural stereoisomers were essentially devoid of activity. These results demonstrate that 17,18-EpETE derived from dietary EPA is converted to a potent bioactive metabolite 12-OH-17,18-EpETE, which may generate an endogenous anti-inflammatory metabolic pathway.