Pigment epithelium-derived factor (PEDF) prevents advanced glycation end products (AGEs)-elicited endothelial nitric oxide synthase (eNOS) reduction through its anti-oxidative properties

PEDF is an effective inhibitor of angiogenesis in the eye with neuronal differentiating activity. Here, we show that PEDF prevents the AGEs-elicited eNOS reduction through its anti-oxidative properties. Our present study suggests that PEDF may also play a protective role against atherosclerosis.     

Pigment epithelium-derived factor (PEDF) inhibits angiotensin II-induced smooth muscle cell proliferation through its anti-oxidative properties

Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis, suggesting that loss of this factor may be involved in angiogenic eye disease. Here, we show that PEDF inhibits angiotensin II-induced smooth muscle cell proliferation through its anti-oxidative properties. Our present study suggests that PEDF may play a protective role against atherosclerosis.     

Molecular breeding of a novel herbicide-tolerant rice by gene targeting

We have previously reported the production of a rice cell line tolerant to the acetolactate synthase (ALS)-inhibiting herbicide bispyribac (BS), and demonstrated that the BS-tolerant phenotype was due to a double mutation in the rice ALS gene. We further indicated that while changing either of the two amino acids (W548 L or S627I) individually resulted in a BS-tolerant phenotype, conversion of both amino acids simultaneously conferred increased tolerance to BS. As the BS-tolerant cell line had lost the ability to regenerate during two years of tissue culture selection, we attempted to introduce these two point mutations into the rice ALS gene via gene targeting (GT). Using our highly efficient Agrobacterium-mediated transformation system in rice, we were able to regenerate 66 independent GT rice plants from 1500 calli. Furthermore, two-thirds of these plants harbored the two point mutations exclusively, without any insertion of foreign DNA such as border sequences of T-DNA. The GT plants obtained in the present study are therefore equivalent to non-GM herbicide-tolerant rice plants generated by conventional breeding approaches that depend on spontaneous mutations. Surprisingly, GT rice homozygous for the modified ALS locus showed hyper-tolerance to BS when compared to BS-tolerant plants produced by a conventional transgenic system; ALS enzymatic activity in plants homozygous for the mutated ALS gene was inhibited only by extremely high concentrations of BS. These results indicate that our GT method has successfully created novel herbicide-tolerant rice plants that are superior to those produced by conventional mutation breeding protocols or transgenic technology.     

Novel linear polymers bearing thiosialosides as pendant-type epitopes for influenza neuraminidase inhibitors

A conventional synthesis of alpha-thioglycoside of sialic acid as a glycomonomer was accomplished. Radical copolymerization of the glycomonomer with vinyl acetate proceeded smoothly to afford a new class of glycopolymers having thiosialoside residues, in which all protection was removed by a combination of transesterification and saponification to provide a water-soluble thiosialoside cluster. The results of a preliminary study on biological responses against influenza virus neuraminidases using the thiosialoside polymer as a candidate for a neuraminidase inhibitor showed that the glycopolymer has potent inhibitory activity against the neuraminidases.     

Regulation of rice NADPH oxidase by binding of Rac GTPase to its N-terminal extension

Reactive oxygen species (ROS) produced by NADPH oxidase play critical roles in various cellular activities, including plant innate immunity response. In contrast with the large multiprotein NADPH oxidase complex of phagocytes, in plants, only the homologs of the catalytic subunit gp91phox and the cytosolic regulator small GTPase Rac are found. Plant homologs of the gp91phox subunit are known as Rboh (for respiratory burst oxidase homolog). Although numerous Rboh have been isolated in plants, the regulation of enzymatic activity remains unknown. All rboh genes identified to date possess a conserved N-terminal extension that contains two Ca2+ binding EF-hand motifs. Previously, we ascertained that a small GTPase Rac (Os Rac1) enhanced pathogen-associated molecular pattern-induced ROS production and resistance to pathogens in rice (Oryza sativa). In this study, using yeast two-hybrid assay, we found that interaction between Rac GTPases and the N-terminal extension is ubiquitous and that a substantial part of the N-terminal region of Rboh, including the two EF-hand motifs, is required for the interaction. The direct Rac-Rboh interaction was supported by further studies using in vitro pull-down assay, a nuclear magnetic resonance titration experiment, and in vivo fluorescence resonance energy transfer (FRET) microscopy. The FRET analysis also suggests that cytosolic Ca2+ concentration may regulate Rac-Rboh interaction in a dynamic manner. Furthermore, transient coexpression of Os Rac1 and rbohB enhanced ROS production in Nicotiana benthamiana, suggesting that direct Rac-Rboh interaction may activate NADPH oxidase activity in plants. Taken together, the results suggest that cytosolic Ca2+ concentration may modulate NADPH oxidase activity by regulating the interaction between Rac GTPase and Rboh.     

Purification and characterization of the Thermus thermophilus HB8 RecX protein

The RecA protein plays a central role in homologous recombination by promoting strand exchange between ssDNA and homologous dsDNA. Since RecA alone can advance this reaction in vitro, it is widely used in gene manipulation techniques. The RecX protein downregulates the function of RecA, indicating that it could be used as an inhibitor to control the activities of RecA in vitro. In this study, the RecX protein of the hyper-thermophilic bacterium Thermus thermophilus (ttRecX) was over-expressed in Escherichia coli and purified by heat treatment and several column chromatography steps. Size-exclusion chromatography indicated that purified ttRecX exists as a monomer in solution. Circular dichroism measurements indicated that the alpha-helical content of ttRecX is 54% and that it is stable up to 80 degrees C at neutral pH. In addition, ttRecX inhibited the DNA-dependent ATPase activity of the T. thermophilus RecA protein (ttRecA). The stable ttRecX may be applicable for variety of techniques using the ttRecA reaction.     

Molecular cloning and histological localization of LH-like substances in a bottlenose dolphin (Tursiops truncatus) placenta

All mammals exhibit pituitary-specific expression of LH and FSH, whereas placental expression of gonadotropins has been reported only in primates and equids. Some cetaceans, such as dolphins, have a long gestational period and a sexual cycle of about 27 days almost comparable with that of humans. Histologically, dolphins have an epitheliochorial placentae that resembles placentas of Perissodactyla including horses. In the present study, we cloned cDNAs encoding gonadotropins and observed their immunohistochemical localization in the placenta of bottlenose dolphin. The cDNAs obtained encoded 120 amino acids for the alpha-subunit (including 96 amino acids of mature proteins), and 141 amino acids for the beta-subunit (including 121 amino acids of mature proteins). The sequence of the alpha-subunit was similar to that in the pig (Artiodactyla) pituitary glycoprotein hormone [96.7% homology at amino acids (aa) level], and the sequence of the beta-subunit was similar to that of luteinizing hormone (LH) in the pig [94.3% homology at aa level] and white rhinoceros (Perissodactyla) [93.3% homology at aa level]. Of interest, dolphin LHbeta lacks carboxyl-terminal-peptides (CTP). This fact suggests that CTP are not essential for placental expression of gonadotropin in dolphins. Immunohistochemical observations employing anti-ovine LHbeta antibody revealed positive staining in the villositycal tissue. Our observations suggest placental expression of gonadotropin homologues in cetaceans and possible evolutionary conservation of placentae-derived hormonal control of ovarian functions during pregnancy.     

Development of enzyme-linked immunosorbent assay for prostaglandin D2 using the stable isosteric analogue as a hapten mimic and its application

For the determination of prostaglandin (PG) D(2) produced by cultured cells in response to external stimuli, immunological methods would be convenient and useful. However, PGD(2) is unstable under the physiological conditions, so that it has been difficult to get a specific antibody for the parent PGD(2). In an attempt to get a specific antibody for PGD(2), we tried to prepare monoclonal antibodies for 11-deoxy-11-methylene-PGD(2), a novel, chemically stable, isosteric analogue of PGD(2). We successfully cloned a hybridoma cell line secreting a monoclonal antibody reacting specifically with the parent PGD(2). To develop the enzyme-linked immunosorbent assay (ELISA) for PGD(2), the immobilized antigen using the stable PGD(2) derivative was immunoreacted in a competitive manner with the monoclonal antibody in presence of free PGD(2). The optimization of the assay provided a sensitive calibration curve for PGD(2) from 0.32 pg to 0.18 ng with a value of 7.6 pg at 50% displacement. PGD(2) was almost stable during the ELISA condition. The developed assay method was useful for applying to the direct determination of PGD(2) in the culture medium of mouse 3T3-L1 adipocytes. The incubation of PGD(2) in the maturation medium of adipocytes at 37 degrees C caused the chemical conversion into PGJ(2) derivatives. The conversion became more evident after 6 h of the incubation. These findings indicate the importance of considering the optimal time for collecting the samples to be determined for PGD(2) before the conversion starts to occur.     

Staphylococcal α-hemolysin does not induce cell damage in murine mast cells but it augments the degranulation induced by FcεRI cross-linking and ionomycin

Staphylococcal α-hemolysin (Hla) is a principal small β-barrel pore forming toxin. It targets a variety of mammalian cells including immune cells; however little is known about its effects on mast cells. In this study, we examined whether Hla affects the degranulation of mast cells. Although Hla bound to the surface of bone marrow-derived mast cells (BMMCs) and formed SDS-stable oligomers on the cells, Hla alone induced neither cytotoxicity nor obvious release of a granule enzyme, β-hexosaminidase. However, Hla more than doubled the releases of β-hexosaminidase from BMMCs induced by FcεRI cross-linking or treatment with ionomycin. The augmentation of the enzyme release by rHla was impaired in the presence of 130?mM of extracellular KCl. The mutants of Hla that lacked pore-formation did not augment the release of the enzyme. These findings demonstrate that Hla is able to enhance the degranulation of mast cells induced by FcεRI cross-linking and ionomycin, although it alone does not induce the degranulation, and the pore-formation of Hla followed by potassium efflux is involved in the augmentation. These findings propose a previously unrecognized role for Hla in S. aureus-associated allergic and inflammatory processes via augmentation of mast cell responses.