Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure

Cardiolipin (CL) is responsible for modulation of activities of various enzymes involved in oxidative phosphorylation. Although energy production decreases in heart failure (HF), regulation of cardiolipin during HF development is unknown. Enzymes involved in cardiac cardiolipin synthesis and remodeling were studied in spontaneously hypertensive HF (SHHF) rats, explanted hearts from human HF patients, and nonfailing Sprague Dawley (SD) rats. The biosynthetic enzymes cytidinediphosphatediacylglycerol synthetase (CDS), phosphatidylglycerolphosphate synthase (PGPS) and cardiolipin synthase (CLS) were investigated. Mitochondrial CDS activity and CDS-1 mRNA increased in HF whereas CDS-2 mRNA in SHHF and humans, not in SD rats, decreased. PGPS activity, but not mRNA, increased in SHHF. CLS activity and mRNA decreased in SHHF, but mRNA was not significantly altered in humans. Cardiolipin remodeling enzymes, monolysocardiolipin acyltransferase (MLCL AT) and tafazzin, showed variable changes during HF. MLCL AT activity increased in SHHF. Tafazzin mRNA decreased in SHHF and human HF, but not in SD rats. The gene expression of acyl-CoA: lysocardiolipin acyltransferase-1, an endoplasmic reticulum MLCL AT, remained unaltered in SHHF rats. The results provide mechanisms whereby both cardiolipin biosynthesis and remodeling are altered during HF. Increases in CDS-1, PGPS, and MLCL AT suggest compensatory mechanisms during the development of HF. Human and SD data imply that similar trends may occur in human HF, but not during nonpathological aging, consistent with previous cardiolipin studies.     

The application of a potential-sensitive cyanine dye to rat small intestinal brush border membrane vesicles

The sensitivity of the fluorescent dye, 3,3′-diethylthiadicarbocyanine (DiS-C₂(5)), was too low for the detection of membrane potential changes in rat small intestinal membrane vesicles. Only after adding LaCl₃ or after fractionation of the intestinal membranes by free-flow electrophoresis could the dye be used to monitor electrogenic Na⁺-dependent transport systems. It is concluded that the response of this potential-sensitive dye is influenced by the negative surface charge density of the vesicles.     

Detection of a calmodulin-sensitive cyclic nucleotide phosphodiesterase in rat parotid gland

Calmodulin coupled to Sepharose has provided a rapid and sensitive means of isolating a cyclic nucleotide phosphodiesterase activity which is stimulated by the calmodulin-Ca²⁺ complex, from rat parotid gland. Initial experiments established that phosphodiesterase activity sensitive to calmodulin and Ca²⁺ could not be demonstrated in crude extracts of rat parotid gland or after partial purification of rat parotid phosphodiesterase over DEAE-cellulose. However, it was possible to readily demonstrate the presence of a cyclic nucleotide phosphodiesterase activity regulated by calmodulin if the extracts were first purified by batch ion-exchange chromatography over DEAE-cellulose followed by affinity chromatography with calmodulin coupled to Sepharose. The batch ion-exchange chromatography step removed the major portion of free parotid calmodulin which could compete with calmodulin-coupled Sepharose for the proteins regulated by calmodulin. Thus, by employing an initial chromatography step over DEAE-cellulose to separate phosphodiesterase activity from calmodulin, it was possible to increase the recovery of calmodulin-sensitive phosphodiesterase after affinity chromatography with calmodulin coupled to Sepharose. This approach should be useful for demonstrating the presence of and for purifying other parotid proteins regulated by calmodulin.     

Developmental patterns of galactosyltransferase activity in various regions of rat brain

Abstract: The developmental pattern of glycoprotein-galactosyltransferase activity was determined in the microsomal fractions of three regions of the embryonic rat brain and in parts of the visual system and the cerebellum postnatally. It could be shown that the enzyme activity was highest in the embryonic brain, where regional differences were apparent, and decreased progressively after birth. The enzyme profile in the cerebellum showed no marked postnatal changes.     

Fine root distribution of trees and understory in mature stands of maritime pine (Pinus pinaster) on dry and humid sites

Maritime pine (Pinus pinaster) is the main tree cropping species in the Landes of Gascogne forest range in south western France. Soils are nutrient poor, sandy podzosols and site fertility is determined essentially by organic matter content and depth of water table, which is known to limit root growth. We hypothesised, with an ultimate goal of constructing a nutrient uptake model applicable to this region, that the organic top horizons together with the depth of the water table should be the most important parameters related to fine root distribution and presence of associated mycorrhiza. To test this hypothesis, we compared two adult Pinus pinaster stands, contrasting in depth of water table and soil fertility and evaluated fine roots (diameter ≤2 mm) of understory species and fine roots and ectomycorrhizal morphotypes of Pinus pinaster down to 1.2 m, using a soil corer approach. Total fine root biomass of Pinus pinaster was not significantly different between both sites (3.6 and 4.5 t ha⁻¹ for the humid, respectively, dry site), but root distribution was significantly shallower and root diameter increased more with depth at the humid site, presumably due to more adverse soil conditions as related to the presence of a hardpan, higher amount of aluminium oxides and / or anoxia. Fine roots of Pinus pinaster represented only about 30% of total fine root biomass and 15% of total fine root length, suggesting that the understory species cannot be ignored with regards to competition for mineral nutrients and water. A comparison of the ectomycorrhizal morphotypes showed that the humid site could be characterised by a very large proportion of contact exploration types, thought to be more relevant in accessing organic nutrient sources, whereas the dry site had a significantly higher proportion of both long-distance and short-distance exploration types, the latter of which was thought to be more resistant to short-term drought periods. These results partly confirm our hypothesis on root distribution as related to the presence of soil mineral nutrients (i.e. in organic matter), point out the potential role of understory plant species and ectomycorrhizal symbiosis and are a valuable step in building a site-specific nutrient uptake model.     

Impact on energy metabolism of quantitative and functional cyclosporine-induced damage of kidney mitochondria

In this study we have measured, under experimental conditions which maintained efficient coupling, respiratory intensity, respiratory control, oxidative phosphorylation capacity and protonmotive force. Succinate cytochrome-c reductase and cytochrome-c oxidase activities were also studied. These investigations were carried out using kidney mitochondria from cyclosporine-treated rats (in vivo studies) and from untreated rats in the presence of cyclosporine (in vitro studies). Inhibition of respiratory intensity by cyclosporine did not exceed 21.1% in vitro and 15.9% in vivo. Since there was no in vitro inhibition of succinate cytochrome-c reductase and cytochrome-c oxidase activities, the slowing of electron flow observed can be interpreted as a consequence of an effect produced by cyclosporine between cytochromes b and c₁. Cyclosporine had no effect on respiratory control either in vitro or in vivo. Statistically significant inhibition of the oxidative phosphorylation was observed both in vitro (6.6%) and in vivo (12.1%). Moreover, cyclosporine did not induce any change of membrane potential either in vivo or in vitro. Our findings show that cyclosporine is neither a protonophore, nor a potassium ionophore. In cyclosporine-treated rats we noticed a decrease of protein in subcellular fraction, including the mitochondrial fraction. The role of the inhibition respiratory characteristics by cyclosporine in nephrotoxicity in vivo must take account of these two parameters: inhibition of the respiratory characteristics measured in vitro and diminution of mitochondrial protein in cyclosporine-treated rats.