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61.
62.
Alexander Mironov Antonino Colanzi Maria Giuseppina Silletta Giusy Fiucci Silvio Flati Aurora Fusella Roman Polishchuk Alexander Mironov Jr. Giuseppe Di Tullio Roberto Weigert Vivek Malhotra Daniela Corda Maria Antonietta De Matteis Alberto Luini 《The Journal of cell biology》1997,139(5):1109-1118
We have investigated the role of the ADP- ribosylation induced by brefeldin A (BFA) in the mechanisms controlling the architecture of the Golgi complex. BFA causes the rapid disassembly of this organelle into a network of tubules, prevents the association of coatomer and other proteins to Golgi membranes, and stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kD (GAPDH and BARS-50; De Matteis, M.A., M. DiGirolamo, A. Colanzi, M. Pallas, G. Di Tullio, L.J. McDonald, J. Moss, G. Santini, S. Bannykh, D. Corda, and A. Luini. 1994. Proc. Natl. Acad. Sci. USA. 91:1114–1118; Di Girolamo, M., M.G. Silletta, M.A. De Matteis, A. Braca, A. Colanzi, D. Pawlak, M.M. Rasenick, A. Luini, and D. Corda. 1995. Proc. Natl. Acad. Sci. USA. 92:7065–7069). To study the role of ADP-ribosylation, this reaction was inhibited by depletion of NAD+ (the ADP-ribose donor) or by using selective pharmacological blockers in permeabilized cells. In NAD+-depleted cells and in the presence of dialized cytosol, BFA detached coat proteins from Golgi membranes with normal potency but failed to alter the organelle's structure. Readdition of NAD+ triggered Golgi disassembly by BFA. This effect of NAD+ was mimicked by the use of pre–ADP- ribosylated cytosol. The further addition of extracts enriched in native BARS-50 abolished the ability of ADP-ribosylated cytosol to support the effect of BFA. Pharmacological blockers of the BFA-dependent ADP-ribosylation (Weigert, R., A. Colanzi, A. Mironov, R. Buccione, C. Cericola, M.G. Sciulli, G. Santini, S. Flati, A. Fusella, J. Donaldson, M. DiGirolamo, D. Corda, M.A. De Matteis, and A. Luini. 1997. J. Biol. Chem. 272:14200–14207) prevented Golgi disassembly by BFA in permeabilized cells. These inhibitors became inactive in the presence of pre–ADP-ribosylated cytosol, and their activity was rescued by supplementing the cytosol with a native BARS-50–enriched fraction. These results indicate that ADP-ribosylation plays a role in the Golgi disassembling activity of BFA, and suggest that the ADP-ribosylated substrates are components of the machinery controlling the structure of the Golgi apparatus. 相似文献
63.
Contribution of Mutation and RNA Recombination to the Evolution of a Plant Pathogenic RNA 总被引:10,自引:0,他引:10
Miguel A. Aranda Aurora Fraile Joaquín Dopazo José M. Malpica Fernando García-Arenal 《Journal of molecular evolution》1997,44(1):81-88
The nucleotide sequence of 17 variants of the satellite RNA of cucumber mosaic virus (CMV-satRNA) isolated from field-infected
tomato plants in the springs of 1989, 1990, and 1991 was determined. The sequence of each of the 17 satRNAs was unique and
was between 334 and 340 nucleotides in length; 57 positions were polymorphic. There was much genetic divergence, ranging from
0.006 to 0.141 nucleotide substitutions per site for pairwise comparisons, and averaging 0.074 for any pair. When the polymorphic
positions were analyzed relative to a secondary structure model proposed for CMV-satRNAs, it was found that there were significantly
different numbers of changes in base-paired and non–base-paired positions, and that mutations that did not disrupt base pairing
were preferred at the putatively paired sites. This supports the concept that the need to maintain a functional structure
may limit genetic divergence of CMV-satRNA. Phylogenetic analyses showed that the 17 CMV-satRNA variants clustered into two
subgroups, I and II, and evolutionary lines proceeding by the sequential accumulation of mutations were apparent. Three satRNA
variants were outliers for these two phylogenetic groups. They were shown to be recombinants of subgroup I and II satRNAs
by calculating phylogenies for different molecular regions and by using Sawyer's test for gene conversion. At least two recombination
events were required to produce these three recombinant satRNAs. Thus, recombinants were found to be frequent (∼17%) in natural
populations of CMV-satRNA, and recombination may make an important contribution to the generation of new variants. To our
knowledge this is the first report of data allowing the frequency of recombinant isolates in natural populations of an RNA
replicon to be estimated.
Received: 14 May 1996 / Accepted: 17 July 1996 相似文献
64.
Shu’a Yagev Michael Heller Arié Pinson 《In vitro cellular & developmental biology. Plant》1984,20(12):893-898
Summary Postnatal rat heart cells in culture enriched with respect to muscle cells were obtained by either high density seeding or
by the replating technique. [3H]Thymidine incorporation to DNA and the enzymatic pattern of cytoplasmic and lysosomal enzymes have been studied as a function
of the culture’s age, of seeding density, and replating. It was shown that (a) replating maintains predominance of myocyte
population for at least 2 wk in culture; (b) heavy seeding density allows homogeneous myocyte population for the 1st wk in
culture; and (c) the enzyme profile of the culture may serve as an indicator for the type of cell population in culture and
its state of differentiation.
This study was done as partial fulfilment of the M.Sc. thesis in Biochemistry (SY). Supported by grants from The Chief Scientist,
Ministry of Health, State of Israel; The Ministry of Education and Sciences, State of Niedersachssen (FRG); and The Foundation
for Heart Research from Mr. and Mrs. D. Vidal-Madjar, Paris, France. 相似文献
65.
66.
3H-thymidine incorporation and DNA-polymerase activity during early hours of wheat embryo germination at two viability levels
have been studied. The patterns of two biosynthetic activities, as well as the dependence of DNA synthesis on protein synthesis,
indicated the presence of a delay in the early phase of imbibition of the aged embryos with respect to viable germs. 相似文献
67.
68.
69.
Keith Paige Melanie Palomares Patricia A. D’Amore Susan J. Braunhut 《In vitro cellular & developmental biology. Animal》1991,27(2):151-157
Summary The growth of the endothelial cell (EC) is tightly regulated throughout the body. Many factors have been implicated in modulating
EC growth including diffusible compounds, cell-to-cell interactions, and the extracellular matrix (ECM). Retinol, or vitamin
A alcohol, has recently been shown to inhibit the growth of bovine capillary ECs, in vitro. Retinoids are known to modify
ECM in other cell systems, and pure ECM components have been shown to effect EC growth rates. We, therefore, examined the
role of the matrix in the retinol-induced inhibition of ECs. Cell-free matrices from control and vitamin A-treated ECs were
prepared by removing cells with EGTA treatment after 7 d of culture. Matrix proteins were analyzed by solubilizing the matrices
in 5M quanidine-HCl and performing Western blot analysis using specific antibodies to matrix proteins. In isolating the ECM, we
observed that retinol-treated cultures of ECs were resistant to EGTA removal; retinol-treated ECs required twice the exposure
time to EGTA to detach from their matrix than did controls cells. Western blot analysis of matrix proteins derived from control
and retinol-treated EC cultures demonstrated a 1.6-fold increase in lamininβ chains and a 2.5-fold increase in fibronectin in the ECM of retinol-treated EC compared to control cell matrix. Functional
properties of these matrices were assessed by plating control and Day 6 retinol-treated ECs onto the matrices and measuring
attachment and growth by determining cell numbers at 24, 72, and 144 h. These studies revealed that control cells attached
in greatest numbers to a control matrix whereas retinol-treated ECs preferentially attached to a matrix derived from retinol-treated
cells. Furthermore, control ECs which grew rapidly on a control matrix were growth inhibited on a retinol-derived matrix.
These data indicate that vitamin A treatment of ECs effects both their phenotype and influences the composition and the functional
properties of their underlying ECM. These studies also demonstrate that alterations of the matrix are at least in part responsible
for the growth inhibition of EC by retinol. 相似文献
70.
Kim B. Saunders Patricia A. D’Amore 《In vitro cellular & developmental biology. Animal》1992,28(7-8):521-528
Summary Heterotypic cell-cell interactions appear to be involved in the control of development and function in a wide variety of tissues.
In the vasculature, endothelial cells and mural cells (smooth muscle cells or pericytes) make frequent contacts, suggesting
a role for intercellular interactions in the regulation of vascular growth and function. We have previously grown endothelial
cells and mural cells together in mixed cultures and found that heterocellular contact led to endothelial growth inhibition.
However, this mixed culture system does not lend itself to the examination of the effects of contact on the phenotype of the
individual cell types. We have therefore developed a co-culture system in which cells can be co-cultured across a porous membrane,
permitting intercellular contact while maintaining pure cell populations. Co-culture of endothelial cells and smooth muscle
cells across membranes with pore sizes of 0.02, 0.4, 0.6, and 0.8μm maintained the two cell types as homogeneous populations, whereas smooth muscle cells migrated across the membrane through
pores of 2.0μm. Vascular cell co-culture across membranes with 0.8-μm pores resulted the inhibition of endothelial cell proliferation and the generation of conditioned media which inhibited
endothelial cell growth. The arrangement of the cells in this co-culture system mimics thein vivo orientation of vascular cells in which mural cells are separated from the abluminal surface of the endothelium by a fenestrated
internal elastic lamina or basement membrane. Because this co-culture system maintains separable populations of cells in contact
or close proximity allowing for biochemical and molecular analyses of pure populations, it should prove useful for the study
of cell-cell interactions in a variety of systems. 相似文献