Evaluation of disinfective potential of reactivated free chlorine in pooled tap water by electrolysis

Tap water is one of the causative factors of hospital infections. We examined the disinfective potential of electrolysis and mechanism of disinfection, and clarified the disinfective effect of electrolysis on tap water contaminated with bacteria, and discussed its clinical applications. Tap waters artificially contaminated with Pseudomonas aeruginosa, Escherichia coli, Legionella pneumophila, and Staphylococcus aureus could be sterilized by electrolysis at 20-30 mA for 5 min. A high-density suspension (10(6) CFU/ml) of a spore forming bacterium, Bacillus subtilis was not completely sterilized by electrolysis at 50 mA up to 30 min, but a low-density suspension (10(5) CFU/ml) was totally sterilized by electrolysis at 50 mA for 5 min. Electrolyzed P. aeruginosa changed morphologically, that is, there was bleb formation on the cell wall and irregular aggregation of cytoplasmic small granules. Moreover, cytoplasmic enzyme, nitrate reductase, was inactivated by the electrolysis. On the other hand, genomic DNA of the electrolyzed bacteria was not degenerated, therefore, their DNA polymerase activity was not completely inactivated. Consequently, the major agent in electrolysis for bactericidal action was considered to be free chlorine, and the possible bactericidal mechanism was by destruction of bacterial membranes, followed by the aggregation of peripheral cytoplasmic proteins. Electrolysis of tap water for both disinfecting contaminating bacteria and increasing the disinfectant capacity was considered effective with some limitations, particularly against high-density contamination by spore-forming bacteria. In clinical settings, electrolysis of tap water is considered effective to disinfect water for hand washing in operation theatres, and bathing water for immunocompromised hosts.     

Isolation of an Arabidopsis thaliana mutant in which accumulation of cucumber mosaic virus coat protein is delayed

Cucumber mosaic virus (CMV) is known to systemically infect Arabidopsis thaliana ecotype Columbia plants. In order to identify the host factors involved in the multiplication of CMV, we isolated an A. thaliana mutant in which the accumulation of the coat protein (CP) of CMV in upper uninoculated leaves was delayed. Genetic analyses suggested that the phenotype of delayed accumulation of CMV CP in the mutant plants was caused by a single, nuclear and recessive mutation designated cum1-1, which was located on chromosome IV. The cum1-1 mutation did not affect the multiplication of tobacco mosaic virus, turnip crinkle virus or turnip yellow mosaic virus, which belong to different taxonomic groups from CMV. Accumulation of CMV CP in the inoculated leaves of cum1-1 plants was also delayed either when CMV virion or CMV virion RNA was inoculated. On the other hand, when cum1-1 and the wild-type Col-0 protoplasts were inoculated with CMV virion RNA by electroporation, the accumulations of CMV-related RNAs and the coat protein were similar. These results suggest that the cum1-1 mutation did not affect the uncoating of CMV virion and subsequent replication in an initially infected cell but affected the spreading of CMV within an infected leaf, possibly the cell-to-cell movement of CMV in a virus-specific manner.     

Contrasted Effects of CO2 on the Regulation of Dormancy and Germination in Xanthium pennsylvanicum and Setaria faberi Seeds

The effects of CO₂ on dormancy and germination were examinedusing seeds of cocklebur (Xanthium pennsylvanicum Wallr.) andgiant foxtail (Setaria faberi Herrm.). The rate of germinationof the giant foxtail seeds as well as cocklebur was promotedby exogenously applied CO₂ at a concentration of 30 mmol mol^-1regardless of the sowing conditions. However, seeds which failedto germinate in the presence of CO₂, entered a secondary phaseof dormancy under unfavourable germination conditions. If CO₂was applied to seeds under conditions such as water stress imposedwith a 200 mol m^-3 mannitol solution, a hypoxic atmosphere of100 mmol mol^-1 O₂ or a treatment of 0·1 mol m^-3 ABA,development of secondary dormancy was accelerated. These contrastedeffects of CO₂ were observed in ecological studies. Under naturalfield conditions germination of buried giant foxtail seeds respondedpositively to CO₂ during a period of release from primary dormancyfrom Feb. to May, but CO₂ accelerated secondary dormancy commencingin early Jun. In other words, in the presence of CO₂, both theenvironmental conditions and the germination states of the seedsclearly showed secondary dormancy-inducing effects. Thus, itseems that CO₂ has contrasted effects on regulation of dormancyand germination of seeds depending on the germination conditions.Copyright1995, 1999 Academic Press Xanthium pennsylvanicum, cocklebur, Setaria faberi, giant foxtail, CO₂, water stress, hypoxia, ABA, germination, secondary dormancy     

Helper virus-independent trans-replication of hepatitis C virus-derived minigenome

In Paramecium, ciliary reversal is coupled with voltage-gated Ca(2+) channels on the ciliary membrane. We previously isolated a P. caudatum mutant, cnrC, with a malfunction of the Ca(2+) channels and discovered that the channel activity of cnrC was restored by transfection of the P. caudatum centrin (Pccentrin1p) gene, which encodes a member of the Ca(2+)-binding EF-hand protein family. In this study, we injected various mutated Pccentrin1p genes into cnrC and investigated whether these genes restore the Ca(2+) channel activity of cnrC. A Pccentrin1p mutant gene lacking Ca(2+) sensitivity of the third and fourth EF-hands lost the ability to restore the channel function of cnrC, and mutation of the fourth EF-hand caused more serious impairment than mutation of the third EF-hand. Moreover, a Pccentrin1p gene lacking the N-terminal 34-amino acid sequence also lost the ability to restore the channel activity. Native-PAGE analysis demonstrated that the N-terminal sequence is important for the Ca(2+)-dependent structural change of Pccentrin1p. These results demonstrate that Pccentrin1p Ca(2+)-dependently regulates the Ca(2+) channel activity in vivo.     

Stability of membrane potential in heart mitochondria: single mitochondrion imaging

Mitochondrial membrane potential (delta psi(m)) plays an important role in cellular activity. Although delta psi(m) of intracellular mitochondria are relatively stable, the recent experiments with isolated mitochondria demonstrate that individual mitochondria show frequent fluctuations of delta psi(m). The current study is performed to investigate the factors that stabilize delta psi(m) in cells by observing delta psi(m) of individual isolated mitochondria with fluorescence microscopy. Here, we report that (1) the transient depolarizations are also induced for mitochondria in plasma membrane permeabilized cells, (2) almost all mitochondria isolated from porcine hearts show the transient depolarizations that is enhanced with the net efflux of protons from the matrix to the intermembrane space, and (3) ATP and ADP significantly inhibit the transient depolarizations by plural mechanisms. These results suggest that the suppression of acute alkalinization of the matrix together with the presence of ATP and ADP contributes to the stabilization of delta psi(m) in cells.     

The Arabidopsis cucumovirus multiplication 1 and 2 loci encode translation initiation factors 4E and 4G

The cum1 and cum2 mutations of Arabidopsis thaliana inhibit cucumber mosaic virus (CMV) multiplication. In cum1 and cum2 protoplasts, CMV RNA and the coat protein accumulated to wild-type levels, but the accumulation of the 3a protein of CMV, which is necessary for cell-to-cell movement of the virus, was strongly reduced compared with that in wild-type protoplasts. In cum2 protoplasts, the accumulation of turnip crinkle virus (TCV)-related RNA and proteins was also reduced. Positional cloning demonstrated that CUM1 and CUM2 encode eukaryotic translation initiation factors 4E and 4G, respectively. Unlike most cellular mRNA, the CMV RNA lacks a poly(A) tail, whereas the TCV RNA lacks both a 5′-terminal cap and a poly(A) tail. In vivo translation analyses, using chimeric luciferase mRNA carrying the terminal structures and untranslated sequences of the CMV or TCV RNA, demonstrated that these viral untranslated sequences contain elements that regulate the expression of encoded proteins positively or negatively. The cum1 and cum2 mutations had different effects on the action of these elements, suggesting that the cum1 and cum2 mutations cause inefficient production of CMV 3a protein and that the cum2 mutation affects the production of TCV-encoded proteins.     

The catalytic mechanism of serine proteases: single proton versus double proton transfer

The two possible mechanisms of proton transfer on the catalytic process of serine proteases (single or double proton transfer) have been analysed. Intermediate neglect of differential overlap calculations have been performed in the absence and in the presence of the substrate molecule and one water molecule localized in the active site. It is shown that, in the absence of the substrate and water, double proton transfer seems to be the most feasible mechanism. However, when these molecules are introduced in the calculation, the role played by them is to facilitate the formation of the zwitterionic structure (single proton transfer) and to destabilize the intermediate structure which leads to double proton transfer. All calculations were made in vacuo.     

Immunohistochemical examination of Peyer's patches in autoimmune mice

Summary The distribution of T-cells and B-cells in Peyer's patches was examined in three autoimmune model mice, MRL/Mp-lpr/lpr, BXSB, NZBWF1/J mice and normal BALB/c mice, between one and ten months old. A multiple layering technique was used for immunohistochemical detection of lymphocyte surface antigens of T-cells (Thy1.2, Lyt1, Lyt2) and B-cells (surface IgM) and peanut agglutinin receptor for germinal center cells. The T-cell population of female MRL/Mp-lpr/lpr mice increased markedly with age, and the B-cell population of the male BXSB mouse tended to increase. However, little change was observed with age in the NZBWF1/J mice. The immunohistochemical properties of the Peyer's patches in the three autoimmune model mice were different.