pH-induced conformational changes in chromatin subunits measured by circular dichroism and immunochemical reactivity

Changes in the conformational state of chromatin core particles from chicken erythrocytes were studied by both immunochemical and biophysical methods as a function of pH and ionic strength. When the pH of core particles in a solution of ionic strength 3, 60 or 220 mM was lowered from pH 7.5, a sharp transition in the circular dichroism spectrum of DNA monitored between 320 and 260 nm was observed at pH 6.65. This change in DNA ellipticity was totally reversible. Binding to core particles of antibodies specific for histones H2B, H2A, H3 and for the IRGERA (synthetic C-terminal) peptide of H3 was used to follow changes in histone antigenicity. Binding was studied in the pH range 7.5-5.35, and at ionic strength of 60 and 220 mM. A change in reactivity of some histone epitopes was observed around pH 6.2–6.5. However, the changes observed by circular dichroism and antibody binding pertain to different components of chromatin subunits and they probably reflect independent phenomena. The alteration in accessibility of these determinants at the surface of core particles was completely reversible and was dependent on ionic strength. The conformation changes in core particles occurring near physiological ionic strength and pH may reflect dynamic changes in chromatin structure that possess functional significance.     

Involvement of the globular domain of histone H1 in the higher order structures of chromatin

We have attacked H1-containing soluble chromatin by α-chymotrypsin under conditions where chromatin adopts different structures.Soluble rat liver chromatin fragments depleted of non-histone components were digested with α-chymotrypsin in NaCl concentrations between 0 mm and 500 mm. at pH 7, or at pH 10, or at pH 7 in the presence of 4 m-urea. α-Chymotrypsin cleaves purified rat liver histone H1 at a specific initial site (CT) located in the globular domain and produces an N-terminal half (CT-N) which contains most of the globular domain and the N-terminal tail, and a C-terminal half (CT-C) which contains the C-terminal tail and a small part of the globular domain. Since in sodium dodecyl sulfate/polyacrylamide-gel electrophoresis CT-C migrates between the core histones and H1, cleavage of chromatin-bound H1 by α-chymotrypsin can be easily monitored.The CT-C fragment was detected under conditions where chromatin fibers were unfolded or distorted: (1) under conditions of H1 dissociation at 400 mm and 500 mm-NaCl (pH 7 and 10); (2) at very low ionic strength where chromatin is unfolded into a filament with well-separated nucleosomes; (3) at pH 10 independent of the ionic strength where chromatin never assumes higher order structures; (4) in the presence of 4 m-urea (pH 7), again independent of the ionic strength. However, hardly any CT-C fragment was detected under conditions where fibers are observed in the electron microscope at pH 7 between 20 mm and 300 mm-NaCl. Under these conditions H1 is degraded by α-chymotrypsin into unstable fragments with a molecular weight higher than that of CT-C. Thus, the data show that there are at least two different modes of interaction of H1 in chromatin which correlate with the physical state of the chromatin.Since the condensation of chromatin into structurally organized fibers upon raising the ionic strength starts by internucleosomal contacts in the fiber axis (zig-zag-shaped fiber), where H1 appears to be localized, it is likely that in chromatin fibers the preferential cleavage site for α-chymotrypsin is protected because of H1-H1 contacts. The data suggest that the globular part of H1 is involved in these contacts close to the fiber axis. They appear to be hydrophobic and to be essential for the structural organization of the chromatin fibers. Based on the present and earlier observations we propose a model for H1 in which the globular domains eventually together with the N-terminal tails form a backbone in the fiber axis, and the nucleosomes are mainly attached to this polymer by the C-terminal tails.     

Investigations on chromatin condensation of hen erythrocyte nuclei in vitro. An ultrastructural and biochemical study

Hen erythrocyte nuclei, isolated in non-buffered sucrose, (3 mM Mg²⁺, at low ionic strength) have a condensed chromatin to which hemoglobin is bound. Incubation of the isolated nuclei in 0.125 M phosphate-buffer solutions of increasing pH induces a release of the bound hemoglobin and a swelling of the nuclei. Between pH 6.4 and 7.0 both processes reach a plateau and above pH 7.0 a second steep increase in nuclear volume is observable, leading to nuclear disruption above pH 7.4. Titration at low ionic strength shifts the process of hemoglobin release and nuclear swelling to higher pH values. Hemoglobin release and nuclear swelling are fully reversible by backtitration to pH 5.8. The nuclear swelling and shrinking is observed electron-microscopically as decondensation and condensation of the chromatin. The results of these investigations suggest that hemoglobin acts like a cation in the maintenance of nuclear condensation under the conditions used for the isolation of hen erythrocyte nuclei, but that this action is unlikely the physiological mechanism causing chromatin condensation during the maturation of erythroid cells.     

Ionic milieu and volume adjustments in detergent-extracted thymic nuclei

Detergent-extracted isolated thymic lymphocyte nuclei were incubated in buffers containing 3 mM Ca++ and Mg++ and varying concentrations of Na+ and K+. Nuclei in 15 mM K+, 15 mM Na+ had a smaller size, smaller interchromatin spaces, and less packed chromatin than nuclei in the absence of these ions, but their water content relative to dry mass was not significantly different. NMR relaxation properties of water protons in these different nuclei were different, and nuclei in 15 mM K+, 15 mM Na+ contained twice as much K and Na as in the buffer solution. These findings indicate that the hydration of chromatin bodies and the size of the interchromatin spaces are sensitive to the free monovalent ion concentrations. When isolated nuclei were exposed to solutions containing 150 mM total concentration of K + Na, the nucleoplasm became disrupted and the hydration index was greater. The results are discussed in regard to possible mechanisms of nuclear volume control in cells.     

Protoplasmic pH modifies water and solute transfer in beta vulgaris root vacuoles

Volume changes were studied in Beta vulgaris storage root vacuoles, using video microscopy, when exposed to hypotonic conditions. The osmotic gradient was either step-applied or progressively imposed in perfusion experiments. Preincubation at low pH (6.6) or with HgCl2 strongly reduced the vacuoles' water permeability, measured in step experiments. Furthermore, the volumetric response depended on the rate with which the aniso-osmotic condition was established. In perfusion experiments a "plateau value" (osmotic equilibrium or steady-state volume value) was observed, which was significantly lower than the theoretically expected one. Furthermore, if vacuoles were preincubated in presence of HgCl2 or at low pH and then the hypo-osmotic challenge was applied in perfusion experiments, a still lower "plateau value" was observed. This reduction was concentration-dependent and completely reversible. In these conditions, when HgCl2 concentration was 300 mM or medium pH was 6.6, the volume change was abolished. In other experiments, when urea iso-osmotically replaced mannitol, a reversible, pH-dependent volumetric response was observed. These results can be interpreted accepting that 1) mercury-sensitive water channels, present in the studied structure, were blocked by low pH during the hypo-osmotic challenge; 2) modification of water permeability prevents excessive swelling during the osmotic shock; 3) the effectiveness of this last mechanism depended on the osmotic challenge rate; and 4) additionally, urea reflection coefficients were also modified by reduced medium pH.     

Effect of chromatin decondensation on the intranuclear matrix

We have studied the effect of chromatin condensation on the morphology of the residual structures isolated from rat liver nuclei. DNAse I digestion followed by high salt extraction of nuclei in the presence of Mg++ yields residual structures consisting of a dense peripheral layer surrounding an internal network, similar to those described by Berezney and Coffey [6]. These structures are stable at low ionic strength in the presence of EDTA. When nuclei swollen in EDTA are digested with DNAse II in the presence of EDTA, structures devoid of internal network are obtained even without subsequent treatment with high salt. When swollen nuclei are exposed to Mg++ a specific recondensation of chromatin takes place. The residual structures from recondensed nuclei are similar to those isolated from control nuclei in the presence of Mg++. The results suggest that the integrity and stability of the intranuclear matrix are acquired in the course of the isolation procedure and this is favoured by chromatin condensation.     

Divalent cations enhance ammonia excretion in Lahontan cutthroat trout in highly alkaline water

The Lahontan cutthroat trout lives under highly alkaline and saline conditions in Pyramid Lake, Nevada (pH 9.4; 0.2 mmol 1⁻¹ Ca⁺⁺; 7.3 mmol 1⁻¹ Mg⁺⁺). These experiments were conducted to study the possible roles of water Ca⁺⁺ and Mg⁺⁺ concentrations on ammonia excretion in the Lahontan cutthroat trout under highly alkaline conditions. The basic protocol of the experiments was to determine ammonia excretion rates during the following three exposure periods (each of 3-h duration) in sequence: (a) in normal lake water; (b) in soft lake water with the divalent cation concentrations reduced; and (c) in the soft lake water with either Ca⁺⁺ or Mg⁺⁺ (or no divalent cations added) added back at the appropriate lake water concentration. The soft-water exposure caused a significant reduction in ammonia excretion to about half of the control (original lake water) levels. When either Ca⁺⁺ or Mg⁺⁺ was added to the soft water in the third exposure period, the ammonia excretion rates were increased more than twofold back to lake water levels.     

Increases in internal Ca2+ and decreases in internal H+ are induced by general anesthetics in squid axons

Squid axons were injected with arsenazo III and treated with sea water containing compounds usually classified as general anesthetics, (pentanol-decanol and a variety of hydrocarbons and their derivatives). Such treatment led to an increase in absorbance by arsenazo III at wavelengths sensitive to [Ca]i. The effect was independent of the presence or absence of Ca++ in sea water and it was not modified by substances that release Ca from internal stores. The effect was easily reversible. In axons injected with phenol red or impaled with a glass electrode sensitive to H+, a similar treatment led to an alkalinization that was also readily reversible. Both Ca release and the change to an alkaline pH had identical time courses. The dose required for action by all of the chemical agents studied could be predicted from a knowledge of their fractional saturation in sea water, i.e. from their thermodynamic activity. For compounds with 8-10 carbon atoms, Ca-release effects can occur at concentration less than those necessary to block either conduction or Na/Ca exchange. A special chemical agent was octylamine, which induced a marked rise in pHi and in addition its nonionic form produced the typical Ca release associated with general anesthetics.     

Intramolecular esterification by lipase powder in microaqueous benzene: effect of moisture content

In view of the biochemical reaction catalyzed by enzyme powder suspended in a water-insoluble organic solvent, an equation was derived to estimate the amount of water bound to the enzyme powder. With this equation, an apparent adsorption isotherm between free water (water freely dissolved in benzene) and bound water (water bound to crude lipase powder of Pseudomonas fluorescens) was obtained. A direct lactonization reaction (synthesis of cyclopentadenolide from 15-hydroxypen-tadecanoic acid) catalyzed by crude lipase powder of Pseudomonas fluorescens was carried out batchwise in microaqueous benzene at 40oC. A kinetic model of the enzymatic reversible lactonization reaction was derived, from which the effect of moisture content on the initial reaction rate with a fully hydrated enzyme was mathematically expressed. The observed initial reaction rate first increased, then decreased with increasing moisture content, giving rise to the maximum rate at a certain level of the moisture content. The drop in the reaction rate at lower moisture content was due to a lesser hydration of the enzyme molecule (hydration-limited) and the decrease in the reaction rate at higher moisture content was attributed to the dependence of the true initial rate of the reversible reaction on the moisture content (true reversible reaction limited), and could be simulated by the kinetic model. The equilibrium yield approached 100% at a lower moisture content.     

Effects of exposure to sublethal levels of cadmium upon water-electrolyte status in the goldfish (Carassius auratus)

Goldfish were exposed to sublethal levels of cadmium (means of 44.5 and 380 μg Cd⁺⁺/l) for periods of 25 and 50 days, and their water-electrolyte status evaluated by reference to plasma and muscle levels of sodium, potassium and chloride and muscle water content. Significant changes in plasma chloride, tissue potassium and tissue water content were observed after 25 days in both test solutions. Specimens held at the more dilute cadmium concentration were apparently able to compensate for most of the initial cadmium effect and, after 50 days exposure, were characterized only by a continuing depression in plasma sodium level. This suggests that the cadmium MATC value for this species under the conditions employed is probably less than 45 μg Cd⁺⁺/l. Goldfish exposed to 380 μg Cd⁺⁺/l for 50 days exhibited significant deviations in plasma sodium and chloride levels as well as in tissue sodium and water content, and these parameters may provide useful indices of cadmium effects at sublethal concentrations.     

Recognition of acidic phospholipase A2 activity in plasma membranes of resident peritoneal macrophages

Phospholipase (PLase) activities in the plasma membrane of guinea pig peritoneal macrophages were studied, as these enzymes having such activity may be candidates for the release of arachidonic acid (AA) from phosphatidylcholine (PC). An AA release system operating at acidic pH was identified in the macrophage plasma membrane and characterized. This membrane-bound acidic PLase A2 had an optimum pH at 4.5, and enzyme activation was observed in Ca++-free medium; but the maximum activity was found at 0.5 mM Ca++ concentration. The Km value for PC of acidic PLase A2 was 4.2 microM, and a Michaelis-Menten relationship was evident. Calcium might act as a cofactor at some intermediate step during the activation of acidic PLase A2 in light of the uncompetitive manner of Ca++ action. Furthermore, the release of [3H]-AA from preradiolabelled macrophage plasma membranes occurred with the addition of Ca++ at pH 4.5. These data suggest that the acid PLase A2 is a component of the plasma membrane and is not due to lysosomal contamination since membrane-bound acidic PLase A2 properties are opposite to those found for lysosomal PLase A2.     

Chromatin organization in isolated nuclei: flow cytometric characterization employing forward and perpendicular light scatter

Flow cytometric perpendicular and forward light scatters have been employed to evaluate whether the changes in chromatin organization due to ionic strength, Mg++ concentration and pH, visible in electron microscopy, can be monitored by flow cytometry. The average intensity of the perpendicular light scatter signal increased as nuclear chromatin became decondensed by lowering the ionic strength or releasing H1 histone at low pH values. These results indicate that flow cytometry signals and in particular the perpendicular light scatter allow the detection of the conformational transitions in chromatin and may therefore be useful for studying cell cycle associated morphological changes in isolated nuclei.     

Characterization of histones and chromatin of the hepatopancreas in Palaemon serratus (Crustacea Natantia)]

The chromatin of shrimp hepatopancreas has been extracted from isolated nuclei and characterized. Nuclei were prepared in the presence of Cu++ and phenyl methyl sulfonyl fluoride in order to inhibit the nuclease and protease activities throughout the different purification steps. The purified nuclei are heterogenous in size and show a density of 1,367 g/ml determined on saccharose - glucose gradients. After washing in 0,14 M NaCl and then in 10(-2) M Tris-HCL, pH = 7,6, the nuclei were disrupted in water. The solubilized chromatin was precipitated in 0,15 M.NaCl. This chromatin is characterized by a high level of RNA (RNA/DNA = 0,38) and of non histone proteins (NHP/DNA = 0,6). The denaturation curve showed only one Tm at 69 degrees in 2.10(-4) M.EDTA. When the chromatin was extracted in the presence of staphylococcal nuclease, the Tm reached 80 degrees C. The kinetics of the digestion by the staphylococcal nuclease have been studied and show that 10 per cent of hydrolysis occurs within the first minute. The repeat length of DNA as determined with the polymers of higher order is 189 +/- 5 base pairs. The existence of nucleosomes was confirmed by electron microscopy. The superstructure of chromatin was not completely destroyed after solubilisation with a Potter. The histones were studied by gel electrophoresis after differential staining. The most important feature consists in the presence of two H1, two H2A and two H4. The acetylation levels of the histones were followed after injection of 14C-acetate in vivo. The subfraction H1, 0 was acetylated. Only one H3 was present and the two H2A fractions showed the same level of acetylation. H2B migrated faster than the H2A fractions like in Echinoderms. The two H4 fractions corresponded to two differently acetylated forms. Shrimp hepatopancreas histones were fractionated by molecular sieving on Biogel P 100 and characterized according to their electrophoretic properties as well as their amino-acid content. The amino-acid compositions of the different histone fractions were nearer to Echinoderm and Sipunculid histones, than Calf thymus homologue histones. All the fractions show a weaker basicity. The H3 fraction was the only one showing a lesser variability when compared to Calf thymus H3. The non histone proteins were extracted in 10(-2) M Tris-HCL, pH = 8 and 0.1 per cent SDS. A series of 50 proteins was detected. 80 per cent of the total amount of protein was localized in a molecular weight range comprised between 40 000 and 80 000 daltons. These proteins were compared to the histones and total proteins of sonicated chromatin solubilized by SDS in order to detect proteasic effects.     

MRI and biochemical investigation of the temporomandibular joint of juvenile pigs after sagittal advancement of the mandibulae

Orthodontic treatment leads to changes of the temporomandibular joint (TMJ). The extent and manner of alterations are not yet clarified completely. The non-invasive diagnostic methods do not provide any precise data about the extent of alterations. The aim of the present investigation was to analyse the TMJ cartilage after orthodontic treatment with non-invasive and invasive methods.

For the present investigation, twelve 10-week-old pigs including six control animals were used. Treatment was carried out with build-ups on the molars for sagittal advancement of the mandibulae. Magnetic resonance imaging (MRI) and determination of the Ca⁺⁺ and Mg⁺⁺ content were carried out 4 weeks after implantation of the build-ups. Tissue samples for ion determination were taken from the posterior area of the TMJ (condyle and fossa). During condylar growth a significant increase in cartilage volume and decrease of Ca⁺⁺ and Mg⁺⁺ content was observed. The newly formed tissue had a water content of 80–90%. On MR images of animals with build-ups this area was visible as a bright zone with increased signal intensity.

The determined stress potential of the condylar cartilage was reduced as a result of less functional stress in the posterior area accompanied by water intake. The newly formed cartilage matrix contained significantly less Ca⁺⁺ and Mg⁺⁺, and cannot be defined as permanently stable cartilage.     

Polarized monolayers formed by epithelial cells on a permeable and translucent support

An epithelial cell line (MDCK) was used to prepare monolayers which, in vitro, develop properties of transporting epithelia. Monolayers were formed by plating cells at high densities (10(6) cells/cm2) on collagen- coated nylon cloth disks to saturate the area available for attachment, thus avoiding the need for cell division. An electrical resistance developed within 4-6 h after plating and achieved a steady-state value of 104 +/- 1.8 omega-cm2 after 24 h. Mature monolayers were morphologically and functionally polarized. They contained junctional complexes composed of desmosomes and tight junctions with properties similar to those of "leaky" epithelia. Monolayers were capable of maintaining a spontaneous electrical potential sensitive to amiloride, produced a net water flux from the apical to basal side, and discriminated between Na+ and Cl- ions. The MDCK permeability barrier behaves as a "thin" membrane with negatively charged sites. It has: (a) a linear conductance/concentration relationship; (b) an asymmetric instantaneous current/voltage relationship; (c) a reduced ability to discriminate between Na+ and Cl- caused by lowering the pH; and (d) a characteristic pattern of ionic selectivity which suggests that the negatively charged sites are highly hydrates and of medium field strength. Measurements of Na+ permeability of electrical and tracer methods ruled out exchange diffusion as a mechanism for ion permeation and the lack of current saturation in the I/deltapsi curves does not support the involvement of carriers. The discrimination between Na+ and Cl- was severely but reversibly decreased at low pH, suggesting that Na+-specific channels which exclude Cl- contain acidic groups dissociated at neutral pH. Bound Ca++ ions are involved in maintaining the integrity of the junctions in MDCK monolayers as was shown by a reversible drop of resistance and opening of the junctions in Ca++-free medium containing EGTA. Several other epithelial cell lines are capable of developing a significant resistance under the conditions used to obtain MDCK monolayers.     

Effect of hydrogen ion concentration in the medium on the state of chromatin in lymphoid cells

Effect of medium acidity (pH 7.2 divided by 6.6) on the state of lymphoid tissue cells was studied by microspectrofluorimetry, electrophoresis and IR-spectroscopy. Two stages of the cell structural-functional changes were found at H2CO3 acidification of the medium. The first one is a reversible transition of chromatin from the state of dense sphere to the margination state when chromatin is located along the membrane. The second one is an irreversible destruction of chromatin and the cell death. The margination stage is related to the formation of non-specific DNA-membrane contacts, while the chromatin destruction stage to the "proton breakdown" of the macromolecules and their supramolecular complexes. A relationship between the degree of margination reversibility and the value of the medium acidification and the time of cell incubation in it is shown. Different functional significance of chromatin marginated state is discussed.     

Alterations in tissue Mg++, Na+ and spinal cord edema following impact trauma in rats

Alterations in water content and total tissue Na+ and Mg++ of rat spinal cord tissue were followed over time after a 100 g-cm impact injury to the T-9 spinal cord segment. Rats subjected to laminectomy but not trauma served as controls. In the injured segment there was a progressive increase in water content with increased Na+ and decreased Mg++ at 1 hour and 24 hours after trauma. At seven days, water and Na+ content remained elevated, whereas Mg++ levels had returned to preinjury baseline values. Because of its important role in many metabolic and physiological regulatory processes the early decline in Mg++ concentration after trauma may contribute to the development of secondary tissue damage after spinal cord injury.     

A novel magnesium-dependent structural transition of chicken erythrocyte chromatin in situ

Lowering magnesium concentration below the value of 1 mM leads to a structural transition of chicken erythrocyte chromatin in situ, which results in a change in its fragmentation by pancreatic DNAase (DNAase I) from double-nucleosome to 100-basepairs mode. At 0.75 mM MgCl2, the pattern of chromatin fragmentation by DNAase I is similar to that generated by DNAase II, and it is further changed at lower concentrations of magnesium. This transition is, at least partly, reversible, and is, presumably, related to packing of the 25-30 nm chromatin fiber into higher-order structures.