Partial Characterization of K and Ca Uptake Systems in the Halotolerant Alga Dunaliella salina

The uptake of K⁺ and Ca²⁺ in Dunaliella salina is mediated by two distinct carriers: a K⁺ carrier with a high selectivity against Na⁺, Li⁺, and choline⁺ but not towards Rb⁺, K⁺, Cs⁺, or NH₄⁺, and a Ca²⁺ carrier with a high selectivity against Mg²⁺. The latter is specifically blocked by La³⁺ and by Cd²⁺. Apparent K_m values for K⁺ and Ca²⁺ uptake are 2.5 and 0.8 millimolar, respectively, and their maximal calculated fluxes are 22 and 0.8 nanomoles per square meter per second, respectively. Effects of permeable ions and ionophores on K⁺ and Ca²⁺ uptake suggest that the driving force for their uptake is the transmembrane electrical potential. Inhibitors of ATP production, typical inhibitors of plasma membrane H⁺-ATPases and protonionophores inhibit K⁺ and Ca²⁺ uptake and accelerate K⁺ efflux. The results suggest that an H⁺-ATPase in the cell membrane provides the driving force for K⁺ and Ca²⁺ uptake. Efflux measurements from ⁸⁶Rb⁺ and ⁴⁵Ca²⁺ loaded cells suggest that part of the intracellular K⁺ and most of the intracellular Ca²⁺ is nonexchangeable with the extracellular pool. Correlations between phosphate and K⁺ contents and the effect of phosphate on K⁺ efflux suggest intracellular associations between K⁺ and polyphosphates. On the basis of these results, it is suggested that: (a) K⁺ and Ca²⁺ uptake in D. salina is driven by the transmembrane electrical potential which is generated by the action of an H⁺-ATPase of the plasma membrane. (b) Part of the intracellular K⁺ is associated with polyphosphate bodies, while most of the intracellular Ca²⁺ is accumulated in intracellular organelles in the algal cells.     

Fetuin in human bone marrow: detection in foetal tissue and patients with mastocytosis

Fetuin, a foetal protein of unknown function, has been shown to be expressed in both the immune and nervous systems, especially during development. Here, we show for the first time, that fetuin is abundantly present in many cells of the foetal human bone marrow, but is restricted to cells of the monocytic lineage in the adult. Fetuin's immunoreactivity increased considerably in adult human bone marrow in some pathological conditions, particularly in mastocytosis and was also increased in bone marrows in some cases of acute leukaemias, especially in acute myeloid leukaemia. This increase in the presence of fetuin in neoplastic bone marrows is not reflected by an increased level of circulating fetuin. This last observation contradicts earlier suggestions that fetuin is specifically reduced in cancer patients. A consistent increase in fetuin immunoreactivity in bone marrow of most cases of mastocytosis, as demonstrated in this paper, could become a useful tool in the diagnosis of this disease.     

Effect of dexmedetomidine on the release of [3H]-noradrenaline from rat kidney cortex slices: characterization of alpha2-adrenoceptor

The presynaptic modulation of [3H]-noradrenaline (NA) release from rat kidney cortex slices, a method used for the first time, was investigated. Rat kidney cortex slices were loaded with [3H]-NA and the release of radioactivity at rest and in response to field stimulation was determined. The alpha(2)-adrenoceptor agonist, dexmedetomidine inhibited the stimulation-evoked release of NA from kidney slices in a concentration-dependent manner, whereas alpha(2)-adrenoceptor antagonist CH-38083 (7,8-methyenedioxy-14-alpha-hydroxyalloberbane HCl), an alpha(2)-adrenoceptor antagonists, enhanced it. When dexmedetomidine and BRL-44408, a selective alpha(2A) antagonist, were added together, the effect of dexmedetomidine was significantly antagonized. In contrast, ARC-239 (2-(2,4-(o-piperazine-1-yl)-ethyl-4,4-dimethyl-1,3-(2H, 4H)disoguinolinedione chloride), a selective alpha(2B)-antagonist, had no effect on the release and failed to prevent the effect of dexmedetomidine. Prazosin, an alpha(1)- and alpha(2B/C)-adrenoceptor antagonist enhanced the release evoked by field stimulation. It is therefore suggested that there is a negative feedback modulation of NA release at the sympathetic innervation of kidney cortex, and dexmedetomidine, a clinically used anesthetic adjunct inhibits the release via activation of alpha(2C)-adrenoceptors.     

Role of glutathione S-transferases in protection against lipid peroxidation. Overexpression of hGSTA2-2 in K562 cells protects against hydrogen peroxide-induced apoptosis and inhibits JNK and caspase 3 activation

The physiological significance of the selenium-independent glutathione peroxidase (GPx) activity of glutathione S-transferases (GSTs), associated with the major Alpha class isoenzymes hGSTA1-1 and hGSTA2-2, is not known. In the present studies we demonstrate that these isoenzymes show high GPx activity toward phospholipid hydroperoxides (PL-OOH) and they can catalyze GSH-dependent reduction of PL-OOH in situ in biological membranes. A major portion of GPx activity of human liver and testis toward phosphatidylcholine hydroperoxide (PC-OOH) is contributed by the Alpha class GSTs. Overexpression of hGSTA2-2 in K562 cells attenuates lipid peroxidation under normal conditions as well as during the oxidative stress and confers about 1.5-fold resistance to these cells from H(2)O(2) cytotoxicity. Treatment with 30 microm H(2)O(2) for 48 h or 40 microm PC-OOH for 8 h causes apoptosis in control cells, whereas hGSTA2-2-overexpressing cells are protected from apoptosis under these conditions. In control cells, H(2)O(2) treatment causes an early (within 2 h), robust, and persistent (at least 24 h) activation of JNK, whereas in hGSTA2-2-overexpressing cells, only a slight activation of JNK activity is observed at 6 h which declines to basal levels within 24 h. Caspase 3-mediated poly(ADP-ribose) polymerase cleavage is also inhibited in cells overexpressing hGSTA2-2. hGSTA2 transfection does not affect the function of antioxidant enzymes including GPx activity toward H(2)O(2) suggesting that the Alpha class GSTs play an important role in regulation of the intracellular concentrations of the lipid peroxidation products that may be involved in the signaling mechanisms of apoptosis.     

The phylogenetic analysis of variable-length sequence data: elongation factor-1alpha introns in European populations of the parasitoid wasp genus Pauesia (Hymenoptera: Braconidae: Aphidiinae)

Elongation factor-1alpha (EF-1alpha) is a highly conserved nuclear coding gene that can be used to investigate recent divergences due to the presence of rapidly evolving introns. However, a universal feature of intron sequences is that even closely related species exhibit insertion and deletion events, which cause variation in the lengths of the sequences. Indels are frequently rich in evolutionary information, but most investigators ignore sites that fall within these variable regions, largely because the analytical tools and theory are not well developed. We examined this problem in the taxonomically problematic parasitoid wasp genus Pauesia (Hymenoptera: Braconidae: Aphidiinae) using congruence as a criterion for assessing a range of methods for aligning such variable-length EF-1alpha intron sequences. These methods included distance- and parsimony-based multiple-alignment programs (CLUSTAL W and MALIGN), direct optimization (POY), and two "by eye" alignment strategies. Furthermore, with one method (CLUSTAL W) we explored in detail the robustness of results to changes in the gap cost parameters. Phenetic-based alignments ("by eye" and CLUSTAL W) appeared, under our criterion, to perform as well as more readily defensible, but computationally more demanding, methods. In general, all of our alignment and tree-building strategies recovered the same basic topological structure, which means that an underlying phylogenetic signal remained regardless of the strategy chosen. However, several relationships between clades were sensitive both to alignment and to tree-building protocol. Further alignments, considering only sequences belonging to the same group, allowed us to infer a range of phylogenetic relationships that were highly robust to tree-building protocol. By comparing these topologies with those obtained by varying the CLUSTAL parameters, we generated the distribution area of congruence and taxonomic compatibility. Finally, we present the first robust estimate of the European Pauesia phylogeny by using two EF-1alpha introns and 38 taxa (plus 3 outgroups). This estimate conflicts markedly with the traditional subgeneric classification. We recommend that this classification be abandoned, and we propose a series of monophyletic species groups.     

Multiple roles of mouse Numb in tuning developmental cell fates.

BACKGROUND: Notch signaling regulates multiple differentiation processes and cell fate decisions during both invertebrate and vertebrate development. Numb encodes an intracellular protein that was shown in Drosophila to antagonize Notch signaling at binary cell fate decisions of certain cell lineages. Although overexpression experiments suggested that Numb might also antagonize some Notch activity in vertebrates, the developmental processes in which Numb is involved remained elusive. RESULTS: We generated mice with a homozygous inactivation of Numb. These mice died before embryonic day E11.5, probably because of defects in angiogenic remodeling and placental dysfunction. Mutant embryos had an open anterior neural tube and impaired neuronal differentiation within the developing cranial central nervous system (CNS). In the developing spinal cord, the number of differentiated motoneurons was reduced. Within the peripheral nervous system (PNS), ganglia of cranial sensory neurons were formed. Trunk neural crest cells migrated and differentiated into sympathetic neurons. In contrast, a selective differentiation anomaly was observed in dorsal root ganglia, where neural crest--derived progenitor cells had migrated normally to form ganglionic structures, but failed to differentiate into sensory neurons. CONCLUSIONS: Mouse Numb is involved in multiple developmental processes and required for cell fate tuning in a variety of lineages. In the nervous system, Numb is required for the generation of a large subset of neuronal lineages. The restricted requirement of Numb during neural development in the mouse suggests that in some neuronal lineages, Notch signaling may be regulated independently of Numb.     

Cytochrome b561 is fatty acylated and oriented in the chromaffin granule membrane with its carboxyl terminus cytoplasmically exposed

Two polyclonal antibodies were raised to synthetic peptides corresponding to amino acids Ser21-Tyr35 and Lys247-Phe261 of cytochrome b561. These antibodies were used to test the native orientation of the amino and carboxyl termini of this transmembrane electron transport protein. Carboxyl-terminal epitopes were lost when intact chromaffin granules were treated with Pronase. This result indicates that the carboxyl terminus is cytoplasmically exposed and confirms a theoretical prediction obtained from hydropathy plots. Epitopes that were recognized by an amino-terminal antipeptide antibody were not removed under the same conditions. This finding implied that the amino terminus was not proteolytically accessible on the exterior of the granule. The abundance of threonine and serine residues in the amino-terminal region suggested that the amino terminus could be held in the membrane by covalent fatty acylation. Treatment of purified delipidated cytochrome b561 with hydroxylamine resulted in the release of a fatty acid hydroxamate. Sulfhydryl analysis of purified cytochrome b561 showed that all 3 cysteine residues were in the free sulfhydryl form. These observations indicate that cytochrome b561 is covalently fatty acylated and that the lipid is bound through ester linkages of serine or threonine residues.     

Extraction and analysis of superoxide free radicals (·O−2) from whole mammalian liver

Extraction of whole lobes of normal rat liver with dimethyl sulphoxide (DMSO) under N₂ gives extracts which contain 5—10 μmol/l·O^?₂ (50-100 nmol·O^?₂ per 10 ml extract per 4 g liver; 1.25-2.50 nmol·O^?₂ per millilitre per gram liver). Evidence for ·O^?₂ in the extracts is given by: (1) electron spin resonance signals (ESR), (2) differential pulse polarography (DPP), (3) chemiluminescence (CL), and (4) nitroblue tetrazolium reduction (NBT). All tests yield results identical with those obtained with authentic ·O^?₂. Extraction of ·O^?₂ is enhanced by tetrabutyl ammonium ion, and is maximal at 1-3 min. These results raise the possibility that substantial amounts of ·O^?₂ are normally sequestered in protective membranous sites in vivo.