Early-stage apoptosis is associated with DNA-damage-independent ATM phosphorylation and chromatin decondensation in NIH3T3 fibroblasts

Chromatin condensation and degradation of DNA into internucleosomal DNA fragments are key hallmarks of apoptosis. The phosphorylation of protein kinase ataxia telangiectasia mutated (ATM) and histone H2A.X was recently shown to occur concurrently with apoptotic DNA fragmentation. We have used immunofluorescence microscopy, Western blot analysis and alkali comet assays to show that phosphorylation of ATM in NIH3T3 fibroblasts occurs prior to apoptotic DNA fragmentation, nuclease degradation and phosphorylation of histone H2A.X in cells treated with low levels of either staurosporine (STS) or tumor necrosis factor-alpha mixed with cycloheximide (TNF-alpha/CHX). In extension to previous findings, ATM phosphorylation was associated with chromatin decondensation, i.e., by loss of dense foci of constitutive heterochromatin. These results suggest that chromatin is decondensed and that ATM is activated independently of DNA damage signaling pathways during the very early stages of apoptosis.     

Berry productivity and molluscicidal saponin yield ofPhytolacca Dodecandra (Phytolaccaceae) under different sunlight,watering and nutrient conditions

The cultivation ofPhytolacca dodecandra has been studied in Zimbabwe in order to initiate local production of the berries from which molluscicidal saponins can be extracted and used in schistosomiasis control programs. The effect of shading, water and nutrient type on growth and berry yield of an imported Ethiopian and a Zimbabwean cultivar were monitored. The molluscicidal potency and saponin concentration of the aqueous extracts of berries, harvested at the full grown, unripe development stage, were determined. There was a marked seasonal variation in berry production with both cultivars being highly productive in the dry season. The best growth and the highest berry yield was obtained with plants grown in full sunlight, under irrigation and with application of cattle manure. The Ethiopian cultivar showed in general better growth and higher berry yield than the Zimbabwean cultivar. Cultivation in shade gave a substantially lower berry yield and saponin concentration in both cultivars, compared to full sunlight. Irrigation was beneficial to the berry yield in both cultivars but lowered the saponin concentration in berries of the Ethiopian cultivar. However, it did not counteract the advantage of irrigation on the total yield. Addition of manure did significantly increase the growth and the berry yield of both cultivars compared to fertilizer application and to the control. The saponin concentration was in general lower with addition of manure, with exception of irrigated Ethiopian plants. The combination of cattle manure and irrigation resulted in the highest saponin yield. Although the two cultivars had different saponin patterns, these patterns and the relative proportions of the saponins were constant, irrespective of the treatments.     

Early Cretaceous mesofossils from Portugal and eastern North America related to the Bennettitales-Erdtmanithecales-Gnetales group

Four new genera and six new species of fossil seed (Buarcospermum tetragonium, Lignierispermum maroneae, Lobospermum glabrum, L. rugosum, L. stampanonii, Rugonella trigonospermum) are described from five Early Cretaceous mesofossil floras from Portugal and eastern North America. The four genera are distinguished by differences in size, shape, and details of seed anatomy, but all are unusual in having an outer seed envelope with a distinctive anatomical structure that surrounds the nucellus and the integument. The integument is extended apically into a long, narrow micropylar tube. The four new genera are part of a diverse, but previously unrecognized, complex of extinct plants that was widespread in Early Cretaceous vegetation and that coexisted in similar habitats with early angiosperms. The distinctive structure of these seeds, and the strong similarities to other fossil seeds (Ephedra, Ephedripites, Erdtmanispermum, Raunsgaardispermum, and some Bennettitales) already known from the Early Cretaceous, suggests that this newly recognized complex of extinct plants, together with Bennettitales, Erdtmanithecales, and Gnetales (the BEG group), is phylogenetically closely related.     

Membrane potential,anion and cation conductances in Ehrlich ascites tumor cell

Summary The fluorescence intensity of the dye 1,1-dipropyloxadicarbocyanine (DiOC₃-(5)) has been measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potential (V _m ) under different ionic conditions, after treatment with cation ionophores and after hypotonic cell swelling. Calibration is performed with gramicidin in Na⁺-free K⁺/choline⁺ media, i.e., standard medium in which NaCl is replaced by KCl and cholineCl and where the sum of potassium and choline is kept constant at 155mm. Calibration by the valinomycin null point procedure described by Lariset al. (Laris, P.C., Pershadsingh, A., Johnstone, R.M., 1976,Biochim. Biophys. Acta 436:475–488) is shown to be valid only in the presence of the Cl^–-channel blocker indacrinone (MK196). Distribution of the lipophilic anion SCN^– as an indirect estimation of the membrane potential is found not to be applicable for the fast changes inV _m reported in this paper. Incubation with DiOC₃-(5) for 5 min is demenstrated to reduce the Cl^– permeability by 26±5% and the NO ₃ ^– permeability by 15±2%, while no significant effect of the probe could be demonstrated on the K⁺ permeability. Values forV _m , corrected for the inhibitory effect of the dye on the anion conductance, are estimated at –61±1 mV in isotonic standard NaCl medium, –78±3 mV in isotonic Na⁺-free choline medium and –46±1 mV in isotonic NaNO₃ medium. The cell membrane is depolarized by addition of the K⁺ channel inhibitor quinine and it is hyperpolarized when the cells are suspended in Na⁺-free choline medium, indicating thatV _m is generated partly by potassium and partly by sodium diffusion. Ehrlich cells have previously been shown to be more permeable to nitrate than to chloride. Substituting NO ₃ ^– for all cellular and extracellular Cl^– leads to a depolarization of the membrane, demonstrating thatV _m is also generated by the anions and that anions are above equilibrium. Taking the previously demonstrated single-file behavior of the K⁺ channels into consideration, the membrane conductances in Ehrlich cells are estimated at 10.4 S/cm² for K⁺, 3.0 S/cm² for Na⁺, 0.6 S/cm² for Cl^– and 8.7 S/cm² for NO ₃ ^– . Addition of the Ca²⁺-ionophore A23187 results in net loss of KCl and a hyperpolarization of the membrane, indicating that the K⁺ permeability exceeds the Cl^– permeability also after the addition of A23187. The K⁺ and Cl^– conductances in A23187-treated Ehrlich cells are estimated at 134 and 30 S/cm², respectively. The membrane potential is depolarized in hypotonically swollen cells, confirming that the increase in the Cl^– permeability following hypotonic exposure exceeds the concommitant increase in the K⁺ permeability. In control experiments where the membrane potentialV _m =E _K =E _Cl =E _Na , it is demonstrated that cell volume changes has no significant effect on the fluorescence signal, apparently because of a large intracellular buffering capacity. The increase in the Cl^– conductances is 68-fold when cells are transferred to a medium with half the osmolarity of the standard medium, as estimated from the net Cl^– efflux and the change inV _m . The concommitant increase in the K⁺ conductance, as estimated from the net K⁺ efflux, is only twofold.     

The EGF-TM7 family: a postgenomic view

With the human and mouse genome projects now completed, the receptor repertoire of mammalian cells has finally been elucidated. The EGF-TM7 receptors are a family of class B seven-span transmembrane (TM7) receptors predominantly expressed by cells of the immune system. Within the large TM7 superfamily, the molecular structure and ligand-binding properties of EGF-TM7 receptors are unique. Derived from the processing of a single polypeptide, they are expressed at the cell surface as heterodimers consisting of a large extracellular region associated with a TM7 moiety. Through a variable number of N-terminal epidermal growth factor (EGF)-like domains, EGF-TM7 receptors interact with cellular ligands such as CD55 and chondroitin sulfate. Recent in vivo studies demonstrate a role of the EGF-TM7 receptor CD97 in leukocyte migration. The different number of EGF-TM7 genes in man compared with mice, the chimeric nature of EMR2 and the inactivation of human EMR4 point toward a still-evolving receptor family. Here we discuss the currently available information on this intriguing receptor family.     

Decreased root hydraulic conductivity reduces leaf water potential, initiates stomatal closure and slows leaf expansion in flooded plants of castor oil (Ricinus communis) despite diminished delivery of ABA from the roots to shoots in xylem sap

Soil flooding reduced stomatal conductance (gs) and slowed transpiration, CO₂ uptake and leaf elongation in Ricinus communis within 2–6 h. These flood-induced responses developed further over the next 21 h. They were not associated with increased delivery of abscisic acid (ABA) in xylem sap. Instead, ABA delivery from flooded roots decreased 6-fold within 3 h, and remained low thereafter. Root hydraulic conductance (Lp) was depressed 47% below control values within 2 h of soil flooding, and declined further during the next 21 h. The smaller Lp temporarily decreased leaf water potentials (ΨL) by up to −0.4 MPa, and caused visible wilting 3 h into the flooding treatment at 80% relative humidity. Consequently, ABA concentrations in the shoot were increased, as indicated by analyses of phloem sap. Wilting, fall in ΨL and a reduction in gs were delayed for 6 h when 0.6 MPa pneumatic pressure (technical maximum) was applied to the roots. In flooded plants, phloem sap ABA concentrations returned to normal after 24 h. The initial stomatal closure, caused by soil flooding in R. communis , is attributed to decreased leaf hydration arising from the reduced LP of oxygen-deficient roots. Continued stomatal closure and slow leaf expansion beyond 24 h were presumably achieved by non-hydraulic means.     

Modulation of KCNQ4 channel activity by changes in cell volume

KCNQ4 channels expressed in HEK 293 cells are sensitive to cell volume changes, being activated by swelling and inhibited by shrinkage, respectively. The KCNQ4 channels contribute significantly to the regulatory volume decrease (RVD) process following cell swelling. Under isoosmotic conditions, the KCNQ4 channel activity is modulated by protein kinases A and C, G protein activation, and a reduction in the intracellular Ca2+ concentration, but these signalling pathways are not responsible for the increased channel activity during cell swelling.     

Phenotypic characterisation of porcine counterparts of human NK cell populations--implications for pre-clinical studies

The phenotype of cryopreserved crossbred porcine PBMC, with special emphasis on NK cell related surface markers, was characterised. The phenotype expressed prior to stimulation with rHu IL2 and rHu IL12 was related to the in vitro cytotoxic capacity estimated in a 4-h or 21-h 51Cr-release assay. PBMC incubated in growth medium without addition of cytokines were used to investigate the spontaneous cytotoxic capacity. The results of this study suggest that crossbred porcine PBMC comprise a specific subset expressing the phenotype CD2+CD3-CD4-CD8+SWC3-CD16+CD21-. No spontaneous cytotoxicity of the PBMC could be estimated, but the expression of CD16 seems to be a basic marker of the cytokine induced cytotoxic activity. This study suggests that cryopreserved porcine PBMC can be used when possible influences on the porcine NK cell system are investigated in relation to disease models conducted experimentally in crossbred pigs.