Age-dependent declines in proteasome activity in the heart.

The proteasome is a major intracellular proteolytic system involved in the removal of oxidized and ubiquitinated protein and the induction of certain stress response pathways. In this study, age-dependent alterations in proteasome function were investigated to gain insight into potential factors which contribute to increased susceptibility to various forms of heart disease during aging. Proteasome activity in cellular extracts prepared from Fisher 344 rat hearts was found to decrease with age. These declines in activity were associated with a decreased 20S proteasome content and loss of specific activities. As determined by two-dimensional gel electrophoresis of purified 20S proteasome, the distribution and silver staining intensities of enzyme subunits were found to vary with age, suggesting that alterations in proteasome subunit composition and/or structure are involved in age-related declines in proteasome activity. In addition, age-dependent increases in the levels of oxidized and ubiquitinated proteins, known substrates of the proteasome, were observed. Thus, loss in proteasome function may impair the ability of myocytes to mount an appropriate response to stress, thereby enhancing the susceptibility of the aging heart to cardiovascular disease.     

Enhanced selective oxidation of trimethylolpropane to 2,2-bis(hydroxymethyl)butyric acid using Corynebacterium sp. ATCC 21245

2,2-Bis(hydroxymethyl)butyric acid (BHMB) is an important multifunctional chemical for the emerging bio-based polymer industry. It can be produced from trimethylolpropane (TMP) by selective oxidation using growing cells of Corynebacterium sp. ATCC 21245. However, this process is limited by the low volumetric productivity and low concentration of the final product. In the present study, we performed sequential batch operation with cell recycling in media containing glycerol, acetic acid, and increasing concentrations of yeast extract. This approach enhanced the conversion of 10 and 15 g/L TMP to 11.0 and 16.3 g/L BHMB at rates of 0.50 and 0.20 g/L^.h, respectively. Applying a cell bleeding strategy resulted in an overall 10-fold improvement in productivity. The consequently prolonged biocatalyst viability resulted in a quantitative conversion of 20 g/L TMP to 22.3 g/L BHMB and a yield of 1.10 g_BHMB/g_TMP (100% molar yield). This work facilitates further studies of the selective oxidation on other industrially important polyols.     

Growth of a manganese oxidizing Pseudomonas sp. in continuous culture

Strain S-36, a marine Pseudomonas sp., was grown under manganese limitation in continuous culture. At dilution rates below a maximal growth rate of 0.066 h^-1, the rate at which the organism fixed CO₂ into macromolecules was equal to the cell carbon production rate. In addition, the total amount of cell carbon or CO₂ fixed at steady-state was in proportion to the amount of energy available from the oxidation of Mn²⁺ in the medium. These data suggest that the organism can grow by obtaining the energy for CO₂ fixation from manganese oxidation.     

Evidence for altered structure and impaired mitochondrial electron transport function in selenium deficiency

Selenium (Se) deficiency in the experimental models,Coturnix coturnix japonica andCorcyra cephalonica, resulted in impaired mitochondrial substrate oxidations and lowered thiol levels. Studies with respiratory inhibitors confirmed reduced mitochondrial electron transport enzyme activities, especially at cytochromec oxidase (COX), the terminal segment. Enhanced mitochondrial lipid peroxidation in Se deficiency was more pronounced in the heart tissue of the quail compared to other tissues. Glutathione peroxidase (GSH-Px) activity toward H₂O₂ and cumene hydroperoxide were generally low in the insect muscle tissue and activity toward H₂O₂ was maximal in the quail heart mitochondria that was not very sensitive to Se status. Lowered COX activity in Se deficiency was more directly correlated with the increased level of lipid peroxidation than with the GSH-Px activity measured, suggestive of Se mediated protective mechanisms independent of GSH-Px. Electron microscopic observations revealed structural changes such as loss of cristae with proliferative and degenerative changes of the mitochondria in Se deficiency. Involvement of Se in maintaining structure and functional efficiency of mitochondria is evident from the present study.     

Endogenous ubiquinol prevents protein modification accompanying lipid peroxidation in beef heart submitochondrial particles

This article is a study of the relationship between lipid peroxidation and protein modification in beef heart submitochondrial particles, and the protective effect of endogenous ubiquinol (reduced coenzyme Q) against these effects. ADP-Fe^3· and ascorbate were used to initiate lipid peroxidation and protein modification, which were monitored by measuring TBARS and protein carbonylation, respectively. Endogenous ubiquinone was reduced by the addition of succinate and antimycin. The parameters investigated included extraction and reincorporation of ubiquinone, and comparison of the effect of ubiquinol with those of various antioxidant compounds and enzymes, as well as the iron chelator EDTA. Under all conditions employed there was a close correlation between lipid peroxidation and protein carbonylation, and the inhibition of these effects by endogenous ubiquinol. SDS-PAGE analysis revealed a differential effect on individual protein components and its prevention by ubiquinol. Conceivable mechanisms behind the observed oxidative modifications of membrane phospholipids and proteins and of the role of ubiquinol in preventing these effects are considered.     

Characterization of main sulfur source of wood-degrading basidiomycetes by S K-edge X-ray absorption near edge spectroscopy (XANES)

The main wood degraders in aerobic terrestrial ecosystems belong to the white- and brown-rot fungi, where their biomass can be created on wood decay only. However, total sulfur (S) concentration in wood is very low and only little is known about the different sulfur compounds in wood today. Sulfur-starved brown-rot fungi Gloeophyllum trabeum and Oligoporus placenta were incubated on sterilized pine wood blocks whereas Lentinus cyathiformis and the white-rot fungi Trametes versicolor were incubated on sterilized beech wood blocks. After 19 weeks of incubation, the S oxidation status was analyzed in wood, in degraded wood, and in biomass of wood-degrading fungi by synchrotron based S K-edge XANES, and total S and sulfate were quantified. Total sulfur and sulfate content in pine wood blocks were approximately 50 and 1 ??g g⁻¹, respectively, while in beech wood approximately 100 and 20 ??g g⁻¹ were found, respectively. Sulfur in beech was dominated by sulfate-esters. In contrast, pine wood also contained larger amounts of reduced S. Three out of four selected fungi caused a reduction of the S oxidation state in wood from oxidized S (sulfate-ester, sulfate) to intermediate S (sulfonate, sulfoxide) or reduced S (thiols, e.g., proteins, peptides, enzyme cofactors). Only O. placenta shifted thiol to sulfonate. Growth experiments of these fungi on selective minimal media showed that in particular cysteine (thiol), sulfonates, and sulfate enhanced total mycelium growth. Consequently, wood-degrading fungi were able to utilize a large variety of different wood S sources for growth but preferentially transformed in vivo sulfate-esters and thiol into biomass structures.     

Proper calibration of ultrasonic power enabled the quantitative analysis of the ultrasonication-induced amyloid formation process

To elucidate the mechanisms of ultrasonication on the amyloid fibril formation, we quantitatively determined the ultrasonic power using both calorimetry and potassium iodide (KI) oxidation, and under the properly calibrated ultrasonic power, we investigated the ultasonication-induced amyloid formation process of the mouse prion protein (mPrP(23-231)). These methods revealed that the ultrasonic power in our system ranged from 0.3 to 2.7 W but entirely dependent on the positions of the ultrasonic stage. Intriguingly, the nucleation time of the amyloid fibrils was found to be shortened almost proportionally to the ultrasonic power, indicating that the probability of the occurrence of nucleus formation increases proportionally to the ultrasonic power. Moreover, mPrP(23-231) formed two types of aggregates: rigid fibrils and short fibrils with disordered aggregates, depending on the ultrasonic power. The nucleation of rigid fibrils required an ultrasonic power larger than 1.5 W. While at the strong ultrasonic power larger than 2.6 W, amyloid fibrils were formed early, but simultaneously fine fragmentation of fibrils occurred. Thus, an ultrasonic power of approximately 2.0 W would be suitable for the formation of rigid mPrP(23-231) fibrils under the conditions utilized (ultrasonication applied for 30 s every 9 min). As ultrasonication has been widely used to amplify the scrapie form of the prion protein, or other amyloids in vitro, the calorimetry and KI oxidation methods proposed here might help determining the adequate ultrasonic powers necessary to amplify them efficiently.     

Modification of swelling–contraction–aggregation processes in rat muscle mitochondria by the 1,4-dihydropyridines,cerebrocrast and glutapyrone,themselves and in the presence of azidothymidine

The influence of the 1,4-dihydropyridines (DHPs), water-soluble glutapyrone available as sodium, potassium and ammonium salts of 2-(2,6-dimethyl-3,5-diethoxycarbonyl-1,4-DHP-4-carboxamide)glutaric acid, from one side, and a lipophylic cerebrocrast, 2-propoxyethyl 2,6-dimethyl-4-(2-difluoromethoxyphenyl)-1,4-DHP-3,5-dicarboxylate, from the other side, on partially damaged mitochondria of the Wistar rat hindlimb muscle was also studied. The following tests were made: (1) rates of endogenous respiration and substrate (succinate) oxidation and oxidative phosphorylation; (2) rates and amplitudes of high-amplitude swelling and contraction after the addition of ATP, ADP and succinate to the previously swollen mitochondria and (3) rate of reversible self-aggregation of mitochondria isolated in salt media after ATP-induced contraction without and in the presence of azidothymidine (AZT). Cerebrocrast (10–100 μM ) partially normalized the endogenous respiration rate and slightly augmented the respiration rate after the addition of succinate and to lesser extent ADP. Cerebrocrast in a concentration-dependent manner (2·5–50 μM ) increased (two-fold at 20–50 μM ) the active contraction amplitude of swollen mitochondria, induced by single or repeated additions of ATP. The influence of cerebrocrast on the ADP- and succinate-induced contractions was less obvious. Unlike cerebrocrast glutapyrone caused a reduction of the ATP-induced contraction amplitude (two-fold at 0·5–5·0 mM ), not impairing the mitochondrial contraction ability in response to ATP or succinate. Pre-exposure to 2·5 mM glutapyrone resulted in at least a 10-fold inhibition of the reversible aggregation rate in the presence of 99 and 198 μM AZT. The results suggest the usefulness of further study of cerebrocrast and glutapyrone in preventing AZT-induced and some other mitochondrial myopathies. © 1997 John Wiley & Sons, Ltd.     

Efficient BiVO4 Photoanode with an Excellent Hole Transport Layer of CuSCN for Solar Water Oxidation

Bismuth vanadate (BiVO₄) is reported as a key material in photoelectrocatalysis owing to high theoretical efficiency, relatively narrow band gap of 2.4 eV, and favorable conduction band edge position for hydrogen evolution. However, the sluggish hole transport dynamics lead to slow photogenerated charge separation and transport efficiencies, which result in charge recombination due to aggregation. Herein, a novel hole transport layer of copper(I) thiocyanate (CuSCN) with the aim of significantly enhancing the efficiency of charge transport and stability of BiVO₄ photoanodes is reported. The introduction of the hole transport layer could provide an appropriate intermediate energy level for photogenerated hole transfer and avoid charge recombination and trapping. After a photoassisted electrodeposition process of NiCoFe-B_i catalysis, the obtained photoanode achieves a photocurrent density of 5.6 mA cm⁻² at 1.23 V versus reversible hydrogen electrode under AM 1.5 G simulated solar radiation, and an applied bias photon to current efficiency of 2.31%. With the CuSCN layer, BiVO₄ photoanode presented impressive stable photocurrent during 50 h continuous illumination. Meanwhile, the unbiased tandem device of the NiCoFe-B_i/CuSCN/BiVO₄ photoanode and the Si solar cell exhibit a solar-to-hydrogen efficiency of 5.75% and excellent stability for 14 h.     

Arabidopsis UDP-sugar pyrophosphorylase: Evidence for two isoforms

Arabidopsis UDP-sugar pyrophosphorylase (AtUSP, EC 2.7.7.64) is a broad substrate pyrophosphorylase that exhibits activity with GlcA-1-P, Gal-1-P and Glc-1-P. Immunoblots using polyclonal antibodies raised to recombinant AtUSP demonstrated the presence of two USP isoforms of approximately 70 kDa (USP1) and 66 kDa (USP2) in crude extracts of Arabidopsis tissues. The 66 kDa isoform was not the result of proteolytic cleavage of USP1 during extraction. Trypsin digestion of bands on SDS gels corresponding to the location of the two isoforms followed by tandem mass spectrometry confirmed that USP peptides were present in both bands. Both USP isoforms were detected in the cytosol as determined by immunoblots of cellular fractions obtained by differential centrifugation. However, some USP1 was also detected in the microsomal fraction. Immunoprecipitation assays demonstrated that AtUSP antibodies removed USP activity (UDP-GlcA→GlcA-1-P) measured in floret extracts. These results indicate that USP is the only pyrophosphorylase that utilizes UDP-GlcA as a substrate and suggest that it serves as the terminal enzyme of the myo-inositol oxidation pathway.