Biomedical subjects

M Maccarrone

Publications and source records attributed to M Maccarrone.

88 records · Page 5Linked to original sources

PubMed1995

Inhibition of soybean lipoxygenase-1 by chain-breaking antioxidants.

The aim of this investigation was to determine whether chain-breaking antioxidants able to prevent lipid peroxidation can inhibit lipoxygenase-1 (EC 1.13.11.12). Therefore, the effects of ascorbic acid, 6-palmitoylascorbic acid and trolox on the enzyme activity were analyzed by means of Lineweaver-Burk double reciprocal plots and Yoshino's graphical method. The effect of these compounds on the formation of free radicals during lipoxygenase-1 reaction was investigated as well, by monitoring the enzymic formation of oxodienes. We present evidence that the chain-breaking antioxidants ascorbic acid, 6-palmitoylascorbic acid and trolox inhibit soybean lipoxygenase-1 in the micromolar concentration range (Ki 27, 3 and 18 microM, respectively). The inhibition is competitive, complete and reversible. All three compounds trap the free radicals formed during the lipoxygenase-catalyzed reaction, which might substantially contribute to their inhibitory ability. These findings can have physiological significance in the light of the lipoxygenase involvement in biomembrane remodelling.

Antioxidants↗

PubMed1994

In vitro oxygenation of soybean biomembranes by lipoxygenase-2.

The ability of soybean lipoxygenases-1 and -2 to oxygenate biomembranes isolated from soybean seedlings has been investigated. Constituents of the lipid bilayer were analyzed by means of reversed phase and chiral phase high performance liquid chromatography, gas chromatography/mass spectrometry, high performance thin layer chromatography and uv spectroscopy. Evidence is presented that soybean lipoxygenase-2, at variance with the type-1 enzyme, oxygenates the esterified unsaturated fatty acid moieties in biomembranes, whereas membrane-embedded free unsaturated fatty acid moieties were not a suitable substrate for either isoenzyme. The oxygenation products derived from the biomembranes were the 9- and 13-hydroperoxides of linoleic acid residues, in a molar ratio of 1.0 to 1.7, and the 9- and 13-hydroperoxides of alpha-linolenic acid residues, in a molar ratio of 1.0 to 0.1. The R/S ratios of 13-hydroperoxy-9Z,11E-octadecadienoic acid and 9-hydroperoxy-10E,12Z,15Z-octadecatrienoic acid were found to be 0.5 and 25.0, respectively. These stereospecificity values were much higher than those of hydroperoxides isolated after incubation of lipoxygenase-2 with non-membraneous fatty acids or their methyl esters. The hydroperoxy fatty acids produced were distributed in neutral lipids and phospholipids isolated from soybean membranes, the former being oxidized to a larger extent. Furthermore, both intracellular and plasma membranes were substrates for the enzymic oxygenation, with a preference for those of chloroplasts followed by those of Golgi apparatus, endoplasmic reticulum, plasma membrane and mitochondria. These data point towards a different action of the two lipoxygenases in soybean cells. We suggest that the type-2 enzyme plays a role in the in vivo remodelling of biomembranes. The physiological relevance of these findings is discussed.

Lipid Bilayers↗

PubMed1992

Thermal injury and ozone stress affect soybean lipoxygenases expression.

The effects of thermal injury (cold and heat shock) and ozone treatment on lipoxygenases 1 (LOX-1) and 2 (LOX-2) of soybean seedlings have been investigated. Cold stress led to a decrease of the specific activities of both isoenzymes, attributable at least in part to a down-regulation of gene expression at the translational level. Both heat shock and ozone treatment enhanced lipoxygenases-specific activities, acting at the level of transcription of the genes. It is proposed that LOX-1 and LOX-2 are involved in the thermotolerance of soybeans and in the precocious aging induced by ozone.

Base Sequence↗

PubMed1992

Interaction of DNA with cationic liposomes: ability of transfecting lentil protoplasts.

The vesicle made of dipalmitoylphosphatidylcholine and stearylamine (9:1) were multilamellar and rather homogeneous in shape as seen by transmission electron microscopy. Upon addition of circular DNA plasmids of different lengths to the liposomes, the formation of vesicle clusters around the DNA filament was observed, with dimensions dictated by the ratio DNA/lipid. These liposomes were able to transfect lentil (Lens culinaris) protoplasts inside the cells two different reporter genes, chloramphenicol-acetyltransferase and beta-glucuronidase. The activity of these two enzymes could be found in the cell lysates after 24 h from the incubation of protoplasts with the lipid-DNA complexes.

Chloramphenicol O-Acetyltransferase↗

PubMed1992

Inhibition of lipoxygenase activity in lentil protoplasts by monoclonal antibodies introduced into the cells via electroporation.

The isolation of lentil protoplasts and the transfer of anti-lipoxygenase monoclonal antibodies into plant protoplasts by electroporation is reported. The dependence of the efficiency of monoclonal antibody incorporation on the field strength is shown as well. The transferred immunoglobulins retained their functional and structural integrity and were able to inhibit the intracellular target enzyme, with a linear relationship between inhibition of lipoxygenase activity and amount of incorporated monoclonal antibody. Moreover, the inhibition of lipoxygenase activity was well correlated with the increase of protoplast viability.

Antibodies, Monoclonal↗

PubMed1991

An investigation on the quinoprotein nature of some fungal and plant oxidoreductases.

The presence of pyrroloquinoline quinone (PQQ) as the organic cofactor of Dactylium dendroides galactose oxidase and lentil (Lens culinaris) seedling amine oxidase, purported PQQ-containing oxidoreductases (Van der Meer, R. A., Jongejan, J. A., and Duine, J. A. (1989) J. Biol. Chem. 264, 7792-7794; Citro, G., Verdina, A., Galati, R., Floris, G., Sabatini, S., and Finazzi-Argo', A. (1989) FEBS Lett. 247, 201-204), was reinvestigated using the nitro blue tetrazolium redoxcycling method (Paz, M. A., Gallop, P. M., Torrelio, B. M., and Flückiger, R. (1988) Biochem. Biophys. Res. Commun. 154, 1330-1337; Paz, M. A., Flückiger, R., Boak, A., Kagan, H. M., and Gallop, P. M. (1991) J. Biol. Chem. 266, 689-692) and the enzyme-linked immunosorbent assay with polyclonal antibodies against PQQ. The possible quinoprotein nature of the laccases from Polyporus versicolor and Rhus vernicifera was also investigated because of the similarities in spectroscopic and kinetic features of these enzymes and the laccase from Phlebia radiata, reported to be a PQQ protein (Karhunen, E., Niku-Paavola, M.-L., Viikari, L., Haltia, T., Van der Meer, R. A., and Duine, J. A. (1990) FEBS Lett. 267, 6-8). The presence of a quinonoid cofactor in lentil seedling amine oxidase is confirmed, whereas galactose oxidase and both laccases do not display any quinoprotein nature.

Amine Oxidase (Copper-Containing)↗

PubMed1991

Phytochrome control and anoxia effect on the activity and expression of soybean seedling lipoxygenases 1 and 2.

The effects of different light irradiations and anoxia treatment on the activity and expression of the lipoxygenases 1 (LOX-1) and 2 (LOX-2) of soybean seedlings have been investigated. A phytochrome-mediated decrease of the specific activities of both isoenzymes was found which was due to the regulation of the gene expression at the transcriptional level. The anoxia treatment of the seedlings led to a decrease of both lipoxygenase specific activities as well, but this effect was attributable to the reduction of the amount of the two isoenzymes, without changes in the steady-state levels of specific mRNAs. Therefore, light and anoxia are both regulators of soybean lipoxygenases, but their mechanism of action at the molecular level is different.

Anaerobiosis↗

PubMed1991

Proteolytic interconversion and N-terminal sequences of the Citrobacter diversus major beta-lactamases.

The N-terminal sequences of the two major beta-lactamases produced by Citrobacter diversus differed only by the absence of the first residue in form II and the loss of five amino acid residues at the C-terminal end. Limited proteolysis of the homogeneous form I protein yielded a variety of enzymatically active products. In the major product obtained after the action of papain, the first three N-terminal residues of form I had been cleaved, whereas at the C-terminal end the treated enzyme lacked five residues. However, this cannot explain the different behaviours of form I, form II and papain digestion product upon chromatofocusing. Form I, which was sequenced up to position 56, exhibited a very high degree of similarity with a Klebsiella oxytoca beta-lactamase. The determined sequence, which contained the active serine residue, demonstrated that the chromosome-encoded beta-lactamase of Citrobacter diversus belong to class A.

Amino Acid Sequence↗

PubMed1990

Soybean lipoxygenase-1 is not a quinoprotein.

Soybean lipoxygenase-1 was reinvestigated with respect to its quinoprotein nature. It has been reported previously that soybean lipoxygenase-1 contains pyrroloquinoline quinone as the organic cofactor. Because spectroscopic data were found to be inconsistent with the evidence presented in [1], we sought to reproduce the published data by carefully following the procedures described in [1] and supplementing them with new analytical results. The combined data lead us to conclude that soybean lipoxygenase-1 is not a quinoprotein.

Coenzymes↗

PubMed1990

Electrophoretic detection of ascorbate oxidase activity by photoreduction of nitroblue tetrazolium.

A method for the detection of ascorbate oxidase in electrophoretic gels is described. This method relies on the ability of the enzyme to prevent the photoreduction of nitroblue tetrazolium (NBT). The method is based on that described by C. Beauchamp and I. Fridovich (1971, Anal. Biochem. 44, 276-287) for the superoxide dismutase and was made specific for ascorbate oxidase detection by treating the gel with 0.1 M hydrogen peroxide. Ascorbate (25 microM) or riboflavin (500 microM) was used as the electron donor. The possible reaction mechanism in the presence of ascorbate has been investigated. Western and Northern blot analyses confirmed the results obtained from the NBT staining procedure.

Ascorbate Oxidase↗

PubMed1989

Kinetic features of ascorbic acid oxidase after partial deglycation.

By means of specific exoglycosidases, sugars have been removed under non-denaturing conditions from ascorbic acid oxidase (AAO), different deglycation schedules being followed. Our results indicate that deglycation clearly affects the kinetic features of AAO, leading to an increase of 'affinity' and 'catalytic ability' of the enzymic forms so generated. A better exposure of the catalytic-site residues could be supposed to occur upon treatment with exoglycosidases. This is supported by the three-dimensional X-ray structure of zucchini (Cucurbita pepo medullosa; courgette) AAO.

Ascorbate Oxidase↗

PubMed1988

Citrobacter diversus ULA-27 beta-lactamases. Improved purification and general properties.

Two chromosome-encoded beta-lactamases have been purified from Citrobacter diversus ULA-27. They exhibited slightly different isoelectric points (6.8 and 6.2) and very similar Mr values (congruent to 29,000). Their specificity spectrum was rather wide, since they hydrolysed some cephalosporins with kcat: values similar to those observed with the best penicillin substrates. Cloxacillin, methicillin and imipenem were hydrolysed very slowly. Hydrolysis of azthreonam could not be detected.

Amino Acids↗

PubMed1988

On the kinetic interaction between ceftriaxone and some beta-lactamases.

The activity of beta-lactamases from Citrobacter diversus ULA-27 on ceftriaxone, a widely recognized third-generation cephalosporin, has been examined and compared to the activity of various other beta-lactamases from different sources. Ceftriaxone (Roche S.p.A. Milan) was found to be resistant to hydrolysis by beta-lactamases from Enterobacter cloacae and Bacillus cereus, but susceptible to beta-lactamases from Mycobacterium fortuitum strain Cow 18 and, mostly, to beta-lactamases from various strains of Citrobacter diversus. Derivatives with substituents in the 3-position of ceftriaxone, namely cefotaxime (Roussel Maestretti S.p.A., Milan) and ceftizoxime (Farmitalia Carlo Erba, Milan), were much less susceptible to hydrolysis by C. diversus ULA-27 enzymes (22 and 6% of ceftriaxone hydrolysis, respectively), the hydrolysis rate being paralleled by differences in MIC values. Ceftriaxone inhibited the activity of E. cloacae beta-lactamases toward cefazolin as substrate, but the inhibition was totally abolished by preincubation of ceftriaxone with the enzyme before addition of the substrate. Overall, the data point to a relevance of C. diversus ULA-27 beta-lactamases in the mechanism of resistance of this strain to the various third-generation cephalosporins.

Bacillus↗

PubMedDate not supplied

Lipoxygenase products induce ultraweak light emission from human erythroleukemia cells.

Several lipoxygenase products were able to enhance ultraweak light emission and membrane permeability of human erythroleukemia K562 cells. In particular, 13-hydroperoxyoctadecadienoic acid (13-HPOD) was more effective than hydrophilic hydroperoxides, like H2O2 and tert-butyl hydroperoxide. The enhancement of luminescence induced by 13-HPOD was inhibited by superoxide and hydroxyl radical scavengers. The effect of 13-HPOD was inhibited by superoxide and hydroxyl radical scavengers. The effect of 13-HPOD was potentiated by the calcium ionophore A23187 and inhibited by the calcium chelator EDTA, and was observed also in liposomes containing unsaturated lipids. Cholesterol enrichment, which decreases the membrane fluidity, did not modify the effect of 13-HPOD on K562 cells.

Calcimycin↗

PubMedDate not supplied

Ultraweak light emission is a common response of bacterial cells to chemico-physical stress.

Escherichia coli JM101 cells were subjected to pore-forming electric fields, irradiation with ultraviolet light or oxidative stress by either the lipoxygenase products 9- and 13-hydroperoxyoctadecadienoic acids (9- and 13-HPOD) or hydrogen peroxide. It was found that all chemico-physical stresses enhanced ultraweak light emission from the bacterial cells, the most effective treatment being electroporation (up to 20-fold increase in luminescence compared to the control value), followed by oxidative stress with 9- or 13-HPOD (up to 4-fold increase) and irradiation with UV light (up to 2.8-fold increase). Bacterial luminescence was always in the red edge of the spectrum and was paralleled by changes in membrane oxidative index and specific activity of catalase and superoxide dismutase.

Catalase↗

PubMedDate not supplied

Endocannabinoids, hormone-cytokine networks and human fertility.

Anandamide (N -arachidonoylethanolamine, AEA) is a major endocannabinoid, shown to impair mouse pregnancy and embryo development and to induce apoptosis in blastocysts. Here, we review the roles of AEA, of the AEA-binding cannabinoid (CB) receptors, of the selective AEA membrane transporter (AMT), and of the AEA-hydrolyzing enzyme fatty acid amide hydrolase (FAAH), in human gestation. In particular, we discuss the interplay between the endocannabinoid system and the hormone-cytokine array involved in the control of human pregnancy, showing that the endocannabinoids take part in the immunological adaptation occurring during early pregnancy. In this line, we discuss the critical role of FAAH in human peripheral lymphocytes, showing that the expression of this enzyme is regulated by progesterone, Th1 and Th2 cytokines, which also regulate fertility. Moreover, we show that AEA and the other endocannabinoid, 2-arachidonoylglycerol, inhibit the release of the fertility-promoting cytokine leukemia inhibitory factor from human lymphocytes. Taken together, low FAAH and consistently high blood levels of AEA, but not CB receptors or AMT, can be early (<8 weeks of gestation) markers of spontaneous abortion, potentially useful as diagnostic tools for large-scale, routine monitoring of gestation in humans.

Animals↗