Differential Roles of Hsp70 and Hsp90 in the Assembly of the Replicase Complex of a Positive-Strand RNA Plant Virus

Assembly of viral replicase complexes of eukaryotic positive-strand RNA viruses is a regulated process: multiple viral and host components must be assembled on intracellular membranes and ordered into quaternary complexes capable of synthesizing viral RNAs. However, the molecular mechanisms underlying this process are poorly understood. In this study, we used a model virus, Red clover necrotic mosaic virus (RCNMV), whose replicase complex can be detected readily as the 480-kDa functional protein complex. We found that host heat shock proteins Hsp70 and Hsp90 are required for RCNMV RNA replication and that they interact with p27, a virus-encoded component of the 480-kDa replicase complex, on the endoplasmic reticulum membrane. Using a cell-free viral translation/replication system in combination with specific inhibitors of Hsp70 and Hsp90, we found that inhibition of p27-Hsp70 interaction inhibits the formation of the 480-kDa complex but instead induces the accumulation of large complexes that are nonfunctional in viral RNA synthesis. In contrast, inhibition of p27-Hsp90 interaction did not induce such large complexes but rendered p27 incapable of binding to a specific viral RNA element, which is a critical step for the assembly of the 480-kDa replicase complex and viral RNA replication. Together, our results suggest that Hsp70 and Hsp90 regulate different steps in the assembly of the RCNMV replicase complex.     

Effect of Substituted β-Phenoxyacrylates on Shoot Elongation of Barnyard-grass

Twenty-seven substituted ethyl β-phenoxyacrylates were prepared to clarify the relationship between chemical structure and inhibition of the shoot elongation of barnyard-grass. ortho-Halo-substitution greatly enhanced the inhibitory activity, the order of which was Cl>F≧Br>I.

The cis/trans ratio of each preparation was approximately 1:4 on the basis of the NMR spectrum, and the activity of trans isomers was about 100 times higher than that of cis isomers in the case of the 2-chloro- and 2,4-dichloro-derivatives. Among the compounds tested, ethyl trans- β-(2,4-dichlorophenoxy) acrylate was found to be the most effective in inhibiting shoot elongation.     

Co-Processed Excipients for Dispersible Tablets–Part 1: Manufacturability

Co-processed excipients may enhance functionality and reduce drawbacks of traditional excipients for the manufacture of tablets on a commercial scale. The following study aimed to characterise a range of co-processed excipients that may prove suitable for dispersible tablet formulations prepared by direct compression. Co-processed excipients were lubricated and compressed into 10.5-mm convex tablets using a Phoenix compaction simulator. Compression profiles were generated by varying the compression force applied to the formulation and the prepared tablets were characterised for hardness, friability, disintegration and fineness of dispersion. Our data indicates that CombiLac, F-Melt type C and SmartEx QD100 were the top 3 most suitable out of 16 co-processed excipients under the conditions evaluated. They exhibited good flow properties (Carr’s index ? 20), excellent tabletability (tensile strength >?3.0 MPa at 0.85 solid fraction), very low friability (<?1% after 15 min), rapid disintegration times (27–49 s) and produced dispersions of ideal fineness (<?250 μm). Other co-processed excipients (including F-Melt type M, Ludiflash, MicroceLac, Pharmaburst 500 and Avicel HFE-102) may be appropriate for dispersible tablets produced by direct compression providing the identified disintegration and dispersion risks were mitigated prior to commercialisation. This indicates that robust dispersible tablets which disintegrate rapidly could be manufactured from a range of co-processed excipients.     

Chloroplast position and photosynthetic characteristics in two monostromatic species,Monostroma angicava and Protomonostroma undulatum (Ulvophyceae), having a shared ecological niche

Monostroma angicava and Protomonostroma undulatum are monostromatic green benthic algae (Ulvophyceae), which grow together in the same intertidal habitat of Muroran, Hokkaido, Japan, during the spring season. Commonly, both species have a single chloroplast with one pyrenoid per cell. The parietal chloroplast is located on the periphery of the thallus in both species, although the location of the chloroplast differs in the two. In M. angicava , the chloroplast was observed to be arranged on one‐side of the thallus surface, whereas, in P. undulatum , it was dispersed and randomly located on either side of the thallus or on the lateral face. The density of chlorophylls (Chls) assessed from the absorption spectra of the thallus and its solvent extract was higher in M. angicava , which appeared dark‐green in color, than in the light‐green colored P. undulatum . The maximum photosynthetic rate per thallus area (μmol O₂ m^?2 s^?1) was higher in M. angicava , whereas, per total chlorophyll content (μmol O₂ g Chl a + Chl b ^?1 s^?1) was higher in P. undulatum . Both species showed similar efficiency of photosynthesis at light‐limiting conditions. The efficiency of light absorption by photosystem II (PSII ) in P. undulatum was higher than M. angicava , whereas the photoprotective response was higher in M. angicava . This indicates that more energy is utilized in M. angicava to protect its PSII due to the chloroplast position, which has more direct exposure to light and, therefore, lowers the efficiency of light absorption by PSII . The higher density of chlorophylls in M. angicava could explain higher photosynthesis per thallus area, whereas, higher efficiency of light absorption by PSII in P. undulatum could explain higher photosynthesis per total chlorophyll content. The differences in light absorption efficiency and quantum efficiency of PSII might be an important ecological strategy in these two species for their coexistence in the intertidal area.     

Antioxidant status and lipid peroxidation in hemodialysis patients undergoing erythropoietin and erythropoietin-vitamin E combined therapy.

In this study, plasma and red blood cell (RBC) antioxidant status and plasma lipid peroxidation were investigated in 46 hemodialysis patients. In addition, the effect of erythropoietin (EPO) and EPO-vitamin E combination therapy on plasma and RBC antioxidant status, and plasma lipid peroxidation were examined. There were 10 healthy subjects in the control group and 10 hemodialysis patients in the untreated group. The third group included 36 hemodialysis patients that were given EPO (100 U/kg) for 3 months, 3 times per week. The fourth group included 36 hemodialysis-patients from the EPO group that were given EPO at a 50% decreased dose + vitamin E (300 mg/day) for 3 months. MDA levels in the untreated group, the EPO group and the EPO + vitamin E groups were found to be higher than the control group (p < 0.001, in both). Furthermore, MDA levels in both of the treatment groups were lower when compared to the untreated group (p < 0.001, in both). Plasma vitamin E levels in the untreated, the EPO group and EPO + vitamin E groups were lower than the control group (p < 0. 001). In contrast, plasma vitamin E levels in the treatment groups were higher in comparison with the control group (p < 0.05). SOD activities in the untreated, the EPO group and the EPO + vitamin E groups were found to be lower than the control group (p < 0.001). SOD activities in the treatment groups were higher than the control group (p<0.001). The SOD activities in the EPO+vitamin E group increased when compared to the EPO group (p < 0.001). CAT activities in the untreated, the EPO group and the EPO + vitamin E groups were found to be lower than the control group (p < 0.001 in untreated and EPO groups, p <0.01 in EPO+ vitamin E group). CAT activities in EPO and EPO+ vitamin E groups were increased when compared to the untreated group (p < 0.01). In conclusion, our findings have shown that antioxidant status decreased and lipid peroxidation increased in hemodialysis patients. EPO has an antioxidant effect on the RBC and plasma antioxidant status, and plasma lipid peroxidation. These effects were moderately increased by the combination of vitamin E and EPO.     

Characterization of residues in human IgE and IgG binding site by chemical modification of ovomucoid third domain.

Chemical modification of ovomucoid third domain (DIII) has been conducted to characterize the binding site residues that determine antigenecity and allergenecity of DIII. Nitration of Tyr, ethoxyformylation of His and succinylation of Lys residues led to a decrease of alpha-helix content of DIII. Modification of His, Tyr, Glu, Asp and Lys residues on DIII resulted in a reduction of human IgG binding activity, but little effect on IgE binding activity. These results suggest that hydrophilic residues appear to be more critical for human IgG binding site, whereas hydrophobic residues may be more important for IgG binding site.     

Variation in growth rate,carbon assimilation,and photosynthetic efficiency in response to nitrogen source and concentration in phytoplankton isolated from upper San Francisco Bay

Six species of phytoplankton recently isolated from upper San Francisco Bay were tested for their sensitivity to growth inhibition by ammonium (NH₄⁺), and for differences in growth rates according to inorganic nitrogen (N) growth source. The quantum yield of photosystem II (F_v/F_m) was a sensitive indicator of NH₄⁺ toxicity, manifested by a suppression of F_v/F_m in a dose‐dependent manner. Two chlorophytes were the least sensitive to NH₄⁺ inhibition, at concentrations of >3,000 μmoles NH₄⁺ · L^?1, followed by two estuarine diatoms that were sensitive at concentrations >1,000 μmoles NH₄⁺ · L^?1, followed lastly by two freshwater diatoms that were sensitive at concentrations between 200 and 500 μmoles NH₄⁺ · L^?1. At non‐inhibiting concentrations of NH₄⁺, the freshwater diatom species grew fastest, followed by the estuarine diatoms, while the chlorophytes grew slowest. Variations in growth rates with N source did not follow taxonomic divisions. Of the two chlorophytes, one grew significantly faster on nitrate (NO₃^?), whereas the other grew significantly faster on NH₄⁺. All four diatoms tested grew faster on NH₄⁺ compared with NO₃^?. We showed that in cases where growth rates were faster on NH₄⁺ than they were on NO₃^?, the difference was not larger for chlorophytes compared with diatoms. This holds true for comparisons across a number of culture investigations suggesting that diatoms as a group will not be at a competitive disadvantage under natural conditions when NH₄⁺ dominates the total N pool and they will also not have a growth advantage when NO₃^? is dominant, as long as N concentrations are sufficient.     

Immunological evaluation of personalized peptide vaccination in combination with UFT and UZEL for metastatic colorectal carcinoma patients

To investigate the safety and immunological responses of personalized peptide vaccination in combination with oral administration of UFT and UZEL for metastatic colorectal carcinoma (mCRC), fourteen patients were enrolled in the present study. Peptides were determined based on the presence of peptide-specific cytotoxic T lymphocyte precursors and IgG in each patient. A maximum of four peptides were subcutaneously administered weekly with UFT (300 mg/m² day⁻¹) and UZEL (75 mg/day) for 4 weeks, followed by 1 week of rest. This therapy was well-tolerated although there was a grade-3 skin reaction at the vaccination site in one patient. An increase in peptide-specific interferon-γ production or peptide-specific IgG after the tenth vaccination was observed in nine of ten or eight of ten patients tested, respectively. IgG responses were well correlated with overall survival (P = 0.0215). The safety and immunological responsiveness of the present therapy suggest that this combination would be of clinical benefit for mCRC patients, and further trials are merited. T. Hattori and T. Mine equally contributed to this work.     

The Decay of the Chromosomally Encoded ccdO157 Toxin–Antitoxin System in the Escherichia coli Species

The origin and the evolution of toxin–antitoxin (TA) systems remain to be uncovered. TA systems are abundant in bacterial chromosomes and are thought to be part of the flexible genome that originates from horizontal gene transfer. To gain insight into TA system evolution, we analyzed the distribution of the chromosomally encoded ccd_O157 system in 395 natural isolates of Escherichia coli. It was discovered in the E. coli O157:H7 strain in which it constitutes a genomic islet between two core genes (folA and apaH). Our study revealed that the folA–apaH intergenic region is plastic and subject to insertion of foreign DNA. It could be composed (i) of a repetitive extragenic palindromic (REP) sequence, (ii) of the ccd_O157 system or subtle variants of it, (iii) of a large DNA piece that contained a ccdA_O157 antitoxin remnant in association with ORFs of unknown function, or (iv) of a variant of it containing an insertion sequence in the ccdA_O157 remnant. Sequence analysis and functional tests of the ccd_O157 variants revealed that 69% of the variants were composed of an active toxin and antitoxin, 29% were composed of an active antitoxin and an inactive toxin, and in 2% of the cases both ORFs were inactive. Molecular evolution analysis showed that ccdB_O157 is under neutral evolution, suggesting that this system is devoid of any biological role in the E. coli species.