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.

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.

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.

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.

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.

[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.

Localization of monoamine oxidase A and B and semicarbazide-sensitive amine oxidase in human peripheral tissues.

Monoamine oxidase (MAO) A and B and semicarbazide-sensitive amine oxidase (SSAO) localizations in peripheral human tissues were compared by immunohistochemistry. The primary antibodies used were mouse monoclonal anti-human MAO-A (6G11/E1) and anti-human MAO-B (3F12/G10/2E3) and a rabbit polyclonal anti-bovine SSAO antibody. Immunoreactivities of the samples, obtained from 6 routine autopsy cases, showed different distributions in the tissues studied (heart, lung, duodenum, liver, pancreas, spleen, thyroid gland, adrenal gland and kidney). The relative MAO-A, MAO-B and SSAO distributions indicated a widespread distribution of these enzymes in the human body that is characterized by a matching cellular pattern in only few tissues. These differences suggest that each amine oxidase may play a specific function in, at least some, peripheral tissues.

Characterization of semicarbazide-sensitive amine oxidase in human subcutaneous adipocytes and search for novel functions.

Numerous studies have characterized semicarbazide-sensitive amine oxidase activity (SSAO) in rat fat cells but this oxidase is scarcely documented in human adipose tissue. Our aim was to further characterize SSAO in human adipose tissue (activity, mRNA and protein abundance) and to investigate whether SSAO activity can interplay with glucose and lipid metabolism in human adipocytes via the hydrogen peroxide it generates. Polyclonal antibodies directed against bovine lung SSAO allowed the detection of a substantial amount of immunoreactive protein (apparent molecular mass 100 kDa) in human subcutaneous adipocytes from either mammary or abdominal fat depots. A 4-kb mRNA was detected in fat depots using a cDNA probe designed from the placenta SSAO sequence. Almost all the oxidation of benzylamine found in adipose tissue homogenates was due to fat cells and was located in the adipocyte membrane fraction. The oxidation of benzylamine and methylamine were similar and totally inhibited by semicarbazide or hydralazine but resistant to pargyline. Histamine was poorly oxidized. Benzylamine and methylamine dose-dependently stimulated glucose transport in intact adipocytes. This insulin-like effect of amines did not increase in the presence of 0.1 mM vanadate but was inhibited by semicarbazide and antioxidants. Benzylamine and methylamine also exhibited antilipolytic effects, with complete inhibition of lipolysis at 1 mM. These results show that fat cells from non-obese subjects express a membrane-bound SSAO which readily oxidizes exogenous amines, generates hydrogen peroxide and exerts short-term insulin-like actions on glucose and lipid metabolism.

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 substrates mimic several of the insulin effects on adipocyte differentiation in 3T3 F442A cells.

We have previously reported that substrates of monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) exert short-term insulin-like effects in rat adipocytes, such as stimulation of glucose transport. In the present work, we studied whether these substrates could also mimic long-term actions of insulin. Adipose differentiation of 3T3 F442A cells, which is highly insulin-dependent, served as a model to test the effects of sustained administration of amine oxidase substrates. Daily treatment of confluent cells with 0.75 mM tyramine (a substrate of MAO and SSAO) or benzylamine (a substrate of SSAO) over 1 week caused the acquisition of typical adipocyte morphology. The stimulation of protein synthesis and triacylglycerol accumulation caused by tyramine or benzylamine reached one half of that promoted by insulin. This effect was insensitive to pargyline (an MAO inhibitor), but was inhibited by semicarbazide (an SSAO inhibitor) and by N-acetylcysteine (an antioxidant agent), suggesting the involvement of the H(2)O(2) generated during SSAO-dependent amine oxidation. Chronic administration of amine oxidase substrates also induced the emergence of adipose conversion markers, such as aP2, glycerol-3-phosphate dehydrogenase, the glucose transporter GLUT4, and SSAO itself. Moreover, cells treated with amines acquired the same insulin sensitivity regarding glucose transport as adipocytes classically differentiated with insulin. In all, most of the adipogenic effects of amines were additive to insulin. Our data reveal that amine oxidase substrates partially mimic the adipogenic effect of insulin in cultured preadipocytes. Furthermore, they suggest that SSAO not only represents a novel late marker of adipogenesis, but could also be directly involved in the triggering of terminal adipocyte differentiation.

Phosphorylation of the protein kinase mutated in Peutz-Jeghers cancer syndrome, LKB1/STK11, at Ser431 by p90(RSK) and cAMP-dependent protein kinase, but not its farnesylation at Cys(433), is essential for LKB1 to suppress cell vrowth.

Peutz-Jeghers syndrome is an inherited cancer syndrome that results in a greatly increased risk of developing tumors in those affected. The causative gene is a protein kinase termed LKB1, predicted to function as a tumor suppressor. The mechanism by which LKB1 is regulated in cells is not known. Here, we demonstrate that stimulation of Rat-2 or embryonic stem cells with activators of ERK1/2 or of cAMP-dependent protein kinase induced phosphorylation of endogenously expressed LKB1 at Ser(431). We present pharmacological and genetic evidence that p90(RSK) mediated this phosphorylation in response to agonists that activate ERK1/2 and that cAMP-dependent protein kinase mediated this phosphorylation in response to agonists that activate adenylate cyclase. Ser(431) of LKB1 lies adjacent to a putative prenylation motif, and we demonstrate that full-length LKB1 expressed in 293 cells was prenylated by addition of a farnesyl group to Cys(433). Our data suggest that phosphorylation of LKB1 at Ser(431) does not affect farnesylation and that farnesylation does not affect phosphorylation at Ser(431). Phosphorylation of LKB1 at Ser(431) did not alter the activity of LKB1 to phosphorylate itself or the tumor suppressor protein p53 or alter the amount of LKB1 associated with cell membranes. The reintroduction of wild-type LKB1 into a cancer cell line that lacks LKB1 suppressed growth, but mutants of LKB1 in which Ser(431) was mutated to Ala to prevent phosphorylation of LKB1 were ineffective in inhibiting growth. In contrast, a mutant of LKB1 that cannot be prenylated was still able to suppress the growth of cells.

Semicarbazide-sensitive amine oxidase substrates stimulate glucose transport and inhibit lipolysis in human adipocytes.

Semicarbazide-sensitive amine oxidases (SSAO) are widely distributed enzymes scavenging biogenic or exogenous amines and generating hydrogen peroxide. We asked whether human adipose tissue could express SSAO. Since hydrogen peroxide exhibits pharmacological insulin-like effects, we also tested whether its endogenous production by SSAO could mimic several insulin effects on adipocytes, such as stimulation of glucose uptake and inhibition of lipolysis. The benzylamine oxidation by human adipose tissue was inhibited by semicarbazide or hydralazine and resistant to pargyline or selegiline. It was due to an SSAO activity localized in adipocyte membranes. A protein of 100-kDa and a 4-kb mRNA corresponding to SSAO were identified in either mammary or abdominal subcutaneous fat depots. In isolated adipocytes, SSAO oxidized similarly benzylamine and methylamine that dose dependently stimulated glucose transport in a semicarbazide-sensitive manner. Antioxidants also inhibited the benzylamine and methylamine effects. Moreover, the ability of diverse substrates to be oxidized by adipocytes was correlated to their effect on glucose transport. Benzylamine and methylamine exerted antilipolytic effects with a maximum attained at 1 mM. These results show that human adipocytes express a membrane-bound SSAO that not only readily oxidizes exogenous amines and generates H(2)O(2), but that also interplays with glucose and lipid metabolism by exerting insulin-like actions. Based on these results and the fact that variations in plasma levels of the soluble form of SSAO have been previously reported in diabetes, we propose that determination of adipocyte SSAO, feasible on subcutaneous microbiopsies, could bring relevant information in pathologies such as obesity or diabetes.

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)

Time-dependent activation of the semicarbazide-sensitive amine oxidase (SSAO) from ox lung microsomes.

The activity of ox lung microsomal semicarbazide-sensitive amine oxidase (EC 1.4.3.6; SSAO) towards benzylamine increased 20-fold during incubation at 37 degrees C. After an initial lag-period, activation was first-order with time and complete after approx. 20 h. No significant changes in activity towards methylamine, histamine or 2-phenylethylamine were observed, although mixed-substrate experiments were consistent with the same enzyme being involved in the oxidation of all these substrates, both before and after time-dependent activation. The enzyme-tryptophan fluorescence increased on incubation at 37 degrees C in parallel with the increase in activity towards benzylamine. Treatment of the activated-enzyme preparation with 6 M guanidinium chloride followed by dialysis, caused both the activity towards benzylamine and the fluorescence to fall to that occurring before activation. However, incubation of this preparation at 37 degrees C resulted in increases in fluorescence and activity similar to those seen with the unactivated enzyme. Benzylamine oxidation was inhibited, uncompetitively with respect to oxygen, by high substrate concentrations but no such inhibition was observed with the other amines. Activation resulted in an increase in V(max) for benzylamine oxidation, with no significant alterations in the K(m) or the K(si) for high-substrate inhibition. Kinetic studies were consistent with sequential mechanisms being followed for the oxidation of both benzylamine and methylamine but the dependence on oxygen concentration was complex. These results might indicate that benzylamine follows a different reaction pathway from the other substrates, with substrate-specific activation involving a reaction step that is rate-limiting for benzylamine oxidation but not for the others.

A spectrophotometric method for determining the oxidative deamination of methylamine by the amine oxidases.

Previously published studies on the oxidative deamination of methylamine by the amine oxidases have determined the formation of radioactively labeled formaldehyde from [(14)C]methylamine. The present work describes a coupled spectrophotometric assay, using formaldehyde dehydrogenase, for the continuous determination of the oxidative deamination of methylamine by semicarbazide-sensitive amine oxidase (SSAO) and its potential use for determining methylamine concentrations in plasma. In this assay, the formaldehyde produced by methylamine deamination is further oxidized to formate, with the reduction of NAD(+), by formaldehyde dehydrogenase. The NADH generated is monitored continuously at 340 nm. Interference from the presence of a rotenone-insensitive NADH oxidase activity in crude tissue homogenates and microsomal fractions can be minimized by pretreating samples with Triton X-100 or substituting NAD(+) by APAD(+) in the coupled assay. This relatively inexpensive and reproducible assay procedure avoids the use of radioactively labeled material.

On the primary structure of membrane-bound semicarbazide-sensitive amine oxidase (SSAO).

Mammalian semicarbazide-sensitive amine oxidase (SSAO) activities are a diverse group of copper dependent enzymes within the classification EC 1.4.3.6. [amine:oxygen oxidoreductase (deaminating) (copper-containing)]. They include plasma amine oxidase and the membrane-bound enzyme. Although soluble plasma SSAO from several species have been successfully purified and cloned, relatively little work has been carried out on the molecular properties of the tissue-bound enzyme. At present there is not conclusive evidence that allows to conclude whether or not plasma SSAO is released from the tissue-bound enzyme. This review focuses upon recent progress made in determining the primary structure of the membrane-bound SSAO.

Regulation of BAD by cAMP-dependent protein kinase is mediated via phosphorylation of a novel site, Ser155.

The interaction of BAD (Bcl-2/Bcl-X(L)-antagonist, causing cell death) with Bcl-2/Bcl-X(L) is thought to neutralize the anti-apoptotic effects of the latter proteins, and may represent one of the mechanisms by which BAD promotes apoptosis. A variety of survival signals are reported to induce the phosphorylation of BAD at Ser(112) or Ser(136), triggering its dissociation from Bcl-2/Bcl-X(L). Ser(136) is thought to be phosphorylated by protein kinase B (PKB, also called Akt), which is activated when cells are exposed to agonists that stimulate phosphatidylinositol 3-kinase (PI3K). In contrast, Ser(112) is reported to be phosphorylated by mitogen-activated protein (MAP) kinase-activated protein kinase-1 (MAPKAP-K1, also called RSK) and by cAMP-dependent protein kinase (PKA). Here we identify Ser(155) as a third phosphorylation site on BAD. We find that Ser(155) is phosphorylated preferentially by PKA in vitro and is the only residue in BAD that becomes phosphorylated when cells are exposed to cAMP-elevating agents. The phosphorylation of BAD at Ser(155) prevents it from binding to Bcl-X(L) and promotes its interaction with 14-3-3 proteins. We also provide further evidence that MAPKAP-K1 mediates the phosphorylation of Ser(112) in response to agonists that activate the classical MAP kinase pathway. However insulin-like growth factor 1, a potent activator of PI3K and PKB does not increase the phosphorylation of Ser(136) in BAD-transfected HEK-293 cells, and nor is the basal level of Ser(136) phosphorylation suppressed by inhibitors of PI3K.

Experiencia de tutorías de alumnos con gran fracaso académico en su primer año en los estudios de medicina / Tutorship experience with medical students who had drastically failed the first year of their course

El presente artículo describe una experiencia de tutorías personalizadas llevadas a cabo en la Facultad de Medicina de la Universidad Autónoma de Barcelona con el objetivo de aumentar el éxito académico de los alumnos de primer curso que, durante el año anterior, habían tenido un gran fracaso académico. En la experiencia, llevada a cabo durante el curso 1997-1998, participaron de forma voluntaria 13 profesores y 19 alumnos. Los estudiantes fueron asignados de forma aleatoria a los diferentes tutores. Éstos debían orientar a los alumnos para optimizar sus aprendizajes y evitar un nuevo fracaso. La mayoría de los estudiantes participantes tuvieron un éxito total y sólo uno de ellos volvió a tener un gran fracaso. Así mismo, la mayoría de estudiantes en la misma situación que no habían participado en la experiencia volvieron a fracasar. A partir de los resultados obtenidos y de la satisfacción de los participantes, la experiencia fue valorada como muy positiva (AU)