Scythe regulates apoptosis through modulating ubiquitin-mediated proteolysis of the Xenopus elongation factor XEF1AO

Scythe was originally identified as a novel Reaper-binding anti-apoptotic protein, although the mechanisms of its functions remain largely obscure. Our previous analysis revealed that Scythe can bind to a proteasomal subunit via N-terminal domains and that the domains are required for appropriate development of Xenopus embryos. In the present study, we show evidence that the N-terminus of Scythe interacts with XEF1AO, a maternal form of Xenopus laevis EF1A that was suggested to be a potential inducer of apoptosis in vertebrates, and that the binding enhances the poly-ubiquitin modification and subsequent degradation of XEF1AO. Scythe is required for degradation of XEF1AO, since immunodepletion of Scythe from embryonic extracts stabilized XEF1AO significantly. Furthermore, we show that apoptosis induced by accumulation of XEF1AO can be suppressed by co-expression of the full-length form of Scythe. These observations indicate that the proteolytic regulation of XEF1AO, mediated through Scythe, is essential to prevent inappropriate accumulation of XEF1AO and resulting apoptotic events during the course of Xenopus development.     

Association of Rpn10 with high molecular weight complex is enhanced during retinoic acid-induced differentiation of neuroblastoma cells

The ubiquitin-binding Rpn10 protein serves as an ubiquitin receptor that delivers client proteins to the 26S proteasome, the protein degradation complex. It has been suggested that the ubiquitin-dependent protein degradation is critical for neuronal differentiation and for preventing neurodegenerative diseases. Our previous study indicated the importance of Rpn10 in control of cellular differentiation (Shimada et al., Mol Biol Cell 17:5356–5371, 2006), though the functional relevance of Rpn10 in neuronal cell differentiation remains a mystery to be uncovered. In the present study, we have examined the level of Rpn10 in a proteasome-containing high molecular weight (HMW) protein fraction prepared from the mouse neuroblastoma cell line Neuro2a. We here report that the protein level of Rpn10 in HMW fraction from un-differentiated Neuro2a cells was significantly lower than that of other cultured cell lines. We have found that retinoic acid-induced neural differentiation of Neuro2a cells significantly stimulates the incorporation of Rpn10 into HMW fractions, although the amounts of 26S proteasome subunits were not changed. Our findings provide the first evidence that the modulation of Rpn10 is linked to the control of retinoic acid-induced differentiation of neuroblastoma cells.     

Promotion of insulin aggregation by protein disulfide isomerase

We examined the aggregation of insulin as a result of reduction of disulfide bonds catalyzed by protein disulfide isomerase (PDI) using various techniques. We demonstrated the kinetic correlation between PDI-catalyzed insulin reduction and the aggregate formation, the relationship between aggregation and amyloid formation, and the structural information on the secondary structure of the aggregates. The initial rate of PDI-catalyzed reduction of insulin, apparent rate constants of aggregate growth for sigmoidal features, and lag times were obtained by changing the PDI concentration, temperature, and insulin concentration. In situ kinetics were studied using the dyes; thioflavin T (ThT) and Congo red (CR) in addition to turbidimetry with the insulin reduction by PDI. The ThT and CR binding assay revealed sigmoidal kinetics, and the spectrum of binding CR showed a red shift against time, suggesting an orderly formation of insulin aggregates. The secondary structure of the PDI-promoted insulin aggregates showed antiparallel beta-sheet conformation by FT-IR measurement.     

Biocidal effect of cathodic protection on bacterial viability in biofilm attached to carbon steel

Biofilm formed on carbon steel by various species of bacterial cells causes serious problems such as corrosion of steel, choking of flow in the pipe, deterioration of the heat-transfer efficiency, and so on. Cathodic protection is known to be a reliable method for protecting carbon steel from corrosion. However, the initial attachment of bacteria to the surface and the effects of cathodic protection on bacterial viability in the biofilm have not been clarified. In this study, cathodic protection was applied to an artificial biofilm containing Pseudomonas aeruginosa (PAO1), a biofilm constituent, on carbon steel. The aims of this study were to evaluate the inhibition effect of cathodic protection on biofilm formation and to reveal the inhibition mechanisms. The viability of PAO1 in artificial biofilm of 5 mm thickness on cathodically protected steel decreased to 1% of the initial cell concentration. Analysis of pH distribution in the artificial biofilm by pH microelectrode revealed that pH in proximity to carbon steel increased to approximately 11 after cathodic protection for 5 h. Moreover, 99% of region in the artificial biofilm was under the pH conditions of over nine. A simulation of pH profile was shown to correspond to experimental values. These results indicate cells in the artificial biofilm were killed or damaged by cathodic protection due to pH increase.     

Hepcidin expression in the liver: relatively low level in patients with chronic hepatitis C

Patients with chronic hepatitis C frequently have serum and hepatic iron overload, but the mechanism is unknown. Recently identified hepcidin, exclusively synthesized in the liver, is thought to be a key regulator for iron homeostasis and is induced by infection and inflammation. This study was conducted to determine the hepatic hepcidin expression levels in patients with various liver diseases. We investigated hepcidin mRNA levels of liver samples by real-time detection-polymerase chain reaction; 56 were hepatitis C virus (HCV) positive, 34 were hepatitis B virus (HBV) positive, and 42 were negative for HCV and HBV (3 cases of auto-immune hepatitis, 7 alcoholic liver disease, 13 primary biliary cirrhosis, 9 nonalcoholic fatty liver disease, and 10 normal liver). We analyzed the relation of hepcidin to clinical, hematological, histological, and etiological findings. Hepcidin expression levels were strongly correlated with serum ferritin (P < 0.0001) and the degree of iron deposit in liver tissues (P < 0.0001). Hepcidin was also correlated with hematological parameters (vs. hemoglobin, P = 0.0073; vs. serum iron, P = 0.0012; vs. transferrin saturation, P < 0.0001) and transaminase levels (P = 0.0013). The hepcidin-to-ferritin ratio was significantly lower in HCV(+) patients than in HBV(+) patients (P = 0.0129) or control subjects (P = 0.0080). In conclusion, hepcidin expression levels in chronic liver diseases were strongly correlated with either the serum ferritin concentration or degree of iron deposits in the liver. When adjusted by either serum ferritin values or hepatic iron scores, hepcidin indices were significantly lower in HCV(+) patients than in HBV(+) patients, suggesting that hepcidin may play a pivotal role in the pathogenesis of iron overload in patients with chronic hepatitis C.     

Function of the Herpes Simplex Virus 1 Small Capsid Protein VP26 Is Regulated by Phosphorylation at a Specific Site

Replacement of the herpes simplex virus 1 small capsid protein VP26 phosphorylation site Thr-111 with alanine reduced viral replication and neurovirulence to levels observed with the VP26 null mutation. This mutation reduced VP26 expression and mislocalized VP26 and its binding partner, the major capsid protein VP5, in the nucleus. VP5 mislocalization was also observed with the VP26 null mutation. Thus, we postulate that phosphorylation of VP26 at Thr-111 regulates VP26 function in vitro and in vivo.     

A unique mechanism of curcumin inhibition on F1 ATPase

ATP synthase (F-ATPase) function depends upon catalytic and rotation cycles of the F₁ sector. Previously, we found that F₁ ATPase activity is inhibited by the dietary polyphenols, curcumin, quercetin, and piceatannol, but that the inhibitory kinetics of curcumin differs from that of the other two polyphenols (Sekiya et al., 2012, 2014). In the present study, we analyzed Escherichia coli F₁ ATPase rotational catalysis to identify differences in the inhibitory mechanism of curcumin versus quercetin and piceatannol. These compounds did not affect the 120° rotation step for ATP binding and ADP release, though they significantly increased the catalytic dwell duration for ATP hydrolysis. Analysis of wild-type F₁ and a mutant lacking part of the piceatannol binding site (γΔ277–286) indicates that curcumin binds to F₁ differently from piceatannol and quercetin. The unique inhibitory mechanism of curcumin is also suggested from its effect on F₁ mutants with defective β–γ subunit interactions (γMet23 to Lys) or β conformational changes (βSer174 to Phe). These results confirm that smooth interaction between each β subunit and entire γ subunit in F₁ is pertinent for rotational catalysis.     

Effects of phylogeny,leaf traits,and the altitudinal distribution of host plants on herbivore assemblages on congeneric Acer species

Historical, niche-based, and stochastic processes have been proposed as the mechanisms that drive community assembly. In plant–herbivore systems, these processes can correspond to phylogeny, leaf traits, and the distribution of host plants, respectively. Although patterns of herbivore assemblages among plant species have been repeatedly examined, the effects of these factors among co-occurring congeneric host plant species have rarely been studied. Our aim was to reveal the process of community assembly for herbivores by investigating the effects of phylogeny, leaf traits, and the altitudinal distribution of closely related host plants of the genus Acer. We sampled leaf functional traits for 30 Acer species in Japan. Using a newly constructed phylogeny, we determined that three of the six measured leaf traits (leaf thickness, C/N ratio, and condensed tannin content) showed a phylogenetic signal. In a field study, we sampled herbivore communities on 14 Acer species within an elevation gradient and examined relationships between herbivore assemblages and host plants. We found that herbivore assemblages were significantly correlated with phylogeny, leaf traits, phylogenetic signals, and the altitudinal distribution of host plants. Our results indicate that the interaction between historical and current ecological processes shapes herbivore community assemblages.