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.     

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.     

Precise quantification of pressure flow waveforms of a pulsatile ventricular assist device

Unreliable quantification of flow pulsatility has hampered many efforts to assess the importance of pulsatile perfusion. Generation of pulsatile flow depends upon an energy gradient. It is necessary to quantify pressure flow waveforms in terms of hemodynamic energy levels to make a valid comparison between perfusion modes during chronic support. The objective of this study was to quantify pressure flow waveforms in terms of energy equivalent pressure (EEP) and surplus hemodynamic energy (SHE) levels in an adult mock loop using a pulsatile ventricle assist system (VAD). A 70 cc Pierce-Donachy pneumatic pulsatile VAD was used with a Penn State adult mock loop. The pump flow rate was kept constant at 5 L/min with pump rates of 70 and 80 bpm and mean aortic pressures (MAP) of 80, 90, and 100 mm Hg, respectively. Pump flows were adjusted by varying the systolic pressure, systolic duration, and the diastolic vacuum of the pneumatic drive unit. The aortic pressure was adjusted by varying the systemic resistance of the mock loop EEP (mm Hg) = (integral of fpdf)/(integral of fdt) SHE (ergs/cm3) = 1,332 [((integral of fpdt)/(integral of fdt))--MAP] were calculated at each experimental stage. The difference between the EEP and the MAP is the extra energy generated by this device. This difference is approximately 10% in a normal human heart. The EEP levels were 88.3 +/- 0.9 mm Hg, 98.1 +/- 1.3 mm Hg, and 107.4 +/- 1.0 mm Hg with a pump rate of 70 bpm and an aortic pressure of 80 mm Hg, 90 mm Hg, and 100 mm Hg, respectively. Surplus hemodynamic energy in terms of ergs/cm3 was 11,039 +/- 1,236 ergs/cm3, 10,839 +/- 1,659 ergs/cm3, and 9,857 +/- 1,289 ergs/cm3, respectively. The percentage change from the mean aortic pressure to EEP was 10.4 +/- 1.2%, 9.0 +/- 1.4%, and 7.4 +/- 1.0% at the same experimental stages. Similar results were obtained when the pump rate was changed from 70 bpm to 80 bpm. The EEP and SHE formulas are adequate to quantify different levels of pulsatility for direct and meaningful comparisons. This particular pulsatile VAD system produces near physiologic hemodynamic energy levels at each experimental stage.