Production of Hybrid-IgG/IgA Plantibodies with Neutralizing Activity against Shiga Toxin 1

Shiga toxin 1 (Stx1) is a virulence factor of enterohemorrhagic Escherichia coli, such as the O157:H7 strain. In the intestines, secretory IgA (SIgA) is a major component of the immune defense against pathogens and toxins. To form SIgA, the production of dimeric IgA that retains biological activity is an important step. We previously established hybrid-IgG/IgA having variable regions of the IgG specific for the binding subunit of Stx1 (Stx1B) and the heavy chain constant region of IgA. If hybrid-IgG/IgA cDNAs can be expressed in plants, therapeutic or preventive effects may be expected in people eating those plants containing a “plantibody”. Here, we established transgenic Arabidopsis thaliana expressing dimeric hybrid-IgG/IgA. The heavy and light chain genes were placed under the control of a bidirectional promoter and terminator of the chlorophyll a/b-binding protein of Arabidopsis thaliana (expression cassette). This expression cassette and the J chain gene were subcloned into a single binary vector, which was then introduced into A. thaliana by means of the Agrobacterium method. Expression and assembly of the dimeric hybrid-IgG/IgA in plants were revealed by ELISA and immunoblotting. The hybrid-IgG/IgA bound to Stx1B and inhibited Stx1B binding to Gb₃, as demonstrated by ELISA. When Stx1 holotoxin was pre-treated with the resulting plantibody, the cytotoxicity of Stx1 was inhibited. The toxin neutralization was also demonstrated by means of several assays including Stx1-induced phosphatidylserine translocation on the plasma membrane, caspase-3 activation and 180 base-pair DNA ladder formation due to inter-nucleosomal cleavage. These results indicate that edible plants containing hybrid-IgG/IgA against Stx1B have the potential to be used for immunotherapy against Stx1-caused food poisoning.     

Intermolecular Interaction of Fluoro Propanes

Abstract

Intermolecular interaction is investigated for an isomeric pair of fluoro propane, CH₃CF₂CF₃ (HFC–245cb, CB) and CH₂FCF₂CHF₂ (HFC–245ca, CA). CB has a larger dipole moment than CA. This may suggest that CB has a larger intermolecular attractive interaction than CA; the reverse is, however, found from the experimental data: normal boiling point, critical temperature, and heat of vaporization. Systematic ab initio calculations have been done for both CB dimer and CA dimer, and confirmed that the former has a smaller attractive interaction than the latter.

On the basis of these calculations, analytic functions have been constructed as the pair potential models for the two isomers. Each of these models has 11 Lennard-Jones and Coulomb interaction sites in the molecule. The present models can explain why CB dimer has a smaller attractive interaction than CA dimer, and they will easily be extended to a series of fluoro propanes, and make it possible to perform the systematic molecular simulation studies.     

The Stability of Clathrate Hydrates: Temperature Dependence of Dissociation Pressure in Xe and Ar Hydrate

Abstract

The thermodynamic stability of clathrate hydrates I and II encaging xenon or argon has been investigated by examining the temperature dependence of the dissociation pressure. The evaluation of the stability is made based on the generalized van der Waals and Platteeuw theory developed by Tanaka and Kiyohara [J. Chem. Phys. 98, 4098 (1993)]. In the new treatment, the free energy of formation of hydrates in equilibrium with ice is calculated by taking the coupling of the host lattice vibrations with guests into consideration. The predicted dissociation pressures of Xe and Ar hydrates agree well with experiments in higher temperature range. A poor agreement between experiment and calculation for Ar clathrate hydrate at low temperature is improved by the use of a quantum mechanical partition function for a harmonic oscillator in evaluating the free energy difference between ice and empty hydrate.     

Yeast Cell Wall Bound Proteolytic Enzymes

Nearly all the amino group-producing activity of the autolysate of cells of Saccharomyces sake was recovered in the cell wall fraction obtained from the autolysis residue. The activity of the cell wall fraction was not lost even after repeated use.

The proteolytic activity of the fraction was not solubilized by incubation with detergents, disruption with cell mill or by freezing and thawing method, but was solubilized to some extent by incubation with a commercial yeast cell-lytic enzyme preparation.

The cell wall fraction hydrolysed casein to about 50%. When casein was previously treated with certain proteinases, more than 60% was digested. The activity of the fraction was significantly increased by the addition of Zn²⁺ while it was decreased by several proteolytic enzyme inhibitors. The interesting fact was that in the presence of EDTA the cell wall fraction showed only carboxypeptidase-like activity, and attacked the oxidized insulin B-chain to release two amino acids from the carboxyl terminal in known order.     

Establishment of a new method to quantitatively evaluate hyphal fusion ability in Aspergillus oryzae

Hyphal fusion is involved in the formation of an interconnected colony in filamentous fungi, and it is the first process in sexual/parasexual reproduction. However, it was difficult to evaluate hyphal fusion efficiency due to the low frequency in Aspergillus oryzae in spite of its industrial significance. Here, we established a method to quantitatively evaluate the hyphal fusion ability of A. oryzae with mixed culture of two different auxotrophic strains, where the ratio of heterokaryotic conidia growing without the auxotrophic requirements reflects the hyphal fusion efficiency. By employing this method, it was demonstrated that AoSO and AoFus3 are required for hyphal fusion, and that hyphal fusion efficiency of A. oryzae was increased by depleting nitrogen source, including large amounts of carbon source, and adjusting pH to 7.0.     

Purification and Properties of Three Types of Xylanases Induced by Methyl β-Xyloside from Streptomyces sp.

When mycelia of Streptomyces sp. No. 3137 were cultivated in a medium containing methyl β-xyloside, xylanases (EC 3.2.1.8) were inductively produced into the medium. Three types of enzyme from the culture filtrate have been purified by ultrafiltration with DIAFLO UM-10, chromatography on DEAE-Sephadex A-25, gel filtration on Bio Gel P-100, and isoelectric focusing with Servalyt 6~8 or 9~11. The three purified enzymes, tentatively named X-I, X-II-A, and X-II-B, were homogeneous by Polyacrylamide gel electrophoresis at pH 4.3. The molecular weight of X-I was about 50,000 by SDS-polyacrylamide gel electrophoresis or gel filtration on Bio Gel P-100. The molecular weight of X-II-A and X-II-B were both approximately 25,000 by SDS-polyacrylamide gel electrophoresis and that of X-II-B was 25,680 by the sedimentation-equilibrium method. X-I had an isoelectric point at 7.10, and X-II-A and X-II-B had different isoelectric points, 10.06 and 10.26, respectively. The three enzymes were optimally active at 60~65°C and stable to 55°C. The optimal pH of X-I, X-II-A, and X-II-B were pH 5.5~6.5, 5.0~6.0, and 5.0~6.0, respectively. The ranges of two X-I’s pH stability (pH 1.5 ~ 11.5) were wider than that of X-I’s (pH 3.0 ~ 10.5). These purified preparations hydrolyzed xylotriose, xylotetraose, and xylan but not xylobiose, cellobiose, maltose, carboxymethyl cellulose, or soluble starch. Their actions were inhibited by Hg²⁺ and Fe³⁺ ions, sodium dodecyl sulfate, and N-bromosuccinimide.     

Synthesis and biological evaluation of C-glucosides with azulene rings as selective SGLT2 inhibitors for the treatment of type 2 diabetes mellitus: Discovery of YM543

Here, a series of C-glucosides with azulene rings in the aglycon moiety was synthesized and the inhibitory activities toward hSGLT1 and hSGLT2 were evaluated. Starting from the azulene derivative 7 which had relatively good SGLT2 inhibitory activity, compound 8a which has a 3-[(azulen-2-yl)methyl]phenyl group was identified as a lead compound for further optimization. Introduction of a phenolic hydroxyl group onto the central benzene ring afforded a potent and selective SGLT2 inhibitor 8e, which reduced blood glucose levels in a dose-dependent manner in rodent diabetic models. A mono choline salt of 8e (YM543) was selected as a clinical candidate for use in treating type 2 diabetes mellitus.