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Biomedical subjects

L Ulloa

Publications and source records attributed to L Ulloa.

At least 19 recordsLinked to original sources
PubMed2002

High mobility group box chromosomal protein 1: a novel proinflammatory mediator in synovitis.

OBJECTIVE: High mobility group box chromosomal protein 1 (HMGB-1) is a ubiquitous chromatin component expressed in nucleated mammalian cells. It has recently and unexpectedly been demonstrated that stimulated live mononuclear phagocytes secrete HMGB-1, which then acts as a potent factor that causes inflammation and protease activation. Macrophages play pivotal roles in the pathogenesis of arthritis. The aim of this study was to determine whether synovial macrophage expression of HMGB-1 is altered in human and experimental synovitis. METHODS: Intraarticular tissue specimens were obtained from healthy Lewis rats, Lewis rats with Mycobacterium tuberculosis-induced adjuvant arthritis, and from patients with rheumatoid arthritis (RA). Specimens were immunohistochemically stained for cellular HMGB-1. Extracellular HMGB-1 levels were assessed in synovial fluid samples from RA patients by Western blotting. RESULTS: Immunostaining of specimens from normal rats showed that HMGB-1 was primarily confined to the nucleus of synoviocytes and chondrocytes, with occasional cytoplasmic staining and no extracellular matrix deposition. In contrast, inflammatory synovial tissue from rats with experimental arthritis as well as from humans with RA showed a distinctly different HMGB-1 staining pattern. Nuclear HMGB-1 expression was accompanied by a cytoplasmic staining in many mononuclear cells, with a macrophage-like appearance and an extracellular matrix deposition. Analysis of synovial fluid samples from RA patients further confirmed the extracellular presence of HMGB-1; 14 of 15 samples had HMGB-1 concentrations of 1.8-10.4 microg/ml. CONCLUSION: The proinflammatory mediator HMGB-1 was abundantly expressed as a nuclear, cytoplasmic, and extracellular component in synovial tissues from RA patients and from rats with experimental arthritis. These findings suggest a pathogenetic role for HMGB-1 in synovitis and indicate a new potential therapeutic target molecule.

Adult↗
PubMed2001

Lefty proteins exhibit unique processing and activate the MAPK pathway.

Lefty polypeptides, novel members of the transforming growth factor-beta (TGF-beta) superfamily, are involved in the formation of embryonic lateral patterning. Members of the TGF-beta superfamily require processing for their activation, suggesting cleavage to be an essential step for lefty activation. Transfection of different cell lines with lefty resulted in expression of a 42-kDa protein, which was proteolytically processed to release two polypeptides of 34 and 28 kDa. Since members of the proprotein convertase (PC) family cleave different TGF-beta factors and are involved in the establishment of embryonic laterality, we studied their role in lefty processing. Cotransfection analysis showed that PC5A processed the lefty precursor to the 34-kDa form in vivo, whereas furin, PACE4, PC5B, and PC7 had a limited activity. None of these PCs showed activity in the processing of the lefty polypeptide to the 28-kDa lefty form. The mutation of the consensus sequences for PC cleavage in the lefty protein allowed the lefty cleavage sites to be identified. Mutations of the sequence RGKR to GGKG (amino acids 74-77) and of RHGR to GHGR (amino acids 132-135) prevented the proteolytic processing of the lefty precursor to the 34- and 28-kDa forms, respectively. To identify the biologically active form of lefty, we studied the effect of lefty treatment on pluripotent P19 cells. Lefty did not induce Smad2 or Smad5 phosphorylation, Smad2/Smad4 heterodimerization, or nuclear translocation of Smad2 or Smad4, but activated the MAPK pathway in a time- and dose-dependent fashion. Further analysis showed the 28-kDa (but not the 34-kDa) polypeptide to induce MAPK activity. Surprisingly, the 42-kDa lefty protein was also capable of inducing MAPK activity, indicating that the lefty precursor is biologically active. The data support a molecular model of processing as a mechanism for regulation of lefty signaling.

3T3 Cells↗
PubMed2001

Lefty inhibits receptor-regulated Smad phosphorylation induced by the activated transforming growth factor-beta receptor.

Transforming growth factor-beta (TGF-beta) is a pleiotropic cytokine that regulates growth and differentiation of diverse types of cells. TGF-beta actions are directed by ligand-induced activation of TGF-beta receptors with intrinsic serine/threonine kinase activity that trigger phosphorylation of receptor-regulated Smad (R-Smad) protein. Phosphorylated R-Smad proteins bind to Smad4, and the complexes formed move into the nucleus, where they act as components of a transcriptional complex. Here, we show that TGF-beta signaling is inhibited by lefty, a novel member of the TGF-beta superfamily. Lefty perturbed TGF-beta signaling by inhibiting the phosphorylation of Smad2 following activation of the TGF-beta receptor. Moreover, lefty inhibited the events that lie downstream from R-Smad phosphorylation, including heterodimerization of R-Smad proteins with Smad4 and nuclear translocation of the R-Smad.Smad4 complex. Lefty repressed TGF-beta-induced expression of reporter genes for the p21, cdc25, and connective tissue growth factor promoters and of a reporter gene driven by the Smad-binding element. Similarly, lefty inhibited both BMP-mediated Smad5 phosphorylation and gene transcription. The action of lefty does not appear to depend on protein synthesis, including synthesis of inhibitory Smad proteins. Thus, lefty provides a repressed state of TGF-beta- or BMP-responsive genes and participates in negative modulation of TGF-beta and BMP signaling by inhibition of phosphorylation of R-Smad proteins.

Animals↗
PubMed1999

Inhibition of transforming growth factor-beta/SMAD signalling by the interferon-gamma/STAT pathway.

Transforming growth factor-beta (TGF-beta) and interferon-gamma (IFN-gamma) have opposite effects on diverse cellular functions, but the basis for this antagonism is not known. TGF-beta signals through a receptor serine kinase that phosphorylates and activates the transcription factors Smads 2 and 3, whereas the IFN-gamma receptor and its associated protein tyrosine kinase Jak1 mediate phosphorylation and activation of the transcription factor Stat1. Here we present a basis for the integration of TGF-beta and IFN-gamma signals. IFN-gamma inhibits the TGF beta-induced phosphorylation of Smad3 and its attendant events, namely, the association of Smad3 with Smad4, the accumulation of Smad3 in the nucleus, and the activation of TGFbeta-responsive genes. Acting through Jak1 and Stat1, IFN-gamma induces the expression of Smad7, an antagonistic SMAD, which prevents the interaction of Smad3 with the TGF-beta receptor. The results indicate a mechanism of transmodulation between the STAT and SMAD signal-transduction pathways.

Cell Line↗
PubMed1998

The phosphorylated isoform of microtubule associated protein 1B (MAP1B) is expressed in the visual system of the tench (Tinca tinca, L) during optic nerve regeneration.

By using Western blot analysis and immunohistochemistry we have demonstrated that microtubule associated protein 1B (MAP1B)-phos is present in growing and regenerating axons of retinal ganglion cells of fish (Tinca tinca, L). We have found that the levels of MAP1B-phos substantially increase in regenerating optic nerves. Our observations suggest that MAP1 B-phos plays an important role in regeneration processes in the central nervous system (CNS) of the fish. These results are compared in the present paper with that found in the regenerating peripheral nervous system (PNS) of mammals.

Animals↗
PubMed1998

Interactions between cellular actin and human respiratory syncytial virus (HRSV).

Actin the main component of the cellular microfilament network, is present in human respiratory syncytial virus (HRSV) purified virions, as an internal component. This fact and the results of immunoprecipitation studies indicate that during HRSV infection in HEp-2 cells there are interactions between cellular actin and viral components, that can promote a transitory increase in the polymerization of synthetized actin, mainly of the beta isotype. This increased actin polymerization can be related with the formation of cytoplasmic extensions, that contain beta actin and viral particles observed in the HRSV infected HEp-2 cells. The formation of these structures may indicate that HRSV has developed an actin-based motility system similar to that described for other viral and bacterial systems.

Actins↗
PubMed1997

NMDA-glutamate receptors regulate phosphorylation of dendritic cytoskeletal proteins in the hippocampus.

Most forms of synaptic potentiation need the activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors which generate changes in dendritic morphology of postsynaptic neurons. Since microtubule proteins have an essential role in dendritic morphology, they may be involved and regulated during the modifications of dendritic morphology associated with synaptic potentiation. The phosphorylation of the microtubule-associated proteins (MAPs) has been analyzed in situ after activation or blockade of NMDA-glutamate receptors in hippocampal slices. The phosphorylation of MAP1B and MAP2 has been studied by using several antibodies raised against phosphorylation-sensitive epitopes. Whereas antibodies 125 and 305 recognize phosphorylated epitopes on MAP1B and MAP2, respectively, Ab 842 recognizes a phosphorylatable sequence on MAP1B only when it is dephosphorylated. NMDA treatment decreased the phosphorylation state of the epitope recognized by the antibody 305 on MAP2 and caused a slight dephosphorylation of MAP1B sequences recognized by Ab 125 and 842. Moreover, exposure to APV (an antagonist of NMDA-glutamate receptors) counteracted the effect of NMDA and induced an increase in the phosphorylation state of these sequences in MAP2. Since phosphorylation regulates the interaction of MAPs with cytoskeleton, the results suggest that the modulation of the phosphorylated state of MAP2 by NMDA-glutamate receptors may be implicated in dendritic plasticity.

2-Amino-5-phosphonovalerate↗
PubMed1997

Analysis of the expression, distribution and function of cyclin dependent kinase 5 (cdk5) in developing cerebellar macroneurons.

Cultures of cerebellar macroneurons were used to study the expression, activity, subcellular localization, and function of cdk5 during neuronal morphogenesis. The results obtained indicate that in non-polarized neurons cdk5 is restricted to the cell body but as soon as polarity is established it becomes highly concentrated at the distal tip of growing axons where it associates with microtubules and the subcortical cytoskeleton. In addition, we show that laminin, an extracellular matrix molecule capable of stimulating axonal extension and promoting MAP1b phosphorylation (DiTella et al., 1996), accelerates the redistribution of cdk5 to the axonal tip and dramatically increases its activity. Finally, our results indicate that cdk5 suppression by antisense oligonucleotide treatment selectively reduces axonal elongation and decreases the phosphorylation status of MAP1b, as well as its binding to microtubules. Taken collectively, our observations suggest that cdk5 may serve as an important regulatory linker between environmental signals (e.g. laminin) and constituents of the intracellular machinery (e.g. MAP1b) involved in axonal formation.

Animals↗
PubMed1996

Characterization of microtubule-associated protein MAP1B: phosphorylation state, light chains, and binding to microtubules.

We have recently described a procedure for the purification of microtubule associated protein 1B (MAP1B) from calf brain [Pedrotti, B., & Islam K. (1995) Cell Motil. Cytoskeleton 30, 301-309], and this study further characterizes the purified protein and its interaction with microtubules. We show that purified MAP1B (1) is thermostable; (2) is mainly phosphorylated at the casein kinase II (CKII) sites but only partially phosphorylated at the proline-directed protein kinase (PDPK) sites; (3) both the CKII and PDPK sites can be dephosphorylated by alkaline phosphatase; and (4) dephosphorylation results in an increased mobility on SDS-PAGE gels. The ability of MAP1B to interact with microtubules was also examined and shows that (1) phosphorylated (1B-P), alkaline phosphatase-treated (1B-AP), and heat-treated (1B-P), alkaline phosphatase-treated (1B-AP), and heat-treated (1B-HT) MAP1B bind to taxol-stabilized microtubules; (2) 1 mol of 1B-P, 1B-AP, or 1B-HT each binds about 13-14 tubulin dimers; (3) light chain interaction with MAP1B heavy chain is not affected by AP- or heat-treatment; (4) MAP1B can be displaced from taxol-stabilized microtubules by titration with salt; (5) higher salt concentrations are required to displace 1B-AP compared with 1B-P from taxol-stabilized microtubules; and (6) MAP2 is able to displace both 1B-P and 1B-AP from taxol-stabilized microtubules. The role of phosphorylation in regulating MAP1B interaction with microtubules and light chains is discussed.

Alkaline Phosphatase↗
PubMed1996

Involvement of gamma and beta actin isoforms in mouse neuroblastoma differentiation.

Two actin isoforms, gamma and beta, are contained within neuroblastoma cells. However, the relative amount and distribution of both isoforms within the cells are differentially regulated during neurite extension. The proportion of gamma-actin isoform became about four times greater than that of beta actin during neuroblastoma cell differentiation. Additionally, whereas beta actin appears to be concentrated in the cell cortex, gamma actin is also present throughout the cell body. Upon differentiation, neuroblastoma cells reorganize their actin cytoskeleton and gamma actin is induced to polymerize whereas beta actin polymers are partially disassembled. Moreover, both actin isoforms are differentially distributed within differentiated cells. Thus, gamma actin polymers are located both in the soma and proximal regions of extended neurites, whereas beta actin is enriched in the terminal tip of the neurites. Our results strongly suggest that both actin isoforms are involved in a different way in neuroblastoma cell differentiation.

Actins↗
PubMed1994

Microtubule-associated protein 1B (MAP1B) is present in glial cells phosphorylated different than in neurones.

A panel of four anti-MAP1B antibodies have been used to study the presence and post-translational modification of MAP1B in primary cultures of glial cells. Two antibodies (150 and 125) recognize phosphorylated epitopes whereas the other two (531 and 842) recognize non-phosphorylated phosphorylatable epitopes on the MAP1B molecule. Immunofluorescence and Western blot analysis with antibodies 531 and 842 revealed the presence of small amounts of MAP1B-like immunoreactivity in type 1 astrocytes and a greater content in more differentiated glial cells found in long-term cultures. By immunofluorescence, these latter cells gave positive immunostaining with antibody 125, which recognizes a phosphorylated epitope phosphorylated by casein kinase II. Antibody 150, which reacts to a phosphorylated epitope on the MAP1B molecule, did not show any detectable immunoreactivity in glial cells cultures, either by immunofluorescence or Western blot. All four antibodies recognized hippocampal neurones in culture, with especially intense immunostaining in cell bodies and axons, and reacted strongly with protein present in hippocampal neurones extracts showing an electrophoretic mobility similar to that of brain MAP1B. In mixed optic nerve glial cell cultures, anti-galactocerebroside (GalC) positive cells gave also positive staining with antibodies 531 and 125. We propose that MAP1B is present in cultures of glial cells in moderate amounts and with a phosphorylation state different than in neurones. Thus, less differentiated glial cells, such as type 1 astrocytes, have a small amount of MAP1B, mainly in a non-phosphorylated form, which is spread diffusely in the cytoplasm and probably does not interact with microtubules. More differentiated glial cells, such as oligodendrocytes, show a greater content in MAP1B which, at least in part, is phosphorylated by a casein kinase II-like activity.

Amino Acid Sequence↗
PubMed1994

Role of phosphorylated MAPlB in neuritogenesis.

The distribution of microtubule-associated protein lB (MAPlB) phosphorylated by either proline-directed protein kinase (PDPK) or casein kinase II (CK II) in neuroblastoma cells and hippocampal neurons has been studied by immunofluorescence using specific antibodies to distinct phosphorylation-sensitive epitopes. A proximo-distal gradient of increasing PDPK-catalyzed phosphorylation of MAPlB is superimposed on a proximo distal gradient of decreasing CK II-catalyzed MAPlB phosphorylation within growing axon-like neurites. Additionally, CK II-phosphorylated MAPlB is present in cell bodies and dendrites where no PDPK-phosphorylated MAPlB is observed. These results suggest distinct roles for both types of modifications of MAPlB in developing neurons.

Animals↗
PubMed1994

Depletion of catalytic and regulatory subunits of protein kinase CK2 by antisense oligonucleotide treatment of neuroblastoma cells.

1. The use of antisense oligonucleotides to inhibit expression of the genes coding for the catalytic (alpha/alpha') and regulatory (beta) subunits of protein kinase casein kinase 2 (CK2) has allowed study of the role of this enzyme in mouse neuroblastoma cells. 2. Selective depletion of catalytic (alpha/alpha') subunits results in the blocking of neuritogenesis. The depletion of catalytic subunits also affects the sorting of the regulatory (beta) subunit of CK2, as the absence of catalytic subunits prevents the translocation of the regulatory subunit to the nuclei. These results emphasize the existence of a control mechanism linking the expression and sorting of CK2 catalytic and regulatory subunits. 3. Selective depletion of the regulatory (beta) subunit of protein kinase CK2 by an specific antisense oligonucleotide causes partial inhibition of neurite extension.

Allosteric Site↗
PubMed1994

Microtubule-associated protein MAP1B showing a fetal phosphorylation pattern is present in sites of neurofibrillary degeneration in brains of Alzheimer's disease patients.

Alzheimer's disease results in the appearance of cytoskeletal disorders yielding pathological structures such a neurofibrillary tangles or dystrophic neurites. It has been previously described that the microtubule-associated protein, tau, modified by phosphorylation in serines adjacent to prolines, is a major component of these structures. Here, we show that another microtubule associated protein, MAP1B, aberrantly phosphorylated by a proline-dependent protein kinase, is a component of these previously mentioned structures. Thus, a possible common phosphorylation of axonal MAPs such as tau or MAP1B may correlate with their association with those aberrant cytoskeletal structures present in AD.

Aged↗
PubMed1994

Localization of differentially phosphorylated isoforms of microtubule-associated protein 1B in cultured rat hippocampal neurons.

The development and plasticity of axons and dendrites in mammalian neurons may depend on the presence and phosphorylation state of cytoskeletal proteins, including certain microtubule-associated proteins. One of these proteins, microtubule-associated protein 1B, is modified by different protein kinases, which give rise to two major types of phosphorylated isoforms. The distribution of these isoforms in cultured hippocampal neurons has been studied using antibodies to specific phosphorylation-sensitive epitopes. Mode I-phosphorylated MAP1B is largely restricted to developing axonal processes, particularly at their distal regions including their growth cones where no mode I-dephosphorylated MAP1B is present. Axonal maturation is accompanied by dephosphorylation of MAP1B at mode I sites. Thus, mode I-phosphorylated MAP1B may be a marker for axonal growth. In contrast, mode II-phosphorylated MAP1B is abundant in the axonal and somatodendritic compartments, and no increased dephosphorylation occurs during maturation. These results are compatible with a role for the mode I phosphorylation of MAP1B (which might be catalysed by proline-directed protein kinases) in supporting a rapid axonal-specific growth mechanism and a more general role for the mode II phosphorylation of MAP1B (which seems to be catalysed by casein kinase II) in controlling axonal and dendritic growth and remodeling.

Amino Acid Sequence↗
PubMed1994

Phosphorylation of microtubule-associated proteins by protein kinase CK2 in neuritogenesis.

Phosphorylation of microtubule-associated protein MAP1B and the neuronal-specific beta III-tubulin isoform takes place during neurite growth in neuroblastoma cells. Protein kinase CK2 (formerly referred to as casein kinase 2) is possibly involved in beta III-tubulin phosphorylation. As for MAP1B, there are at least two types of phosphorylation; one catalyzed by proline-directed protein kinases and another catalyzed by CK2. Protein kinase CK2 is primarily localized to the nuclei in proliferating neuroblastoma cells, whereas an increased amount of the enzyme is present in the cytoplasm of postmitotic cells bearing neurites. Treatment of neuroblastoma cells with an antisense oligonucleotide which specifically results in CK2 catalytic subunit depletion inhibits neuritogenesis. CK2 depletion is accompanied by dephosphorylation of MAP1B on the corresponding phosphorylatable sites. This dephosphorylation is paralleled by a release of MAP1B from microtubules. These results suggest that MAP1B phosphorylation by CK2 may be required for the assembly of microtubules within neurites. Other neuronal cytoskeletal proteins including MAP1A and tau are also substrates for CK2, indicating a role for the enzyme in the regulation of cytoskeletal functions also in mature neurons.

Amino Acid Sequence↗
PubMed1993

Dephosphorylation of distinct sites on microtubule-associated protein MAP1B by protein phosphatases 1, 2A and 2B.

Rat brain microtubule-associated protein MAP1B has been tested as a substrate for Ser/Thr protein phosphatases (PP). The dephosphorylation reactions were followed by specific antibodies recognizing phosphorylated and phosphorylatable epitopes. One set of phosphorylation sites on MAP1B are referred to as mode I sites, and their phosphorylation is presumably catalyzed by proline-directed protein kinases. These mode I sites are efficiently dephosphorylated by PP2B and 2A but not by PP1. Another set of phosphorylation sites on MAP1B are named mode II sites, and their phosphorylation is possibly due to casein kinase II. These mode II sites are dephosphorylated by PP2A and PP1, the PP2B being ineffective. The selectivity of phosphatases for different sites within the same protein indicates the complexity of the dephosphorylation reactions regulating the functionality of MAP1B in neurons.

Animals↗