Growth and Development, and Auxin Polar Transport in Higher Plants under Microgravity Conditions in Space: BRIC-AUX on STS-95 Space Experiment

Received 13 September 1999/ Accepted in revised form 12 October 1999     

Dissociation-independent selection of high-affinity anti-hapten phage antibodies using cleavable biotin-conjugated haptens

The engineering of hapten-specific antibodies with affinity constant higher (K(a) values >10(10)M(-1)) than those of conventional antibodies promises hapten immunoassays exhibiting sub-femtomole range sensitivity, based on the conventional competitive assay principle. Here we report a simple method to select phage particles displaying anti-hapten antibody fragments with exceptionally high affinity. 11-Deoxycortisol (11-DC), selected as a model target hapten, was covalently conjugated to biotin via a spacer that included a reductively cleavable disulfide bond. Phage particles displaying high-affinity, single-chain Fv fragments (scFvs) specific for 11-DC (K(a)1.3 x 10(10)M(-1)) were incubated with the "cleavable biotin"-conjugated 11-DC, and the resulting complexes was captured on immobilized NeutrAvidin. Mild reductive conditions that did not decrease phage infectivity easily cleaved the disulfide bond, allowing the recovery of target phage particles; this process is fully independent of the dissociation of the antigen-antibody interaction. Five serial rounds of selection enabled the isolation and enrichment of the anti-11-DC phage (specific phage ratio >90%) from among a 100,000-fold excess of nonspecific phage particles. This method will be applicable for selection of extra-high-affinity phage antibodies against a wide variety of haptens.     

Production of long-chain levan by a sacC insertional mutant from Bacillus subtilis 327UH

A hyper extracellular protein producer, Bacillus subtilis 327UH, produced large amounts of levan in a medium containing 20% sucrose, and the yield of levan after 10 hours was more than 60%, when based on the fructose amount of sucrose. After transformation of 327UH with a levanase-deficient 168SC (sacC::Cm(r)) chromosomal DNA, a Cm(r) transformant 327UHSC (sacC::Cm(r) degSU(Hy)) produced 3 times longer levan than that of the wild type.     

Induced response to herbivory in stinging hair traits of Japanese nettle (Urtica thunbergiana) seedlings in two subpopulations with different browsing pressures by sika deer

Evolutionary theories predict that natural selection favors inducible defense when the risk of predation is unpredictable. In this context, the magnitude of the induced defense in populations experiencing intermittent herbivory is predicted to be larger than that in populations experiencing constant herbivory when there is genetic differentiation between populations. To test this prediction, we conducted a clipping experiment to investigate induced response to shoot damage by the stinging hair traits of Japanese nettle (Urtica thunbergiana) seedlings. For this purpose, we studied two nettle subpopulations, one under constant browsing and another under intermittent browsing by sika deer in Nara Park, central Japan. The clipping experiment demonstrates that both subpopulations exhibited induced defenses in response to the clipping of the shoot apex as the number and length of stinging hairs increased after clipping. The subpopulation experiencing intermittent browsing exhibited smaller trait values and larger induced defenses, indicated by the number of stinging hairs on the upper leaf surface and the length of stinging hairs on both leaf surfaces compared with the subpopulation experiencing constant browsing. These results are consistent with the prediction and suggest that genetic differentiation of the induced defense between subpopulations is caused by adaptation to the herbivory regime. We discuss other plausible factors affecting the magnitude of the induced defense of the nettle subpopulations.     

JAB1 participates in unfolded protein responses by association and dissociation with IRE1

Recent papers have reported that neuronal death in patients with Alzheimer's disease, Parkinson's disease, and cerebral ischemia has its origin in the endoplasmic reticulum (ER). IRE1alpha is one of the ER stress transducers that detect the accumulation of unfolded proteins in the ER. IRE1alpha mediates two major cellular responses, which are the unfolded protein response (UPR), a defensive response, and apoptosis that leads to cell death. However, little is known about the regulatory mechanisms that select between the UPR and apoptosis. We identified Jun activation domain-binding protein-1 (JAB1) as a molecule that interacts with IRE1alpha using a yeast two-hybrid system. We demonstrated that JAB1 binds to IRE1alpha in the absence of stress, but that binding is decreased by ER stress inducers. Moreover, mutant JAB1 down-regulates the UPR signaling pathway through tight binding with IRE1alpha. These results suggested that JAB1 may act as a key molecule in selecting the UPR or cell death by association and dissociation with IRE1alpha.     

Gemcitabine enhances Wilms�� tumor gene WT1 expression and sensitizes human pancreatic cancer cells with WT1-specific T-cell-mediated antitumor immune response

Wilms' tumor gene (WT1), which is expressed in human pancreatic cancer (PC), is a unique tumor antigen recognized by T-cell-mediated antitumor immune response. Gemcitabine (GEM), a standard therapeutic drug for PC, was examined for the regulation of WT1 expression and the sensitizing effect on PC cells with WT1-specific antitumor immune response. Expression of WT1 was examined by quantitative PCR, immunoblot analysis, and confocal microscopy. Antigenic peptide of WT1 presented on HLA class I molecules was detected by mass spectrometry. WT1-specific T-cell receptor gene-transduced human T cells were used as effecter T cells for the analysis of cytotoxic activity. GEM treatment of human MIAPaCa2 PC cells enhanced WT1 mRNA levels, and this increase is associated with nuclear factor kappa B activation. Tumor tissue from GEM-treated MIAPaCa2-bearing SCID mice also showed an increase in WT1 mRNA. Some human PC cell lines other than MIAPaCa2 showed up-regulation of WT1 mRNA levels following GEM treatment. GEM treatment shifted WT1 protein from the nucleus to the cytoplasm, which may promote proteasomal processing of WT1 protein and generation of antigenic peptide. In fact, presentation of HLA-A*2402-restricted antigenic peptide of WT1 (CMTWNQMNL) increased in GEM-treated MIAPaCa2 cells relative to untreated cells. WT1-specific cytotoxic T cells killed MIAPaCa2 cells treated with an optimal dose of GEM more efficiently than untreated MIAPaCa2 cells. GEM enhanced WT1 expression in human PC cells and sensitized PC cells with WT1-specific T-cell-mediated antitumor immune response.     

Identification of the composite parameters of the BBH-B model specifying the effects of biohydrologic processes on the water balance of crop fields

A model of soil hydrology incorporating rainfall interception and macropore flow, which are representative biohydorologic processes, (BBH-B model) has two composite parameters, Π and Φ. These parameters express the ratios of rainfall interception and macropore flow to gross rainfall, respectively. Their values, however, change widely with the vegetation, soil texture and wetness. The results of experiments that have been carried out for various objectives under various conditions by the present authors were reanalyzed to evaluate these two parameters. Further, two supplemental experiments were designed to identify the two parameters. In the experiments, the monthly mean of Π for a cornfield ranged from 0.18 to 0.64 in the summer months, while Φ for a weed-grown field reached a maximum of 0.8 when daily rainfall was more than 40 mm day⁻¹. From these analyses and experiments, we concluded that the effects of biohydrologic processes on the water balance of crop fields are rather large and not negligible.     

A rapid screening method for cell lines producing singly-tagged recombinant proteins using the “TARGET tag” system

Recombinant production of extracellular glycoproteins in stable mammalian cell lines is an ideal technique for obtaining a large quantity of high-quality proteins. In most cases, however, current methodologies do not allow for sufficiently rapid cell line development and protein purification. Here, we describe a 21-residue peptide tag (designated as TARGET tag) and its use for rapid stable cell line development and purification. The ability of the anti-tag antibody P20.1 to withstand repetitive regeneration cycles has enabled the development of a sensitive surface plasmon resonance-based screening format that requires only 20 µl of cell culture supernatants. Direct and semi-quantitative screening at the 96-well culture scale eliminated the need for a second screening, re-cloning, or sorting, thereby minimizing culture pre-production time. Using this system, “high producer” cell lines were established in less than a month, and milligram quantities of target proteins could be purified with a standardized protocol.