RESUMEN

This review focuses on the impact of bacteriophages on the manufacture of dairy foods. Firstly, the impact of phages of lactic acid bacteria in the dairy industry, where they are considered enemies, is discussed. The sources of phage contamination in dairy plants are detailed, with special emphasis on the rise of phage infections related to the growing use of cheese whey as ingredient. Other topics include traditional and new methods of phage detection, quantification and monitoring, and strategies of phage control in dairy plants, either of physical, chemical or biological nature. Finally, the use of phages or purified phage enzymes as allies to control pathogenic bacteria in the food industry is reviewed.

Asunto(s)

Bacteriófagos/aislamiento & purificación , Productos Lácteos/virología , Bacteriófagos/clasificación , Bacteriófagos/genética , Productos Lácteos/microbiología , Industria de Alimentos , Lactobacillales/metabolismo , Lactobacillales/virología

RESUMEN

Neuroblastoma is an embryonic tumour of the sympathetic nervous system and is one of the most common cancers in childhood. A high differentiation stage has been associated with a favourable outcome; however, the mechanisms governing neuroblastoma cell differentiation are not completely understood. The MYCN gene is considered the hallmark of neuroblastoma. Even though it has been reported that MYCN has a role during embryonic development, it is needed its decrease so that differentiation can be completed. We aimed to better define the role of MYCN in the differentiation processes, particularly during the early stages. Considering the ability of MYCN to regulate non-coding RNAs, our hypothesis was that N-Myc protein might be necessary to activate differentiation (mimicking embryonic development events) by regulating miRNAs critical for this process. We show that MYCN expression increased in embryonic cortical neural precursor cells at an early stage after differentiation induction. To investigate our hypothesis, we used human neuroblastoma cell lines. In LAN-5 neuroblastoma cells, MYCN was upregulated after 2 days of differentiation induction before its expected downregulation. Positive modulation of various differentiation markers was associated with the increased MYCN expression. Similarly, MYCN silencing inhibited such differentiation, leading to negative modulation of various differentiation markers. Furthermore, MYCN gene overexpression in the poorly differentiating neuroblastoma cell line SK-N-AS restored the ability of such cells to differentiate. We identified three key miRNAs, which could regulate the onset of differentiation programme in the neuroblastoma cells in which we modulated MYCN. Interestingly, these effects were accompanied by changes in the apoptotic compartment evaluated both as expression of apoptosis-related genes and as fraction of apoptotic cells. Therefore, our idea is that MYCN is necessary during the activation of neuroblastoma differentiation to induce apoptosis in cells that are not committed to differentiate.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Diferenciación Celular , Neuroblastoma/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/metabolismo , Apoptosis , Biomarcadores de Tumor/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/metabolismo , Proteína Proto-Oncogénica N-Myc , Células-Madre Neurales/metabolismo , Neuroblastoma/genética , Neuroblastoma/patología , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Interferencia de ARN , Factores de Tiempo , Transfección , Tretinoina/farmacología

RESUMEN

UNLABELLED: Two bacteriophages, isolated from faeces, were assayed as biocontrol agents of pathogenic Escherichia coli during milk fermentation. Phage DT1 was tested on the strain E. coli DH5α, one enteropathogenic E. coli (EPEC) strain and one Shiga toxigenic E. coli O157:H7 (STEC) strain. Phage DT6 was tested on two STEC strains (O157:H7 and non-O157). One additional assay was performed by using a cocktail of both phages against the O157:H7 STEC strain. Streptococcus thermophilus 10-C, the strain used as lactic starter, reached 10(9)  CFU ml(-1) after 4 h, while pH values fell to 4·5 after 8 h, regardless of the presence of E. coli strains and/or phages. In absence of phages, E. coli strains reached 4-6 log CFU ml(-1) at 5-6 h. Escherichia coli DH5α and O157:H7 STEC strains were rapidly and completely inactivated by phage DT1 and phage cocktail, respectively, while O157:H7 STEC was completely inactivated either by DT1 or by DT6, after 8 h. The EPEC strain was not detected at 1 h (<10 CFU ml(-1) ) but grew afterwards, though at lower rates than without phage. For non-O157:H7 STEC, reductions lower than 1 log CFU ml(-1) were observed for all sampling times. Phages DT1 and DT6, either individually or as a cocktail, effectively reduce O157:H7 STEC counts during milk fermentation, without compromising the starter culture performance. SIGNIFICANCE AND IMPACT OF THE STUDY: Coliphages DT1 and DT6, isolated from faeces and selected on the basis of their host range, showed to be valuable tools for the control of pathogenic Escherichia coli during milk fermentation, without compromising the starter culture performance. Both phages, either individually or as a cocktail, may function as an extra safety barrier beyond traditional pasteurization, effectively reducing O157:H7 Shiga toxin-producing Escherichia coli (STEC) counts during early growth, thus avoiding Shiga toxin production and accumulation.

Asunto(s)

Agentes de Control Biológico , Colifagos , Escherichia coli O157/virología , Heces/virología , Leche/microbiología , Escherichia coli Shiga-Toxigénica/virología , Animales , Bovinos , Escherichia coli O157/crecimiento & desarrollo , Fermentación , Escherichia coli Shiga-Toxigénica/crecimiento & desarrollo , Streptococcus thermophilus/crecimiento & desarrollo

RESUMEN

Temperate bacteriophages ф iLp84 and ф iLp1308, previously isolated from mitomycin C-induction of Lactobacillus paracasei strains 84 and CNRZ1308, respectively, were tested for their resistance to several physical and chemical treatments applied in dairy industry. Long-term survival at 4 °C, -20 °C and -80 °C, resistance to either thermal treatments of 63 °C, 72 °C and 90 °C, high pressure homogenization (HPH, 100 MPa) or classic (ethanol, sodium hypochlorite and peracetic acid) and new commercial sanitizers, namely A (quaternary ammonium chloride), B (hydrogen peroxide, peracetic acid and peroctanoic acid), C (alkaline chloride foam), D (p-toluensulfonchloroamide, sodium salt) and E (ethoxylated nonylphenol and phosphoric acid), were determined. Phages were almost completely inactivated after eight months of storage at 25 °C, but viability was not affected at 4 °C, -20 °C or -80 °C. Both phages tolerated well HPH treatments. Phage iLp1308 showed higher thermal resistance than ф iLp84, but neither resisted 90 °C for 2 min. Best chemical inactivation was accomplished using peracetic acid or biocides A, C and E, whereas biocides B and D were completely ineffective. These results help to improve selection of chemical agents and physical treatments to effectively fight against phage infections in dairy plants.

Asunto(s)

Bacteriófagos/química , Bacteriófagos/efectos de los fármacos , Desinfectantes/farmacología , Lactobacillus/virología , Esterilización/métodos , Bacteriófagos/crecimiento & desarrollo , Contaminación de Alimentos/prevención & control , Microbiología de Alimentos , Calor , Presión , Inactivación de Virus/efectos de los fármacos

RESUMEN

AIMS: To isolate and characterize bacterial strains derived from Lactobacillus casei and Lactobacillus paracasei strains and resistant to phage MLC-A. METHODS AND RESULTS: Two of nine assayed strains rendered resistant mutants with recovery efficiencies of 83% (Lact. paracasei ATCC 27092) and 100% (Lact. casei ATCC 27139). DNA similarity coefficients (RAPD-PCR) confirmed that no significant genetic changes occurred while obtaining resistant mutants. Neither parent nor mutant strains spontaneously released phages. Phage-resistant mutants were tested against phages PL-1, J-1, A2 and MLC-A8. Lactobacillus casei ATCC 27092 mutants showed, overall, lower phage resistance than Lact. paracasei ATCC 27092 ones, but still higher than that of the parent strain. Lactobacillus paracasei ATCC 27092 mutants moderately adsorbed phage MLC-A only in calcium presence, although their parent strain successfully did it with or without calcium. Adsorption rates for Lact. casei ATCC 27139 and its mutants were highly influenced by calcium. Again, phage adsorption was higher on the original strain. CONCLUSIONS: Several isolates derived from two Lact. casei and Lact. paracasei strains showed resistance to phage MLC-A but also to other Lact. casei and Lact. paracasei phages. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights isolation of spontaneous bacteriophage-resistant mutants from Lact. casei and Lact. paracasei as a good choice for use in industrial rotation schemes.

Asunto(s)

Bacteriófagos/fisiología , Lacticaseibacillus casei/aislamiento & purificación , Lactobacillus/aislamiento & purificación , Tipificación de Bacteriófagos , Calcio/metabolismo , ADN Bacteriano/genética , Lactobacillus/genética , Lactobacillus/virología , Lacticaseibacillus casei/genética , Lacticaseibacillus casei/virología , Mutación , Fenotipo , Técnica del ADN Polimorfo Amplificado Aleatorio , Internalización del Virus

RESUMEN

The term trophic is widely used to indicate a general pro-survival action exerted on target cells by different classes of extracellular messengers, including neurotrophins (NTs), a family of low-molecular-weight proteins whose archetypal member is the nerve growth factor (NGF). The pro-survival action exerted by NTs results from a coordinated activation of multiple metabolic pathways, some of which have only recently come to light. NGF has been shown to exert a number of different, experimentally distinguishable effects on neurons, such as survival, differentiation of target neurons, growth of nerve fibers and their guidance (tropism) toward the source of its production. We have proposed a more complete definition of the NGF trophic action that should also include its newly discovered property of inhibiting the amyloidogenic processing of amyloid precursor protein (APP), which is among the first hypothesized primary trigger of Alzheimer's disease (AD) pathogenesis. This inhibitory action appears to be mediated by a complex series of molecular events and by interactions among NGF receptors (TrkA and p75), APP processing and tau metabolic fate and function.

Asunto(s)

Precursor de Proteína beta-Amiloide/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Apoptosis , Factor de Crecimiento Nervioso/farmacología , Factores de Crecimiento Nervioso/farmacología , Neuronas/citología , Neuronas/metabolismo , Ratas , Receptor trkA/metabolismo , Receptor trkA/fisiología

RESUMEN

AIMS: To evaluate the phage diversity in the environment of a dairy industry which manufactures a product fermented with a probiotic strain of Lactobacillus paracasei. METHODS AND RESULTS: Twenty-two Lact. paracasei phages were isolated from an industrial plant that manufactures a probiotic dairy product. Among them, six phages were selected based on restriction profiles, and two phages because of their notable thermal resistance during sample processing. Their morphology, host range, calcium dependency and thermal resistance were investigated. All phages belonged to the Siphoviridae family (B1 morphotype), were specific for Lact. casei and paracasei strains showing identical host spectrum, and only one phage was independent of calcium for completing its lytic cycle. Some of the phages showed an extraordinary thermal resistance and were protected by a commercial medium and milk. CONCLUSIONS: Phage diversity in a probiotic product manufacture was generated to a similar or greater extent than during traditional yogurt or cheese making. SIGNIFICANCE AND IMPACT OF THE STUDY: This work emphasizes probiotic phage infections as a new ecological situation beyond yogurt or cheese manufactures, where the balanced coexistence between phages and strains should be directed toward a favourable state, thus achieving a successful fermentation.

Asunto(s)

Bacteriófagos/aislamiento & purificación , Industria Lechera , Lactobacillus/virología , Apoptosis/efectos de los fármacos , Bacteriófagos/genética , Bacteriófagos/crecimiento & desarrollo , Calcio/farmacología , ADN Viral/análisis , Electroforesis en Gel de Agar , Microbiología Ambiental , Manipulación de Alimentos , Calor , Cinética , Microscopía Electrónica , Mapeo Restrictivo , Esterilización/métodos

RESUMEN

The present study shows that increased Abeta production in hippocampal neurons, due to a failure of NGF signal, induces an unexpected phosphorylation of tyrosine kinase receptor A (TrkA), followed by activation of the phospholipase C gamma (PLCgamma) pathway and neuronal death. Such phosphorylation seems causally connected with 2 kinases known be involved in amyloidogenesis, Src and CDK5, and associated with alpha and gamma secretase-mediated p75 processing. Pharmacologic inhibition of TrkA phosphorylation and partial silencing of TrkA and/or p75 receptors prevent PLCgamma activation and protect neurons from death. Concomitantly with these events, TrkA, p75, Abeta peptides, and PS1 protein coimmunoprecipitate, suggesting their direct interplay in the subsequent onset of apoptotic death. Together, these findings depict a cellular mechanism whereby the same cellular transducing system may invert its intracellular message from trophic and antiapoptotic to a death signaling, which could also have relevance in the onset of Alzheimer's disease.

Asunto(s)

Péptidos beta-Amiloides/metabolismo , Apoptosis , Receptor trkA/metabolismo , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Núcleo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quinasa 5 Dependiente de la Ciclina/metabolismo , Activación Enzimática/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Hipocampo/citología , Inmunoprecipitación , Factor de Crecimiento Nervioso/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/enzimología , Fosfolipasa C gamma/metabolismo , Fosforilación/efectos de los fármacos , Presenilina-1/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Familia-src Quinasas/metabolismo

RESUMEN

The wish of this work is the study of the effect of electromagnetic (EMF) radiations at a frequency of 50 Hz on the development of cerebellar granule neurons (CGN). Granule neurons, prepared from newborn rat cerebellum (8 days after birth), were cultured after plate-seeding in the presence of EMF radiations, with the plan of characterizing their cellular and molecular biochemistry, after exposure to the electromagnetic stimulus. Five days challenge to EMF radiations showed, by the cytotoxic glutamate (Glu) pulse test, a 30% decrease of cells survival, while only 5% of mortality was reported for unexposed sample. Moreover, blocking the glutamate receptor (GluR) with the Glu competitor MK-801, no toxicity effect after CGN challenge to EMF radiations and Glu was detected. By patch-clamp recording technique, the Kainate-induced currents from 6 days old exposed CGN exhibited a significant increase with respect to control cells. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses show that EMF exposure of rats CGN, induces a change in both GluRs proteins and mRNAs expression with respect to control. In addition, the use of monoclonal antibody raised against neurofilament protein (NF-200) reveals an increase in NF-200 synthesis in the exposed CGN. All these results indicate that exposure to non-ionizing radiations contribute to a premature expression of GluRs reducing the life span of CGN, leading to a more rapid cell maturation.

Asunto(s)

Diferenciación Celular/efectos de la radiación , Senescencia Celular/efectos de la radiación , Cerebelo/citología , Neuronas/citología , Neuronas/fisiología , Radiación , Animales , Animales Recién Nacidos , Western Blotting , Células Cultivadas , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas de Neurofilamentos/metabolismo , Neuronas/metabolismo , Ratas , Ratas Wistar , Distribución Tisular

RESUMEN

We show that treatment of cerebellar granules with interleukin-8 (IL-8), growth-related gene product beta (GRObeta) or AMPA induced activation of PI3-K/Akt and of ERK pathways, the latter being independent of PI3-K and dependent on PTX-sensitive G proteins. We also show that AMPA-mediated neuron survival was abolished both by ERK kinase inhibitor PD98059 and AMPA-Rs blocker CNQX, and that chemokine-mediated survival was blocked by the PI3-K inhibitors LY294002 and wortmannin. We conclude that the neurotrophic effects of AMPA need the contemporary activation of ERKs and stimulation of AMPA-Rs, and that PI3-K/Akt activation is a determinant pathway for the IL-8/GRObeta anti-apoptotic activity.

Asunto(s)

Cerebelo/citología , Quimiocinas CXC , Péptidos y Proteínas de Señalización Intercelular , Proteínas Serina-Treonina Quinasas , Receptores AMPA/fisiología , Receptores de Interleucina-8B/fisiología , Transducción de Señal , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Supervivencia Celular , Factores Quimiotácticos/farmacología , Activación Enzimática , Sustancias de Crecimiento/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas Wistar , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacología

RESUMEN

157Gd is a potential agent for neutron capture cancer therapy (GdNCT). We directly observed the microdistribution of Gd in cultured human glioblastoma cells exposed to Gd-diethylenetriaminepentaacetic acid (Gd-DTPA). We demonstrated, with three independent techniques, that Gd-DTPA penetrates the plasma membrane, and we observed no deleterious effect on cell survival. A systematic microchemical analysis revealed a higher Gd accumulation in cell nuclei compared with cytoplasm. This is significant for prospective GdNCT because the proximity of Gd to DNA increases the cell-killing potential of the short-range, high-energy electrons emitted during the neutron capture reaction. We also exposed Gd-containing cells to thermal neutrons and demonstrated the GdNC reaction effectiveness in inducing cell death. These results in vitro stimulated in vivo Gd-DTPA uptake studies, currently underway, in human glioblastoma patients.

Asunto(s)

Gadolinio/farmacocinética , Gadolinio/uso terapéutico , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Terapia por Captura de Neutrón , Muerte Celular/efectos de la radiación , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Gadolinio DTPA/farmacocinética , Gadolinio DTPA/toxicidad , Humanos , Isótopos , Espectrometría de Masas , Espectrometría por Rayos X , Células Tumorales Cultivadas

RESUMEN

One objection to using cell cultures for studying the proliferation of tumors is the potential for phenotypic changes that may occur in vitro. Here, we compared the antigen pattern expression of cultured meningioma cells with that of the primary tumor. Cell cultures established from 9 intracranial meningiomas and deparaffinized sections of the resected tumors were analyzed for immunophenotyping with the following antibodies: vimentin, cytokeratin, epithelial membrane antigen, S-100, neuron-specific enolase, synaptophisin, factor VIII-related antigen, CD4, CD31, CD34, CD45RB, CD68-PGM1, CD68-KP, and myeloid/histiocyte antigen (MAC387). Overall, the cultured meningioma cells retained the main feature of the primary tumor, being positive both for mesenchymal antigens and for epithelial antigens. Interestingly, the cultured meningioma cells abundantly expressed the CD68 antigens at early passage. The CD68 antigens, which are normally found on hematopoietic cells like macrophages and monocytes, were not detectable on meningioma cells in situ. Our results show that phenotypic changes on human meningioma cells may occur in vitro. This phenomenon suggests caution when transposing the in vitro results to the in vivo condition.

Asunto(s)

Neoplasias Meníngeas/genética , Meningioma/genética , Anciano , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Western Blotting , Femenino , Humanos , Inmunohistoquímica/métodos , Masculino , Neoplasias Meníngeas/metabolismo , Neoplasias Meníngeas/patología , Meningioma/metabolismo , Meningioma/patología , Persona de Mediana Edad , Fenotipo , Coloración y Etiquetado , Células Tumorales Cultivadas

RESUMEN

Boron neutron capture therapy (BNCT) is an experimental, binary treatment for brain cancer which requires as the first step that tumor tissue is targeted with a boron-10 containing compound. Subsequent exposure to a thermal neutron flux results in destructive, short range nuclear reaction within 10 microm of the boron compound. The success of the therapy requires than the BNCT agents be well localized in tumor, rather than healthy tissue. The MEPHISTO spectromicroscope, which performs microchemical analysis by x-ray absorption near edge structure (XANES) spectroscopy from microscopic areas, has been used to study the distribution of trace quantities of boron in human brain cancer tissues surgically removed from patients first administered with the compound Na2B12H11SH (BSH). The interpretation of XANES spectra is complicated by interference from physiologically present sulfur and phosphorus, which contribute structure in the same energy range as boron. We addressed this problem with the present extensive set of spectra from S, B, and P in relevant compounds. We demonstrate that a linear combination of sulfate, phosphate and BSH XANES can be used to reproduce the spectra acquired on boron-treated human brain tumor tissues. We analyzed human glioblastoma tissue from two patients administered and one not administered with BSH. As well as weak signals attributed to BSH, x-ray absorption spectra acquired from tissue samples detected boron in a reduced chemical state with respect to boron in BSH. This chemical state was characterized by a sharp absorption peak at 188.3 eV. Complementary studies on BSH reference samples were not able to reproduce this chemical state of boron, indicating that it is not an artifact produced during sample preparation or x-ray exposure. These data demonstrate that the chemical state of BSH may be altered by in vivo metabolism.

Asunto(s)

Borohidruros/metabolismo , Terapia por Captura de Neutrón de Boro , Boro/análisis , Neoplasias Encefálicas/radioterapia , Glioblastoma/radioterapia , Compuestos de Sulfhidrilo/metabolismo , Borohidruros/análisis , Borohidruros/química , Borohidruros/uso terapéutico , Boro/química , Boro/metabolismo , Compuestos de Boro/análisis , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Humanos , Microtomía , Análisis Espectral , Compuestos de Sulfhidrilo/análisis , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/uso terapéutico , Azufre , Rayos X

RESUMEN

The functional expression of the seven-transmembrane domain G protein-coupled chemokine receptor CXCR-4/fusin in rat nerve cell was demonstrated by staining with a polyclonal anti-CXCR-4 Ab, and by evaluating the calcium responses to the physiological agonist stromal-derived cell factor-1alpha (SDF-1alpha) in both cerebellar granule cells in culture and Purkinje neurons (PNs) in cerebellar slices. Cerebellar glial, granule and Purkinje cells showed a pronounced staining for CXCR-4. Furthermore, cultured granule cells exhibited Ca2+ transients elicited by the application of SDF-1alpha, both in cell bodies and in neuronal processes. Whole-cell patch-clamped PNs in cerebellar slices responded to SDF-1alpha application by a slow inward current followed by an increase of both intracellular Ca2+ level and spontaneous synaptic activity. In particular, the SDF-1alpha-induced slow inward current was considerably reduced by ionotropic glutamate receptor blockers, but developed fully in a medium in which synaptic transmission was inhibited, indicating that this current might be, at least in part, mediated by extrasynaptic glutamate, possibly released from the surrounding glial and/or nerve cells. Taken together, these findings indicate a functional involvement of CXCR-4 in the modulation of synaptic transmission, adding another member to the repertoire of the chemokine receptors exerting a neuromodulatory role in the cerebellum.

Asunto(s)

Quimiocinas CXC/farmacología , Células de Purkinje/fisiología , Transmisión Sináptica/efectos de los fármacos , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Anticuerpos , Calcio/metabolismo , Células Cultivadas , Quimiocina CXCL12 , Electrofisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Microglía/química , Microglía/citología , Microglía/fisiología , Microscopía Confocal , Neuroinmunomodulación/fisiología , Neuronas/química , Neuronas/citología , Neuronas/fisiología , Células de Purkinje/química , Células de Purkinje/citología , Ratas , Ratas Wistar , Receptores CXCR4/análisis , Receptores CXCR4/inmunología , Transmisión Sináptica/fisiología , Tetrodotoxina/farmacología

RESUMEN

Cultured cerebellar granule neurons are widely used as a cellular model to study mechanisms of neuronal cell death because they undergo programmed cell death when switched from a culture medium containing 25 mM to one containing 5 mM K(+). We have found that the growth-related gene product beta (GRObeta) partially prevents the K(+)-depletion-induced cell death, and that the neuroprotective action of GRObeta on granule cells is mediated through the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type of ionotropic glutamate receptors. GRObeta-induced survival was suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione, which is a specific antagonist of AMPA/kainate receptors; it was not affected by the inhibitor of N-methyl-D-aspartate receptors, 2-amino-5-phosphonopentanoic acid, and was comparable to the survival of granule cells induced by AMPA (10 microM) treatment. Moreover, GRObeta-induced neuroprotection was abolished when granule cells were treated with antisense oligonucleotides specific for the AMPA receptor subunits, which significantly reduced receptor expression, as verified by Western blot analysis with subunit-specific antibodies and by granule cell electrophysiological sensitivity to AMPA. Our data demonstrate that GRObeta is neurotrophic for cerebellar granule cells, and that this activity depends on AMPA receptors.

Asunto(s)

Apoptosis/efectos de los fármacos , Benzodiazepinas , Corteza Cerebelosa/efectos de los fármacos , Quimiocinas CXC , Factores Quimiotácticos/farmacología , Sustancias de Crecimiento/farmacología , Péptidos y Proteínas de Señalización Intercelular , Proteínas del Tejido Nervioso/fisiología , Fármacos Neuroprotectores/farmacología , Receptores AMPA/fisiología , 2-Amino-5-fosfonovalerato/farmacología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Ansiolíticos/farmacología , Corteza Cerebelosa/citología , ADN Complementario/genética , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-8/farmacología , Activación del Canal Iónico , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Oligodesoxirribonucleótidos Antisentido/farmacología , Potasio/farmacología , Potasio/fisiología , Ratas , Ratas Wistar , Receptores AMPA/efectos de los fármacos , Receptores AMPA/genética , Receptores de N-Metil-D-Aspartato/efectos de los fármacos

RESUMEN

UV/ozone ashing of thin tissue sections and cell cultures is a simple technique to enhance relative elemental concentrations, while maintaining their spatial location at the sub-micron level. This approach may enhance the capability of spatially resolved analysis techniques to detect the distribution of trace elements in biological matrices. We present results from light microscopy and x-ray spectromicroscopy studies of tissues and cells demonstrating that the micro-structure is very well conserved. We show the signal enhancement resulting from the removal of carbon, which allows otherwise undetectable gadolinium to be mapped in cancer tissue for a novel neutron capture therapy.

Asunto(s)

Carbono , Glioblastoma/química , Meningioma/química , Microscopía Electrónica/métodos , Ozono , Espectrofotometría/métodos , Oligoelementos/análisis , Rayos Ultravioleta , Carbono/análisis , Neoplasias del Sistema Nervioso Central/química , Gadolinio/análisis , Humanos , Neoplasias Meníngeas/química , Ozono/química , Células Tumorales Cultivadas

RESUMEN

The pheochromocytoma PC12 cell line that develops neuronal characteristics of sympathetic cells after treatment with nerve growth factor (NGF) represents a well-established cellular model system for studying NGF signalling. Interesting information on the different mechanistic pathways of NGF can be obtained by adopting the pharmacological approach of inhibiting P2 receptors, expressed in naive PC12 cells and recognised as important biological mediators of neurotransmitters and growth factors. We show here that Basilen Blue, an antagonist of P2 receptor, reversibly prevents NGF-dependent neurite outgrowth with an IC(50) in the 5-10 microM range. Suramin, oxidised-ATP and diisothiocyanatostilbene-disulfonic acid, differently from other purinoceptor ligands, are also effective in this regard. NGF-dependent regeneration and stability of neurites, selected NGF-dependent extracellular and intracellular protein phosphorylations, binding of [(3)H] ATP to PC12 cell membranes are also modulated by Basilen Blue. On the contrary, cell adhesion, cellular duplication, 5'-nucleotidase activity, NGF-induced tyrosine autophosphorylation of TrkA receptors are not affected. NGF furthermore directly modulates the extracellular release of ATP and especially the levels of P2X(2) receptor protein in PC12 cells. In addition, extracellular ATP improves the neuritogenic effect of sub-optimal concentrations of NGF. Our study identifies P2 receptor ligands, particularly Basilen Blue, as useful tools to dissect different NGF-evoked functions, suggesting a mechanistic role for P2 receptors in the signalling pathways of NGF.

Asunto(s)

Factor de Crecimiento Nervioso/fisiología , Neuritas/fisiología , Antagonistas del Receptor Purinérgico P2 , 5'-Nucleotidasa/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Autorradiografía , Western Blotting , Electroforesis en Gel de Poliacrilamida , Ligandos , Factor de Crecimiento Nervioso/farmacología , Neuritas/efectos de los fármacos , Células PC12 , Fosforilación , Pruebas de Precipitina , Unión Proteica , Ratas , Receptor trkA/metabolismo

RESUMEN

The growth-related gene product beta (GRObeta) is a small chemoattractant cytokine that belongs to the CXC chemokine family, and GRObeta receptors are expressed in the brain, including the cerebellum. We demonstrate that rat cerebellar granule neurones express the GRObeta receptor CXCR2. We also show that, in addition to the known stimulation of a phosphoinositide-specific phospholipase C, GRObeta activates both neutral (N-) and acidic (A-) sphingomyelinases (SMase) and the stress-activated c-Jun N-terminal kinase 1 (JNK1). Although both exogenous ceramide and bacterial SMase stimulate JNK1, GRObeta-induced JNK1 activation is an event probably independent of ceramide generated by A-SMase, since it is maintained in the presence of compounds that block A-SMase activity. This is the first report on the activation of the SMase pathway by chemokines.

Asunto(s)

Cerebelo/metabolismo , Quimiocinas CXC , Factores Quimiotácticos/metabolismo , Sustancias de Crecimiento/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/metabolismo , Esfingomielinas/metabolismo , Animales , Factores Quimiotácticos/genética , Activación Enzimática , Regulación de la Expresión Génica , Sustancias de Crecimiento/genética , Hidrólisis , Proteínas Quinasas JNK Activadas por Mitógenos , Ratas , Ratas Wistar , Transducción de Señal

RESUMEN

We studied a new approach to cell ashing based on illuminating the specimens with a low-pressure mercury discharge lamp. We analyzed with synchrotron spectromicroscopy its effects on different physiological elements in neurobiological specimens. Our results demonstrate that carbon is removed, whereas phosphorus, calcium, potassium, and sulfur are retained and their relative concentrations are enhanced. Applied to trace elements, this technique will enhance their practical detectability.

Asunto(s)

Técnicas de Química Analítica/métodos , Neuronas/química , Oligoelementos/análisis , Animales , Células Cultivadas , Aumento de la Imagen , Ozono/química , Ratas , Espectrofotometría Ultravioleta

RESUMEN

We quantified the effect of the excitatory amino acids kainate and glutamate on the uptake of cobalt in primary rat cerebellar granule neurons, by using inductively coupled plasma-atomic emission spectroscopy (ICP-AES). We quantitatively demonstrated that Co2+ uptake, although enhanced by glutamate and kainate also takes place in the absence of excitatory amino acids. We also found that cobalt uptake is not significantly altered by the presence of glutamate receptor competitive or noncompetitive antagonists, indicating that cobalt uptake in granule neurons does not require glutamate receptor stimulation. Our results suggest, therefore, that Co2+ may enter the cell by passive diffusion through the plasma membrane.

Asunto(s)

Cerebelo/metabolismo , Cobalto/metabolismo , Aminoácidos Excitadores/farmacología , Animales , Células Cultivadas , Cerebelo/citología , Cerebelo/efectos de los fármacos , Ácido Glutámico/farmacología , Ácido Kaínico/farmacología , Ratas , Espectrometría por Rayos X