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M Mata

Publications and source records attributed to M Mata.

At least 37 records · Page 2Linked to original sources
PubMed2000

Neuronal interaction determines the expression of the alpha-2 isoform of Na, K-ATPase in oligodendrocytes.

Na,K-ATPase is an integral membrane enzyme responsible for maintenance of the transmembrane Na+/K+ gradient which generates membrane excitability. Previous studies showed that oligodendrocytes within the CNS robustly expressed the alpha2 isoform of the Na,K-ATPase while oligodendrocytes in isolated cultures did not. We tested whether the levels of this isoform might be modulated by interactions with neurons. Western blots showed alpha2 protein expression was very low in rat optic nerve immediately after birth, but that expression was greatly increased by days 5 and 14. In adult optic nerves, levels were barely detectable. Since the first myelinated axons are observed in rat optic nerve at day 5, and the next 2 weeks are considered the period of peak myelination, this timing suggested a relationship between oligodendrocyte-neuron contact, myelination onset and the upregulation of the alpha2 isoform. In further experiments we plated oligodendrocytes in isolation or in co-culture with neurons dissociated from cerebral cortex at the day of birth. After 6 days in vitro, 45% of oligodendrocytes co-cultured with neurons expressed abundant alpha2 protein which was detected by immunohistochemistry, a six-fold increase over cells expressing alpha2 protein in isolated cultures. Conditioned medium from neuronal cultures did not affect alpha2 levels in oligodendrocytes. These results suggest that neurons may play a role in upregulating glial expression of the alpha2 isoform during peak periods of myelination, and that the effect is likely to be dependent on contact.

Animals↗
PubMed2000

Characterization of Mucor pusillus rennin expressed in Pichia pastoris: enzymic, spectroscopic and calorimetric studies.

The aspartic proteinase gene of Mucor pusillus rennin expressed in Pichia pastoris was characterized in terms of structural and conformational stability induced by temperature. This enzyme is 12% glycosylated, with a similar specific activity to the native fungal enzyme. The secondary structure determined by CD is mainly due to beta-sheet structures with an important contribution of aromatic components. The calorimetric studies were carried out in the temperature range in which the enzyme is most stable. The enzyme undergoes an irreversible, highly scan-rate-dependent thermal denaturation under all the experimental conditions investigated. Between pH 3.0 and 7.0, only one endotherm characterized the thermal denaturation of enzyme. At pH 5.0, the most stable condition found, the denaturation can be fitted to the two-state irreversible model. Thus the kinetic constant and activation parameters of the denaturation process could be obtained. Upon reaching pH 7.5, the denaturation is characterized by two endotherms. This evidence indicates the complex tridimensional structure of this enzyme. Finally, taking into account the conservative tertiary structure of the aspartic proteinase family we comment on our results with reference to the crystallographic structure of M. pusillus pepsin [Newman, Watson, Roychowdhury, Badasso, Cleasby, Wood, Tickle and Blundell (1993) J. Mol. Biol. 221, 1295-1309].

Biotechnology↗
PubMed2000

Herpes simplex virus type 1 ICP0 protein does not accumulate in the nucleus of primary neurons in culture.

Infected-cell protein 0 (ICP0), the product of the herpes simplex virus (HSV) immediate-early (IE) alpha0 gene, is a promiscuous transactivator of viral early (E) and late (L) gene expression. HSV mutants lacking ICP0 function are severely deficient in viral growth and protein synthesis, particularly at low multiplicities of infection. Early in the infectious process in vitro, ICP0 protein accumulates in distinct domains within the nucleus to form characteristic structures active in the transcription of viral genes. However, following infection of primary trigeminal ganglion cells in vitro with a recombinant HSV mutant that expresses only ICP0, we observed that ICP0 protein accumulated in the characteristic intranuclear distribution only in the nuclei of Schwann cells; neurons in the culture did not accumulate ICP0 despite expression of ICP0 RNA in those cells. The same phenomenon was observed in PC12 cells differentiated to assume a neuronal phenotype. In primary neurons in culture, the amount of ICP0 protein could be increased by pharmacologic inhibition of calcium-activated protease (calpain) activity or by inhibition of protein phosphatase 2B (calcineurin). The failure of ICP0 protein to accumulate in the nucleus of neurons suggests that one mechanism which may impair efficient replication of the virus in neurons, and thus favor the establishment of viral latency in those cells, may be found in the cell-specific processing of that IE gene product.

Animals↗
PubMed2000

Listeria monocytogenes as an alternative vaccine vector for HIV.

The necessity for an HIV vaccine and a brief review of current strategies towards this aim are given here to set into context contemporary studies towards exploiting the bacterium Listeria monocytogenes as an HIV vaccine vector. The cell biology and immunology of this unusual intracellular organism are also reviewed, in addition to its application to introducing viral antigens, including HIV antigens, to the immune system.

AIDS Vaccines↗
PubMed1999

Th1 T cell responses to HIV-1 Gag protein delivered by a Listeria monocytogenes vaccine are similar to those induced by endogenous listerial antigens.

Listeria monocytogenes is a facultative intracellular bacterium that lives and grows in the cytoplasm of the host cell. The hallmark of a listerial infection is a cell-mediated immune response to its own secreted virulence factors. Thus, L. monocytogenes vaccines engineered to secrete HIV proteins may be ideal vectors for boosting cellular immune responses against HIV. Using strains of L. monocytogenes that stably express and secrete HIV Gag (Lm-Gag) to deliver this Ag to the immune system, we have previously shown strong MHC class I-restricted cytotoxic T cell responses to this protein. In this study, we examine MHC class II-restricted T cell responses to HIV-Gag delivered by Lm-Gag. We demonstrate the induction of CD4+ T cells that are HIV-Gag specific and identify three epitopes in two strains of mice, BALB/c (H-2d) and C57BL/6 (H-2b), two of which are both H-2d and H-2b restricted, but are not immunodominant for both haplotypes. In addition, we show that the CD4+ T cells induced are of the Th1 phenotype that produce IFN-gamma at levels similar to CD4+ T cells induced to endogenous listerial Ags. These studies suggest that chromosomally modified strains of L. monocytogenes may be useful as vaccine vectors for the induction of Th1 T cell responses against HIV.

Amino Acid Sequence↗
PubMed1999

[Severe liver failure in erythropoietic protoporphyria. A report of a case treated by liver transplantation].

Severe liver failure is a rare complication of erythropoietic protoporphyria (PEP), which is associated with a high rate mortality. Until now, 31 patients with this hepatic complication had underwent a liver transplantation, with a high rate of survival, but their long-term outcome is not well established. We report the first case in Spain of PEP in 59-year-old, whose acute liver failure was treated with liver transplantation, without postoperative complications. The patient is in good clinical condition 30 months later. Nevertheless during the first eleven months of follow-up the plasma levels of protoporphyrin remained elevated, which was accompanied of biochemical and histological evidence of relapse of the metabolic disease in the graft. Cases such as this stress the usefulness of liver transplantation, but also the need of more efficient measures to decrease the protoporphyrin levels before and after the transplant, in order to prevent hepatic and extrahepatic complications in these patients.

Biopsy↗
PubMed1999

The mixed lineage kinase DLK utilizes MKK7 and not MKK4 as substrate.

Mixed lineage kinases DLK (dual leucine zipper-bearing kinase) and MLK3 have been proposed to function as mitogen-activated protein kinase kinase kinases in pathways leading to stress-activated protein kinase/c-Jun NH2-terminal kinase activation. Differences in primary protein structure place these MLK (mixed lineage kinase) enzymes in separate subfamilies and suggest that they perform distinct functional roles. Both DLK and MLK3 associated with, phosphorylated, and activated MKK7 in vitro. Unlike MLK3, however, DLK did not phosphorylate or activate recombinant MKK4 in vitro. In confirmatory experiments performed in vivo, DLK both associated with and activated MKK7. The relative localization of endogenous DLK, MLK3, MKK4, and MKK7 was determined in cells of the nervous system. Distinct from MLK3, which was identified in non-neuronal cells, DLK and MKK7 were detected predominantly in neurons in sections of adult rat cortex by immunocytochemistry. Subcellular fractionation experiments of cerebral cortex identified DLK and MKK7 in similar nuclear and extranuclear subcellular compartments. Concordant with biochemical experiments, however, MKK4 occupied compartments distinct from that of DLK and MKK7. That DLK and MKK7 occupied subcellular compartments distinct from MKK4 was confirmed by immunocytochemistry in primary neuronal culture. The dissimilar cellular specificity of DLK and MLK3 and the specific substrate utilization and subcellular compartmentation of DLK suggest that specific mixed lineage kinases participate in unique signal transduction events.

Animals↗
PubMed1999

Herpes simplex virus vector-mediated expression of Bcl-2 prevents 6-hydroxydopamine-induced degeneration of neurons in the substantia nigra in vivo.

6-Hydroxydopamine (6-OHDA) is widely used to selectively lesion dopaminergic neurons of the substantia nigra (SN) in the creation of animal models of Parkinson's disease. In vitro, the death of PC-12 cells caused by exposure to 6-OHDA occurs with characteristics consistent with an apoptotic mechanism of cell death. To test the hypothesis that apoptotic pathways are involved in the death of dopaminergic neurons of the SN caused by 6-OHDA, we created a replication-defective genomic herpes simplex virus-based vector containing the coding sequence for the antiapoptotic peptide Bcl-2 under the transcriptional control of the simian cytomegalovirus immediate early promoter. Transfection of primary cortical neurons in culture with the Bcl-2-producing vector protected those cells from naturally occurring cell death over 3 weeks. Injection of the Bcl-2-expressing vector into SN of rats 1 week before injection of 6-OHDA into the ipsilateral striatum increased the survival of neurons in the SN, detected either by retrograde labeling of those cells with fluorogold or by tyrosine hydroxylase immunocytochemistry, by 50%. These results, demonstrating that death of nigral neurons induced by 6-OHDA lesioning may be blocked by the expression of Bcl-2, are consistent with the notion that cell death in this model system is at least in part apoptotic in nature and suggest that a Bcl-2-expressing vector may have therapeutic potential in the treatment of Parkinson's disease.

Animals↗
PubMed1998

The MHC class I-restricted immune response to HIV-gag in BALB/c mice selects a single epitope that does not have a predictable MHC-binding motif and binds to Kd through interactions between a glutamine at P3 and pocket D.

Using a strain of Listeria monocytogenes that stably expresses and secretes HIV gag to deliver this Ag to the MHC class I pathway of Ag processing, we have identified the immunodominant CTL epitope to gag in the BALB/c mouse and shown that it is Kd restricted. The specific motif for the peptides that bind the MHC class I molecule H-2 Kd is believed to be a nonamer with residues tyrosine or phenylalanine in the second amino acid position and leucine or isoleucine in the carboxyl-terminal or ninth amino acid position as dominant anchoring positions. Surprisingly, the identified gag peptide, AMQMLKETI, does not contain an anchoring aromatic residue in position two although competition assays with other Kd-restricted epitopes indicated that it binds to Kd with comparable affinity. Using a theoretical molecular dynamics approach to probe the stability of peptide binding to MHC class I molecules, we show that the absence of an appropriate anchor residue at P2 in AMQMLKETI is compensated by favorable interactions of the glutamine at P3 with pocket D of Kd. These findings were verified experimentally, demonstrating the predictive power of this theoretical approach in analyzing MHC class I/peptide interactions. These studies also indicate that CTL epitope prediction that relies on dominant peptide motifs may not always identify the correct epitope.

Amino Acid Sequence↗
PubMed1998

Deletion of multiple immediate-early genes from herpes simplex virus reduces cytotoxicity and permits long-term gene expression in neurons.

Herpes simplex virus type 1 (HSV-1) has many attractive features that suggest its utility for gene transfer to neurons. However, viral cytotoxicity and transient transgene expression limit practical applications even in the absence of viral replication. Mutant viruses deleted for the immediate early (IE) gene, ICP4, an essential transcriptional transactivator, are toxic to many cell types in culture in which only the remaining IE genes are expressed. In order to test directly the toxicity of other IE gene products in neurons and develop a mutant background capable of longterm transgene expression, we generated mutants deleted for multiple IE genes in various combinations and tested their relative cytotoxicity in 9L rat gliosarcoma cells, Vero monkey kidney cells, and primary rat cortical and dorsal root neurons in culture. Viral mutants deleted simultaneously for the IE genes encoding ICP4, ICP22 and ICP27 showed substantially reduced cytotoxicity compared with viruses deleted for ICP4 alone or ICP4 in combination with either ICP22, ICP27 or ICP47. Infection of neurons in culture with these triple IE deletion mutants substantially enhanced cell survival and permitted transgene expression for over 21 days. Such mutants may prove useful for efficient gene transfer and extended transgene expression in neurons in vitro and in vivo.

Animals↗
PubMed1997

Modulation of phosphorylation of neuronal cytoskeletal proteins by neuronal depolarization.

1. The neuronal cytoskeletal protein tau and the carboxy tails of cytoskeletal proteins neurofilament-M (NF-M) and neurofilament-H (NF-H) are phosphorylated on serine residues by the cyclin-dependent kinase cdk-5. 2. In aggregating neuronal-glial cultures we show that veratridine-mediated cation influx causes dephosphorylation of tau, NF-M and NF-H. Dephosphorylation was blocked specifically by cyclosporine A but not by okadiac acid at concentrations up to 200 nM. 3. These results suggest that veratridine-triggered cation influx causes activation of PP-2B (calcineurin) leading to dephosphorylation of these cytoskeletal proteins.

Animals↗
PubMed1996

[Adequacy of the pharmacologic treatment to the recommendations of a performance protocol in hypercholesterolemia in primary care].

OBJECTIVE: To evaluate whether hypolipaemiant treatment in patients with hypercholesterolaemia (HC) corresponds to the recommendations in a primary care diagnosis and treatment protocol. DESIGN: Descriptive observational study. SETTING: Primary care centre. PATIENTS: 304 patients whose long-term prescription records recorded hypolipaemiant drugs. RESULTS: Average age was 62.7 and 198 were women. 176 presented simple HC and 125 mixed dyslipaemia. 46 had a history of ischaemic cardiopathy. Pre-treatment LDL-cholesterol (LDL-c) was recorded for 224; 168 of them had 2 or more cardiovascular risk factors. Of the 202 who had a record of post-treatment LDL-c, 163 were at level III of risk, which went down to 47 at 6 months. The values before and after medication were as follows: total cholesterol (TC) 290.3 mg/dl and 246.2 mg/dl; LDL-c 207.8 mg/dl and 166.0 mg/dl; HDL-cholesterol 50.5 mg/dl and 53.1 mg/dl; and triglycerides (TG) 156.1 mg/dl and 133.6 mg/dl. Mean reduction of TC and LDL-c was greater in the patients treated with statins that in those treated with fibrates. Decrease in TG was greater with fibrates. CONCLUSIONS: Treatment was initiated when LDL-c figures were over the limit specified in the protocol. In most cases this was because of the accumulation of cardiovascular risk factors.

Adult↗
PubMed1996

Characterization of dual leucine zipper-bearing kinase, a mixed lineage kinase present in synaptic terminals whose phosphorylation state is regulated by membrane depolarization via calcineurin.

The biochemistry and regulation of dual leucine zipper bearing kinase (DLK), a member of the mixed lineage kinase or MLK subfamily of protein kinases, was examined in the nervous system. DLK transcript expression in the nervous system was predominantly neuronal. DLK protein was present in synaptic terminals where it was associated with both plasma membrane and cytosol fractions. Within these two fractions, DLK had differing characteristics. Cytosolic DLK existed in both a phosphorylated and dephosphorylated state; DLK associated with plasma membrane existed in the dephosphorylated state only. On nonreducing SDS-polyacrylamide gel electrophoresis, cytosolic DLK migrated at 130 kDa, while membrane associated DLK migrated with an apparent Mr >/= 260,000. Similarly, DLK transiently expressed in COS 7 cells autophosphorylated in vivo and migrated at approximately 260 kDa when separated by nonreducing SDS-polyacrylamide gel electrophoresis. In cotransfection experiments, FLAG-tagged DLK or a FLAG-tagged truncated DLK mutant (F-Delta520) was coimmunoprecipitated with Myc-tagged DLK and formed complexes under nonreducing conditions consistent with the conclusion that DLK formed covalently associated homodimers in overexpressing COS 7 cells. In aggregating neuronal-glial cultures, depolarization of plasma membrane lead to dephosphorylation of DLK. Treatment of aggregates with 5 nM or 200 nM okadaic acid lead to a shift in electrophoretic mobility consistent with phosphorylation of DLK. Treatment with cyclosporin A, a specific inhibitor of the calcium/calmodulin-dependent protein phosphatase 2B (calcineurin), had no effect on DLK phosphorylation under basal conditions. However, cyclosporin A completely inhibited DLK dephosphorylation upon membrane depolarization.

Animals↗
PubMed1996

Differential expression of Na,K-ATPase isoforms in oligodendrocytes and astrocytes.

Using Western blot and immunocytochemistry to detect protein, and in situ hybridization and Northern blot to detect RNA, we studied the expression of Na,K-ATPase subunit isoforms in cultured astrocytes and oligodendrocytes in vitro, and in glial cells in the brain. We found that astrocytes in culture express alpha 1 and alpha 2 isoforms of the catalytic subunit and both beta 1 and beta 2 isoforms of the regulatory subunit. Oligodendrocytes in culture express only alpha 1, but in vivo high levels of alpha 2 are seen in oligodendrocytes of white matter tracts. These results suggest that expression of the alpha 2 isoform in mature oligodendrocytes may be regulated by axonal contact and related to myelination.

Animals↗