Linkage of the efflux-pump expression level with substrate extrusion rate in the MexAB-OprM efflux pump of Pseudomonas aeruginosa

The amount of the subunit proteins of the MexAB-OprM efflux pump in Pseudomonas aeruginosa was quantified by the immunoblotting method. A single cell of the wild-type strain contained about 2500, 1000, and 1200 copies of MexA, MexB, and OprM, respectively, and their stoichiometry therefore was 2:1:1. The mexR mutant produced an eightfold higher level of these proteins than did wild-type cells. Assuming that MexB and OprM exist as a trimer in a pump assembly, the total number of MexAB-OprM per wild-type cell was calculated to be about 400 assemblies. The substrate efflux rate of MexAB-OprM was calculated from the fluorescent intensity of ethidium in intact cells that a single cell extruded ethidium at a maximum of about 3 x 10(-19) mol s(-1) and, therefore, the turnover rate of a single pump unit was predicted to be about 500 s(-1).     

Increased rigidity of domain structures enhances the stability of a mutant enzyme created by directed evolution

A mutant of kanamycin nucleotidyltransferase (KNT) was previously created by directed evolution. This mutant, HTK, has 19 amino acid substitutions, which increase the thermostability by 20 degrees C. In this study, we have examined to what extent each mutation contributes to the increased stability and analyzed how the mutations affect the structure of KNT at 72 degrees C using molecular dynamics simulations. The effects of some mutations on the stability are simply additive, but those of others are cooperative. Mutations with large effects on the stability are introduced into the N-terminal domain, which appears to be less stable than the C-terminal domain. Results of the molecular dynamics simulations have indicated that the rigidity of the domain structures is increased by the mutations: at 72 degrees C, the intradomain fluctuations of HTK are decreased, and in turn, its interdomain motions are pronounced, whereas the structure of the preevolved KNT fluctuates randomly. Chemical modification experiments of cysteine residues have shown that the cysteine residues of HTK are less accessible to an SH reagent than those of the preevolved KNT. The present results suggest that the 19 mutations of HTK stabilize KNT by affecting the dynamic behavior of the structure of this enzyme without significantly changing its static overall structure.     

Production of herbicide-tolerant zoysiagrass by Agrobacterium-mediated transformation

Herbicide-resistant zoysiagrass (Zoysia japonica Steud.) has been developed by Agrobacterium-mediated transformation. A callus-type transformation system was established by optimizing several factors that affect the rate of transformation, including co-cultivation period and concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), CaCl2 and acetosyringone. Maximal GUS expression was observed when a Type 3 callus was co-cultivated on 2,4-D-free co-cultivation medium for 9 d. In addition, removal of calcium and addition of 50-100 mg/L acetosyringone during co-cultivation enhanced GUS expression. When this optimized protocol was applied to the transformation of the bialaphos resistance gene (bar), four plants per 700 mg of infected calluses survived on the selective medium. DNA gel-blot analysis showed that two copies of the transgene had been integrated. After application of 2 g/L bialaphos for a week the transgenic plants survived herbicide spraying, while untransformed zoysiagrasses and invading weeds died. The herbicide-tolerant zoysiagrass will permit more efficient weed control in this widely cultivated turf grass.     

Rescue of infertile transgenic rat lines by intracytoplasmic injection of cryopreserved round spermatids

Transgenic male rats carrying human alpha-lactalbumin with thymidine kinase gene (line name; LAC3) were found to be infertile due to expression of the transgene in the testes. Furthermore, it was not possible to maintain the line even by the use of intracytoplasmic sperm injection (ICSI). Therefore, round spermatids prepared from the LAC3 rats were microinjected into strontium-activated oocytes using a Piezo-driven micromanipulator. Of 263 oocytes microinjected with LAC3 spermatids, 244 (92.8%) survived the injection and 96 (39.3%) developed to the 2-cell stage. Three viable offspring were born after transfer (1.4%, 3/219), and two offspring carried the LAC3 transgene. In the control experiment using spermatids of Wistar rats, similar proportions of post-injection survival (91.3%, 241/264), cleavage (40.2%, 97/241), and development into offspring (0.5%, 1/206) were obtained. Thus, this paper reports not only the first rat offspring derived from round spermatid injection but also the practical application of the microinsemination technique to the rescue of transgenes of infertile transgenic male rats.     

Additive effect of pronase on the efficacy of eradication therapy against Helicobacter pylori

Background. Helicobacter pylori (H. pylori) colonizes not only the surface of the surface mucous cells but also the surface mucous gel layer (SMGL). Thus, we examined the possible value of pronase, a mucolytic agent, as a potential eradication therapy. Materials and Methods. One hundred and thirty‐five patients were randomly assigned to two treatment groups. Sixty‐eight patients received 30 mg of lansoprazole once daily, 500 mg of amoxicillin and 250 mg of metronidazole thrice daily for 2 weeks (LAM group), while the other 67 patients received the same dosage of those agents plus 18,000 tyrosine units of pronase thrice daily for 2 weeks (LAMP group). Eradication was assessed 4–6 weeks after treatment by immunohistochemical tests and cultures. We also determined the in vitro activity of pronase against H. pylori, and evaluated the synergistic effects between pronase and the other three drugs. To investigate the effect of pronase on the structure of the SMGL, surgically removed stomachs obtained from patients who had taken pronase were examined histopathologically. Results. The cure rates for H. pylori infection in the LAMP group were significantly higher than those in the LAM group (intention to treat analysis: 94.0 vs. 76.5%, p = .0041). Pronase exhibited no antibacterial activity against H. pylori., and no in vitro synergistic effects were observed. In the patients who took pronase before surgery, the SMGL was thinner than in the patients who did not take pronase, and the structure of the SMGL was markedly disrupted. Conclusions. Pronase has an additive effect in curing H. pylori infection. Pronase has no apparent in vitro activity against H. pylori, but may improve the local delivery of antibiotics by virtue of its removal and disruption of the SMGL.     

Functional adaptation of cancellous bone in human proximal femur predicted by trabecular surface remodeling simulation toward uniform stress state

Two-dimensional simulation of trabecular surface remodeling was conducted for a human proximal femur to investigate the structural change of cancellous bone toward a uniform stress state. Considering that a local mechanical stimulus plays an important role in cellular activities in bone remodeling, local stress nonuniformity was assumed to drive trabecular structural change to seek a uniform stress state. A large-scale pixel-based finite element model was used to simulate structural changes of individual trabeculae over the entire bone. As a result, the initial structure of trabeculae changed from isotropic to anisotropic due to trabecular microstructural changes caused by surface remodeling according to the mechanical environment in the proximal femur. Under a single-loading condition, it was shown that the apparent structural property evaluated by fabric ellipses corresponded to the apparent stress state in cancellous bone. As is observed in the actual bone, a distributed trabecular structure was obtained under a multiple-loading condition. Through these studies, it was concluded that trabecular surface remodeling toward a local uniform stress state at the trabecular level could naturally bring about functional adaptation phenomenon at the apparent tissue level. The proposed simulation model would be capable of providing insight into the hierarchical mechanism of trabecular surface remodeling at the microstructural level up to the apparent tissue level.     

Identification of the outer membrane protein of E.coli that produces transmembrane channels in reconstituted vesicle membranes

Outer membrane of $\text{Escherichia}$$\text{coli}$ allows a rapid diffusion of saccharides of molecular weights less than 550. This permeability property could be restored in vesicle membranes reconstituted from isolated phospholipids, lipopolysaccharide, and an outer membrane protein. The active protein aggregates were isolated from the insoluble material left after solubilization of cell envelope of $\text{Escherichia}$$\text{coli}$ B with sodium dodecyl sulfate at 35°. Analysis by acrylamide gel electrophoresis, isoelectric focusing and amino terminal amino acid determination revealed that only a single species of protein, with a molecular weight of 36,500 forms the oligoprotein aggregates which produces diffusion channels.     

Nisin Production by a Mixed-Culture System Consisting of Lactococcus lactis and Kluyveromyces marxianus

To control the pH during antimicrobial peptide (nisin) production by a lactic acid bacterium, Lactococcus lactis subsp. lactis (ATCC11454), a novel method involving neither addition of alkali nor a separation system such as a ceramic membrane filter and electrodialyzer was developed. A mixed culture of L. lactis and Kluyveromyces marxianus, which was isolated from kefir grains, was utilized in the developed system. The interaction between lactate production by L. lactis and its assimilation by K. marxianus was used to control the pH. To utilize the interaction of these microorganisms to maintain high-level production of nisin, the kinetics of growth of, and production of lactate, acetate, and nisin by, L. lactis were investigated. The kinetics of growth of and lactic acid consumption by K. marxianus were also investigated. Because the pH of the medium could be controlled by the lactate consumption of K. marxianus and the specific lactate consumption rate of K. marxianus could be controlled by changing the dissolved oxygen (DO) concentration, a cascade pH controller coupled with DO control was developed. As a result, the pH was kept constant because the lactate level was kept low and nisin accumulated in the medium to a high level compared with that attained using other pH control strategies, such as with processes lacking pH control and those in which pH is controlled by addition of alkali.     

Crystal structure of the drug discharge outer membrane protein, OprM, of Pseudomonas aeruginosa: dual modes of membrane anchoring and occluded cavity end

The OprM lipoprotein of Pseudomonas aeruginosa is a member of the MexAB-OprM xenobiotic-antibiotic transporter subunits that is assumed to serve as the drug discharge duct across the outer membrane. The channel structure must differ from that of the porin-type open pore because the protein facilitates the exit of antibiotics but not the entry. For better understanding of the structure-function linkage of this important pump subunit, we studied the x-ray crystallographic structure of OprM at the 2.56-angstroms resolution. The overall structure exhibited trimeric assembly of the OprM monomer that consisted mainly of two domains: the membrane-anchoring beta-barrel and the cavity-forming alpha-barrel. OprM anchors the outer membrane by two modes of membrane insertions. One is via the covalently attached NH(2)-terminal fatty acids and the other is the beta-barrel structure consensus on the outer membrane-spanning proteins. The beta-barrel had a pore opening with a diameter of about 6-8 angstroms, which is not large enough to accommodate the exit of any antibiotics. The periplasmic alpha-barrel was about 100 angstroms long formed mainly by a bundle of alpha-helices that formed a solvent-filled cavity of about 25,000 angstroms(3). The proximal end of the cavity was tightly sealed, thereby not permitting the entry of any molecule. The result of this structure was that the resting state of OprM had a small outer membrane pore and a tightly closed periplasmic end, which sounds plausible because the protein should not allow free access of antibiotics. However, these observations raised another unsolved problem about the mechanism of opening of the OprM cavity ends. The crystal structure offers possible mechanisms of pore opening and pump assembly.