Subcellular localisation of FLAG tagged enzymes of the dynamic protein S-palmitoylation cycle of Trypanosoma cruzi epimastigotes

Dynamic S-palmitoylation of proteins is the addition of palmitic acid by zDHHC palmitoyl transferases (PATs) and depalmitoylation by palmitoyl protein thioesterases (PPTs). A putative PAT (TcPAT1) has been previously identified in Trypanosoma cruzi, the etiological agent of Chagas disease. Here we analyse other 14 putative TcPATs and 2 PPTs in the parasite genome. T. cruzi cell lines expressing TcPATs and TcPPTs plus a FLAG tag at the C terminus were produced for most enzymes, with positive detection by indirect immunofluorescence. Overexpressed TcPATs were mostly found as single spots at the parasite anterior end, while the TcPPTs were dispersed throughout the parasite body.

Bioelectrical Impedance May Predict Cell Viability During Ischemia and Reperfusion in Rat Liver

Ischemia and reperfusion (I/R) injury is a major cause of hepatic failure after liver surgery, but no method could monitor or predict it real-time during surgery. We measured bioelectrical impedance (BEI) and cell viability to assess the usefulness of BEI during I/R in rat liver. A 70% partial liver ischemia model was used. BEI was measured at various frequencies. Adenosine triphosphate (ATP) content, and palmitic acid oxidation rate were measured, and histological changes were observed in order to quantify liver cell viability. BEI changed significantly during ischemia at low frequency. In the ischemia group, BEI increased gradually during 60 min of ischemia and had a tendency to plateau thereafter. The ATP content decreased below 20% of the baseline level. In the I/R group, BEI recovered to near baseline level. After 24 hr of reperfusion, the ATP contents decreased to below 50% in 30, 60 and 120 min of ischemia and the palmitic acid metabolic rates decreased to 91%, 78%, and 74%, respectively, compared with normal liver. BEI may be a good tool for monitoring I/R during liver surgery. The liver is relatively tolerant to ischemia, however after reperfusion, liver cells may be damaged depending upon the duration of ischemia.

Retainment of membrane binding capacity of non-palmitoylated Gs alpha mutants expressed in COS-1 cells

Heterotrimeric guanine nucleotide binding regulatory proteins (G proteins) transduce extracellular signals into intracellular signals by coupling receptors and effectors. Because most of the G protein-coupled receptors are integral proteins, the G proteins need to have a membrane binding capacity to receive signals from the receptors. The alpha subunit of G protein binds tightly to the cytoplasmic face of the plasma membrane without any membrane spanning domain. Fatty acylation of G alpha with myristic acid or palmitic acid, in addition to the beta gamma subunits, plays an important role in anchoring the G alpha subunit. The reversible and dynamic palmitoylation of the alpha subunit of stimulatory G protein (Gs alpha) has been suggested as essential for its membrane attachment. However, in our previous experiments, Gs alpha deleted in the amino terminus containing palmitoylation site, retained its binding capacity when expressed in COS cells. Thus, to evaluate the role of palmitoylation in Gs alpha membrane binding, we constructed and expressed non-palmitoylated mutants of Gs alpha and analyzed their subcellular distributions in COS-1 cells. We found that non-palmitoylated mutants of Gs alpha, C3S- and G2A/C3S Gs alpha, retained their membrane binding capacities in COS-1 cells, demonstrating that palmitoylation is not essential for membrane binding of Gs alpha in COS-1 cells. We also found that the palmitoylation did not change significantly the distribution of Gs alpha in Triton X-114 partition. These results suggest that the palmitoylation of Gs alpha may produce different effects on membrane binding depending on cell types.

Comparative analysis of erythrocyte fatty acid composition in a sample of Mexico city children and young adults on a free diet

In the present study the influence of age on red blood cell fatty acid (RBCFA) composition was analyzed in a sample of Mexico City children and young people on a free diet, as there is scarce information about RBCFA composition in the Mexican population. Erythrocyte lipids were extracted with isopropyl alcohol and fatty acid methyl esters were prepared to be analyzed by gas liquid chromatography. The 1. to 2-year-old group showed a higher percent level of C18:0 (34.73 ñ 2.5 vs. 29.67 ñ 1.3, p<0.002) and lower of C16:1 (0.58 ñ 0.2 vs. 1.09 ñ 0.2, p<0.005), C20:4 (14.08 ñ 4.1 vs, 18.20 ñ 1.2, p<0.05) and C22:5 (2.79 ñ 1.7 vs. 7.68 ñ 0.8, p<0.001) than the 20- to 25-year-old group. Both groups showed a very low linoleic acid proportion, children 0.48 percent and young adults 0.54 percent. The unsaturated/saturated fatty acid ratio was found to be 0.55 ñ 0.2 in children and 0.91 ñ 0.1 in adults (p<0.001). These findigs indicate the presence of factors related to age that affect the fatty acid composition in the erythrocyte membrane different from diet habits in the sample analyzed. Results are compared with reports in the literature

The influence of albumin on palmitate transport in the isolated perfused rat liver

Unidirectional fluxes (influx and efflux) and the net flux of palmitate across the hepatocyte membrane were measured in the intact rat liver employing the multiple indicator dilution technique. At albumin concentrations in the range between 0.1 and 0.5 mM the influx rate was 2.3 times greater than that of the net flux. The rate of efflux was somewhat higher than the net flux, indicating that palmitate undergoes an exchange process across the liver cell membranes with efflux to the extracellular albumin site being significant. At. lower albumin concentrations, hower, the influx/net flux approached unit, implying that transport becomes a rate-limiting factor for metabolism

Increased fat degradation in the denervated muscle of frog.

The total lipids triglycerides, free fatty acids, glycerol and acetoaectate levels in the gastrocnemius muscle of frog denervated for 1 month were compared. A significant atrophy without any change in total DNA content per whole muscle was noted on denervation. No significant change in the mitochondrial protein content in the atrophied muscle was noted. Relative to total muscle mass, the total fat, triglyceride, free fatty acids and glycerols decreased on denervation. This suggests the increased degradation of fat during denervation. Concomittantly lypolytic and esterase activities in the atrophied muscle increased. Palmitate and pyruvate oxidations in the mitochondria of atrophied muscle and also the enzymes of beta-oxidative scheme increased. 14C-acetate incorporation rates revealed that there is a decrease in fatty acid synthesis in the atrophied muscle. It was suggested that the increased fatty acid oxidation and decreased fatty acid synthesis in the atrophic process may not be the result of a simple functional demand; but may involve more factors in terms of neuro-muscular functions.