Electron transfer from menaquinol to fumarate. Fumarate reductase anchor polypeptide mutants of Escherichia coli

Fumarate reductase (FRD) of Escherichia coli is a four-subunit membrane-bound complex that is synthesized during anaerobic growth when fumarate is available as a terminal oxidant. The two subunits that comprise the catalytic domain, FrdA and FrdB, are anchored to the cytoplasmic membrane surface by two small hydrophobic polypeptides, FrdC and FrdD, which are also required for the enzyme to interact with quinone. To better define the individual roles of the FrdC and FrdD polypeptides in FRD complex formation and quinone binding, we selectively mutagenized the frdCD genes. Frd- strains were identified by their inability to grow on restrictive media, and the resulting mutant FRD complexes were isolated and biochemically characterized. The majority of the frdC and frdD mutations were identified as single base deletions that caused premature termination in either FrdC or FrdD and resulted in the loss of one or more of the predicted transmembrane helices. Two additional frdC mutants were characterized that contained single base changes resulting in single amino acid substitutions. All mutant enzyme complexes were incapable of oxidizing the physiological electron donor, menaquinol-6, in the presence of fumarate. Additionally, the ability of the mutant complexes to oxidize reduced benzyl viologen or reduce the ubiquinone analogue 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzoquinone and phenazine methosulfate with succinate as electron donor were also affected but to varying degrees. The separation of oxidative and reductive activities with quinones suggests there are two quinone binding sites in the fumarate reductase complex and that electron transfer occurs in two le- steps carried out at these separate sites.     

Routine colposcopic survey of patients with squamous atypia. A method for identifying cases with false-negative smears

Routine colposcopy was performed on 376 women with cervical squamous atypia (originally reported as "inflammatory atypia"). Colposcopy showed no abnormalities in 240 cases and a lesion in 136 cases; the latter were sampled by colposcopy-guided biopsy. The biopsy samples showed evidence of human papillomavirus (HPV) infection and/or grade I cervical intraepithelial neoplasia (CIN I) in 42 cases (11.1%), CIN II in 4 cases (1.1%) and CIN III in 5 cases (1.3%); the other 85 biopsied cases were histologically negative. Most cases of HPV/CIN I (35 of 42) and all of the cases of CIN II-III occurred in women under the age of 40. The detection rates were 4.4% for CIN II-III in women under the age of 40, 4.0% for HPV/CIN I in women 40 and older and 17.2% for HPV/CIN I in women under the age of 40 (P less than .001). It thus appears that women under the age of 40 who show cytologic evidence of squamous atypia would benefit from colposcopic examination.     

Transport of amino acids in intact 3T3 and SV3T3 cells. Binding activity for leucine in membrane preparations of ehrlich ascites tumor cells

Transport of amino acids into 3T3 and SV3T3 (SV40 virus-transformed 3T3) cells was measured on glass cover slips. The 3T3 and SV3T3 cells contain both A (alanine preferring) and L (leucine preferring) systems for neutral amino acid transport. Initial rates of uptake of amino acids are about twofold higher in SV3T3 than in 3T3 cells. Other parameters measured, however, do not indicate marked differences in the transport of amino acids by the two cell types. L-system amino acids, such as leucine, are subject to trans-stimulation in both cell lines, whereas A-system amino acids, such as alanine and glycine, are not. Leucine was transported to higher levels in confluent cells than in nonconfluent cells. Glycine, however, shows distinctly less transport activity as the cells become confluent. Ehrlich ascites cell plasma membranes were prepared and assayed for amino acid-binding activity. Leucine-binding activity was detected by equilibrium dialysis in Triton X-100-treated membrane preparations.     

The Action of Hydrogen Peroxide on the Formation of Thiobarbituric Acid-Reactive Material From Microsomes, Liposomes Or From Dna Damaged By Bleomycin Or Phenanthroline. Artefacts in the Thiobarbituric Acid Test

Incubation of rat-liver microsomes, previously azide-treated to inhibit catalase, with H₂O₂ caused a loss of cytochrome P-450 but not of cytochrome b₅. This loss of P-450 was not prevented by scavengers of hydroxyl radical, chain-breaking antioxidants or metal ion-chelating agents. Application of the thiobarbituric acid (TBA) assay to the reaction mixture suggested that H₂O₂ induces lipid peroxidation, but this was found to be due largely or completely to an effect of H²O₂ on the TBA assay. By contrast, addition of ascorbic acid and Fe(III) to the microsomes led to lipid peroxidation and P-450 degradation: both processes were inhibited by chelating agents and chain-breaking antioxidants, but not by hydroxyl radical scavengers. H₂O₂ inhibited ascorbate/Fe (III)-induced microsomal lipid peroxidation, but part of this effect was due to an action of H₂O₂ in the TBA test itself. H₂O₂ also decreased the colour measured after carrying out the TBA test upon authentic malondialdehyde, tetraethoxypropane, a DNA-Cu²⁺/o-phenanthroline system in the presence of a reducing agent, ox-brain phospholipid liposomes in the presence of Fe(III) and ascorbate, or a bleomycin-iron ion/DNA/ascorbate system. Caution must be used in interpreting the results of TBA tests upon systems containing H₂O₂.     

Correction to: Relative Importance of Climate,Soil and Plant Functional Traits During the Early Decomposition Stage of Standardized Litter

Ecosystems - A correction to this paper has been published: https://doi.org/10.1007/s10021-021-00614-y     

Fabrication,Operation and Flow Visualization in Surface-acoustic-wave-driven Acoustic-counterflow Microfluidics

Surface acoustic waves (SAWs) can be used to drive liquids in portable microfluidic chips via the acoustic counterflow phenomenon. In this video we present the fabrication protocol for a multilayered SAW acoustic counterflow device. The device is fabricated starting from a lithium niobate (LN) substrate onto which two interdigital transducers (IDTs) and appropriate markers are patterned. A polydimethylsiloxane (PDMS) channel cast on an SU8 master mold is finally bonded on the patterned substrate. Following the fabrication procedure, we show the techniques that allow the characterization and operation of the acoustic counterflow device in order to pump fluids through the PDMS channel grid. We finally present the procedure to visualize liquid flow in the channels. The protocol is used to show on-chip fluid pumping under different flow regimes such as laminar flow and more complicated dynamics characterized by vortices and particle accumulation domains.     

Factors Controlling the Redox Potential of ZnCe6 in an Engineered Bacterioferritin Photochemical ‘Reaction Centre’

Photosystem II (PSII) of photosynthesis has the unique ability to photochemically oxidize water. Recently an engineered bacterioferritin photochemical ‘reaction centre’ (BFR-RC) using a zinc chlorin pigment (ZnCe₆) in place of its native heme has been shown to photo-oxidize bound manganese ions through a tyrosine residue, thus mimicking two of the key reactions on the electron donor side of PSII. To understand the mechanism of tyrosine oxidation in BFR-RCs, and explore the possibility of water oxidation in such a system we have built an atomic-level model of the BFR-RC using ONIOM methodology. We studied the influence of axial ligands and carboxyl groups on the oxidation potential of ZnCe₆ using DFT theory, and finally calculated the shift of the redox potential of ZnCe₆ in the BFR-RC protein using the multi-conformational molecular mechanics–Poisson-Boltzmann approach. According to our calculations, the redox potential for the first oxidation of ZnCe₆ in the BRF-RC protein is only 0.57 V, too low to oxidize tyrosine. We suggest that the observed tyrosine oxidation in BRF-RC could be driven by the ZnCe₆ di-cation. In order to increase the efficiency of tyrosine oxidation, and ultimately oxidize water, the first potential of ZnCe₆ would have to attain a value in excess of 0.8 V. We discuss the possibilities for modifying the BFR-RC to achieve this goal.     

Cardiac Per2 Functions as Novel Link between Fatty Acid Metabolism and Myocardial Inflammation during Ischemia and Reperfusion Injury of the Heart

Disruption of peripheral circadian rhyme pathways dominantly leads to metabolic disorders. Studies on circadian rhythm proteins in the heart indicated a role for Clock or Per2 in cardiac metabolism. In contrast to Clock^−/−, Per2^−/− mice have larger infarct sizes with deficient lactate production during myocardial ischemia. To test the hypothesis that cardiac Per2 represents an important regulator of cardiac metabolism during myocardial ischemia, we measured lactate during reperfusion in Per1^−/−, Per2^−/− or wildtype mice. As lactate measurements in whole blood indicated an exclusive role of Per2 in controlling lactate production during myocardial ischemia, we next performed gene array studies using various ischemia-reperfusion protocols comparing wildtype and Per2^−/− mice. Surprisingly, high-throughput gene array analysis revealed dominantly lipid metabolism as the differentially regulated pathway in wildtype mice when compared to Per2^−/−. In all ischemia-reperfusion protocols used, the enzyme enoyl-CoA hydratase, which is essential in fatty acid beta-oxidation, was regulated in wildtype animals only. Studies using nuclear magnet resonance imaging (NMRI) confirmed altered fatty acid populations with higher mono-unsaturated fatty acid levels in hearts from Per2^−/− mice. Unexpectedly, studies on gene regulation during reperfusion revealed solely pro inflammatory genes as differentially regulated ‘Per2-genes’. Subsequent studies on inflammatory markers showed increasing IL-6 or TNFα levels during reperfusion in Per2^−/− mice. In summary, these studies reveal an important role of cardiac Per2 for fatty acid metabolism and inflammation during myocardial ischemia and reperfusion, respectively.