All genomic mutations in the antimicrobial surfactant-resistant mutant, Escherichia coli OW66, are involved in cell resistance to surfactant

The spontaneous antimicrobial surfactant-resistant mutant, Escherichia coli OW66, has been isolated, and its physiological properties have been characterized in our previous paper (Ishikawa et al., J Appl Microbiol 92:261–268, 2002b). This report revealed that strain OW66 had seven mutations in their chromosomal DNA by comparative genomic hybridization microarray, and that their alternative functions were involved in cell resistance to antimicrobial surfactants. These mutations were located in oppB, ydcR, IVR(vacJ-yfdC), rpoN, rpoB, rpoC, and soxR. Furthermore, seven of the single-mutated isogenic strains and seven of the six-mutated isogenic strains were constructed from strains OW6 (NBRC106482) and OW66, respectively, through homologous recombination, and their resistances to an antimicrobial surfactant were measured using the minimum inhibitory concentration method. These results revealed that all six-mutated strains were more sensitive than strain OW66, and that the soxR66 mutation was independently involved in antimicrobial surfactant resistance of E. coli cells. Expression of soxR66 and soxS was increased in both strains OW66 and OW6-soxR66 without the surfactant treatment by the quantitative real time-polymerase chain reaction analysis, compared with strain OW6. Two-dimensional polyacrylamide gel electrophoresis analysis also revealed that some proteins in the soxRS regulon, including Mn-SOD, were overexpressed in both strains OW66 and OW6-soxR66. These results indicate that the soxR66 mutation leads to the constitutive expression of the soxRS regulon, resulting in the acquired resistance of E. coli cells to an antimicrobial surfactant.     

Stochastic simulation of biological reactions, and its applications for studying actin polymerization

Molecular events in biological cells occur in local subregions, where the molecules tend to be small in number. The cytoskeleton, which is important for both the structural changes of cells and their functions, is also a countable entity because of its long fibrous shape. To simulate the local environment using a computer, stochastic simulations should be run. We herein report a new method of stochastic simulation based on random walk and reaction by the collision of all molecules. The microscopic reaction rate P(r) is calculated from the macroscopic rate constant k. The formula involves only local parameters embedded for each molecule. The results of the stochastic simulations of simple second-order, polymerization, Michaelis-Menten-type and other reactions agreed quite well with those of deterministic simulations when the number of molecules was sufficiently large. An analysis of the theory indicated a relationship between variance and the number of molecules in the system, and results of multiple stochastic simulation runs confirmed this relationship. We simulated Ca2(+) dynamics in a cell by inward flow from a point on the cell surface and the polymerization of G-actin forming F-actin. Our results showed that this theory and method can be used to simulate spatially inhomogeneous events.     

Genetic verification of Bordetella pertussis seed strains used for production of Japanese acellular pertussis vaccines

In Japan, the Bordetella pertussis strain Tohama provided by the National Institute of Health, Japan has been used for the production of acellular pertussis (aP) vaccines since 1981. In the present study, in order to verify the genetic consistency of B. pertussis vaccine seed strains, we analyzed the genetic properties of the working seeds obtained from five Japanese vaccine manufacturers, and compared them with those of B. pertussis Tohama reference strains (NIID L-7 and ATCC BAA-589). Genetic analyses with pulsed-field gel electrophoresis and allele typing showed 100% genetic identity among the five seed strains and the Tohama reference strains. In addition, Southern blot analyses revealed the absence of four orthologous genes (BB0537, BB0920, BB1149 and BB4885), which are specifically absent in the strain Tohama, and in the genome of all seed strains tested, suggesting that the regions of difference (RD11–RD14) are absent in their genomes. Consequently, no genetic difference was observed among the working seeds and Tohama reference strains. Our observations indicate that B. pertussis seed strains for Japanese aP vaccine production are genetically comparable with B. pertussis Tohama.     

Antibody recycling by engineered pH-dependent antigen binding improves the duration of antigen neutralization

For many antibodies, each antigen-binding site binds to only one antigen molecule during the antibody's lifetime in plasma. To increase the number of cycles of antigen binding and lysosomal degradation, we engineered tocilizumab (Actemra), an antibody against the IL-6 receptor (IL-6R), to rapidly dissociate from IL-6R within the acidic environment of the endosome (pH 6.0) while maintaining its binding affinity to IL-6R in plasma (pH 7.4). Studies using normal mice and mice expressing human IL-6R suggested that this pH-dependent IL-6R dissociation within the acidic environment of the endosome resulted in lysosomal degradation of the previously bound IL-6R while releasing the free antibody back to the plasma to bind another IL-6R molecule. In cynomolgus monkeys, an antibody with pH-dependent antigen binding, but not an affinity-matured variant, significantly improved the pharmacokinetics and duration of C-reactive protein inhibition. Engineering pH dependency into the interactions of therapeutic antibodies with their targets may enable them to be delivered less frequently or at lower doses.     

Nitrated fatty acids: Endogenous anti-inflammatory signaling mediators

Nitroalkene derivatives of linoleic acid (LNO2) and oleic acid (OA-NO2) are present; however, their biological functions remain to be fully defined. Herein, we report that LNO2 and OA-NO2 inhibit lipopolysaccharide-induced secretion of proinflammatory cytokines in macrophages independent of nitric oxide formation, peroxisome proliferator-activated receptor-gamma activation, or induction of heme oxygenase-1 expression. The electrophilic nature of fatty acid nitroalkene derivatives resulted in alkylation of recombinant NF-kappaB p65 protein in vitro and a similar reaction with p65 in intact macrophages. The nitroalkylation of p65 by fatty acid nitroalkene derivatives inhibited DNA binding activity and repressed NF-kappaB-dependent target gene expression. Moreover, nitroalkenes inhibited endothelial tumor necrosis factor-alpha-induced vascular cell adhesion molecule 1 expression and monocyte rolling and adhesion. These observations indicate that nitroalkenes such as LNO2 and OA-NO2, derived from reactions of unsaturated fatty acids and oxides of nitrogen, are a class of endogenous anti-inflammatory mediators.     

Ambroxol-induced modification of ion transport in human airway Calu-3 epithelia

Ambroxol is often used as a mucolytic agent in various lung diseases. However, it is unclear how ambroxol acts on bronchial epithelial cells. To clarify the action of ambroxol, we studied the effects of ambroxol on the ion transport in human Calu-3 cells, a human submucosal serous cell line, measuring the transepithelial short-circuit current and conductance across monolayers of Calu-3 cells. Ambroxol of 100 microM diminished the terbutaline (a beta2-adrenergic agonist)-stimulated Cl-/HCO3(-)-dependent secretion without any decreases in the conductance of cystic fibrosis transmembrane conductance regulator (CFTR) channel locating on the apical membrane. On the other hand, under the basal (unstimulated) condition ambroxol increased the Cl(-)-dependent secretion with no significant change in the apical CFTR channel conductance and decreased the HCO3- secretion associated with a decrease in the apical CFTR channel conductance. Ambroxol had no major action on the epithelial Na+ channel (ENaC) or the ENaC-mediated Na+ absorption. These results indicate that in Calu-3 cells: (1) under the basal (unstimulated) condition ambroxol increases Cl- secretion by stimulating the entry step of Cl- and decreases HCO3- secretion by diminishing the activity of the CFTR channel and/or the Na+/HCO3(-)-dependent cotransporter, (2) under the adrenergic agonist-stimulated condition, ambroxol decreases Cl- secretion by acting on the Cl-/HCO3- exchanger, and (3) ambroxol has a more powerful action than the adrenergic agonist on the Cl-/HCO3- exchanger, leading fluid secretion to a moderately stimulated level from a hyper-stimulated level.     

The proteasome activator PA28 functions in collaboration with Hsp90 in vivo

We have previously shown that the proteasome activator PA28 is essential to Hsp90-dependent protein refolding in vitro, where PA28 mediates transfer of the Hsp90-bound substrate protein to the Hsc70/Hsp40 chaperone machine for its correct refolding. This observation suggests that PA28 may also collaborate with Hsp90 in cells. To examine this possibility, here we have used double-stranded RNA interference (RNAi) against PA28 in Caenorhabditis elegans mutants of daf-21, which encodes Hsp90. We show that C. elegans PA28 facilitates Hsp90-initiated protein refolding, albeit with an activity lower than that of mouse PA28 proteins. RNAi-mediated knockdown of PA28 significantly suppresses the Daf-c (dauer formation constitutive) phenotype of the daf-21 mutant, but it has no affect on the distinct defects of this mutant in sensing odorants. Taking these results together, we conclude that PA28 is likely to function in collaboration with Hsp90 in vivo.     

Involvement of mitogen-activated protein kinases in class B scavenger receptor type I-induced phagocytosis of apoptotic cells

Class B scavenger receptor type I (SR-BI) is a multiligand membrane protein expressed in a variety of cell types. This receptor is responsible for the incorporation of lipids from high density lipoprotein (HDL) by steroidogenic cells, as well as for the phosphatidylserine (PS)-mediated phagocytosis of apoptotic cells by some phagocytic cell types, such as testicular Sertoli cells. Although SR-BI directly binds to PS present on the surface of apoptotic cells, as to whether SR-BI transmits signals to induce engulfment has not been clear. In the present study, we examined this issue using a monoclonal antibody that neutralizes SR-BI activity and a chemical known to be an inhibitor of the SR-BI-mediated incorporation of HDL lipids. The chemical compound inhibited the incorporation of HDL lipids and PS-containing liposomes by an SR-BI-expressing culture cell line, with no effect on the binding of these targets. Similarly, the phagocytosis of PS-exposing apoptotic cells by primary cultured rat Sertoli cells was inhibited in the presence of either reagent, not at the recognition but at the engulfment step. The addition of apoptotic cells or PS-containing liposomes caused a temporal increment of the phosphorylation of all three mitogen-activated protein kinases, p38, extracellular-signal-regulated kinase (ERK) and c-Jun amino-terminal kinase (JNK), in Sertoli cells. The increase of phosphorylated p38 and ERK, but not of phosphorylated JNK, was cancelled in the presence of the monoclonal antibody. Furthermore, the level of Sertoli cell phagocytosis of PS-exposing apoptotic cells, as well as that of PS-containing liposomes, was reduced only when the actions of p38 and ERK were simultaneously repressed. In conclusion, these results indicate that SR-BI, when it binds to PS, transmits signals to activate the mitogen-activated protein kinase pathway, which leads to the induction of the engulfment of PS-exposing apoptotic cells by phagocytic cells.