N-terminal vacuolar sorting signal at the mouse antibody alters the N-linked glycosylation pattern in suspension-cultured tobacco BY2 cells

Recombinant DNA technology enables the use of plants as the host for the production of pharmaceutical proteins, such as antibodies. The glycosylation of recombinant proteins plays physiological and biological roles. However, because glycosylation in plants is different from that in human cells, the development of glycoengineering is required. In plant cells, glycan structures are shown to correlate with the localization of the recombinant protein produced. In this study, the vacuolar sorting signal (VSS) of sporamin was fused to the heavy (H) and light (L) chains of a mouse monoclonal antibody (mAb), and the mAb was produced in suspension-cultured tobacco BY2 cells. The sugar chain structures were determined by high-performance liquid chromatography, exoglycosidase digestion, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Typical plant glycans with α1,3-fucosylation and/or β1,2-xylosylation derived from mAb with the VSS-fused H-chain (mIgG1000) and mAb with the VSS-fused H- and L-chain (mIgG1010) occupied the large amount of the total N-glycans, 72.1% and 85.0%, respectively, such as those derived from mAb without VSS (mIgG0000), 74.6% (Fujiyama et al., J. Biosci. Bioeng., 101, 212-218, 2006). In contrast, the typical plant glycan structure Man?FucXylGlcNAc? particularly in vacuoles accounted for 37.8% of the total sugar chains derived from mIgG1000 and 58.5% of those derived from mIgG1010 compared with 24.3% of those derived from mIgG0000. These results suggest that the sporamin signal peptide fused to mAb acts as a VSS and leads to the increase in the amount of Man?FucXylGlcNAc?, which is the main N-glycan structure in vacuoles.     

Probabilistic models of combinations of stochastic loads for limit state design

When more than one load act on a structure, the maximum intensity of the combined load effect needs to be evaluated. Turkstra's rule is often used in reliability-based limit state design because of its simplicity; however, the approximation is unconservative. This paper presents the theoretical expressions of the cumulative distribution function (cdf) of the maximum intensity during a reference period taking the temporal variation of load intensity into account. Theoretical investigation and numerical examples show that the theoretical expression provides a fairly good estimate regardless of the statistical characteristics of the load processes. To avoid considering time-dependency in determining a design point, it is proposed to consider the intensities of the load processes at the time when the combination takes its maximum value. Based on the observation that the cross section of the joint probability density function (pdf) of these intensities cut by the limit state surface is similar to that of the joint pdf of the basic random variables, the design point is determined in the basic variable space using AFOSM.     

Effect of FGF-2 and melatonin on implant bone healing: a histomorphometric study

Melatonin influences the release of growth hormone and cortisol in humans, and it was recently reported that it promoted bone formation. On the other hand, fibroblast growth factor-2 (FGF-2) was reported to facilitate the proliferation of osteoblasts. In the present study, we examined the effect of recombinant human FGF-2 and melatonin on the promotion of osteogenesis around titanium implants. Twenty-four 10-week-old female rats of the Wistar strain received titanium implants in both tibiae. In the experimental groups, 100 mg/kg body weight of melatonin was administered by intraperitoneal injection for 4 weeks after implantation and 10 microg of FGF-2 was locally injected around the implant sites 5 days after implantation. The control groups were administered saline only. In the control group, few newly formed bone could be seen around the implants. It was observed to be in direct contact with the implant surface, but otherwise unmineralized connective tissue was occasionally interposed. In the experimental group, newly formed bone was observed around the titanium implant. In addition, in contrast to the control group, abundant bone trabeculae were seen in the medullary canal region. Bone trabeculae were directly connected to existing cortical bone. These results strongly suggested that melatonin and FGF-2 have the potential to promote osseointegration.     

Effects of human hair and nail proteins and their films on rat mast cells

Human hair and nail are valuable materials for producing individual corresponding biocompatible materials. A rapid and convenient protein extraction method (Shindai method) and novel procedures for preparing their protein films from their extracts have been developed using human hair and nail. The effects of the human hair and nail proteins and their films on histamine release from rat peritoneal mast cells were investigated. Both protein solutions and their films, mainly consisting of keratins and matrix proteins, did not induce histamine release from the mast cells. Scanning electron microscopy (SEM) also showed that the mast cells were only slightly affected by adding the human hair and nail proteins or by incubating on their protein films. The IgE-dependent histamine release was inhibited by the hair and nail proteins and their films. Incubation of the mast cells with the hair and nail proteins prior to the addition of the IgE serum resulted in a high inhibition (50%) of the histamine release, while the inhibition was approximately 10% when the protein solutions were mixed with the mast cells after incubation with the IgE serum. These results suggest that the human hair and nail proteins and their films will be useful materials for antiallergic actions.     

In Planta production of immunogenic poliovirus peptide using tobacco mosaic virus-based vector system

The tobacco mosaic virus (TMV) provides an attractive means of producing foreign peptides in plants. In this study, a TMV-based vector was designed such that a fragment encoding 15 amino acids of the poliovirus peptide (PVP) derived from the viral capsid proteins VP3 and VP1 of poliovirus type 1 Sabin was inserted downstream of the six-base 3' context nucleotide sequence of the TMV coat protein (CP) gene. This design allowed readthrough at the amber stop codon, thereby producing the chimeric TMV particle with both intact CP and CP-fusion protein (CP-PVP) in Nicotiana tabacum cv. Samsun infected with the TMV vector. The TMVCP-PVP virus particle induced antibodies against PVP as well as TMVCP in mice after intraperitoneal immunization. These data illustrate the potential of the readthrough translation system with TMVCP for antigen presentation and vaccine production.