Self-assembly DNA-conjugated polymer for detection of single nucleotide polymorphism

We developed a self-assembly DNA-conjugated polymer based on polyacrylic acid (PAA) for DNA chip fabrication. A 20-mer single-stranded DNA (ssDNA, probe-1), and 3-(2-pyridyldithio)propionyl hydrazide (PDPH), for promoting self-assembled immobilization, were both covalently attached to PAA as sidechains. This DNA-conjugated PAA was then spontaneously immobilized on a gold substrate. Probe-1 on the immobilized polymer was hybridized to a 34-mer ssDNA (probe-2), which had the sequence desired for analyzing the target DNA. The fluorescence intensity after incubating the P-1 DNA-conjugated polymer with probe-2 DNA was much higher than with control sequence in the first hybridization. The interactions between target DNA and the DNA-conjugated PAA were investigated by fluorescence measurement. The interaction of fully matched target DNA with this immobilized DNA conjugated polymer has been studied at different ion strength conditions. SNP sequences as targets showed less than 15% the intensity of fully matched target DNA in the second hybridization, indicating that the gold surfaces coated with the DNA-conjugated PAA was highly specific to fully matched DNA. The DNA-conjugated PAA immobilized on a gold substrate is characterized by reduced nonspecific adsorption, due to less electrostatic repulsion as well as the polymer coating. Therefore, DNA-conjugated PAA can be used for probe DNA immobilization method.     

Systematic single base-pair substitution analysis of DNA binding by the cAMP receptor protein in cyanobacterium Synechocystis sp. PCC 6803

The cAMP receptor protein SYCRP1 in cyanobacterium Synechocystis sp. PCC 6803 is a regulatory protein that binds to the consensus DNA sequence (5'-AAATGTGATCTAGATCACATTT-3') for the cAMP receptor protein CRP in Escherichia coli. Here we examined the effects of systematic single base-pair substitutions at positions 4-8 (TGTGA) of the consensus sequence on the specific binding of SYCRP1. The consensus sequence exhibited the highest affinity, and the effects of base-pair substitutions at positions 5 and 7 were the most deleterious. The result is similar to that previously reported for CRP, whereas there were differences between SYCRP1 and CRP in the rank order of affinity for each substitution.     

Slow ADP-dependent acceleration of microtubule translocation produced by an axonemal dynein

Dynein has four nucleotide binding sites, of which the functional significance is unknown except for the single catalytic site. To obtain clues to the function of non-catalytic nucleotide binding, we examined the effect of ADP on the in vitro motility of Chlamydomonas inner-arm dynein species 'a'. Upon continuous perfusion with ATP and ADP, microtubules glided on a dynein-coated glass surface with a velocity that gradually increased over a few minutes. The velocity increased faster at higher ADP concentrations. These results suggest that this dynein is activated by nucleotide binding to regulatory site(s) through an extremely slow process.     

Identification of binding domains in the sodium channel Na(V)1.8 intracellular N-terminal region and annexin II light chain p11

In human brain the Aβ peptide is produced mainly by neurons and the overexpression of amyloid precursor protein (APP) that involves an increase in Aβ secretion, has been observed in some areas of the Alzheimer's disease patients brain. We have generated two stably transfected human neuroblastoma lines which overexpress APP; both of them secreted Aβ and showed morphological changes and cell death with apoptotic program characteristics. Interestingly, coculture experiments with the untransfected human neuroblastoma cell line showed that the Aβ peptide was not responsible for the death in those cell lines; additionally, we indicate that upon cell death, Aβ peptide is secreted into cell medium.     

Molecular cloning and functional analysis of zebrafish neutral ceramidase

Almost all observations on the functions of neutral ceramidase have been carried out at cellular levels but not at an individual level. Here, we report the molecular cloning of zebrafish neutral ceramidase (znCD) and its functional analysis during embryogenesis. We isolated a cDNA clone encoding znCD by 5' and 3' rapid amplification of cDNA ends-PCR. It possessed an open reading frame of 2,229 base pairs encoding 743 amino acids. A possible signal/anchor sequence near the N terminus and four potential O-glycosylation and eight potential N-glycosylation sites were found in the putative sequence. The enzyme activity at neutral pH increased markedly after transformation of Chinese hamster CHOP and zebrafish BRF41 cells with the cDNA. The overexpressed enzyme was found to be distributed in endoplasmic reticulum/Golgi compartments as well as the plasma membranes. The antisense morpholino oligonucleotide (AMO), which was designed based on the sequence of znCD mRNA, successfully blocked the translation of znCD in a wheat germ in vitro translation system. The knockdown of znCD with AMO led to an increase in the number of zebrafish embryos with severe morphological and cellular abnormalities such as abnormal morphogenesis in the head and tail, pericardiac edema, defect of blood cell circulation, and an increase of apoptotic cells, especially in the head and neural tube regions, at 36 h post-fertilization. The ceramide level in AMO-injected embryos increased significantly compared with that in control embryos. Simultaneous injection of both AMO and synthetic znCD mRNA into one-cell-stage embryos rescued znCD activity and blood cell circulation. These results indicate that znCD is essential for the metabolism of ceramide and the early development of zebrafish.     

Involvement of LMO7 in the association of two cell-cell adhesion molecules, nectin and E-cadherin, through afadin and alpha-actinin in epithelial cells

Nectins are Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules that are involved in formation of cadherin-based adherens junctions (AJs). The nectin-based cell-cell adhesion induces activation of Cdc42 and Rac small G proteins, which eventually enhances the formation of AJs through reorganization of the actin cytoskeleton. Although evidence has accumulated that nectins recruit cadherins to the nectin-based cell-cell adhesion sites through their cytoplasm-associated proteins, afadin and catenins, it is not fully understood how nectins are physically associated with cadherins. Here we identified a rat counterpart of the human LIM domain only 7 (LMO7) as an afadin- and alpha-actinin-binding protein. Rat LMO7 has two splice variants, LMO7a and LMO7b, consisting of 1,729 and 1,395 amino acids, respectively. LMO7 has calponin homology, PDZ, and LIM domains. Western blotting revealed that LMO7 was expressed ubiquitously in various rat tissues. Immunofluorescence and immunoelectron microscopy revealed that LMO7 localized at cell-cell AJs, where afadin localized, in epithelial cells of rat gallbladder. In addition, LMO7 localized at the cytoplasmic faces of apical membranes in the same epithelial cells. We furthermore revealed that LMO7 bound alpha-actinin, an actin filament-bundling protein, which bound to alpha-catenin. Immunoprecipitation analysis revealed that LMO7 was associated with both the nectin-afadin and E-cadherin-catenin systems. LMO7 was assembled at the cell-cell adhesion sites after both the nectin-afadin and E-cadherin-catenin systems had been assembled. These results indicate that LMO7 is an afadin- and alpha-actinin-binding protein that connects the nectin-afadin and E-cadherin-catenin systems through alpha-actinin.     

Suppression of adriamycin-induced apoptosis by sustained activation of the phosphatidylinositol-3'-OH kinase-Akt pathway

The mechanisms by which growth factors trigger signal transduction pathways leading to protection against apoptosis are of great interest. In this study, we investigated the effect of hepatocyte growth factor (HGF/SF) and epidermal growth factor (EGF) on adriamycin (ADR)-induced apoptosis. Treatment of human epithelial MKN74 cells with ADR, a DNA topoisomerase IIalpha inhibitor, caused apoptosis. However, cells pretreated with HGF/SF, but not those pretreated with EGF, were resistant to this apoptosis. The protective effect of HGF/SF against the ADR-induced apoptosis was abolished in the presence of either LY294002, an inhibitor of phosphatidylinositol-3'-OH kinase (PI3-K) or 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, an inhibitor of Akt, thus implicating the activation of PI3-K-Akt signaling in the antiapoptotic action of HGF/SF. Immunoblotting analysis revealed that HGF/SF stimulated the sustained phosphorylation of Akt for several hours but that EGF stimulated the phosphorylation only transiently. Furthermore, ADR-induced activation of caspase-9, a downstream molecule of Akt, was inhibited for at least 24 h after HGF/SF stimulation, but it was not affected by EGF stimulation. Cell-surface biotin-labeling analysis showed that the HGF/SF receptor remained on the cell surface until at least 30 min after HGF/SF addition but that the EGF receptor level on the cell surface was attenuated at an earlier time after EGF addition. These results indicate that HGF/SF, but not EGF, transmitted protective signals against ADR-induced apoptosis by causing sustained activation of the PI3-K-Akt signaling pathway. Furthermore, the difference in antiapoptotic capacity between HGF/SF and EGF is explained, at least in part, by the delayed down-regulation of the HGF/SF receptor.     

Inhibitory effects of trientine, a copper-chelating agent, on induction of DNA strand breaks in hepatic cells of Long-Evans Cinnamon rats

Effects of treatment with trientine, a specific copper-chelating agent, on accumulation of copper and induction of DNA strand breaks were investigated in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson's disease. Copper accumulated in the livers of LEC rats in an age-dependent manner from 4 to 13 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, hepatic copper contents did not increase and were maintained at the same levels as those in 10-week-old LEC rats. When the amounts of DNA single-strand breaks (SSBs) were estimated by a comet assay, SSBs of DNA were induced in a substantial population of LEC rat hepatic cells around 8 weeks of age and the amounts of SSBs increased in an age-dependent manner from 8 to 15 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, the observed number of cells with DNA damage decreased dramatically, suggesting that induction of SSBs of DNA was inhibited and/or SSBs were repaired during the period of treatment with trientine. The results show that treatment of LEC rats with trientine decreases the number of DNA strand breaks observed, although copper contents remain high in the liver.     

Two extracellular proteins with alkaline peroxidase activity, a novel cytochrome c and a catalase-peroxidase, from Bacillus sp. No.13

A novel cytochrome c and a catalase-peroxidase with alkaline peroxidase activity were purified from the culture supernatant of Bacillus sp. No.13 and characterized. The cytochrome c exhibited absorption maxima at 408 nm (Soret band) in its oxidized state, and 550 (alpha-band), 521 (beta-band), and 415 (Soret band) nm in its reduced state. The native cytochrome c with a relative molecular mass of 15,000 was composed of two identical subunits. The cytochrome c showed over 50 times higher peroxidase activity than those of known c-type cytochromes from various sources. The optimum pH and temperature of the peroxidase activity were about 10.0 and 70 degrees C, respectively. The peroxidase activity is stable in the pH range of 6.0 to 10.8 (30 degrees C, 1-h treatment), and at temperatures up to 80 degrees C (pH 8.5, 20-min treatment). The heme content was determined to be 1 heme per subunit. The amino acid sequence of the cytochrome c showed high homology with those of the c-type cytochromes from Bacillus subtilis and Bacillus sp. PS3. The catalase-peroxidase showed high catalase activity and considerable peroxidase activity, the specific activities being 55,000 and 0.94 micromol/min/mg, respectively. The optimum pH and temperature of the peroxidase activity were in the range of 6.4 to 10.1 and 60 degrees C, respectively. The catalase-peroxidase showed a lower K(m) value (0.67 mM) as to H(2)O(2) than known catalase-peroxidases.