Systematic approaches to using the FOX hunting system to identify useful rice genes

Ectopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23 000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis . This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.     

High‐throughput screening of optimal solution conditions for structural biological studies by fluorescence correlation spectroscopy

Protein aggregation is an essential molecular event in a wide variety of biological situations, and is a causal factor in several degenerative diseases. The aggregation of proteins also frequently hampers structural biological analyses, such as solution NMR studies. Therefore, precise detection and characterization of protein aggregation are of crucial importance for various research fields. In this study, we demonstrate that fluorescence correlation spectroscopy (FCS) using a single‐molecule fluorescence detection system enables the detection of otherwise invisible aggregation of proteins at higher protein concentrations, which are suitable for structural biological experiments, and consumes relatively small amounts of protein over a short measurement time. Furthermore, utilizing FCS, we established a method for high‐throughput screening of protein aggregation and optimal solution conditions for structural biological experiments.     

Polyamine compound deoxyspergualin inhibits heat shock protein-induced activation of immature dendritic cells

Polyamine compound deoxyspergualin (DSG) is a potent immunosuppressive agent that has been applied clinically for protecting graft rejection and treatment of Wegener's granulomatosis. Though DSG can bind to heat-shock proteins (HSPs) in cells, its mechanism of immunosuppressive action remains unknown. It is widely accepted that extracellular HSPs are capable of stimulating dendritic cells (DC) through cell surface receptors, leading to DC activation and cytokine release. In this study, we examined if DSG analogs could inhibit HSP70-induced DC activation. Bone marrow derived immature mouse DCs and peripheral blood mononuclear cell-derived immature human DCs were generated and incubated with Alexa 488-labeled Hsp70 in the presence of methoxyDSG (Gus-1) that had comparable HSP70-binding affinity to DSG or DSG analog GUS-7, which had much more reduced binding affinity for HSP70. The binding of HSP70 to immature DCs was analyzed by laser microscopy and flow cytometry. HSP70-induced DC activation was assessed by TNF-α release by enzyme-linked immunosorbent assay. Binding of Hsp70 to the cell surface of immature DCs was inhibited under the presence of Gus-1, but not under the presence of Gus-7. Immature DCs were activated and released TNF-α by the stimulation with HSP70 for 12 hours; however, the HSP70-induced TNF-α release was suppressed under the presence of Gus-1, and partially suppressed under the presence of Gus-7. Similar results were observed when immature human DCs were stimulated under the same conditions. Immunosuppressive mechanism of DSG may be explained, at least in part, by the inhibition of extracellular HSP70-DC interaction and HSP70-induced activation of immature DCs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Alpha-synuclein overexpression and aggregation exacerbates impairment of mitochondrial functions by augmenting oxidative stress in human neuroblastoma cells

Overexpression of alpha-synuclein and oxidative stress has been implicated in the neuronal cell death in Parkinson's disease. Alpha-synuclein associates with mitochondria and excessive accumulation of alpha-synuclein causes impairment of mitochondrial functions. However, the mechanism of mitochondrial impairment caused by alpha-synuclein is not fully understood. We recently reported that alpha-synuclein associates with mitochondria and that overexpression of alpha-synuclein causes nitration of mitochondrial proteins and release of cytochrome c from the mitochondria [Parihar M.S., Parihar A., Fujita M., Hashimoto M., Ghafourifar P. Mitochondrial association of alpha-synuclein causes oxidative stress. Cell Mol Life Sci. 2008a;65:1272–1284]. The present study shows that overexpression of alpha-synuclein A53T or A30P mutants or wild-type in human neuroblastoma cells augmented aggregation of alpha-synuclein. Immunoblotting and immuno-gold electron transmission microscopy show localization of alpha-synuclein aggregates within the mitochondria of overexpressing cells. Overexpressing cells show increased mitochondrial reactive oxygen species, increased protein tyrosine nitration, decreased mitochondrial transmembrane potential, and hampered cellular respiration. These findings suggest an important role for mitochondria in cellular responses to alpha-synuclein.     

Segregation of GM1 and GM3 clusters in the cell membrane depends on the intact actin cytoskeleton

Gangliosides have been implicated in exerting multiple physiological functions, and it is important to understand how their distribution is regulated in the cell membrane. By using freeze-fracture immunolabeling electron microscopy, we showed that GM1 and GM3 make independent clusters that are significantly reduced by cholesterol depletion. In the present study, we examined the effects of actin depolymerization/polymerization and Src-family kinase inhibition on the GM1 and GM3 clusters. Both GM1 and GM3 clustering was reduced when the actin cytoskeleton was perturbed by latrunculin A or jasplakinolide, but the decrease was less significant than that induced by cholesterol depletion. On the other hand, inhibition of Src-family kinases decreased GM3 clustering more drastically than did cholesterol depletion, whereas its effect on GM1 clustering was less significant. GM1 and GM3 were segregated from each other in unperturbed cells, but co-clustering increased significantly after actin depolymerization. Our results indicate that the GM1 and GM3 clusters in the cell membrane are regulated in different ways and that segregation of the two gangliosides depends on the intact actin cytoskeleton.     

The expense for one implantation of a banked bone allograft from a cadaveric donor and the issues affecting current advanced medical treatment in the Japanese orthopaedic field

Demand for banked bone allografts is increasing in Japan; however, there are too few bone banks and the bone bank network is not well-established. One reason for this was lack of funding for banks. Bone banks had to bear all material expenses of banked bone allografts themselves because this was not designated a covered expense. In December 2004, the Japanese government started a new “Advanced Medical Treatment” administration system which allowed an approved institution to charge the expense of authorized advanced medical treatments directly to patients. The treatment named “Cryopreserved allogenic bone and ligamentous tissue retrieved from cadaveric donor” was approved as an advanced medical treatment in March 2007. We present the calculation method and the expense per implantation of a banked bone allograft from a cadaveric donor under this treatment and raise issues which affect this advanced medical treatment and remain to be resolved in the Japanese orthopaedic field.