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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., (© 2022 The Authors. Cell Biochemistry and Function published by John Wiley & Sons Ltd.)

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., (© 2021 International Union of Biochemistry and Molecular Biology, Inc.)

There is a paradox in the oxidizing phase of the phosphate pentose pathway that has not yet been solved. The flow through the pathway is reduced in basal conditions; however, it must rise notably when a NADPH supplement is required. The paradox consists of the strong inhibition that the NADPH exerts on the both dehydrogenases of the pathway, especially on the regulating enzyme glucose-6-phosphate dehydrogenase (G6PD). Theoretically, in anabolic situations, the increase of gene expression of G6PD and 6-phosphogluconate dehydrogenase can induce a rise in the production of NADPH, which would cause the immediate inhibition of the enzyme and a drastic flow reduction. However, increasing the flow through oxidative phase of the pentose phosphate pathway (OPPP) has been experimentally demonstrated in many physiological states. However, this situation will be resolved if the NADPH metabolized or otherwise sufficient NADPH is sequestered to relax the inhibition of the dehydrogenases of OPPP and to maintain high ratio of NADPH/NADP(+) needed to ensure the reducing environment of the cell cytoplasm and the contribution of NADPH for anabolic processes. In 1974, the presence of a protein capable of reversing the inhibition of G6PD by NADPH was detected; however, to date, this paradox remains undisclosed. This review deals with the possibility that such reverting action might be similar to the activity of a protein named HSCARG, which is responsible for the abduction of NADPH, thus keeping a portion of the coenzyme away from the catalytic action and, simultaneously, the immune response through the NF-κB (nuclear factor kappa light-chain enhancer of activated B cells) system. The model has many similarities with the hypothesis proposed some 40 years back on the reversion of G6PD inhibition by NADPH., (© 2014 International Union of Biochemistry and Molecular Biology.)

Previous works revealed the presence of a Ca(2+)-dependent protein kinase (p60) and a Ca(2+)-independent protein kinase (p105) in the mantle tissue from the sea mussel Mytilus galloprovincialis Lmk. The expression of both isoforms shows a balance between cytosolic and membrane fractions in mantle, gills, and hepatopancreas, whereas, in hemocytes, their expression is mainly cytosolic, as happens in muscle tissues with p60 alone. Both enzymatic forms contain phosphorylated serines, and no phosphorylation was detected in tyrosines. Only the form p105 mediates the PMA-induced activation of the hemocytes of M. galloprovincialis, and it does so by a process of down-regulation. The form p60 does not respond to the presence of the phorbol ester, suggesting structural differences related to the binding sites of the diacylglycerol.

The innate immune system of marine mussels (Mytilus galloprovincialis) is operated by phagocytic cells termed hemocytes. Lipopolysaccharide (LPS), interleukin-2 (IL-2), or platelet-derived growth factor (PDGF) increase biogenic amine synthesis in these cells, and the enzymes Ca(2+)-independent protein kinase C (PKC) (p105/108) and Ca(2+)-dependent PKC (p60) are involved in these processes. Stimulation by PDGF induces a down-regulation process affecting the form p108 of the Ca(2+)-independent PKC. In addition, PDGF produces the increase of expression of p60 in the membrane fraction. IL-2 induces the disappearance of p108 from the membrane but does not affect the presence of p60 in cytosol or membrane. For its part, LPS activates exclusively p60 by a down-regulation mechanism. The ensemble of results suggests that each agonist starts a pathway that implicates the PKC isoenzymes that mediate the regulation of the activities dopa decarboxylase, dopamine beta-hydroxilase, and phenyletanolamine N-methyltranferase, which lead to different actions related to biogenic amine synthesis.

An enzyme that can be included into the so-called conventional PKCs has been purified to homogeneity from the mantle tissue of the sea mussel Mytilus galloprovincialis. This enzyme has a molecular weight of 60 kDa, which is DAG-dependent, PS-activated, and Ca2+-dependent. It was separated from a Ca2+-independent PKC (p105) (Mercado et al., Mol Cell Biochem 233:99-105, 2002) by means of an ionic exchange chromatography on DE-52 cellulose. The molecular weights and kinetic properties of both the enzymes are different. The protein p60 is broadly distributed among the tissues, which suggests that it may carry out specific functions, different from those performed by p105.

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.

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.

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.

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 exposure of organisms to stressing agents may affect the level and pattern of protein expression. Certain proteins with an important role in protein homeostasis and in the tolerance to stress, known as stress proteins, are especially affected. Different tissues and cells show a range of sensitivities to stress, depending on the habitat to which organisms have adapted. The response of different tissues and cells from the mussel Mytilus galloprovincialis Lmk. to heat shock has been studied in this work using different exposure times and temperatures. During the assays, protein expression was observed to vary depending on the tissue studied, the temperature or the exposure time used. But maybe the most prominent thing is the different response obtained from the cultured haemocytes and those freshly obtained from stressed mussels, which makes us think that the extraction procedure is the main cause of the response of non-cultured cells, although the haemolymph may contain components that modulate haemocyte response.

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.

The change in the content of cyclic GMP, cyclic AMP, ATP, ADP, AMP and fructose-2,6-bisphosphate that occurred in the mantle of the mussel Mytilus galloprovincialis Lmk when specimens of this mollusk were subjected to a hypoxia/anoxia situation were assessed. After the early 24 h in anaerobiosis, a clear decrease was observed in the ATP content, which remained close to that value for the rest of the time. AMP content doubled during the early 24 h in anaerobiosis and, from that time on, it remained close to that value. Fructose-2,6-bisphoshate and cyclic GMP showed a similar behavior. The levels of these compounds rose significantly during the early hours in anaerobiosis, and then fell to values similar to those of aerobiosis, remaining constant for the rest of the time. Neither ADP nor cAMP showed significant variations.

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.

Two fast and sensitive methods for the determination of cAMP and cGMP levels in mantle tissue of the sea mussel Mytilus galloprovincialis Lmk. are described. Both methods use ion-pair high-performance liquid chromatography with diode array detection. The use of the diode array detector permitted the simultaneous detection of the absorbance at two different wavelengths and the obtaining of the UV absorption spectrum for each detected peak, confirming peak purity and identity. Method precision was good. The detection limit for both nucleotides was 2.5 pmol (signal-to-noise ratio = 4 at 254 nm). Previous to the injection onto the chromatograph, both nucleotides were purified by precipitation of the adenine and guanine 5'-ribonucleotides with ZnSO(4)-Na(2)CO(3). The supernatant obtained was subsequently passed over an anion-exchange column (AG l-X8 formate form resin). Early results seem to indicate that there is a seasonal variation in the contents of both cyclic nucleotides in mantle tissue. Such variation is probably related to the annual gametogenic cycle., (Copyright 2000 Academic Press.)

Cytosolic extracts from the posterior adductor muscle of the bivalve mollusk Mytilus galloprovincialis contain significant amounts of both cGMP-binding and cGMP-stimulated protein kinase activities. However, photoaffinity labeling with 8-azido-[32P]cGMP revealed only a major cGMP-binding protein with an apparent molecular mass of 54 kDa (p54), lacking protein kinase activity itself. Instead, the purified and cGMP-free p54 protein has the ability to inhibit a mussel protein kinase homologous to the mammalian cAMP-dependent protein kinase (cAPK) catalytic subunit, the inhibition being relieved by cAMP or cGMP, which suggests that it can act as a regulatory subunit of cAPK. However, p54 failed to be recognized by a specific antibody against the regulatory subunit (type RII) previously isolated from mussel. Therefore, p54 must be a novel isoform of cAPK regulatory subunit that seems to have high affinity for both cGMP and cAMP., (Copyright 1998 Academic Press.)

Electrophoresis on agarose/formaldehyde gels of rRNA in molluscs display a pattern of bands which could suggest a RNase action due to incorrect manipulation of the samples. This study shows that the disappearance of the band corresponding to the 28S fraction is due to the denaturing conditions used when electrophoresis is carried out and not to RNases action.

Several proteins with M(r) > 70 kDa from various tissues of the sea mussel Mytilus galloprovincialis were specifically recognized in vitro by the regulatory subunit (type RII alpha) of cAMP-dependent protein kinase (cAPK) from porcine heart. However, none of these proteins interacted with the regulatory subunit of cAPK from the mollusc itself. The results suggest that, unlike mammalian RII, regulatory subunit from mussel lacks the specific residues responsible for interaction with R-binding proteins. Consequently, the identified molluscan RII alpha-binding proteins should play a distinct role from cAPK anchoring.

The glucose-6-phosphate dehydrogenase from mouse liver is fully inhibited in vitro by physiological concentrations of NADPH. This suggests that the oxidative phase of the pentose phosphate pathway requires some deinhibitory system. In order to investigate regulation of the pentose phosphate pathway, various parameters (intermediate concentrations, mass-action ratios of reactions, etc.) were measured in liver from control mice and from meal-fed mice. Assays were also carried out to detect any molecules causing the reverse of glucose-6-phosphate dehydrogenase inhibition by NADPH. The liver of meal-fed mice show greater glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities. They also had greater concentrations of several metabolic intermediates and triglycerides than the control animals (P < 0.001). These results prove that the diet increases the flow of the pentose phosphate pathway in a lipogenic sense. The glutathione reductase does not change with the diet, suggesting that this enzyme does not participate in the modulating process. Unlike rat liver, no molecules causing the reverse of glucose-6-phosphate dehydrogenase inhibition by NADPH were detected. These data suggest that the increase of flow of the pentose phosphate pathway during lipogenesis is obtained by an increase in enzyme synthesis.

Phosphofructokinase purified from mantle tissue of the sea mussel Mytilus galloprovincialis, was phosphorylated "in vitro" by the catalytic subunit of cyclic AMP-dependent protein kinase. The incorporation of phosphate gave rise to an activation of the enzyme by increasing its affinity for fructose-6-phosphate, by decreasing its sensitivity to the inhibition by ATP and by enhancing the effect of allosteric activators (5'-AMP and fructose-2,6-bisphosphate). In addition, the effects of phosphorylation on the catalytic activity are pH-dependent.

PKF-2 from mussel mantle was phosphorylated by cAMP-dependent protein kinase. The phosphorylation does not change the enzyme activity at neutral pH values, but at acid pH the activity of the phosphorylated form is higher than the native PFK-2. With respect to the native enzyme, the activation consisted of a reduction in the Km for Fru-6-P and a decrease in the inhibitory effect of PEP. These results are in keeping with the stabilized concentration of Fru-2,6-P2 found in the mussel mantle during the physiological hypoxia caused by the closure of the valves.

Fructose-2,6-bisphosphatase (FBPase-2) from the mantle tissue of the mussel Mytilus galloprovincialis shows a hyperbolic kinetic with a Km value (0.40 mM) for its substrate, that suggest that the "in vivo" Fru-2,6-P2 concentration is not a limiting factor for activity. The enzyme possesses an optimum pH for activity between 6 and 7 units, similar to the reached in mussel mantle during physiological hypoxia. The modulation of activity by the pH, and in addition, the positive effect of ATP are in keeping with the little decrease in concentration of the Fru-2,6-P2 that occurs during the first hour of hypoxia due to the valve closure.

In the absence of AMP and Fru-2,6-P2, several amino-acids such as histidine, lysine, alanine, aspartic acid, and other molecules, as reduced glutathione or citrate, activate FBPase-1 from Mytilus galloprovincialis mantle. AMP decreases Vmax and Km for Fru-1,6-P2 both in the absence and in the presence of activators; but the addition of Fru-2,6-P2 decreases the affinity of the enzyme by its substrate. Na+ acts as a inhibitor reducing both Vmax and Km. The Km value is lower than the physiological level of Fru-1,6-P2, suggesting that the enzyme is operative but its activity is very reduced.

Concentrations of glycolytic intermediates and adenine nucleotides have been estimated in adductor muscle and hepatopancreas from the sea mussel Mytilus galloprovincialis Lmk. after various periods of valve closure. Mass action ratios of enzyme steps involved in the metabolism of these components are compared with their equilibrium constants. This reveals hexokinase, phosphofructokinase, pyruvate kinase and fructose-1,6-bisphosphatase catalyze non-equilibrium reactions. The changes in the concentrations of the glycolytic intermediates and in the rate M.A.R./Keq during hypoxia suggest that the carbon flow after valve closure is first controlled by phophofructokinase, but later on the rate of transformation of phosphoenolyruvate regulates this flow.

A strong inhibitor of G6PDH has been detected in rat liver homogenates. The inhibitor, isolated by ultrafiltration methods, proved to be very stable under incubation with trypsin and high temperatures. Gel-filtration through Sephadex G-75 showed it to have a molecular weight of 3,500 daltons, though perchloric acid treatment produced a light form of 900 daltons. Both forms of inhibitor act as competitive inhibitor with respect to G6P and exhibit non-competitive inhibition pattern with respect to NADP+. Physical and kinetic properties, and the increase of G6PDH activity at low NADP+ concentrations, in the presence of NADPH and inhibitor or palmitoyl-CoA, in relation to the G6PDH activity in presence of NADPH, lead to the identification of the low-molecular weight inhibitor as palmitoyl-CoA.