Juan Ignacio Ramos-Martínez

• PhD
• Head of Department at University of Santiago de Compostela

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...

Eggleston and Krebs pointed to a paradox in G6PD regulating process that has not yet been solved, and which originated the term “fine regulation” of G6PD and, therefore, of 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 NADP...

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, especi...

Experimental evidence shows that the hemocytes of Mytilus galloprovincialis Lamark have a multivalent receptor that responds to diverse agonists provoking the activation of the immune response. The same such agonists have the property of modulating the operability of the endocrine system. The relationship between the neuroendocrine and immune syste...

In molluscs, hemocytes perform the molecular mechanisms related to immunity. These cells have the ability to respond to the different varieties of stress by modulating their responses. The stressors may be bacterial toxins, cytokines or growth factors, and even physical agents such as changes in temperature or oxygen partial pressure. In the first...

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...

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, t...

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...

The hemocytes are the cells responsible for the immunity in molluscs. Cytokines, growth factors, etc, present in the soluble fraction of the hemolymph modulate the immune response. The inflammatory cytokine interleukin-2 (IL-2) induces the synthesis of nitric oxide (NO), responsible for oxidizing processes. In hemocytes of mollusks, the presence of...

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...

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...

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 mi...

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 gallo...

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...

In the marine mussel Mytilus galloprovincialis Lmk., the cells in charge of the immune response are the haemocytes. This work focused on the response capacity of these cells throughout the year, as significant differences between summer and winter months had been detected. The haemocytes were kept in culture for three days in order to obtain certai...

A quick and sensitive method for the determination of AMP, ADP, and ATP levels in mantle tissue of the sea mussel Mytilus galloprovincialis Lmk. is described. The method uses ion-pair high-performance liquid chromatography with diode array detection, allowing obtaining of the UV absorption spectrum for each peak. Method precision was good. The dete...

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 galloprovin...

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...

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 exp...

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...

Hemocytes from the mollusk Mytilus galloprovincialis Lmk. were maintained for more than 20 days in Leibovitz L-15 medium supplemented with salts and antibiotics. Survival of cells cultured in foetal calf serum (FCS)-supplemented L-15 medium was similar to that of those cultured in inactivated hemolymph-supplemented L-15 medium. Cell activation by l...

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, wh...

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. T...

Previous results suggested operative similarities between Apl II from nervous cells of Aplysia californica, epsilonPKC from brain of vertebrates, and p105 from mantle tissue of Mytilus galloprovincialis Lmk., all of them belonging to the nPKC family. The optimal substrate for Apl II and p105 from mussel is protamine sulfate. In contrast, Ca2+ inhib...

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 d...

Carbohydrate metabolism in mussels shows two phases separated seasonally. During summer and linked to food supply, carbohydrates, mainly glycogen, are accumulated in the mantle tissue. During winter, mantle glycogen decreases concomitantly with an increase in triglyceride synthesis. In spring, after spawning, the animals go in to metabolic rest unt...

In the hemolymph of the sea mussel Mytilus galloprovincialis, two different cell types have been found. Rounded (RH) cells display a nucleus that is very large in relation to the cell size; spread (SH) cells have an expanded cytoplasm and multiple granules. We determined by flow cytometry that only the SH cell types express three interleukin-2 rece...

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), lack...

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 RNa...

In this paper we have examined the distribution of some isoforms of protein kinase C in different tissues from the sea mussel Mytilus galloprovincialis Lmk.. By immunoblot analysis, we have detected the presence of at least three PKC isoforms, all preferably associated with the cellular cytosolic fraction. The Ca(2+)-independent form PKC delta was...

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...

Three cAMP-binding proteins have been identified by photoaffinity labeling with 8-azido[32P]cAMP and purified from the mantle tissue of the sea mussel Mytilus galloprovincialis. Their molecular masses, determined by SDS/PAGE, were 54, 42 and 37 kDa. The purified 54-kDa protein, which had two cAMP-binding sites/monomer, was judged to be a regulatory...

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,...

Three cAMP-binding proteins have been identified by photoaffinity labeling with 8-azido[32P]cAMP and purified from the mantle tissue of the sea mussel Mytilus galloprovincialis. Their molecular masses, determined by SDS/PAGE, were 54, 42 and 37 kDa. The purified 54-kDa protein, which had two cAMP-binding sites/monomer, was judged to be a regulatory...

The catalytic subunit of cyclic AMP-dependent protein kinase was isolated from mantle tissue of the sea mussel Mytilus galloprovincialis and purified to apparent homogeneity using a simple two-step procedure. The purified enzyme had a molecular weight of 40 ± 1.5 kDa on electrophoresis under denaturing conditions, Stokes' radius 25.8 Å and isoelect...

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...

1.1. Glucose-6-phosphate dehydrogenase (G6PDH EC 1.1.1.49) from mouse liver has been purified 1100-fold by extraction, ion-exchange chromatography on DE-52, absorption chromatography on Bio-Gel HTP and gel filtration through sepharose 6 HR 10/30. The purified enzyme showed a single band in silver stained SDS-PAGE.2.2. The native and subunit molecul...

1.1. Fru-2,6-P2 plays an important modulating role in the glycolysis/gluconeogenesis of sessile marine molluscs, indicating by its content the change in both the flow direction and the glycogen reserves.2.2. PFK-1 is activated, while FBPase-1 is inhibited by Fru-2,6-P2. The phosphoester synergically enhances the effect caused by AMP on both enzymes...

6-phosphofructo-2-kinase (PFK-2) from the mantle of the sea musselMytilus galloprovincialis Lmk, collected from the Ra de Arosa (NW Spain) in 1990, was purified 550-fold by extraction and sequential affinity chromatography on Affi-gel Blue and ATP-agarose columns. The enzyme was a dimer with a native molecular weight of 100 kilodaltons (KDa) and a...

1.1. Fructose-2,6-bisphosphate and AMP each acted as mixed inhibitors of FBPase 1 from mussel mantle, whereas the mechanism of inhibition in each case is different.2.2. The joint action of Fru-2,6-P2 and AMP is a synergistic inhibition of FBPase 1.3.3. In the absence of effectors, EDTA activated FBPase 1 from the mantle of mussels. The inhibitors c...

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 t...

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 dec...

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 d...

The dependence of the initial rate on the concentration of MgATP2− and fructose-6-phosphate, its behaviour with the alternative substrates MgGTP2− and MgITP2- and the inhibition patterns by its products, suggest that the reaction catalysed by phosphofructokinase from the mantle tissue of the mussel Mytilus galloprovincialis Lmk, involves a Theorell...

Phosphofructokinase (PFK) from the mantle of Mytilus galloprovincialis Lmk. was purified 302-fold with a yield of 27%. The enzyme proved to be a 340,000-dalton oligomer comprising four identical 85,000-dalton subunits. Like other phosphofructokinases, the enzyme behaved cooperatively with fructose 6-phosphate and was inhibited by high concentration...

The effects of different modulators on the phosphofructokinase (PFK) activity from the posterior adductor muscle of the sea musselMytilus galloprovincialis Lmk. were studied in mussels collected from N.W. Spain in spring/summer, 1988. Adenosine monophosphate (AMP), fructose 2,6-bisphosphate (Fru 2,6-P2) and ammonium ions individually activated PFK....

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...

The effect of pH on the phosphofructokinase activity from the posterior adductor muscle of the sea mussel Mytilus galloprovincialis Lmk., is dependent on the concentrations of MgATP2- and free ATP. Reducing pH from pH 8.0 to pH 6.9 alters the equilibria among the various forms of free ATP, greatly increasing the concentration of HATP3-, without app...

Aliphatic polyamines are able to relieve the inhibition of glucose-6-phosphate dehydrogenase (G6PDH) by palmitoyl-coenzyme A (CoA). Of the polyamines assayed, spermine was the most effective in relieving this inhibition, followed by spermidine and ethylenediamine. These data suggest that the extent of the ability to relieve the inhibition depends o...

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 o...

1.1. The kinetics of the all-year (I) form of glutathione reductase from mantle tissue of mussel alternate between a ping-pong and a sequential mechanism as the concentration of oxidized glutathione varies.2.2. The winter form (II) exhibits sequential behaviour.3.3. The inhibition of form I by NADPH depends on the time of incubation and is relieved...

1.1. Glutathione reductase (GSSG-R) from mantle tissue of Mytilus edulis exists in two forms (I and II). GSSG-R I is present throughout the year and in the present study was purified 92-fold by extraction, ammonium sulphate fractionation, ion-exchange chromatography on DEAE-Sephadex, absorption chromagraphy on hydroxyapatite and gel-filtration on S...

Human platelet glutathione peroxidase (GSH-Px) exhibits a ping-pong type kinetic mechanism. Peroxide changes the redox state of the enzyme and renders it highly stable against attack by alkylating agents. Peroxide seems also to act as a positive homotropic modulator modifying the allosteric kinetics of the enzyme. The combination of these two mecha...

An enzyme with a glutathione peroxidase activity was partially purified from human platelets. [75Se] cochromatographed with the enzyme activity which was optimal at pH 8.5 and inhibited by N-ethylmaleimide or copper ions. The energy of activation was 8.0 kcal. Kinetics appeared michaelian with an apparent Km of 55 μM for t-butylhydroperoxide and no...

The method proposed for measuring glutathione peroxydase (GSH-Px) activity is based on the determination of oxidized glutathione (GSSG) using o-phtalaldehyde (OPT) as a fluorescent reagent. This method makes it possible to study the kinetics of both substrates (peroxide and reduced glutathione, GSH), and allosteric kinetics were found for GSH, with...

1.1. The enzyme fructose-1,6-bisphosphatase was purified from the mantle of the sea mussel Mytilus galloprovincialis Lmk. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis. The mol. wt and subunit mol. wt of the enzyme were 105,000 and 27,000, respectively.2.2. Divalent cations are essential for the enzyme activity....