Biomedical subjects

F J Moreno

Publications and source records attributed to F J Moreno.

At least 19 recordsLinked to original sources

PubMed2001

Heterogeneity of caprine kappa-casein macropeptide.

The heterogeneity of caprine caseinmacropeptide (CMP) was determined by means of treatments with neuraminidase and acid phosphatase and analyses by anion exchange FPLC and reversed-phase (RP)-HPLC, with on-line and off-line electrospray ionization mass spectrometry. The main CMP components were two non-glycosylated and di-phosphorylated forms, as well as two other mono-phosphorylated species, each corresponding to a genetic variant of caprine kappa-casein due to the silent substitution Ile/Val at position 119. Asialo-aglyco mono- and di-phosphorylated forms were found in the ratios 8-14% and 86-92%, respectively. Approximately 36% of caprine CMP was glycosylated. Based on the obtained molecular masses, the occurrence of tri-, di- and monosaccharide-containing di-phosphorylated CMP are reported, assuming that N-acetylgalactosamine, galactose, N-acetyl and N-glycolylneuraminic acids would constitute the main monosaccharides of caprine CMP. CMP microheterogeneity due to the genetic polymorphism was also observed in the glycosylated forms.

Acid Phosphatase↗

PubMed2000

Chromatographic characterization of ovine kappa-casein macropeptide.

Ovine casein macropeptide (CMP) was characterized by anion-exchange FPLC and reversed-phase (RP) HPLC. To study heterogeneity (the degree of glycosylation and phosphorylation), CMP was desialylated with neuraminidase and dephosphorylated with acid phosphatase. Following RP-HPLC, the main CMP components were identified using either on-line or off-line mass spectrometry. The most abundant ovine CMP component was a diphosphorylated carbohydrate-free form, followed by one or two monophosphorylated and a non-phosphorylated asialo-aglyco species. Aglyco non-phosphorylated, monophosphorylated and diphosphorylated forms were in the ratio 3:20:77. Only approximately 30% of ovine CMP was glycosylated. Assuming that the monosaccharide fraction of ovine CMP is composed of N-acetylgalactosamine, galactose and N-glycolylneuraminic acid, molecular masses consistent with the presence of CMP containing tetra-, tri-, di- and monosaccharide were identified.

Acid Phosphatase↗

PubMed1999

Downregulation of glycogen synthase kinase-3beta (GSK-3beta) protein expression during neuroblastoma IMR-32 cell differentiation.

Glycogen synthase kinase-3gamma (GSK-3beta) is a multifunctional protein kinase that phosphorylates a variety of substrates including the neuronal-specific microtubule-associated protein tau. Here we report that the down-regulation of the GSK-3beta protein is an early event in the course of the differentiation of human neuroblastoma IMR-32 cells. This decline in GSK-3beta is accompanied by a significant decrease in the phosphorylation state of tau protein. A noteworthy increase in tau protein expression also takes place later during the differentiation of IMR-32 cells. The augmented expression and diminished phosphorylation of tau protein in differentiated IMR-32 cells can be correlated with increments in the assembly of microtubules and in the association of tau with microtubules. These results suggest a contribution of a decrease in GSK-3beta to molecular events leading to neuroblastoma cell differentiation. Among these, tau protein dephosphorylation might favor microtubule stabilization within neurites.

Bucladesine↗

PubMed1999

OP18/stathmin binds near the C-terminus of tubulin and facilitates GTP binding.

It is has been previously suggested that the protein Op18/stathmin may interact with tubulin via the alpha-tubulin subunit [Larsson, N., Marklund, U., Melander Gradin, H., Brattsand, G. & Gullberg, M. (1997) Mol. Cell. Biol. 17, 5530-5539]. In this study we have used limited proteolysis and cross-linking analysis to localize further the stathmin-binding site on alpha-tubulin. Our results indicate that such a binding site is in a region close to the C-terminus of the molecule comprising residues 307 to the subtilisin-cleavage site on the alpha-tubulin subunit. Based on a recent model of the structure of tubulin [Nogales, E., Wolf, S.G. & Dowing, D.H. (1998) Nature (London) 391, 199-203], we found that this region contained the same areas that may be involved in longitudinal contacts of alpha-tubulin subunits within the microtubule. We also observed that the binding of stathmin to tubulin can modulate the binding of GTP to tubulin, as a consequence of a conformational change in the beta-tubulin subunit that occurs upon interaction of stathmin with tubulin.

Adenosine Triphosphate↗

PubMed1999

Binding of polylysine to protein kinase CK2, measured by Surface Plasmon Resonance.

The interaction between protein kinase CK2 and polylysine has been studied by Surface Plasmon Resonance (SPR). The binding process has a very low energy of activation, it is irreversible, and too slow as to explain the enzyme activity stimulation as a direct consequence of the polylysine binding. The polylysine interaction with a peptide substrate and with casein are faster, and in agreement with a substrate-mediated mechanism of activity stimulation. After several hours of incubation, the binding of polylysine to CK2 produces the loss of enzymatic activity.

Casein Kinase II↗

PubMed1999

Distribution of CK2, its substrate MAP1B and phosphatases in neuronal cells.

Neuronal morphogenesis depends on the organization of cytoskeletal elements among which microtubules play a very important role. The organization of microtubules is controlled by the presence of microtubule-associated proteins (MAPs), the activity of which is modulated by phosphorylation and dephosphorylation. One of these MAPs is MAP1B, which is very abundant within growing axons of developing neurons where it is found phosphorylated by several protein kinases including CK2. The expression of MAP1B is notably decreased after neuronal maturation in parallel with a change in the localization of the protein, which becomes largely concentrated in neuronal cell bodies and dendrites. Interestingly, MAP1B remains highly phosphorylated at sites targeted by protein kinase CK2 in mature neurons. We have analyzed the expression and localization of CK2 catalytic subunits along neuronal development. CK2alpha subunit appears early during development whereas CK2alpha' subunit appears within mature neurons at the time of dendrite maturation and synaptogenesis, in parallel with the change in the localization of MAP1B. CK2alpha subunit is found associated with microtubule preparations obtained from either grey matter or white matter from adult bovine brain, whereas CK2alpha' subunit is highly enriched in microtubules obtained from grey matter. These results lend support to the hypothesis that CK2alpha' subunit is concentrated in neuronal cell bodies and dendrites, where it associates with microtubules, thus contributing to the increased phosphorylation of MAP1B in this localization in mature neurons.

Amino Acid Sequence↗

PubMed1998

Phosphorylation of stathmin modulates its function as a microtubule depolymerizing factor.

Oncoprotein 18 or stathmin was isolated from bovine brain, characterized and novel features of its function as a microtubule depolymerizing factor were tested. The effect of phosphorylation of stathmin on its function as a microtubule depolymerizing factor has been tested in vitro. Five different protein kinases, protein kinase A, MAP kinase, cdc2 kinase, glycogen synthase kinase 3 and casein kinase 2, were used to modify stathmin, since it is known that these kinases could phosphorylate several residues that are modified in vivo and could have important roles in stathmin function. The residues phosphorylated in vitro by the different protein kinases were identified and in some cases they correspond to those modified in vivo. Recombinant unphosphorylated stathmin and native stathmin, which was previously dephosphorylated with alkaline phosphatase, showed similar microtubule depolymerizing activity. This activity is higher than that of stathmin phosphorylated by protein kinase A, MAP kinase or cdc 2 kinase, whereas phosphorylation of the protein with casein kinase 2 or glycogen synthase kinase 3 resulted in a slight increase of the depolymerizing activity.

Animals↗

PubMed1998

[Hepatocellular carcinoma associated to porphyria cutanea tarda and hepatitis C virus infection without cirrhosis].

The molecular basis of porphyria cutanea tarda is defective activity of hepatic uroporphyrinogen decarboxylase. A common characteristic is liver dysfunction than goes from cirrhosis to hepatocellular carcinoma. A high prevalence of hepatitis C virus markers has been demonstrated. We study a patient with PCT and hepatitis C virus markers that finally develops to hepatocellular carcinoma without previous cirrhosis. Previous studies with association of PCT and hepatitis C virus markers, have found liver diseases ranging from cirrhosis to hepatocellular carcinoma. The importance of this study is the absence of cirrhosis.

Carcinoma, Hepatocellular↗

PubMed1998

Inhibition of nucleolar protein nucleolin by electroporation with anti-nucleolin antibodies results in an increase of the nucleolar size.

Electroporation of exponentially growing human larynx epidermoid carcinoma cells (HEp-2) with a serum against nucleolin, one of the most abundant non-histone nuclear proteins, has shown, 24 h after electroporation, a significant increase in the size of the nucleolus of these cells compared with normal HEp-2 cells (non-electroporated) and electroporated HEp-2 cells in the absence of anti-nucleolin serum (P < 0.01). Image analysis evaluation of the different nucleolar components proved a major contribution of the dense fibrillar component to the total nucleolar size in cells electroporated with anti-nucleolin antibodies, more than that corresponding to the dense fibrillar component in cells from any of the control groups (P < 0.01), indicating that the reported increase in nucleolar size was due to a marked enlargement of the dense fibrillar regions. These results, in agreement with previous biochemical and molecular biology studies, suggest a pivotal role for nucleolin in pre-rRNA processing and constitute morphological evidence supporting this role. Following nucleolin inhibition, impaired pre-rRNA processing might result in an accumulation of this molecular species in the dense fibrillar component of the nucleolus, where pre-rRNA is first present.

Antibodies↗

PubMed1997

Lithium inhibits Alzheimer's disease-like tau protein phosphorylation in neurons.

In Alzheimer's disease, tau protein becomes hyperphosporylated, which can contribute to neuronal degeneration. However, the implicated protein kinases are still unknown. Now we report that lithium (an inhibitor of glycogen synthase kinase-3) causes tau dephosphorylation at the sites recognized by antibodies Tau-1 and PHF-1 both in cultured neurons and in vivo in rat brain. This is consistent with a major role for glycogen synthase kinase-3 in modifying proline-directed sites on tau protein within living neurons under physiological conditions. Lithium also blocks the Alzheimer's disease-like proline-directed hyperphosphorylation of tau protein which is observed in neurons treated with a phosphatase inhibitor. These data raise the possibility of using lithium to prevent tau hyperphosphorylation in Alzheimer's disease.

Alzheimer Disease↗

PubMed1997

A surface-plasmon-resonance analysis of polylysine interactions with a peptide substrate of protein kinase CK2 and with the enzyme.

The mechanism of protein kinase CK2 (CK2) activity stimulation by polylysine has been studied by surface plasmon resonance (SPR). The kinetics of the polylysine interaction with a peptide substrate of the enzyme, and with the enzyme itself, have been investigated. A peptide containing a threonine (T) residue surrounded by a cluster of negatively charged acidic [arginine (R) and glutamic acid (E)] residues, RRREEETEEE, and specifically phosphorylated by CK2, was selected. Polylysine interacts with both the enzyme and the peptide substrate. The rate constant, the stoichiometry of the polylysine-peptide substrate interaction and the kinetic parameters of the stimulated enzyme were used to calculate the polylysine-dependent stimulation of CK2. The results are in agreement with experimentally determined polylysine-dependent stimulation. The polylysine-enzyme interaction is too slow to account for enzyme stimulation. The behaviour of polylysine is not reproduced by the polyamine spermine. The results are consistent with a substrate-mediated mechanism of CK2 stimulation by polylysine, and they suggest that the CK2 stimulation by polyamines occurs by a different mechanism.

Amino Acid Sequence↗

PubMed1996

Glycogen synthase kinase 3 phosphorylation of different residues in the presence of different factors: analysis on tau protein.

Several peptides derived from microtubule-associated tau protein, have been tested as substrates for glycogen synthase kinase 3 (GSK 3). In the absence of cofactors, GSK 3 can modify serines or threonines followed by prolines. In other cases, a phosphorylation in position +4 is required for the phosphorylation of threonine/serine residues. A third type of substrate can be modified by GSK 3 in the presence of heparin. The comparison of GSK 3 with other kinases suggests some similar features of this kinase with proline-directed protein kinases, such as cdc-2 or mitogen-activated protein kinase (MAP Kinases,) and also with casein kinase 2 (CK 2). Thus, all these kinases are specifically inhibited by 5,6-Dichloro-1-(beta-D-ribofuranosyl)-benzimidazole (DRB). However, heparin is an inhibitor of CK 2 whereas it activates the modification of certain substrates by GSK 3. A possible explanation for the obtained results is that the consensus sequence for GSK 3 phosphorylation is a serine/threonine adjacent to a proline or other beta-turn former residue and that such recognition could be favoured by the presence of adjacent negative charges or the addition of polyanions.

Amino Acid Sequence↗

PubMed1995

Casein kinase 2 inactivation by Mg2+, Mn2+ and Co2+ ions.

Mg2+ as well as Mn2+, and Co2+, which may substitute Mg2+ in the mental ion requirement of casein kinase 2 (Gatica et al., FEBS Lett: 315:173-173, 1993), have been repeatedly reported to display an optimal concentration at which activity of casein kinase 2 is maximal. As far as we know this intriguing property has always been observed with casein as substrate. This phosphoprotein is not the natural substrate of the enzyme, and it is well known that it binds divalent metal ions, which provoke the aggregation and precipitation of the protein. Since an optimal concentration of metal ion might have a regulatory role, we have examined if it is a consequence of the particular properties of casein, or it is an inherent property of the enzyme, extensive to other substrates. We have used the type II regulatory subunit of protein kinase A which is a physiological substrate of the enzyme, and the peptide RRREEETEEE as a specific substrate. No optimal concentration of Mg2+ is observed when these two substrates are used. The results explain, however, why that optimum is observed with casein. Although low concentration of Mn2+, and Co2+ render about 25% of the maximal activity found with Mg2+, they inactivate the enzyme almost fully at concentrations at which Mg2+ yield the maximal activity.

Amino Acid Sequence↗

PubMed1995

Glycogen synthase kinase 3 phosphorylates recombinant human tau protein at serine-262 in the presence of heparin (or tubulin).

Tau protein, the major component of the aberrant structures termed paired helical filaments (PHFs) present in the brain of Alzheimer's disease patients, is pathologically phosphorylated in sites in and around the tubulin-binding sites. A single protein kinase, glycogen synthase kinase 3 (GSK 3), is able to phosphorylate tau at the flanking regions and, additionally, at the tubulin-binding motifs if heparin or tubulin is present. Serines-262 and -324 have been found to be modified at the tubulin-binding region of tau protein by GSK 3 in the presence of heparin or tubulin.

Amino Acid Sequence↗

PubMed1994

Bromocriptine treatment increases lipolysis and steady-state levels of G proteins in adipocytes from lactating rats.

An increase in the rate of lipogenesis and a decrease in the lipolytic response to catecholamines can be observed in adipocytes after weaning or litter removal. Bromocriptine treatment of lactating rats also produces an increase in lipogenesis but fails to decrease the lipolytic response of adipocytes to catecholamines seen after weaning or litter removal. No changes in total number or affinity of beta-adrenergic receptors or adenosine A1 receptors were detected by bromocriptine treatment using radioligand binding assays. However, we observed an increase in the amount of radioactivity from [32P]NAD+ incorporated into alpha-Gs (192 +/- 26%) and alpha-Gi (178 +/- 33%) by cholera- and pertussis-toxin-catalyzed ADP-ribosylation, respectively, with the same treatment, Immunoblotting using RM/1 and AS/7 antisera, which specifically recognize alpha-Gs and alpha-Gi 1,2, respectively, confirmed the increase in the steady-state levels of these G-protein alpha-subunits. The increase in the steady-state levels of alpha-Gs may account, at least in part, for the increased lipolytic response of adipocytes to catecholamines in bromocriptine-treated rats.

Adenosine↗

PubMed1993

A polylysine-induced aggregation of substrate accompanies the stimulation of casein kinase II by polylysine.

Casein kinase II (CK-II) activation by polylysine parallels an aggregation of substrates promoted by the polycation. CK-II is known to be stimulated by basic polypeptides and polyamines. The mechanism by which this stimulation takes place, however, is not yet fully understood. Here we show that, in the usual CK-II assay, polylysine induces the aggregation of casein. This aggregation has been monitored by turbidimetry, electron microscopy and gel filtration. The polylysine-concentration-dependence of the casein aggregation parallels the polylysine-concentration-dependence of the enzyme stimulation. In the presence of polylysine the enzyme is incorporated into the casein aggregates promoted by the polycation, thus supporting the view that this substrate aggregation is directly related to the mechanism of CK-II stimulation. Preliminary results show that a similar parallelism occurs with other natural substrates of the enzyme. The physiological meaning of this substrate aggregation, and its possible relation to other polylysine-stimulated enzymes and polylysine-aggregated proteins, are discussed.

Animals↗

PubMed1992

Dipyridamole stimulates types II cAMP-dependent protein kinase in vitro.

Dipyridamole activates in vitro type II cAMP-dependent protein kinase. This agent stimulates the autophosphorylation of the regulatory subunit in the presence of cAMP but not so in the absence of the cyclic nucleotide. The activation was also observed with exogenous substrates such as casein, histone 2A and MAP2. This stimulation did not seem to be related to the cAMP binding to the R II subunit of the enzyme. Competition binding experiments showed that dipyridamole does not compete with adenosine for the A1 receptor. The results suggest that the reported regulatory properties of dipyridamole on lipid metabolism (González-Nicolás et al. Int J Biochem 21: 883-888, 1989) might be mediated through a direct action--an activation--on the catalytic subunit of a cAMP-dependent protein kinase.

Adipose Tissue↗

PubMed1992

Effects of litter removal on the lipolytic response and the regulatory components of the adenylate cyclase in adipocytes isolated from lactating rats.

The effects of litter removal on the status of different components of the hormone-sensitive adenylate cyclase system were analysed in plasma membranes of rat adipocytes. These effects were correlated with the decreased lipolytic response of adipose tissue. No changes in total number of A1 adenosine receptors or their affinity were detected in response to litter removal. In contrast, beta-adrenergic receptors showed a decrease (35%) in total number of receptors, without any significant change in their affinity. The status of alpha-GS and alpha-Gi, the alpha-subunits of G proteins which mediate stimulation and inhibition respectively of adenylate cyclase, were probed by cholera- and pertussis-toxin-catalysed ADP-ribosylation respectively and by immunoblot. Associated with litter removal, decreases of 63% and 62% in the incorporation of [alpha 32P]ADP-ribose catalysed by cholera toxin and pertussis toxin into alpha-Gs and alpha-Gi respectively were detected. Immunoblotting using RM/1 (anti-alpha-Gs) and AS/7 (anti-alpha-Gi) antisera also showed decreases in the levels of alpha-Gs (52%) and alpha-Gi (55%) in adipocyte membranes from litter-removed rats compared with lactating rats. Alterations in the status of hormone-sensitive adenylate cyclase components, such as those described herein, may be biochemical mechanism(s) by which adipose tissue shows a decreased lipolytic response during recovery from lactation.

Adenylyl Cyclases↗