Isolation of Sclerosporin,a Sporogenic Substance,from Sclerotinia fructicola

The folate-hydrolyzing enzyme was purified 49-fold from the crude extract of Crithidia fasciculata ATCC 12857 by heat treatment, column chromatographies on DEAE-cellulose and Sephadex G-200, and preparative polyacrylamide gel electrophoresis. The final preparation was electrophoretically homogeneous. The enzyme had a molecular weight of 200,000 daltons and consisted of 4 identical subunits of which the molecular weight was about 51,000 daltons. The enzyme hydrolyzed aminopterin, methotrexate, and pABG more effectively than folate. The enzyme hydrolyzed the reduced folates, dihydrofolate and 10-formyltetrahydrofolate, more weakly than folate. The enzyme did not act on pteroly-γ,γ-diglutamylglutamate. The optimum pH for the reaction with each substrate described above was 7.0. Km values for folate, methotrexate, aminopterin, and pABG were 0.13, 0.46, 0.40, and 0.43 mM, respectively. The enzyme activity was inhibited by 2-mercaptoethanol, pCMB, chelating reagents such as α,α′α′′-tripyridyl and bathophenanthroline, divalent cations such as Hg²⁺, Cu^{2 +}, Cd²⁺, Pb²⁺, and Zn²⁺, and by pyrophosphate and orthophosphate.     

Solution structure of the N‐terminal domain of a replication restart primosome factor,PriC, in Escherichia coli

In eubacterial organisms, the oriC‐independent primosome plays an essential role in replication restart after the dissociation of the replication DNA‐protein complex by DNA damage. PriC is a key protein component in the replication restart primosome. Our recent study suggested that PriC is divided into two domains: an N‐terminal and a C‐terminal domain. In the present study, we determined the solution structure of the N‐terminal domain, whose structure and function have remained unknown until now. The revealed structure was composed of three helices and one extended loop. We also observed chemical shift changes in the heteronuclear NMR spectrum and oligomerization in the presence of ssDNA. These abilities may contribute to the PriC‐ssDNA complex, which is important for the replication restart primosome.     

Assembly of anthrax toxin pore: Lethal‐factor complexes into lipid nanodiscs

We have devised a procedure to incorporate the anthrax protective antigen (PA) pore complexed with the N‐terminal domain of anthrax lethal factor (LF_N) into lipid nanodiscs and analyzed the resulting complexes by negative‐stain electron microscopy. Insertion into nanodiscs was performed without relying on primary and secondary detergent screens. The preparations were relatively pure, and the percentage of PA pore inserted into nanodiscs on EM grids was high (～43%). Three‐dimensional analysis of negatively stained single particles revealed the LF_N‐PA nanodisc complex mirroring the previous unliganded PA pore nanodisc structure, but with additional protein density consistent with multiple bound LF_N molecules on the PA cap region. The assembly procedure will facilitate collection of higher resolution cryo‐EM LF_N‐PA nanodisc structures and use of advanced automated particle selection methods.     

Occurrence and reduction of human viruses,F‐specific RNA coliphage genogroups and microbial indicators at a full‐scale wastewater treatment plant in Japan

Aims

To evaluate and compare the reductions of human viruses and F‐specific coliphages in a full‐scale wastewater treatment plant based on the quantitative PCR (qPCR) and plate count assays.

Methods and Results

A total of 24 water samples were collected from four locations at the plant, and the relative abundance of human viruses and F‐RNA phage genogroups were determined by qPCR. Of the 10 types of viruses tested, enteric adenoviruses were the most prevalent in both influent and effluent wastewater samples. Of the different treatment steps, the activated sludge process was most effective in reducing the microbial loads. Viruses and F‐RNA phages showed variable reduction; among them, GI and GIII F‐RNA phages showed the lowest and the highest reduction, respectively.

Conclusions

Ten types of viruses were present in wastewater that is discharged into public water bodies after treatment. The variability in reduction for the different virus types demonstrates that selection of adequate viral indicators is important for evaluating the efficacy of wastewater treatment and ensuring the water safety.

Significance and Impact of the Study

Our comprehensive analyses of the occurrence and reduction of viruses and indicators can contribute to the future establishment of appropriate viral indicators to evaluate the efficacy of wastewater treatment.     

Utilization of transposable element mPing as a novel genetic tool for modification of the stress response in rice

Transposable elements (TEs) are DNA fragments that have the ability to move from one chromosomal location to another. The insertion of TEs into gene-rich regions often affects changes in the expression of neighboring genes. Miniature Ping (mPing) is an active miniature inverted-repeat TE discovered in the rice genome. It has been found to show exceptionally active transposition in a few japonica rice varieties, including Gimbozu, where mPing insertion rendered adjacent genes stress-inducible. In the Gimbozu population, it is highly possible that several genes with modified expression profiles are segregating due to the de novo mPing insertions. In our study, we utilized a screening system for detecting de novo mPing insertions in the upstream region of target genes and evaluated the effect of mPing on the stress response of the target genes. Screening for 17 targeted genes revealed five genes with the mPing insertion in their promoters. In most cases, the alteration of gene expression was observed under stress conditions, and there was no change in the expression levels of those five genes under normal conditions. These results indicate that the mPing insertion can be used as a genetic tool to modify an expression pattern of a target gene under stress conditions without changing the expression profiles of those under natural conditions.     

Role of cytosolic phospholipase A(2)α in cell rounding and cytotoxicity induced by ceramide-1-phosphate via ceramide kinase

Type II phosphatidylinositol (PtdIns) 4-kinases produce PtdIns 4-phosphate, an early key signaling molecule in phosphatidylinositol cycle, which is indispensable for T cell activation. Type II PtdIns 4-kinase alpha and beta have similar biochemical properties. To distinguish these isoforms Epigallocatechin gallate (EGCG) has been evaluated as a specific inhibitor. EGCG is the major active catechin in green tea having anti-inflammatory, antiatherogenic and cancer chemopreventive properties. The precise mechanism of actions and molecular targets of EGCG in early signaling cascades are not well understood. In the present study, we have shown that EGCG inhibits type II PtdIns 4-kinases (α and β isoforms) and PtdIns 3-kinase activity in vitro. EGCG directly bind to both alpha and beta isoforms of type II PtdIns 4-kinases with a Kd of 2.62 μM and 1.02 μM, respectively. Type II PtdIns 4-kinase-EGCG complex have different binding pattern at its excited state. Both isoforms showed significant change in helicity upon binding with EGCG. EGCG modulates its effect by interacting with ATP binding pocket; the residues likely to be involved in EGCG binding were predicted by Autodock. Our findings suggest that EGCG inhibits two isoforms and could be a key to regulate T cell activation.     

Role of cytosolic phospholipase A2α in cell rounding and cytotoxicity induced by ceramide-1-phosphate via ceramide kinase

Ceramide-1-phosphate (C1P), produced by ceramide kinase (CERK), is implicated in the regulation of many biological functions including cell growth and inflammation. C1P is a direct activator of group IVA cytosolic phospholipsase A₂ (PLA2G4A or cPLA₂α). Although activation of the CERK–C1P pathway causes mitogenic and cytoprotective responses in many cells, the pathway shows cytotoxicity in several cells and the precise mechanism has not been elucidated. In the present study, we examined the effect of human CERK (hCERK) expression on cytotoxicity in two cell lines. Expression of hCERK in CHO cells caused cell rounding and lactate dehydrogenase (LDH) leakage, and co-addition of ceramide enhanced these responses. Expression of hCERK enhanced C1P formation and release of arachidonic acid in Ca²⁺ ionophore-stimulated cells. Treatment with 20 μM C2-C1P for 24 h caused cell rounding, and the response was significantly decreased by an inhibitor of cPLA₂α. In L929 cells, expression of hCERK with and without ceramide caused cell rounding and LDH leakage, respectively, and the responses were significantly less in a stable clone of L929 cells lacking cPLA₂α. These findings suggest the involvement of cPLA₂α in CERK–C1P pathway-induced cytotoxicity.     

Effect of insecticides on the mortalities of three whitefly parasitoid species, Eretmocerusmundus, Eretmoceruseremicus and Encarsiaformosa (Hymenoptera: Aphelinidae)

The toxicities of 24 insecticides for the biological control of whiteflies were evaluated for Eretmocerus mundus (Mercet), Eretmocerus eremicus Rose and Zolnerowich and Encarsia formosa Gahan using the residual film method (for adults) and the dipping method (for pupae). Mortalities from insect growth regulators (IGRs) (flufenoxuron and lufenuron), Bacillus thuringiensis (Bt), pymetrozine and sulfur were <30% for both pupae and adults of all three species, indicating that the parasitoids were not seriously affected by these insecticides. Neonicotinoids (acetamiprid, clothianidin, dinotefuran, imidacloprid and nitenpyram), synthetic pyrethroids (etofenprox and permethrin), organophosphates (acephate and fenitrothion), chlorphenapyr, emamectin benzoate, spinosad and tolfenpyrad were seriously harmful (100% mortality) and acaricides (chinomethionat, milbemectin and pyridaben) were moderately harmful or seriously harmful to adult parasitoids (leading to mortalities of >92%). For each insecticide, the mortality of pupae was generally lower than that of adults, even though the toxicity classification for the two groups was similar. The results indicate that IGRs, Bt, pymetrozine and sulfur are relatively harmless, and are compatible with the use of parasitoids to help control whiteflies for integrated pest management in greenhouses.