The use of zinc(II) to probe iron binding and oxidation by the ferritin (EcFtnA) of Escherichia coli

 The ferritin of Escherichia coli (EcFtnA) is similar to human H-chain ferritin (HuHF) in having 24 subunits, each containing a dinuclear site at which two iron atoms can be oxidised (the diiron centre). In EcFtnA, unlike HuHF, fluorescence quenching of Trp122, located near site A of the dinuclear centre, can be used to monitor metal binding (this tryptophan is absent from HuHF). Metal binding also perturbs the UV absorbance spectrum of Trp122 and that of Tyr24 (a conserved residue near site B of the dinuclear centre). Using UV-difference spectroscopy and fluorescence quenching it is shown that Fe(II) and Zn(II) bind at the same sites, A and B. Sequential stopped-flow studies of Fe(II) binding and oxidation also show that Zn(II) is an effective competitor of Fe(II) binding and an inhibitor of its oxidation. Received: 10 June 1998 / Accepted: 18 September 1998     

Oxidation-deficient silkworm hemolymph as a medium supplement for insect cell culture

Hemolymph is oxidized and darkens visibly during the collection from silkworms due to the activity of tyrosinase in it. Toxic quinones are produced by the oxidation and consequently inhibit the cell growth. Heat treatment can be used to prevent the oxidation; however, the oxidation may occur during the collection of hemolymph before it is heat-treated. It makes the hemolymph collection difficult especially on a large-scale preparation. Hemolymphs collected from 257 different strains of silkworms were examined to select the slowly oxidized hemolymphs. Hemolymphs collected from mutant strains such as Lemone, TBO, Cre, Y4, and wE^b showed relatively slow color changes. Oxidation rates of the hemolymphs were measured by the absorbance change using a spectrophotometer. The hemolymph of wE^b showed the slowest oxidation. The absorbance of this mutant hemolymph reached the saturation value at 20°C in 450 min, whereas the total oxidation time of the wild-type (Baekokjam) hemolymph at the same temperature was 120 min. We tested if this mutant hemolymph is useful as a medium supplement for insect cell culture. Cell growth rate and final cell concentration in the medium supplemented with the wE^b hemolymph were almost same as those in the medium supplemented with the wild-type hemolymph. Hemolymph is collected on a small scale by clipping the abdominal leg; however, this method is not appropriate for large scale preparation. Centrifugation after chopping the silkworm hemolymph by a blending mixer is a more appropriate procedure for large scale collection. Slowly oxidized wE^b hemolymph resulted in higher cell concentration than the wild-type hemolymph when hemolymph was collected by the large scale preparation method.     

Plant Functional Type Effects on Trace Gas Fluxes in the Shortgrass Steppe

Plant community structure is expected to regulate the microbial processes of nitrification and denitrification by controlling the availability of inorganic N substrates. Thus it could also be a factor in the concomitant release of NO and N2O from soils as a result of these processes. C3 and C4 plants differ in several attributes related to the cycling of nitrogen and were hypothesized to yield differences in trace gas exchange between soil and atmosphere. In this study we estimated fluxes of NO, N2O and CH4 from soils of shortgrass steppe communities dominated by either C3 plants, C4 plants or mixtures of the two types. We collected gas samples weekly from two sites, a sandy clay loam and a clay, throughout the growing seasons of 1995 and 1996. Plant functional type effects on gas fluxes at the clay site were not apparent, however we found several differences among plant communities on the sandy clay loam. CH4 uptake from atmosphere to soil was significantly greater on C4 plots than C3 plots in both years. NO fluxes were significantly greater from C4 plots than from C3 plots in 1995. NO fluxes from C3 and mixed plots were not significantly different between 1995 and 1996, however fluxes from C4 plots were significantly greater in 1995 compared to 1996. Results indicate that under certain environmental conditions, particularly when factors such as moisture and temperature are not limiting, plant community composition can play an important role in regulating trace gas exchange.     

Studies on the relation between the fast phase of millisecond delayed light emission and the proton released from oxidation of water in spinach chloroplast

The relation between the fast phase of ms-DLE (delayed light emission measured with a phosphoroscope) and the proton released from water oxidation in spinach chloroplasts was studied in several aspects. When photophosphorylation was allowed to be coupled to the Hill reaction the intensity of the fast phase of ms-DLE of chloroplast was lowered more at 1 °C than at 25 °C, and the photophosphorylation rate within 40 ms of flashing light was higher at 1 °C than at 25 °C. Adding the subunit of ATP synthase to the chloroplast preparation to block the leakage of protons through ATP synthase, the intensity of the fast phase of ms-DLE was enhanced, to a larger extent at 1 °C than at 25°C. When the ms-DLE was measured under isotonic conditions, the intensity of fast phase of ms-DLE enhanced by proton released from oxidation of water was more pronounced. The above results support the suggestion that under lower temperature and isotonic conditions, the proton released from water oxidation was liable to be localized and could enhance the intensity of the fast phase of ms-DLE more effectively.     

Molecular ecology of a facultative swine waste lagoon

Aims:  To investigate the microbial ecology of three facultative swine waste lagoons.
Methods and Results:  Phylogenetic analysis of sequences in a 16S rRNA gene clone library and fluorescence in situ hybridization (FISH) analyses were used to assess bacterial diversity in a swine waste lagoon. FISH analysis and Gram-staining were used to compare the microbial communities of all three swine waste lagoons. Six operational taxonomic units were in high relative abundance and corresponded to the following phylotypes; Thiolamprovum , Verrucomicrobia , Acholeplasma , Turicibacter , Clostridium and Bacteroides . PCR was employed to detect the genes apsA and dsrAB which encode for enzymes specifically associated with dissimilatory sulfate-reduction within sulfate-reducing bacteria (SRB). Amplification of these genes confirmed their presence within the lagoons.
Conclusions:  All lagoons were dominated by purple sulfur bacteria, affiliated to Thiolamprovum pedioforme . The molecular identification of fermentative bacteria and SRB indicate the following metabolic processes within such facultative ponds: sulfur-cycling, fermentation, inter-species hydrogen transfer and carbon cycling.
Significance and Impact of the Study:  This study provides the first molecular evidence for the existence of a sulfur cycle which is linked to phototrophic sulfide oxidation by purple bacteria and organotrophic sulfate-reduction by SRB.     

Effect of modification of soybean lipoxygenase-1 with N-bromosuccinimide on linoleate oxidation,pigment bleaching and carbonyl production

Essential tryptophan residues were specifically modified in soybean lipoxygenase-1 by N-bromosuccinimide (NBS). Both linoleate oxidation and pigment bleaching (β-carotene or chlorophyll a) activities were significantly reduced with the modified enzyme under aerobic conditions. However, the effect on the reduction of linoleate oxidation was more marked. Pigment bleaching under anaerobic conditions was almost completely blocked with the modified enzyme. On the basis of spectral studies it was elucidated that soybean lipoxygenase-1 contains two essential tryptophan residues in its active site.     

Post‐translational modifications of transthyretin affect the triiodonine‐binding potential

Transthyretin (TTR) is a visceral protein, which facilitates the transport of thyroid hormones in blood and cerebrospinal fluid. The homotetrameric structure of TTR enables the simultaneous binding of two thyroid hormones per molecule. Each TTR subunit provides a single cysteine residue (Cys₁₀), which is frequently affected by oxidative post‐translational modifications. As Cys₁₀ is part of the thyroid hormone‐binding channel within the TTR molecule, PTM of Cys₁₀ may influence the binding of thyroid hormones. Therefore, we analysed the effects of Cys₁₀ modification with sulphonic acid, cysteine, cysteinylglycine and glutathione on binding of triiodothyronine (T3) by molecular modelling. Furthermore, we determined the PTM pattern of TTR in serum of patients with thyroid disease by immunoprecipitation and mass spectrometry to evaluate this association in vivo. The in silico assays demonstrated that oxidative PTM of TTR resulted in substantial reorganization of the intramolecular interactions and also affected the binding of T3 in a chemotype‐ and site‐specific manner with S‐glutathionylation as the most potent modulator of T3 binding. These findings were supported by the in vivo results, which indicated thyroid function‐specific patterns of TTR with a substantial decrease in S‐sulphonated, S‐cysteinylglycinated and S‐glutathionylated TTR in hypothyroid patients. In conclusion, this study provides evidence that oxidative modifications of Cys₁₀ seem to affect binding of T3 to TTR probably because of the introduction of a sterical hindrance and induction of conformational changes. As oxidative modifications can be dynamically regulated, this may represent a sensitive mechanism to adjust thyroid hormone availability.     

Antioxidant enzymes as redox-based biomarkers: a brief review

The field of redox proteomics focuses to a large extent on analyzing cysteine oxidation in proteins under different experimental conditions and states of diseases. The identification and localization of oxidized cysteines within the cellular milieu is critical for understanding the redox regulation of proteins under physiological and pathophysiological conditions, and it will in turn provide important information that are potentially useful for the development of novel strategies in the treatment and prevention of diseases associated with oxidative stress. Antioxidant enzymes that catalyze oxidation/reduction processes are able to serve as redox biomarkers in various human diseases, and they are key regulators controlling the redox state of functional proteins. Redox regulators with antioxidant properties related to active mediators, cellular organelles, and the surrounding environments are all connected within a network and are involved in diseases related to redox imbalance including cancer, ischemia/reperfusion injury, neurodegenerative diseases, as well as normal aging. In this review, we will briefly look at the selected aspects of oxidative thiol modification in antioxidant enzymes and thiol oxidation in proteins affected by redox control of antioxidant enzymes and their relation to disease. [BMB Reports 2015; 48(4): 200-208]     

Structural insight into activation of homoserine dehydrogenase from the archaeon Sulfolobus tokodaii via reduction

Homoserine dehydrogenase (HSD; 305 amino acid residues) catalyzes an NAD(P)-dependent reversible reaction between l-homoserine and aspartate 4-semialdehyde and is involved in the aspartate pathway. HSD from the hyperthermophilic archaeon Sulfolobus tokodaii was markedly activated (2.5-fold) by the addition of 0.8 mM dithiothreitol. The crystal structure of the homodimer indicated that the activation was caused by cleavage of the disulfide bond formed between two cysteine residues (C303) in the C-terminal regions of the two subunits.