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171.
Development and evaluation of an automated workstation for single nucleotide polymorphism discrimination using bacterial magnetic particles 总被引:4,自引:0,他引:4
Tanaka T Maruyama K Yoda K Nemoto E Udagawa Y Nakayama H Takeyama H Matsunaga T 《Biosensors & bioelectronics》2003,19(4):325-330
We designed an automated workstation for magnetic particle-based single nucleotide polymorphism (SNP) discrimination of ALDH genotypes. Bacterial magnetic particles (BMPs) extracted from Magnetospirillum magneticum AMB-1 were used as DNA carriers. The principle for SNP discrimination in this study was based on fluorescence resonance energy transfer (FRET) between FITC (donor) and POPO-3 (acceptor) bound to double-stranded DNA. The workstation is equipped with a 96-way automated pipetter which collects and dispenses fluids as it moves in x- and z-directions. The platform contains a disposable tip rack station, a reagent vessel serving as a stock for POPO-3 and FITC-labeled probes and a reaction station for a 96-well microtiter plate. BMPs were collected by attaching a neodymium iron boron sintered (Nd-Fe-B) magnet on the bottom of the microtiter plate. This system permits the simultaneous heating and magnetic separation of 96 samples per assay. The genotypes ALDH2*1 and ALDH2*2 were discriminated by calculating the relative fluorescence intensities on BMPs. 相似文献
172.
Chosa E Hamada H Kitamura K Kuwasako K Yanagita T Eto T Tajima N 《Biochemical and biophysical research communications》2003,303(1):379-386
For clarifying a process of de-differentiation in culturing chondrocytes, the present study was undertaken to investigate the secretion of adrenomedullin (AM) by chondrocyte phenotype cells and whether or not AM effects this proliferation in a cAMP-dependent fashion. Chondrocyte phenotype cells expressed AM and the AM receptor, and secreted high concentration of AM into the culture medium. When added to cultures, AM increased the intracellular cAMP level and decreased the number of these cells in a similar concentration-dependent fashion. Addition of forskolin and dibutyryl-cAMP caused a significant decrease in the number of these cells. Furthermore, the effect of AM was inhibited by a cAMP-dependent protein kinase A inhibitor (H89). The present findings indicate that AM has an autocrine/paracrine type of anti-proliferative effect on these cells mediated via a cAMP-dependent pathway and raise the possibility that AM plays a role in the local modulation of a process of de-differentiation by culturing chondrocyte phenotype cells. 相似文献
173.
Yoshida Y Ito N Shimakawa S Niki E 《Biochemical and biophysical research communications》2003,305(3):747-753
Lipid peroxidation is an old and yet novel subject. It induces membrane disturbance and damage and its products are known to induce the generation of various cytokines and cell signaling. In the present work, the susceptibility and specificity of human plasma lipids to oxidation were studied, aiming specifically at elucidating the effects of oxidation milieu and oxidants. Cholesteryl esters (CEs) and phosphatidylcholines (PCs) were more readily oxidized in plasma than in organic solution under similar conditions. The susceptibilities of PC and free cholesterol (FC) relative to CE to free radical-mediated lipid peroxidation induced by peroxyl radicals and peroxynitrite were smaller in plasma than in organic solution. The higher rate of CE oxidation by free radicals than PC may be accounted for by the physical effects as well as higher content of polyunsaturated lipids in CE than PC. On the contrary, PC was more readily oxidized than CE by lipoxygenases. The lipid hydroperoxides were stable in organic solution but reduced to the corresponding hydroxides in plasma, the rate being much faster for PC hydroperoxides than for CE and FC hydroperoxides. It was confirmed that free radical-mediated oxidation gave both cis,trans and trans,trans, racemic, random hydroperoxides, while that by lipoxygenase gave only regio- and stereo-specific cis,trans-hydroperoxide. 相似文献
174.
The crystal structure of DJ-1, a protein related to male fertility and Parkinson's disease 总被引:8,自引:0,他引:8
Honbou K Suzuki NN Horiuchi M Niki T Taira T Ariga H Inagaki F 《The Journal of biological chemistry》2003,278(33):31380-31384
DJ-1 is a multifunctional protein that plays essential roles in tissues with higher order biological functions such as the testis and brain. DJ-1 is related to male fertility, and its level in sperm decreases in response to exposure to sperm toxicants. DJ-1 has also been identified as a hydroperoxide-responsive protein. Recently, a mutation of DJ-1 was found to be responsible for familial Parkinson's disease. Here, we present the crystal structure of DJ-1 refined to 1.95-A resolution. DJ-1 forms a dimer in the crystal, and the monomer takes a flavodoxin-like Rossmann-fold. DJ-1 is structurally most similar to the monomer subunit of protease I, the intracellular cysteine protease from Pyrococcus horikoshii, and belongs to the Class I glutamine amidotransferase-like superfamily. However, DJ-1 contains an additional alpha-helix at the C-terminal region, which blocks the putative catalytic site of DJ-1 and appears to regulate the enzymatic activity. DJ-1 may induce conformational changes to acquire catalytic activity in response to oxidative stress. 相似文献
175.
176.
Yoshida Y Noguchi N Watanabe A Iwaki Y Yanagisawa M Kodama T Cynshi O Niki E 《Free radical research》2002,36(11):1171-1178
With increasing evidence suggesting the involvement of oxidative stress in various disorders and diseases, the role of antioxidants in vivo has received much attention. 2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butylbenzofuran (BO-653) was designed, synthesized and has been evaluated as a novel antiatherogenic drug. In order to further understand the action of BO-653 and also radical-scavenging antioxidants in general, the dynamics of inhibition of oxidation by BO-653 were compared with those of the related compounds, 2,3-dihydro-5-hydroxy-2,2-dimethyl-4,6-di-tert-butylbenzofuran (BOB), 2,3-dihydro-5-hydroxy-2,2,4,6-tetramethylbenzofuran (BOM), alpha-tocopherol and 2,2,5,7,8-pentamethyl-6-chromanol (PMC), aiming specifically at elucidating the effects of substituents and side chain length of the phenolic antioxidants. These five antioxidants exerted substantially the same reactivities toward radicals and antioxidant capacities against lipid peroxidation in organic solution. When compared with di-methyl side chains, the di-pentyl side chains of BO-653 reduced its inter-membrane mobility but exerted less significant effect than the phytyl side chain of alpha-tocopherol on the efficacy of radical scavenging within the membranes. Di-tert-butyl groups at both ortho-positions made BO-653 and BOB more lipophilic than di-methyl substituents and reduced markedly the reactivity toward Cu(II) and also the synergistic interaction with ascorbate. The results of the present study together with those of the previous work on the effect of substituents on the stabilities of aryloxyl radicals suggest that tert-butyl group is more favorable than methyl group as the substituent at the ortho-positions and that di-pentyl side chains may be superior to a phytyl side chain. 相似文献
177.
Beene DL Brandt GS Zhong W Zacharias NM Lester HA Dougherty DA 《Biochemistry》2002,41(32):10262-10269
A series of tryptophan analogues has been introduced into the binding site regions of two ion channels, the ligand-gated nicotinic acetylcholine and serotonin 5-HT(3A) receptors, using unnatural amino acid mutagenesis and heterologous expression in Xenopus oocytes. A cation-pi interaction between serotonin and Trp183 of the serotonin channel 5-HT(3A)R is identified for the first time, precisely locating the ligand-binding site of this receptor. The energetic contribution of the observed cation-pi interaction between a tryptophan and the primary ammonium ion of serotonin is estimated to be approximately 4 kcal/mol, while the comparable interaction with the quaternary ammonium of acetylcholine is approximately 2 kcal/mol. The binding mode of nicotine to the nicotinic receptor of mouse muscle is examined by the same technique and found to differ significantly from that of the natural agonist, acetylcholine. 相似文献
178.
The object of this paper is to provide an enzymatic means to attain faster swelling or shrinking kinetics of polyelectrolyte gels that undergo volume phase transition as an immobilized enzyme reaction sets in. For this, we studied the coimmobilization of gluconolactonase (GL) with glucose oxidase (GOD). A gel used was in the shape of a small cylinder (several hundred micrometers in diameter) and composed of a lightly cross-linked copolymer of N-isopropylacrylamide and acrylic acid. GL was isolated from Aspergillus niger and purified about 100-fold. It was found that in a substrate solution containing glucose, the gel with the coimmobilized GL and GOD shrinks very rapidly. The shrinking rate was identical to that of the enzyme-free gel that undergoes a shrinking transition in response to a sudden pH change of the outer medium from 7 to 5. This indicates the rate-limiting step in the shrinking process to be diffusion of the networks, but not the enzyme reaction. In the gel with singly immobilized GOD, a very slow shrinking was observed because the process is governed by the enzyme reaction. These results were discussed in full in connection with an enzymatically induced decrease in pH within and in the vicinity of the gel phase. As a result, it has become apparent that the faster shrinking kinetics in the coimmobilized enzyme system is attained by the GL-catalyzed hydrolysis of D-glucono-delta-lactone resulting from the oxidation of glucose with GOD. 相似文献
179.
A 135-kD actin-bundling protein was
purified from pollen tubes of lily (Lilium longiflorum)
using its affinity to F-actin. From a crude extract of the pollen
tubes, this protein was coprecipitated with exogenously added F-actin
and then dissociated from F-actin by treating it with
high-ionic-strength solution. The protein was further purified
sequentially by chromatography on a hydroxylapatite column, a
gel-filtration column, and a diethylaminoethyl-cellulose ion-exchange
column. In the present study, this protein is tentatively referred to
as P-135-ABP (Plant 135-kD
Actin-Bundling Protein). By the
elution position from a gel-filtration column, we estimated the native
molecular mass of purified P-135-ABP to be 260 kD, indicating that it
existed in a dimeric form under physiological conditions. This protein
bound to and bundled F-actin prepared from chicken breast muscle in a
Ca2+-independent manner. The binding of 135-P-ABP to actin
was saturated at an approximate stoichiometry of 26 actin monomers to 1
dimer of P-135-ABP. By transmission electron microscopy of thin
sections, we observed cross-bridges between F-actins with a
longitudinal periodicity of 31 nm. Immunofluorescence microscopy using
rhodamine-phalloidin and antibodies against the 135-kD polypeptide
showed that P-135-ABP was colocalized with bundles of actin filaments
in lily pollen tubes, leading us to conclude that it is the factor
responsible for bundling the filaments.Actin filaments, one of the major components of the cytoskeleton,
are organized into a highly ordered architecture and are involved in
various kinds of cell motility. Their architecture is regulated by
several kinds of actin-binding proteins, including cross-linking
proteins, severing proteins, end-capping proteins, and
monomer-sequestering proteins in animal, protozoan, and yeast cells
(Stossel et al., 1985; Pollard and Cooper, 1986; Vandekerckhove
and Vancompernolle, 1992). In plant cells the organization of the actin
cytoskeleton also changes remarkably during the cell cycle or during
developmental processes, and it is suggested that actin-binding
proteins are involved in their dynamic change. However, little is known
about actin-binding proteins in plant cells.Only a low-Mr actin-binding and -depolymerizing
protein, profilin, in white birch (Betula verrucosa;
Valenta et al., 1991), maize (Zea mays; Staiger
et al., 1993; Ruhlandt et al., 1994), bean (Phaseolus
vulgaris; Vidali et al., 1995), tobacco (Nicotiana
tabacum; Mittermann et al., 1995), tomato (Lycopersicon
esculentum; Darnowski et al., 1996), Arabidopsis
(Arabidopsis thaliana; Huang et al., 1996), and lily
(Lilium longiflorum; Vidali and Hepler, 1997), and an ADF in
lily (Kim et al., 1993), rapeseed (Brassica napus; Kim
et al., 1993), and maize (Rozycka et al., 1995; Lopez et al., 1996),
have been identified by biochemical or molecular biological means.The native and recombinant forms of these proteins are capable of
binding to animal or plant actin (Valenta et al., 1993; Giehl et al.,
1994; Ruhlandt et al., 1994; Lopez et al., 1996; Perelroizen et al.,
1996; Carlier et al., 1997). Plant profilin expressed in mammalian
BHK-21 cells (Rothkegel et al., 1996) or profilin-deficient Dictyostelium discoideum cells (Karakesisoglou et al., 1996) was
able to functionally substitute for endogenous profilin in these cells.
The introduction of plant profilin into living stamen hair cells by
microinjection caused the rapid reduction of the number of actin
filaments (Staiger et al., 1994; Karakesisoglou et al., 1996; Ren et
al., 1997). These results indicate that plant profilin and ADF share
many functional similarities with other eukaryote profilins and
ADFs.It is well known that the actin cytoskeleton undergoes dynamic changes
in organization during hydration and activation of the vegetative cells
of pollen grains (Pierson and Cresti, 1992). Before hydration actin
filaments exist as fusiform or spiculate structures (a storage form),
but they are rearranged to form a network upon hydration
(Heslop-Harrison et al., 1986; Tiwari and Polito, 1988). In the growing
pollen tube there are strands or bundles of actin filaments parallel to
the long axis (Perdue et al., 1985; Pierson et al., 1986; Miller et
al., 1996) that are involved in cytoplasmic streaming (Franke et al.,
1972; Mascarenhas and Lafountain, 1972) and transport of vegetative
nuclei and generative cells to the growing tip (Heslop-Harrison et al.,
1988; Heslop-Harrison and Heslop-Harrison, 1989). Characterization of
the function of actin-binding proteins is essential to understanding
the regulation of actin organization during the developmental process
of pollen. Since only a small number of vacuoles containing proteases
develop in pollen grains and pollen tubes at a younger stage, pollen
tubes are suitable materials for isolating and biochemically studying
actin-binding proteins responsible for organizing actin filaments into
various forms.In a previous paper we reported that several components in a crude
extract prepared from lily pollen tubes, including a 170-kD myosin
heavy chain and 175-, 135-, and 110-kD polypeptides, could be
coprecipitated with exogenously added F-actin (Yokota and Shimmen,
1994). We also found that rhodamine-labeled F-actin was tightly bound
to the glass surface treated with the fraction containing the 135- and
110-kD polypeptides (Yokota and Shimmen, 1994). These results suggested
that either one or both of the 135- and 110-kD polypeptides possesses
an F-actin-binding activity. In the present study, we purified the
135-kD polypeptide from lily pollen tubes by biochemical procedures and
then characterized its F-actin-binding properties and distribution in
the pollen tubes. This protein was able to bundle F-actin isolated from
chicken breast muscle and colocalized with actin-filament bundles in
pollen tubes. We refer to this protein as P-135-ABP (Plant
135-kD Actin-Bundling
Protein). 相似文献
180.
Dynamics of Vitamin E Action against LDL Oxidation 总被引:5,自引:0,他引:5
Vitamin E acts as an important antioxidant against oxidative modification of low density lipoprotein (LDL) which is accepted as an initial event in the pathogenesis of atherosclerosis. In spite of the numerous studies and reports, the action and role of vitamin E have not been fully elucidated yet. In this brief overview, the dynamics of action of vitamin E as an antioxidant have been discussed and it is emphasized that the total antioxidant potency is determined by the relative importance of many competing reactions which is determined by the reactivities and concentrations of substrates, radicals and antioxidant and by physical factors of the environment. 相似文献