A novel procedure for the assessment of the antioxidant capacity of food components

Carbonylation, an oxidative modification of the amino group of arginine and lysine residues caused by reactive oxygen species, has emerged as a new type of oxidative damage. Protein carbonylation has been shown to exert adverse effects on various protein functions. Recently, the role of food components in the attenuation of oxidative stress has been the focus of many studies. Most of these studies focused on the chemical properties of food components. However, it is also important to determine their effects on protein functions via post-translational modifications. In this study, we developed a novel procedure for evaluating the antioxidant capacity of food components. Hydrogen peroxide (H₂O₂)-induced protein carbonylation in HL-60 cells was quantitatively analyzed by using fluorescent dyes (Cy5–hydrazide dye and IC3–OSu dye), followed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and fluorescence determination. Among a panel of food components tested, quinic acid, kaempferol, saponin, squalene, trigonelline, and mangiferin were shown to be capable of suppressing protein carbonylation in HL-60 cells. Our results demonstrated that this fluorescence labeling/SDS–PAGE procedure allows for the detection of oxidative stress-induced protein carbonylation with high sensitivity and quantitative accuracy. This method should be useful for the screening of new antioxidant food components as well as the analysis of their suppression mechanism.     

Mouse geminin inhibits not only Cdt1-MCM6 interactions but also a novel intrinsic Cdt1 DNA binding activity

DNA replication is controlled by the stepwise assembly of a pre-replicative complex and the replication apparatus. Cdt1 is a novel component of the pre-replicative complex and plays a role in loading the minichromosome maintenance (MCM) 2-7 complex onto chromatin. Cdt1 activity is inhibited by geminin, which is essential for the G(2)/M transition in metazoan cells. To understand the molecular basis of the Cdt1-geminin regulatory mechanism in mammalian cells, we cloned and expressed the mouse Cdt1 homologue cDNA in bacterial cells and purified mouse Cdt1 to near homogeneity. We found by yeast two-hybrid analysis that mouse Cdt1 associates with geminin, MCM6, and origin recognition complex 2. MCM6 interacts with the Cdt1 carboxyl-terminal region (amino acids 407-477), which is conserved among eukaryotes, whereas geminin associates with the Cdt1 central region (amino acids 177-380), which is conserved only in metazoans. In addition, we found that Cdt1 can bind DNA in a sequence-, strand-, and conformation-independent manner. The Cdt1 DNA binding domain overlaps with the geminin binding domain, and the binding of Cdt1 to DNA is inhibited by geminin. Taken together, we have defined structural domains and novel biochemical properties for mouse Cdt1 that suggest that Cdt1 behaves as an intrinsic DNA binding factor in the pre-replicative complex.     

Responses of soil microbial communities to prescribed burning in two paired vegetation sites in southern China

Prescribed burning is a common site preparation practice for forest plantation in southern China. However, the effects of prescribed burning on soil microbial communities are poorly understood. This study examined changes in microbial community structure, measured by phospholipid fatty acids (PLFAs), after a single prescribed burning in two paired vegetation sites in southern China. The results showed that the total amount of PLFA (totPLFA) was similar under two vegetation types in the wet season but differed among vegetation type in the dry season, and was affected significantly by burning treatment only in the wet season. Bacterial PLFA (bactPLFA) and fungal PLFA (fungPLFA) in burned plots all decreased compared to the unburned plots in both seasons (P = 0.059). Fungi appeared more sensitive to prescribed burning than bacteria. Both G⁺ bacterial PLFA and G⁻ bacterial PLFA were decreased by the burning treatment in both dry and wet seasons. Principal component analysis of PLFAs showed that the burning treatment induced a shift in soil microbial community structure. The variation in soil microbial community structure was correlated significantly to soil organic carbon, total nitrogen, available phosphorus and exchangeable potassium. Our results suggest that prescribed burning results in short-term changes in soil microbial communities but the long-term effects of prescribed burning on soil microbial community remain unknown and merit further investigation.     

Elongated mouse chromosomes suitable for enhanced molecular cytogenetics

Characterization of genetic disorders in humans and animal models requires identification of chromosomal aberrations. However, identifying fine deletions or insertion in metaphase chromosomes has been always a challenge due to limitations of resolution. In this study we developed a rapid method for chromosome elongation using two different intercalating agents: ethidium bromide and 5-bromo-2′-deoxyuridine (BrdU), together with a short-term mitotic block using colcemid. About 70% of the chromosomes from cells that underwent this elongation procedure reached three times longer than those prepared from control cells. FISH experiments using elongated chromosomes revealed a duplicated region of chromosome 11 that was not visible in cells prepared with conventional methods.     

Anti-Arrhythmic Effect of Verapamil Is Accompanied by Preservation of Cx43 Protein in Rat Heart

The present study was to test the hypothesis that anti-arrhythmic properties of verapamil may be accompanied by preserving connexin43 (Cx43) protein via calcium influx inhibition. In an in vivo study, myocardial ischemic arrhythmia was induced by occlusion of the left anterior descending (LAD) coronary artery for 45 min in Sprague-Dawley rats. Verapamil, a calcium channel antagonist, was injected i.v. into a femoral vein prior to ischemia. Effects of verapamil on arrhythmias induced by Bay K8644 (a calcium channel agonist) were also determined. In an ex vivo study, the isolated heart underwent an initial 10 min of baseline normal perfusion and was subjected to high calcium perfusion in the absence or presence of verapamil. Cardiac arrhythmia was measured by electrocardiogram (ECG) and Cx43 protein was determined by immunohistochemistry and western blotting. Administration of verapamil prior to myocardial ischemia significantly reduced the incidence of ventricular arrhythmias and total arrhythmia scores, with the reductions in heat rate, mean arterial pressure and left ventricular systolic pressure. Verapamil also inhibited arrhythmias induced by Bay K8644 and high calcium perfusion. Effect of verapamil on ischemic arrhythmia scores was abolished by heptanol, a Cx43 protein uncoupler and Gap 26, a Cx43 channels inhibitor. Immunohistochemistry data showed that ischemia-induced redistribution and reduced immunostaining of Cx43 were prevented by verapamil. In addition, diminished expression of Cx43 protein determined by western blotting was observed following myocardial ischemia in vivo or following high calcium perfusion ex vivo and was preserved after verapamil administration. Our data suggest that verapamil may confer an anti-arrhythmic effect via calcium influx inhibition, inhibition of oxygen consumption and accompanied by preservation of Cx43 protein.     

Vanadate‐Based Materials for Li‐Ion Batteries: The Search for Anodes for Practical Applications

While the practical application of electrode materials depends intensively on the Li⁺ ion storage mechanisms correlating ultimately with the coulombic efficiency, reversible capacity, and morphology variation of electrode material upon cycling, only intercalation‐type electrode materials have proven viable for commercialization up to now. This paper reviews the promising anode materials of metal vanadates (M_xV_yO_z, M = Co, Cu, Mn, Fe, Zn, Ni, Li) that have high capacity, low cost, and abundant resource, and also discusses the related Li⁺ ion storage mechanism. It is concluded that most of these (M_xV_yO_z, M = Co, Cu, Mn, Fe, Zn, Ni) exhibit irreversible redox reactions upon lithiation/delithiation accompanied by large volume expansion, which is not favorable for industrial applications. In particular, Li₃VO₄ with specific intercalation Li⁺ ion storage mechanism and compatible merits of safety and energy density exhibits great potential for practical application. This review systematically summarizes the latest progress in Li₃VO₄ research, including the representative fabrication approaches for advanced morphology and state‐of‐the‐art technologies to boost performance and the morphology variation associated with Li⁺ ion storage mechanisms. Furthermore, an outlook on where breakthroughs for Li₃VO₄ may be most likely achieved will be provided.     

DNA polymerization-independent functions of DNA polymerase epsilon in assembly and progression of the replisome in fission yeast

DNA polymerase epsilon (Pol ε) synthesizes the leading strands, following the CMG (Cdc45, Mcm2-7, and GINS [Go-Ichi-Nii-San]) helicase that translocates on the leading-strand template at eukaryotic replication forks. Although Pol ε is essential for the viability of fission and budding yeasts, the N-terminal polymerase domain of the catalytic subunit, Cdc20/Pol2, is dispensable for viability, leaving the following question: what is the essential role(s) of Pol ε? In this study, we investigated the essential roles of Pol ε using a temperature-sensitive mutant and a recently developed protein-depletion (off-aid) system in fission yeast. In cdc20-ct1 cells carrying mutations in the C-terminal domain of Cdc20, the CMG components, RPA, Pol α, and Pol δ were loaded onto replication origins, but Cdc45 did not translocate from the origins, suggesting that Pol ε is required for CMG helicase progression. In contrast, depletion of Cdc20 abolished the loading of GINS and Cdc45 onto origins, indicating that Pol ε is essential for assembly of the CMG complex. These results demonstrate that Pol ε plays essential roles in both the assembly and progression of CMG helicase.     

Suppressive effects of 4-phenylbutyrate on the aggregation of Pael receptors and endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress is defined as an accumulation of unfolded proteins in the endoplasmic reticulum. 4-phenylbutyrate (4-PBA) has been demonstrated to promote the normal trafficking of the DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) mutant from the ER to the plasma membrane and to restore activity. We have reported that 4-PBA protected against cerebral ischemic injury and ER stress-induced neuronal cell death. In this study, we revealed that 4-PBA possesses chemical chaperone activity in vitro, which prevents the aggregation of denatured alpha-lactalbumin and bovine serum albumin (BSA). Furthermore, we investigated the effects of 4-PBA on the accumulation of Parkin-associated endothelin receptor-like receptor (Pael-R) pathologically relevant to the loss of dopaminergic neurons in autosomal recessive juvenile parkinsonism (AR-JP). Interestingly, 4-PBA restored the normal expression of Pael-R protein and suppressed ER stress induced by the overexpression of Pael-R. In addition, we showed that 4-PBA attenuated the activation of ER stress-induced signal transduction pathways and subsequent neuronal cell death. Moreover, 4-PBA restored the viability of yeasts that fail to induce an ER stress response under ER stress conditions. These results suggest that 4-PBA suppresses ER stress by directly reducing the amount of misfolded protein, including Pael-R accumulated in the ER.