Effect of cytoplasmic acidification on the membrane potential of T-lymphocytes: Role of trace metals

Summary The effect of lowering intracellular pH on the membrane potential (E _m ) of rat thymic lymphocytes was studied using the potential-sensitive dyebis-oxonol. Cells were acid loaded by addition of the electroneutral K⁺/H⁺ exchanging ionophore nigericin. Acidification to pH 6.3 in Na⁺-free solution resulted in a biphasic change inE _m : an early transient hyperpolarization followed by a sustained depolarization. These changes were associated with a rise in cytosolic free Ca²⁺ ([Ca²⁺] _i ). The hyperpolarization was eliminated when the change in [Ca²⁺] _i was prevented using BAPTA, an intracellular Ca²⁺ chelator. Moreover, a similar hyperpolarization was elicited by elevation of [Ca²⁺] _i at physiological pH _i using ionomycin, suggesting involvement of Ca²⁺-activated K⁺ channels. In contrast, the depolarization phase could not be mimicked by raising [Ca²⁺] _i with ionomycin. However, intracellular BAPTA effectively inhibited the acidificationinduced depolarization. Inhibition was also obtained by extracellular addition of EGTA or dithiothreitol, even when the external free Ca²⁺ concentration remained unaltered. These observations suggested a possible role of contaminating trace metals. Cytosolic acidification is envisaged to induce intracellular accumulation of one or more trace metals, which induces the observed changes inE _m . Accordingly, similar changes inE _m can be induced without acidification by the addition of small amounts of Cu²⁺ to the medium. The ionic basis of theE _m changes induced by acidification and the significance of these observations are discussed.     

Response to osmotic medium and fusicoccin by seeds of radish (Raphanus sativus) in the early phase of germination

The effect of increasing osmotic values of the medium (mannitol) on the growth and the response mechanisms of seeds of radish ( Raphanus sativus L., cv. Ton do Rosso Quarantino) during the early phase of germination was investigated in the presence or absence of fusicoccin (FC). Decreasing the water potential in the medium inhibited the growth and the evolution of protein synthesis and enhanced H⁺ extrusion, net uptake of K⁺ and malic acid synthesis. FC, which stimulates these latter functions, counteracted the inhibitory effect of the decreasing water potential of the medium on growth and protein synthesis. Neither in the absence nor in the presence of FC did decreasing water potential of the medium enhance the synthesis of soluble sugars and amino acids to support the osmotic pressure of the seeds. The osmotic and water potentials of the seeds increased during germination. FC made the increase more rapid, while mannitol kept both potentials low. The pressure potentials of the seeds also decreased with time, and both FC and mannitol enhanced this change. If the seeds were without turgor, the development of protein synthesis was blocked. The seeds counteract the effect of decreasing water potentials in the medium by: a) enhancing H⁺ extrusion (and, as a consequence, wall loosening and transport mechanisms) and the synthesis of malic acid as apparent in the presence of FC; b) regulating the osmotic potentials of the cells (with a lower dilution of the osmotic compounds present in the seeds due to the diminished uptake of water); c) controlling the growth through the effects of a) and b) on the pressure potentials (internal hydrostatic pressure) of the seeds and on protein synthesis.     

GABAA Receptor Complex in an Experimental Model of Hepatic Encephalopathy: Evidence for Elevated Levels of an Endogenous Benzodiazepine Receptor Ligand

The involvement of the gamma-aminobutyric acidA (GABAA) receptor complex in the pathogenesis of hepatic encephalopathy was examined in thioacetamide-treated rats with fulminant hepatic failure. Partially purified extracts from encephalopathic rat brain were approximately three times more potent in inhibiting [3H]Ro 15-1788 binding to benzodiazepine receptors than identically prepared extracts from control rats. High levels of inhibitory activity were also found in extracts of plasma, heart, and liver from thioacetamide-treated rats. The inhibition of [3H]Ro 15-1788 binding by brain extracts appeared to be competitive and reversible and was unaffected by treatment with either proteolytic enzymes or boiling. Further, GABA significantly enhanced the potency of these extracts in inhibiting [3H]flunitrazepam binding. In contrast, no differences were found in radioligand binding to the constituent recognition sites of the GABAA receptor complex in well-washed brain membranes prepared from control and encephalopathic animals. These findings suggest that the recognition-site qualities of the constituent proteins of the GABAA receptor complex are unchanged in an experimental model of hepatic encephalopathy. However, significant elevations in the level of a substance or substances with neurochemical properties characteristic of a benzodiazepine receptor agonist may contribute to the electrophysiological and behavioral manifestations of hepatic encephalopathy.     

A new enzymatic route to the synthesis of 12-ketoursodeoxycholic acid

Summary Cholic acid (3,7,12-trihydroxy-5-cholanoic acid) was completely and selectively transformed into 12-ketoursodeoxycholic acid (3,7-dihydroxy-12-oxo-5-cholanoic acid) by means of two consecutive enzymatic steps catalyzed, the first, by 7- and 12-hydroxysteroid dehydrogenase and, the second, by 7-hydroxysteroid dehydrogenase. Coenzyme regeneration was carried out with -ketoglutarate-glutamate dehydrogenase and glucose-glucose dehydrogenase, respectively.     

Aspects of Gondwana paleobotany: gymnosperms of the Paleozoic—Mesozoic transition

During the late Paleozoic and early Mesozoic the Gondwana Supercontinent underwent dramatic geographic and climatic changes. Geologic and biologic factors concurrently played an important role modelling the vegetation of that time. The gymnospermic component of plant assemblages shows significant variations in composition and in the distribution of different taxa. Analysis of the assemblages shows that some plant groups dominated the scenario, such as the pteridosperms, glossopterids, corystosperms and, to a lesser degree, cordaites and conifers. Ginkgophytes, bennettites or cycads were less important in the Paleozoic but their numbers increased in the Triassic. Paleozoic assemblages were extensively dominated by glossopterids that became extinct in the earliest Mesozoic. Pteridosperms crossed the P—M barrier and became dominant during the Triassic, at a time when corystosperms evolved and radiated. Other groups became extinct in the Paleozoic, namely dicranophylls and cordaites. Conifers were represented by different families, restricted either to the Paleozoic or the Mesozoic. They were not conspicuous in the analysed assemblages. In some areas of Gondwana, taxa of the Euramerican alliance are present through a migrational mechanism that occurred during continental displacements which produced global climatic changes. Recent studies have shown that there are far more common elements between Euramerica and Gondwana than suspected up to now. These elements find their distribution especially in the western part of Gondwana (Africa—South America).     

The proliferating cell marker monoclonal antibody Ki-67 recognizes specific antigens associated with the nuclear envelope of the early Drosophila embryo

Summary— Immunofluorescence and immunoelectron microscopy indicated that the antibody raised against the nuclear antigen Ki-67 of mammalian cells recognized antigenic determinants of early Drosophila embryos, localized on the outside of the nuclear envelope. Hence, the nuclear envelope of Drosophila appears to share a similar epitope with the chromosome scaffold of mitotic mammalian cells. With the progression of mitosis the antigen persisted around the mitotic spindle region and was also found in the pole regions at metaphase and anaphase. The antibody also stained the equatorial regions of the spindles from anaphase to late telophase. The antibody may therefore be used as a biochemical marker of the nuclear envelope for studying nuclear membrane biogenesis and behavior during the mitotic divisions of the Drosophila embryo.     

cAMP-dependent protein phosphorylation in mitochondria of bovine heart

A study is presented of the cAMP-dependent phosphorylation in bovine heart mitochondria of three proteins of 42, 16 and 6.5 kDa associated to the inner membrane. These proteins are also phosphorylated by the cytosolic cAMP-dependent protein kinase and by the purified catalytic subunit of this enzyme. In the cytosol, proteins of 16 and 6.5 kDa are phosphorylated by the cAMP-dependent kinase. It is possible that cytosolic and mitochondrial cAMP-dependent kinases phosphorylate the same proteins in the two compartments.     

Characterization of a wilty sunflower (Helianthus annuus L.) mutant II. Water relations, stomatal conductance, abscisic acid content in leaves and xylem sap of plants subjected to water deficiency

The response of w-1, a wilty sunflower (Helianthus annuus L.)mutant, to water stress is described in comparison with thecontrol line (W-1). Detached leaves of w-1 strongly dehydratedduring the first 30 min without significant changes in leafconductance, whereas W-1 responded rapidly to water loss byreducing stomatal aperture. After 2 h stress ABA increased slightlyin w-1, while W-1 leaves showed a 20-fold increase. When waterstress was imposed to potted plants by water withholding, w-1quickly dehydrated, and lost turgor, while W-1 maintained positiveturgor values for a longer period. Wild-type plants respondedto small changes in leaf water potential by accumulating ABAand by closing stomata, whereas in the mutant significant changesin ABA content and in stomatal conductance were found only atvery low water potentials. In another experiment in which waterwas withheld under high relative humidity, when soil water contentstarted to decrease W-1 rapidly closed stomata in the absenceof any change in leaf water status and the reduction in conductancewas paralleled by a rise in xylem sap ABA concentration. Bycontrast the mutant started to accumulate ABA in the xylem sapand to close stomata when soil water content and leaf waterpotential were dramatically reduced. The low endogenous ABAlevels and the inability to synthesize the hormone rapidly eitherin the leaves or in the roots seem to be responsible for thehigh sensitivity of w-1 to water stress. Key words: ABA, Helianthus annuus L, water relations, stomatal conductance, drought, wilty mutant     

Changes in drinking water selenium and mortality for coronary disease in a residential cohort

In a part of the municipal territory of Reggio Emilia, northern Italy, selenium in drinking water decreased from 7 μg/L to less than 1 μg/L. In a cohort of 4419 individuals, previously exposed for at least 5 yr to the drinking water with higher selenium content, the 7-yr temporal distribution of deaths for coronary disease and for stroke was analyzed to examine a possible relationship with changes in drinking water selenium. From January 1986 until August 1988, when tap water selenium was 7 μg/L, deaths for coronary disease were one in males and two in females. After the decrease in drinking water selenium, 21 and 10 coronary deaths were observed, respectively, in males and in females from September 1988 to December 1992. No significant difference in the temporal distribution of stroke deaths was observed both in males and in females. Even if an effect of chance and aging in the temporal distribution of coronary deaths may not be excluded, findings of the study seem to be consistent with the hypothesis of a beneficial effect of selenium on coronary disease mortality.