Tissue responses against Cladosporium trichoides and its parasitic forms in congenitally athymic nude mice and their heterozygous littermates

The tissue responses against Cladosporium trichoides and its parasitic forms were studied using nude (nu/nu) mice and their heterozygous (nu/+) littermates of BALB/c background.1.0,0.1 and 0.01% cell suspensions were prepared from a culture broth which had been inoculated with the C. trichoides and cultured with reciprocal shaking at 27 ° C for 7 days. Sixty nu/nu or 60 nu/+ mice were divided into three groups consisting of 20 each which was allotted to one of the three cell suspensions. Each mouse was inoculated intravenously with 0.1 ml of either the cell suspensions. Two mice from each of the six groups were sacrificed at adequate intervals until 30 days after inoculation and histopathologic sections stained with H & E or by PAS were prepared from their visceral organs.There were no characteristic findings in the nu/nu and nu/+ mice inoculated with the 0.01% cell suspension. When inoculated with the 1.0% cell suspension, the brain was the favorite target organ in both groups of mice and the kidney was the second. When inoculated with the 0.1% cell suspension, brain lesions were observed only in the nu/nu mice. The susceptibility of the nu/nu mice was higher than that of the nu/+ mice.The parasitic forms in the brain of the nu/nu and nu/+ mice were slender septate true hyphae with or without polymorphonuclear leucocyte infiltrate, while in the liver, spleen and lung of both groups of mice the parasitic forms were short thick hyphae, moniliform hyphae, chlamydospores or round cells (sclerotic cells). Many giant cells containing fungal elements appeared in the liver of the nu/nu mice.     

Immunopotentiator separated from hot water extract of the seed of Benincasa cerifera Savi (Tohgashi)

Summary The separation and properties of a new immunopotentiator, Benincasa cerifera mitogen (BCM) fraction, were investigated. BCM fraction was separated from hot water extract of the seed of Benincasa cerifera Savi (Tohgashi) by gel filtration using Sepharose 4B. BCM fraction is a heteropolymer consisting of uronic acid, neutral sugars, protein, and phosphorus. The proliferation and differentiation of murine B cells were markedly stimulated by BCM fraction. The in vitro development of peritoneal macrophages into antitumor macrophages was also activated by the addition of BCM fraction to cultures. BCM fraction augmented the IgM and IgG antibody responses against sheep erythrocytes (SRBC) and the induction of delayed-type footpad reaction against SRBC. The antitumor activity of BCM fraction was observed in terms of prolongation of the survival period of mice bearing Meth A fibrosarcoma. After hydrolysis with 1% acetic acid at 100° C for 4 h, marked mitogenic activity was found in a precipitate composed of 29% neutral sugars, 50% uronic acid, 1% protein, and 0.1% phosphorus. The precipitate did not contain detectable amino sugar. The possibility that the biological activities of BCM fraction may be due to contamination by bacterial lipopoly-saccharide was ruled out on the basis of the results of chemical analysis and of marked mitogenicity noted in C3H/HeJ spleen cell cultures. Abbreviations used: BCM, Benincasa cerifera mitogen; SRBC, sheep erythrocytes; PFC, plaque-forming cells; TNP-HRBC, trinitrophenylated horse erythrocytes; PBA, polyclonal B-cell activation; SI, stimulation index     

Photosynthetic electron transfer system is inoperative in anaerobic cells of Erythrobacter species strain OCh 114

Some of the photosynthetic reactions were measured under aerobic and anaerobic conditions in intact cells of an aerobic photosynthetic bacterium Erythrobacter species strain OCh 114 (ATCC No. 33942). In intact cells, the flash-light induced oxidation of cytochrome c-551, the continuous light-induced oxidation of reaction center bacteriochlorophyll and the continuous light-induced pH change ( ) of the suspension decreased on aerobic-anaerobic transition and almost disappeared under anaerobic conditions. These photosynthetic reactions reappeared when the suspension was aerated again. These phenomena were reconciled with the fact that Erythrobacter sp. cannot grow anaerobically even in the light. The incompetence of photoanaerobic growth of this bacterium was explained by the reduction of the primary electron acceptor (Q_I) before illumination, resulting partly from the relatively high midpoint potential of Q_I of this bacterium.Abbreviations Q_I Primary electron acceptor - E_h ambient redox potential - E_m midpoint redox potential     

Ralfuranones contribute to mushroom‐type biofilm formation by Ralstonia solanacearum strain OE1‐1

After invasion into intercellular spaces of tomato plants, the soil‐borne, plant‐pathogenic Ralstonia solanacearum strain OE1‐1 forms mushroom‐shaped biofilms (mushroom‐type biofilms, mBFs) on tomato cells, leading to its virulence. The strain OE1‐1 produces aryl‐furanone secondary metabolites, ralfuranones (A, B, J, K and L), dependent on the quorum sensing (QS) system, with methyl 3‐hydroxymyristate (3‐OH MAME) synthesized by PhcB as a QS signal. Ralfuranones are associated with the feedback loop of the QS system. A ralfuranone productivity‐deficient mutant (ΔralA) exhibited significantly reduced growth in intercellular spaces compared with strain OE1‐1, losing its virulence. To analyse the function of ralfuranones in mBF formation by OE1‐1 cells, we observed cell aggregates of R. solanacearum strains statically incubated in tomato apoplast fluids on filters under a scanning electron microscope. The ΔralA strain formed significantly fewer microcolonies and mBFs than strain OE1‐1. Supplementation of ralfuranones A, B, J and K, but not L, significantly enhanced the development of mBF formation by ΔralA. Furthermore, a phcB‐ and ralA‐deleted mutant (ΔphcB/ralA) exhibited less formation of mBFs than OE1‐1, although a QS‐deficient, phcB‐deleted mutant formed mBFs similar to OE1‐1. Supplementation with 3‐OH MAME significantly reduced the formation of mBFs by ΔphcB/ralA. The application of each ralfuranone significantly increased the formation of mBFs by ΔphcB/ralA supplied with 3‐OH MAME. Together, our findings indicate that ralfuranones are implicated not only in the development of mBFs by strain OE1‐1, but also in the suppression of QS‐mediated negative regulation of mBF formation.     

Bioluminescence‐based cytotoxicity assay for simultaneous evaluation of cell viability and membrane damage in human hepatoma HepG2 cells

We have developed a bioluminescence‐based non‐destructive cytotoxicity assay in which cell viability and membrane damage are simultaneously evaluated using Emerald luciferase (ELuc) and endoplasmic reticulum (ER)‐targeted copepod luciferase (GLuc‐KDEL), respectively, by using multi‐integrase mouse artificial chromosome (MI‐MAC) vector. We have demonstrated that the time‐dependent concentration response curves of ELuc luminescence intensity and WST‐1 assay, and GLuc‐KDEL luminescence intensity and lactate dehydrogenase (LDH) activity in the culture medium accompanied by cytotoxicity show good agreement in toxicant‐treated ELuc‐ and GLuc‐KDEL‐expressing HepG2 stable cell lines. We have clarified that the increase of GLuc‐KDEL luminescence intensity in the culture medium reflects the type of cell death, including necrosis and late apoptosis, but not early apoptosis. We have also uncovered a strong correlation between GLuc‐KDEL luminescence intensity in the culture medium and the extracellular release of high mobility group box 1 (HMGB1), a representative damage‐associated molecular pattern (DAMP) molecule. The bioluminescence measurement assay using ELuc and GLuc‐KDEL developed in this study can simultaneously monitor cell viability and membrane damage, respectively, and the increase of GLuc‐KDEL luminescence intensity in the culture medium accompanied by the increase of cytotoxicity is an index of necrosis and late apoptosis associated with the extracellular release of DAMP molecules.     

Highly Reversible Oxygen‐Redox Chemistry at 4.1 V in Na4/7−x[□1/7Mn6/7]O2 (□: Mn Vacancy)

Increasing the energy density of rechargeable batteries is of paramount importance toward achieving a sustainable society. The present limitation of the energy density is owing to the small capacity of cathode materials, in which the (de)intercalation of ions is charge‐compensated by transition‐metal redox reactions. Although additional oxygen‐redox reactions of oxide cathodes have been recognized as an effective way to overcome this capacity limit, irreversible structural changes that occur during charge/discharge cause voltage drops and cycle degradation. Here, a highly reversible oxygen‐redox capacity of Na₂Mn₃O₇ that possesses inherent Mn vacancies in a layered structure is found. The cross validation of theoretical predictions and experimental observations demonstrates that the nonbonding 2p orbitals of oxygens neighboring the Mn vacancies contribute to the oxygen‐redox capacity without making the Mn?O bond labile, highlighting the critical role of transition‐metal vacancies for the design of reversible oxygen‐redox cathodes.     

A luciferase complementation assay system using transferable mouse artificial chromosomes to monitor protein–protein interactions mediated by G protein-coupled receptors

G protein-coupled receptors (GPCRs) are seven-transmembrane domain receptors that interact with the β-arrestin family, particularly β-arrestin 1 (ARRB1). GPCRs interact with 33% of small molecule drugs. Ligand screening is promising for drug discovery concerning GPCR-related diseases. Luciferase complementation assay (LCA) enables detection of protein–protein complementation via bioluminescence following complementation of N- and C-terminal luciferase fragments (NEluc and CEluc) fused to target proteins, but it is necessary to co-express the two genes. Here, we developed LCAs with mouse artificial chromosomes (MACs) that have unique characteristics such as stable maintenance and a substantial insert-carrying capacity. First, an NEluc-ARRB1 was inserted into MAC4 by Cre-loxP recombination in CHO cells, named ARRB1-MAC4. Second, a parathyroid hormone receptor 2 (PTHR2)-CEluc or prostaglandin EP4 receptor (hEP4)-CEluc were inserted into ARRB1-MAC4, named ARRB1-PTHR2-MAC4 and ARRB1-hEP4-MAC4, respectively, via the sequential integration of multiple vectors (SIM) system. Each MAC was transferred into HEK293 cells by microcell-mediated chromosome transfer (MMCT). LCAs using the established HEK293 cell lines resulted in 35,000 photon counts upon somatostatin stimulation for ARRB1-MAC4 with transient transfection of the somatostatin receptor 2 (SSTR2) expression vector, 1800 photon counts upon parathyroid hormone stimulation for ARRB1-PTHR2-MAC4, and 35,000 photon counts upon prostaglandin E2 stimulation for ARRB1-hEP4-MAC4. These MACs were maintained independently from host chromosomes in CHO and HEK293 cells. HEK293 cells containing ARRB1-PTHR2-MAC4 showed a stable reaction for long-term. Thus, the combination of gene loading by the SIM system into a MAC and an LCA targeting GPCRs provides a novel and useful platform to discover drugs for GPCR-related diseases.     

Survival strategy of the salt-tolerant lactic acid bacterium,Tetragenococcus halophilus,to counteract koji mold,Aspergillus oryzae,in soy sauce brewing

In soy sauce brewing, the results of the fermentation of lactic acid greatly affect the quality of soy sauce. The soy sauce moromi produced with Aspergillus oryzae RIB40 allows the growth of Tetragenococcus halophilus NBRC 12172 but not T. halophilus D10. We isolated and identified heptelidic acid (HA), an inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), produced by A. oryzae RIB40 as the growth inhibitor of the salt-tolerant lactic acid bacteria. The growth inhibition of T. halophilus D10 by HA was suggested to be associated with the direct inhibition of GAPDH activity under high salt environment. The difference in the susceptibility to HA among various strains of T. halophilus was caused by the mutations in the gene encoding GAPDH.     

Male origin determines satyrization potential of <Emphasis Type="Italic">Aedes aegypti</Emphasis> by invasive <Emphasis Type="Italic">Aedes albopictus</Emphasis>

Rapid displacements of resident Aedes aegypti by invasions of Aedes albopictus have been documented in the southeastern United States and Bermuda. Interspecific mating has been detected in nature between these species and proposed as a likely mechanism for these displacements by means of asymmetric reproductive interference, or satyrization. However, rapid displacements of A. aegypti have not been detected in most localities where these two invasive species are known to co-occur. Aedes albopictus invaded the United States and Brazil at approximately the same time, in the mid-1980s, but the origins of the invading strains are known to be different. Here we tested the hypothesis, in standardized cage environments, that A. albopictus males from Brazil are less capable of satyrizing A. aegypti females than A. albopictus males from the United States. Using strains of A. aegypti and A. albopictus from the United States of known susceptibility to and capacity for interspecific mating, we demonstrate that A. albopictus colonized from collections in the Brazilian cities of Rio de Janeiro and Manaus were relatively unsuccessful in inseminating virgin female A. aegypti from Key West Florida compared to A. albopictus from peninsular Florida. We suggest that the low satyrization potential of Brazilian A. albopictus males may contribute to the lack of documented competitive displacements of A. aegypti in that country.