X-ray microanalysis of unfixed chromatin in dinoflagellate cells prepared by a monolayer cryotechnique

The fine structural appearance and elemental composition of unfixed chromatin is described for cells of the dinoflagellate Prorocentru micans prepared by a freeze-drying technique. This involves rapid freezing, cryo-dehydration and resin infiltration of a monolayer of cells dispersed over a resin base.The fine structure of the nucleus appears quite different from chemically fixed and dehydrated cells. In stained sections, the chromosomes are seen as pale areas containing diffuse chromatin, and are surrounded by electron-dense nucleoplasm which has a reticulate substructure.X-ray microanalysis reveals the presence of high levels of chromatin-associated Ca and transition metals Fe, Ni, Cu and Zn, in accordance with previous observations on chemically processed cells. Calculation of elemental mass fractions by on-line computer demonstrates an overall ratio of one divalent cation per two phosphorus groups (or per two nucleotides). This ratio is similar to that obtained previously for chemically fixed chromatin, and shows that the precise overall association of metals is not primarily determined by the process of fixation.     

Some observations on the structure, cation content and possible evolutionary status of dinoflagellate chromosomes

SICEE, D. C., 1984. Some observations on the structure, cation content and possible evolutionary status of dinoflagellate chromosomes. Dinoflagellate chromosomes have a well-ordered structure, as observed in living cells, glutaraldehyde/osmium tetroxide-fixed cells, ultrathin cryosections and freeze-etch preparations. It is suggested that the stabilization of this chromatin in the living cell is largely mediated by divalent cations, acting as bridging molecules between the DNA superstructure and the protein matrix. Studies using X-ray micro-analysis and autoradiography have shown that these chromosomes have high levels of bound Ca and transition metals, and that these are associated with both the DNA and surrounding proteins.
The organization and stabilization of chromatin in dinoflagellate chromosomes is quite different from that of the cells of other eukaryotes, but shows some resemblance to the dispersed chromatin of bacteria. The evolution of dinoflagellate chromosomes from a prokaryote-like ancestral genome is attributed to two main factors–the retention of a primitive cationic non-histone stabilization system, and a pronounced evolutionary trend towards high DNA values. On this theory, dinoflagellate chromosomes are phylogenetically distinct from all other eukaryote chromosomes, and provide a separate evolutionary route for the attainment of high DNA levels and increased cell size.     

X-ray microanalysis of toluidine blue stained chromosomes: a quantitative study of the metachromatic reaction of chromatin

The stoichiometry of metachromatic staining of chromatin by toluidine blue was investigated in isolated metaphase chromosomes from L929 cells using X-ray microanalysis. Microspectrophotometric measurements revealed that a hypsochromic shift (from 595 to 570 nm) occurs in toluidine blue stained chromosomes in relation to the staining solution. Under the electron microscope, stained chromosomes. After toluidine blue staining, X-ray microanalysis of chromosomes revealed a large increase for sulphur counts and a considerable increase for Fe and Cu counts, while the signal of Mg, Ca, Cl, K and Zn was reduced. After subtraction of the intrinsic sulphur signal, S/P ratios of 0.82--for euchromatic arms--and 0.85--for centromeric heterochromatin--were obtained. They are considered representative of dye/DNA phosphate ratios. These results indicate the occurrence of a nearly stoichiometric binding of toluidine blue to chromatin DNA and suggest that an external dye stacking is responsible for the metachromatic staining of metaphase chromosomes.     

X-ray microanalysis of toluidine blue stained chromosomes: a quantitative study of the metachromatic reaction of chromatin

Summary The stoichiometry of metachromatic staining of chromatin by toluidine blue was investigated in isolated metaphase chromosomes from L929 cells using X-ray microanalysis. Microspectrophotometric measurements revealed that a hypsochromic shift (from 595 to 570 nm) occurs in toluidine blue stained chromosomes in relation to the staining solution. Under the electron microscope, stained chromosomes showed higher electron density than control chromosomes. After toluidine blue staining, X-ray microanalysis of chromosomes revealed a large increase for sulphur counts and a considerable increase for Fe and Cu counts, while the signal of Mg, Ca, Cl, K and Zn was reduced. After subtraction of the intrinsic sulphur signal, S/P ratios of 0.82 — for euchromatic arms — and 0.85 — for centromeric heterochromatin — were obtained. They are considered representative of dye/DNA phosphate ratios. These results indicate the occurrence of a nearly stoichiometric binding of toluidine blue to chromatin DNA and suggest that an external dye stacking is responsible for the metachromatic staining of metaphase chromosomes.     

Kinetics of metal elimination in oysters from a contaminated estuary

In oysters Crassostrea gigas translocated from a metal-enriched estuary (Gironde, France) to a comparatively clean site, the Bay of Bourgneuf (France), Cd, Cu and Zn concentrations were determined monthly in the whole soft tissues, or in different fractions (cytosolic or insoluble) of gills and digestive glands. In all cases, the concentrations of all of the three metals decreased logarithmically and half-lives were always shortest for Cd (86-251 days). After 4 months, the Cd concentration had become not significantly different from the threshold considered safe for human consumption (1 mg kg(-1) wet wt.). In the digestive gland, half-lives were similar in cytosolic and insoluble fractions. In contrast, in the gills, elimination patterns differed markedly between these fractions. The long half-lives calculated for divalent metals in the insoluble fraction of the gills (1505 and 3010 days for Zn and Cu, respectively) is possibly due to a fossilization of metals in intracellular membrane-bound inclusions as shown previously in Ostrea edulis. It is interesting to underline that elimination is fastest for cytosolic metals compared to the insoluble fraction.     

CT banding of human chromosomes

Summary This paper decribes a study of the role of certain cations in the alkaline pretreatment step used in the CT technique for chromosome band formation. Treatment of human chromosomes with ammonium bases or with the hydroxides of the monovalent alkali metals Na, K, or Li resulted in their rapid disintegration, unless very short treatment periods or diluted solutions were used. In the latter cases a subsequent staining produced a weak G-banding pattern. The chromosomes appeared to be much less sensitive to treatment with the hydroxides of the divalent alkaline earch metals Ba, Sr, Ca, and Mg. Staining after exposure to these hydroxides yielded R-banding patterns. The reduced alkali sensitivity of the chromosomes and the reverse banding pattern formation observed are probably the result of a chromatin stabilization by the divalent cations of the alkaline earth metals. It is proposed that not only in the R-band formation with the hydroxides of the alkaline earth metals but also in that obtained by other techniques, chromosome stabilization plays an important role.     

解磷微生物修复土壤重金属污染研究进展

土壤重金属污染问题日益严重,具有普遍性、隐蔽性、表聚性、不可逆性等特点,已经成为环境污染治理中的热点、难点问题。解磷微生物能够依靠自身的代谢产物或通过与其他生物的协同作用,将土壤中的难溶性磷转化为可供植物吸收利用的磷,具有多重植物促生长功能和重金属解毒能力,可在重金属毒害水平下,促进植物生长、提高植物抗病能力、克服重金属对植物生长的不利影响,从而增强重金属修复植物的生存竞争力。从解磷微生物的研究现状入手,介绍了解磷微生物对土壤重金属污染的修复能力,综述了解磷微生物对土壤重金属污染修复的作用机制,分析了目前解磷微生物在重金属修复过程中存在的问题,并提出了今后研究的方向,为重金属污染土壤的修复提供了新思路。     

Interaction of divalent cations with beta-galactosidase (Escherichia coli).

Although the addition of various divalent metals to beta-galactosidase resulted in apparent activation, only Mg2+ and Mn2+ actually did activate. The apparent activation by the other divalent metals was shown to be due to Mg2+ impurities. Calcium did not activate, but experiments suggested that it did bind. Other divalent metals which were studied failed to bind. The dissociation constants for Mg2+ and Mn2+ were 2.8 X 10(-7) and 1.1 X 10(-8) M, respectively, and in each case one ion bound per monomer. These constants corresponded very closely to apparent values which were obtained from activation studies. The apparent binding constant for Ca2+, obtained from competition studies, was 1.5 X 10(-5) M. Data were obtained which showed that Mg2+, Mn2+, and Ca2+ all compete for binding at a single site. Of interest and of possible molecular biological importance was the observation that, while Mg2+ bound noncooperatively (n = 1.0), Mn2+ did so in a highly cooperative manner (n = 3.4). The binding of Mn2+ (as compared to Mg2+) resulted in a twofold drop in the Vmax for the hydrolysis and transgalactosylis reactions of lactose but had little effect on the Vmax of hydrolysis of allolactose, p-nitrophenyl beta-D-galactopyranoside (PNPG), or o-nitrophenyl beta-D-galactopyranoside (ONPG); Km values were not effected differently for any of the substrates by Mn2+ as compared to Mg2+. When very low levels of divalent metal ions were present (0.01 M EDTA added) or when Ca2+ was bound with lactose as the substrate, a greater decrease was observed in the rate of the transgalactosylic reaction than in the rate of the hydrolytic reaction, and the Km values for lactose and ONPG were increased. Of the three divalent metal ions which bound to beta-galactosidase, only Mn2+ had significant stabilizing effects toward denaturing urea and heat conditions.     

Dinoflagellate chromosome behaviour during stages of replication.

In most dinoflagellate species, chromosomes are characterized by an almost continuous condensation of the nucleofilaments throughout the cell cycle and the absence of longitudinal differentiation as Q, G, or C banding. Their supercoiled architecture is maintained by divalent cations and structural RNAs. Their chromatin is devoid of histones and nucleosomes and their DNA composition is distinctive: in several species, more than 60% of thymines are replaced by a rare base, hydroxymethyluracil. We report here an immunofluorescence (conventional and confocal laser scanning microscopy, CLSM) and immunogold transmission electron microscopy (TEM) analysis of some stages of the early replication process in Prorocentrum micans dinoflagellate cells, after long pulse incorporation (3, 6 or 9 days) with 50 micrograms/ml bromodeoxyuridine (BrdU) in the presence of 5-fluoro-2'-deoxyuridine (FUdR) and BrdU antibody technique (BAT) detection. The large DNA content (45 pg per nucleus) of P. micans cells is compacted on 100 chromosomes, 10 microns in length. In early S-phase, DNA replication sites are revealed as fluorescent domains organized in clusters, which appear in the periphery of the nucleus unlike other eukaryotes. In late S-phase, the number of labelled clusters increased; helically distributed, they did not appear synchronously in the whole chromosome. Under TEM, spherical domains of equivalent diameter appeared located all along the chromosomes after 6 days BrdU pulse. Replication occurs, but in our experimental conditions, segregation of daughter chromosomes was never observed. The blockade of the cell cycle after BrdU incorporation intervening just before the segregation of daughter chromosomes is discussed.     

Intrinsic phosphatase activity of bovine brain calcineurin requires a tightly bound trace metal

The divalent metal requirement of intrinsic phosphatase activity was investigated using native and trypsinized calcineurin. This was assessed by examining (1) the stimulation of the enzyme by various metals, (2) the inhibition of the enzyme activity by metal chelators (EDTA and EGTA), and (3) the restoration by various metals of the activity of the EDTA-inhibited calcineurin phosphatase. The results supported the view that a tightly bound trace metal is necessary for expression of the phosphatase activity of calcineurin and implicate Mn2+ as the tightly bound metal.     

Distribution of tightly bound non-histone proteins in chromatin fractions obtained by DNAase II digestion

Digestion of pig liver chromatin with DNAse II afforded three different fractions which were characterized in terms of their DNA, RNA and tightly bound non-histone protein content, their DNA fragment size and their template activity. Two of these fractions are soluble after digestion with DNAase II and have been separated on the basis of their different solubility in MgCl2. A third fraction is not solubilized even after extensive digestion, although the size of its DNA is comparable to that of the enzyme solubilized fractions. The three fractions show qualitatively and quantitatively different distribution of tightly bound non-histone proteins, with specific protein components in each fraction; furthermore the non-solubilized fraction is greatly enriched in proteins tightly bound to DNA. From all the data obtained it can be suggested that the tightly bound proteins of the insoluble fraction may play, directly or indirectly, a role in maintaining an organized chromatin structure.     

Nuclear assembly of purified Crythecodinium cohnii chromosomes in cell—free extracts of Xenopus laevis eggs

Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation,nuclear envelope assembly,and nuclear reconstitution.Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle.The assembled nuclei,being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii.However,incubation of dinoflagellate Cyrthecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinium cohnii cells does not induce nuclear reconstitution.     

Dielectric properties of polyelectrolytes. III. Effect of divalent cations on dielectric increment of polyacids

Dielectric dispersion of polyacrylic acid (PAA) and polystyrene sulfonic acid (PSS) was measured in the presence of divalent cations. Effects of divalent ions were studied by neutralization with varying ratios of sodium hydroxide and divalent base concentration, addition of salts of divalent cations, and neutralization with divalent bases only. Two dispersion regions were observed in all cases, i.e., low-frequency dispersion (10²–10⁴ Hz) and high-frequency dispersion (10⁵–10⁶ Hz). The dielectric increment increases in the presence of sodium and alkaline earth metal ions together, but not with sodium and transition metal ions. This is due to the increment of low-frequency dispersion and is attributable to the fluctuation of bound counterions which is explained by our theory previously reported.¹ In the case of PAA neutralized with large fractions of divalent ions, or with divalent ions only, the increment is very small because of reduction of the fluctuation by interaction between bound ions at the neighboring sites and reduction of the effective length of polyion probably due to chelation by divalent ions. There are some differences among the effects of Mg⁺⁺, Ca⁺⁺, and Ba⁺⁺ on dielectric increment which may result from affinity or chelating ability of these ions.     

Kinetic properties of microsomal UDP-glucuronyltransferase. Regulation by metal ions

Treatment of microsomes with EDTA abolishes the stimulation of glucuronidation produced by UDP-N-acetylglucosamine. Addition of divalent metal ions to EDTA-treated microsomes restores the sensitivity of UDP-glucuronyltransferase to UDP-N-acetylglucosamine. Regulation of the activity of this enzyme by UDP-N-acetylglucosamine depends, therefore, on the presence of divalent metal ions. In addition, divalent metals increase the rate of glucuronidation of p-nitrophenol at V_max. The data indicate that metals are essential for the efficient function of UDP-glucuronyltransferase.     

Binding of transferrin and metal ions by suspensions of reticulocyte-rich rabbit blood

Reticulocyte binding of Fe(III)_-transferrin and transferrin complexes with other metal ions have been compared by different investigators. The functional relevance of this comparison is not clear, therefore transferrin complexes with Fe(III), Cu(II), Mn(II) and Zn(II) have been studied further by DEAE-cellulose chromatography and by measurement of transferrin and metal uptakes by rabbit reticulocytes.Human Fe-transferrin behaved as a weaker anion than apotransferrin during DEAE-cellulose chromatography; since Fe-transferrin has a higher negative charge than apotransferrin and behaves a as stronger anion in electrophoretic systems, the chromatographic result was the opposite of that anticipated. The lower affinity of human Fe-transferrin for DEAE-cellulose is probably caused by a redistribution of charged groups on the surface of transferrin molecules when Fe(III) ions are bound and is therefore considered to be dependent on molecular conformation. Apotransferrin and divalent metal-transferrin complexes were found to have nearly equal affinities for DEAE-cellulose, thus the effect on surface charge of human transferrin molecules induced by binding Fe(III) appeared to be limited to that metal ion.Iron uptake by reticulocytes was associated with increased binding of transferrin to the cell surface: uptake of divalent metals occured without a concomitant increase in transferrin uptake or evidence of a specific metal-transfer process. Cu-transferrin was rapidly dissociated during incubation with cells.The effect of Fe(III)_binding on human transferrin molecules was to alter the molecular affinity for charged surfaces, namely DEAE-cellulose and reticulocyte membranes. This was less apparent with rabbit transferrin. Transferrin complexes with divalent metals behaved as apotransferrin in the process of association with reticulocytes.     

X-ray microanalysis of chromatin-bound period IV metals inGlenodinium foliaceum: A binucleate dinoflagellate

Summary Each vegetative cell of the dinoflagellateGlenodinium foliaceum possesses two distinct types of nucleus, both of which have high levels of chromatin-bound Period IV (Periodic Table) metal elements.The typical dinoflagellate (dinocaryotic) nucleus has chromatin which differs from the atypical (supernumerary) nucleus in its high degree of condensation and in the related high levels of P, Ca, and Transition metals Fe, Ni, Cu, and Zn. The complete absence of detectable Fe and Ni in the supernumerary chromatin represents a major difference which may relate to differences in phyllogenetic origin of the two nuclei.The two types of chromatin show close similarities at the molecular level, including the possession of 40 atoms of Period IV elements per 100 atoms of P—of which approximately half are Ca atoms, and half Transition metals. In both cases, the levels of Ca and Zn show a high correlation with the level of P, suggesting a direct association of these particular metal atoms with nucleic acid phosphate groups. The close similarity in metal binding at the molecular level suggests that the association of Period IV elements with the two types of chromatin is unrelated to any differences in chromatin proteins—such as the presence or absence of histones.     

Evaluation of Phosphorus Species in the Bed Sediments of an Atlantic Basin: Bioavailability and Relation with Surface Active Components of the Sediment

In this study, total phosphorus (P) concentrations and P fractions in the bed sediments of the Anllóns River (NW Spain) have been examined. The Lowest Effect Level of the Ontario Sediment Quality Guidelines was trespassed for total phosphorus (600 mg kg^?1) at three out of the five sampling sites which, in addition, showed an average of 43% of bioavailable phosphate. The inorganic P fractions (loosely bound P, redox-sensitive P, metal oxide-bound P, carbonate and apatite P, and residual P) showed that inorganic P was mostly found as metal oxide bound P. The difference between total phosphorus and the sum of the inorganic P forms determined in these extracts is attributed to P associated to organic compounds being solubilized in some of the extractants, and represented in average 58% of total phosphorus. With the exception of loosely bound P, all the P fractions showed significant positive correlations with each other and with total phosphorus.     

The potential use of metallothionein in the clam Ruditapes decussatus as a biomarker of in situ metal exposure

This work aimed to validate the relationship between metallothioneins (MTs) and metals (Cd, Cu and Zn) in field conditions. Specimens of the marine bivalve Ruditapes decussatus (Linné, 1758) from Gargour were transferred in two sites: Gargour and Sidi Mansour, both situated along the south-eastern coast of Tunisia. The bivalves were removed from pairs of cages at day 0 (date of transplantation), day 62 and day 132. Metals (Cd, Cu and Zn) and MTs were determined in the subcellular fractions of the digestive gland. In Gargour, metal and MT levels increased significantly after 62 days of transplantation. However, they showed modest and non-significant variations in Sidi Mansour. Zn was mainly associated with the insoluble fraction, whereas Cd and Cu percentages in the soluble and the insoluble fractions were equivalent. Simple correlation analysis showed a positive and significant relationship between MTs and each metal. If all metals were taken together, multiple correlations showed that MTs were significantly correlated with Cd and Zn, with an important coefficient for Cd, but no significant relationship was observed for Cu. Gel filtration chromatography showed that in the heat stable fraction, the only cytosolic SH rich compounds have an apparent low molecular mass (about 15 kDa), which could correspond to metallothioneins. In the digestive gland of R. decussatus MTs responded to moderate increases of metal contamination, without interference with other factors, and could be a promising biochemical indicator of metal exposure.     

The distribution of quinacrine on chromosomes as determined by X-ray microanalysis

The distribution of quinacrine and protein sulphur has been compared with that of DNA in euchromatic and heterochromatic regions of mouse chromosomes stained with the fluorescent dye quinacrine, using X-ray microanalysis. Heterochromatin tends to bind relatively more quinacrine than euchromatin, and contains a greater concentration of sulphur. Measurements of quinacrine fluorescence, when compared with quinacrine binding, show that the excitation of fluorescence is more efficient when the dye is bound to euchromatin than when it is bound to heterochromatin. Although this observation is consistent with the hypothesis that the dull quinacrine fluorescence of mouse centromeres is due to quenching by guanine residues, two other factors should also be considered: the lower absolute amount of dye bound to the centromeres, and a concentration-dependent quenching of fluorescence.