Changes in some chromatin and cytoplasmic enzymes of perinatal rat hepatocytes during culture

Summary Hepatocytes prepared from rats at various perinatal stages were cultured in selective medium that does not allow fibroblastic cell growth. Cell population remained homogeneous during the culture. Hepatocytes undergo divisions for a period, which varies according to the stage of development of the rat. Light and electron microscope observations showed the presence of numerous cytoplasmic organelles; moreover, hydrocortisone-induced structures similar to bile canaliculi. Chromatin protein kinase decreased rapidly during culture except in samples prepared from 17-day fetuses in which it remained unchanged for 2 days and decreased to a lesser extent afterwards. Chromatin nonhistone proteins were incubated with (γ-³²P) ATP and the phosphorylation pattern analyzed on polyacrylamide gels. Many radioactive peaks were observed in chromatin proteins from 17-day fetuses; they were much lower in proteins from 19-day fetuses. The phosphorylation pattern was analyzed in hepatocytes after 2 days of culture. Many radioactive peaks were observed with proteins from heapatocytes taken from 17-day fetuses; no radioactivity was observed in proteins from 19-day fetuses. This is in contrast with the absence of radioactive peaks in chromatin proteins from adult rat hepatocytes. In cytoplasm, aldolase and pyruvate kinase specific activities varied according to the age of the rat. They strongly decreased during culture except in hepatocytes from 15-and 17-day fetuses, in which they remained stable for at least 5 days. The stability of chromatin and cytoplasmic enzymes in hepatocytes from 17-day fetuses could result from their ability to be regulated by hormones that are secreted at this stage of development.     

Phospholipids in chromatin: incorporation of [32P]O4(2-) in different subcellular fractions of hepatocytes

Nuclear, chromatin and microsomal fractions were isolated from hepatocytes prepared from rats injected with [32P]O4(2-) and killed subsequently at times between 1 and 48 h. Specific activities of the total phospholipids (PL) were determined for each subcellular fraction. The major points noted were the initial specific activity of the chromatin PL was half that of both nuclear and microsomal PL at 1 h; the first peak of labelling occurred at 6 h in both nuclear and microsomal PL, but was 3 h later (9h) in the chromatin PL; and a second peak of labelling occurred in the chromatin and microsomal PL, but not in those of the nuclei. On fractionation of the PL, the major and most metabolically active components were phosphatidylcholine + phosphatidylethanolamine, whilst sphingomyelin accounted for only about 8 per cent of the total PL. The chromatin and microsomal fractions were somewhat similar in their labelling patterns though with a delayed peaking of activity in the chromatin. This is indicative of a synthesis and transport of PL from the microsomes to the chromatin.     

Dynamics of chromatin position in the interphase nucleus volume

In this article, we attempt to analyze the relationship between the processes which occur in the nucleus and the dynamics of chromatin, as well as to classify the changes in the position of chromatin in the cell nucleus during the lifetime of the cell. The proposed concept integrates possible types of chromatin movement within the nucleus.     

Reactive oxygen species and nitric oxide are involved in ABA inhibition of stomatal opening

Although nitric oxide (NO) and reactive oxygen species (ROS) are essential signalling molecules required for mediation of abscisic acid (ABA)-induced stomatal closure, it is not known whether these molecules also mediate the ABA inhibition of stomatal opening. In this study, we investigated the role of NO and ROS in the ABA inhibition of stomatal opening in Vicia faba. ABA induced both NO and ROS synthesis, and the NO scavenger reduced the ABA inhibition of stomatal opening. Exogenous NO and hydrogen peroxide (H2O2) also inhibited stomatal opening, indicating that NO and ROS are involved in the inhibition signalling process. An inhibitor of nitric oxide synthase (NOS) reversed the ABA inhibition of stomatal opening. Either the NO scavenger or the NOS inhibitor also reversed the process in the H2O2 inhibition of stomatal opening. We found that in the ABA inhibition of stomatal opening, NO is downstream of ROS in the signalling process, and NO is synthesized by a NOS-like enzyme.     

动物早期胚胎发育中染色质结构的继承和重编程

染色质开放性和染色质三维高级结构在基因表达和调控中发挥着非常重要的作用,广泛参与分化、发育、肿瘤发生等细胞生理过程,是表观遗传研究的热点领域之一。动物胚胎发育起始于终端分化的卵子受精形成全能性的受精卵。在精卵结合的过程中,染色质开放性和染色质三维高级结构发生了剧烈的变化,经历继承、重编程、重新建立的过程,并指导调控受精卵分化发育最终成为多细胞、多器官组织的新生命个体。本文介绍了近年来研究染色质开放性和染色质三维高级结构的实验分析技术手段,染色质结构在动物早期胚胎发育过程中的变化规律及其在早期胚胎发育中的作用,染色质结构与其他表观遗传信息(甲基化、组蛋白修饰等)关系方面的重要研究进展和存在的科学问题,以期为表观遗传调控早期胚胎发育的研究提供参考。     

Changes in ribonucleoprotein particle and chromatin organization induced by liposomes in isolated nuclei

Nuclei isolated from rat liver, incubated in the presence of liposomes of different phospholipids, undergo typical modifications: chromatin dispersion and reduction of the interchromatin granules in nuclei incubated with negatively charged liposomes and increase of the chromatin density and of the number and size of the interchromatin granules in nuclei incubated with neutral liposomes. The possibility that the observed modifications are caused by an impairment of the transport and translocation of ribonucleoproteins belonging to the inner nuclear matrix, is suggested by the results obtained by radiotracer techniques on the release of RNA from liposome-incubated nuclei.     

Endogenous degradation of rat liver chromatin studied by agar gel electrophoresis of nuclei

Direct agar gel electrophoresis of incubated rat liver nuclei revealed that most of the chromatin is rapidly converted to stable, large fragments, showing identical electrophoretic mobility. Short and long term incubation gave the same results. The analysis of deproteinized DNA under nondenaturing as well as denaturing conditions showed, however, a correlation between the DNA size pattern and the time of incubation. Our data on the persistance of large and uniform in size chromatin fragments despite the presence of cleaved DNA in them may indicate naturally footprinted regions of chromatin, implying most probably some strong ordered interaction of chromatin constituents. It seems that some substantial unknown features of higher order structure of chromatin are preserved in rat liver nuclei isolated and digested under the experimental conditions used.     

Cadmium induced apoptotic changes in chromatin structure and subphases of nuclear growth during the cell cycle in CHO cells

CHO cells were grown in the presence of 1 M CdCl₂ and subjected to ATP-dependent replicative DNA synthesis after permeabilization. By decreasing the density of the cell culture replicative DNA synthesis was diminishing. At higher than 2 × 10⁶ cell/ml concentration Cd had virtually no effect on the rate of DNA replication. Growth at higher cell concentrations could be supressed by increasing Cd concentration. After Cd treatment cells were synchronized by counterflow centrifugal elutriation. Cadmium toxicity on cell growth in early and mid S phase led to the accumulation of enlarged cells in late S phase. Flow cytometry showed increased cellular and nuclear sizes after Cd treatment. As the cells progressed through the S phase, 11 subpopulations of nuclear sizes were distinguished. Apoptotic chromatin changes were visualized by fluorescent microscopy in a cell cycle dependent manner. In the control untreated cells the main transitory forms of chromatin corresponded to those we have published earlier (veil-like, supercoiled chromatin, fibrous, ribboned structures, chromatin strings, elongated prechromosomes, precondensed chromosomes). Cadmium treatment caused: (a) the absence of decondensed veil-like structures and premature chromatin condensation in the form of apoptotic bodies in early S phase (2.2–2.4 average C-value), (b) the absence of fibrous structures, the lack of supercoiled chromatin, the appearance of uncoiled ribboned chromatin and perichromatin semicircles, in early mid S phase (2.5–2.9 C), (c) the presence of perichromatin fibrils and chromatin bodies in mid S phase (2.9–3.2 C), (d) early intra-nuclear inclusions, elongated forms of premature chromosomes, the extrusion and rupture of nuclear membrane later in mid S phase (3.3–3.4 C), (e) the exclusion of chromatin bodies and the formation of clusters of large-sized perichromatin granules in late S phase (3.5–3.8 C) and (f) large extensive disruptions and holes in the nuclear membrane and the clumping of incompletely folded chromosomes (3.8–4. C).     

Canopy development of a determinate and an indeterminate cultivar of Vicia faba L. under contrasting plant distributions and densities

Field experiments were conducted in 1987 and 1988 to quantify differences in canopy formation between an indeterminate and a determinate genotype of Vicia faba L., grown at two plant densities and three spatial distributions. The number of stems per unit area produced by determinate plants was related to the growth rate before flowering. Leaf production per stem per unit of thermal time was similar in both plant types, but twice as many leaves per stem were produced by the indeterminate cultivar. The indeterminate cultivar produced fewer and smaller leaves in the warmer and drier weather of 1988 than in 1987. The determinate genotype produced similar sizes and numbers of leaves in both years, but fewer tillers developed in 1988 than in 1987. Accordingly, leaf mass per unit ground area was greater in 1987 than in 1988 in both genotypes. Except during early flowering, relationships between leaf mass and leaf area were constant, with higher specific leaf areas in the determinate than the indeterminate genotype. Shoot dry matter partitioning into leaves was identical in both years for indeterminate plants, but differed in determinate ones.
It is concluded that canopy development is regulated through individual leaf weight and leaf number per stem in non-tillering indeterminate, and by stem numbers per unit area in tillering determinate plants.     

Gamma irradiation-induced apoptosis in murine pre-B cells prevents the condensation of fibrillar chromatin in early S phase

Local changes in chromatin structure leading to temporally distinct geometric forms were characterized in nuclei of reversibly permeabilized cells. Reversal of permeabilization was tested by 3H-thymidine incorporation and trypan blue dye exclusion. Apoptotic changes were visualized in a cell cycle dependent manner at the chromatin level by fluorescent microscopy in non-irradiated cells and after 400 rad Co60 irradiation. Fluorescent microscopy of chromatin structures belonging mainly to the interphase of the cell cycle confirmed the existence of specific geometric forms in nuclei of non-irradiated cells. In this control population, the following main transitory forms of condensing chromatin were distinguished: decondensed veil-like structures and fibrous structures in early and mid S phase (2.0-2.5 average C-value), chromatin bodies, semicircles later in mid S phase (3.0-3.5 C), precondensed chromosomes in late S (3.5-3.7 C) and metaphase chromosomes at the end and after S phase (3.7-4.0 C). Our results show that upon gamma-irradiation (a) the cellular and nuclear sizes were increased, (b) the DNA content was lower in each elutriated subpopulation of cells, (c) the progression of the cell cycle was arrested in the early S phase at 2.4 C value, (d) the chromatin condensation was blocked between the fibrillar chromatin and precondensed elongated chromosomal forms, and (e) the number and size of apoptotic bodies were inversely correlated with the progression of the cell cycle, with many small apoptotic bodies in early S phase and less and larger apoptotic bodies in late S phase.     

Differential effects of ecotoxicological substances on lipid peroxidation of plant cell protoplasts

Abstract The determination of ethane formation in the light and dark in the presence and absence of SHAM provides a memthod for the elucidation of the mechanism of membrane destruction in plant cell protoplasts, induced by chemical treatment. Accordingly, PCP is postulated to act via radical and enzymic-mediated lipid peroxidation. In contrast, HgCl₂ appears to inhibit the enzymic pathway, and causes damage to the membranes via a radical mediated mechanism alone.     

A protein fraction stably linked to DNA in plant chromatin

DNA from the chromatin of roots and shoots of maize seedlings was isolated and extensively deproteinized by repeated high-salt extractions, by subsequent deproteinizations eliminating noncovalently associated proteins and by CsC1 density gradient centrifugation. Nevertheless, a protein component resisting all extraction procedures was found firmly associated to plant nuclear DNA. This component was responsible for the ¹²⁵I uptake when a DNA preparation had been labeled by the chloramine-T method.A residual oligodeoxynucleotide-oligopeptide complex was obtained after extensive digestions of the initial DNA-protein complex with proteases and nucleases. The stability of this complex to different chemical treatments suggested a phosphoester type of a linkage. The hydrolysis of this complex by phosphodiesterases indicated that the protein component was linked to plant chromosomal DNA through a phosphodiester bond formed by a hydroxyaminoacid and a 5-end DNA phosphate. Two-dimensional tryptic peptide mapping of the proteins isolated from the two maize chromatins revealed a high degree of similarity to the corresponding proteins of animal origin. Its conservative structure suggests an important role for this protein component in the functioning of the eukaryotic genome.     

Synthesis of lipids in isolated nuclei from rat thymus and liver cells

Synthesis of lipids was studied in isolated nuclei from rat thymus and liver cells. On incubation of the isolated nuclei with [2-¹⁴C]acetate and [1-¹⁴C]glycerol, the label was intensively incorporated into phospholipids and with a significantly lower intensity into fatty acids and cholesterol. Only trace amounts of radioactivity were detected in the lipids of chromatin prepared from isolated thymus nuclei after their incubation, and this suggested that lipids were mainly synthesized on the nuclear membrane. On the preincubation of thymus tissue homogenate with [2-¹⁴C]acetate and the subsequent isolation of the nuclei and chromatin, the radioactivity of chromatin lipids was comparable to the radioactivity of nuclear lipids. The findings suggested that in the isolated nuclei the newly synthesized lipids were not transported into chromatin from the nuclear membrane. The specific radioactivities of individual phospholipids and fatty acids were different in the isolated nuclei and in nuclei obtained from preincubated homogenate. Mechanisms of lipid synthesis in isolated nuclei and causes of the different radioactivities of lipids in the isolated nuclei and in the nuclei obtained from the preincubated homogenate are discussed.     

The superstructure of chromatin and its condensation mechanism

Changes in the structure of chicken erythrocyte chromatin fibres at low ionic strength resulting from enzymatic digestion, thermal denaturation and binding of Netropsin and Distamycin were monitored by synchrotron X-ray solution scattering. Digestion with micrococcal nuclease confirms the previous assignment of the 0.05 nm^-1 band to an interference between nucleosomes with an average distance of 23 nm. The results of thermal denaturation indicate that above 40°C there is a progressive increase of the internucleosomal distance and that above 60°C the characteristic structure of the chromatin fibre is destroyed. Binding of Netropsin and Distamycin also results in an increase of the internucleosomal distance which can be estimated to correspond to about 0.2 nm/mol.     

Nucleotides induce higher order chromatin degradation

Higher order chromatin degradation (HOCD) is a stepwise dismantling of the genome through the excision of chromatin loops and their oligomers at matrix attachment regions (MARs) during the early stages of programmed cell death. Although HOCD ultimately leads to the inactivation of the genome and cell death, a partial HOCD in cells receiving sublethal signals may result in the loss of genetic stability leading to neoplasia, degeneration, and aging. The present study was undertaken to determine the role of protein poly(ADP-ribosyl)ation in HOCD. Nuclei isolated from rat glioma C6 cells were able to carry poly(ADP-ribosyl)ation as assessed by the incorporation of ³²P-NAD⁺ into TCA-insoluble fraction. Under the same experimental conditions, millimolar NAD⁺ induced rapid HOCD in nuclei. However, while poly(ADP-ribosyl)ation was totally abrogated by specific inhibitor, benzamide, NAD⁺-induced HOCD was unaffected. Benzamide also failed to inhibit HOCD induced by H₂O₂ exposure in intact cells. These results indicate that HOCD is not mediated through chromatin poly(ADP-ribosyl)ation, and that NAD⁺ activates MAR-associated endonuclease or facilitates the access of the enzyme to DNA by other mechanisms. Furthermore, other nucleotides including NADP⁺, ATP, UTP, GTP, and CTP were also found to induce HOCD in isolated nuclei indicating that HOCD is controlled by nucleotide-related ligands.     

植物三维染色质构型研究进展

染色质在细胞核内的缠绕、折叠及其在细胞核内的空间排布是真核生物染色质构型的主要特征。在经典DNA探针荧光原位杂交显微观察的基础上,基于新一代测序技术的Hi-C及ChIA-PET染色质构型捕获技术已经被广泛应用于动物及植物细胞核染色质构型的研究中,并以新的角度定义了包括:染色体(质)域(chromosome territory)、A/B染色质区室(compartment A/B)、拓扑偶联结构域(topological associated domains,TADs)、染色质环(chromatin loops)等在内的多个更为精细的染色质构型。利用以上两种主流技术,越来越多的植物物种染色质构型特征被鉴定、分析和比较。本文系统分析和总结了近年来以植物细胞为模型的细胞核染色构型领域取得的重要成果,包括各级染色质构型特征的组成、建立机制和主要影响因素等。在此基础上,分析了目前研究植物染色质构型技术的瓶颈和突破性的技术进展,并对后续研究主要关注的问题和研究内容进行了展望,以期为相关领域的研究提供更多的理论参考和依据。     

Distribution of nitrate reductase activity in Vicia faba: Effect of nitrate and plant genotype

The short term effect of NO₃⁻ (12 mM) on nitrate reductase (NR. EC 1.6.6.1) activity has been studied in the roots, nodules and leaves of different genotypes of Vicia faba L. at the end of vegetative growth. Root and leaf NR activity responded positively to NO₃⁻ while nodule activity, where detected, proved to he strongly inhibited. The withdraw of this NO₃⁻ from the solution consistently reduced activity in the roots and leaves but surprising, promoted a significant increase in nodule activity, which matched or surpassed that of control plants On the other hand, nodules developed in the presence of 8 mM NO₃⁻ expressed an on average 141% higher level of NR activity than did controls. This effect was observed even in nodules with negligible control activity. In any case, a naturally occurring mutant (VF17) lacking root and nodule NR activity is described. The results indicate that in V. faba. the effects of NO₃⁻ and plant genotype on NR activity depended on plant organ and time of NO₃⁻ application, hut the distribution of NO₃⁻ reduction through the plain was mainly dependent on plant genotype, and to a lesser extent on NO: supply and plant age.