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Dina Raveh Eliezer Huberman Esra Galun 《In vitro cellular & developmental biology. Plant》1973,9(3):216-222
Summary Tobacco protoplasts obtained from leaf mesophyll cells and suspended in agar nutrient medium divided and produced colonies
only when plated at high densities, above 104 cells per ml. At such densities coalescence of the expanding colonies occurred at high frequency.
Nondividing X-irradiated protoplasts used as feeder cells supported division of viable protoplasts plated at densities as
low as 102 cells per ml. The feeder cell technique should thus facilitate the application of screening procedures for the isolation
of colonies originating from single mutated cells occurring in a suspended population. 相似文献
4.
Proteins are active, flexible machines that perform a range of different
functions. Innovative experimental approaches may now provide limited partial
information about conformational changes along motion pathways of proteins.
There is therefore a need for computational approaches that can efficiently
incorporate prior information into motion prediction schemes. In this paper, we
present PathRover, a general setup designed for the integration
of prior information into the motion planning algorithm of rapidly exploring
random trees (RRT). Each suggested motion pathway comprises a sequence of
low-energy clash-free conformations that satisfy an arbitrary number of prior
information constraints. These constraints can be derived from experimental data
or from expert intuition about the motion. The incorporation of prior
information is very straightforward and significantly narrows down the vast
search in the typically high-dimensional conformational space, leading to
dramatic reduction in running time. To allow the use of state-of-the-art energy
functions and conformational sampling, we have integrated this framework into
Rosetta, an accurate protocol for diverse types of structural modeling. The
suggested framework can serve as an effective complementary tool for molecular
dynamics, Normal Mode Analysis, and other prevalent techniques for predicting
motion in proteins. We applied our framework to three different model systems.
We show that a limited set of experimentally motivated constraints may
effectively bias the simulations toward diverse predicates in an outright
fashion, from distance constraints to enforcement of loop closure. In
particular, our analysis sheds light on mechanisms of protein domain swapping
and on the role of different residues in the motion. 相似文献
5.
Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model
Amyloid aggregates of the calcium-binding EF-hand proteins, S100A8 and S100A9, have been found in the corpora amylacea of patients with prostate cancer and may play a role in carcinogenesis. Here we present a novel model system using the yeast Saccharomyces cerevisiae to study human S100A8 and S100A9 aggregation and toxicity. We found that S100A8, S100A9 and S100A8/9 cotransfomants form SDS-resistant non-toxic aggregates in yeast cells. Using fluorescently tagged proteins, we showed that S100A8 and S100A9 accumulate in foci. After prolonged induction, S100A8 foci localized to the cell vacuole, whereas the S100A9 foci remained in the cytoplasm when present alone, but entered the vacuole in cotransformants. Biochemical analysis of the proteins indicated that S100A8 and S100A9 alone or coexpressed together form amyloid-like aggregates in yeast. Expression of S100A8 and S100A9 in wild type yeast did not affect cell viability, but these proteins were toxic when expressed on a background of unrelated metastable temperature-sensitive mutant proteins, Cdc53-1p, Cdc34-2p, Srp1-31p and Sec27-1p. This finding suggests that the expression and aggregation of S100A8 and S100A9 may limit the capacity of the cellular proteostasis machinery. To test this hypothesis, we screened a set of chaperone deletion mutants and found that reducing the levels of the heat-shock proteins Hsp104p and Hsp70p was sufficient to induce S100A8 and S100A9 toxicity. This result indicates that the chaperone activity of the Hsp104/Hsp70 bi-chaperone system in wild type cells is sufficient to reduce S100A8 and S100A9 amyloid toxicity and preserve cellular proteostasis. Expression of human S100A8 and S100A9 in yeast thus provides a novel model system for the study of the interaction of amyloid deposits with the proteostasis machinery. 相似文献
6.
Yeast mating switch Ho endonuclease is rapidly degraded by the ubiquitin system and this depends on the DNA damage response functions, MEC1, RAD9, and CHK1. A PEST sequence marks Ho for degradation. Here we show that the novel F-box receptor, Ufo1, recruits phosphorylated Ho for degradation. Mutation of PEST residue threonine 225 stabilizes Ho, yet HoT225A still binds Ufo1 in vitro. Stable HoT225A accumulates within the nucleus, whereas HoT225E is degraded. Deletion of the nuclear exportin Msn5 traps native Ho in the nucleus and extends its half-life. These experiments suggest that Ho is degraded in the cytoplasm. In mec1 mutants stable Ho accumulates within the nucleus; Ho produced in mec1 cells does not bind Ufo1. Thus the MEC1 pathway has functions both in phosphorylation of Thr-225 for nuclear export and in additional phosphorylations for binding Ufo1. Cells with HO under its genomic promoter, but stabilized by deletion of the Msn5 exportin, proliferate, but are multibudded. These experiments elucidate some of the links between the DNA damage response and degradation of Ho by the ubiquitin system. 相似文献
7.
Raveh Tilleman T Tilleman MM Krekels GA Neumann MH 《Plastic and reconstructive surgery》2004,113(3):857-861
The common excision skin pattern is either a fusiform ellipse or another pattern with dissimilar length and width. The purpose of this study was to define the most advantageous skin pattern regarding skin waste, vertex angle, and scar length. Five skin excision patterns used traditionally for closure of round lesions were analyzed: fusiform ellipse, fusiform circle, rhomboid, mosque, and S-shaped. In the analysis, the pattern characteristics were formulated by geometric principles, from which the results were compared. The smallest skin waste was found in rhomboid and mosque patterns, whereas the largest skin waste was found in the fusiform circle and ellipse. The vertex angle was found to decrease monotonously with the excision length-to-width ratio for all patterns except the mosque shape, which is zero per definition. The paradigm stating that a vertex angle of 30 degrees or less is maintained for length-to-width ratios below 4 in the surgical ellipse was found incorrect. It holds only for rhomboid and S-shaped excisions. The scar length was found almost independent of the pattern, with a variance of 3 percent. The authors conclude that the most advantageous surgical skin patterns are the rhomboid and mosque excisions. 相似文献
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9.
Amitai G Adani R Limanovich O Teitlboim S Yishay S Tveria L Yacov G Meshulam H Raveh L 《Chemico-biological interactions》2008,175(1-3):249-254
Organophosphorus hydrolases (OPH) such as mammalian plama paraoxonase (PON1) detoxify asymmetric toxic organophosphorus (OP) nerve agents by preferentially hydrolyzing the less toxic P(+) optical isomer. In order to develop new OPHs with broader stereoselectivity we have prepared a series of asymmetric fluorogenic organophosphonates (Flu-OPs). Such Flu-OPs may serve as molecular probes for screening large libraries of OP hydrolases during directed evolution. Flu-OPs were prepared as methylphosphonates (MPs) diesters containing either ethyl (E), isopropyl (I), cyclohexyl (C) or pinacolyl (P) groups that are structural congeners of the nerve agents VX, sarin, cyclosarin and soman, respectively. The second ester bond was formed with fluorescent moieties that are either 3-cyano-4-methyl-7-hydroxy coumarin (MeCyC) or 1,3-dichloro-7-hydroxy 9,9-dimethyl-9H-acridin-2-one (DDAO). To further characterize the Flu-OPs as surrogates of their respective nerve agents, we have studied the reactivation of Flu-OP-inhibited AChE using 2-PAM and toxogonin (TOX). AChE was 90–95% inhibited by all Flu-OPs (0.36–0.9 (M) and then was reactivated by either 2-PAM or TOX. TOX caused a more rapid reactivation than 2-PAM with the following rank order; EMP > IMP > CMP. TOX was also shown to be a better reactivator than 2-PAM for AChE inhibited by the nerve agents VX and cyclosarin. PMP-AChE could not be reactivated by either TOX or 2-PAM, similarly to aging of PMP-AChE formed by inhibition with soman.Racemic CMP-MeCyC was used for screening two new PON1 variants from a neutral library of PON1. These multiple mutation variants include replacement of active site amino acid residues. Neither mutation in these new variants appeared in PON1 variants previously discovered by directed evolution using symmetric Flu-OP. Detoxification rate of cylcosarin by these new PON1 variants was rather slow indicating the need to further screen PON1 clones using optically active Flu-OPs. Therefore, we have separated enzymatically the P(−) enantiomer of CMP-MeCyC and determined its 98% purity using chiral HPLC. 相似文献
10.
Nucleolar development in the cleaving chick germ up to the formation of the primary hypoblast was followed through a series of well-defined uterine and early incubated stages both by light and electron microscopy. Well-established criteria of nucleolar morphology were used for determining the developmental stage of onset of rRNA synthesis. By these criteria rRNA synthesis was first observed at midcleavage in uterine stage VII [1] germs. This could be correlated with the first morphogenetic event—the posterio-anteriorly orientated formation of the area pellucida which results in a bilaterally symmetrical blastoderm. 相似文献