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1.
The synthesis of chromosomal proteins and the incorporation of labelled proteins into chromosomes in the mitotic cell cycle
ofHaplopappus gracilis, 2n=4, were traced autoradiographically with3H-arginine,3H-lysine, and3H-tryptophane. The duration of the mitotic cell cycle in the root tip cells was determined by3H-thymidine autoradiography and was measured to be 13.0 hr (G1 1.3 hr, S 6.5 hr, G2 3.8 hr and M 1.4 hr).3H-arginine labelled proteins which were synthesized at S and G2 were found to be incorporated into chromosomes to a greater extent than proteins which were synthesized either at G1, at the transition phase from late S to early G2, or at the mitotic phase. Such varied incorporation was also found in3H-lysine labelled proteins, but not in3H-tryptophane labelled proteins. These findings indicate that the chromosomal proteins are synthesized mainly at S and G2. Some of the3H-arginine labelled proteins which were synthesized during the first mitotic cell cycle, were found to be incorporated into
the chromosomes of the second mitotic cell cycle. The incorporation of the proteins synthesized at one stage of the mitotic
cell cycle was found to occur locally in some regions of the chromosomes, while the pattern of incorporation was observed
to be similar between euchromatic and heterochromatic regions. 相似文献
2.
Smooth muscle cell (SMC) proliferation plays an important role in the pathogenesis of vascular diseases such as atherosclerosis and postangioplasty restenosis. Recently we demonstrated the thiol antioxidantN-acetylcysteine (NAC) inhibits constitutive NF-κB/Rel activity and growth of vascular SMCs. Here we show that treatment of human and bovine aortic SMC with the thiol antioxidant NAC causes cells to exit the cell cycle and remain quiescent as determined by a greatly reduced incorporation of [3H]thymidine and G0/G1DNA content. Removal of NAC from the culture medium stimulates SMCs to synchronously reenter the cell cycle as judged by induction of cyclin D1 and B-mybgene expression during mid and late G1phase, respectively, and induction of histone gene expression and [3H]thymidine incorporation during S phase. The time course of cyclin D1, B-myb,and histone gene expression after NAC removal was similar to that of serum-deprived cells induced to resume cell cycle progression by the addition of fetal bovine serum to the culture medium. Taken together, these results indicate that NAC treatment causes SMCs to enter a reversible G0quiescent, growth-arrested state. Thus, NAC provides an important new method for synchronizing SMCs in culture. 相似文献
3.
R. D. MacLeod 《Chromosoma》1969,27(3):327-337
Roots of Vicia faba were given a one hour pulse label with. 3H-TdR (1 C/ml), either before or after a three hour treatment with a 10–5 M solution of 2,4,5-trichlorophenoxyacetic acid (TCPA). The durations of the various phases of the mitotic cycle were derived from labeled prophase curves, prepared from autoradiographs of lateral root apical meristems. — TCPA was found to lengthen the duration of the mitotic cycle, primarily because it extended the duration of the period of DNA synthesis (S), though post-synthetic interphase (G2) was also longer. No measurements could be made with respect to the duration of presynthetic interphase (G1), because of rapid changes in the lengths of the G2 and S periods following treatment. — As well as extending the duration of S, TCPA treatment also resulted in at least an initial increase in the rate of DNA synthesis and a decrease in the actual number of cells in S. These results have been discussed with respect to the control of the organization of the root apical meristem.Supported by a grant from the Assistant Professor Research Fund of the University of Missouri. 相似文献
4.
Summary Incorporation of15NO3- into amino acids was studied in 3-day-old aerobic rice seedlings (with coleoptile and root) subjected for 24h to anaerobic conditions. The incorporation of15N into glutamate, glutamine and alanine accounted for 89% and 84% of total incorporation in coleoptile and root, respectively. These findings indicate that, after the primary incorporation of15N into glutamate and glutamine, the main fate of nitrate nitrogen in rice seedlings subjected to anoxia is alanine. 相似文献
5.
《FEBS letters》2003
The role of RGS proteins on dopaminergic D2S receptor (D2SR) signalling was investigated in Chinese hamster ovary (CHO)-K1 cells, using recombinant RGS protein- and PTX-insensitive Gαo proteins. Dopamine-mediated [35S]GTPγS binding was attenuated by more than 60% in CHO-K1 D2SR cells coexpressing a RGS protein- and PTX-insensitive GαoGly184Ser:Cys351Ile protein versus cells coexpressing a similar amount of PTX-insensitive GαoCys351Ile protein. Dopamine-agonist-mediated Ca2+ responses were dependent on the coexpression with a GαoCys351Ile protein and were fully abolished upon coexpression with a GαoGly184Ser:Cys351Ile protein. These results suggest that interactions between the Gαo protein and RGS proteins are involved in efficient D2SR signalling. 相似文献
6.
The stimulatory effects of gibberellic acid (GA3) and the inhibitory effects of polyethylene glycol 4000 (PEG) on hypocotyl elongation and cell cycle kinetics in subapical pith cells of dwarf watermelon seedlings (Citrullus lanatus [Thunb.] Matsu and Nakai) were investigated. Mitotic indices (MI) were determined from direct counts of pith cells stained by a modified Feulgen technique. Labeling indices (LI) were determined from direct counts of labeled pith cells sampled 1.5 h after apical applications of3H-thymidine. Root application of 0.32 mM GA3 at 96, 120, or 144 h after sowing resulted in significant increases in both mitotic and labeling indices within 4.5 to 7.5 h following treatment. A single mitotic peak at 13.5 h occurred in all three treatment periods. Labeling peaks were often less defined than mitotic peaks; however, a relatively high proportion of labeled nuclei were usually observed between 7.5 and 9 h after GA3 treatment and at 16.5 h, the latter period coinciding with progression of cells into S phase from the peak period of mitosis. The results suggest that GA3 increases the proportion of rapidly dividing cells in the subapical meristem by increasing the probability that slowly cycling or nonproliferative cells in both 2C and 4C DNA states will enter the proliferative pool. The addition of PEG (200 g/l, = 1.5 mPA) to the rooting medium of dwarf watermelon seedlings inhibited hypocotyl elongation and reduced both mitotic and labeling indices simultaneously within 4.5 h after treatment. Within 24–28 h after PEG treatment, mitotic and labeling indices approached 0. Seedlings transferred from PEG to either water or GA3 exhibited rapid recovery of cell division and hypocotyl elongation. Mitotic and labeling indices increased within 4.5–7.5 h into the recovery period in either water or GA3 and reached control values within 10.5 h. GA3 hastened the recovery from PEG-induced stress. It is concluded that water stress imposed by PEG 4000 causes arrest of cell division in meristematic cells of watermelon seedlings in both G1 and G2 periods. PEG and GA treatments resulted in only a partial and transitory synchronization of the cell cycle. 相似文献
7.
Seven ascidiacyclamide [cyclo(–Ile–oxazoline–d ‐Val–thiazole–)2] (ASC) analogues incorporating the β‐amino acids βIle, βoxazoline, and/or d ‐βVal were synthesized. We then investigated the effects of the position and number of incorporated β‐amino acids on the structure, cytotoxicity, and copper binding by these seven analogues. The structural analyses revealed that both βIle and d ‐βVal favor a gauche‐type θ torsion angles, while βoxazoline favors a trans‐type θ torsion angle. Expansion of the macrocycle by incorporation of βIle or d ‐βVal readily induced molecular folding. On the other hand, the incorporation of two βoxazoline residues strongly extended the peptide conformation, and the incorporation of one was sufficient for the moderate restriction important for conformational equilibrium and cytotoxicity. Despite expansion of the macrocycles, the structure‐cytotoxicity relationships were largely maintained. In studies of complexation of the analogues with Cu (II) ion, the position and number of incorporated β‐amino acids had a large impact on the structure of the metal complex and may contribute to its stabilization. 相似文献
8.
Yohei Miyanoiri Mitsuhiro Takeda Kosuke Okuma Akira M. Ono Tsutomu Terauchi Masatsune Kainosho 《Journal of biomolecular NMR》2013,57(3):237-249
The 1H–13C HMQC signals of the 13CH3 moieties of Ile, Leu, and Val residues, in an otherwise deuterated background, exhibit narrow line-widths, and thus are useful for investigating the structures and dynamics of larger proteins. This approach, named methyl TROSY, is economical as compared to laborious methods using chemically synthesized site- and stereo-specifically isotope-labeled amino acids, such as stereo-array isotope labeling amino acids, since moderately priced, commercially available isotope-labeled α-keto acid precursors can be used to prepare the necessary protein samples. The Ile δ1-methyls can be selectively labeled, using isotope-labeled α-ketobutyrates as precursors. However, it is still difficult to prepare a residue-selectively Leu and Val labeled protein, since these residues share a common biosynthetic intermediate, α-ketoisovalerate. Another hindering drawback in using the α-ketoisovalerate precursor is the lack of stereo-selectivity for Leu and Val methyls. Here we present a differential labeling method for Leu and Val residues, using four kinds of stereo-specifically 13CH3-labeled [U–2H;15N]-leucine and -valine, which can be efficiently incorporated into a protein using Escherichia coli cellular expression. The method allows the differential labeling of Leu and Val residues with any combination of stereo-specifically isotope-labeled prochiral methyls. Since relatively small amounts of labeled leucine and valine are required to prepare the NMR samples; i.e., 2 and 10 mg/100 mL of culture for leucine and valine, respectively, with sufficient isotope incorporation efficiency, this approach will be a good alternative to the precursor methods. The feasibility of the method is demonstrated for 82 kDa malate synthase G. 相似文献
9.
Nathalie Sibille Xavier Hanoulle Fanny Bonachera Dries Verdegem Isabelle Landrieu Jean-Michel Wieruszeski Guy Lippens 《Journal of biomolecular NMR》2009,43(4):219-227
Adding the 13C labelled 2-keto-isovalerate and 2-oxobutanoate precursors to a minimal medium composed of 12C labelled glucose instead of the commonly used (2D, 13C) glucose leads not only to the 13C labelling of (I, L, V) methyls but also to the selective 13C labelling of the backbone Cα and CO carbons of the Ile and Val residues. As a result, the backbone (1H, 15N) correlations of the Ile and Val residues and their next neighbours in the (i + 1) position can be selectively identified in HN(CA) and HN(CO) planes. The availability of a selective HSQC spectrum corresponding
to the sole amide resonances of the Ile and Val residues allows connecting them to their corresponding methyls by the intra-residue
NOE effect, and should therefore be applicable to larger systems. 相似文献
10.
Proliferation of mesangial cells is a common feature of renal disease, and conditioned media from glomerular epithelial and endothelial cells have been found to contain heparin-like molecules that suppress proliferation of rat mesangial cells (RMC). We have partially characterized the glycosaminoglycans that are labeled with 35SO42? by RMC in culture at early passage and examined their ability to inhibit mitogenic stimulation of the cells. Four chondroitin/dermatan sulfate proteoglycans (CS/DSPG) were identified, the largest and smallest of which (Kd of 0.04 and 0.26 on Superose 6) were retained in the cell layer while the other two (Kd = 0.17 and 0.22) were secreted into the medium. Heparan sulfate proteoglycans (HSPG) with Kd values of 0.09, 0.13, and 0.39 were minor components of the cell layer, while a single heparan sulfate (Kd = 0.17) was recovered from the medium. After 16 h of labeling in serum-free medium, about 60% of macromolecular 35S was cell-associated and 40% was in the medium. Cell-associated label consisted of 7% CS/DSPG, 9% HSPG, and 84% free glycosaminoglycan chains (mostly CS/DS), whereas the medium contained 52% CS/DSPG, 17% HSPG, and approximately equal amounts of free HS and CS/DS chains. Bovine lung heparin (1 μg/ml) decreased by 45% the incorporation of [3H]-thymidine into DNA after release of serum-starved RMC from growth arrest. Heparin acted prior to the G1/S interface; arrest of the cells in early S phase with aphidicolin abrogated the heparin response. The endogenous HSPGs had a slight antimitogenic effect on the RMC, but heparan sulfate chains from both the medium and cell layer had a potent effect. On an equivalent mass basis, only the free glycosaminoglycan chains were more potent than heparin in this regard, decreasing thymidine incorporation by over 90% when present at 1 μg/ml. These results demonstrate that heparan sulfate glycosaminoglycans derived from mesangial proteoglycans are potential negative autocrine growth regulators. Proteoglycan metabolism releases these soluble heparan sulfate chains, determining the level of this activity. © 1994 wiley-Liss, Inc. 相似文献
11.
Kawana S Nakagawa K Hasegawa Y Yamaguchi S 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2010,878(30):3113-3118
A simple and rapid method for quantitative analysis of amino acids, including valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met) and phenylalanine (Phe), in whole blood has been developed using GC/MS. In this method, whole blood was collected using a filter paper technique, and a 1/8 in. blood spot punch was used for sample preparation. Amino acids were extracted from the sample, and the extracts were purified using cation-exchange resins. The isotope dilution method using 2H8-Val, 2H3-Leu, 2H3-Met and 2H5-Phe as internal standards was applied. Following propyl chloroformate derivatization, the derivatives were analyzed using fast-GC/MS. The extraction recoveries using these techniques ranged from 69.8% to 87.9%, and analysis time for each sample was approximately 26 min. Calibration curves at concentrations from 0.0 to 1666.7 μmol/l for Val, Leu, Ile and Phe and from 0.0 to 333.3 μmol/l for Met showed good linearity with regression coefficients = 1. The method detection limits for Val, Leu, Ile, Met and Phe were 24.2, 16.7, 8.7, 1.5 and 12.9 μmol/l, respectively. This method was applied to blood spot samples obtained from patients with phenylketonuria (PKU), maple syrup urine disease (MSUD), hypermethionine and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), and the analysis results showed that the concentrations of amino acids that characterize these diseases were increased. These results indicate that this method provides a simple and rapid procedure for precise determination of amino acids in whole blood. 相似文献
12.
《Nucleosides, nucleotides & nucleic acids》2013,32(4-7):1399-1402
The influence of amino acid side chains [derived from: Ala, Val, Leu, Ile, Phe, Tyr(Bzl), Ser(Bzl), Thr(Bzl), Pro, Trp], incorporated into “aminoalkyl” part of PNA monomers, on the temperature-dependent distributions of rotamers about the tertiary amide bond was studied by means of 1H NMR at 0, 25 and 40°C in CDCl3. The ΔG0 values of the energy differences between individual rotamers were calculated. The results may be helpful in the designing of monomers with desirable properties. 相似文献
13.
Treatment of Vicia faba lateral roots with a range of concentrations of 5-aminouracil (5-AU) indicate that cells are stopped at a particular point in interphase. The timing of the fall in mitotic index suggests that cells are held at the S - G2 transition. When cells are held at this point, treatments with 5-AU can be used to estimate the duration of G2 + mitosis/2 of proliferating cells. Treatment with 5-AU can also be used to demonstrate the presence of subpopulations of dividing cells that differ in their G2 duration. Using this method, 5-AU-induced inhibition, we have confirmed that in V. faba lateral roots there are two populations of dividing cells: (a) a fast-dividing population, which makes up ~85% of the proliferating cell population and has a G2 + mitosis/2 duration of 3.3 hr, and (b) a slow-dividing population, which makes up ~15% of dividing cells and has a G2 duration in excess of 12 hr. These estimates are similar to those obtained from percentage labeled mitosis (PLM) curves after incorporation of thymidine-3H. 相似文献
14.
Heterotrophically grown cells ofChlorella protothecoides were transferred to autotrophic medium and allowed to green at 25°C. The protein synthetic activity of the greening cells
measured in terms of incorporation of [35S]-methionine showed a maximum around 20 h of greening and thereafter started declining. Similarly, an analysis of densitometric
tracings of the fluorographic profile of the polypeptides associated with both total cellular fraction and membrane fractions
during different hours of greening revealed that maximum number of polypeptides were getting labelled around 20 h of greening.
At 20 h of greening, the cells were shifted to 40°C and the effect of heat shock on protein synthesis was studied. The heat
shock treatment caused a definite decrease in the incorporation of [35S]-methionine into proteins. Due to heat shock, the synthesis of total soluble proteins was affected much more than that of
the thylakoid membrane bound proteins. When the cells were transferred back to 25°C after a brief period of heat shock at
40°C, there was a considerable recovery in the protein synthesis and this recovery was found to be significant in the case
of soluble proteins, while there was no such definite recovery in the synthesis of thylakoid membrane bound proteins. 相似文献
15.
The time and duration of each phase of the premeiotic interphase were determined in microsporocytes of two clones (S and K
clones) ofTrillium kamtschaticum. After collectionTrillium plants were stored at 3 C or 7 C prior to completion of premeiotic mitosis in archesporial cells. For autoradiography, cells
were explanted in the presence of3H-thymidine to identify the interval of the premeiotic DNA synthesis. Approximate durations of the G1, S and G2 phases for the K clone stored at 3 C were estimated to be 12, 12 and 14 days, respectively. The interval of premeiotic development
was markedly different between clones. A high degree of synchrony in meiotic development, which is usually observed within
anthers up to late meiotic prophase, was confirmed at the S phase, suggesting that synchrony is established during the G1 interval. 相似文献
16.
Derepression of the cell cycle by starvation is involved in the induction of tobacco pollen embryogenesis 总被引:1,自引:0,他引:1
V. Žárský D. Garrido L. Říhová J. Tupý O. Vicente E. Heberle-Bors 《Sexual plant reproduction》1992,5(3):189-194
Summary Microspectrophotometry following Feulgen staining and autoradiography following (3H)-thymidine labelling were used to study cell-cycle events during pollen development in tobacco (Nicotiana tabacum L.). During normal gametophytic pollen development in the anther and in vitro the generative nucleus passes through the S phase to the G2 phase soon after microspore mitosis, while the vegetative nucleus remains arrested in G1 (=G0). During embryogenie induction by an in vitro starvation treatment of immature pollen ongoing DNA replication in the generative nucleus is completed and followed by DNA replication in the vegetative cell in a large fraction of the pollen grains. Addition of the DNA replication inhibitor hydroxyurea to the starvation medium postpones S phase entry until the pollen is transferred to a rich medium and does not affect embryo formation. These results demonstrate that one of the crucial events of embryogenic induction is the derepression of the G1 arrest in the cell cycle of the vegetative cell. 相似文献
17.
Ascidiacyclamides containing oxazoline and thiazole motifs assume square conformations and show high cytotoxicity
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Akiko Asano Takeshi Yamada Taizo Taniguchi Masahiro Sasaki Kenji Yoza Mitsunobu Doi 《Journal of peptide science》2018,24(10)
Four cyclic octapeptides were designed from ascidiacyclamide [cyclo(–Ile–Oxz–D ‐Val– Thz–)2] (ASC, 1 ) to investigate the effects of oxazoline (Oxz) and thiazole (Thz) rings on the structures and cytotoxicities of the peptides. cyclo(–Ile–Thz–D ‐Val–Oxz–)2 ( 2 ) had the same number of Oxz and Thz rings as ASC, but the ring positions were switched. cyclo(–Ile–Oxz–D ‐Val–Thz–Ile–Thz–D ‐Val–Thz–) ( 3 ) and cyclo(–Ile–Thz–D ‐Val–Oxz–Ile–Thz–D ‐Val–Thz–) ( 4 ) contained one Oxz and three Thz rings within the molecule. All Oxz rings were substituted with Thz in cyclo(–Ile–Thz–D ‐Val–Thz–)2 ( 5 ). These analogues had new Oxz and Thz blocks forming the 24‐membered ring. Based on CD spectra and X‐ray diffraction analyses, the structures of all four analogues were classified as square ASC forms. But the structures of 2 and 5 differed from the original square form of 1 , and they showed no cytotoxicity. The structure of 3 was very similar to that of 1 , and 3 showed 10 times greater cytotoxicity than 1 . Although no definite structure of 4 was obtained, it showed three times greater cytotoxicity than 1 . It appears that the position and number of Oxz residues are essential determinants in the structure‐cytotoxicity relationship of ASC analogues. 相似文献
18.
Ongena Marc Jacques Philippe de Pauw Edwin Thonart Philippe 《International journal of peptide research and therapeutics》2001,8(1):21-27
Summary The pattern of pyoverdin-type siderophores produced byPseudomonas putida BTP16 in iron-limited succinate medium supplemented or not with isoleucine was characterized by electrospray ionization mass
spectrometry. Two new compounds were observed in culture supernatants from BTP16 grown in the presence of isoleucine. For
both molecules, mass differences with pyoverdins originally synthesized by the strain were only interpreted considering the
incorporation of Ile into the peptide chain. Further evidence supporting this phenomenon was provided by radioactivity incorporation
in pyoverdins produced in the presence of14C-labeled Ile. Our results are discussed in relation to the non-ribosomal mechanism supposed to be involved in the synthesis
of these molecules. 相似文献
19.
Near-ultraviolet and visible radiations increased the duration of the mitotic cycle in excised pea root meristems primarily by lengthening the duration of the pre-DNA synthetic period (G1). All radiations tested shortened the duration of the post-DNA synthetic period (G2). The most pronounced effects were exhibited by green radiation, which lengthened the duration of the cell cycle, G1, DNA synthesis (S), and mitosis (M), and shortened the duration of G2. Progression of cells arrested by starvation in G1 and G2 into DNA synthesis and mitosis was also affected by light treatments. Green radiation appeared to arrest a group of cells in DNA synthesis as well as in G1 and G2. Meristems receiving green and near-ultraviolet radiations exhibited the most rapid progression of G1 cells through S and G2. 相似文献
20.
Flemming Kristensen Christoph Walker Florence Bettens Franziska Joncourt Alain L. de Weck 《Cellular immunology》1982,74(1):140-149
When mouse thymocytes are stimulated with PHA, the proliferative response is very low, unless the culture medium is enriched with interleukin 1 (IL-1)- or interleukin 2 (IL-2)-containing supernatants. Cytofluorometric analyses show, however, that PHA stimulation generates a significant number of cells with increased RNA content (transition from the G0 to G1 phase of the cell cycle). If IL-2 is added to such cultures, the activated cells complete their process of RNA synthesis and then enter the S phase. The use of IL-2-containing culture medium thus permits one to obtain a high correlation between the number of g1 cells and [3H]thymidine incorporation (r = 0.97). Enrichment with IL-1-containing supernatants also results in a statistically significant correlation (r = 0.68), but the regression lines are markedly different for the two interleukins (s = 20.3 for IL-2 and s = 9.2 for IL-1), when analyzed after 48 hr of incubation. These observations suggest that the G1 phase must be divided into two subcompartments, G1a and G1b, the G1a-G1b transition being an IL-2-dependent event. If the number of G1b cells is used to establish correlations with [3H]thymidine incorporation, all values fall on the same regression line, regardless of culture conditions and of the addition of interleukins. It is concluded that IL-2 regulates lymphocyte proliferation at the level of RNA synthesis (G1a-G1b transition) rather than that of DNA synthesis (G1-S transition). 相似文献