Shiga toxin type 2 B subunit protects mice against toxin challenge when leashed and bundled by a stable pentameric coiled-coil molecule.

Vaccines against Shiga toxin (Stx)-producing Escherichia coli (STEC) have not yet been developed. Two immunologically distinct serotypes of Stx (Stx1 and Stx2) are the main virulence factors of STEC. Thus, blocking their B subunits (StxB) from binding to the cell surface receptor globotriaosylceramide (Gb3) efficiently prevents the action of these toxins. We expressed Stx1B and Stx2B in E. coli inclusion bodies and reassembled them into pentamers by a stepwise dialysis. Stx1B pentamer fully protected mice against Stx1 challenge, but Stx2B pentamer failed to protect mice against Stx2 challenge. To explain those observations, we proposed that the pentamer of Stx2B readily dissociates into its constituent monomers, especially under in vivo conditions, thus being unable to induce pentamer-specific immunity. To increase pentamer stability, we fused the B subunit to a pentameric coiled-coil domain of the cartilage oligomeric matrix protein (COMP). This "five-to-five" fusion hybrid molecule (Stx2B-COMP) was shown to be protective against Stx2 challenge, demonstrating that the Stx2B subunit when leashed and bundled by a rigid pentameric coiled-coil domain mount a pentamer-specific immune response and efficiently neutralize the toxin both in vitro and in vivo. Our data strongly suggest that the Stx2B subunit moiety fluctuates between a pentameric and monomeric state within the fusion protein, which may increase the likelihood of the immune system recognizing the pentameric conformation for toxin neutralization.

Highly efficient protein expression of Plasmodium vivax surface antigen, Pvs25, by silkworm and its biochemical analysis.

Plasmodium vivax ookinete surface protein, Pvs25, is a candidate for a transmission-blocking vaccine (TBV) for malaria. Pvs25 has four EGF-like domains containing 22 cysteine residues forming 11 intramolecular disulfide bonds, a structural feature that makes its recombinant protein expression difficult. In this study, we report the high expression of recombinant Pvs25 as a soluble form in silkworm, Bombyx mori. The Pvs25 protein was purified from hemolymphs of larvae and pupae by affinity chromatography. In the Pvs25 expressed by silkworm, no isoforms with inappropriate disulfide bonds were found, requiring no further purification step, which is necessary in the case of Pichia pastoris-based expression systems. The Pvs25 from silkworm was confirmed to be molecularly uniform by sodium dodecyl sulfate gel electrophoresis and size-exclusion chromatography. To examine the immunogenicity, the Pvs25 from B. mori was administered to BALB/c mice subcutaneously with oil adjuvant. The Pvs25 produced by silkworm induced potent and robust immune responses, and the induced antisera correctly recognized P. vivax ookinetes in vitro, demonstrating the potency of Pvs25 from silkworm as a candidate for a malaria TBV. To the best of our knowledge, this is the first study to construct a system for mass-producing malaria TBV antigens using silkworm.

Fiber knob domain lacking the shaft sequence but fused to a coiled coil is a candidate subunit vaccine against egg-drop syndrome.

Egg-drop syndrome (EDS) virus is an avian adenovirus that causes a sudden drop in egg production and in the quality of the eggs when it infects chickens, leading to substantial economic losses in the poultry industry. Inactivated EDS vaccines produced in embryonated duck eggs or cell culture systems are available for the prophylaxis of EDS. However, recombinant subunit vaccines that are efficacious and inexpensive are a desirable alternative. In this study, we engineered chimeric fusion proteins in which the trimeric fiber knob domain lacking the triple ß-spiral motif in the fiber shaft region was genetically fused to trimeric coiled coils, such as those of the engineered form of the GCN4 leucine zipper peptide or chicken cartilage matrix protein (CMP). The fusion proteins were expressed predominantly as soluble trimeric proteins in Escherichia coli at levels of 15-80mg/L of bacterial culture. The single immunization of chickens with the purified fusion proteins, at a dose equivalent to 10µg of the knob moiety, elicited serum antibodies with high hemagglutination inhibition (HI) activities, similar to those induced by an inactivated EDS vaccine. A dose-response analysis indicated that a single immunization with as little as 1µg of the knob moiety of the CMP-knob fusion protein was as effective as the inactivated vaccine in inducing antibodies with HI activity. The immunization of laying hens had no apparent adverse effects on egg production and effectively prevented clinical symptoms of EDS when the chickens were challenged with pathogenic EDS virus. This study demonstrates that the knob domain lacking the shaft sequence but fused to a trimeric coiled coil is a promising candidate subunit vaccine for the prophylaxis of EDS in chickens.

Cholera toxin B subunit pentamer reassembled from Escherichia coli inclusion bodies for use in vaccination.

The cholera toxin B subunit (CTB) is secreted in its pentameric form from Escherichia coli if its leader peptide is replaced with one of E. coli origin. However, the secretion of the pentamer is generally severely impaired when the molecule is mutated or fused to a foreign peptide. Therefore, we attempted to regenerate pentameric CTB from the inclusion bodies (IBs) of E. coli. Stepwise dialysis of the IBs solubilized in guanidine hydrochloride predominantly generated soluble high-molecular-mass (HMM) aggregates and only a small fraction of pentamer. Three methods to reassemble homogeneous pentameric molecules were evaluated: (i) using a pentameric coiled-coil fusion partner, expecting it to function as an assembly core; (ii) optimizing the protein concentration during refolding; and (iii) eliminating contaminants before refolding. Coiled-coil fusion had some effect, but substantial amounts of HMM aggregates were still generated. Varying the protein concentration from 0.05 mg/mL to 5mg/mL had almost no effect. In contrast, eliminating the contaminants before refolding had a robust effect, and only the pentamer was regenerated, with no detectable HMM aggregates. Surprisingly, the protein concentration at refolding was up to 5mg/mL when the contaminants were removed, with no adverse effects on refolding. The regenerated pentamer was indistinguishable in its biochemical and immunological characteristics from CTB secreted from E. coli or choleragenoid from Vibrio cholerae. This study provides a simple but very efficient strategy for pentamerizing CTB with a highly homogeneous molecular conformation, with which it may be feasible to engineer CTB derivatives and CTB fusion antigens.

Structure of a novel α-glucan substitute with the rare 6-deoxy-D-altrose from Lactarius lividatus (mushroom).

A novel α-glucan substituted rare 6-deoxy-D-altropyranose was isolated from edible fruiting bodies of a mushroom (Lactarius lividatus) grown in Okinawa, Japan. The polysaccharide consists of D-glucose, D-galactose and 6-deoxy-D-altrose in a molar ratio of 3.0:1.0:1.0. The specific rotation [α](589) was estimated as +64.3° (0.2% in water) at 25 °C. Based on results of IR, NMR ((1)H, (13)C, 2D-COSY, 2D-HMQC, 2D-ROESY and 2D-HMBC), and methylation analyses, the structure of the polysaccharide was determined as [formula, see text] This work is the first demonstration of rare 6-deoxy-D-altropyranose moiety on polysaccharides.

Identification of rare 6-deoxy-D-altrose from an edible mushroom (Lactarius lividatus).

6-Deoxy-L-altrose is well known as a constituent sugar moiety of lipopolysaccharides in Gram-negative bacteria. However, its isomer, 6-deoxy-D-altrose, is little known. Identification of 6-deoxy-D-altrose isolated from a polysaccharide extracted from an edible mushroom (Lactarius lividatus), its comparison with chemically synthesized 6-deoxy-D-altrose using (1)H and (13)C NMR including COSY, HMQC spectroscopy, and investigation of its specific optical rotation were all conducted in this study. The 6-deoxy-hexose isolated from acid hydrolysate of the polysaccharide extracted from L. lividatus was involved in four anomeric isomers (α-pyranose and ß-pyranose, and α-furanose and ß-furanose), as was chemically synthesized 6-deoxy-d-altrose in an aqueous solution because of mutarotation. Almost all signals of 1D ((1)H NMR and (13)C NMR) and 2D (COSY and HMQC)-NMR spectra agreed with those of the authentic 6-deoxy-D-altrose. The specific optical rotation [α](589) of 6-deoxy-sugar showed a value of +18.2°, which was in agreement with that of authentic 6-deoxy-D-altrose. Consequently, 6-deoxy-hexose was identified as the 6-deoxy-D-altrose. This work is the first complete identification of 6-deoxy-D-altrose in a natural environment.

Rapid determination of carbohydrates, ash, and extractives contents of straw using attenuated total reflectance fourier transform mid-infrared spectroscopy.

Analysis of the chemical components of lignocellulosic biomass is essential to understanding its potential for utilization. Mid-infrared spectroscopy and partial least-squares regression were used for rapid measurement of the carbohydrate (total glycans; glucan; xylan; galactan; arabinan; mannan), ash, and extractives content of triticale and wheat straws. Calibration models for total glycans, glucan, and extractives showed good and excellent predictive performance on the basis of slope, r², RPD, and R/SEP criteria. The xylan model showed good and acceptable predictive performance. However, the ash model was evaluated as providing only approximate quantification and screening. The models for galactan, arabinan, and mannan indicated poor and insufficient prediction for application. Most models could predict both triticale and wheat straw samples with the same degree of accuracy. Mid-infrared spectroscopic techniques coupled with partial least-squares regression can be used for rapid prediction of total glycans, glucan, xylan, and extractives in triticale and wheat straw samples.

Production of carbohydrates, lignins, and minor components from triticale straw by hydrothermal treatment.

The effects of temperature (116 °C, 150 °C, and 183 °C) and flow rate (66, 150, and 234 mL/min) on the fractionation of triticale straw into different products was determined using a flow-through pressurized low polarity water reactor. The greatest concentration of biomass was hydrolyzed and extracted in the first two of eight 600 mL fractions (1.2 L), after which dry matter yield decreased. Carbohydrate, lignin, acetyl group, and uronic acid yield increased with temperature, but there was no effect due to flow rate. Most dry matter extracted at 116 °C was probably associated with the extractives. Xylan yields decrease slightly at the highest flow rate due to a decrease in the residence time of the acids produced in situ. Carbohydrates were extracted mostly as oligosaccharides, and the highest processing temperature resulted in the production of furans from the xylose and arabinose in the liquid extracts.

Rapid determination of lignin content of straw using fourier transform mid-infrared spectroscopy.

To determine lignin content in triticale and wheat straws, calibration models were built using Fourier transform mid-infrared spectroscopy combined with partial least-squares regression. The best model for triticale and wheat straws was built using averaged spectra with raw spectrum in spectrum format and constant in path length as spectral pretreatments. The values of r(2), root-mean-square error of prediction (RMSEP), and residual predictive deviation (RPD) for the triticale straw model were 0.935, 0.305, and 3.89, respectively. The r(2), RMSEP, and RPD values for the wheat straw model were 0.985, 0.163, and 8.50, respectively. Both models showed good predictive ability. A model built using both triticale and wheat straws indicated that the values of r(2), RMSEP, and RPD were 0.952, 0.27, and 4.63, respectively. This model also showed good predictive ability and could predict lignin contents in triticale and wheat straws with the same high accuracy.

Gelation and Retrogradation Mechanism of Wheat Amylose.

The flow behavior, dynamic viscoelasticity, and optical rotation of aqueous solutions of wheat amylose were measured using a rheogoniometer and a polarimeter. The amylose solutions, at 25 °C, showed shear-thinning behavior at a concentration of 1.2%, but plastic behavior at 1.4 and 1.6%, the yield values of which were estimated to be 0.6 and 1.0 Pa, respectively. The viscosity of the wheat amylose increased a little with increase in temperature up to 10 or 20 °C at 1.2% or 1.4 and 1.6%, which was estimated to be a transition temperature. The elastic modulus increased with increase in concentration, and increased with increasing temperature up to 20, 25 and 30 °C, which was estimated to be a transition temperature, respectively, then decreased gradually but stayed at a large value even at high temperature (80 °C). A very low elastic modulus of the wheat amylose was observed upon addition of urea (4.0 M) and in alkaline solution (0.05 M NaOH) even at low temperature. The optical rotation of wheat amylose solution increased a little with decreasing temperature down to 25 °C, then increased rapidly with further decrease in the temperature. The mode of gelation mechanism of amylose molecules, which was previously proposed, was confirmed and a retrogradation mechanism of wheat amylose was proposed.

Fucoidan isolated from Laminaria angustata var. longissima induced macrophage activation.

We investigated macrophage activation by fucoidan from Laminaria angustata var. longissima in a murine macrophage cell line, RAW 264.7. The ratio of the chemical composition of the fucoidan was L-fucose:D-galactose:D-glucose:D-xylose:uronic acid:sulfate=1.00:0.54:0.08:0.08:0.64:0.87. The fucoidan induced production of nitric oxide, tumor necrosis factor-α, and interleukin-6 in RAW 264.7 cells. These results indicate that the fucoidan induced macrophage activation.

The chemical structure of galactomannan isolated from seeds of Delonix regia.

A galactomannan isolated from seeds of Delonix regia was investigated. The specific rotation of the galactomannan was +15 degrees at 25 degrees C. The molecular mass of the galactomannnan was estimated to be 2.5 x 10(5). The ratio of D-mannose to D-galactose was estimated approximately to be 4.0:1.0 by HPLC and (1)H-NMR. NMR ((1)H and (13)C) spectra, and methylation analysis of the galactomannan indicated that it was composed of 1,4-linked beta-D-mannose, 1,4,6-linked beta-D-mannose, and terminal alpha-D-galactose. It was composed of pentasaccharide repeating-units, the chemical structure of which is proposed below.

Lignin extraction from straw by ionic liquids and enzymatic hydrolysis of the cellulosic residues.

Lignocellulose is a promising starting material for bioproducts, ranging from biofuels to specialty chemicals; however, lignocellulose is resistant to enzymatic degradation. Overcoming this resistance is therefore an important priority for the development of the lignocellulosic biorefinery concept. In this work, 1-ethyl-3-methylimidazolium acetate ([emim]Ac) was selected from six ionic liquid candidates for the extraction of lignin from triticale and wheat straw and flax shives. Lignin extractability, composition, and cellulose enzymatic digestibility of the residues after extraction by [emim]Ac were determined at various temperatures (70-150 degrees C) and time intervals (0.5-24 h). The optimal result (52.7% of acid insoluble lignin in triticale straw) was obtained at 150 degrees C after 90 min, yielding >95% cellulose digestibility of the residue. Little cellulose was extracted, and the extracted lignin was recovered by acid precipitation. Selective extraction of lignin by ionic liquids is a potentially efficient technique for the comprehensive utilization of lignocellulose.

Isolation and characterization of pectin from peel of Citrus tankan.

A pectin was extracted from the peel of Citrus tankan with a yield of 2.75%. The uronic acid content was 80.0%, and the degree of methoxylation was 63.2%. The pectin was composed of D-GalA, D-Gal, L-Ara and L-Rha in the molar ratio of 100:11.3:3.6:2.6. The molecular weight was estimated to be approximately 9.2 x 10(4). The pectin formed a gel by conventional procedures.