Solution structures and DNA binding properties of the N-terminal SAP domains of SUMO E3 ligases from Saccharomyces cerevisiae and Oryza sativa

SUMO E3 ligase of the Siz/PIAS family that promotes sumoylation of target proteins contains SAP motif in its N-terminal region. The SAP motif with a consensus sequence of 35 residues was first proposed to be as a new DNA binding motif found in diverse nuclear proteins involved in chromosomal organization. We have determined solution structures of the SAP domains of SUMO ligases Siz1 from yeast and rice by NMR spectroscopy, showing that the structure of the SAP domain (residues 2-105) of rice Siz1 is a four-helix bundle with an up-down-extended loop-down-up topology, whereas the SAP domain (residues 1-111) of yeast Siz1 is comprised of five helices where the fifth helix alpha5 causes a significant change in the alignment of the four-helix bundle characteristic to the SAP domains of the Siz/PIAS family. We have also demonstrated that both SAP domains have binding ability to an A/T-rich DNA, but that binding affinity of yeast Siz1 SAP is at least by an order of magnitude higher than that of rice Siz1 SAP. Our NMR titration experiments clearly showed that yeast Siz1 SAP uses alpha2-helix for DNA binding more effectively than rice Siz1 SAP, which would result from the dislocation of this helix due to the existence of the extra helix alpha5. In addition, based on the structures of the SAP domains determined here and registered in Protein Data Bank, general features of structures of the SAP domains are discussed in conjunction with equivocal nature of their DNA binding.     

Effect of a conjugated quercetin metabolite, quercetin 3-glucuronide, on lipid hydroperoxide-dependent formation of reactive oxygen species in differentiated PC-12 cells

To assess the efficacy of conjugated quercetin metabolites as attenuators for oxidative stress in the central nervous system, we measured the 13-hydroperoxyoctadecadienoic acid (13-HPODE)-dependent formation of reactive oxygen species (ROS) in pheochromocytoma PC-12 cells in the presence of quercetin 3-O-β-glucuronide (Q3GA) and related compounds. A 2',7'-dichlorofluorescin (DCFH) assay showed that Q3GA significantly suppressed the formation of ROS, when it was coincubated with 13-HPODE (coincubation system). However, it was less effective than quercetin aglycon in the concentration range from 0.5 to 10 μM. In an experiment in which the cells were incubated with the test compounds for 24 h before being exposed to 13-HPODE, Q3GA was also effective in suppressing the formation of ROS in spite that little Q3GA was taken up into the cells. These results suggest that antioxidative metabolites of quercetin are capable of protecting nerve cells from attack of lipid hydroperoxides.     

The kanamycin resistance transposon Tn2680 derived from the R plasmid Rts1 and carried by phage P1Km has flanking 0.8-kb-long direct repeats

Phage P1Km carries within the invertible DNA segment a 5-kb insertion with 0.8-kb terminal direct repeats flanking the kanamycin resistance determinant. The same structure was also found on the R plasmid Rts1, from which the Km resistance segment of P1Km was derived. Obviously, this Km resistance segment translocated as a unit to the P1 genome and it is therefore called Tn2680. Loss of the Km resistance determinant due to recombination between the flanking direct repeats occurs during vegetative growth of P1Km. Amplification of Tn2680 to tandem oligomers is documented and is thought to result from recombination between the flanking direct repeats. The flanking 0.8-kb repeats are different from known IS elements.     

Localization of lipocaline-type prostaglandin D synthase in rat brain: immunoelectron microscopic study

The distribution of lipocaline-type prostaglandin D synthase (L-PGDS) in rat brain was investigated by immunoelectron microscopy using a protein A-gold technique. In perivascular cells adjacent to the basement membrane of arterioles in the pia-arachnoid and of blood vessels in the subpial cortex, gold labeling was confined to the lumen of the dilated rough endoplasmic reticulum, and not found in the few lysosomes present in the cytoplasm. The results suggest that the perivascular cells secrete L-PGDS and seem not to degrade lipophilic molecules carried by L-PGDS. Moreover, gold particles representing the antigenic sites of L-PGDS were found in the Golgi apparatus, rough endoplasmic reticulum, vesicles, and nuclear envelope of arachnoid trabecular cells, arachnoid barrier cells, and arachnoid pia mater cells. The labeling was less detectable in the same organelles of choroid plexus epithelial cells, compared with leptomeningeal cells. In meningeal macrophages and parenchymal microglia, L-PGDS was detected in lysosomes, multivesicular bodies, and endocytic vesicles. The production of L-PGDS in perivascular cells is important to the various functions of this enzyme in brain parenchyma.     

Effect of quercetin and its conjugated metabolite on the hydrogen peroxide-induced intracellular production of reactive oxygen species in mouse fibroblasts

To clarify the antioxidative role of quercetin metabolites in cellular oxidative stress, we measured the inhibitory effects of the quercetin aglycon and quercetin 3-O-beta-D-glucuronide (Q3GA), which is one of the quercetin metabolites in the blood after an intake of quercetin-rich food, on the production of hydrogen peroxide (H2O2)-induced intracellular reactive oxygen species in mouse fibroblast 3T3 cultured cells. When the cells were exposed to H2O2 in the presence of quercetin or Q3GA, Q3GA was found to be less effective than quercetin. In the case of a pretreatment with quercetin or Q3GA before the exposure, Q3GA, but not the quercetin aglycon, exerted an inhibitory effect, although its cellular uptake was unlikely. The quercetin aglycon appeared to fail in its antioxidative effect due to metabolic conversion into isorhamnetin conjugates, with substantial oxidative degradation resulting from the pretreatment. It is, therefore, suggested that quercetin metabolites take part in the protection of intracellular oxidative stress induced by the extraneous attack of H2O2.     

Transport of maternal transthyretin to the fetus in the viviparous teleost Neoditrema ransonnetii (Perciformes,Embiotocidae)

Journal of Comparative Physiology B - The molecular basis of viviparity in non-mammalian species has not been widely studied. Neoditrema ransonnetii, a surfperch, is a matrotrophic teleost whose...