Biomedical subjects
Publications and source records attributed to G Lenaz.
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Explore the source record for details and available documents.
Explore the source record for details and available documents.
Lipid-soluble spin labels were used to probe the fluidity of membrane lipids in erythrocyte membranes from patients with Duchenne muscular dystrophy. A greater mobility of spin label motion was noticed at the surface of the Duchenne erythrocyte membranes in comparison with the similary labeled normal human erythrocytes. Changes in the protein conformation and/or protein-lipid organization, as a consequence of an altered membrane lipid fluidity, have been demonstrated by the use of a sulfhydryl group specific protein spin label. Strongly immobilized sulfhydryl groups appear to be located both at the membrane surface and deep within the lipid bilayer. The ratio of the spectral amplitude of the spin label attached to weakly immobilized sulfhydryl groups to that of strongly immobilized SH-groups is significantly greater in Duchenne membranes, compared to those of controls. These studies suggest alterations in lipid-protein organization at the surface of Duchenne erythrocytes, indicating that Duchenne muscular dystrophy may be a disease resulting from a membrane abnormality at the molecular level.
We have found that dicyclohexylcarbodiimide (DCCD) inhibits both the succinate-cytochrome c and the ubiquinol-cytochrome c reductases in cytochrome c-depleted mitochondria. On the other hand the succinate-ubiquinone reductase is not decreased at the same levels of the inhibitor. The inhibition curve of DCCD results sigmoidal for succinate-cytochrome c reductase, whereas it is hyperbolic for the ubiquinol-1-cytochrome c reductase, with also a lower apparent KI. The inhibition appears dependent both on the time of preincubation and on the mitochondrial concentration. The apparent Km for ubiquinol-1 is increased and the maximal velocity of ubiquinol-cytochrome c reductase is decreased by DCCD. The effects do not appear to be caused by unspecific modification of the physicochemical state of the bc1 region of the respiratory chain. The results therefore suggest the presence of a DCCD-sensitive electron transfer step in the redox pathways from ubiquinol to cytochrome c.
The effects of extraction and reincorporation of ubiquinone on succinate dehydrogenase of mitochondrial membranes have been studied. The succinate dehydrogenase activity, measured with ferricyanide as electron acceptor, was diminished by approximatively 75% upon the extraction of ubiquinone and was restored when ubiquinone was reincorporated into the membranes. A study in a model system represented by ubiquinols incorporated in liposomes shows that the initial rates of ubiquinol oxidation by external ferricyanide are almost two order of magnitude lower than the rates of succinate-ferricyanide reductase in mitochondria. It is therefore concluded that the compound feeding electrons to ferricyanide in damaged mitochondria is either ubiquinone in a bound form or a compound between UQ and the antimycin block.
The percentage of echinocytes in the blood from patients affected by Duchenne muscular dystrophy, in mothers carriers of the dystrophic state, and in Steinert dystrophic patients, has been investigated after addition of low levels of lysolecithin. The echinocyte content was significantly higher in both Duchenne patients and in carriers, and lower in Steinert patients than in controls. The extents of the differences suggest the possibility to use this methodology as a diagnostic test in Duchenne muscular dystrophy.
In order to achieve a better elucidation of the physico-chemical properties of ubiquinones (Qs) in natural membranes, we have investigated the UV spectral features of Q-homologs in different model systems. In phospholipid monolamellar vesicles the UV spectra of physiological ubiquinones resemble those in isooctane, whereas in detergent micelles the spectra appear similar to those of the hydrophilic Q1 in water. For short-chain Qs it is possible to describe a linear dependence of the absorption parameters upon the polarity of the media. Long-chain Qs exhibit strong deviations due to aggregation states. In aqueous media a pH dependence of the spectral properties of ubiquinones is also observed. The findings in model systems can provide useful correlations between the spectroscopic properties of ubiquinones, their physico-chemical characteristics in the natural membranes, and rapid spectrophotometric detections of their redox changes.
The first paper of this series describes a working hypothesis for the action of general anesthetics. According to such hypothesis, anesthetics, by inducing a labilisation of lipid-protein interactions in biomembranes, affect the conformation, and hence the activity of membrane-bound catalytic proteins. It is conceivable that such changes in ionic channels in neuronal membranes will abolish the transmission of nervous impulses and give rise to anesthesia. The hypothesis is discussed on the basis of previously known experimental data and of theoretical considerations. Thermodynamic considerations are in favour of the idea that a rupture of lipid-protein interactions will expose protein groups to water destabilising helical structures. A large decrease of alpha-helical content after lipid removal had been previously found. Furthermore lipids affect the kinetics of membrane-bound enzymes, suggesting that conformational changes occur in the catalytic site after lipid removal or perturbation.
We have investigated the effect of general anesthetics (the normal alcohol series up to pentanol, halothane, pentrane, ether, chloroform, and ketamine) on lipid fluidity of phospholipid vesicles and mitochondrial and erythrocyte membranes by using spin labels and fluorescent probes. The spin labels used (5- and 16-doxyl stearic acids) show that all anesthetics tested have a slight fluidizing effect on lipid vesicles but induce a very strong increase in mobility of spin labels in mitochondria and lower in erythrocyte ghosts. These results are interpreted as a labilization of lipid protein interactions at all depths in the bilayer. The fluorescent molecules ANS and NPN, which probe the glycerol region and the core of the bilayer respectively, show a decrease of fluorescence induced by alcohols, halothane, ether, chloroform in both lipid vesicles and membranes. The decrease of fluorescence is due to decreased quantum yield as shown by double reciprocal plots of probe fluorescence against membrane concentration. The fluorescence decrease is interpreted mainly as an increase in fluidity of the lipid bilayer and not as an increase of polarity of the probe environment. The effect of ketamine is that of fluidization in the bilayer core (NPN) but of increased rigidity in the glycerol region (ANS) perhaps due to the amphipathic character of this anesthetic, that is supposed to bind in the polar region of the bilayer. Pentrane also induces fluidization in the bilayer core (NPN) but has a peculiar effect near the surface (ANS): in lipid vesicles it induces a fluorescence decrease, whereas an increase is seen in mitochondrial membranes. These complex effects are considered as the result of some specific change in the lipid protein interactions in the region probed by ANS. The effects of anesthetics on maximal NPN fluorescence (Fo) have been usually found to be stronger in mitochondrial membranes than in lipid vesicles, thus confirming the results of the spin label studies, showing a labilization of lipid protein interactions induced by anesthetics. The effects on Fo of ANS, however, appear to be stronger in lipid vesicles than in membranes. These findings indicate that the presence of the proteins counteracts the perturbation induced by anesthetics at the level of the membrane surface, in contrast with the disruption of lipid protein interactions observed in the membrane hydrophobic areas.
BACKGROUND: The aim of the study is to evaluate the limits of the compensatory mechanisms and the tissue damages caused by the low oxygen content during severe normovolemic hemodilution in pigs. METHODS: The experimental procedure was performed in 10 animals after general anaesthesia was induced and iso-hypervolemic hemodilution to Hct 10% was maintained for five hours without any intensive care. Hemodynamic, biochemical and ultrastructural parameters were detected before and at the end of hemodilution in addition to analysis of oxygen delivery/uptake and mitochondrial enzymes function. RESULTS: The collected data show: the initial good compensatory mechanism was subsequently exhausted; five animals demonstrated cardiac ischemia and low CO and two of them died before the end of the experiment; no hemodynamic and hemoxymetric data predicted the cardiac ischemia; the dilution caused alterations of some detected biochemical parameters such as hemocoagulation; no evidence of morphologic and ultrastructural tissue damage or interstitial edema; decreasing in mitochondrial enzymes activity significant only for NADH-related. CONCLUSIONS: In conclusion, it seems that, in pigs at least, the compensatory mechanisms can keep a sufficient tissue oxygen supply throughout the experimental time with the exception of cardiac muscle.