Different effects of Hg2+ and Cu2+ on mussel (Mytilus galloprovincialis) plasma membrane Ca2+-ATPase: Hg2+ induction of protein expression.

Deregulation of Ca2+ homeostasis can produce serious effects on cell functioning due to an alteration of Ca2+ signaling. The aim of this study was to evaluate variations in plasma membrane Ca2+-ATPase (PMCA) induced in mussels by in vivo exposure to Cu2+ or Hg2+. PMCA activity was assayed using a cytochemical method allowing localization and in situ quantification of Ca2+-ATPase on cryostat tissue sections. The effects of fixed concentrations of Cu2+ (0.6 microM) or Hg2+ (1.3 microM) were evaluated after different times of exposure (1, 4, 6 days), while those of increasing amounts of Cu2+ (0.3, 0.6, 1.3 microM) or of Hg2+ (0.6, 1.3, 2.4 microM) were evaluated after 4 days. Cu2+ produces dose-dependent inhibition of PMCA in the digestive gland, with a minimum at the fourth day of treatment and a recovery at the sixth day. Conversely, Hg2+ induces a significant rise of PMCA activity, with a maximum at the fourth day. Similar results have been found after biochemical assay of PMCA, using plasma membranes obtained from density-gradient separation of gill homogenates. PMCA expression has been assessed by immunoprecipitation and Western immunoblotting on digestive gland homogenates, showing an induction after exposure to Hg2+ but not to Cu2+. In conclusion, Cu2+ does not vary PMCA expression but reduces PMCA activity, indicating PMCA inhibition; conversely, Hg2+ increases PMCA expression more than PMCA activity, suggesting that it also produces PMCA inhibition, but the induction of PMCA expression leads to a net increase in enzyme activity.

Cascading effects of predatory fish exclusion on the detritus-based food web of a lake littoral zone (Lake Vico, central Italy).

An exclosure experiment was carried out in the reed-dominated littoral zone of a volcanic lake (Lake Vico, central Italy) to test whether the impact of predatory fish on benthic invertebrates cascades on fungal colonisation and breakdown of leaf detritus. The abundance, biomass, and Shannon diversity index of the invertebrate assemblage colonising Phragmites australis leaf packs placed inside: (1) full-exclosure cages, (2) cages allowing access only to small-sized fish predators, and (3) cageless controls, were monitored over a 45-day period together with the mass loss and associated fungal biomass of leaf packs. The species composition of the fungal assemblage was further assessed at the end of the manipulation. In general, invertebrate predators did not show any significant response to fish exclusion, either on a trophic guild or on a single taxon level. In contrast, the exclusion of large predatory fish induced a diverse spectrum of changes in the abundance and population size-structure of dominant detritivore taxa, ultimately increasing the biomass and Shannon diversity index of the whole detritivorous guild. These changes corresponded with significant variations in leaf detritus decay rates as well as in the biomass and assemblage structure of associated fungal colonisers. Our experimental findings provide evidence that in Lake Vico effects of fish predators on invertebrate detritivores influence the fungal conditioning and breakdown of the detrital substrate. We conclude that in lacustrine littoral zones predator-driven constraints may structure lower trophic levels of detritus-based food webs and affect the decomposition of leaf detritus originated from the riparian vegetation.

[Prediction of outcome in critically ill patients using haemodynamic and oxyphoretic parameters]. / Predizione dell'esito dei pazienti critici utilizzando i parametri emodinamici ed ossiforetici.

OBJECTIVE: To find a predictive model for short time mortality and survival of haemodinamically instable patients. DESIGN: Prospective study on two consecutive series of critically ill patients admitted to ICU. SUBJECTS: 115 critically ill patients, subdivided in two series of 83 (47 survivors and 36 non survivors after 20 days from admission) and 32 patients (19 survivors and 13 non survivors), respectively. INTERVENTIONTS: All the patients have been monitored with a Swan-Ganz catheter. MEASUREMENTS: Oxyphoretic parameters were measured at 7 different times (T): T0 (start), T1 (after 12 hours from T0, T2 (24 hrs), T3 (48 hrs), T4 (72 hrs), T5 (96 hrs) and T6 (120 hrs). A total number of 401 recorded values of the first patients' series were used to create the predictive model for outcome (0 non survivors, 1 survivors). Stepwise logistic regression was used to create the model. Model calibration and discrimination was assessed. The model was validated using data collected from the second series. RESULTS: The probability of survival after 20 days was Pr (survive) = e(logit)/1+e(logit), with "logit" = -5,106+(-6,58E-02 SVI)+(-2,76E-03 SVR)+(0,1379 LVSWI)+(0,8933 Hb)+(-3,25E-02 VO2I)+(9,09E-02 O2ER)+(-7,89E-02 SHUNT)+PAT. For medical patients PAT = -0.568; for surgical patients PAT = -1.0525 and for politrauma PAT = 1.593. Goodness-of-fit test showed a good calibration: chi 2 = 4.267, p = 0.832. The area under the ROC curve was 0.831. The model used in the validation data set, with 199 recorded values, also showed a good calibration and discrimination (Goodness-of-fit test: chi 2 = 15.65, p = 0.111; area under the curve 0.798). CONCLUSIONS: The mathematical model we found has been validated also in the second series and the discrimination capability increases with time. Using this model we can evaluate the probability of survival at every time. Its application at different times permits a better evaluation of haemodynamically instable patient trend.

Effects of heavy metals on the Ca(2+)-ATPase activity present in gill cell plasma-membrane of mussels (Mytilus galloprovincialis Lam.).

1. Heavy metals (Hg2+, Cu2+, Cd2+, Zn2+, Pb2+) at micromolar concentrations strongly inhibit the Ca(2+)-ATPase activity present in the plasma-membrane obtained from the gill cells of Mytilus galloprovincialis Lam. Heavy metals act through inhibition of the formation of the phosphorylated intermediate. 2. All the heavy metals tested inhibit the Ca(2+)-ATPase activity, the effect following the order: Hg2+ > Pb2+ > Cu2+ > Cd2+ > Zn2+; the simultaneous addition of different heavy metals causes a summatory inhibition of the enzyme activity; addition to the reaction mixture of GSH at a final concentration of 0.5 mM, reverses inhibitory effects of heavy metals. 3. The inhibitory effects of Cu2+ on Ca(2+)-ATPase are highly enhanced by addition of ascorbate to the reaction mixture. In the presence of ascorbate (100 microM), copper strongly stimulates the lipid peroxidation damage of the gill plasma-membranes, a result that may explain the high copper cytotoxicity.

Reduction of sperm whale ferrylmyoglobin by endogenous reducing agents: potential reducible loci of ferrylmyoglobin.

The reactivity of the endogenous antioxidants ascorbate, ergothioneine, and urate toward the high oxidation state of sperm whale myoglobin, ferrylmyoglobin-formed upon oxidation of metmyoglobin by H2O2--was evaluated by optical spectroscopy and SDS-PAGE analysis. Depending on whether these antioxidants were present in the reaction mixture before or after the addition of H2O2 to a metmyoglobin suspension, two different effects were observed: (a) In the former instances, ascorbate, ergothioneine, and urate reduced efficiently the oxoferryl moiety in ferrylmyoglobin to metmyoglobin and prevented dimer formation, a process which requires intermolecular cross-link involving specific tyrosyl residues. In addition, all the reducing compounds inhibited--albeit with different efficiencies--dityorosine-dependent fluorescence build up produced via dimerization of photogenerated tyrosyl radicals. (b) In the latter instances, the antioxidants reduced the preformed sperm whale ferrylmyoglobin to a modified metmyoglobin, the spectral profile of which was characterized by a blue shift of the typical 633 nm absorbance of native metmyoglobin. In addition, under these experimental conditions, the antioxidants did not affect dimer formation, thus indicating the irreversible character of the process. The dimeric form of sperm whale myoglobin--separated from the monomeric form by gel electrophoresis of a solution in which ergothioneine was added to preformed ferrylmyoglobin--revealed optical spectral properties in the visible region identical to that of the modified myoglobin. This suggests that the dimeric form of the hemoprotein is redox active, inasmuch as the oxoferryl complex can be reduced to its ferric form. These results are discussed in terms of the potential reactivity of these endogenous antioxidants toward the reducible loci of ferrylmyoglobin, the oxoferryl moiety, and the apoprotein radical.

Role of carnitine and carnitine palmitoyltransferase as integral components of the pathway for membrane phospholipid fatty acid turnover in intact human erythrocytes.

The deacylation and reacylation process of phospholipids is the major pathway of turnover and repair in erythrocyte membranes. In this paper, we have investigated the role of carnitine palmitoyltransferase in erythrocyte membrane phospholipid fatty acid turnover. The role of acyl-L-carnitine as a reservoir of activated acyl groups, the buffer function of carnitine, and the importance of the acyl-CoA/free CoA ratio in the reacylation process of erythrocyte membrane phospholipids have also been addressed. In intact erythrocytes, the incorporation of [1-14C]palmitic acid into acyl-L-carnitine, phosphatidylcholine, and phosphatidylethanolamine was linear with time for at least 3 h. The greatest proportion of the radioactivity was found in acyl-L-carnitine. Competition experiments using [1-14C]palmitic and [9,10-3H]oleic acid demonstrated that [9,10-3H]oleic acid was incorporated preferentially into the phospholipids and less into acyl-L-carnitine. When an erythrocyte suspension was incubated with [1-14C]palmitoyl-L-carnitine, radiolabeled palmitate was recovered in the phospholipid fraction, and the carnitine palmitoyltransferase inhibitor, 2-tetradecylglycidic acid, completely abolished the incorporation. ATP depletion decreased incorporation of [1-14C]palmitic and/or [9,10-3H]oleic acid into acyl-L-carnitine, but the incorporation into phosphatidylcholine and phosphatidylethanolamine was unaffected. In contrast, ATP depletion enhanced the incorporation into phosphatidylcholine and phosphatidylethanolamine of the radiolabeled fatty acid from [1-14C]palmitoyl-L-carnitine. These data are suggestive of the existence of an acyl-L-carnitine pool, in equilibrium with the acyl-CoA pool, which serves as a reservoir of activated acyl groups. The carnitine palmitoyltransferase inhibition by 2-tetradecylglycidic acid or palmitoyl-D-carnitine caused a significant reduction of radiolabeled fatty acid incorporation into membrane phospholipids, only when intact erythrocytes were incubated with [9,10-3H]oleic acid. These latter data may be explained by the differences in rates and substrates specificities between acyl-CoA synthetase and the reacylating enzymes for palmitate and oleate, which support the importance of carnitine palmitoyltransferase in modulating the optimal acyl-CoA/free CoA ratio for the physiological expression of the membrane phospholipids fatty acid turnover.

Possible mechanism of inhibition of nitrite-induced oxidation of oxyhemoglobin by ergothioneine and uric acid.

The time course of oxyhemoglobin oxidation by nitrite consisted of a kinetic lag followed by a transition phase which progressed into a rapid autocatalytic phase. The imidazolthione and imidazolone derivatives, ergothioneine and uric acid, respectively, caused an increase in the duration of the lag phase in a concentration-dependent manner, without affecting the onset and rate of the autocatalytic phase. Neither compound reacted with H2O2 or nitrite, oxidizing species required in the initiation steps of oxyhemoglobin oxidation. On the other hand, both compounds reduced effectively and at comparable rates the high oxidation state of hemoglobin, i.e., ferrylhemoglobin, which is an intermediate species occurring in the autocatalytic phase. In addition, the rate of ergothioneine oxidation, upon its reaction with ferrylmyoglobin, was accelerated by nitrite, thus suggesting a reaction between the thione and nitrogen dioxide. Nitrogen oxide and ferrylhemoglobin are key species in the free radical chain propagation leading to oxyhemoglobin oxidation by nitrite. These data support the view that ergothioneine and urate delay oxyhemoglobin oxidation by nitrite upon the temporary removal of the propagating species, i.e., nitrogen dioxide and, secondarily, ferrylhemoglobin, and within a mechanism encompassing alterations of the nitrite in equilibrium with nitrogen dioxide and ferrylhemoglobin in equilibrium with methemoglobin redox transitions.

Measurement and analysis of triplet-state lifetimes by multifrequency cross-correlation phase and modulation phosphorimetry.

In this paper we describe a novel approach to study the triplet-state lifetimes by a conventional multifrequency cross-correlation phase and modulation apparatus. The analysis of phase and modulation data of eosin-labeled band 3 erythrocyte ghosts revealed the existence of two phosphorescence lifetime values of 2700 and 750 microseconds, with a fractional contribution of 78 and 22%, respectively, which are in good agreement with those reported in the literature. Differential polarization phase analysis, which facilitates the study of the rotational properties of band 3, provided data in good agreement with those reported in the literature. The method proposed in this paper to study the radiative emission from the triplet state may represent a convenient alternative to the pulse laser flash technique.

Carnitine palmitoyltransferase in human erythrocyte membrane. Properties and malonyl-CoA sensitivity.

Carnitine palmitoyltransferase located in the erythrocyte plasma membrane is sensitive to inhibition by malonyl-CoA and 2-bromopalmitoyl-CoA plus carnitine. Although this inhibition and other properties suggest similarities to the intracellular enzymes in other tissues, no cross-reaction was observed with antisera to the peroxisomal or to the mitochondrial inner-membrane enzyme. The activity was solubilized by and was stable in Triton X-100, which destroys the enzymes found in microsomes and in the mitochondrial outer membrane. The substrate specificity is broader than for the intracellular enzymes, the activities with stearoyl-CoA (114%) and arachidonoyl-CoA (97%) being equal to that with palmitoyl-CoA, and the activities with linoleoyl-CoA (44%) and erucoyl-CoA (46%) about half that with palmitoyl-CoA. The function of this carnitine palmitoyltransferase is probably to buffer the acyl-CoA present in the erythrocyte for turnover of the fatty acyl groups of the membrane lipids.

Angiodysplasia of the colon, Meckel's diverticulum and cardiac malformation. A case report.

A clinical case of angiodysplasia of the colon, Meckel's diverticulum and mitral valve prolapse is reported in a 20 year old man. Association of vascular ectasia of the colon and Meckel's diverticulum has been reported in a few cases but rarely with other kinds of malformation. This brief clinical paper reports a congenital aetiology of an uncommon disease of the colon, such as angiodysplasia in a young male.

Palmitoyl-L-carnitine, a metabolic intermediate of the fatty acid incorporation pathway in erythrocyte membrane phospholipids.

In this paper we report that palmitoyl-L-carnitine can be a metabolic intermediate of the fatty acid incorporation pathway into erythrocyte membrane phosphatidylcholine, and phosphatidylethanolamine. Phospholipid acylation was evaluated by measuring the incorporation of radioactive [1-14C]-palmitoyl-L-carnitine in membrane erythrocyte ghost phospholipids in the presence or absence of CoA. CoA highly stimulated the incorporation of [1-14C]-palmitic acid into both the phospholipids examined, although the incorporation was also evident in the absence of added CoA. Incorporation of [1-14C]-palmitic acid into phosphatidylcholine was greater than into phosphatidylethanolamine. 2-Bromo-palmitoyl-CoA, an irreversible inhibitor of the erythrocyte carnitine palmitoyltransferase, inhibited the acylation process.

Metabolic effects induced by L-carnitine and propionyl-L-carnitine in human hypoxic muscle tissue during exercise.

An experimental model was developed to investigate some metabolic effects of strenuous exercise in hypoxic muscle tissue of human volunteers. The incidence of carnitine supplementation was studied, assuming as marker the thiobarbituric acid reaction products analysed in plasma samples collected during the course of the protocol programme. Propionyl-L-carnitine appears to antagonize in a significant degree the damaging effects of muscle fatigue combined with hypoxic status. Under these conditions the detoxifying role played by propionyl-L-carnitine, previously reported in various tissues and in other pathological conditions, appears to be relevant, although further studies are needed to elucidate the pharmacodynamics of this molecule.

Effect of L-carnitine and acetyl-L-carnitine on the human erythrocyte membrane stability and deformability.

In this study we examined the effect of carnitine and acetylcarnitine on the human erythrocyte membrane stability and membrane deformability. Since erythrocyte membranes are impermeable to these compounds, we resealed erythrocyte ghosts in the presence of different concentrations of carnitine or acetylcarnitine. Resealed ghosts can be adequately studied in their cellular deformability and membrane stability properties by means of ektacytometry. Both carnitine and acetylcarnitine alter the membrane stability but not membrane deformability of the red cell membrane. Resealed ghosts containing 20, 50, 150, and 300 microM carnitine had 1.1, 1.6, 0.9, and 0.7 times the normal stability. While resealed ghosts containing 20, 50, 150, and 300 microM acetylcarnitine had 1.1, 1.5, 1.3, and 1.2 times the normal stability. Such changes were found to be reversible. We also conducted SDS PAGE of cytoskeletal membrane proteins from membrane fragments and residual membranes produced during membrane stability analysis, and unsheared resealed membranes in those samples where we observed an increase or a decrease of membrane stability. No changes in the cytoskeletal membrane proteins were noticed, even when the samples, prior SDS PAGE analysis, were treated with or without dithiothreitol. In addition, fluorescence steady state anisotropy of DPH in the erythrocyte membrane treated with carnitine or acetylcarnitine shows no modification of the lipid order parameter. Our results would suggest that both carnitine and its acetyl-ester, at physiological concentrations, may increase membrane stability in mature erythrocytes, most likely via a specific interaction with one or more cytoskeletal proteins, and that this effect would manifest when the erythrocytes are subjected to high shear stress.

Effect of propionyl-L-carnitine on rat spinal cord ischaemia and post-ischaemic reperfusion injury.

In this study we have examined the effect of propionyl-L-carnitine (PC) on rat spinal cord ischaemia and post-ischaemic reperfusion injury by evaluating two lipid peroxidation indices, thiobarbituric acid reactive substances (TBARS) and diene conjugation, before and after the addition of an ADP-Fe+2 complex to spinal cord homogenates. Aerobic, ischaemic, and post ischaemic reperfusion rat spinal cord homogenates from PC treated and untreated animals did not show any statistically significant difference in their TBARS and conjugated diene content. The addition of the ADP-Fe+2 complex to these homogenates resulted in an increased production of both the lipid peroxidation indices, though the magnitude of such formation was related to the type of experimental intervention. The post-ischaemic reperfusion samples of untreated rats showed the highest TBARS and conjugated diene content, while ischaemic samples in either treated and untreated rats did not show any statistically significant difference with respect to the aerobic samples. The post-ischaemic reperfusion samples of treated rats showed a statistically significant decrease of TBARS and conjugated diene production in comparison to the untreated samples. In addition, PC was also able to partially inhibit TBARS and conjugated diene formation in linoleic acid micelles exposed to hemoglobin, though it did not protect albumin fragmentation from the irradiation of water with an X-ray source.

Effect of oxidative stress on membrane phospholipid and protein organization in human erythrocytes.

Membrane phospholipid and protein organization was studied in intact human erythrocytes exposed to phenylhydrazine, an oxidative agent inducer. The evaluation of the membrane phospholipid and protein organization was carried out in terms of asymmetric distribution across the membrane bilayer for the phospholipids, and in terms of accessibility of cleavable sites present on the outer membrane surface for the proteins. Treatment of phenylhydrazine-exposed erythrocytes either with bee venom phospholipase A2 or with trinitrobenzenesulfonic acid indicated that phosphatidylserine (PS), which is the only phospholipid not formally present on the outer leaflet of the membrane, was translocated to the outer surface of the cell membrane. The extent of this phenomenon was directly proportional to the concentration of the oxidant having a peak value at 0.1 mM. Phosphatidylcholine and phosphatidylethanolamine conserved their original distribution across the erythrocyte membrane throughout the study. The oxidant, at a dose which did not induce any modification of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis cytoskeleton membrane protein pattern, did not provoke any alteration of the membrane protein surface architecture, although the translocation of PS to the membrane outer leaflet in intact erythrocytes was present.

Mechanism of spectrin degradation induced by phenylhydrazine in intact human erythrocytes.

The exposure of human erythrocytes to phenylhydrazine results in the degradation of both monomers of spectrin, a major cytoskeleton membrane protein. The degradative process, characterized by a loss of spectrin without the appearance of high-molecular-weight products, either under reducing conditions or not, is almost complete in 10 min when a 5% erythrocyte suspension is treated with 1 mM phenylhydrazine. Under these conditions, we found a loss of 62.3 and 48.5% for the alpha and beta monomer, respectively. A similar degradative extent was obtained when the membrane ghost plus cellular free extracts, were dialyzed, and the membrane ghost plus hemoglobin was exposed to 1 mM phenylhydrazine for 10 min. The presence of different proteinase inhibitors and effectors, such as EDTA, diethylenetriaminepentaacetic acid, EGTA, leupeptin, aprotinin, phenylmethylsulfonyl fluoride, pepstatin, Ca2+ and ATP plus Mg2+, in the membrane ghost plus cellular free extract system (undialyzed) did not affect the degree of the spectrin-degradative process induced by phenylhydrazine. In addition, a purified spectrin tetramer preparation exposed to 1 mM phenylhydrazine in the presence of hemoglobin was degraded to an extent comparable to that with intact cells. Our data suggest that the initial degradative step of spectrin induced by phenylhydrazine in intact erythrocytes may be ascribed more to a direct oxidative breakdown, probably involving main-chain cleavage and side-chain cleavage processes, than to an eventual proteolytic system.

NPGB-induced inhibition of superoxide anion production by normal Lewis rat macrophages.

The treatment of Lewis rat peritoneal macrophages with p1-nitrophenyl p-guanidinobenzoate (NPGB) inhibited the superoxide anion production stimulated with phorbol myristate acetate (PMA). The addition of NPGB at the time of maximum superoxide generation was still able to block the superoxide release. It appears from these findings that NPGB may block either the activation process of the membrane bound NAD(P)H oxidase or directly on the active enzyme. Other protease inhibitors such as, epsilon-amino caproic acid (EACA), pepstatin, trans aminomethyl cyclohexane carboxylic acid (AMCA), aprotinin, and leupeptin did not inhibit the superoxide release. The superoxide anion release by the xanthine-xanthine oxidase system was not inhibited by NPGB. This finding indicates that NPGB does not itself react with superoxide. It has been also demonstrated that NPGB is a good reactant toward sulfhydryl group. The relevance of these finding to experimental allergic encephalomyelitis (EAE) is discussed.

Isolation and biochemical characterization of the microsomal fraction from the digestive gland of mussel Mytilus galloprovincialis Lam.

A procedure to prepare microsomes from the mussel digestive gland is proposed. The data concerning the biochemical characterization of this subcellular fraction shows a typical RNA:protein ratio, but the presence of hydrolytic enzymes was also found; therefore a mixture of hydrolase inhibitors to study the different biochemical characteristics was used. The biochemical data demonstrate that glucose-6-phosphatase activity (G6Pase), a typical microsomal marker in mammalian cells, is not present in mussel digestive gland microsomes but a high non-specific phosphatase activity was detected. Benzo[a]pyrene hydroxylase activity was found to be present although in a minimal amount. The evaluation of the molecular weight of the rRNA demonstrates that the larger ribosomal subunit contains RNA of Mr 1.40 X 10(-6) (approximately 26S) and the smaller subunit is composed of RNA of Mr 0.65 X 10(-6) (18S). The data from mussel digestive gland microsomes was compared with that experimentally obtained from rat liver microsomes and discussed from a functional or an evolutionary point of view.