Real-time digital holographic microscopy of multiple and arbitrarily oriented planes.

Digital holographic microscopy is used to numerically refocus a recorded hologram at an arbitrary axial distance. However, as a straightforward property of coherent light fields, image reconstruction on an arbitrary tilted plane could be directly obtained by a rotation in k-space. We demonstrate that this property allows the real-time microscopic inspection of particle distribution over three mutually orthogonal planes at the same time. As a straightforward application we use the proposed technique for real-time monitoring of fluid flow over the three cross sections of a microfluidic channel.

[Nephrogenic systemic fibrosis]. / Fibrosi nefrogenica sistemica.

Nephrogenic systemic fibrosis (NSF) is a new, rare, and severe disease occurring in patients with renal failure who have been exposed to gadolinium. The pathogenesis of NSF is not completely known. In fact, the first warning about a significant relationship between NSF and gadolinium (a contrast medium used in magnetic resonance imaging) was only issued in 2006. No cases of NSF have been reported in Italy to date. A nationwide investigation should therefore be carried out to assess the real prevalence of NSF within the Italian uremic population. Furthermore, we need guidelines to reduce the risk of NSF in renal patients undergoing MRI with contrast medium.

NADH and NADPH inhibit lipid peroxidation promoted by hydroperoxides in rat liver microsomes.

Lipid peroxidation induced through cytochrome P-450 activation of cumene hydroperoxide, linolenic acid hydroperoxide and peroxidized phosphatidylcholine in rat liver microsomes is markedly inhibited by either NADH or NADPH. This inhibition is not due to an antioxidant effect. Conversely, cumene hydroperoxide decomposition is stimulated by the reduced pyridine nucleotides but not by some modifiers of cytochrome P-450 (SKF-525A, metyrapon and aniline). The mechanism by which NADH and NADPH prevent lipid peroxidation may involve a reduction of the hydroperoxides mediated by cytochrome P-450 and occurring without formation of free radical forms that are usual sparkers of lipid peroxidation.

Comparison of cumene hydroperoxide- and NADPH/Fe3+/ADP-induced lipid peroxidation in heart and liver submitochondrial particles. Mechanisms of protection by succinate.

The NADPH/Fe3+ /ADP system stimulates lipid peroxidation both in rat liver and bovine heart submitochondrial particles, while cumene hydroperoxide is active only in rat liver submitochondrial particles. The lack of a peroxidizing effect of cumene hydroperoxide in heart submitochondrial particles was related to the absence of cytochrome P-450. When ubiquinones are extracted from rat liver and bovine heart submitochondrial particles, succinate can still partially protect the cumene hydroperoxide-induced lipid peroxidation but not the peroxidation induced by NADPH/Fe3+ /ADP. The protective effect of succinate in lipid peroxidation was referred either to the reduction of ubiquinones that can act as antioxidants in the NADPH/Fe3+ /ADP system, or to the reduction of cytochrome P-450 that acts as a peroxidase in the cumene hydroperoxide system.

Mitochondrial lipid peroxidation by cumene hydroperoxide and its prevention by succinate.

Rat liver mitochondria form lipid hydroperoxides when they are incubated aerobically with cumene hydroperoxide. The rate of reaction is dependent on the initial concentration of the latter and involves the consumption of oxygen. Gradient-separated and cytochrome c-depleted mitochondria, mitoplasts and submitochondrial fractions also undergo this peroxidation. Mitochondrial lipid peroxidation by cumene hydroperoxide is strongly inhibited by SKF52A (an inhibitor of cytochrome P-450), by antioxidants and to a lesser extent by the enzymes superoxide dismutase and catalase. Conversely, rotenone and N-ethylmaleimide stimulate the reaction. Succinate protects against the lipid peroxidation and in some mitochondrial fractions the associated oxygen uptake is also inhibited. This protection by succinate is prevented by malonate but not by N-ethylmaleimide or antimycin. Lipid hydroperoxides present in previously peroxidised mitochondria are partly lost on reincubation with succinate and this reaction is also unaffected by N-ethylmaleimide but inhibited by both malonate and antimycin. The results suggest that reduction of mitochondrial ubiquinone may prevent the generation of lipid hydroperoxides but that their subsequent removal may require reduction at or beyond cytochrome b.

Hydrolysis of ITP generates a membrane potential in submitochondrial particles.

ITP hydrolysis catalysed by the ATPase of submitochondrial particles from both bovine heart and rat liver is shown to be linked to the generation of a membrane potential, and therefore also to proton translocation. The magnitude of the membrane potential is similar to that observed during ATP hydrolysis at equivalent concentrations of phosphate and nucleoside tri- and diphosphates. An explanation is suggested for why in other reports ITP was found to be a poor substrate for supporting energy-linked reactions that are driven by the membrane potential.

Platelet responses promoted by the activation of protein kinase C or the increase of cytosolic Ca2+ are potentiated by adrenaline. Effects of cAMP and staurosporine.

We studied the action of the alpha 2 adrenergic agonist adrenaline on the platelet responses evoked by the activation of protein kinase C or by the ionophore induced increase of cytosolic Ca2+. Both the phorbol ester and ionomycin-induced aggregation are strongly potentiated by adrenaline which per se does not behave as an activating agonist. The potentiation by adrenaline is observed both when added before and after the aggregating agent; in the latter case the effect increases on increasing the delay of adrenaline addition. Adrenaline also reverses the inhibition by cAMP of the PMA (or ionomycin) induced aggregation. It also has a strong potentiating effect (over 100%) on the phorbol ester induced ATP secretion and a weaker effect on the secretion induced by ionomycin. The effect on secretion is visible only when adrenaline is added prior to the stimulus. The inhibition by cAMP of the PMA or ionomycin induced secretion is also counteracted by adrenaline. In no case adrenaline modifies the pattern of platelet phosphoproteins. Ionomycin induces some platelet aggregation also in the presence of the protein kinase inhibitor staurosporine; also this phosphoprotein independent aggregation is strongly stimulated by adrenaline.

Further purification and characterization of the succinyl-CoA:3-hydroxy-3-methylglutarate coenzyme A transferase from rat-liver mitochondria.

Succinyl-CoA:3-hydroxy-3-methylglutarate coenzyme A transferase, previously identified in rat-liver mitochondria (Deana et al. (1981), Biochim. Biophys. Acta 662, 119-124), was purified to near homogeneity and further characterized. After the last purification steps consisting of Ultrogel AcA-44 filtration and agarose-hexane-coenzyme A chromatography, the enzyme was apparently tetrameric with a mass of 48-52 kDa determined by gel filtration on Sephadex G-75, ultracentrifugation through a sucrose gradient and SDS-gel electrophoresis. By means of a HPLC technique developed for measuring the CoA esters we could determine the enzyme activity in both forward and reverse directions and show that the kinetic constants, i.e., Km of reactants and Vmax, are not too different for the two reactions. Double-reciprocal plots of the enzyme velocities versus the concentration of one substrate at different fixed concentrations of the other substrate gave families of straight lines converging below the substrate-abscissa for both forward and backward reactions, indicating a kinetic mechanism of rapid equilibrium random Bi-Bi type. The competitive inhibition of the product succinate with respect to both reactants, 3-hydroxy-3-methylglutarate and succinyl-CoA, as well as the Haldane relationships are consistent with this conclusion. An inhibitory effect on CoA transferase activity by acetate, acetoacetate, acetyl-CoA, acetoacetyl-CoA, coenzyme A, carnitine, ZnCl2 and high concentrations of the monovalent anions ClO4-, F-, I- and Cl- was also found.

The adenosine inhibition of glutamate exocytosis in synaptosomes is removed by the collapse of the vesicle-cytosol deltapH plus the opening of farnesol-sensitive Ca(2+) channels.

Adenosine inhibits synaptosomal exocytosis of glutamate, triggered by KCl or by the K(+) channel inhibitor, 4-aminopyridine (4-AP), without affecting Ca(2+) influx. Its effect is removed by the activation of protein kinase C (PKC). We show that in the presence of the protein kinase inhibitor, staurosporine, the adenosine inhibition is removed also by collapsing deltapH between secretory vesicle and the cytosol with methylamine (MA), provided that exocytosis is triggered by KCl (which activates an initial transient spike of Ca(2+) influx) but not by 4-AP. If KCl is supplied prior to Ca(2+), the spike of Ca(2+) influx is absent and the adenosine inhibition is maintained. MA can remove the adenosine inhibition also with 4-AP, provided that tetraethylammonium (TEA), an inhibitor of a different class of K(+) channels, is supplied together with 4-AP. TEA promotes a further increase of cytosolic free Ca(2+) concentration ([Ca(2+)](i)), which adds to the 4-AP-induced Ca(2+) influx. Farnesol (5-10 microM), a physiological derivative of farnesyl pyrophosphate of the sterol biosynthetic pathway, specifically inhibits the Ca(2+) spike after KCl as well as the TEA-promoted Ca(2+) increase. At the same time, it prevents the removal of the adenosine inhibition by MA. We conclude that the adenosine inhibition is removed by the coincidence of two signals, the alkalinization of secretory vesicles and the opening of a particular class of Ca(2+) channels associated to the TEA-sensitive K(+) channels, equivalent to the Ca(2+) spike after KCl, and sensitive to farnesol.

Effects of calcium chelators, divalent cations and sulfhydryl reagents on calcium uptake and motility of bovine spermatozoa.

Addition of 1 mM Ca/EGTA complex (1:1 ratio) to an incubation medium containing 1.5 mM Ca2+ produced a notable increase in the Ca2+ cycling in ejaculated bovine spermatozoa. Similar results were also obtained with the Ca/EDTA and Ca/EDTA complexes or with the heavy metal chelator DTPA (50 microM). Ba2+, Ni2+ or Co2+ added at 0.1 mM concentration abolished the stimulatory effect of the Ca/EGTA complex on Ca2+ cycling, whereas it did not affect the calcium movement in the absence of the calcium chelator complex. It is concluded that small amounts of these cations should be bound to the plasma membrane of bovine spermatozoa and inhibit the cellular calcium influx. 0.1 mM Cd2+ and NEM or 1 mM diamide produced a calcium efflux from the spermatozoa together with an inhibition of cellular motility and an increase in glutamate-oxaloacetate transaminase release. Conversely the impermeant sulfhydryl reagent mersalyl caused a net calcium efflux but did not alter the cellular motility nor the transaminase release. It is suggested that the permeant thiol reagents could decrease the spermatozoal mobility by impairing the mitochondrial ATP-synthesis.

Diacylglycerol mediates the thrombin-induced, protein kinase C and Ca2+ independent activation of the Na+/H+ exchanger in platelets.

Treatment of aspirinated platelets with the electroneutral K+/H+ exchanger nigericin induces a decrease in intraplatelet pH as measured with the intracellular fluorescent indicator BCECF. Under these conditions, the proton permeability of the plasma membrane is unaffected. The addition of thrombin induces a rapid partial recovery of pH(i), which is completely abolished by the Na+/H+ exchanger inhibitor NHA. The effect is also evident in the presence of the PKC inhibitors GF 109203X or staurosporine and in the absence of both external (EGTA-chelated) and internal (BAPTA-chelated) Ca2+. This makes the thrombin-induced activation of the exchanger independent of the involvement of the hitherto described activators, namely PKC and the increase in [Ca2+]i, as well of the recently reported activator arachidonic acid [Cavallini, L., Coassin, M., Borean, A., and Alexandre, A. (1996) Biochem. J. 319, 567-574], whose production requires a high [Ca2+]i. The thrombin-dependent recovery of pH(i) is prevented by the phospholipase C inhibitor ET 18 O-CH3 and is mimicked by the addition of the permeable diglyceride dioctanoyl glycerol (DiC8) exogenously supplied. The effect of thrombin and DiC8 is unaffected by inhibition of diacylglycerol lipase and diacylglycerol kinase. These experiments identify diglyceride as a novel activator of the Na+/H+ exchanger in platelets.

Modification of the xanthine-converting enzyme of perfused rat heart during ischemia and oxidative stress.

The reversible and irreversible conversion of xanthine dehydrogenase to xanthine oxidase during ischemia/reperfusion and oxidative stress induced by hydrogen peroxide or diamide and its relationship with glutathione and protein SH groups were studied. The direct spectrophotometric measurement of the various forms of the xanthine-converting enzyme indicates that, in the fresh rat heart or after normoxic perfusion, there always is a basal level of 80% xanthine dehydrogenase and 20% of xanthine oxidase (15% irreversible and 5% reversible) that could contribute to the background production of free radicals. There is no significant increase of irreversible xanthine oxidase during ischemia nor during reperfusion. After global ischemia the reversible oxidase shows almost no increase while, when ischemia is followed by reperfusion, there is a limited increase (less then 9%) of the reversible xanthine oxidase. In the latter conditions there is a decrease of glutathione and of SH groups of about 70% and 25%, respectively. Perfusion for 1 h with oxidizing agents like hydrogen peroxide (60 microM) or diamide (100 microM) determines a marked conversion of xanthine dehydrogenase to reversible xanthine oxidase of about 40% and 60%, respectively; this oxidase activity partially reconverts to the dehydrogenase after withdrawing the oxidizing agents from the perfusion medium. The level of irreversible xanthine oxidase remains unchanged in all the conditions tested. Both hydrogen peroxide and diamide induce a strong decrease in SH groups and depletion of glutathione. The xanthine dehydrogenase----xanthine oxidase conversion thus appears to be sensitive to the redox state of thiol groups.

Inhibition of rat liver hydroxymethylglutaryl-CoA reductase by sulfhydryl reagents, coenzyme A esters and synthetic compounds.

The activity of the microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase was assayed with a procedure based on the extraction of the product mevalonolactone in a benzene phase. Diamide is an uncompetitive inhibitor of the reaction, while coenzyme A disulfide and tetraethylthiouram disulfide act as non-competitive inhibitors. Diamide inhibition cooperatively increases with the inhibitor concentration. HMG produces a decrease in enzyme activity that combines with that of coenzyme A disulfide. Both CoASH and coenzyme A esters strongly inhibit the reductase activity. Three new synthetic compounds with either thio-ether or thio-ester groups also show inhibitory effect on the enzyme activity.

Fructose-1,6-diphosphate inhibits platelet activation.

Fructose-1,6-diphosphate (FDP) is a physiological product which exhibits pharmacological properties. This study shows that FDP (1-3 mM) inhibits platelet aggregation induced by the agonists thrombin, vasopressin, platelet activating factor, ADP, adrenaline, arachidonate and the stable thromboxane analogue U 44069. Thrombin-promoted ATP secretion and cytosolic Ca2+ rise are also drastically inhibited by FDP, which decreases, although to a lesser extent, the protein kinase C-dependent phosphorylation of the 47 kDa protein. The inhibition on thrombin-induced aggregation is shared, albeit less efficiently, by glucose-1,6-diphosphate and fructose-2,6-diphosphate but not by other phosphorylated monosaccharides (fructose-1:2 cyclic,6-diphosphate, glucose-1- and glucose-6-phosphate, fructose-1- and fructose-6-phosphate, mannose-6-phosphate and 5-phosphoryl ribose-1-pyrophosphate). FDP does not affect platelet activation induced by the protein kinase C activators dioctanoylglycerol or phorbol 12-myristate 13-acetate. No increase of cAMP concentration is observed in FDP-treated platelets. Altogether, these results indicate that FDP inhibits platelet activation at a level preceding phospholipase C. The data are consistent with a general inhibitory action of FDP on signal transmission.

Involvement of nuclear factor-kappa B (NF-kappaB) activation in mitogen-induced lymphocyte proliferation: inhibitory effects of lymphoproliferation by salicylates acting as NF-kappaB inhibitors.

The transcription factor nuclear factor-kappa B (NF-kappaB) is involved in the production of inflammatory cytokines and in the control of the inflammatory response. Some nonsteroidal anti-inflammatory drugs such as acetylsalicylic acid (ASA) or salicylate are known to exert some of their anti-inflammatory pharmacological properties independently of cyclooxygenase inhibition. For ASA and salicylate, an NF-kappaB inhibitory effect at mM concentrations (pharmacological plasma concentrations reached in vivo) has been shown. We studied the action of ASA, salicylate, and several NF-kappaB inhibitors on the mitogen-induced activation of peripheral blood lymphocytes (PBL) and purified T cells. We showed that ASA and salicylate (1-3 mM) (but not indomethacin, a specific cyclooxygenase inhibitor) as well as a group of chemically unrelated inhibitors of NF-kappaB (including the sesquiterpene lactone parthenolide, Bay 11-7082, sulfasalazine, the proteasome inhibitor MG-132 and the peptide SN-50, an inhibitor of the nuclear transfer of the p50 subunit of NF-kappaB), were potent inhibitors of phytohemoagglutinin-activated PBL and T cell proliferation. At the same concentrations, they inhibited NF-kappaB binding to DNA in nuclear extracts. The inhibition of proliferation was not relieved by exogenous interleukin (IL)-2. We concluded that NF-kappaB activation has a fundamental role in T cell proliferation independently of IL-2 production. Some pharmacological actions of ASA may be ascribed to the inhibition of immune cell proliferation via the inhibition of the transcription factor NF-kappaB.

Evidence that long-term administration of a methionine-enkephalin analogue stimulates the growth and steroidogenic capacity of rat inner adrenocortical cells.

A prolonged infusion with D-ala2-met-enkephalinamide (DALA) caused a significant increase in both basal and ACTH-stimulated corticosterone secretion by dispersed inner adrenocortical cells of rats whose hypothalamo-hypophyseal axis was pharmacologically interrupted. This effect of DALA was associated with a notable hypertrophy of isolated cells. These findings suggest that enkephalins are involved in the stimulation of the growth and steroidogenic capacity of the cells of the inner layers of rat adrenal cortex.

Adenosine inhibits glutamate exocytosis largely without interfering with Ca2+ influx in rat cerebrocortical synaptosomes.

Adenosine is an inhibitor of glutamate release in synaptosomes. The inhibition is removed by the A(1) adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). We monitored the variations of cytoplasmic free Ca(2+) concentrations ([Ca(2+)](i)) in KCl or 4-aminopyridine-stimulated synaptosomes, in the presence of adenosine or adenosine plus DPCPX. The increment of [Ca(2+)](i) upon stimulation was unmodified by adenosine (up to 400-500 microM) while it was strongly decreased when exocytosis was decreased to a similar extent by lowering KCl or 4-aminopyridine. Adenosine also inhibited glutamate release induced by the Ca(2+) ionophore ionomycin. Increasing adenosine to 1.5 mM resulted in a decrease of the stimulus-induced increase of [Ca(2+)](i) and in the further potentiation of the adenosine inhibition of exocytosis from 41+/-3 to 51+/-4%. We conclude that adenosine affects glutamate exocytosis mostly in a Ca(2+) independent mode.

Modulation of glutamate exocytosis by redox changes of superficial thiol groups in rat cerebrocortical synaptosomes.

The treatment of cerebral cortex synaptosomes with the membrane impermeable thiol reagent 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) induces a long-lasting partial inhibition (about 40%) of the KCl-stimulated Ca2+-dependent exocytosis of glutamate. Synaptosomes are not damaged by the treatment. The increase of cytoplasmic free Ca2+ concentration ([Ca2+]i) upon depolarization is not affected by DTNB. The inhibition is observed also if exocytosis is induced with the Ca2+-ionophore ionomycin. In all cases the inhibition is reversed by the impermeable reductant glutathione (GSH). Similarly the inhibition of exocytosis by H2O2 (Zoccarato, F., Valente, M. and Alexandre, A., Hydrogen peroxide induces a long-lasting inhibition of the Ca2+-dependent glutamate release in cerebrocortical synaptosomes without interfering with cytosolic Ca2+. J. Neurochem., 64 (1995) 2552-2558.) is reversed by GSH. It is concluded that redox changes (possibly thiol-disulfide transitions) of superficial groups modulate the exocytotic apparatus directly. In an attempt to identify the protein(s) involved in this novel type of control, we evidenced DTNB (H2O2) reactive bands at 35 and at 85-150 kDa which can be labeled with a monobromotrimethylammoniobimane bromide (qBBr) derivatization.

A procedure allowing measurement of cytosolic Ca2+ in rat platelets. Inhibition of a plasma lipoprotein on fura 2-AM loading.

Loading of the fluorescent Ca2+ probe fura 2 in rat platelets is highly inhibited by a plasmatic factor that is removed by gel filtration through a Sepharose C-2B column. Rat plasma also inhibits fura 2 loading in human platelets. The inhibitory effect is abolished by perchloric acid-deproteinization or heat denaturation of plasma suggesting a proteic nature of the inhibitory compound. Indeed the 10,000 x g supernatant of the heat denaturated plasma shows a positive effect on fura 2 accumulation, most likely by partially inhibiting its cellular effux. These effects are only negligibly shown by the corresponding fractions of human plasma. Results obtained by fractionation of rat plasma proteins by means of ion exchange DEAE Sepharose C-6B chromatography and ultracentrifugation through high density saline solutions indicate that the inhibition of cellular fura 2 loading is due to the HDL fraction of rat plasma.

Decrease of serum malondialdehyde in patients treated with chlorpromazine.

Malondialdehyde determination in serum from schizophrenic patients before and after treatment with chlorpromazine showed that, after treatment, patients had significantly lower values than before. The antioxidant properties of chlorpromazine can be related to its effect on the level of serum lipid peroxides and possibly to its neuroleptic action.