Hydroxyl Radical Generation Caused by the Reaction of Singlet Oxygen with a Spin Trap,DMPO, Increases Significantly in the Presence of Biological Reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (¹O²) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ¹O² with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (?OH) adduct of DMPO (DMPO-OH) resulting from ¹O²-dependent generation of ?OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its ?OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of ?OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of ?OH from ¹O², and that spin trap-mediated ?OH generation hardly occurs with DEPMPO.     

Increase of salt dependence of halophilic nucleoside diphosphate kinase caused by a single amino acid substitution

Nucleoside diphosphate kinase (HsNDK) from an extremely halophilic archaea, Halobacterium salinarum, is composed of a homo hexamer, assembled as a trimer of basic dimeric units. It requires >2 M NaCl for refolding, although it does not require NaCl for stability or enzymatic activity below 30 °C. A HisN111L mutant with an N-terminal extension sequence containing hexa-His tag, in which Asn111 was replaced with Leu, was designed to be less stable between basic dimeric units. This mutant can lose between 6 and 12 hydrogen bonds between basic dimeric units in the hexamer structure. The HisN111L mutant had enhanced salt requirements for enzymatic activity and refolding even though the secondary structure of the HisN111L mutant was confirmed to be similar to the control, HisNDK, in low and high salt solutions using circular dichroism. We reported previously that G114R and D148C mutants, which had enhanced interactions between basic dimeric units, showed facilitated refolding and stabilization in low salt solution. The results of this study help to elucidate the process for engineering industrial enzymes by controlling subunit–subunit interactions through mutations.     

Reduced Serum Selenium Concentration in Miscarriage Incidence of Indonesian Subjects

Selenium is an essential nutrient for human health, and maternal selenium concentration has been reported to be associated with pregnancy outcome. To further investigate the possible role of selenium (Se) in miscarriage, we conducted a case–control study to evaluate the correlations among selenium status, glutathione peroxidase activity, and spontaneous abortion. A total of 46 subjects with normal pregnancies and 25 subjects with spontaneous abortion were recruited, and their serum selenium concentrations and serum glutathione peroxidase activities were analyzed. The total serum selenium concentrations in subjects with normal pregnancies were significantly higher than those of subjects with spontaneous abortion; however, the glutathione peroxidase activities were similar in both groups. We further separated the subjects into smoking and nonsmoking groups, and the logistic regression analysis suggested that total serum selenium concentration, but not serum glutathione peroxidase activity or smoking, was significantly correlated with the incidence of miscarriage. The present study thus reaffirms that low serum selenium levels are associated with miscarriage and that selenium plays an important role in pregnancy maintenance.     

Comparisons of the transferability of plasmids pCAR1, pB10, R388, and NAH7 among Pseudomonas putida at different cell densities

The transferability of plasmids pCAR1, pB10, R388, and NAH7 was compared using the same donor-recipient system at different cell density combinations in liquid or on a solid surface. pCAR1 was efficiently transferred in liquid, whereas the other plasmids were preferentially transferred on a solid surface. Difference of liquid or solid affected the transfer frequency especially at lower cell densities.     

Mouse knockout of guanylyl cyclase C: Recognition memory deficits in the absence of activity changes

Guanylyl cyclase C (GC‐C) is found in brain regions where dopamine is expressed. We characterized a mouse in which GC‐C was knocked out (KO) that was reported to be a model of attention deficit hyperactivity disorder (ADHD). We re‐examined this model and controlled for litter effects, used 16 to 23 mice per genotype per sex and assessed an array of behavioral and neurochemical outcomes. GC‐C KO mice showed no phenotypic differences from wild‐type mice on most behavioral tests, or on striatal or hippocampal monoamines, and notably no evidence of an ADHD‐like phenotype. KO mice were impaired on novel object recognition, had decreased tactile startle but not acoustic startle, and females had increased latency on cued training trials in the Morris water maze, but not hidden platform spatial learning trials. Open‐field activity showed small differences in females but not males. The data indicate that the GC‐C KO mouse with proper controls and sample sizes has a moderate cognitive and startle phenotype but has no ADHD‐like phenotype.     

Single giant vesicle rupture events reveal multiple mechanisms of glass-supported bilayer formation

The formation of supported lipid bilayers (SLBs) on glass from giant unilamellar vesicles (GUVs) was studied using fluorescence microscopy. We show that GUV rupture occurs by at least four mechanisms, including 1), spontaneous rupture of isolated GUVs yielding almost heart-shaped bilayer patches (asymmetric rupture); 2), spontaneous rupture of isolated GUVs yielding circular bilayer patches (symmetric rupture); 3), induced rupture of an incoming vesicle when it contacts a planar bilayer edge; and 4), induced rupture of an adsorbed GUV when a nearby GUV spontaneously ruptures. In pathway 1, the dominant rupture pathway for isolated GUVs, GUVs deformed upon adsorption to the glass surface, and planar bilayer patch formation was initiated by rupture pore formation near the rim of the glass-bilayer interface. Expanding rupture pores led to planar bilayer formation in approximately 10-20 ms. Rupture probability per unit time depended on the average intrinsic curvature of the component lipids. The membrane leaflet adsorbed to the glass surface in planar bilayer patches originated from the outer leaflet of GUVs. Pathway 2 was rarely observed. We surmise that SLB formation is predominantly initiated by pathway 1 rupture events, and that rupture events occurring by pathways 3 and 4 dominate during later stages of SLB formation.     

Double-layered collagen gel hemisphere for cell invasion assay: successful visualization and quantification of cell invasion activity

Although various methods for collagen gel-based cell invasion assays have been described, there continues to be a need for a simpler and more objective assay. Here, we describe an easy-to-prepare double-layered collagen gel hemisphere (DL-CGH) system that satisfies these requirements, and we demonstrate the advantages of this new system for visualizing cell movements during invasion. DL-CGH consists of a central core collagen layer surrounded by an outer cover collagen layer. A droplet of collagen I solution (containing cells to be examined) naturally forms a small hemisphere on the bottom of the culture dish. After this central core layer gels, a second droplet is placed atop the first gel, encapsulating it completely. The hemisphere is submerged in the medium and cultured. The invasive activity of cells that infiltrate from the inner to the outer layer can be evaluated optically. Using this in vitro system, we measured the inhibitory effect of E-cadherin expression on cancer cell invasion. DL-CGH also allowed visualization of interactions between invading cancer cells and the stroma. Cancer cells, which lack the proteases required for direct entrance into the three-dimensional collagen matrix, were seen to slip like amoebas through matrix gaps generated by the pericellular proteolytic activity of fibroblasts. [Supplementary materials are available for this article. Go to the publisher's online edition of Cell Communication and Adhesion for the following free supplemental resources: Movies 1-3; 4a and b].     

New highly active taxoids from 9 beta-dihydrobaccatin-9,10-acetals. Part 5

To improve the metabolic stability of 3, which exhibited both in vitro antitumor activity and in vivo efficacy by both iv and po administration, we designed and synthesized new taxane analogues. Most of the synthetic compounds maintained excellent antitumor activity and were scarcely metabolized by human liver microsomes. And some compounds exhibited potent antitumor effects against B16 melanoma BL6 in vivo by both iv and po administration similarly to 3.     

Hydroxyl radical generation caused by the reaction of singlet oxygen with a spin trap, DMPO, increases significantly in the presence of biological reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (₁O₂) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ₁O₂ with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (•OH) adduct of DMPO (DMPO-OH) resulting from ₁O₂-dependent generation of •OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its •OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of •OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of •OH from ₁O₂, and that spin trap-mediated •OH generation hardly occurs with DEPMPO.