N-Terminal domain of annexin 2 regulates Ca(2+)-dependent membrane aggregation by the core domain: a site directed mutagenesis study.

Annexin 2 binds and aggregates biological membranes in a Ca(2+)-dependent manner. This protein exists as a monomer (p36) or as a heterotetramer (p90) in which two p36 chains are associated with a dimer of p11, a member of the S100 protein family. Protein kinase C phosphorylates the protein at the level of the N-terminal tail on serines 11 and 25, thereby modifying its oligomeric structure and its properties of membrane aggregation. To analyze these effects, the properties of a series of mutants in which serines 11 and 25 were replaced by alanine and/or glutamic acid were investigated. The affinity for p11 light chain was decreased in the S11E mutants. Glutamic acid residues in positions 11 or 25 did not change membrane binding, either in the tetrameric or in the monomeric form. On the other hand, these mutations affected the aggregation properties of the two forms. For the tetramer, the aggregation efficiency was decreased but not the Ca(2+) sensitivity, whereas the latter was affected in the case of the monomer. The effects were stronger in the S11E mutants, and they were cumulative in the double mutant. They suggest a different conformation of the N-terminal domain in the mutants (and in the phosphorylated protein), a hypothesis which is supported by proteolysis experiments. This conformational change would affect aggregation by the monomer through a dimerization step.

Differential potentiation of arachidonic acid release by rat alpha2 adrenergic receptor subtypes.

CHO transfectants expressing the three subtypes of rat alpha2 adrenergic receptors (alpha2AR): alpha2D, alpha2B, alpha2C were studied to compare the transduction pathways leading to the receptor-mediated stimulation of phospholipase A2 (PLA2) in the corresponding cell lines CHO-2D, CHO-2B, CHO-2C. The alpha2B subtype stimulated the arachidonic acid (AA) release after incubation of the cells with 1 microM epinephrine, whereas alpha2D and alpha2C gave no stimulation. Calcium ionophore A23187 (1 microM) increased the release by a factor of 2-4 in the three strains. When cells were incubated with both epinephrine and Ca2+ ionophore, the AA release differed greatly between cell lines with strong potentiation in CHO-2B (2-3 times greater than Ca2+ ionophore alone), moderate potentiation in CHO-2D, and no potentiation in CHO-2C. The three cell lines each inhibited adenylylcyclase with similar efficiencies when 1 microM epinephrine was used as the agonist. The potentiation depended on both alpha2AR and Gi proteins since yohimbine and pertussis toxin inhibited the process. Pretreatment of CHO-2B cells with MAFP which inhibits both cytosolic and Ca2+-independent PLA2, reduced the release of AA induced by epinephrine+Ca2+ ionophore to basal value, whereas bromoenol lactone, a specific Ca2+-independent PLA2 inhibitor, had no effect. Preincubation of the cells with the intracellular calcium chelator BAPTA gave a dose-dependent inhibition of the arachidonic acid (AA) release. In CHO cells expressing the angiotensin II type 1 receptor, coupled to a Gq protein, the agonist (10-7 M) produced maximal AA release: there was no extra increase when angiotensin and Ca2+ ionophore were added together. There was no increase in the amount of inositol 1,4, 5-triphosphate following stimulation of CHO-2B, -2C, -2D cells with 1 microM epinephrine. Epinephrine led to greater phosphorylation of cPLA2, resulting in an electrophoretic mobility shift for all three cell lines, so inadequate p42/44 MAPKs stimulation was not responsible for the weaker stimulation of cPLA2 in CHO-2C cells. Therefore, the stimulation of cPLA2 by Gi proteins presumably involves another unknown mechanism. The differential stimulation of cPLA2 in these transfectants will be of value to study the actual involvement of the transduction pathways leading to maximal cPLA2 stimulation.

Alpha 2 adrenergic receptor subtypes expressed in Chinese hamster ovary cells activate differentially mitogen-activated protein kinase by a p21ras independent pathway.

Epinephrine stimulation of rat alpha 2D, alpha 2B, and alpha 2C adrenergic receptor subtypes, expressed stably in Chinese hamster ovary (CHO) cells, caused a rapid, transient activation of mitogen-activated protein kinase (MAPK), with subtype-specific different efficiencies. The order of activation was CHO-2B approximately CHO-2D much greater than CHO-2C. Pertussis toxin blocked the stimulation of MAPK enzymatic activity and the parallel MAPK phosphorylation, demonstrating that these responses are mediated by pertussis toxin-sensitive Gi proteins. Contrary to what has been reported for the alpha 2A subtype expressed in rat-1 fibroblasts, epinephrine did not cause any detectable activation of p21ras in the CHO transfectants. Furthermore, combined application of epinephrine and phorbol myristate acetate had a potent cooperative but not additive effect in clones CHO-2D and CHO-2B but not in CHO-2C, suggesting that protein kinase C is probably differently involved in the signaling by the three alpha 2 receptor subtypes. These results show that in CHO cells, the different alpha 2 adrenergic receptor subtypes utilize differential pathways to activate MAPK in a p21ras-independent way.

Re-expression of thyroxine-binding globulin in post-weaning rats during protein or energy malnutrition.

Thyroxine-binding globulin, the highest affinity thyroid hormone binder of rat serum, was studied during 28 days of dietary protein restriction (6% protein vs 18% protein in isocaloric control diet) or energy restriction (60% intake of control diet). Studies were performed on male rats aged four weeks at the beginning of experiments: the animals had reached the ontogenic stage when the thyroxine-binding globulin had declined, after its high postnatal surge, to undetectable levels. Short-term administration (seven days) of one or the other restricted diet similarly induced resynthesis of the protein. Its serum concentrations reached 26-46% of those measured in eight-day pups (peak of the neonatal surge) and its liver mRNAs showed corresponding enhanced signals. Serum T4 binding activities were increased, although concomitantly transthyretin, second specific T4 carrier of the rat serum, decreased markedly (65-75% of controls) in response to the dietary restrictions. Longer-term diet administration (14 or 28 days) resulted in the further increase of the thyroxine-binding globulin in the protein-restricted rats, in contrast to its decline and eventual disappearance in the energy-restricted animals. Protein restriction was associated with increased total and free T3 serum concentrations, in contrast to energy restriction which little affected these parameters. These studies reveal rat thyroxine-binding globulin as a positive (increasing), highly sensitive reactant of malnutrition, able to discriminate between energy deficiency and composition dysequilibrium of diets. They suggest that up-regulation of its synthesis in the two dietary models involves differential mechanisms.

Regulation of hepatic transthyretin messenger RNA levels during moderate protein and food restriction in rats.

This study was designed to determine whether the decrease in serum transthyretin that occurs during food restriction results from gross energy reduction or from depressed protein or lipid intake and to examine the relationship between serum transthyretin and hepatic transthyretin mRNA during moderate protein or food deficiency. Groups of young rats were allowed free access to either a 18% (control) or a 6% protein diet (protein-restricted), or reduced intakes. The food-restricted groups received 60% of control intake from the control diet, a 40% protein-enriched diet, or a 40% lipid-enriched diet, for 28 d. Serum transthyretin concentrations were lower in all experimental groups on d 7 relative to the control group. Control values were reached only in the protein-restricted group by d 14. The low serum transthyretin levels, which were similar in the food-restricted groups, likely resulted from gross energy restriction. Hepatic transthyretin mRNA levels were determined in the control, protein-restricted and food-restricted groups. They were unchanged relative to controls in the protein-restricted group but declined moderately in the food-restricted group on d 7 and 14, before returning to control values by d 28. Thus, the changes in liver transthyretin mRNA levels could partially explain the changes in serum transthyretin in food-restricted rats.

Changes in rat plasma apolipoproteins and lipoproteins during moderate protein deficiency: potential use in the assessment of nutritional status.

To test apolipoprotein sensitivity as protein deficiency markers, concomitant evolution of plasma apolipoproteins (apo) and usual nutritional markers (transthyretin, albumin, transferrin) were followed during a 28-d protein restriction in young male Wistar rats. In addition, plasma lipids and chemical composition of lipoproteins were assayed by d 28. The control and the deficient groups were fed 18% and 6% casein diets, respectively. By d 28 the protein-deficient group exhibited hypotriglyceridemia resulting from the decrease in VLDL triacylglycerols; free cholesterol and phospholipids were increased, reflecting the increment in LDL-HLDL1. In plasma total lipoproteins, apo BH, AI and E were not different than controls in the deficient group. Apolipoprotein AIV decreased after d 14 and was significantly less than in controls at d 28. Apolipoprotein BI was considerably reduced by d 14 (43% less) and d 28 (52% less) compared with the control group. Apolipoproteins C + AII were significantly lower in the protein-deficient group by d 14 (43%). By d 28, VLDL apo C were decreased 60% by protein restriction. Transthyretin level was 20% lower in the protein-deficient group by d 7 but returned to control values by d 14. A moderately lower value was observed for albumin by d 7 and d 14 and for transferrin by d 28. These results indicate that, in this model, apo BI and C are more sensitive to protein depletion than usual nutritional markers.

[Influence of different types of experimental obesity on resistance of the mouse to infection by Salmonella typhimurium and Klebsiella pneumoniae]. / Influence de divers types d'obésité expérimentale sur la résistance de la souris à l'infection par Salmonella typhimurium et Klebsiella pneumoniae.

Resistance to infections inducing two types of immune response, humoral and cell-mediated, has been measured in mice after Salmonella typhimurium and Klebsiella pneumoniae inoculation; the animals exhibited different kinds of obesity: genetic, ob/ob and db/db mutants, induced by fat diet or gold thioglucose (aurothioglucose) injection (determining obesity of central origin). Klebsiella infection was aggravated in all types of obesity. Salmonella infection was aggravated in genetically diabetic and dietary-obese mice. The two kinds of genetically obese mice show an important functional decrease in splenic lymphocytes. In contrast, aurothioglucose-obese mice were more resistant than controls.

[Effect of experimental alcoholic intoxication on the cell-mediated immune response in mice]. / Effet de l'intoxication expérimentale par l'alcool sur la réponse d'immunite à médiation cellulaire de la souris.

Repeated ip injection of ethyl alcohol in a relatively high dose depressed the immune response, to a greater degree in Swiss mice than in C57BL/6 mice, as shown by a diminution in granuloma size and in the hypersensitivity reaction to tuberculin compared with untreated controls. The control of bacillary multiplication in the popliteal lymph node was more efficient in alcohol-treated Swiss mice than in the corresponding controls, but was less efficient in alcohol-treated C57BL/6 animals than in their controls. Alcohol treatment caused no reduction in the number of circulating lymphocytes, and no modification of the distribution of B and T lymphocytes in the spleen, or of the stimulation of T lymphocytes in the presence of mitogens.

[Effect of moderate protein-calorie deprivation on cell-mediated immunity in mice]. / Effet d'une carence protéino-calorique modérée sur l'immunité à médiation cellulaire de la souris.

Mild protein-calorie deprivation in mice decreases their early nonspecific inflammatory response to an injection of BCG or tuberculin into the footpad. After an injection of BCG, the size of the granuloma and the delayed hypersensitivity reaction to tuberculin significantly decreased. This decrease in cell-mediated immunity was paradoxically accompanied by a fall in bacillus multiplication at the popliteal ganglion, i.e. the site of BCG injection. A reduction in microbial multiplication was also observed in the spleen after intraperitoneal injection of Listeria and in the liver after intravenous injection.

[Comparative effects of a diet enriched in live or heated yogurt on the immune system of the mouse]. / Effets comparé d'un régime enrichi en yoghourt vivant ou thermisé sur le système immunitaire de la souris.

Growth was fastest in mice fed yogurt. There was no difference in the effects of live or heated yogurt on the ponderal curve or the organ weight of vaccinated or unvaccinated mice. Sera antitoxin levels were slightly elevated in animals fed live yogurt and given an antitetanus vaccination at the particular concentrations used. Significantly high IgG2a immunoglobulin levels were found only in unvaccinated mice fed live yogurt. Histological examination before, and particularly after, antitetanus vaccination confirmed these findings in the thymus and especially in the spleen.