Combining proteomics and lipid analysis to unravel Confidor stress response in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful model for studying the influence of different stress factors on eukaryotic cells. In this work we used the pesticide imidacloprid, in the Confidor formulation, as the stress factor and analyzed its influence on the metabolic activity, proteome and lipid content and composition of Saccharomyces cerevisiae yeast. During the cultivation of yeast, the lowest recommended application dose of Confidor (0.025%, v/v) was added to the growth media and its influence on the mitochondria, cytosol with microsomes, and the whole yeast cells was monitored. The results show that under the stress provoked by the toxic effects of Confidor, yeast cells density significantly decreased and the percentage of metabolically disturbed cells significantly increased comparing with untreated control. Also, there was a downregulation of majority of glycolytic, gluconeogenesis, and TCA cycle enzymes (Fba1, Adh1, Hxk2, Tal1, Tdh1,Tdh3, Eno1) thus providing enough acetyl‐CoA for the lipid restructuring and accumulation mechanism since we have found the changes in the cell and mitochondrial lipid content and FA composition. This data suggest that lipids could be the molecules that orchestrate the answer of the cells in the stress response to the Confidor treatment.     

An antiserum to a disease-associated factor from the cells of an HLA-B27 positive patient with ankylosing spondylitis specifically recognizes an HLA-B27 associated determinant

Antiserum raised to a factor elaborated by a lymphoblastoid cell line derived from the peripheral blood cells of an HLA-B27 positive patient with ankylosing spondylitis specifically lyses the B27 positive, but not the B27 negative, cells of ankylosing spondylitis patients. The cells of B27 positive and B27 negative normal controls are not lysed. This serum has similar specificity to antisera against cross-reactive bacteria.     

A factor shed by lymphoblastoid cell lines of HLA-B27 positive patients with ankylosing spondylitis, specifically modifies the cells of HLA-B27 positive normal individuals.

Epstein-Barr virus transformed lymphoblastoid cell lines from HLA-B27 positive individuals with ankylosing spondylitis (B27+AS+) release, into the culture medium, a factor capable of specifically modifying the HLA-B27 positive lymphocytes of normal individuals (B27+AS-); this modification results in a phenotypic change similar to that seen on B27+AS+ lymphocytes. This lymphoblastoid cell line derived factor appears to be physically and functionally similar to a factor present in the culture filtrate of certain Klebsiella isolates. Biogel P-100 chromatography of the material released from the cell line indicated a mol.wt of 25,000-30,000, similar to that of the Klebsiella derived factor. Chromatofocusing on a PBE 94 column revealed that cell line derived factor had an isoelectric point of 5.5 (cf. pI 5.4 for the Klebsiella derived factor). Immunoadsorption experiments suggest that the factor from the B27+AS+ cell line shares antigenic determinants with a cell surface component present on certain Klebsiella isolates. These results will form the basis for future studies on the nature of the interaction between HLA-B27 and certain enteric organisms and their products. A better understanding of this association should elucidate some of the early events in the pathogenesis of the seronegative arthropathies.     

Transgenic expression of a CD46 (membrane cofactor protein) minigene: Studies of xenotransplantation and measles virus infection

CD46 (membrane cofactor protein) is a human cell-surface regulator of activated complement and a receptor for the measles virus. A CD46 transgenic mouse line with an expression pattern similar to that of human tissues has been produced, to develop an animal model of (i) the control of complement activation by complement regulators in hyperacute rejection of xenografts, and (ii) measles virus infection. The mouse line was made using a CD46 minigene that includes promoter sequence and the first two introns of genomic CD46, which was coinjected into mouse ova with chicken lysozyme matrix attachment region DNA. A high level of CD46 expression in homozygotic transgenic mice was obtained with spleen cells having approximately 75% of the level found on human peripheral blood mononuclear cells. CD46 was detected in all tissues examined by immunohistochemistry, radioimmunoassay and Western blotting, showing that these mice were suitable for transplantation and measles virus infection studies. It also indicated that the transgene included the important regulatory elements of the CD46 promoter. Transgenic spleen cells were significantly protected in vitro from human complement activated by either the classical or alternative pathways and from alternative pathway rat complement. Furthermore, transgenic mouse hearts transplanted to rats regulated complement deposition in an in vivo model of antibody-dependent hyperacute xenograft rejection. Similar to human lymphocytes, transgenic lymphoblasts could be infected in vitro with measles virus; infected cells expressed viral proteins and produced infectious viral particles. The data demonstrate the suitability of this minigene for obtaining high-level CD46 expression sufficient for enhanced resistance of transgenic cells to complement attack and for obtaining wide tissue distribution of CD46, analogous to human tissues and, therefore, useful for comparative studies.     

Factors influencing the outcome of mark-release-recapture studies with Culex tarsalis (Diptera: Culicidae)

Three potentially important aspects of mark-release-recapture experimentation were addressed: 1) source of mosquitoes for release, 2) time of release, and 3) weather during recapture. Culex tarsalis Coquillett mosquitoes collected as adult host-seeking females from dry ice-baited traps (CO2 traps) operated within the study area (local) were recaptured more frequently than females collected from traps operated outside the study area (foreign) or reared from field-collected immatures (reared). These results supported published studies on Anopheles and Ochlerotatus that indicated mosquitoes may "memorize" flight paths within their environment. Releasing gravid females provided a potentially useful replacement for reared females, because these gravids oviposited at wetlands and then dispersed to upland traps. Releasing local, foreign, or reared mosquitoes just after sunrise or just before sunset did not alter recapture success or the distance dispersed. Elevated wind speeds inhibited dispersal from protected microhabitats with citrus orchards and resulted in most recaptures being found at the leeward portion of the orchard.     

The time-course of ribavirin-provoked changes of basal and AMPH-induced motor activities in rats

The time-course of changes of basal and amphetamine (AMPH)-induced locomotor and stereotypic activities in adult male Wistar rats after a single ribavirin injection was studied. In the first set of experiments, 10, 20 or 30 mg ribavirin/kg body weight (b.w.) were injected i.p. to rats and their basal motor activities were recorded every 10 min for 2 h and compared with those of saline-treated controls. In the second set of experiments, the animals were pretreated with ribavirin and 20 min later i.p. injected with AMPH (1.5 mg/kg b.w.). The controls received AMPH 20 min after the saline injection. Motor activity was recorded after the first injection and until 120 min after AMPH administration. Ribavirin did not significantly affect the time-course of either basal locomotor or stereotypic activities. Pretreatment with any of the applied ribavirin doses decreased the AMPH-induced hyperlocomotor response. However, the most pronounced effect was observed with ribavirin doses of 20 mg/kg and 30 mg/kg when administered during the first 10 min and 30 min after the AMPH injection respectively. In contrast, the stereotypic activities of these animals were only slightly changed. These results indicate a different susceptibility of regions in the basal ganglia to ribavirin.     

Induction of delayed hypersensitivity by dinitrophenylated lymphocytes

Erythrocytes, serum proteins and both living and killed lymphocytes from the peripheral blood of guinea-pigs were dinitrophenylated and then injected intraperitoneally into either the donor or other guinea-pigs. Animals sensitized by intradermal DNCB and unsensitized normal guinea-pigs served as positive and negative controls respectively. Eleven to 14 days after injection, the animals were tested with topically applied DNCB and the intensity of the reactions assessed by the local exudation of bovine serum albumin labelled with radio-iodine ([¹³¹I]BSA.)

Sensitization was achieved with dinitrophenylated living autologous lymphocytes, to a lesser degree with homologous lymphocytes, but not with erythrocytes, serum proteins or killed lymphocytes.