Activation of human lung semicarbazide sensitive amine oxidase by a low molecular weight component present in human plasma.

Semicarbazide-sensitive amine oxidase (SSAO) encodes a wide family of enzymes named E.C.1.4.3.6 [amine:oxygen oxidoreductase (deaminating) (copper containing)] that metabolises primary aliphatic and aromatic amines. It is present in almost all vascularised and nonvascularised mammalian tissues, and it is also present in soluble form in plasma. SSAO appears to show different functions depending on the tissue where it is expressed. Here we describe, for the first time, the activation of the SSAO from human lung by human plasma. The extent of activation was greater when the human plasma came from diabetic and heart infarcted patients. A kinetic mechanism of such effect is proposed. The activation was lost after the plasma was dialysed, indicating a low molecular weight component (MW <3800 Da) to be responsible. The activator component is heat stable and resistant to proteolysis by chymotrypsin and trypsin and also resistant to perchloric acid treatment. However, treatment with 35% formic acid, completely abolished activation, suggesting involvement of lipid material. The possibility of that lysophosphatidylcholine (LPC), an amphiphilic phospholipid derived from the phosphatidylcholine, the major component in plasma accumulated in pathological conditions, was studied. LPC was shown to behave as a "competitive activator" of human lung SSAO at concentrations below its critical micellar concentration (CMC value=50 microM). Thus LPC may be a component of the SSAO activatory material present in human plasma.

Tissue activity and cellular localization of human semicarbazide-sensitive amine oxidase.

Semicarbazide-sensitive amine oxidase (SSAO), widely distributed in highly vascularized mammalian tissues, metabolizes endogenous and xenobiotic aromatic and aliphatic monoamines. To assess whether its physiological role in humans is restricted to oxidation, we used an immunohistochemical approach to examine the cellular localization of SSAO in human peripheral tissues (adrenal gland, duodenum, heart, kidney, lung, liver, pancreas, spleen, thyroid gland, and blood vessels) and also analyzed its subcellular localization. The results are in agreement with the specific activities also determined in the same samples and are discussed with reference to the tissue distribution of monoamine oxidase A and B. Together with the oxidative deamination of monoamines, SSAO cellular localization indicates that, in most human peripheral tissues, it might participate in the regulation of physiological processes via H(2)O(2) generation. (J Histochem Cytochem 49:209-217, 2001)

Inhibition of monoamine oxidase A and B activities by imidazol(ine)/guanidine drugs, nature of the interaction and distinction from I2-imidazoline receptors in rat liver.

1. I2-Imidazoline sites ([3H]-idazoxan binding) have been identified on monoamine oxidase (MAO) and proposed to modulate the activity of the enzyme through an allosteric inhibitory mechanism (Tesson et al., 1995). The main aim of this study was to assess the inhibitory effects and nature of the inhibition of imidazol(ine)/guanidine drugs on rat liver MAO-A and MAO-B isoforms and to compare their inhibitory potencies with their affinities for the sites labelled by [3H]-clonidine in the same tissue. 2. Competition for [3H]-clonidine binding in rat liver mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the pharmacological profile of the interaction (2-styryl-2-imidazoline, LSL 61112 > idazoxan > 2-benzofuranyl-2-imidazoline, 2-BFI = cirazoline > guanabenz > oxymetazoline > > clonidine) was typical of that for I2-sites. 3. Clonidine inhibited rat liver MAO-A and MAO-B activities with very low potency (IC50S: 700 microM and 6 mM, respectively) and displayed the typical pattern of competitive enzyme inhibition (lineweaver-Burk plots: increased K(m) and unchanged Vmax values). Other imidazol(ine)/guanidine drugs also were weak MAO inhibitors with the exception of guanabenz, 2-BFI and cirazoline on MAO-A (IC50S: 4-11 microM) and 2-benzofuranyl-2-imidazol (LSL 60101) on MAO-B (IC50: 16 microM). Idazoxan was a full inhibitor although with rather low potency, on both MAO-A and MAO-B isoenzymes (IC50S: 280 microM and 624 microM, respectively). Kinetic analyses of MAO-A inhibition by these drugs revealed that the interactions were competitive. For the same drugs acting on MAO-B the interactions were of the mixed type inhibition (increased K(m) and decreased Vmax values), although the greater inhibitory effects on the apparent value of Vmax/K(m) than on the Vmax value indicated that the competitive element of the MAO-B inhibition predominated. 4. Competition for [3H]-Ro 41-1049 binding to MAO-A or [3H]-Ro 19-6327 binding to MAO-B in rat liver mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the drug inhibition constants (Ki values) were similar to the IC50 values displayed for the inhibition of MAO-A or MAO-B activities In fact, very good correlations were obtained when the affinities of drugs at MAO-A or MAO-B catalytic sites were correlated with their potencies in inhibiting MAO-A (r = 0.92) or MAO-B (r = 0.99) activity. This further suggested a direct drug interaction with the catalytic sites of MAO-A and MAO-B isoforms. 5. No significant correlations were found when the potencies of imidazol(ine)/guanidine drugs at the high affinity site (pKiH, nanomolar range) or the low-affinity site (pKiL, micromolar range) of I2-imidazoline receptors labelled with [3H]-clonidine were correlated with the pIC50 values of the same drugs for inhibition of MAO-A or MAO-B activity. These discrepancies indicated that I2-imidazoline receptors are not directly related to the site of action of these drugs on MAO activity in rat liver mitochondrial fractions. 6. Although these studies cannot exclude the presence of additional binding sites on MAO that do not affect the activity of the enzyme, they would suggest that I2-imidazoline receptors represent molecular species that are distinct from MAO.

Contribution of different amine oxidases to the metabolism of dopamine in bovine retina.

The contribution of monoamine oxidase (MAO) A, MAO B and semicarbazide-sensitive amine oxidase (SSAO) to the metabolism of dopamine in the bovine retina was studied. These activities were present in the optic nerve, iris, choroid and bovine retina, but they were absent in the lens. SSAO activity towards dopamine was present in the choroid and the retina, but not in the iris or the optic nerve. The corresponding kinetic values for this substrate in the retina and the choroid showed higher affinity for MAO A (Km 271 and 197 microM, respectively) than for MAO B (Km 861 and 404 microM, respectively). This effect was counteracted by the higher Vmax value for MAO B resulting in the Vmax/Km ratio being similar for both cases. The absence of detectable SSAO activity towards dopamine in these last two tissues contrasts with the presence of that enzyme when benzylamine was studied as substrate. These results indicate that two different SSAO activities could be present in the bovine eye.

Amine oxidase activities in rat breast cancer induced experimentally with 7,12-dimethylbenz(alpha)anthracene.

The activities and distribution of monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) in solid breast tumour induced in the rat by treatment with 7,12-dimethylbenz(alpha)anthracene (DMBA) were studied. The mammary tumours were classified according to anatomopathological criteria into: the benign fibroadenoma (FAD) and the malignant adenocarcinoma (ADC) and infiltrant adenocarcinoma (I-ADC). The proportions of total MAO (15%) and SSAO activities (85%) did not change with malignancy. However, an increasing degree of malignancy was associated with an increase in MAO-A activity and a decrease in MAO-B and SSAO activities. Kinetic constants were calculated for SSAO and for each MAO form separately, using specific substrates. The Km values did not change significantly with the degree of malignancy, but Vmax values for MAO-A increased whereas Vmax for SSAO and MAO-B diminished with malignancy. The dependence of SSAO activity on protein concentration indicated the presence of endogenous reversible inhibitory material in extracts from the more malign tumours. This inhibitor was associated with the microsomal fraction and was not removed by dialysis. It was also present in detergent-solubilized extracts, suggesting that the phenomenon might be due to an association of the enzyme itself producing an inactive species.

The oxidation of dopamine by the semicarbazide-sensitive amine oxidase (SSAO) from rat vas deferens.

The activities of monoamine oxidase A and B and the semicarbazide-sensitive amine oxidase from rat vas deferens were compared towards benzylamine and dopamine. The selective inhibitors (-)-deprenyl and clorgyline were used to allow the contributions of the A and B forms of monoamine oxidase to be determined separately. Comparison of the kinetic constants of the three enzymes towards dopamine indicated that, although each of them had activity towards this substrate, their relative contributions to the total oxidative deamination would depend on the substrate concentration. At all concentrations in the range 1 microM to 10 mM monoamine oxidase-B would contribute about 50% of the total activity. In the range 1 to 10 microM the contributions made by activities of monoamine oxidase-A and the semicarbazide-sensitive enzyme were similar but at higher concentrations the activity of the latter enzyme became more important, its contribution to the total activity rising to some 35% of the total at 500 microM dopamine. The activity of the semicarbazide-sensitive enzyme towards dopamine might thus be important under conditions where either or both the monoamine oxidases were inhibited in pharmacological studies. Its possible relevance to Norrie disease, in which both forms of the human enzyme are deficient, requires further examination.

Inhibition of bovine lung semicarbazide-sensitive amine oxidase (SSAO) by some hydrazine derivatives.

Microsomal semicarbazide-sensitive amine oxidase (SSAO) from bovine lung was shown to be inhibited by a number of hydrazine derivatives, but the mechanisms of inhibition were found to differ. Hydralazine behaved as an irreversible and partially time-dependent inhibitor with an IC50 value of 1 microM under the conditions used. Phenylhydrazine was found to be a potent irreversible inhibitor of SSAO (IC50 30 nM). Semicarbazide behaved as a specific irreversible inhibitor (active-site-directed irreversible inhibitor) in first forming a non-covalent enzyme-semicarbazide complex (with a Ki value of 85 microM), which then reacted to give an irreversibly inhibited enzyme species in a reaction defined by the first-order rate constant k2 = 0.065 min-1. Phenelzine behaved as a reversible inhibitor, but dialysis at 37 degrees C was found to be necessary to obtain full recovery of enzyme activity. The dependence of inhibition on phenelzine concentration was complex and consistent with multiple binding sites for this inhibitor. This diversity in the action of a family of compounds with the same functional group must be taken into account in attempts to design more specific inhibitors of this enzyme.

Semicarbazide-sensitive amine oxidase (SSAO) from human and bovine cerebrovascular tissues: biochemical and immunohistological characterization.

Semicarbazide-sensitive amine oxidase (SSAO) is widely distributed in almost all tissues, especially in vascularized ones. However, its presence in brain microvessels is still controversial. We have investigated the presence of SSAO in human and bovine brain microvessels by biochemical and immunohistological techniques, and we have compared it with SSAO present in meninges from the same species. SSAO metabolizes benzylamine and methylamine in all tissues tested and possibly dopamine and octopamine as well, as shown in competition studies. Kynuramine inhibited the metabolism of benzylamine by SSAO with high affinity in a non-competitive manner. Western-blot analysis rendered a positive staining of a 100 kDa band, in tissues from both species. These results were confirmed by immunohistological studies: the tunica media and intima of the meninges from both species were positively stained, and so was the endothelial layer of microvessels. SSAO was absent in brain parenchyma. These results definitively confirm the presence of SSAO in human and bovine cerebrovascular tissues and they demonstrate for the first time, the presence of this amine oxidase in endothelial cells from microvessels, through biochemical and immunological approaches.

Overexpression of semicarbazide sensitive amine oxidase in the cerebral blood vessels in patients with Alzheimer's disease and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy.

Semicarbazide sensitive amine oxidase (SSAO) metabolizes oxidative deamination of primary aromatic and aliphatic amines, and, in the brain, it is selectively expressed in blood vessels. SSAO expression is examined, by immunohistochemistry with a purified polyclonal antibody to SSAO from bovine lung, in the brains of subjects with Alzheimer disease (AD; n=10), cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL; n=2), and age-matched controls (n=8). SSAO immunoreactivity is restricted to meningeal and parenchymal blood vessels in control and diseased brains. Yet, a marked and selective increase in SSAO immunoreactivity occurs in association with betaA4 vascular amyloid deposits in patients with AD, and in the vicinity of the typical granular deposits in the blood vessels of gray and white matter in patients with CADASIL. Oxidative deamination of primary aromatic and aliphatic amines by SSAO produces ammonia, hydrogen peroxide and the corresponding aldehyde. Moreover, increased SSAO immunoreactivity is associated with increased Cu/Zn superoxide dismutase 1 expression restricted to abnormal blood vessels in diseased brains. Therefore, it is suggested that increased SSAO expression is a source of oxidative stress in the blood vessel wall in AD and CADASIL.

Amine oxidase activities in chemically-induced mammary cancer in the rat.

The activities and distributions of monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) were studied in solid breast tumours induced in rat by treatment with 7,12-dimethylbenz(alpha)anthracene (DMBA). It was observed that increasing degree of malignancy was associated with an increase of MAO-A activity an decrease of MAO-B and SSAO activities. The Km values did not change significantly with malignancy but Vmax values for MAO-A increased whereas Vmax for MAO-B and SSAO diminished with malignancy. It was detected in the more malignant tumours the presence of endogenous reversible inhibitor of SSAO activity not removed by dialysis.

Amine oxidase activities in bovine lung.

The subcellular distributions of amine oxidase activities MAO-A, MAO-B and SSAO, in bovine lung have been determined. MAO activities are largely confined to the mitochondria whereas SSAO activity is concentrated in the microsomal fraction. The contribution of different type of amine oxidase present in the microsomal fraction to the metabolism of the benzylamine, tyramine, phenylethylamine, tryptamine and dopamine were determined. The kinetic constants towards benzylamine showed SSAO to have a higher affinity than MAO-B, and the comparison of the Vmax/Km relationship for both enzymes indicated that benzylamine would be oxidised more efficiently by SSAO than by MAO-B.

Several aspects on the amine oxidation by semicarbazide-sensitive amine oxidase (SSAO) from bovine lung.

The lung has been shown to be potentially important in the metabolism of amines. Since SSAO has been demonstrated to be active towards some volatile short-chain aliphatic amines in other tissues, the current study determined the specificity and kinetic constants for the metabolism by bovine lung SSAO, of several aliphatic and aromatic amines some of which have been suggested to be physiological substrates (e.g. methylamine, aminoacetone and beta-phenylethylamine), and others (e.g. benzylamine) which are non-physiological. In the case of benzylamine, an inhibition at high substrate concentration was observed. Kinetic assays ruled out the possibility that this inhibition was caused by products of the deamination of benzylamine, and consequently it is suggested that these results may indicate the presence of two binding sites for the interaction of benzylamine with SSAO.

Studies on the time-dependent activation of microsomal semicarbazide-sensitive amine oxidase.

The semicarbazide-sensitive amine oxidase (SSAO) from bovine lung microsomes was activated in a temperature- and time-dependent process. This behaviour was observed when the enzyme was preincubated at 25 degrees C, 37 degrees C and 50 degrees C but not at 4 degrees C. This activation was only observed when benzylamine was used as substrate but not when methylamine, histamine or 2-phenylethylamine were used. The activation was independent of pH, ionic strength and the nature of the buffer used. At 37 degrees C the specific activity had risen to a value that was about 7 times higher than that of the starting material after 120 min. This process affected only the maximum velocity of the reaction with the Km value remaining essentially unchanged. Treatment of SSAO with phospholipases and detergents did not affect this behaviour. Incubation of the enzyme with serine proteases, metal chelating agents, reducing agents or protease inhibitors, had no effect on the activation. The fact that both forms of the enzyme (activated and non-activated), showed the same Mr values on gel filtration chromatography excluded the possibility of an enzyme aggregation and/or degradation being involved in this process.

Inhibition of monoamine oxidase from bovine retina by beta-carbolines.

The behaviour of some beta-carboline derivatives as inhibitors of monoamine oxidase has been studied in bovine retina. Inhibition was found not to show any significant time dependence. Di- and tetrahydro-beta-carbolines were shown to behave as reversible and competitive inhibitors. In contrast, the fully unsaturated beta-carbolines harmane, harmine and harmaline, which showed deviation from linearity at high substrate concentrations, behaved as tight-binding inhibitors. In these cases, the concentration of the enzyme and the inhibitor were of the same order. This was confirmed by the Ki values for these compounds in the nanomolar concentration range. Consistent with this was that inhibition was only partly reversed by dialysis for 18 h at 4 degrees C, although complete reversal was observed after dialysis for the same period at 37 degrees C. Structure-activity relationships indicated that substitution of a methoxy group at the C7 position of the aromatic ring is determinant for this tight-binding behaviour; a substitution of this group at the C6 position greatly reduced inhibition. Since beta-carbolines have been reported to be formed endogenously, this suggests that they might have important physiological actions on monoamine oxidase activity in-vivo. In contrast, all the beta-carbolines investigated in this study had low potencies as inhibitors of monoamine oxidase B.

Protective effect of N-(2-propynyl)-2-(5-benzyloxyindolyl) methylamine (PF 9601N), a novel MAO-B inhibitor, on dopamine-lesioned PC12 cultured cells.

Oxidative stress may play a role in the pathogenesis of Parkinson's disease. We have standardised a new model of dopaminergic-cell toxicity that uses dopamine, which is able to generate free radicals, as a toxin. The effect of this catecholamine on cell viability (MTT staining) was dose-dependent, reaching 65% of cell loss at a dopamine concentration of 300 microM. In this model, the protective effect of a novel MAO-B inhibitor, N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine (PF 9601N), was studied and compared with the effect of L-deprenyl assayed under the same experimental conditions. Whereas PF 9601N (50 microM and 100 microM) showed a significant protective effect, this was not the case with L-deprenyl. This different behaviour could be explained in terms of difference in antioxidant capacity. The toxicity induced in PC12 cells by 300 microM dopamine was partially reversed by incubating it in the presence of GBR-12909, a dopamine-transporter blocker. The results indicated that, besides the intracellular toxicity effect of dopamine, another non-specific extracellular mechanism could be involved.

[Expression and clinical significance of collagenase-3 (MMP-13) in gastric cancer]. / Expresión y significación clínica de la MMP-13 (colagenasa-3) en el cáncer gástrico.

BACKGROUND: MMP-13 (collagenase-3) is a metalloproteinase with potent degradative activity against a variety of elements of the extracellular matrix. Its expression has been described in some human carcinomas, where it seems to play a role in tumor progression and metastasis. The objective of this study was to investigate the expression and clinical significance of MMP-13 in gastric carcinomas. PATIENTS AND METHOD: MMP-13 expression was analyzed by immunohistochemistry in resected specimens from 44 patients with gastric adenocarcinoma. The mean ( standard error) follow-up period was 21.4 3.2 months. RESULTS: A total of 14 gastric carcinomas (31.8%) showed positive immunostaining for MMP-13. The percentage of MMP-13-positive tumors was significantly (p = 0.009) higher in stage IV carcinomas (69.2%) than in lower stages (I: 22.2%; II: 12.5%; and III: 14.3%), as well as in nonresectable tumors (R1 and R2) (61.5%) than in resectable carcinomas (R0) (19.4%) (p = 0.017). Likewise, MMP-13 tumor expression was significantly associated with shortened overall survival in both the entire group of patients (p = 0.0006) and in the subgroup of patients with resectable tumors (p = 0.018). CONCLUSIONS: Our results suggest that, in patients harboring gastric adenocarcinoma, MMP-13 tumor expression is associated with higher tumor aggressiveness and a poor prognosis.

PDR-1/hParkin negatively regulates the phagocytosis of apoptotic cell corpses in Caenorhabditis elegans.

Apoptotic cell death is an integral part of cell turnover in many tissues, and proper corpse clearance is vital to maintaining tissue homeostasis in all multicellular organisms. Even in tissues with high cellular turnover, apoptotic cells are rarely seen because of efficient clearance mechanisms in healthy individuals. In Caenorhabditis elegans, two parallel and partly redundant conserved pathways act in cell corpse engulfment. The pathway for cytoskeletal rearrangement requires the small GTPase CED-10 Rac1 acting for an efficient surround of the dead cell. The CED-10 Rac pathway is also required for the proper migration of the distal tip cells (DTCs) during the development of the C. elegans gonad. Parkin, the mammalian homolog of the C. elegans PDR-1, interacts with Rac1 in aged human brain and it is also implicated with actin dynamics and cytoskeletal rearrangements in Parkinsons's disease, suggesting that it might act on engulfment. Our genetic and biochemical studies indicate that PDR-1 inhibits apoptotic cell engulfment and DTC migration by ubiquitylating CED-10 for degradation.

Identification of dual mTORC1 and mTORC2 inhibitors in melanoma cells: prodigiosin vs. obatoclax.

The PI3K/AKT/mTOR signaling pathway regulates cell proliferation, survival and angiogenesis. The mammalian target of rapamycin (mTOR) is a protein kinase ubiquitously expressed within cells that regulates cell growth and survival by integrating nutrient and hormonal signals. mTOR exists in two complexes, mTORC1 and mTORC2. Hyperactivation of the mTOR protein has been linked to development of cancer, raising mTOR as an attractive target for cancer therapy. Prodigiosin (PG) and obatoclax (OBX), two members of the prodiginines family, are small molecules with anticancer properties which are currently under clinical trials. In the present paper, we demonstrate that mTOR is a molecular target of both prodiginines in melanoma, a highly drug-resistant cancer model. The inhibition of mTORC1 and mTORC2 complexes by PG or OBX resulted in a loss of AKT phosphorylation at S473, preventing its full activation, with no significant effect on T308. The strongest activity inhibition (89%) was induced by PG on mTORC2. Binding assays using Surface Plasmon Resonance (SPR) provide kinetic and affinity data of the interaction of these small molecules with mTOR. In addition, in silico modeling produced a detailed atomic description of the binding modes. These results provide new data to understand the mechanism of action of these molecules, and provide new structural data that will allow the development of more specific mTOR inhibitors for cancer treatment.

Akt2 interacts with Snail1 in the E-cadherin promoter.

Snail1 is a transcriptional factor essential for triggering epithelial-to-mesenchymal transition. Moreover, Snail1 promotes resistance to apoptosis, an effect associated to PTEN gene repression and Akt stimulation. In this article we demonstrate that Snail1 activates Akt at an additional level, as it directly binds to and activates this protein kinase. The interaction is observed in the nucleus and increases the intrinsic Akt activity. We determined that Akt2 is the isoform interacting with Snail1, an association that requires the pleckstrin homology domain in Akt2 and the C-terminal half in Snail1. Snail1 enhances the binding of Akt2 to the E-cadherin (CDH1) promoter and Akt2 interference prevents Snail1 repression of CDH1 gene. We also show that Snail1 binding increases Akt2 intrinsic activity on histone H3 and have identified Thr45 as a residue modified on this protein. Phosphorylation of Thr45 in histone H3 is sensitive to Snail1 and Akt2 cellular levels; moreover, Snail1 upregulates the binding of phosphoThr45 histone H3 to the CDH1 promoter. These results uncover an unexpected role of Akt2 in transcriptional control and point out to phosphorylation of Thr45 in histone H3 as a new epigenetic mark related to Snail1 and Akt2 action.