Localization of a Mg- or Ca-activated (“basic”) ATPase in skeletal muscle

A chicken pectoralis muscle membrane fraction enriched in a Mg²⁺- or Ca²⁺-activated (‘basic’) ATPase was obtained by sucrose gradient centrifugation. Enzymatic properties of the ‘basic’ ATPase were determined and used to localize its enzymatic activity in situ by ultrastructural cytochemistry. The enzyme was activated by Mg²⁺ or Ca²⁺ but not by Sr²⁺, Ba²⁺, Co²⁺, Ni²⁺ or Pb²⁺. It was present in a membranous fraction with a buoyant density of 1.10-1.12 (24–27.5% (w/w) sucrose). ‘Basic’ ATPase activity had a sedimentation pattern similar to the putative plasma membrane enzymes, 5′-nucleotidase and leucyl β-naphthylamidase, but different from that of sarcoplasmic reticulum Ca²⁺ ATPase. Also unlike sarcoplasmic reticulum Ca²⁺ ATPase, ‘basic’ ATPase was resistant to N-ethylmaleimide and aldehyde fixatives, was active in a medium containing a high Ca²⁺ concentration (3 mM), and was lost when exposed to Triton X-100 or deoxycholate. In cytochemical studies, a low Pb²⁺ concentration was used to capture the enzymatically released phosphate ions. Under conditions which eliminated interfering (Na⁺ + K⁺) ATPase and sarcoplasmic reticulum Ca²⁺ ATPase activities, electron-dense lead precipitates were present at the plasmalemma and T-system membranes. These studies suggest that ‘basic’ ATPase activity is associated with plasmalemma and T-system membranes of skeletal muscle.     

Membrane effects of retinoids: Possible correlation with toxicity

The mechanism of toxicity of vitamin A and other retinoids is unknown. One theory holds that these compounds disrupt cellular function by acting on membranes as detergents would. Using rat erythrocyte ghosts labeled with fluorescent probes, we studied the changes in fluorescence polarization effected by detergents and retinoids. Fluorescence lifetime measurements allowed us to calculate apparent microviscosities. Sodium dodecyl sulfate, Triton X-100, and lysophosphatidylcholine seemed to cause a structural transition in the membrane where the apparent microviscosity was reduced by half at a detergent concentration of approximately 10^?3m; the same transition point was reported by 1,6-diphenylhexatriene or perylene. In contrast, retinoic acid reduced membrane microviscosity by half at a concentration of only 10^?5m or lower. Furthermore, the results obtained with DPH were very different from those obtained with perylene as the probe. The effect of retinoic acid was prevented by d-α-tocopherol acetate. Other retinoids were also effective in reducing membrane microviscosity, and the order of their effectiveness exactly paralleled the order of their toxicities, as determined by others using bioassays. These results suggest that retinoid toxicity may indeed be related to a membrane effect, but the action of retinoids on membranes is unlike that of simple detergents.     

Thermotropic ‘two-stage’ liquid crystalline crystalline lipid phase separation in microsomal membranes

The effect of temperature on native microsomal membrane vesicles isolated from Tetrahymena is investigated by wide angle X-ray diffraction. A 4.2 Å reflection, typical for lipids in the crystalline state, can be recorded in the temperature range between 0°C and 35°C. Quantitative evaluation of this reflection reveals a broad thermotropic ‘two-stage’ liquid crystallinecrystalline lipid phase separation with a ‘breakpoint’ at approx. 18°C. This ‘breakpoint’ coincides with the emergence of lipid-protein segregations in endomembranes of intact Tetrahymena cells as previously visualized by freeze-etch electron microscopy.     

DNA replication in macronuclei of Tetrahymena pyriformis GL

DNA replication in macronuclei of Tetrahymena pyriformis GL has been studied to discriminate between hypotheses developed for the interpretation of intraclonal differentiation in ciliated protozoa (the diploid subnuclear, and the ‘master’-‘slave’ hypotheses). Tetrahymena cells were grown in a heavy ¹⁵N-³H medium and then transferred to a light ¹⁴N-¹⁴C medium. DNA was isolated after various periods following this transfer and studied in equilibrium CsCl density gradient centrifugation. Time-related changes in the DNA buoyant density pattern were investigated. The data obtained are interpreted to mean that all DNA in macronuclei of asynchronously growing Tetrahymena at exponential phase replicates semiconservatively once in a cell cycle. These data are in good agreement with the findings of Andersen & Zeuthen obtained on synchronous Tetrahymena cultures in the presence of BUdR.These results are not consistent with the ‘master’-‘slave’ hypothesis. The diploid subnuclear hypothesis is not in accord with other experimental evidence. An alternative hypothesis has been proposed concerning the nature of the macronuclear units and the process of determination. The two main points of this hypothesis are: (a) macronuclear units are diploid genome fragments (‘nucleosomes’); (b) determination is a process of haploidization by ‘allelic splitting’ at a definite macronuclear fission. Consistency with experimental data is discussed and some predictions of the hypothesis are given.     

Effects of redox agents on the Ca-activated K channel

The sensitivity to Ca²⁺ of the Ca²⁺-dependent K⁺ channel can be increased by the artificial electron donor system ascorbate + phenazine-methosulphate in a variety of animal cells. In the human erythrocyte the shift from the ‘low’ to the ‘high-affinity’ state seems to depend on the reduction of a membrane component accepting 2 electrons and with an standard redox potential (pH 7.5) of about 47 mV. The relevance of this redox modulation under physiological circumstances is unknown at the moment.     

Specific and Redundant Functions of Retinoid X Receptor/Retinoic Acid Receptor Heterodimers in Differentiation, Proliferation, and Apoptosis of F9 Embryonal Carcinoma Cells

We have generated F9 murine embryonal carcinoma cells in which either the retinoid X receptor (RXR)α and retinoic acid receptor (RAR)α genes or the RXRα and RARγ genes are knocked out, and compared their phenotypes with those of wild-type (WT), RXRα^−/−, RARα^−/−, and RARγ^−/− cells. RXRα^−/−/ RARα^−/− cells were resistant to retinoic acid treatment for the induction of primitive and parietal endodermal differentiation, as well as for antiproliferative and apoptotic responses, whereas they could differentiate into visceral endodermlike cells, as previously observed for RXRα^−/− cells. In contrast, RXRα^−/−/RARγ^−/− cells were defective for all three types of differentiation, as well as antiproliferative and apoptotic responses, indicating that RXRα and RARγ represent an essential receptor pair for these responses. Taken together with results obtained by treatment of WT and mutant F9 cells with RAR isotype– and panRXR-selective retinoids, our observations support the conclusion that RXR/ RAR heterodimers are the functional units mediating the retinoid signal in vivo. Our results also indicate that the various heterodimers can exert both specific and redundant functions in differentiation, proliferation, and apoptosis. We also show that the functional redundancy exhibited between RXR isotypes and between RAR isotypes in cellular processes can be artifactually generated by gene knockouts. The present approach for multiple gene targeting should allow inactivation of any set of genes in a given cell.     

Nucleolus-like bodies in embryonal carcinoma cells of the embryoid bodies isolated from mouse teratocarcinoma

Undifferentiated embryonal carcinoma cells (EC cells) in the embryoid bodies isolated from mouse teratocarcinoma contained nucleolus-like bodies (NLBs) of smaller sizes in their cytoplasm (their sectional area averaged about 0.036 μm²). At the onset of EC cell differentiation, the average sectional area of NLBs significantly increased (about 0.107 μm²). When EC cells had differentiated into mesenchymal cells and endothelial cells of primitive blood vessels, NLBs decreased dramatically both in size and number. The possible role of NLBs in the differentiation process of EC cells is discussed.     

Stimulation of differentiation of several murine embryonal carcinoma cell lines by retinoic acid

Retinoic acid stimulates several murine embryonal carcinoma (EC) cell lines, even those previously considered to be incapable of differentiating, to give rise to cell types distinguishable from the parental phenotype in morphology, production of plasminogen activator and surface protein properties. Retinoic acid promotes these changes over a range of low concentrations (10⁻⁹–10⁻⁵ M) which are generally non-toxic to the cells. The effects are clearly demonstrated when EC cells are aggregated prior to exposure to retinoic acid. It is concluded that the observed phenotypic alterations induced by retinoic acid reflect differentiation of the EC cells since non-EC cell characteristics are maintained by cloned cells several generations after retinoic acid is removed from the cultures. Our studies suggest that although retinoic acid stimulates the conversion of EC cells to differentiated derivatives, it does not influence the direction of differentiation. Furthermore, the effectiveness of retinoic acid in stimulating differentiation of EC cells from lines such as Nulli-SCC1 raises the question of whether true ‘nullipotent’ EC lines really exist.     

Rb fluxes in Chinese hamster ovary cells as a function of membrane cholesterol content

Steady-state fluxes of ⁸⁶Rb⁺ (as a tracer for K⁺) were measured in Chinese hamster ovary cells (CHO-K1) and a mutant (CR1) defective in the regulation of cholesterol biosynthesis; the membrane cholesterol content of this mutant was varied by growing it on a range of cholesterol supplements to lipid-free medium (Sinensky, M. (1978) Proc. Natl. Acad. Sci. U.S. 75, 1247–1249).Analogous to previous findings in ascites tumor cells, ⁸⁶Rb⁺ influx in the parent strain was differentiated into a ouabain-inhibitable ‘pump’ flux, furosemide-sensitive, chloride-dependent exchange diffusion, and a residual ‘leak’ flux.On the basis of this flux characterization, ⁸⁶Rb⁺ pump and leak fluxes were measured in the mutant as a function of membrane cholesterol content. Pump and leak fluxes, when expressed per ml cell water, were independent of the cholesterol content of the mutant. Moreover, ⁸⁶Rb⁺ fluxes in the mutant were equal to those in the parent strain. Our data imply that the flux behavior of K⁺ in the steady state is independent of the ordering of membrane lipid acyl chains.     

Interactions of cardiac glycosides with cells and membranes Properties and structural aspects of two receptor sites for ouabain in erythrocytes

The existence of two types of binding sites for ouabain in human erythrocyte membranes is described. Receptor sites designated as ‘type I’, which may be identical to the K⁺-insensitive sites of intact cells, were detected at concentrations of ouabain as low as 10⁻⁷ M. The ‘type II’ receptor sites require the inclusion of Mg²⁺ + P_i to form complexes with ouabain; they may be identical to the K⁺-sensitive sites of intact cells. These sites were saturated at approx. 5 · 10⁻⁷ M ouabain but could not be detected at higher concentrations. The range of ouabain concentrations at which ‘type I’ receptors start to predominate (i.e. 5 · 10⁻⁸–5 · 10⁻⁷ M) was termed ‘critical digitalis concentrations’. The process of binding reached equilibrium within 1 and 4 h for ‘type I’ and ‘type II’ sites, respectively. The dissociation constant for ‘type II’ receptor-ouabain complexes was 7.6 · 10⁻⁹ M.Under similar experimental conditions, rat erythrocyte membranes exhibited only non-saturable sites.Alterations in the proportions of the two types of receptors were demonstrated by preincubation of the membranes, in the presence or absence of Mg²⁺ + P_i, prior to the addition of ouabain. In the first case, ‘type II receptor-ouabain’ complexes were stabilized at about 50% of the untreated membranes and ‘type I-ouabain’ complexes slowly approached equilibrium over a period of 24 h. In the latter instance, ‘type I’ receptors were not detected, and only ‘type II-ouabain’ complexes prevailed.     

Characterization of membrane permeability alterations induced in vero cells by Clostridium perfringens enterotoxin

Alterations in plasma membrane permeability induced by Clostridium perfringens enterotoxin were studied using Vero (African green monkey kidney) cells which were radioactively labeled with four markers of different molecular size. The markers were α-amino[¹⁴C]isobutyric acid (M_r 103), ³H-labeled nucleotide (M_r approx. 300), ⁵¹Cr label (M_r approx. 3000) and [³H]RNA (M_r > 25 000). Over a 2 h period, enterotoxin caused significant release of aminoisobutyric acid, nucleotides and ⁵¹Cr label but not RNA. The effects of enterotoxin on label release were dose- and time-dependent. The rate of release of markers was dependent upon their size. Permeability alterations could be detected within 15 min with a high dose of enterotoxin. Gel chromatography of released material was used to determine that markers of M_r 3000 but not 25 000 leaked from permeabilized cells. It was concluded that enterotoxin is producing functional ‘holes’ of limited size in the membrane. Permeability changes due to enterotoxin treatment differed between confluent and non-confluent (growing) cells. We propose that the primary action of the enterotoxin is to interact with the plasma membrane and produce functional ‘holes’ of defined size. The resultant alterations in membrane permeability cause the loss of essential cellular substances which inhibits processes such as macromolecular synthesis and eventually leads to cell deterioration and death.     

Electron microscopic visualization of the arrangement of the two protein components of (Na + K)-ATPase

The information obtained by electron microscopic examination of highly purified membrane preparations of (Na⁺ + K⁺)-ATPase after freeze-fracturing or negative staining suggests the following conclusions. The catalytic 100 000 dalton protein component penetrates with its greater ‘globular’ mass the plasma membrane and protudes with its smaller mass from the protoplasmic surface by a stalked knob carrying the catalytic centre. The 40 000 dalton glycoprotein component is anchored in the membrane interior by a non-polar ‘fibrous’ side chain, whereas its major polar mass projects from the outer membrane surface forming a surface coat of ill-definable substructure.     

Cytogenetic stability of <Emphasis Type="Italic">Centaurea ragusina</Emphasis> long-term culture

Centaurea ragusina L. (Asteraceae) is an endemic Croatian plant species, which developed xeromorphic characteristics as a consequence of its natural environment – vertical limestone cliffs above the Adriatic sea. Cytogenetic status of C. ragusina long-term culture (94th subculture) and C. ragusina seedlings was analysed and compared after 4 weeks of growth. Cytogenetic stability was investigated in root meristem cells of C. ragusina cultured plants originated from Pen đa (cliffs near Dubrovnik) and seedlings originated from three different localities in south Adriatic (Penđa, Pasjača – cliffs near Dubrovnik and island of Komiža) using mitotic index and mitotic and chromosomal abnormalities as parameters. Mitotic indices of cultured plants and ‘Penđa’ seedlings were similar and showed significant increase compared to mitotic indices of ‘Komiža’ and ‘Pasjača’ seedlings. Although the highest number of mitotic abnormalities was recorded in root meristem cells of cultured plants, it was only a bit higher than in root tips of ‘Pasjača’ and ‘Penđa’ seedlings, while that of ‘Komiža’ was two times lower compared to cultured plants. Pattern of analysed mitotic abnormalities was very similar in root tips of cultured plants and ‘Pasjača’ and ‘Penđa’ seedlings, with exception of ‘Komiža’ seedlings. Presented results suggest that long-term cultivation of C. ragusina has almost no effect on culture ageing considering similar distribution of scored mitotic abnormalities as in ‘Penđa’ and ‘Pasjača’ seedlings.     

Oxidative stress,antioxidant activity and Fe(III)-chelate reductase activity of five <Emphasis Type="Italic">Prunus</Emphasis> rootstocks explants in response to Fe deficiency

The aim of this work was to investigate whether Fe reduction and antioxidant mechanisms were expressed differently in five Prunus rootstocks (‘Peach seedling,’ ‘Barrier,’ ‘Cadaman,’ ‘Saint Julien 655/2’ and ‘GF-677’). These rootstocks differ in their tolerance to Fe deficiency when grown in the absence of Fe (−Fe) or in presence of bicarbonate (supplied as 5 or 10 mM NaHCO₃). Fe deficiency conditions, especially bicarbonate, were shown to decrease Fe and total chlorophyll (CHL) concentration. In the (−Fe)-treated roots of all rootstocks and in the 5 mM NaHCO₃-treated ones of the tolerant ‘GF-677’ the Fe(III)-chelate reductase (FCR) activity was stimulated. On the contrary, apart from the ‘GF-677,’ FCR activity was greatly inhibited by the 10 mM NaHCO₃. From the results obtained with decapitated rootstocks, it is not entirely clear whether or not the presence of shoot apex was a prerequisite to induce FCR function in all rootstocks tested. In the leaves of rootstocks exposed to the (−Fe) treatment, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities were enhanced whereas the levels of the non-enzymatic antioxidants (FRAP values) were increased in the Fe-deprived leaves, irrespective of the rootstock. Except for ‘Peach seedling,’ foliar SOD activity was stimulated by the presence of NaHCO₃. Furthermore, POD activity was increased in the ‘Saint Julien 655/2’ and ‘GF-677,’ but was depressed in the ‘Barrier’ rootstocks exposed to 10 mM NaHCO₃. As a result of 10 mM NaHCO₃, the expression of a Cu/Zn-SOD and a POD isoform was diminished in the leaves of ‘Peach seedling’ and ‘Barrier,’ respectively. By contrast, an additional isoform of both POD and Mn–SOD were expressed in the leaves of ‘GF-677’ exposed to 10 mM NaHCO₃ suggesting that the tolerance of rootstocks to Fe deficiency is associated with induction of an antioxidant defense mechanism. Although CAT activity was increased in the 5 mM NaHCO₃-treated leaves of ‘GF-677,’ specifically the 10 mM NaHCO₃ treatment resulted in a decrease of CAT activity and an accumulation of H₂O₂, indicating that bicarbonate-induced Fe deficiency may cause more severe oxidative stress in the rootstocks, than the absence of Fe. A general link between Fe deficiency-induced oxidative stress and Fe reduction-sensing mechanism is also discussed.     

Murine embryonal carcinoma cells differentiate in vitro in response to retinol

In an initial effort to determine whether circulating retinol might promote differentiation of embryonal carcinoma (EC) cells in tumor form, we have assessed the ability of retinol to stimulate differentiation of cultured EC cells. We found that retinol induces several murine EC cell lines to differentiate in vitro. Differentiated derivatives were distinguishable from parental EC cells by morphology, cell surface antigenic properties and levels of secretion of plasminogen activator. Retinol effects could be seen at concentrations as low as 8.7 × 10⁻⁸ M (0.025 (μg/ml). Only two of eight EC lines tested failed to differentiate in response to retinol: PCC4-azaIR, which dies at retinol concentrations above 3.5 × 10⁻⁷ M; and PCC4(RA)^-1, a mutant line derived from PCC4-azaIR cells, which also fails to differentiate in response to retinoic acid.     

[Na] in the subsarcolemmal ‘fuzzy’ space and modulation of [Ca]i and contraction in cardiac myocytes

The strength of the heart beat depends on the amplitude and time course of the transient increase in [Ca²⁺] in the myocytes with each cycle. [Na⁺]_i modulates cardiac contraction through its effect on the Ca²⁺ flux through the Na/Ca exchanger. Cardiac excitation–contraction coupling has been postulated to occur in a microdomain or ‘fuzzy’ space at the junction of the T-tubules and the sarcoplasmic reticulum. This ‘fuzzy’ space is well described for the Ca²⁺ fluxes and the interaction between the L-type Ca²⁺ channel, the Ca²⁺ release channel of the sarcoplasmic reticulum and the Na/Ca exchanger. Co-localization of the Na⁺ transporters, in particular the Na/K pump and the Na⁺ channel, within this ‘fuzzy’ space is not as well established. The functional and morphological characteristics of the ‘fuzzy’ space for Na⁺ and its interaction with the Ca²⁺ handling suggest that this space is not strictly co-inciding with the Ca²⁺ microdomain. In this space [Na⁺] can be several-fold higher or lower than [Na⁺] in the bulk cytosol. This has implications for modulation of [Ca²⁺]_i during a single beat as well as during alterations in Na⁺ fluxes seen in pathological conditions.     

Cell surface antigens of clonal teratocarcinoma cells at various stages of differentiation

We have studied cell surface antigen expression of teratocarcinoma cells at various stages of differentiation. These cells can be maintained in the undifferentiated state or will differentiate in vitro in a manner which parallels the early development of the mouse embryo. Three antigens were studied: a stem cell antigen (C); the major histocompatibility alloantigens (H-2); and the alloantigen Thy-1.The stem cell antigen was recognized by an anti-serum raised against a pluripotent teratocarcinoma cell line. This antiserum was shown to label embryonal carcinoma cells and early mouse embryo cells. The activity of the antiserum against embryonal carcinoma cells could be adsorbed with brain, kidney, and sperm from adult mice.The phenotype of the undifferentiated embryonal carcinoma cells is C⁺, H-2⁻, Thy-1⁻ or C⁻, H-2⁻, Thy-1⁻. The first stage in the process of differentiation is the formation of simple embryoid bodies with a layer of endodermal cells surrounding an inner core of embryonal carcinoma cells. The endodermal cells are C⁻, H-2⁻, Thy-1⁻. Further differentiation of the embryoid bodies attached to a substratum is associated with the appearance of H-2⁺ and Thy-1⁺ cells in the cultures.     

Localization of phosphatase activities on mouse teratocarcinoma embryoid bodies

Non-specific alkaline phosphatase and Mg²⁺-dependent adenosine triphosphatase activities were ultracytochemically investigated on embryoid bodies of murine teratocarcinomas, in order to find markers of endodermal cell differentiation of early embryonic cells. The former was localized mainly on the cell surface of inner embryonal carcinoma cells, as already shown by other workers, and weakly on the bound surface of outer endodermal cells of embryoid bodies. The latter, however, was found only on the outer free surface of endodermal cells and never on the surface of embryonal carcinoma cells. It suggests that Mg²⁺-dependent ATP activity might become the marker for early differentiation of embryonal carcinoma cells.     

Natural revegetation on topsoiled mining-spoils according to the exposure

Comparative plant successional studies on derelict sites are providing significant insights into vegetation dynamics to ensure the success of future revegetation projects in these areas and, in the short-term, by using a space-for-time substitution. In this paper we describe, in relation to site exposure, vegetation development on waste rock materials covered with biologically active soil media, and compare this development with that from a previous study at the same mine (CW Spain) on non-biologically modified waste rock materials. The succession under study is faster on the North slope, as expected, and does not differ significantly from the general pattern of primary revegetation, although it was characterised by its own sequence of plant species. The topsoiling of waste increases richness and diversity from the first year of revegetation, reduces the time required for recovery of a terminal stage, and highlights the influence of slope orientation on vegetation dynamics. A total of 237 plant taxa were recorded, showing one of four patterns of change: (1) ‘pioneer’, (2) ‘intermediate’, (3) ‘late coloniser’ and (4) ‘fluctuating’.     

Can elevated CO(2) improve salt tolerance in olive trees?

We compared growth, leaf gas exchange characteristics, water relations, chlorophyll fluorescence, and Na⁺ and Cl⁻ concentration of two cultivars (‘Koroneiki’ and ‘Picual’) of olive (Olea europaea L.) trees in response to high salinity (NaCl 100 mM) and elevated CO₂ (eCO₂) concentration (700 μL L⁻¹). The cultivar ‘Koroneiki’ is considered to be more salt sensitive than the relatively salt-tolerant ‘Picual’. After 3 months of treatment, the 9-month-old cuttings of ‘Koroneiki’ had significantly greater shoot growth, and net CO₂ assimilation (A_CO2) at eCO₂ than at ambient CO₂, but this difference disappeared under salt stress. Growth and A_CO2 of ‘Picual’ did not respond to eCO₂ regardless of salinity treatment. Stomatal conductance (g_s) and leaf transpiration were decreased at eCO₂ such that leaf water use efficiency (WUE) increased in both cultivars regardless of saline treatment. Salt stress increased leaf Na⁺ and Cl⁻ concentration, reduced growth and leaf osmotic potential, but increased leaf turgor compared with non-salinized control plants of both cultivars. Salinity decreased A_CO2, g_s, and WUE, but internal CO₂ concentrations in the mesophyll were not affected. eCO₂ increased the sensitivity of PSII and chlorophyll concentration to salinity. eCO₂ did not affect leaf or root Na⁺ or Cl⁻ concentrations in salt-tolerant ‘Picual’, but eCO₂ decreased leaf and root Na⁺ concentration and root Cl⁻ concentration in the more salt-sensitive ‘Koroneiki’. Na⁺ and Cl⁻ accumulation was associated with the lower water use in ‘Koroneiki’ but not in ‘Picual’. Although eCO₂ increased WUE in salinized leaves and decreased salt ion uptake in the relatively salt-tolerant ‘Koroneiki’, growth of these young olive trees was not affected by eCO₂.