Different functional recognition of basolateral signals in Caco-2 and MDCK cells

Using the basolateral mutant PS of the normally apical neurotrophin receptor p75 (p75NTR) we have identified two cytoplasmic determinants responsible for this reversed localization in the human intestinal cell line, Caco2. These signals are based on two consecutive leucines (322-323) and a tyrosine (Y308). Truncation of the cytoplasmic tail removing the two leucines or their replacement by alanines led to a nonpolarized expression of the resulting mutants in Caco2 cells. To our surprise, the same mutations had no effect on the basolateral localization of the mutant PS in MDCK cells. In MDCK cells, the basolateral localization was entirely dependent on a cytoplasmic tyrosine Y308, while in Caco-2 cells this tyrosine signal was functional as a basolateral signal only when the cytoplasmic domain of PS was truncated shortly after it. These data indicate for the first time that there is a differential recognition of basolateral signals between MDCK and Caco-2 cells.     

31P magnetic resonance spectroscopy study of phosphocreatine recovery kinetics in skeletal muscle: the issue of intersubject variability

We have analyzed by (31)P MRS the relationship between kinetic parameters of phosphocreatine (PCr) recovery and end-of-exercise status under conditions of moderate and large acidosis induced by dynamic exercise. Thirteen healthy subjects performed muscular contractions at 0.47 Hz (low frequency, moderate exercise) and 0.85 Hz (high frequency, heavy exercise). The rate constant of PCr resynthesis (k(PCr)) varied greatly among subjects (variation coefficients: 43 vs. 57% for LF vs. HF exercises) and protocols (k(PCr) values: 1.3+/-0.5 min(-1) vs. 0.9+/-0.5 min(-1) for LF vs. HF exercises, P<0.03). The large intersubject variability can be captured into a linear relationship between k(PCr), the amount of PCr consumed ([PCr(2)]) and pH reached at the end of exercise (pH(end)) (k(PCr)=-3.3+0.7 pH(end)-0.03 [PCr(2)]; P=0.0007; r=0.61). This dual relationship illustrates that mitochondrial activity is affected by end-of-exercise metabolic status and allows reliable comparisons between control, diseased and trained muscles. In contrast to k(PCr), the initial rate of PCr recovery and the maximum oxidative capacity were always constant whatever the metabolic conditions of end-of-exercise and can then be additionally used in the identification of dysfunctions in the oxidative metabolic pathway.     

Analysis of the expression of CLA1, a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis

The discovery of the 2-C-methyl-D-erythritol-4-phosphate pathway for the biosynthesis of isoprenoids raises the important question of the nature and regulation of the enzymes involved in this pathway. CLA1, a gene previously isolated from Arabidopsis, encodes the first enzyme of the 2-C-methyl-D-erythritol-4-phosphate pathway, 1-deoxy-D-xylulose-5-phosphate synthase. We demonstrate this enzyme activity by complementation of the cla1-1 mutant phenotype and by direct enzymatic assays. Based on mRNA and protein expression patterns this enzyme is expressed mainly in developing photosynthetic and non-photosynthetic tissues. The beta-glucuronidase expression pattern driven from the CLA1 gene regulatory region supports the northern and protein data while also showing that this gene has some level of expression in most tissues of the plant. A mutation in the CLA1 gene interferes with the normal development of chloroplasts and etioplasts, but does not seem to affect amyloplast structure. Microscopic analysis also shows a pleiotropic effect of the CLA1 gene mutation in mesophyll tissue formation.     

Peroxynitrite-mediated nitration of the stable free radical tyrosine residue of the ribonucleotide reductase small subunit

Ribonucleotide reductase activity is rate-limiting for DNA synthesis, and inhibition of this enzyme supports cytostatic antitumor effects of inducible NO synthase. The small R2 subunit of class I ribonucleotide reductases contains a stable free radical tyrosine residue required for activity. This radical is destroyed by peroxynitrite, which also inactivates the protein and induces nitration of tyrosine residues. In this report, nitrated residues in the E. coli R2 protein were identified by UV-visible spectroscopy, mass spectrometry (ESI-MS), and tryptic peptide sequencing. Mass analysis allowed the detection of protein R2 as a native dimer with two iron clusters per subunit. The measured mass was 87 032 Da, compared to a calculated value of 87 028 Da. Peroxynitrite treatment preserved the non-heme iron center and the dimeric form of the protein. A mean of two nitrotyrosines per E. coli protein R2 dimer were obtained at 400 microM peroxynitrite. Only 3 out of the 16 tyrosines were nitrated, including the free radical Tyr122. Despite its radical state, that should favor nitration, the buried Tyr122 was not nitrated with a high yield, probably owing to its restricted accessibility. Dose-response curves for Tyr122 nitration and loss of the free radical were superimposed. However, protein R2 inactivation was higher than nitration of Tyr122, suggesting that nitration of the nonconserved Tyr62 and Tyr289 might be also of importance for peroxynitrite-mediated inhibition of E. coli protein R2.     

Plasma membrane coating with cationic silica particles and osmotic shock alters the morphology of bovine aortic endothelial cells

We have used a published method of membrane preparation based on the precoating of the apical membrane of aortic endothelial cells with cationic silica microbeads (with or without polyacrylic acid) in combination with an osmotic shock and mechanical shearing to isolate the apical from the basal plasma membranes of these cells, in vitro. After labeling of the plasma membrane of adherent endothelial cells with a fluorescent derivative of phosphatidylcholine and by using laser confocal fluorescence scanning microscopy, we found that this method of membrane isolation rapidly induced invaginations of the basal plasma membrane to an extent which makes this method unsuitable for further membrane lipid analysis. Morphological analysis of the cells and fluorescence recovery after photobleaching experiments on the plasma membranes were performed at each step of the purification procedure and showed that only hypotonic shock and mechanical shearing of the cells enabled the basal plasma membranes to be purified without significant morphological changes.     

Systemic administration of cationic phosphonolipids/DNA complexes and the relationship between formulation and lung transfection efficiency

Performances of cationic lipid formulations for intravenous gene delivery to mouse lungs have been previously reported. We report in this study that cationic phosphonolipids, when appropriately formulated, can be good synthetic vectors for gene delivery to lung after intravenous administration. One of our reagents, GLB43, was capable of mediating a 500-fold higher expression in the lungs of mice than could be obtained with free pDNA alone (P=0.018). We demonstrate that the most important parameters for cationic phosphonolipid transfection activity after systemic administration are the chemical structure of the cationic phosphonolipid, the lipid to DNA charge ratio and the inclusion of co-lipid in the formulation. We report using a luciferase reporter gene that transfection activity in vivo 24 h after cationic phosphonolipid systemic administration could not be predicted from in vitro analysis. In contrast to in vitro studies, cationic phosphonolipids including the oleyl acyl chains (GLB43) were more effective than its analogue with the myristyl acyl chains (GLB73). Using pathological analysis of animal livers, we demonstrate that the toxicity level was correlated with the lipoplex formulation and the lipid to DNA ratio.     

Stimulation of rat cutaneous fibroblasts and their synthetic activity by implants of powdered nacre (mother of pearl)

The components of the cutaneous envelope, the epidermis and the dermis, change in response to aging or environmental stress factors. The fibroblasts involved in maintaining skin tone are the main targets. Nacre, mother of pearl, from Pinctada maxima, which can stimulate and regulate bone forming cells, was implanted in the dermis of rats to test its action on the skin fibroblasts. This report describes the effect of nacre on the skin fibroblast recruitment and physiological activity. It resulted in enhanced extracellular matrix synthesis and the production of components implicated in cell to cell adhesion and communication (such as decorine) and in tissue regeneration (type I and type III collagens). The nacre implant produced a well vascularized tissue. The physiological conditions in the region around the implant are thus those required for the positive interactions between the dermis and epidermis which are fundamental for the physiological function of the skin.