Characterization of three different single chain antibodies recognizing non-reducing terminal mannose residues expressed in Escherichia coli by an inducible T7 expression system

We previously isolated phage antibodies from a phage library displaying human single chain antibodies (scFvs) by screening with a mannotriose (Man3)-bearing lipid. Of four independent scFv genes originally characterized, 5A3 gene products were purified as fusion proteins such as a scFv-human IgG1 Fc form, but stable clones secreting 1A4 and 1G4 scFv-Fc proteins had never been established. Thus, bacterial expression systems were used to purify 1A4 and 1G4 scFv gene products as soluble forms. Purification of 1A4 and 1G4 scFv proteins from inclusion bodies was also carried out together with purification of 5A3 scFv protein in order to compare their Man3-binding abilities. The present studies demonstrated that 1A4 and 1G4 scFv proteins have a higher affinity for Man3 than 5A3 scFv protein, which may determine whether scFv-Fc proteins expressed in mammalian cells are retained in the ER or secreted. Furthermore, the inhibitory effects of anti-Man3 1G4 scFv and anti-Tn antigen scFv proteins on MCF-7 cell growth were evaluated. Despite the fact that no obvious difference was detected in cell growth, microscopic observations revealed inhibition of foci formation in cells grown in the presence of the anti-carbohydrate scFv proteins. This finding provides a basis for the development of cancer therapeutics.     

Immunocytochemistry for bestatin and its application to drug accumulation studies in rat intestine and kidney

The in vivo role of transporters in drug disposition, in the context of other transporters, and metabolism has not been established. We prepared an anti-bestatin serum against bestatin conjugated to albumin with glutaraldehyde (GA). The antiserum was specific for GA-conjugated bestatin and weakly reacted with free bestatin, but no reaction occurred with structurally unrelated compounds according to both the inhibition and binding ELISAs. The antiserum allowed us to develop an immunocytochemical (ICC) method for detecting the uptake of bestatin in the rat intestine and kidney. Three hours after a single oral administration of bestatin, the ICC method revealed that the drug distributed in the microvilli, cytoplasm and nuclei of the absorptive epithelial cells at much larger amounts than in all other cell types in the small intestine. However, no drug was detected in the mucin goblets in the epithelium. In the kidney, the drug distributed to a greater extent in the S3 segment than in the S1 and S2 segments of the proximal tubule, and also was detected in the microvilli of the proximal tubule cells (S1, S2 and S3). These findings that bestatin accumulated in large amounts, especially in the cells and/or at the sites where the transporters PEPT1 and PEPT2 occur, corresponded well to those observed with β-lactam amoxicillin in the previous ICC studies. Thus, this may indicate a possibility that both the transporters might be involved, at least in part, in the distribution of bestatin in the small intestine and kidney under the conditions examined.     

The endocytic pathway acts downstream of Oskar in Drosophila germ plasm assembly

Cell fate is often determined by the intracellular localization of RNAs and proteins. In Drosophila oocytes, oskar (osk) RNA localization and the subsequent Osk synthesis at the posterior pole direct the assembly of the pole plasm, where factors for the germline and abdomen formation accumulate. osk RNA produces two isoforms, long and short Osk, which have distinct functions in pole plasm assembly. Short Osk recruits downstream components of the pole plasm, whose anchoring to the posterior cortex requires long Osk. The anchoring of pole plasm components also requires actin cytoskeleton, and Osk promotes long F-actin projections in the oocyte posterior cytoplasm. However, the mechanism by which Osk mediates F-actin reorganization remains elusive. Furthermore, although long Osk is known to associate with endosomes under immuno-electron microscopy, it was not known whether this association is functionally significant. Here we show that Rabenosyn-5 (Rbsn-5), a Rab5 effector protein required for the early endocytic pathway, is crucial for pole plasm assembly. rbsn-5(-) oocytes fail to maintain microtubule polarity, which secondarily disrupts osk RNA localization. Nevertheless, anteriorly misexpressed Osk, particularly long Osk, recruits endosomal proteins, including Rbsn-5, and stimulates endocytosis. In oocytes lacking rbsn-5, the ectopic Osk induces aberrant F-actin aggregates, which diffuse into the cytoplasm along with pole plasm components. We propose that Osk stimulates endosomal cycling, which in turn promotes F-actin reorganization to anchor the pole plasm components to the oocyte cortex.     

Environmental risk assessment of transgenic miraculin-accumulating tomato in a confined field trial in Japan

The commercial use of genetically modified (GM) crops requires prior assessment of the risks to the environment when these crops are grown in the field or distributed. Assessments protocols vary across countries and GM crop events, but there is a common need to assess environmental biosafety. In this study, we conducted an environmental risk assessment in a confined field of GM tomato plants that can produce miraculin, a taste-altering protein that causes sour tastes to be perceived as sweet, for practical use in Japan. The evaluation was conducted for 1) competitiveness (the ability to compete with wild plants for nutrients, sunlight, and growing areas and prevent their growth) and 2) the production of toxic substances (the ability to produce substances that interfere with the habitat and growth of wild plants, animals, and microorganisms). Investigations of plant morphology and growth characteristics as well as tolerance to low temperature during early growth and overwintering for assessment endpoints related to competitiveness showed no biologically meaningful difference between GM tomato and non-GM tomato. In addition, harmful substances in plant residues and root secretions were assessed by the plow-in method, succeeding crop test and soil microflora tests, and it was determined that GM tomato does not exhibit an increase in harmful substances. Based on these results, it was concluded that GM miraculin-accumulating tomato is comparable to conventional tomato and is unlikely to have unintended adverse effects in the natural environment of Japan.     

ARHGAP4-SEPT2-SEPT9 complex enables both up- and down-modulation of integrin-mediated focal adhesions,cell migration,and invasion

The Rho family of GTPases are inactivated in a cell context–dependent manner by Rho-GTPase-activating proteins (Rho-GAPs), but their signaling mechanisms are poorly understood. Here we demonstrate that ARHGAP4, one of the Rho-GAPs, forms a complex with SEPT2 and SEPT9 via its Rho-GAP domain and SH3 domain to enable both up- and down-modulation of integrin-mediated focal adhesions (FAs). We show that silencing ARHGAP4 and overexpressing its two mutually independent upstream regulators, SEPT2 and SEPT9, all induce reorganization of FAs to newly express Integrin Beta 1 and also enhance both cell migration and invasion. Interestingly, even if these cell migration/invasion–associated phenotypic changes are induced upon perturbations to the complex, it does not necessarily cause enhanced clustering of FAs. Instead, its extent depends on whether the microenvironment contains ligands suitable for the up-regulated Integrin Beta 1. These results provide novel insights into cell migration, invasion, and microenvironment-dependent phenotypic changes regulated by the newly identified complex.     

Analysis of senescence-responsive stress fiber proteome reveals reorganization of stress fibers mediated by elongation factor eEF2 in HFF-1 cells

Stress fibers (SFs), which are actomyosin structures, reorganize in response to various cues to maintain cellular homeostasis. Currently, the protein components of SFs are only partially identified, limiting our understanding of their responses. Here we isolate SFs from human fibroblasts HFF-1 to determine with proteomic analysis the whole protein components and how they change with replicative senescence (RS), a state where cells decline in the ability to replicate after repeated divisions. We found that at least 135 proteins are associated with SFs, and 63 of them are up-regulated with RS, by which SFs become larger in size. Among them, we focused on eEF2 (eukaryotic translation elongation factor 2) as it exhibited on RS the most significant increase in abundance. We show that eEF2 is critical to the reorganization and stabilization of SFs in senescent fibroblasts. Our findings provide a novel molecular basis for SFs to be reinforced to resist cellular senescence.     

Intraspecific independent evolution of floral spur length in response to local flower visitor size in Japanese Aquilegia in different mountain regions

Geographic differences in floral traits may reflect geographic differences in effective pollinator assemblages. Independent local adaptation to pollinator assemblages in multiple regions would be expected to cause parallel floral trait evolution, although sufficient evidence for this is still lacking. Knowing the intraspecific evolutionary history of floral traits will reveal events that occur in the early stages of trait diversification. In this study, we investigated the relationship between flower spur length and pollinator size in 16 populations of Aquilegia buergeriana var. buergeriana distributed in four mountain regions in the Japanese Alps. We also examined the genetic relationship between yellow‐ and red‐flowered individuals, to see if color differences caused genetic differentiation by pollinator isolation. Genetic relationships among 16 populations were analyzed based on genome‐wide single‐nucleotide polymorphisms. Even among populations within the same mountain region, pollinator size varied widely, and the average spur length of A. buergeriana var. buergeriana in each population was strongly related to the average visitor size of that population. Genetic relatedness between populations was not related to the similarity of spur length between populations; rather, it was related to the geographic proximity of populations in each mountain region. Our results indicate that spur length in each population evolved independently of the population genetic structure but in parallel in response to local flower visitor size in different mountain regions. Further, yellow‐ and red‐flowered individuals of A. buergeriana var. buergeriana were not genetically differentiated. Unlike other Aquilegia species in Europe and America visited by hummingbirds and hawkmoths, the Japanese Aquilegia species is consistently visited by bumblebees. As a result, genetic isolation by flower color may not have occurred.     

Development of motorized plasma lithography for cell patterning

The micropatterning of cells, which restricts the adhesive regions on the substrate and thus controls cell geometry, is used to study mechanobiology-related cell functions. Plasma lithography is a means of providing such patterns and uses a spatially-selective plasma treatment. Conventional plasma lithography employs a positionally-fixed mask with which the geometry of the patterns is determined and thus is not suited for producing on-demand geometries of patterns. To overcome this, we have manufactured a new device with a motorized mask mounted in a vacuum chamber of a plasma generator, which we designate motorized plasma lithography. Our pilot tests indicate that various pattern geometries can be obtained with the control of a shielding mask during plasma treatment. Our approach can thus omit the laborious process of preparing photolithographically microfabricated masks required for the conventional plasma lithography.