Adenosine selectively inhibits noncholinergic transmission in guinea pig bronchi

The neuromodulatory action of adenosine and ATP was investigated in isolated guinea pig bronchial strip chain preparations contracted with electrical field stimulation. The tissues were placed in organ baths containing physiological salt solution and stimulated at 8-Hz frequency, 0.5-ms pulse duration, and 30 V (approximately 100 mA) for 5 s. Electrical field stimulation evoked a biphasic contraction of bronchial muscle, consisting of an initial contraction followed by a sustained contraction, which was mediated by intramural cholinergic and noncholinergic nerve stimulations, respectively. Adenosine, at concentrations greater than M, caused a concentration-dependent inhibition in the height of the noncholinergically mediated contraction, accompanied by a very weak inhibition on the cholinergically mediated response. ATP (10(-5) to 3 x 10(-3) M) also produced a similar inhibitory effect on the noncholinergically mediated contraction, but the inhibitory potency was less than that of adenosine. The inhibitory response to adenosine was enhanced by the pretreatment with dipyridamole (2 x 10(-6) M) but antagonized with aminophylline (10(-5) M). Contractions of bronchial muscle evoked by exogenous acetylcholine (2 x 10(-6) M) or substance P (2 x 10(-7) M) were significantly inhibited by the adenosine (3 x 10(-4) M) pretreatment. These data suggest that in isolated guinea pig bronchi adenosine selectively inhibits noncholinergic neurotransmission through prejunctional P1-purinoceptors.     

Fast protocol for the production of Histoplasma capsulatum antigens for antibody detection in the immunodiagnosis of histoplasmosis

Background

Current methods for the production of Histoplasma capsulatum antigens are problematic in terms of standardization, specificity, stability, repeatability and reproducibility.

Opr86 is essential for viability and is a potential candidate for a protective antigen against biofilm formation by Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that is one of the most refractory to therapy when it forms biofilms in the airways of cystic fibrosis patients. To date, studies regarding the production of an immunogenic and protective antigen to inhibit biofilm formation by P. aeruginosa have been superficial. The previously uncharacterized outer membrane protein (OMP) Opr86 (PA3648) of P. aeruginosa is a member of the Omp85 family, of which homologs have been found in all gram-negative bacteria. Here we verify the availability of Opr86 as a protective antigen to inhibit biofilm formation by P. aeruginosa PAO1 and several other isolates. A mutant was constructed in which Opr86 expression could be switched on or off through a tac promoter-controlled opr86 gene. The result, consistent with previous Omp85 studies, showed that Opr86 is essential for viability and plays a role in OMP assembly. Depletion of Opr86 resulted in streptococci-like morphological changes and liberation of excess membrane vesicles. A polyclonal antibody against Opr86 which showed reactivity to PAO1 cells was obtained. The antibody inhibited biofilm formation by PAO1 and the other clinical strains tested. Closer examination of early attachment revealed that cells treated with the antibody were unable to attach to the surface. Our data suggest that Opr86 is a critical OMP and a potential candidate as a protective antigen against biofilm formation by P. aeruginosa.     

Novel asymmetrically engineered antibody Fc variant with superior FcγR binding affinity and specificity compared with afucosylated Fc variant

Fc engineering is a promising approach to enhance the antitumor efficacy of monoclonal antibodies (mAbs) through antibody-dependent cell-mediated cytotoxicity (ADCC). Glyco- and protein-Fc engineering have been employed to enhance FcγR binding and ADCC activity of mAbs; the drawbacks of previous approaches lie in their binding affinity to both FcγRIIIa allotypes, the ratio of activating FcγR binding to inhibitory FcγR binding (A/I ratio) or the melting temperature (T_M) of the C_H2 domain. To date, no engineered Fc variant has been reported that satisfies all these points. Herein, we present a novel Fc engineering approach that introduces different substitutions in each Fc domain asymmetrically, conferring optimal binding affinity to FcγR and specificity to the activating FcγR without impairing the stability. We successfully designed an asymmetric Fc variant with the highest binding affinity for both FcγRIIIa allotypes and the highest A/I ratio compared with previously reported symmetrically engineered Fc variants, and superior or at least comparable in vitro ADCC activity compared with afucosylated Fc variants. In addition, the asymmetric Fc engineering approach offered higher stability by minimizing the use of substitutions that reduce the T_M of the C_H2 domain compared with the symmetric approach. These results demonstrate that the asymmetric Fc engineering platform provides best-in-class effector function for therapeutic antibodies against tumor antigens.     

Cryobiological properties of immature zebrafish oocytes assessed by their ability to be fertilized and develop into hatching embryos

As a step to develop a cryopreservation method for zebrafish oocytes, we investigated the cryobiological properties of immature oocytes at stage III by examining their ability to mature and to develop into hatching embryos after fertilization. When oocytes were chilled at −5 °C for 30 min, the maturation rate decreased, but the rates of fertilization and hatching were not significantly different from those of controls. When oocytes were exposed to hypotonic solutions for 60 min at 25 °C, the rates of maturation, fertilization, and hatching decreased in a solution with 0.16 Osm/kg or below. When oocytes were exposed to hypertonic solutions (containing sucrose) at 25 °C for 30 min, the maturation rate decreased in solution with 0.51 Osm/kg, whereas the hatching rate decreased with lower osmolality (0.40 Osm/kg). In an experiment on the toxicity of cryoprotectants (∼10%, at 25 °C), it was found that glycerol and ethylene glycol were toxic both by the assessment of maturation and hatching. Propylene glycol, DMSO and methanol were less toxic by the assessment of maturation, but were found to be toxic by the assessment of hatching. Methanol was the least toxic, but it was less effective to make a solution vitrify than propylene glycol. Therefore, a portion of methanol was replaced with propylene glycol. The replacement increased the toxicity, but could be effective to reduce chilling injury at −5 °C. These results clarified the sensitivity of immature oocytes to various cryobiological properties accurately, which will be useful for realizing cryopreservation of zebrafish oocytes.