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91.
The organization of microtubules (MTs) in the cortex of cells at interphase is an important element in morphogenesis. Mechanisms
controlling the initiation of MTs and their spatial ordering, however, are largely unknown. Our recent study concerning the
generation of a radial array of MTs in stomatal guard cells inAllium showed that the MTs initiate in a cortical MT-organizing zone adjacent to the ventral wall separating the two young guard
cells (Marc, Mineyuki and Palevitz, 1989, Planta179, 516, 530). In an attempt to detect MT-ordering mechanisms separate from the sites of MT initiation, we now employ various
drugs to manipulate the geometry and integrity of the ventral wall and thereby also the associated MT-organizing zone. In
the presence of cytochalasin D the ventral wall is tilted away from its normal mid-longitudinal anticlinal alignment, while
treatments with the herbicide chloroisopropyl-N-phenylcarbamate (CIPC) induce the formation of a branched ventral wall. Nonetheless, in either case the MTs still
form a radial array, although this is asymmetric as it is centered in accordance with the misaligned or branched ventral wall.
Since the MTs maintain their original course undisturbed as they extend beyond the abnormal ventral wall, there is no evidence
for the presence of an inherent MT-ordering mechanism at locations remote from MT-initiation sites. Following treatments with
caffeine, which abolishes the formation of the ventral wall, the MTs revert to a transversely oriented cylindrical array as
in normal epidermal cells. Thus the presence of the ventral wall, and presumably also the associated MT-organizing zone, is
essential for the establishment of the radial array. The MT-organizing zone is therefore involved not only in the initiation
of MTs, but also in determining their spatial order throughout the cell cortex.
We thank Drs. Richard J. Cyr and Yoshi Mineyuki for providing valueable suggestions during the course of this work, and Ms.
Elizabeth Bruce printing some of the figures. This research was supported by Funds from the National Science Foundation grants
DCB-8703292 to B.A.P. and DCB-8803286 to B.A.P. and J.M. 相似文献
92.
Cora-Jean S. Edgell Jill E. Haizlip C. Robert Bagnell Joan P. Packenham Paul Harrison Barry Wilbourn Victoria J. Madden 《In vitro cellular & developmental biology. Plant》1990,26(12):1167-1172
Summary Weibel-Palade bodies are ultrastructurally defined organelles found only in vascular endothelial cells. Because endothelium
in corpo is very dispersed, isolation and further characterization of this organelle has been dependent on increasing the
number of cells in culture. However, primary isolates of endothelial cells have a limited replication potential and tend to
senesce in culture. In this report, EA.hy926, a continuously replicating cell line derived from human endothelium, is shown
to contain Weibel-Palade bodies. Electron micrographs demonstrate the ultrastructural characteristics of these tissue-specific
organelles and their cytoplasmic distribution in EA.hy926 cells. Von Willebrand factor, which has been shown to exist in Weibel
Palade bodies, is demonstrated by immunofluorescence in discrete rod-shaped organelles whose size, shape, and distribution
are consistent with that of Weibel-Palade bodies in primary endothelial cell cultures. Rapid release of von Willebrand factor
can be induced by calcium ionophore, and large multimeric forms of the protein are found in EA.hy926 cells. These two properties
are consistent with the function currently ascribed to Weibel Palade bodies: storage of multimerized von Willebrand factor.
Thus ultrastructural, immunologic, and functional data establish the existence of this as yet poorly understood tissue-specific
organelle in a continuous, vigorously replicating human cell line. 相似文献
93.
Incubation of rat-liver microsomes, previously azide-treated to inhibit catalase, with H2O2 caused a loss of cytochrome P-450 but not of cytochrome b5. This loss of P-450 was not prevented by scavengers of hydroxyl radical, chain-breaking antioxidants or metal ion-chelating agents. Application of the thiobarbituric acid (TBA) assay to the reaction mixture suggested that H2O2 induces lipid peroxidation, but this was found to be due largely or completely to an effect of H2O2 on the TBA assay. By contrast, addition of ascorbic acid and Fe(III) to the microsomes led to lipid peroxidation and P-450 degradation: both processes were inhibited by chelating agents and chain-breaking antioxidants, but not by hydroxyl radical scavengers. H2O2 inhibited ascorbate/Fe (III)-induced microsomal lipid peroxidation, but part of this effect was due to an action of H2O2 in the TBA test itself. H2O2 also decreased the colour measured after carrying out the TBA test upon authentic malondialdehyde, tetraethoxypropane, a DNA-Cu2+/o-phenanthroline system in the presence of a reducing agent, ox-brain phospholipid liposomes in the presence of Fe(III) and ascorbate, or a bleomycin-iron ion/DNA/ascorbate system. Caution must be used in interpreting the results of TBA tests upon systems containing H2O2. 相似文献
94.
How to Characterize a Biological Antioxidant 总被引:15,自引:0,他引:15
Barry Halliwell 《Free radical research》1990,9(1):1-32
An antioxidant is a substance that, when present at low concentrations compared to those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate. Many substances have been suggested to act as antioxidants in vivo, but few have been proved to do so. The present review addresses the criteria necessary to evaluate a proposed antioxidant activity. Simple methods for assessing the possibility of physiologically-feasible scavenging of important biological oxidants (superoxide, hydrogen peroxide, hydroxyl radical, hypochlorous acid, haem-associated ferryl species, radicals derived from activated phagocytes, and peroxyl radicals, both lipid-soluble and water-soluble) are presented, and the appropriate control experiments are described. Methods that may be used to gain evidence that a compound actually does function as an antioxidant in vivo are discussed. A review of the pro-oxidant and anti-oxidant properties of ascorbic acid that have been reported in the literature leads to the conclusion that this compound acts as an antioxidant in vivo under most circumstances. 相似文献
95.
96.
The calcium-dependent modulation of the affinity of the cyclic nucleotide-gated (CNG) channels for adenosine 3′,5′-cyclic
monophosphate (cAMP) was studied in enzymatically dissociated rat olfactory receptor neurons, by recording macroscopic cAMP-activated
currents from inside-out patches excised from their dendritic knobs. Upon intracellular addition of 0.2 mm Ca2+ (0.2 Ca) the concentration of cAMP required for the activation of half-maximal current (EC50) was reversibly increased from 3 μm to about 30 μm. This Ca2+-induced affinity shift was insensitive to the calmodulin antagonist, mastoparan, was abolished irreversibly by a 2-min exposure
to 3 mm Mg2++ 2 mm EGTA (Mg + EGTA), and was not restored by the application of calmodulin (CAM). Addition of CAM plus 0.2 mm Ca2+ (0.2 Ca + CAM), further reversibly shifted the cAMP affinity from 30 μm to about 200 μm. This affinity shift was not affected by Mg + EGTA exposure, but was reversed by mastoparan. Thus, the former Ca2+-only effect must be mediated by an unknown endogenous factor, distinct from CAM. Removal of this factor also increased the
affinity of the channel for CAM. The affinity shift induced by Ca2+-only was maintained in the presence of the nonhydrolyzable cAMP analogue, 8-bromo-cAMP and the phosphatase inhibitor, microcystin-LR,
ruling out modulation by phosphodiesterases or phosphatases. Our results indicate that the olfactory CNG channels are modulated
by an as yet unidentified factor distinct from CAM.
Received: 26 December 1995/Revised: 14 March 1996 相似文献
97.
Regional and Ontogenic Expression of the NMDA Receptor Subunit NR2D Protein in Rat Brain Using a Subunit-Specific Antibody 总被引:7,自引:0,他引:7
† Anthone W. Dunah †Robert P. Yasuda Yue-hua Wang Jianhong Luo Martha I. Dávila-García †Muyiwa Gbadegesin †‡Stefano Vicini †Barry B. Wolfe 《Journal of neurochemistry》1996,67(6):2335-2345
Abstract: A polyclonal antibody for the NMDA receptor subunit NR2D has been developed that identifies an ∼160-kDa band on immunoblots from NR2D transfected cells and CNS tissues. No cross-reactivity is seen with other NMDA receptor subunits. The NR2D receptor subunit is N -glycosylated in both brain and transfected cells. Transfected cells expressing NR2D are immunofluorescently labeled, whereas untransfected cells or cells transfected with other NMDA receptor subunit cDNAs are not. Similarly, the NR2D subunit is selectively and quantitatively immunoprecipitated, whereas the NR1, NR2A, or NR2B subunit is not. The relative densities of the NR2D subunit in nine areas of postnatal day 7 and adult rat brains have been determined by quantitative immunoblotting. NR2D was expressed at highest levels in the thalamus, midbrain, medulla, and spinal cord, whereas intermediate levels of this subunit were found in the cortex and hippocampus. Low or undetectable levels were seen in the olfactory bulb, striatum, and cerebellum. Following a peak after the first week of birth, NR2D protein levels decreased by about twofold in adulthood in all rat brain regions examined. More complete ontogenic profiles were determined for the diencephalon, telencephalon, and spinal cord where similar ontogenic patterns were seen. NR2D protein is present at high levels at embryonic stages of development, rises to a peak at postnatal day 7, and decreases but remains measurable during late postnatal life. This study demonstrates the generation and characterization of an antibody selective for the NR2D NMDA receptor subunit as well as a determination of the distribution and ontogenic profile of this subunit in rat brain. The results suggest that native NMDA receptors containing the NR2D subunit may have functional roles not only in the young brain but also in adult brain. 相似文献
98.
99.
Stationary phase-associated protein expression in Mycobacterium tuberculosis: function of the mycobacterial alpha-crystallin homolog. 总被引:8,自引:0,他引:8
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The majority of active tuberculosis cases arise as a result of reactivation of latent organisms which are quiescent within the host. The ability of mycobacteria to survive extended periods without active replication is a complex process whose details await elucidation. We used two-dimensional gel electrophoresis to examine both steady-state protein composition and time-dependent protein synthetic profiles in aging cultures of virulent Mycobacterium tuberculosis. At least seven proteins were maximally synthesized 1 to 2 weeks following the end of log-phase growth. One of these proteins accumulated to become a predominant stationary-phase protein. N-terminal amino acid sequencing and immunoreactivity identified this protein as the 16-kDa alpha-crystallin-like small heat shock protein. The gene for this protein was shown to be limited to the slowly growing M. tuberculosis complex of organisms as assessed by Southern blotting. Overexpression of this protein in wild-type M. tuberculosis resulted in a slower decline in viability following the end of log-phase growth. Accumulation of this protein was observed in log-phase cultures following a shift to oxygen-limiting conditions but not by other external stimuli. The protein was purified to homogeneity from overexpressing M. smegmatis in two steps and shown to have a significant ability to suppress the thermal denaturation of alcohol dehydrogenase. Collectively, these results suggest that the mycobacterial alpha-crystallin protein may play a role in enhancing long-term protein stability and therefore long-term survival of M. tuberculosis. 相似文献
100.
Leaching of soluble substances may be an important first step in leaf litter decomposition in small streams, but recent research has suggested that large leaching losses (up to 30% of initial mass in 48 h) may be an artifact created by using air-dried leaves in decomposition experiments. In laboratory experiments, we compared 3 d leaching losses from freshly fallen and air-dried senescent leaves of 27 tree species from different regions across Canada. Air-dried leaves from all species leached measurable amounts of original mass (3.6–32.8% dry mass), but leaching losses from fresh leaves (0–35%) were detectable in all but two species. Air-drying increased leaching losses in many species, but in others it reduced leaching losses or had no measurable effect. Results for leaves of the same species collected in different regions or in different years were generally similar, but species within the same genus often behaved very differently. Neither moisture content (fresh or air-dried), leaf thickness, nor cuticle thickness proved of any value as predictors of leaching losses or the effect of air-drying. The propensity of autumn-fallen leaves to leach, whether fresh or air-dried, appears to be a property of the individual tree species. 相似文献