Partial characterization of nuclear binding sites for retinol delivered by cellular retinol binding protein

Retinol (vitamin A alcohol), which plays an important role in the differentiation of epithelia, can be transferred to chromatin in vitro. Rat liver chromatin can accept retinol in a specific and saturable manner only when the retinol is presented as a complex with cellular retinol-binding protein (CRBP). A partial characterization of the nuclear components responsible for accepting retinol is reported here. A preparation of solubilized chromatin isolated from liver nuclei was able to accept retinol from its complex with CRBP as described previously for nuclei and chromatin. The binding of retinol to chromatin was noncovalent. However, chromatin prepared from nuclei which were incubated with DNase I or micrococcal nuclease did not accept retinol specifically. Chromatin in the form of mono and dinucleosomes also did not accept retinol. However, treatment of nuclei with RNase did not affect the specific binding of retinol. Furthermore, it has been found that retinol was not transferred to purified double or single stranded DNA. These results are interpreted to indicate that the transfer of retinol to specific nuclear binding sites requires a higher order of chromatin structure than that occurring in nucleosome preparations.     

Fractionation of L-cell chromatin into DNA, RNA, and protein fractions on Cs2SO4 equilibruim density gradients

A new procedure is described for fractionation of chromatin into DNA, RNA, and total chromatin protein. By isopycnic gradient centrifugation of chromatin preparations in Cs₂SO₄ solutions containing dimethylsulfoxide and sodium sarcosyl it is possible to obtain highly purified fractions of these components. The method gives a very high yield of these chromatin fractions unlike some other methods, where irreversible binding to columns occurs. Also with this method it is possible to obtain highly concentrated fractions, which after a simple dialysis step, can be conveniently analyzed by polyacrylamide gel electrophoresis.Nuclei from L-929 cells were isolated by a method involving citric acid or by a method using a nonionic detergent. The yields of DNA obtained by both methods was compared. Chromatin was isolated from purified nuclei (prepared in either of the above ways) in two different ways also. In one method, chromatin was extracted from nuclei with 1 m NaCl. A second method involving fractionation of lysed nuclei in sucrose and metrizamide solutions was also used. The yields of DNA obtained by both methods was compared. There appears to be little nuclear membrane contamination of any of these chromatin preparations.A preliminary analysis of L-929 cell chromatin total RNA and protein fractions on polyacrylamide and agarose gels has been made. Both fractions appear to be quite complex with a wide spectrum of subcomponents of differing S values.     

Isolated thallus-associated compounds from the macroalga Fucus vesiculosus mediate bacterial surface colonization in the field similar to that on the natural alga

This study investigated whether surface-associated compounds isolated from the macroalga Fucus vesiculosus had the potential to mediate microbial and/or macrobial epibiosis similar to that on the natural alga. To selectively yield thallus-associated compounds and avoid contamination by intracellular algal compounds, cell lysis was monitored by surface microscopy of algal cells and chemical profiling of algal surface extracts by coupled gas chromatography mass spectroscopy. The optimized extraction resulted in polar and non-polar algal surface extracts. The non-polar surface extract was immobilized in hydrogel, the polar surface extract was homogeneously perfused through the gel to ensure a temporally constant delivery of polar extract components. During a 7 day field trial, bacterial biofilms were formed on control gels and gels featuring polar and/or non-polar extract components. PERMANOVA revealed that bacterial community profiles on controls and on gels featuring polar or non-polar extract were significantly different from the profile on F. vesiculosus, while the profile on the gels bearing both polar and non-polar extracts was not. Moreover, the polar surface extracts inhibited the settlement of barnacle cyprids. Considering the pronounced effects of bacterial biofilms on invertebrate larval settlement, these results suggest that algal surface chemistry affects macrofouling not only directly but also indirectly, via its control of biofilm formation and composition.     

Screening of oleaginous algal strains from Chlamydomonas reinhardtii mutant libraries via density gradient centrifugation

Microalgae have a high potential to be utilized as feedstock for biofuels because they have high growth rates and do not compromise food production. Commercialized algae-based biofuel production relies on the development of strains with high lipid content. Based on the relatively low density of lipids compared to other cellular components, density gradient centrifugation was used to isolate high lipid content algal strains from Chlamydomonas reinhardtii mutant libraries. The correlation between cell density and lipid content was confirmed by analysis of Nile red fluorescence intensity, total lipids, and total fatty acid methyl ester content. A strain isolated by this screening method had 50% higher lipid content and 7% lower cell density than the parent wild-type strain. Consequently, we demonstrated that screening of algal strains with low cell density via continuous density gradient centrifugation allows simple, rapid, and inexpensive screening for high lipid content strains.     

Quantitative proteomic analysis of yeast DNA replication proteins

Chromatin is dynamically regulated, and proteomic analysis of its composition can provide important information about chromatin functional components. Many DNA replication proteins for example bind chromatin at specific times during the cell cycle. Proteomic investigation can also be used to characterize changes in chromatin composition in response to perturbations such as DNA damage, while useful information is obtained by testing the effects on chromatin composition of mutations in chromosome stability pathways. We have successfully used the method of stable isotope labeling by amino acids in cell culture (SILAC) for quantitative proteomic analysis of normal and pathological changes to yeast chromatin. Here we describe this proteomic method for analyzing changes to Saccharomyces cerevisiae chromatin, illustrating the procedure with an analysis of the changes that occur in chromatin composition as cells progress from a G1 phase block (induced by alpha factor) into S phase (in the presence of DNA replication inhibitor hydroxyurea).     

Flow Cytometry studies of marine phytoplankton populations

Abstract

Flow cytometry has been applied to the study of phytoplankton populations and cell components. In this work, cells cycle studies on three marine diatoms cultures were carried out by a flow cytometer. Phaeodactylum tricomutum, Chaetoceros simplex and Thalassiosira allenii, showed different DNA patterns in G1, G2, S, phases. This situation may be related to the specific algal physiology. Cultures of P. tricomutum were maintained in 4 media with different silicates concentrations. The lack of silicates did not seem produced a significative cells arresting in the cell cycle phases. During the experiment, the cell fraction in S phase, decreased in all media tested. These preliminary results could be further developed to apply flow cytometry to environmental problems in order to identify general algal groups and study algal physiology in different growth conditions.     

Action of heparin on mammalian nuclei. II. Cell-cycle-specific changes in chromatin organization correlate temporally with histone H1 phosphorylation

The interaction of the polyanion heparin with the inner histones of chromatin has been used to detect changes in chromatin organization associated with cell-cycle traverse. Synchronized populations of Chinese hamster cells were obtained either in early G1 or near the G1/S boundary. The rate of interaction of heparin with chromatin-associated inner histones was measured using nuclei isolated from synchronized cell populations in different phases of the cell cycle. A G1-specific decrease in rate of interaction of heparin with inner histones was observed and found to be independent of the presence of hydroxyurea during traverse of G1. A further decrease in heparin-inner histone interaction occurred in late S and G2. These changes correlate temporally with the interphase phosphorylation(s) of histone H1. This correlation is discussed within the framework of current models of higher order chromatin structure (i.e. organization above the nucleosome level). Analysis of the cooperativity of interaction of heparin with inner histones was performed using the kinetic analog of the Hill equation. This analysis suggests that the organization of inner histones on chromatin does not undergo large variations during the cell cycle.     

Study of chromatin decondensation factors in human spermatozoids by flow cytometry

To date, the mechanisms responsible for radical change of chromatin structure in male germ cells during fertilization are unclear. Evidence suggesting the existence of proteolytic nuclear enzymes in mature human spermatozoids are presented in this work. The possible role of these previously unknown proteases in decondensation of chromatin of spermatozoids in a fertilized ovum is discussed. Application of the flow cytometry technique has shown that treatment of human spermatozoid nuclei with SH-reagents leads not only to destruction of disulfide bonds between protamine molecules that is necessary for their effective utilization but also induces specific endogenous proteolytic activity that consequently results in rather fast decondensation of chromatin followed by proteolytic cleavage of nuclear proteins. A chromatin decondensation process can be almost totally blocked by serine protease inhibitors and components of seminal fluid. An original cytochemical approach of binding of fluorescently labeled protease inhibitor to the target of investigation has been used in order to visualize the localization of proteases in male germ cell nuclei. The results of our study suggest that one of the factors of chromatin reorganization involved in the formation of male pronucleus is endogenous nuclear protease of spermatozoids, which is activated by glutathione or other SH-components of ovum cytoplasm.     

小麦珠心细胞衰退过程酸性磷酸酶的超微细胞化学定位(英文)

采用磷酸铅盐沉淀技术对小麦（ Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ Ｌ．） 珠心细胞衰退过程进行了酸性磷酸酶的超微细胞化学定位研究。结果显示,在未有明显衰退迹象的一些珠心细胞中,酸性磷酸酶只出现在细胞核轻微凝聚的染色质上。随珠心细胞衰退程度的逐渐增大,其衰退特征越来越明显,酸性磷酸酶依次在细胞质中较小液泡、细胞壁、线粒体、质体以及内质网等结构上出现活性反应。紧连胚囊的珠心细胞衰退程度最大,细胞严重变形,酸性磷酸酶定位于细胞绝大部分结构中,但此时变形的细胞核则无酸性磷酸酶活性反应。研究结果表明,小麦珠心细胞的衰退过程中,酸性磷酸酶存在一个有规律的变化,支持珠心细胞的衰退是属于细胞程序性死亡类型的观点     

Ultracytochemical Localization of Acid Phosphatase in Nucellar Cells of Wheat During Degeneration

The distribution of acid phosphatase activity in nucellar ceils of wheat ( Triticum aestivum L. ) during degeneration has been studied using the lead precipitation method at the electron microscopic level. Acid phosphatase was localized in the slightly condensed nuclear chromatin in nucellar cells without any sign of ultrastructural degeneration. As the nuceilar cells started degenerating, the enzyme activity in the cell was observed, in the order from small vacuoles to cell walls, mitochondria, plastids and endoplasmic reticulum. Enzyme activity was the highest in most components of the nuceilar ceils adjacent to the embryo sac where the degeneration of nucellar cells was the strongest, but it was not observed in the nuclei of the degenerated nucel]ar cells. The results indicated that the degeneration of nucellar cells was a progressive and orderly process and supported that the degeneration of nuceilar cells was a programmed cell death.     

The 4D Nucleome: Genome Compartmentalization in an Evolutionary Context

4D nucleome research aims to understand the impact of nuclear organization in space and time on nuclear functions, such as gene expression patterns, chromatin replication, and the maintenance of genome integrity. In this review we describe evidence that the origin of 4D genome compartmentalization can be traced back to the prokaryotic world. In cell nuclei of animals and plants chromosomes occupy distinct territories, built up from ~1 Mb chromatin domains, which in turn are composed of smaller chromatin subdomains and also form larger chromatin domain clusters. Microscopic evidence for this higher order chromatin landscape was strengthened by chromosome conformation capture studies, in particular Hi-C. This approach demonstrated ~1 Mb sized, topologically associating domains in mammalian cell nuclei separated by boundaries. Mutations, which destroy boundaries, can result in developmental disorders and cancer. Nucleosomes appeared first as tetramers in the Archaea kingdom and later evolved to octamers built up each from two H2A, two H2B, two H3, and two H4 proteins. Notably, nucleosomes were lost during the evolution of the Dinoflagellata phylum. Dinoflagellate chromosomes remain condensed during the entire cell cycle, but their chromosome architecture differs radically from the architecture of other eukaryotes. In summary, the conservation of fundamental features of higher order chromatin arrangements throughout the evolution of metazoan animals suggests the existence of conserved, but still unknown mechanism(s) controlling this architecture. Notwithstanding this conservation, a comparison of metazoans and protists also demonstrates species-specific structural and functional features of nuclear organization.     

Structural domains and conformational changes in nuclear chromatin: a quantitative thermodynamic approach by differential scanning calorimetry

A good deal of information on the thermodynamic properties of chromatin was derived in the last few years from optical melting experiments. The structural domains of the polynucleosomal chain, the linker, and the core particle denature as independent units. The differential scanning calorimetry profile of isolated chromatin is made up of three endotherms, at approximately 74, 90, and 107 degrees C, having an almost Gaussian shape. Previous work on this matter, however, was mainly concerned with the dependence of the transition enthalpy on external parameters, such as the ionic strength, or with the melting of nuclei from different sources. In this paper we report the structural assignment of the transitions of rat liver nuclei, observed at 58, 66, 75, 92, and 107 degrees C. They are representative of the quiescent state of the cell. The strategy adopted in this work builds on the method developed for the investigation of complex biological macromolecules. The heat absorption profile of the nucleus was related to the denaturation of isolated nuclear components; electron microscopy and electrophoretic techniques were used for their morphological and molecular characterization. The digestion of chromatin by endogenous nuclease mimics perfectly the decondensation of the higher order structure and represented the source of several misinterpretations. This point was carefully examined in order to define unambiguously the thermal profile of native nuclei. The low-temperature transitions, centered around 58 and 66 degrees C, arise from the melting of scaffolding structures and of the proteins associated with heterogeneous nuclear RNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Behavior of filamentous cyanobacterium Anabaena spp. in water column and its cellular characteristics

From a practical viewpoint, cyanobacteria have two opposite aspects: one is that some of the species participate in water bloom, and the other is that they may be used in agriculture as a biofertilizer. Akinete, a dormant cell, is the key to the engineering analysis of both cases. In order to analyze the behavior of cyanobacteria in water, Anabaena cylindrica, a filamentous cyanobacterium, was investigated in terms of the behavior of algal biomass in a water column and its characteristics in akinete formation. The algal culture transferred to a glass cylinder settled down initially and then floated to the surface of the water column, forming an algal mat due to oxygen bubbles generated extracellularly by photosynthesis. The differentiation of vegetative cells into akinetes was not induced by light limitation but iron depletion. Akinetes tended to separate from trichomes and settle down because their density is higher than that of water. This phenomenon suggests an operational method for removing the source of water bloom from an aquatic environment, or harvesting akinetes as the seed of biofertilizers.     

Specific binding of lectins with the nucleus of the sea urchin embryo and changes in the lectin affinity of the embryonic chromatin during the course of development

(1) Embryonic cells of sea urchins were made permeable by treating them with glycerol solution for the purpose of allowing penetration of macromolecules into the cell. With the use of such permeabilized cells, several kinds of fluorescent dye-labeled lectins were introduced into the cell, and it was found that some lectins showed notable affinity with the nucleus as compared with cytoplasmic structures. (2) Isolated chromatin was incubated with several kinds of fluorescent dye-labeled lectins in vitro, and the amount of lectins bound with the chromatin was measured by fluorometry. By means of this method, the lectin-binding capacity of chromatin was estimated and compared at various stages of development. It was found that lectins could be classified into three groups according to the mode of binding with the chromatin: (a) Extent of binding increased notably at the gastrula stage (Con A and RCA-120); (b) extent of binding showed a temporary decrease at the gastrula stage (TTA); and (c) very low level of binding was maintained throughout all stages, and no particular change was observed at any stage of development (WGA, SBA, and UEA-I). (3) These facts seem to suggest that lectin-binding components are contained in sea urchin chromatin, and that drastic changes occur in these components of chromatin at the stage of gastrulation. It was proposed that the lectin-binding components such as proteoglycans and glycoproteins may play regulatory roles in embryonic chromatin at early stages of development.     

Intranuclear relocalization of matrix binding sites during T cell activation detected by amplified fluorescence in situ hybridization

We describe a method for analyzing the nuclear localization of specific DNA sequences, with special emphasis on their binding status to the nuclear matrix, depending on the developmental stage of the cells. This method employs high-resolution fluorescence in situ hybridization procedures. For our studies, it was important to examine the nuclear localization of a particular gene locus. Previously, however, it was not possible to detect a single-copy genomic sequence using a DNA probe less than several kilobases in size. We describe here a signal amplification technique based on tyramide which makes such a task possible. Using this method, we monitored single-copy loci using a short, 509-bp DNA sequence that binds in vivo to the T cell factor SATB1 within T cell nuclei, high-salt-extracted nuclei (histone-depleted nuclei generating "halos" with distended chromatin loops), and the nuclear matrix, before and after T cell activation. We found that these loci were anchored onto the nuclear matrix, creating new bases of chromatin loops, only after T cell activation. This experimental strategy, therefore, enabled us to detect the changes in higher order chromatin structure upon activation and study gene regulation at a new dimension: the loop domain structure. The methods shown here can be widely applied to explore other functions involving chromatin, including recombination and replication.