Immunomodulation of voltage-dependent K+ channels in macrophages: molecular and biophysical consequences

Voltage-dependent potassium (K_v) channels play a pivotal role in the modulation of macrophage physiology. Macrophages are professional antigen-presenting cells and produce inflammatory and immunoactive substances that modulate the immune response. Blockage of K_v channels by specific antagonists decreases macrophage cytokine production and inhibits proliferation. Numerous pharmacological agents exert their effects on specific target cells by modifying the activity of their plasma membrane ion channels. Investigation of the mechanisms involved in the regulation of potassium ion conduction is, therefore, essential to the understanding of potassium channel functions in the immune response to infection and inflammation. Here, we demonstrate that the biophysical properties of voltage-dependent K⁺ currents are modified upon activation or immunosuppression in macrophages. This regulation is in accordance with changes in the molecular characteristics of the heterotetrameric K_v1.3/K_v1.5 channels, which generate the main K_v in macrophages. An increase in K⁺ current amplitude in lipopolysaccharide-activated macrophages is characterized by a faster C-type inactivation, a greater percentage of cumulative inactivation, and a more effective margatoxin (MgTx) inhibition than control cells. These biophysical parameters are related to an increase in K_v1.3 subunits in the K_v1.3/K_v1.5 hybrid channel. In contrast, dexamethasone decreased the C-type inactivation, the cumulative inactivation, and the sensitivity to MgTx concomitantly with a decrease in K_v1.3 expression. Neither of these treatments apparently altered the expression of K_v1.5. Our results demonstrate that the immunomodulation of macrophages triggers molecular and biophysical consequences in K_v1.3/K_v1.5 hybrid channels by altering the subunit stoichiometry.     

A chemogenomic screening of sulfanilamide-hypersensitive Saccharomyces cerevisiae mutants uncovers ABZ2, the gene encoding a fungal aminodeoxychorismate lyase

Large-scale phenotypic analyses have proved to be useful strategies in providing functional clues about the uncharacterized yeast genes. We used here a chemogenomic profiling of yeast deletion collections to identify the core of cellular processes challenged by treatment with the p-aminobenzoate/folate antimetabolite sulfanilamide. In addition to sulfanilamide-hypersensitive mutants whose deleted genes can be categorized into a number of groups, including one-carbon related metabolism, vacuole biogenesis and vesicular transport, DNA metabolic and cell cycle processes, and lipid and amino acid metabolism, two uncharacterized open reading frames (YHI9 and YMR289w) were also identified. A detailed characterization of YMR289w revealed that this gene was required for growth in media lacking p-aminobenzoic or folic acid and encoded a 4-amino-4-deoxychorismate lyase, which is the last of the three enzymatic activities required for p-aminobenzoic acid biosynthesis. In light of these results, YMR289w was designated ABZ2, in accordance with the accepted nomenclature. ABZ2 was able to rescue the p-aminobenzoate auxotrophy of an Escherichia coli pabC mutant, thus demonstrating that ABZ2 and pabC are functional homologues. Phylogenetic analyses revealed that Abz2p is the founder member of a new group of fungal 4-amino-4-deoxychorismate lyases that have no significant homology to its bacterial or plant counterparts. Abz2p appeared to form homodimers and dimerization was indispensable for its catalytic activity.     

Computational approach for identification and characterization of GPI-anchored peptides in proteomics experiments

Genes that encode glycosylphosphatidylinositol anchored proteins (GPI-APs) constitute an estimated 1-2% of eukaryote genomes. Current computational methods for the prediction of GPI-APs are sensitive and specific; however, the analysis of the processing site (omega- or omega-site) of GPI-APs is still challenging. Only 10% of the proteins that are annotated as GPI-APs have the omega-site experimentally verified. We describe an integrated computational and experimental proteomics approach for the identification and characterization of GPI-APs that provides the means to identify GPI-APs and the derived GPI-anchored peptides in LC-MS/MS data sets. The method takes advantage of sequence features of GPI-APs and the known core structure of the GPI-anchor. The first stage of the analysis encompasses LC-MS/MS based protein identification. The second stage involves prediction of the processing sites of the identified GPI-APs and prediction of the corresponding terminal tryptic peptides. The third stage calculates possible GPI structures on the peptides from stage two. The fourth stage calculates the scores by comparing the theoretical spectra of the predicted GPI-peptides against the observed MS/MS spectra. Automated identification of C-terminal GPI-peptides from porcine membrane dipeptidase, folate receptor and CD59 in complex LC-MS/MS data sets demonstrates the sensitivity and specificity of this integrated computational and experimental approach.     

Influence of metabolic status on oocyte quality and follicular characteristics at different postpartum periods in primiparous rabbit does

Low reproductive performance of high-yield primiparous animals is closely associated with the metabolic stress caused by a simultaneous gestation and lactation. The aim of this work was (1) to analyze body composition and metabolic environment at three time points along lactation (at parturition time; in the lactation period [Day 11 postpartum]; and in the postweaning period [Day 32 postpartum]) of primiparous rabbit does (Oryctolagus cuniculus) and (2) to investigate the ovarian status at insemination time and the possible link with metabolic environment and with their reproductive performance. To this end, does were either submitted to a semi-intensive reproductive rhythm (Group S, inseminated on Day 11 postpartum) or an extensive rhythm (Group E, inseminated on Day 32 postpartum). Body energy (P < 0.05) and protein content (P < 0.001) as well as serum leptin (P < 0.05) and protein concentrations (P < 0.001) increased significantly along the postpartum period. At parturition, body lipid content was significantly lower and serum nonesterified fatty acids concentrations were significantly higher than that on Days 11 postpartum and 32 postpartum. Concerning ovarian status at insemination time, no significant differences were found in mean follicular stages, serum estradiol, progesterone, and prolactin (PRL) concentrations or in prolactin receptor (PRL-R) immunostaining. However, follicles in Group S showed a significantly higher apoptosis index than that of Group E (P < 0.001). The nuclear and cytoplasmic oocyte maturation rates of Group S were also significantly lower than that in Group E. In addition, conception rate and prolificacy were improved in Group E (P < 0.001 and P < 0.05, respectively). In conclusion, in the early postpartum period, metabolic status seems to impact negatively on ovarian follicle and oocyte quality leading to a poor reproductive outcome in primiparous rabbit does.     

Temporal speciation pattern in the western Mediterranean genus Tudorella P. Fischer, 1885 (Gastropoda,Pomatiidae) supports the Tyrrhenian vicariance hypothesis

The land snail genus Tudorella shows a peculiar disjunct distribution around the western Mediterranean coasts. Despite high phenotypic plasticity, only two species with a disputed number of subspecific taxa are currently recognised. We delimited the species with mitochondrial (COI & 16S) and nuclear (ITS-1) markers based on the unified species concept and suggested that there are eight species in the genus, two of them currently undescribed. Applying Bayesian phylogenetic model selection, we tested four different biogeographic hypotheses that could be causal for the current distribution pattern of extant Tudorella species. A scenario involving vicariance events resulting from the repeated splits of the Tyrrhenian plate with subsequent dispersal events over land bridges during the Pliocene received greatest support in the data.     

Differential responses of three fungal species to environmental factors and their role in the mycorrhization of<Emphasis Type="Italic"> Pinus radiata</Emphasis> D. Don

Three ectomycorrhizal (ECM) isolates of Rhizopogon luteolus, R. roseolus and Scleroderma citrinum were found to differ markedly in their in vitro tolerance to adverse conditions limiting fungal growth, i.e. water availability, pH and heavy metal pollution. S. citrinum was the most sensitive, R. luteolus intermediate and R. roseolus the most tolerant species. Pinus radiata D. Don seedlings were inoculated in the laboratory and in a containerised seedling nursery with spore suspensions of the three ECM species. Colonisation percentage was considerably lower under nursery conditions, probably due to competition by native fungi. The effects of nursery ECM inoculation on seedling growth depended on the fungal species. Only R. roseolus-colonised plants showed a significantly higher shoot growth than non-mycorrhizal plants. All three fungi induced significantly higher root dry weights relative to control plants. Despite the low mycorrhizal colonisation, mycorrhization with all three species improved the physiological status of nursery-grown seedlings, e.g. enhanced root enzyme activity, shoot nutrient and pigment content, net photosynthesis rate and water use efficiency. Of the three fungal species, R. roseolus was the most effective; this species was also the most adaptable and showed the greatest range of tolerance to adverse environmental conditions in pure culture. It is, therefore, proposed as a promising fungal species for ECM inoculation of P. radiata in the nursery.     

Three biotechnical processes using Ashbya gossypii, Candida famata, or Bacillus subtilis compete with chemical riboflavin production

Chemical riboflavin production, successfully used for decades, is in the course of being replaced by microbial processes. These promise to save half the costs, reduce waste and energy requirements, and use renewable resources like sugar or plant oil. Three microorganisms are currently in use for industrial riboflavin production. The hemiascomycetes Ashbya gossypii, a filamentous fungus, and Candida famata, a yeast, are naturally occurring overproducers of this vitamin. To obtain riboflavin production with the Gram-positive bacterium Bacillus subtilis requires at least the deregulation of purine synthesis and a mutation in a flavokinase/FAD-synthetase. It is common to all three organisms that riboflavin production is recognizable by the yellow color of the colonies. This is an important tool for the screening of improved mutants. Antimetabolites like itaconate, which inhibits the isocitrate lyase in A. gossypii, tubercidin, which inhibits purine biosynthesis in C. famata, or roseoflavin, a structural analog of riboflavin used for B. subtilis, have been applied successfully for mutant selections. The production of riboflavin by the two fungi seems to be limited by precursor supply, as was concluded from feeding and gene-overexpression experiments. Although flux studies in B. subtilis revealed an increase both in maintenance metabolism and in the oxidative part of the pentose phosphate pathway, the major limitation there seems to be the riboflavin pathway. Multiple copies of the rib genes and promoter replacements are necessary to achieve competitive productivity. Received: 19 November 1999 / Accepted: 21 December 1999