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81.
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
82.
Yokota Etsuo; Sonobe Seiji; Igarashi Hisako; Shimmen Teruo 《Plant & cell physiology》1995,36(8):1563-1569
The latent ability of plant microtubules to translocate or slidein the presence of motor proteinwas examined in a motility assayin vitro. Plant microtubules isolated from tobacco BY-2 cellsmoved over a glass surface covered with outer arm 21S dyneinfrom flagella of sea urchinsperm with an average velocity of3.7 µm s-1. This velocity was similar to that of microtubulesisolated from bovine brain under the same conditions (averagevelocity, about 4.1 µm s-1). These results suggest thatplant microtubules have an intrinsic ability to interact withand to be translocated by dynein. It is postulated that microtubule-basedmotor proteins, including dynein ATP-ase,are involved in thefunctioning of microtubules in plant cells. (Received May 20, 1995; Accepted September 18, 1995) 相似文献
83.
Etsuo Hasegawa Yoh-ichi Matsushita Manabu Kaneda Kiyoshi Ejima Eishun Tsuchida 《Biochemical and biophysical research communications》1982,105(4):1416-1419
To prepare a potent synthetic oxygen carrier in aqueous media, the iron(II) picket fence porphyrin complex with one hydrophobic imidazole was incorporated into a lipid bilayer of phosphatidylcholine. The incorporation was confirmed by gel permeation chromatography and ultracentrifugation, which indicates the complex being trapped in the multilayer liposome. The liposomal iron(II) porphyrin complex could bind molecular oxygen reversibly in neutral aqueous media and in the serum of a rat blood at 25°C. 相似文献
84.
Recently, raw fish, sashimi, is becoming a popular dish in countries other than Japan. Therefore, in order to assure that the raw fish and shellfish are safe for human consumption, a quality evaluation sensor, which shows, at a glance, the quality of sashimi, was developed. The proposed sensor is based on the principle that the freshness of sashimi, which is judged from the KI value, can be determined from the degree of color change of thiazole blue (MTT: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) due to the redox reaction of MTT accompanying the oxidation of hypoxanthine (Hx) by xanthine oxidase (XOD). The proposed sensor consists of 5 ml of 80% ethanol-1 M Tris-HCl buffer (pH 7.8) containing 2.0 mg of Hx, 2.0 mg of MTT and 0.38 unit of XOD. The proposed sensor and fish were kept together at 5, -10 and -20 degrees C, and the freshness of sashimi stored at each temperature was determined from the color change of the sensor. The concept "freshness of sashimi" can be expressed as remaining of validity (RDV), which is described in our previous study. A good relationship was obtained between the KI value and the RDV determined by the proposed sensor. From these results, the proposed sensor system can be used to non-destructively determine the fish freshness and RDV. 相似文献
85.
A rat model of a hyperkinetic disorder was used to investigate the mechanisms underlying motor hyperactivity. Rats received an intracisternal injection of 6-hydroxydopamine on post-natal day 5. At 4 weeks of age, the animals showed significant motor hyperactivity during the dark phase, which was attenuated by methamphetamine injection. Gene expression profiling was carried out in the striatum and midbrain using a DNA macroarray. In the striatum at 4 weeks, there was increased gene expression of the NMDA receptor 1 and tachykinins, and decreased expression of a GABA transporter. At 8 weeks, expression of the NMDA receptor 1 in the striatum was attenuated, with enhanced expression of the glial glutamate/aspartate transporter. In the midbrain, a number of genes, including the GABA transporter gene, showed decreased expression at 4 weeks. At 8 weeks, gene expression was augmented for the dopamine transporter, D4 receptor, and several genes encoding peptides, such as tachykinins and their receptors. These results suggest that in the striatum the neurotransmitters glutamate, GABA and tachykinin may play crucial roles in motor hyperactivity during the juvenile period. Several classes of neurotransmitters, including dopamine and peptides, may be involved in compensatory mechanisms during early adulthood. These data may prompt further neurochemical investigations in hyperkinetic disorders. 相似文献
86.
Hashimoto K Igarashi H Mano S Nishimura M Shimmen T Yokota E 《Plant & cell physiology》2005,46(5):782-789
The genome of Arabidopsis thaliana contains 13 myosin XI isoforms. Here we prepared a specific antibody against a peptide that mimics a unique C-terminal region from the myosin XI isoform, MYA2. The resulting antibody was used to demonstrate that MYA2 in Arabidopsis protein extracts co-sedimented with actin filaments and dissociated from the filaments with ATP treatment. Immunolocalization studies showed that MYA2 co-localized predominantly with actin filaments in clustered punctuate dots in leaf epidermal cells, root hair cells and suspension-cultured cells. In a transgenic plant in which peroxisomes are labeled with green fluorescent protein, some MYA2 signals were localized on peroxisomes in an actin-dependent manner. We propose that the peroxisome is one of the cargos translocated by MYA2 on actin filaments. 相似文献
87.
Kohei Kugo Masaki Okuno Koji Kitayama Tatsuro Kitaura Jun Nishino Nobuo Ikuta Etsuo Nishio Makoto Iwatsuki 《Biopolymers》1992,32(3):197-207
Fourier transform ir attenuated total reflectance (FTIR ATR) spectra have been obtained to investigate the secondary structure of poly(γ -methyl L -glutamate) (PMLG) surfaces untreated and treated with formic acid in a quantitative manner. Curve analysis including Fourier self-deconvolution and the band fitting was applied to the ir spectra in the amide II region, revealing that the amide II band of those surfaces consists of five components. The essentially α-helical form in the PMLG surface layer transformed readily into the β-structure by the formic acid treatment, and the β-structure content increased with increasing time of the treatment. The content of random coil structure of treated PMLG was generally very little and/or negligible. The depth profile obtained by considering the depth of ir beam penetration indicated that the β-structure content also increased with approaching the outermost surface. 相似文献
88.
Monochloramine (NH(2)Cl) is a physiological oxidant produced by activated neutrophils. In the present work, we studied the underlying mechanism of cytotoxic effects of NH(2)Cl on an undifferentiated rat pheochromocytoma PC12 cell line and the protective effects of antioxidants. The cells treated with 100 microM NH(2)Cl exhibited signs of apoptotic cell death such as phosphatidylserine exposure and caspase activation. To understand the mechanism of NH(2)Cl cytotoxicity, we examined the effect of various kinds of antioxidants including alpha-tocopherol (alpha-Toc) and beta-tocopherol (beta-Toc). These antioxidants exerted a protective effect against NH(2)Cl-induced cell death, and alpha-Toc exhibited the most potent inhibitory effect among the antioxidants used. A loss of cellular glutathione was observed in the cells treated with 100 microM NH(2)Cl. The formation of reactive oxygen species (ROS) was also measured using the fluorescent probe dichlorofluorescin diacetate. The fluorescence intensity increased prior to cell death and an antioxidant, such as alpha-Toc, suppressed the increase in ROS. Interestingly, beta-Toc also exerted similar inhibitory effects on cytotoxicity and caspase activation. These results suggest that free radical mediated process is involved in NH(2)Cl-induced PC12 cell death and that tocopherols inhibit this cell death via antioxidative function. 相似文献
89.
Saito Y Nishio K Ogawa Y Kimata J Kinumi T Yoshida Y Noguchi N Niki E 《Free radical research》2006,40(6):619-630
The turning point between apoptosis and necrosis induced by hydrogen peroxide (H2O2) have been investigated using human T-lymphoma Jurkat cells. Cells treated with 50 μM H2O2 exhibited caspase-9 and caspase-3 activation, finally leading to apoptotic cell death. Treatment with 500 μM H2O2 did not exhibit caspase activation and changed the mode of death to necrosis. On the other hand, the release of cytochrome c from the mitochondria was observed under both conditions. Treatment with 500 μM H2O2, but not with 50 μM H2O2, caused a marked decrease in the intracellular ATP level; this is essential for apoptosome formation. H2O2-reducing enzymes such as cellular glutathione peroxidase (cGPx) and catalase, which are important for the activation of caspases, were active under the 500 μM H2O2 condition. Prevention of intracellular ATP loss, which did not influence cytochrome c release, significantly activated caspases, changing the mode of cell death from necrosis to apoptosis. These results suggest that ATP-dependent apoptosome formation determines whether H2O2-induced cell death is due to apoptosis or necrosis. 相似文献
90.
Thiol compounds exert diverse functions in the defense network against oxidative stress in vivo. Above all, the role of glutathione in the enzymatic removal of hydrogen peroxide and lipid hydroperoxides has been well established. The scavenging of reactive free radicals is one of the many functions. In this study, the reactivities of several thiol compounds toward oxygen- and nitrogen-centered radicals were measured from their reaction with galvinoxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and also from their sparing effects on the decay of fluorescein, pyrogallol red, and BODIPY induced by peroxyl radicals. Furthermore, the antioxidant capacity against lipid peroxidation was assessed in the oxidation of methyl linoleate induced by free radicals in micelle systems. Cysteine, homocysteine, and glutathione exhibited considerable reactivity toward galvinoxyl, DPPH, and peroxyl radicals in this order but methionine did not. Bovine serum albumin (BSA) was less reactive toward these radicals than cysteine on molar base. Cysteine, homocysteine, and glutathione suppressed the oxidation of methyl linoleate in micelle systems, but methionine did not. The reactivity toward free radicals and antioxidant capacity of these thiol compounds were less than that of ascorbic acid, but higher than that of uric acid. 相似文献