Fluidization of Membrane Lipids Enhances the Tolerance of Saccharomyces cerevisiae to Freezing and Salt Stress

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Δ⁹ position. We expressed two sunflower (Helianthus annuus) oleate Δ¹² desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Δ^9,12, the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15°C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp⁺ or Trp⁻ strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30°C or 15°C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.     

Genetic diversity in Tunisia: a study based on the GM polymorphism of human immunoglobulins

The GM polymorphism of human immunoglobulins is analyzed in three Berber populations of southern Tunisia and compared to other GM data. Genetic diversity among Tunisian populations is higher than that among Europeans but does not exhibit any significant geographic or linguistic structure. This result suggests a complex pattern of genetic differentiation.     

Synthesis and Characterization of Tripodal Iron(II) Complexes Prepared from 2-Pyridinecarboxaldehyde and 1-Methyl-2-Imidazolecarboxaldehyde. Stabilization of Iron(II) Cations with N(6) Donor Sets

Reaction in air of iron(III) salts with tripodal ligands formed from the condensation of tris-(2-aminoethyl)amine (tren) with three equivalents of 2-pyridinecarboxaldehyde (py) or 1-methyl-2-imidazolecarboxaldehyde (NCH₃Im) yielded exclusively the iron(II) complexes, [Fetren(py)₃]X₂ (X=ClO₄ ⁻ or PF₆ ⁻) or [Fetren(NCH₃Im)₃](ClO₄)₂. The complexes were characterized by EA, IR, UV, Mössbauer, and mass spectroscopy. The structure of [Fetren(py)₃](ClO₄)₂ was determined at 100 and 290 K. The structures are essentially the same and feature an octahedral iron with facial coordination of pyridine and imine nitrogen atoms with average bond distances of 1.9747 and 1.9523 Å, respectively, at 290 K. The short Fe-N bond distances and lack of variation with temperature support a low spin, ¹A, assignment for the iron atom. The center nitrogen atom of the tren is essentially planar and is outside of bonding interaction with the iron, 3.45 Å. The low spin assignment is supported by Mössbauer spectroscopy, which reveals the presence of two low spin forms that are not in thermal equilibrium. In contrast, the Mössbauer spectrum of [Fetren(NCH₃Im)₃](ClO₄)₂ reveals the presence of both high spin, ⁵T, and low spin, ¹A, forms at room temperature, which on cooling to 77 K simplify to the low spin form.     

Spatial and temporal regulation of RACK1 function and N-methyl-D-aspartate receptor activity through WD40 motif-mediated dimerization

Efficient signaling requires accurate spatial and temporal compartmentalization of proteins. RACK1 is a scaffolding protein that fulfils this role through interaction of binding partners with one of its seven WD40 domains. We recently identified the kinase Fyn and the NR2B subunit of the N-methyl-D-Aspartate receptor (NMDAR) as binding partners of RACK1. Scaffolding of Fyn near its substrate NR2B by RACK1 inhibits Fyn phosphorylation of NR2B and thereby negatively regulates channel function. We found that Fyn and NR2B share the same binding site on RACK1; however, their binding to RACK1 is not mutually exclusive (Yaka, R., Thornton, C., Vagts, A. J., Phamluong, K., Bonci, A., and Ron, D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5710-5715). We therefore tested the hypothesis that RACK1 forms a homodimer that allows the simultaneous binding of Fyn and NR2B. We found that RACK1 binds to itself both in vitro and in the brain. Deletion analyses identified a RACK1-RACK1 dimer-binding site within the 4th WD40 repeat, and application of the 4th WD40 repeat or a peptide derivative to hippocampal slices inhibited NMDAR activity. We further found that in hippocampal slices, both RACK1 and NR2B associated with another WD40 protein, the beta-subunit of G protein (Gbeta), previously shown to heterodimerize with RACK1 in vitro (Dell, E. J., Connor, J., Chen, S., Stebbins, E. G., Skiba, N. P., Mochly-Rosen, D., and Hamm, H. E. (2002) J. Biol. Chem. 277, 49888-49895). However, activation of the pituitary adenylate cyclase polypeptide (1-38) G protein-coupled receptor, previously found to induce the dissociation of RACK1 from the NMDAR complex (Yaka, R., He, D. Y., Phamluong, K., and Ron, D. (2003) J. Biol. Chem. 278, 9630-9638), attenuated the association of Gbeta with RACK1 and NR2B. Based on these results, we propose that WD40-mediated homo- and heterodimerization of RACK1 mediate the formation of a transient signaling complex that includes the NMDAR, a G protein and Fyn.     

Ion channel activity of membrane vesicles released from sea urchin sperm during the acrosome reaction

The sperm acrosome reaction (AR) involves ion channel activation. In sea urchin sperm, the AR requires Ca2+ and Na+ influx and K+ and H+ efflux. During the AR, the plasma membrane fuses with the acrosomal vesicle membrane forming hybrid membrane vesicles that are released from sperm into the medium. This paper reports the isolation and preliminary characterization of these acrosome reaction vesicles (ARVs), using synaptosome-associated protein of 25 kDa (SNAP-25) as a marker. Isolated ARVs have a unique protein composition. The exocytosis regulatory proteins vesicle-associated membrane protein and SNAP-25 are inside ARVs, as judged by protease protection experiments, and membrane associated based on Triton X-114 partitioning. ARVs fused with planar bilayers display three main types of single channel activity. The most frequently recorded channel is cationic, weakly voltage dependent and has a low open probability that increases with negative potentials. This channel is activated by cAMP, blocked by Ba2+, and has a PK+/PNa+ selectivity of 4.5. ARVs represent a novel membrane preparation suitable to deepen our understanding of ion channel activity in the AR and during fertilization.     

Regulation of prohibitin expression during follicular development and atresia in the mammalian ovary

Prohibitin is a ubiquitous and highly conserved protein implicated as an important regulator in cell survival. Prohibitin content is inversely associated with cell proliferation, but it increases during granulosa cell differentiation as well as in earlier events of apoptosis in a temperature-sensitive granulosa cell line. In the present study, we have characterized the spatial expression patterns for prohibitin using established in vivo models for the induction of follicular development and atresia in the mammalian ovary. Comparative Western blot analyses of granulosa cell lysates from control ovaries and from ovaries primed with eCG or treated with eCG plus anti-eCG (gonadotropin withdrawal) were conducted. Prohibitin was immunolocalized in rat ovarian sections probed with antibodies against either proliferating cell nuclear antigen (PCNA) or cholesterol side-chain cleavage cytochrome P450 (P450(scc)) or in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeled sections. Additionally, porcine oocytes, zygotes, and blastocyts were also immunolocalized with prohibitin antibody. Immunolocalization revealed the presence of prohibitin in granulosa cells, theca-interstitial cells, and the oocyte. The results indicate that prohibitin protein expression in the gonadotropin-treated cells was upregulated. Immunoreactivity of prohibitin was inversely related to PCNA expression during follicular maturation and colocalized with P450(scc). Prohibitin appeared to be translocated from the cytoplasm to the nucleus in atretic follicles, germinal vesicle-stage oocytes, zygotes, and blastocysts. These results suggest that prohibitin has several functional regulatory roles in granulosa and theca-interstitial cells and in the ovum during follicular maturation and atresia. It is likely that prohibitin may play an important role in determining the fate of these cells and eventual follicular destiny.     

Trypanosoma cruzi: partial characterization of minor cruzipain isoforms non-adsorbed to Concanavalin A-Sepharose

The present paper reports the partial characterization of a subset of atypical cruzipain molecules which do not bind to Concanavalin A-Sepharose column. They are present in different strains of epimastigote forms of Trypanosoma cruzi and represent a 2-4% of total cruzipain. They were purified by affinity chromatography on Cystatin-Sepharose, recognized by the polyclonal anti-cruzipain serum, and their activity in gelatin-containing gels was completely abolished by E-64, TLCK, leupeptin, and aprotinin but not by PMSF, pepstatin A, EDTA or 1,10-phenantroline. These cysteine proteinases, as well as cruzipain showed to be endoproteinases able to hydrolize azocasein, hemoglobin, and bovine serum albumin at acidic pHs. However, evidences are presented indicating that this subset of cruzipain isoforms were also able to use the same blocked chromogenic peptidyl substrates than cruzipain at similar optimal alkaline pH values although with a different order of preference. Moreover, they showed a different oligosaccharide pattern after enzymatic treatment by high pH anion exchange chromatography, suggesting that this structural difference may account for the atypical behaviour in the lectin column.     

Mortality rate and relative strain index in Buenos Aires city

Thermal stress may seriously affect human health to the extent of provoking death in those human groups at high risk such as little children and elderly persons. This research studies the climatology of the relative strain index (RSI) in the city of Buenos Aires (Argentina). It is observed that the conditions at 1400 hours local time on more than 75% of summer days cause discomfort, though only on 25% of days are physiological failure levels reached. The nocturnal RSI values show 75% of nights to be comfortable, bringing relief and allowing physiological recuperation. Comparison between the RSI frequency distributions of different decades by the application of the Kolmogorov-Smirnov test shows a possible urban heat-island effect that increases nocturnal RSI values. The relationship between the RSI and the daily number of interments is also analysed. Because of the large number of different causes of mortality, only about 10% of the variance of the daily number of interments can be explained by the RSI value at 1400 hours local time 1 or 2 days before. Comparison of mortality figures with these RSI values shows deaths to be at a minimum for the interval 0.0-0.1 (the RSI comfort range), slightly increasing for negative values that could represent unexpected Summer cold waves. For values of RSI over 0.2 the increment is more abrupt, indicating an impact of a thermal strain on mortality. The relationship between the joint occurrence of the 1400 hour and 0200 hour (local time) RSI values and mortality shows a clear impact of prolonged exposure to uncomfortable conditions.