Exploring the Biological Properties of Zn(II) Bisthiosemicarbazone Helicates.

The design of artificial helicoidal molecules derived from metal ions with biological properties is one of the objectives within metallosupramolecular chemistry. Herein, we report three zinc helicates derived from a family of bisthiosemicarbazone ligands with different terminal groups, Zn2(LMe)2∙2H2O 1, Zn2(LPh)2∙2H2O 2 and Zn2(LPhNO2)23, obtained by an electrochemical methodology. These helicates have been fully characterized by different techniques, including X-ray diffraction. Biological studies of the zinc(II) helicates such as toxicity assays with erythrocytes and interaction studies with proteins and oligonucleotides were performed, demonstrating in all cases low toxicity and an absence of covalent interaction with the proteins and oligonucleotides. The in vitro cytotoxicity of the helicates was tested against MCF-7 (human breast carcinoma), A2780 (human ovarian carcinoma cells), NCI-H460 (human lung carcinoma cells) and MRC-5 (normal human lung fibroblasts), comparing the IC50 values with cisplatin. We will try to demonstrate if the terminal substituent of the ligand precursor exerts any effect in toxicity or in the antitumor activity of the zinc helicates.

Could inhibition of metalloproteinases be used to block the process of metastasis?

Metastasis is a multisequential process that allows tumor cells to migrate to tissues distant from the primary tumor. Only a small number of cells escape from the primary tumor; however, the metastases generated are responsible for more than 90% of cancer deaths. Many metastatic processes initially require the total or partial start-up of a program for the transformation of tumor epithelial cells into mesenchymal cells (EMT). The launching of the EMT program is stimulated by cytokines and other elements produced by the diverse types of cells composing the tumor stroma. In parallel, a process of destabilization of the extracellular matrix (ECM) takes place by means of the synthesis of proteases of the matrix metalloproteinases (MMPs) family. EMC degradation allows the exportation of some tumor cells as mesenchymal cells to the circulatory system and their subsequent implantation in a tissue distant from the primary tumor. The blocking of these both processes appears as a hypothetical stop point in the metastatic mechanism. The present review deals with the different options to achieve the inhibition of MMPs, focusing on MMP7 as a target given its involvement in the metastatic processes of a wide variety of tumors.

Spectroscopic Characterization of Mitochondrial G-Quadruplexes.

Guanine quadruplexes (G4s) are highly polymorphic four-stranded structures formed within guanine-rich DNA and RNA sequences that play a crucial role in biological processes. The recent discovery of the first G4 structures within mitochondrial DNA has led to a small revolution in the field. In particular, the G-rich conserved sequence block II (CSB II) can form different types of G4s that are thought to play a crucial role in replication. In this study, we decipher the most relevant G4 structures that can be formed within CSB II: RNA G4 at the RNA transcript, DNA G4 within the non-transcribed strand and DNA:RNA hybrid between the RNA transcript and the non-transcribed strand. We show that the more abundant, but unexplored, G6AG7 (37%) and G6AG8 (35%) sequences in CSB II yield more stable G4s than the less profuse G5AG7 sequence. Moreover, the existence of a guanine located 1 bp upstream promotes G4 formation. In all cases, parallel G4s are formed, but their topology changes from a less ordered to a highly ordered G4 when adding small amounts of potassium or sodium cations. Circular dichroism was used due to discriminate different conformations and topologies of nucleic acids and was complemented with gel electrophoresis and fluorescence spectroscopy studies.

Inhibition of glutathione reductase uncovers the activation of NADPH-inhibited glucose-6-phosphate dehydrogenase.

Eggleston and Krebs pointed to a paradox in glucose-6-phosphate dehydrogenase (G6PD) regulating process that has not yet been solved, and which originated the term "fine regulation" of G6PD and, therefore, of oxidative phase of pentose phosphate pathway (OPPP). The paradox is that, in basal-like conditions, the activity of G6PD evaluated "in vitro" is very low or nearly null because of the potent inhibiting effect exerted by NADPH, a coenzyme whose concentration in the cell is much higher than that of the substrate NADP+ . However, "in vivo," flow through OPPP occurs in basal conditions. Eggleston and Krebs speculated on the possible existence of a system that would reverse the inhibition by NADPH. Such system would involve oxidized glutathione and exclude the participation of glutathione reductase (GR). The present work confirms the experimental results obtained by Eggleston and Krebs and proves that oxidized glutathione (GSSG) in the absence of NADPH is a direct inhibitor of G6PD. In the presence of GSSG, the G6PD activity recovery system suggested can be observed when GR is previously inhibited by alkylating agents. An unknown element with a molecular weight ranging between 12 and 50 kDa has been found to reverse part of G6PD inhibition by NADPH.

Structural, morphometric and immunohistochemical study of the rabbit accessory olfactory bulb.

The accessory olfactory bulb (AOB) is the first neural integrative centre of the vomeronasal system (VNS), which is associated primarily with the detection of semiochemicals. Although the rabbit is used as a model for the study of chemocommunication, these studies are hampered by the lack of knowledge regarding the topography, lamination, and neurochemical properties of the rabbit AOB. To fill this gap, we have employed histological stainings: lectin labelling with Ulex europaeus (UEA-I), Bandeiraea simplicifolia (BSI-B4), and Lycopersicon esculentum (LEA) agglutinins, and a range of immunohistochemical markers. Anti-G proteins Gαi2/Gαo, not previously studied in the rabbit AOB, are expressed following an antero-posterior zonal pattern. This places Lagomorpha among the small groups of mammals that conserve a double-path vomeronasal reception. Antibodies against olfactory marker protein (OMP), growth-associated protein-43 (GAP-43), glutaminase (GLS), microtubule-associated protein-2 (MAP-2), glial fibrillary-acidic protein (GFAP), calbindin (CB), and calretinin (CR) characterise the strata and the principal components of the BOA, demonstrating several singular features of the rabbit AOB. This diversity is accentuated by the presence of a unique organisation: four neuronal clusters in the accessory bulbar white matter, two of them not previously characterised in any species (the γ and δ groups). Our morphometric study of the AOB has found significant differences between sexes in the numerical density of principal cells, with larger values in females, a pattern completely opposite to that found in rats. In summary, the rabbit possesses a highly developed AOB, with many specific features that highlight the significant role played by chemocommunication among this species.

Sensitivity of the kinase activity of human vaccinia-related kinase proteins to toxic metals.

The human vaccinia-related kinase (VRK) proteins VRK1 and VRK2 regulate different processes, such as the cell cycle, DNA damage response, and signaling by mitogen-activated protein kinases in response to growth factors or cellular stress. Alterations in expression levels of these Ser-Thr kinases are associated with cancer and neurodegenerative diseases. These functions suggest that they might also be targets of toxic metals, and thus contribute to the pathogenic effects associated with metal intoxication. VRK1 is inhibited by cadmium, copper, and mercury, and VRK2 is more sensitive to cadmium and much less sensitive to copper and mercury. Both kinases are insensitive to lead and cobalt. VRK1 is in general more sensitive than VRK2 in the low micromolar range. This inhibitory effect induced by these metals was detected in an autophosphorylation assay, as well as in phosphorylation assays using p53 and histone H3 as substrates. The accumulation of these three metals in cells can contribute, by inhibition of VRKs, to their toxic pathogenic effects, particularly their neurological manifestations. In this context copper has not generally been associated with any intoxication syndrome, except Wilson's syndrome, but it might be implicated in some alterations with which it has not yet been associated.

Differential inhibitor sensitivity between human kinases VRK1 and VRK2.

Human vaccinia-related kinases (VRK1 and VRK2) are atypical active Ser-Thr kinases implicated in control of cell cycle entry, apoptosis and autophagy, and affect signalling by mitogen activated protein kinases (MAPK). The specific structural differences in VRK catalytic sites make them suitable candidates for development of specific inhibitors. In this work we have determined the sensitivity of VRK1 and VRK2 to kinase inhibitors, currently used in biological assays or in preclinical studies, in order to discriminate between the two proteins as well as with respect to the vaccinia virus B1R kinase. Both VRK proteins and vaccinia B1R are poorly inhibited by inhibitors of different types targeting Src, MEK1, B-Raf, JNK, p38, CK1, ATM, CHK1/2 and DNA-PK, and most of them have no effect even at 100 µM. Despite their low sensitivity, some of these inhibitors in the low micromolar range are able to discriminate between VRK1, VRK2 and B1R. VRK1 is more sensitive to staurosporine, RO-31-8220 and TDZD8. VRK2 is more sensitive to roscovitine, RO 31-8220, Cdk1 inhibitor, AZD7762, and IC261. Vaccinia virus B1R is more sensitive to staurosporine, KU55933, and RO 31-8220, but not to IC261. Thus, the three kinases present a different pattern of sensitivity to kinase inhibitors. This differential response to known inhibitors can provide a structural framework for VRK1 or VRK2 specific inhibitors with low or no cross-inhibition. The development of highly specific VRK1 inhibitors might be of potential clinical use in those cancers where these kinases identify a clinical subtype with a poorer prognosis, as is the case of VRK1 in breast cancer.

After the Prestige oil spill modifications in NO production and other parameters related to the immune response were detected in hemocytes of Mytilus galloprovincialis.

In marine mollusks, many physiologic functions are regulated seasonally depending on such factors as the reproductive cycle or the presence of food. The synthesis of nitric oxide by hemocytes of Mytilus galloprovincialis is among the multiple physiologic actions in the immune response, and it is also affected by season. The maximal basal production of NO by hemocytes of M. galloprovincialis was detected in summer, whereas the minimum values were detected in winter. In winter, the presence of IL-2 induced an increase in NO production that was not detected in summer. Three months after the Prestige oil spill (November 2002), basal NO production by the hemocytes of mussels in the Galician coast showed a progressive decrease and stopping, both in summer and in winter. The characteristic increase of NO synthesis induced by IL-2 in winter also disappeared all through 2003 and 2004. The two different nitric oxide synthases previously identified by immunoblotting between 1999 and 2002 were undetectable in both 2003 and 2004. When comparing the data obtained during 2003 and 2004 to those obtained in previous years, an increase in the proportion of SH cells was detected. Also, these cells showed a higher sensitivity to apoptosis- and necrosis-inducing agents than in earlier years.

Implication of PKC in the seasonal variation of the immune response of the hemocytes of Mytilus galloprovincialis Lmk. and its role in interleukin-2-induced nitric oxide synthesis.

The hemocytes are the cells responsible for the immune response in marine mollusks. The role of NO in processes related to the activation of the hemocytes has turned out evident over the late years. In the case of the mussel Mytilus galloprovincialis Lmk., hemocyte NO basal production varies throughout the year, showing a maximum in summer and a minimum in winter. IL-2 reverts the low winter NO basal production through a process mediated by cAMP-dependent protein kinase and by an apparent side effect of protein kinase C. The seasonal variation of NO production in the presence of the PKC inhibitor bisindolylmaleimide (BSM) allows suggesting a model in which PKC would modulate the activity of the enzymes responsible for nitric oxide production.

In hemocytes from Mytilus galloprovincialis Lmk., treatment with corticotropin or growth factors conditions catecholamine release.

The cells in charge of the innate immune response in the sea mussel Mytilus galloprovincialis Lmk. are the hemocytes, which have the capacity to release catecholamines when subjected to stressing conditions. Hemocytes were kept in culture before stimulation. That is, their behaviour was not studied immediately after extraction from the mollusc, as happens in most studies. This avoids the interference and variability caused by the conditions in which mussels may be when collected. This work describes the great variability found in the pattern of catecholamine release when the hemocytes are stimulated with either corticotropins or growth factors. Dopamine, adrenaline and noradrenaline release differs with each of the inducers assayed, with stimulation time and with the season of hemocyte collection. One of the results presented is particularly remarkable; such is the great amount of adrenaline and noradrenaline released to the medium when the hemocytes obtained in summer are stimulated with transforming growth factor-beta1 (TGF-beta1) for 60 min.

Nitric oxide production by haemocytes from Mytilus galloprovincialis shows seasonal variations.

Nitric oxide (NO) has been identified as an important physiological modulator, with evidence of its role as a signalling molecule throughout the whole phylogenetic scale. In marine molluscs, it intervenes in processes related to the immune function of haemocytes. The presented results indicate that basal NO production by haemocytes of Mytilus galloprovincialis shows seasonal variations, with summer values statistically higher than those of winter. The presence of IL-2 increased NO production in winter. In summer, incubating the haemocytes with TNF-alpha for 6h slightly increased NO production. LPS, TGF-beta1 or PDGF did not induce significant effects on NO production by the haemocytes. Immunoblotting experiments detected two proteins that bind to vertebrate iNOS and eNOS antibodies, with different seasonal expression: the protein that binds to anti-iNOS antibody was expressed throughout the year, whereas the anti-eNOS antibody bound with a protein that was only detected in winter. IL-2 is suggested to start a signalling system dependent on the seasonal presence of winter protein. Such a system would activate the enzyme, thus favouring the higher NO production detected in winter.

The vaccinia virus B1R kinase induces p53 downregulation by an Mdm2-dependent mechanism.

Poxvirus infection has a strong effect on cellular functions. To understand viral pathogenesis, it is necessary to know how viral proteins interact with host proteins. The B1R kinase is an early viral gene required for vaccinia virus DNA synthesis and replication, but no cellular substrate is known for this viral kinase. B1R is able to hyperphosphorylate p53 in several residues in the N-terminal transactivation domain, including Ser15 and Thr18. B1R does not phosphorylate Mdm2. B1R promotes an increase in p53 ubiquitination and a reduction of p53 acetylation by p300. The over-expressed B1R protein induces the degradation of p53 in a concentration-dependent manner and is lost when Ser15 and Th18 are changed to alanine or when the B1R kinase is inactivated by introducing the K149Q substitution. The B1R-induced downregulation of p53 requires Mdm2. The hyperphosphorylated p53 is transcriptionally active, and this activity also falls as B1R increases. The BAX gene promoter is more sensitive to this reduction of transcription than p21 or 14-3-3 gene promoters. This effect of B1R on p53 can be one of the mechanisms by which vaccinia virus exerts its role in infected cells.

c-Jun phosphorylation by the human vaccinia-related kinase 1 (VRK1) and its cooperation with the N-terminal kinase of c-Jun (JNK).

The VRK1 kinase is a novel Ser-Thr kinase in the human kinome that diverged from the casein kinase 1 branch. These kinases phosphorylate transcription factors related to stress responses, such as p53. In this report we have studied the phosphorylation of the transcription factor c-Jun in its N-terminal region. The VRK1 protein phosphorylates c-Jun with a Km of 0.4 muM, and is not inhibited by SP600125. VRK1 phosphorylates c-Jun in Ser63 and Ser73 in vitro, the same residues targeted by the N-terminal kinase of c-Jun (JNK). This phosphorylation induces the stabilization and accumulation of the c-Jun protein. VRK1 phosphorylates the endogenous c-Jun in Ser63. VRK1 activates c-Jun dependent transcription, which is dependent on phosphorylation of Ser63 and Ser73. The c-Jun with Ser63Ala and Ser73Ala substitutions is not transcriptionally active when cotransfected with VRK1. VRK1 interacts with c-Jun but not with JNK. The cotransfection of VRK1 and JNK has an additive effect on the transcriptional activation of c-Jun indicating that they can cooperate when both are at suboptimal dose; otherwise, maximum effect by one of them prevents the effect of the other. The VRK1-c-Jun connection represents a component of a new signaling pathway whose upstream elements remain to be identified.

Implication of PKA and PKC in the activation of the haemocytes of Mytilus galloprovincialis Lmk by LPS and IL-2.

LPS and IL-2 play an essential role in the generation of the immune response in diverse eukaryotic species, as they provoke the activation of several pathways of signal transduction in macrophages. Among the kinases related to these pathways, PKA and the PKCs are some of the most important. In the haemolymph of the marine mussel Mytilus galloprovincialis Lmk, the cells responsible for the defence reactions are the haemocytes. These act as active phagocytes, and are able also to secrete humoral factors. The effect of the stimulation of the haemocytes with either LPS or IL-2 on the expression of both a Ca(2+)-independent PKC (p105) and a regulatory subunit (RII) of PKA found in mussel tissues are studied in this work. Also, the effect of inhibitors specific for these kinases on their expression and on their release of catecholamines is reported.

In vitro effects of LPS, IL-2, PDGF and CRF on haemocytes of Mytilus galloprovincialis Lmk.

The cells in charge of the innate immune response in the marine mussel Mytilus galloprovincialis Lmk. are the haemocytes. These cells respond in different ways to agents such as lipopolysaccharide (LPS), interleukin-2 (IL-2), platelet-derived growth factor (PDGF) and corticotropin releasing factor (CRF). After stimulation of the haemocytes, the expression of molecules reactive with monoclonal antibodies raised to the alpha chain of the IL-2 receptor, present in their membrane, differed depending on the agent used. The same happened with regard to the levels of dopamine, adrenaline and noradrenaline released to the medium by the haemocytes. It should also be noted that no catecholamine release was detected and the level of expression of IL-2Ralpha showed no significant variation in cultured cells that had not been treated with inducers. These facts would indicate that most haemocytes were in the same starting condition at the moment that the stimulation was performed. Therefore, cultured haemocytes can be a highly reliable model in the study of the innate immune system.

Nitric oxide release by hemocytes of the mussel Mytilus galloprovincialis Lmk was provoked by interleukin-2 but not by lipopolysaccharide.

As other marine and land mollusks, mussels have special cells in charge of the immune function called hemocytes. The activation of these cells leads to a series of events that end up in phagocytosis and in secretion of digestive enzymes that eliminate the pathogen. The production of nitric oxide is among the early activation processes. Contrary to what happens in cells of vertebrates and of other species of mollusks, in hemocytes of Mytilus galloprovincialis, LPS did not induce secretion of NO to the medium. However, human IL-2 provoked an important increase in NO production. The maximal synthesis of NO was detected after the hemocytes were incubated with the cytokine for 24h. In both stimulated and non-stimulated cells, Western blotting showed the presence of a protein of 130kDa, recognized by anti-mouse iNOS. Therefore, the higher production of NO can only be explained as a direct action of some effector upon the nitric oxide synthetase. NO production decreased by the action of H-89, a powerful inhibitor of the cAMP-dependent protein kinase (PKA). This suggests the involvement of PKA in the pathway of NO synthesis.

Phorbol esters induce translocation of the nPKC p105 to membrane in mussel hemocytes.

Previous works revealed the presence of the nPKC enzyme p105 in hemocytes of M. galloprovincialis Lmk. Specific mussel antibodies were obtained from mouse and used in confocal microscopy and Western blotting. These techniques allowed the observation of p105 cytosol-to-membrane translocation induced by TPA for the first time in hemocytes of molluscs. The incubation of mussel immune cells with TPA for longer than 30 min also triggered a down-regulation process. Mussel hemocytes are an excellent model to study the molecular processes of innate immunity.

Purification of a lipid-activated and Ca2+-independent protein kinase from the mantle tissue of Mytilus galloprovincialis Lmk.

A phospholipid-sensitive Ca2+-independent protein kinase (p105) was purified to homogeneity from mantle tissue of the mussel Mytilus galloprovincialis Lmk., employing consecutively DE-52 cellulose, Sephacryl S-200 and Biogel HTP chromatographies. The purified enzyme appeared as a single band on 10% SDS-PAGE, and had a molecular weight of 105 kDa. The positive Western blotting of the purified eluate for anti-human-PKCdelta and PKCepsilon suggests that the enzyme from mussel mantle may be an ancestral nPKC isoform, with the kinetic properties of the enzyme very close to those of PKCepsilon isoform of vertebrates. Western blotting of samples from different steps of purification using specific mouse anti-p105, showed two protein bands in samples from the initial steps. However, only one band was detected in the Biogel-HTP eluate, the most purified fraction. The purification steps did not affect the presence of P-serine in p105. No P-tyrosine peptides were detected in any of the purification steps. These results open a new field of work on the study of several molecular processes related to energetic metabolism and reproduction in molluscs, whose regulation is associated with the activation of protein kinases.

Kinetic properties of p53 phosphorylation by the human vaccinia-related kinase 1.

The vaccinia-related kinase 1 (VRK1) protein is a nuclear Ser-Thr kinase that phosphorylates p53 in Thr18. We have determined the enzyme properties regarding its different substrates. VRK1 has a high affinity for ATP (K(m) 50 microM) and is thus saturated by the intracellular concentration of ATP in vivo. VRK1 uses preferentially magnesium, but is also functional with manganese and zinc. The VRK1 protein is autophosphorylated in multiple residues without effect on its activity. One autophosphorylated residue, T355, is within the VRK1 regulatory carboxy terminus. The kinase phosphorylates p53 with a K(m) of 1 microM and is well suited to respond to the variations of intracellular p53 concentration, which fluctuates as a response to different types of cellular stress.

Transient activation of the c-Jun N-terminal kinase (JNK) activity by ligation of the tetraspan CD53 antigen in different cell types.

The CD53 antigen is a member of the tetraspanin membrane protein family that is expressed in the lymphoid-myeloid lineage. We have studied the implication of CD53 antigen in signal transduction by determining the effect of its ligation on the c-Jun N-terminal kinase (JNK) in different cell types. Ligation of the rat or human CD53 antigen induces a three- to fourfold transient activation of JNK activity that peaks at 3-5 min. The effect was detected by assaying the endogenous or exogenous (transfected) JNK activity. The JNK response was detected in IR938F cells, a rat B-cell lymphoma, and in Jurkat cells derived from a human T-cell lymphoma. This JNK activation was not mediated by the vav oncogene, and CD53 does not cooperate with CD3 for vav activation. A similar JNK activation was also detected in a human renal carcinoma cell line that was transiently transfected with the human CD53 cDNA to mimic the CD53 ectopic expression in carcinomas. In stable CD53-transfected cells it stimulated Jun-dependent transcriptional activity. We conclude that parts of the cell responses modulated by the CD53 are mediated by JNK activation, and this activation is independent of the different protein interactions that the CD53 protein has on specific cell types.