Halothane induced disorder of red cell membranes of subjects susceptible to malignant hyperthermia

Membrane fluidity of red blood cells drawn from malignant hyperthermic pigs and humans was studied using spin-probes and electron paramagnetic resonance technique. The order parameter and rotational correlation time were determined with 12-doxylstearate and 16-doxylstearate, respectively. It was found that halothane decreased both parameters, but that the decrease of these parameters in subjects susceptible to malignant hyperthermia was much greater than that in normal subjects. The differences were most pronounced at 3 mM halothane. A possibility of using blood for a non-invasive screening for malignant hyperthermia is discussed.     

The analysis of digital-palmar dermatoglyphics in a sample of individuals affected by essential hypertension

In this research the digital and palmar dermatoglyphics of a sample of 97 individuals from Sardinia affected by essential hypertension were examined with the aim of identifying possible peculiarities. As already observed by other authors, the tendency towards a distal position of the axial triradius, a dermatoglyphic characteristic common in many pathologies, was also confirmed in our sample. Furthermore, some characteristics not observed in previous works which are distinctive to hypertensive subjects of both sexes are identified: a transversal tendency of the ridges and a greater asymmetry of the TRC (total ridge count). The asymmetry of the TRC is usually explained as a consequence of disturbances during embryologic development; in our case these disturbances seem to be represented by changes in arterial pressure levels originating during the prenatal period.     

The role of calpain in the selective increased phosphorylation of the anion-transport protein in red cell of hypertensive subjects

The phosphorylation of the anion-transport protein (band 3) is selectively increased in human red cell membrane, following exposure of intact cells to ionophore and micromolar calcium. The phosphorylation is catalyzed by a membrane associated protein kinase distinct from either protein kinase C or Ca2+/calmodulin dependent protein kinase. We show that the increase in phosphorylation of band 3 is abolished if red cells had been pre-loaded with an inhibitor of calpain or with an anticalpain monoclonal antibody. Our findings suggest that calpain activity may control, both at a functional and at a structural level, the activity of this important transmembrane protein through the modulation of its susceptibility as a substrate of membrane bound protein kinase(s). Based on previous observations indicating the presence in erythrocytes from hypertensive patients of an uncontrolled intracellular calpain-mediated proteolytic system accompanied by an increased phosphorylation of band 3 protein(s), we suggest that our results may shed light on the type of molecular alteration which is associated with the hypertensive state.     

Solubility of phenothiazines in red blood cell membranes as evidenced by photoaffinity labeling

Radioactively labeled 7-azido-fluphenazine and 7-azido-triflupromazine methiodide have been synthesized and their binding to membranes of intact red blood cells and to ghosts was compared after irradiation. The results indicated that tertiary phenothiazines react with integral membrane components. We conclude from the results that amphiphilic substances solubilize in biological membranes. This is in contradiction to the proposal that these compounds are excluded from the hydrophobic core of biological membranes (Conrad & Singer (1979) Proc.Natl.Acad.Sci.U.S.A. 76, 5202-5206 and (1981) Biochemistry 20, 808-818).     

Alterations of red cell membranes from phenylhydrazine-treated rabbits

Reticulocytosis was induced in rabbits by two methods: phlebotomy and injection of phenylhydrazine. Normal erythrocytes, reticulocytes from bed rabbits, reticulocytes from phenylhydrazine-treated rabbits, and erythrocytes treated in vitro with phenylhydrazine were compared with respect to their plasma membrane labeling by galactose oxidase and NaB³H₄, and lactoperoxidase-catalyzed incorporation of ¹²⁵I. Normal erythrocyte membranes and membranes from reticulocytes of bled rabbits showed almost identical labeling patterns, the presence of 2–3 glycoproteins with moderate to low mobilities on dodecyl sulfate acrylamide gel electrophoresis. Labeling in the absence of enzyme was negligible. In contrast, the reticulocytes from phenylhydrazine-treated rabbits exhibited a large incorporation of tritium without prior treatment with galactose oxidase. Even after prereduction with unlabeled NaBH₄ to remove this nonspecific labeling, the labeled glycoprotein components found in normal erythrocytes were not detectable. Normal erythrocytes treated in vitro with phenylhydrazine, washed, and labeled with galactose oxidase had labeling patterns, including high nonspecific incorporation of ³H, similar to those observed with in vivo phenylhydrazine treatment.Solubilization of membranes with lithium diiososalicylate followed by partitioning with phenol showed that the same glycoproteins were presented in normal or phenylhydrazine membranes, although only the former extract was labeled by galactose oxidase. Individual carbohydrates from the membranes were analyzed by gas-liquid chromatography and, in the case of hexosamines, on the amino acid analyzer. The results of these analyses indicated a slight decline in galactose content with phenylhydrazine treatment. Reticulocyte membranes from bled rabbits also showed a decrease in galactose content, although it was less pronounced.Most of the label incorporated by nonspecific borohydride labeling of membranes from phenylhydrazine-treated animals was found associated with protein. The modified amino acids from labeled proteins are similar to those formed in reactions of oxidized lipids and proteins in model systems.     

Oxidative stress in subjects affected by celiac disease

In order to study the role of oxidative stress in celiac disease, protein carbonyl groups, thiobarbituric acid-reactive substance and pentosidine were evaluated in the plasma of nine patients with asymptomatic celiac disease and in a control group (n = 25). Plasma alpha-tocopherol, retinol and lipids were determined in the same samples. The levels of markers of oxidative stress derived from both protein (carbonyl groups) and lipids (thiobarbituric acid-reactive substances) were significantly higher in celiac disease patients, whereas lipoproteins and alpha-tocopherol were significantly lower. These data indicate that in celiac disease, even when asymptomatic, a redox imbalance persists; this is probably caused by an absorption deficiency, even if slight. Dietary supplementation with antioxidant molecules may offer some benefit and deserves further investigation.     

Selective solubilization of proteins from red blood cell membranes by protein perturbants

Isolated human erythrocyte membranes were exposed to a series of reagents known to modify or perturb proteins; these included sodium hydroxide, lithium diiodosalicylate, acid anhydrides, and organic mercurials. Each reagent liberated the same set of relatively polar polypeptides from the membrane, while the other, more hydrophobic species invariably remained associated with the membrane residue. The selective elution pattern was precisely that seen previously with 6 M guanidine hydrocloride. The released polypeptides, comprising half of the membrane protein mass, contained no carbohydrate; current evidence indicates that all of these components are confined to the cytoplasmic surface of the membrane. The residue contained all the lipids and all the glycoproteins. The latter are accessible to the outer membrane surface and, in at least two cases, seem to extend asymmetrically across the thickness of the membrane. Thus, the distinctive elution behavior which defines these two groups of polypeptides relates both to their chemical composition and their organizational disposition in the membrane.     

Diphenoloxydase (DPOx) in red cell membranes]

Two diphenoloxydases (DPOx) with similar molecular weights (158 000) are found in normal human red cell hemolysates. One of these enzymes appears to be tightly bound to the membranes while the other is not, or at most only weakly bound.     

Pyruvate kinase-catalyzed ATP-formation in human red blood cell membranes

Previous studies on the linkage between enzymatically catalyzed ATP-generating reactions in the red blood cell membrane and the sodium and potassium transport in the control of overall glycolysis of human erythrocytes were controversial. In this study a significant amount of pyruvate kinase activity is shown to be localized within the membrane. Membrane fragments produce 20.5 mumol of ATP per 10(10) membranes per hour from phosphoenolpyruvate and ADP. The kinetics of the membrane-localized pyruvate kinase do not differ from those of the enzyme from hemolysates. The results clearly document the presence of the second ATP-generating enzyme of glycolysis, pyruvate kinase, in human red blood cell membranes. The main fraction of the enzyme is deeply hidden in the lipid layers of the membrane. It can be demasked by mechanical desintegration of membranes at high levels of activity. It is suggested that the amount of the membrane-localized fraction of pyruvate kinase is related to the clinical severity of the hemolytic process in pyruvate kinase deficiency.