RESUMEN
Consistency in gold chloride staining is essential for anatomical analysis of sensory nerve endings. The gold chloride stain for this purpose has been modified by many investigators, but often yields inconsistent staining, which makes it difficult to differentiate structures and to determine nerve ending distribution in large tissue samples. We introduce additional steps and major changes to the modified Gairns' protocol. We controlled the temperature and mixing rate during tissue staining to achieve consistent staining and complete solution penetration. We subjected samples to sucrose dehydration to improve cutting efficiency. We then exposed samples to a solution containing lemon juice, formic acid and paraformaldehyde to produce optimal tissue transparency with minimal tissue deformity. We extended the time for gold chloride impregnation 1.5 fold. Gold chloride was reduced in the labrum using 25% formic acid in water for 18 h and in the capsule using 25% formic acid in citrate phosphate buffer for 2 h. Citrate binds gold nanoparticles, which minimizes aggregation in the tissue. We stored samples in fresh ultrapure water at 4° C to slow reduction and to maintain color contrast in the tissue. Tissue samples were embedded in Tissue Tek and sectioned at 80 and 100 µm instead of using glycerin and teasing the tissue apart as in Gairns' modified gold chloride method. We attached sections directly to gelatin subbed slides after sectioning with a cryostat. The slides then were processed and coverslipped with Permount. Staining consistency was demonstrated throughout the tissue sections and neural structures were clearly identifiable.
Asunto(s)
Compuestos de Oro/química , Células Receptoras Sensoriales/química , Coloración y Etiquetado , Femenino , Humanos , Masculino , Persona de Mediana Edad , Escleroproteínas/química , Células Receptoras Sensoriales/citología , Hombro/anatomía & histologíaRESUMEN
In the article is conducted assessment of the dental health of adults of Zaporozhye on the basis of epidemiological investigation. It is established that despite of the diversity of all diseases that were revealed during examination, adult population seeks dental help in the event of acute pain. It is shown, that there is dependence of level negotiability for dental help from social status, education and income.
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Servicios de Salud Dental/estadística & datos numéricos , Encuestas de Salud Bucal , Satisfacción del Paciente/estadística & datos numéricos , Clase Social , Odontalgia/prevención & control , Adulto , Femenino , Humanos , Masculino , Enfermedades Estomatognáticas/epidemiología , Ucrania/epidemiologíaRESUMEN
AIM: To study relationship between Yersinia pseudotuberculosis cultivation temperature and immunobiological properties of lypopolysaccharide (LPS). MATERIALS AND METHODS: LPS producing strain--Yersinia pseudotuberculosis 164/84 OX; experimental animal--guinea pigs, rabbits; methods--immunochemical (indirect hemagglutionation assay, RDP), physico-chemical (electrophoresis, gas-liquid chromatography), standard biochemical and statistical methods. RESULTS: It was established that increase of Yersinia pseudotuberculosis cultivation temperature from 10 degrees C to 37 degrees C was associated with attenuation of LPS toxicity in guinea pigs, decrease of its immunochemical activity and contents and degree of acylation of 3OH-14:0 hydoxyacid as well as amount of electrophoretically detected O-side chains. It was assumed that the polysaccharide component plays its role in LPS toxicity. CONCLUSION: Relation between Yersinia pseudotuberculosis cultivation temperature and structural and functional properties of LPS preparations obtained from the culture was determined.
Asunto(s)
Lípido A/inmunología , Yersinia pseudotuberculosis/crecimiento & desarrollo , Yersinia pseudotuberculosis/inmunología , Acilación , Oxidorreductasas de Alcohol/metabolismo , Animales , Precipitación Química , Cobayas , Sueros Inmunes/inmunología , Dosificación Letal Mediana , Lípido A/química , Lípido A/toxicidad , Conejos , TemperaturaRESUMEN
Physical rehabilitation with endurance exercise for patients with Parkinson's disease has not been well established, although some clinical and laboratory reports suggest that exercise may produce a neuroprotective effect and restore dopaminergic and motor functions. In this study, we used a chronic mouse model of Parkinsonism, which was induced by injecting male C57BL/6 mice with 10 doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (25 mg/kg) and probenecid (250 mg/kg) over 5 weeks. This chronic parkinsonian model displays a severe and persistent loss of nigrostriatal neurons, resulting in robust dopamine depletion and locomotor impairment in mice. Following the induction of Parkinsonism, these mice were able to sustain an exercise training program on a motorized rodent treadmill at a speed of 18 m/min, 0 degrees of inclination, 40 min/day, 5 days/week for 4 weeks. At the end of exercise training, we examined and compared their cardiorespiratory capacity, behavior, and neurochemical changes with that of the probenecid-treated control and sedentary parkinsonian mice. The resting heart rate after 4 weeks of exercise in the chronic parkinsonian mice was significantly lower than the rate before exercise, whereas the resting heart rate at the beginning and 4 weeks afterward in the control or sedentary parkinsonian mice was unchanged. Exercised parkinsonian mice also recovered from elevated electrocardiogram R-wave amplitude that was detected in the parkinsonian mice without exercise for 4 weeks. The values of oxygen consumption, carbon dioxide production, and body heat generation in the exercised parkinsonian mice before and during the Bruce maximal exercise challenge test were all significantly lower than that of their sedentary counterparts. Furthermore, the exercised parkinsonian mice demonstrated a greater mass in the left ventricle of the heart and an increased level of citrate synthase activity in the skeletal muscles. The amphetamine-induced, dopamine release-dependent locomotor activity was markedly inhibited in the sedentary parkinsonian mice and was also inhibited in the exercised parkinsonian mice. Finally, neuronal recovery from the loss of nigrostriatal tyrosine hydroxylase expression and dopamine levels in the severe parkinsonian mice after exercise was not evident. Taken all together, these data suggest that 4 weeks of treadmill exercise promoted physical endurance, resulting in cardiorespiratory and metabolic adaptations in the chronic parkinsonian mice with severe neurodegeneration without demonstrating a restorative potential for the nigrostriatal dopaminergic function.
Asunto(s)
Técnicas de Ejercicio con Movimientos/métodos , Frecuencia Cardíaca/fisiología , Degeneración Nerviosa/etiología , Enfermedad de Parkinson/rehabilitación , Resistencia Física/fisiología , Respiración , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Animales , Conducta Animal , Calorimetría Indirecta/métodos , Citrato (si)-Sintasa/metabolismo , Modelos Animales de Enfermedad , Dopamina/metabolismo , Electrocardiografía/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Actividad Motora/fisiología , Neostriado/metabolismo , Enfermedad de Parkinson/complicaciones , Enfermedad de Parkinson/etiología , Enfermedad de Parkinson/patología , Probenecid/toxicidad , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
A lot of attention has been paid lately to the study of risk factors of cardiovascular diseases in patients with diabetes mellitus type 2 (DM2). Numerous investigations have revealed a connection between the intensity of thrombus formation and carbohydrate exchange disturbances. In the present paper, the dependence of the levels of Willebrand factor--glycoproteins IIb/IIIa complex on the clinical forms of coronary artery disease (I-II functional class stenocardia; old myocardial infarction) and lower limb ischemic disease (intermittent claudication) in patients with DM2 were studied. The comparison group consisted of DM2 patients without clinical manifestations of coronary artery disease or lower limb ischemic disease; the control group consisted of practically healthy individuals. The study found that the level of Willebrand factor--glycoproteids IIb/IIIa complex was significantly higher in all DM2 patients vs. healthy controls.
Asunto(s)
Trombosis Coronaria/sangre , Diabetes Mellitus Tipo 2/sangre , Claudicación Intermitente/sangre , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Factor de von Willebrand/metabolismo , Adulto , Anciano , Biomarcadores/sangre , Trombosis Coronaria/complicaciones , Trombosis Coronaria/diagnóstico , Diabetes Mellitus Tipo 2/complicaciones , Electrocardiografía Ambulatoria , Prueba de Esfuerzo , Femenino , Humanos , Claudicación Intermitente/complicaciones , Claudicación Intermitente/diagnóstico , Masculino , Persona de Mediana Edad , Pronóstico , Índice de Severidad de la Enfermedad , Espectrofotometría , Ultrasonografía Doppler DúplexRESUMEN
The focus of this study was to determine whether minimal levels of exercise could halt the formation of diabetes-induced heart pathology. Seven-week-old male rats were divided into four groups: sedentary nondiabetic, exercise-trained non-diabetic, sedentary diabetic and exercise-trained diabetic. Individualised exercise programmes were based on the animal's tolerance, and continued for 7 weeks after the induction of diabetes. At the completion of the study, no differences were found in skeletal muscle citrate synthase activity between diabetic sedentary and exercise-trained rats, indicating that the exercise was low intensity. Diabetes-induced heart hypertrophy was not reversed with exercise as measured by heart-to-body weight ratios and EKG (R wave height). There was no statistical difference between groups in the response to an exercise stress test prior to the induction of diabetes. However, 4 weeks of diabetes resulted in a significant decrease in resting and post-stress test heart rates (9% and 20%, respectively), which remained depressed at week 7. The sedentary diabetic animals demonstrated an abnormal response during the recovery period of the EKG exercise test, which was not present in non-diabetic or exercise-trained diabetic animals. In conclusion, lowintensity exercise training improved the cardiac response to an exercise stress test in diabetic animals.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 1/fisiopatología , Prueba de Esfuerzo , Condicionamiento Físico Animal , Animales , Cardiomegalia/fisiopatología , Citrato (si)-Sintasa/metabolismo , Diabetes Mellitus Experimental/sangre , Angiopatías Diabéticas/fisiopatología , Electrocardiografía , Masculino , Ratas , Ratas Endogámicas , Ratas Sprague-Dawley , CarreraRESUMEN
Endothelial cell dysfunction (ECD) is emerging as a common denominator for diverse cardiovascular abnormalities associated with inhibition of endothelial nitric oxide (NO) synthase (eNOS). Elevated levels of asymmetric dimethylarginine (ADMA), a potent eNOS inhibitor, are common in renal failure and may contribute to ECD. Through DNA microarray screening of genes modulated in human umbilical vein endothelial cells (HUVEC) by N(G)-nitro-l-arginine methyl ester (l-NAME), we found a 1.8-fold increase in low-density lipoprotein receptor-1 (LOX-1) expression. LOX-1 is a major endothelial receptor for oxidized low-density lipoproteins (OxLDL) and is assumed to play a role in the initiation and progression of atherosclerosis. Here, we confirmed the upregulation of LOX-1 mRNA and protein level by quantitative RT-PCR and Western blot analysis. Increased expression of LOX-1 was associated with the accumulation of DiI-labeled OxLDL (DiI-OxLDL) in ADMA- and l-NAME-pretreated HUVEC. To evaluate the contribution of LOX-1 in ADMA-induced accumulation of OxLDL by HUVEC, we used the competitive receptor inhibitor, soluble LOX-1. Treatment of HUVEC with soluble LOX-1 was associated with an approximately two- to threefold inhibition of DiI-OxLDL uptake in l-NAME- or ADMA-treated HUVEC. In conclusion, ADMA- or l-NAME-induced NO deficiency leads to the increased expression of LOX-1 mRNA and protein in HUVEC, which in turn results in the accumulation of OxLDL. Competition with LOX-1-soluble extracellular domain reduces OxLDL accumulation. In summary, elevated ADMA levels, i.e., in patients with renal failure, may be responsible for endothelial accumulation of OxLDL via upregulated LOX-1 receptor, thus contributing to endothelial lipidosis and dysfunction.
Asunto(s)
Arginina/análogos & derivados , Arginina/farmacología , Inhibidores Enzimáticos/farmacología , Fallo Renal Crónico/fisiopatología , Óxido Nítrico/deficiencia , Receptores de LDL/biosíntesis , Arteriosclerosis/fisiopatología , Western Blotting , Técnicas de Cultivo de Célula , Células Endoteliales/fisiología , Regulación de la Expresión Génica , Humanos , Metabolismo de los Lípidos , Óxido Nítrico Sintasa/farmacología , Receptores de LDL Oxidadas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptores Depuradores de Clase E , Regulación hacia ArribaRESUMEN
Intimal infiltration by monocytes and accumulation of lipids represent a critical step in the formation of fatty streaks during atherogenesis. Because elevated plasma levels of asymmetric dimethylarginine (ADMA), a potent nitric oxide (NO) synthase (NOS) inhibitor, are prevalent in diverse cardiovascular diseases, the goal of this study was to examine the contribution of NO deficiency to macrophage lipid accumulation. Inhibition of NO synthesis in PMA-primed human monocytic leukemia HL-60 cells resulted in a twofold increase in expression of the receptor for oxidized LDL (OxLDL), termed the lectin-like OxLDL receptor (LOX-1). Blockade of inducible NOS in activated macrophages resulted in 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-OxLDL accumulation and imparted macrophages with a foamy appearance as detected with oil-red O lipid staining. ADMA (15 microM) or N(G)-nitro-l-arginine methyl ester (l-NAME, 300 microM), both of which suppress inducible NOS activity, increased oil-red staining 1.9- and 2.8-fold, respectively. Macrophages treated with ADMA or l-NAME showed a 2.4-fold increase in accumulation of DiI-OxLDL. To examine the role of LOX-1 in this process, we used small interfering RNA (siRNA) duplex-mediated LOX-1 gene silencing. LOX-1 expression was suppressed twofold by siRNA as shown by Western blot analysis. This suppression was associated with a two- to fourfold decrease in DiI-OxLDL uptake as identified by fluorescence microscopy and decreased oil-red O staining by activated macrophages. In conclusion, accumulation of ADMA (a competitive inhibitor of NOS) in patients with chronic renal failure may be responsible for upregulation of LOX-1 receptor and increased OxLDL uptake, thus contributing to lipidosis and foam cell formation. The data illustrate an additional nonendothelial mode of antiatherogenic action of NO: prevention of LOX-1 induction and lipid accumulation by macrophages.
Asunto(s)
Arginina/análogos & derivados , Arginina/farmacología , Células Espumosas/citología , Macrófagos/citología , Macrófagos/metabolismo , Receptores de LDL/metabolismo , Diferenciación Celular , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Células HL-60 , Humanos , Lipoproteínas LDL/metabolismo , Macrófagos/fisiología , Metaloporfirinas/farmacología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/antagonistas & inhibidores , Donantes de Óxido Nítrico/farmacología , ARN Interferente Pequeño/farmacología , Receptores de Superficie Celular/metabolismo , Receptores de LDL/antagonistas & inhibidores , Receptores de LDL/genética , Venas Umbilicales/citología , Venas Umbilicales/metabolismo , Regulación hacia ArribaRESUMEN
Thrombin, the ultimate protease in the blood coagulation cascade, mediates its known cellular effects by unique proteolytic activation of G-protein-coupled protease-activated receptors (PARs), such as PAR1, PAR3, and PAR4, and a "tethered ligand" mechanism. PAR1 is variably expressed in subpopulations of neurons and largely determines thrombin's effects on morphology, calcium mobilization, and caspase-mediated apoptosis. In spinal cord motoneurons, PAR1 expression correlates with transient thrombin-mediated [Ca(2+)](i) flux, receptor cleavage, and elevation of rest [Ca(2+)](i) activating intracellular proteases. At nanomolar concentrations, thrombin retracts neurites via PAR1 activation of the monomeric, 21 kDa Ras G-protein RhoA, which is also involved in neuroprotection at lower thrombin concentrations. Such results suggest potential downstream targets for thrombin's injurious effects. Consequently, we employed several G-protein-specific modulators prior to thrombin exposure in an attempt to uncouple both heterotrimeric and monomeric G-proteins from motoneuronal PAR1. Cholera toxin, stimulating Gs, and lovastatin, which blocks isoprenylation of Rho, reduced thrombin-induced calcium mobilization. In contrast, pertussis toxin and mastoparan, inhibiting or stimulating G(o)/G(i), were found to exacerbate thrombin action. Effects on neuronal rounding and apoptosis were also detected, suggesting therapeutic utility may result from interference with downstream components of thrombin signaling pathways in human motor neuron disorders, and possibly other neurodegenerative diseases. Published 2001 John Wiley & Sons, Inc.
Asunto(s)
Apoptosis/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Hemostáticos/farmacología , Neuronas Motoras/metabolismo , Trombina/farmacología , Anticolesterolemiantes/farmacología , Inhibidores de Caspasas , Línea Celular , Toxina del Cólera/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas de Unión al GTP/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Lovastatina/farmacología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/ultraestructura , Neuritas/efectos de los fármacos , Neuritas/fisiología , Oligopéptidos/farmacología , Péptidos , Toxina del Pertussis , Receptor PAR-1 , Receptores de Trombina/metabolismo , Factores de Virulencia de Bordetella/farmacología , Venenos de Avispas/farmacologíaRESUMEN
BACKGROUND: Mechanisms underlying neurodegeneration are actively sought for new therapeutic strategies. Transgenic, knockout and genetic mouse models greatly aid our understanding of the mechanisms for neuronal cell death. A naturally occurring, autosomal recessive mutant, known as wobbler, and mice transgenic for familial amyotrophic lateral sclerosis (FALS) superoxide dismutase (SOD)1 mutations are available, but the molecular mechanisms remain equally unknown. Both phenotypes are detectable after birth. Wobbler is detectable in the third week of life, when homozygotes (wr/wr) exhibit prominent gliosis and significant motor neuron loss in the cervical, but not in lumbar, spinal cord segments. To address molecular mechanisms, we evaluated "death signals" associated with the multifunctional serine protease, thrombin, which leads to apoptotic motor neuronal cell death in culture by cleavage of a G-protein coupled, protease-activated receptor 1 (PAR-1). MATERIALS AND METHODS: Thrombin activities were determined with chromogenic substrate assays, Western immunoblots and immunohistochemistry were performed with anti-PAR-1 to observe localizations of the receptor and anti-GFAP staining was used to monitor astrocytosis. PAR-1 mRNA levels and locations were determined by reverse transcription polymerase chain reaction (qRT-PCR) and in situ hybridizations. Cell death was monitored with in situ DNA fragmentation assays. RESULTS: In preliminary studies we found a 5-fold increase in PAR-1 mRNA in cervical spinal cords from wr/wr, compared with wild-type (wt) littermates. Our current studies suggested that reactive astrocytosis and motor neuron cell death were causally linked with alterations in thrombin signaling. PAR-1 protein expression was increased, as demonstrated by immunocytochemistry and confirmed with in situ hybridization, in phenotypic wr/wr motor neurons, compared with wt, but not in astrocytes. This increase was much greater in cervical, compared with lumbar, segments, paralleling motor neuron degeneration. We also found, using reverse transcription polymerase chain reaction (qRT-PCR) with RNA from genotyped embryos, that PAR-1 was already increased in wr/wr cords at E12, the earliest time examined. CONCLUSIONS: Thus, motor neuron degeneration and death follows PAR-1 expression both temporally and topographically in wobbler mice. Since our culture studies show that thrombin mobilized [Ca2+]i by activating PAR-1, eventually leading to motor neuron apoptosis, up-regulation of PAR-1 during development may contribute both to "appropriate" as well as "inappropriate" neuronal death in wobbler.
Asunto(s)
Neuronas Motoras/fisiología , Receptores de Trombina/genética , Médula Espinal/patología , Transcripción Genética , Animales , Muerte Celular , Cruzamientos Genéticos , Sondas de ADN , Femenino , Genotipo , Proteína Ácida Fibrilar de la Glía/análisis , Gliosis , Homocigoto , Humanos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Enfermedad de la Neurona Motora/genética , Enfermedad de la Neurona Motora/fisiopatología , Neuronas Motoras/citología , Neuronas Motoras/patología , ARN Mensajero/análisis , Receptor PAR-1 , Médula Espinal/fisiopatología , Superóxido Dismutasa/genética , Trombina/metabolismoRESUMEN
Metal response element-binding transcription factor-1 (MTF-1) is a unique, zinc-inducible transcription factor that binds to metal response elements in the metallothionein promoter and activates transcription in response to metals and oxidative stress. MTF-1 contains six zinc fingers of the Cys(2)-His(2) type. It was previously shown that MTF-1 is reversibly activated to bind DNA in response to changes in zinc status, unlike other zinc finger transcription factors, which do not appear to be reversibly activated by zinc in the cellular environment. Here we show that zinc fingers 2-4 constitute the core DNA-binding domain, whereas fingers 5 and 6 appear to be unnecessary for DNA binding in vitro. Deletion of finger 1 resulted in a protein that bound DNA constitutively in vitro. Furthermore, transfer of MTF-1 finger 1 to a position immediately preceding the three zinc fingers of Sp1 resulted in a chimeric protein that required exogenous zinc to activate DNA binding in vitro, unlike native Sp1, which binds DNA constitutively. Transient transfection experiments demonstrated that intact MTF-1 activated a reporter 2.5-4-fold above basal levels after metal treatment in mouse MTF-1 knockout cells, Drosophila SL2 cells, and yeast. However, the metal response was lost in all three systems when finger 1 was deleted, but was unaffected by deletion of fingers 5 and 6. These data suggest that finger 1 of MTF-1 constitutes a unique metal-sensing domain that, in cooperation with the transactivation domains, produces a zinc-sensing metalloregulatory transcription factor.
Asunto(s)
ADN/metabolismo , Factores de Transcripción/química , Dedos de Zinc , Animales , Sitios de Unión , Células Cultivadas , Proteínas de Unión al ADN , Drosophila , Metalotioneína/genética , Ratones , Estrés Oxidativo , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae , Factor de Transcripción Sp1/metabolismo , Relación Estructura-Actividad , Activación Transcripcional , Factor de Transcripción MTF-1RESUMEN
Metal response element-binding transcription factor-1 (MTF-1) is a six-zinc finger protein that plays an essential role in activating metallothionein expression in response to the heavy metals zinc and cadmium. Low affinity interactions between zinc and specific zinc fingers in MTF-1 reversibly regulate its binding to the metal response elements in the mouse metallothionein-I promoter. This study examined the subcellular distribution and DNA binding activity of MTF-1 in cells treated with zinc or cadmium. Immunoblot analysis of cytosolic and nuclear extracts demonstrated that in untreated cells, about 83% of MTF-1 is found in the cytosolic extracts and is not activated to bind to DNA. In sharp contrast, within 30 min of zinc treatment (100 microM), MTF-1 is detected only in nuclear extracts and is activated to bind to DNA. The activation to bind to DNA and nuclear translocation of MTF-1 occurs in the absence of increased MTF-1 content in the cell. Furthermore, immunocytochemical localization and immunoblotting assays demonstrated that zinc induces the nuclear translocation of MTF-1-FLAG, expressed from the cytomegalovirus promoter in transiently transfected dko7 (MTF-1 double knockout) cells. Immunoblot analysis of cytosolic and nuclear extracts from cadmium-treated cells demonstrated that concentrations of cadmium (10 microM) that actively induce metallothionein gene expression cause only a small increase in the amount of nuclear MTF-1. In contrast, an overtly toxic concentration of cadmium (50 microM) rapidly induced the complete nuclear translocation and activation of DNA binding activity of MTF-1. These studies are consistent with the hypothesis that MTF-1 serves as a zinc sensor that responds to changes in cytosolic free zinc concentrations. In addition, these data suggest that cadmium activation of metallothionein gene expression may be accompanied by only small changes in nuclear MTF-1.
Asunto(s)
Cadmio/metabolismo , Núcleo Celular/metabolismo , Factores de Transcripción/metabolismo , Zinc/metabolismo , Animales , Especificidad de Anticuerpos , Transporte Biológico , Células Cultivadas , Citosol/metabolismo , Proteínas de Unión al ADN/metabolismo , Sueros Inmunes , Inmunohistoquímica , Ratones , Factores de Transcripción/inmunología , Factor de Transcripción MTF-1RESUMEN
Apoptosis, well-established in development and now also in degenerative disease, occurs with regularity in several cell compartments early after controlled contusion spinal cord injury (SCI). Cell death in astrocytic, microglial, and neuronal populations peaks at 3 days, while oligodendroglial apoptosis is found 10-14 days later. In this regard, the executioners of apoptosis, the caspase proteases, are also activated within 3 days of SCI. On the other hand, serine proteases, which have been shown to initiate apoptosis and activate caspases in culture models, have not been extensively studied in regards to nervous system trauma. As part of an ongoing effort to examine the spectrum of genes that are up- and downregulated in the injured rat spinal cord, we synthesized serine protease family specific primers to take advantage of conserved residues in the charge relay system and the codon preferences of these mammalian genes. These primers were then employed in a modified, family-specific differential mRNA display technique. One specific serine protease gene we found that was upregulated after injury was prothrombin. Qualitative and quantitative RT-PCR techniques indicated that this increase occurred early, already evident at 8 h after injury, and reached a maximum level fourfold above baseline at 24 h. Peak expression for prothrombin mRNA occurred prior to peak levels of apoptosis in astrocytic, microglial and neuronal compartments at 72 h. Of additional interest, gene database mining revealed that prothrombin shared approximately 48% similarity with myelencephalon-specific protease (MSP), a neurotoxic serine protease previously found to be increased two- to threefold at 3 days after excitotoxic SCI. Since thrombin induces apoptosis in murine and chick motor and rat hippocampal neurons by activating a member of the novel protease-activated receptor (PAR) gene family known as PAR-1, we also analyzed PAR-1 by similar techniques and found that it, too, was upregulated after SCI with the same kinetics as prothrombin. We confirmed these results with gene array analyses that revealed more than one trypsin subfamily serine protease was activated by SCI. They imply the possibility of using specific, tissue-directed serine protease inhibition at translational or transcriptional levels, and offer a potential paradigm shift in drug discovery for SCI to limit the extent of apoptosis, and consequent functional loss, in the human spinal cord.
Asunto(s)
Neurotoxinas/metabolismo , Protrombina/metabolismo , Receptores de Trombina/metabolismo , Serina Endopeptidasas/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Secuencias de Aminoácidos , Animales , Muerte Celular , Femenino , Expresión Génica , Familia de Multigenes , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor PAR-1 , Receptores de Trombina/genética , Lugares Marcados de Secuencia , Serina Endopeptidasas/genética , Transducción de Señal , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Trombina/fisiología , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Apoptosis, often also termed "programmed cell death", occurs in normal development in the brain and spinal cord. Important to concepts of disease and potential intervention is the exciting finding that apoptosis is also found after neurotrauma and in a number of neurodegenerative diseases. Although the precise mechanism of neuronal cell loss remains unknown, much emphasis has been placed recently on the activation of cell death protease cascades within the cell. How these cascades may be activated, especially from extracellular influences, is currently poorly understood. Thrombin, the multifunctional coagulation protease, is an early phase modulator at sites of tissue injury and has been shown to induce cell death in neurons by an apoptotic mechanism by activating its receptor, PAR-1. Using a model motor neuronal cell line, NSC19, which we have shown undergoes apoptosis after treatment with classic apoptosis inducers such as the topoisomerase inhibitors camptothecin and etoposide, we unambiguously found that nanomolar thrombin induced characteristic signs of apoptosis. Strikingly, endonucleolysis was accompanied by an increase in caspase-3-like activity in cellular extracts, which correlated with both detection of caspase-induced signature cleavage of the cortical cytoskeleton component nonerythroid spectrin (alpha-fodrin) and identification of increased accessibility of a caspase cleavage domain, using an antibody (Ab127) made against a synthetic peptide KGDEVD. Demonstrating that thrombin activation of death proteases was linked to cell death, we were able to inhibit thrombin-induced apoptosis by using a caspase family inhibitor, benzyloxycarbonyl-Asp-(oMe)-fluoromethyl ketone (Boc-D-FMK). These novel results demonstrate that thrombin serves as an extracellular "death signal" to activate intracellular protease pathways. These pathways lead to apoptotic cell death and can be modulated by inhibiting caspase activity downstream to PAR-1.
Asunto(s)
Apoptosis/fisiología , Endopeptidasas/fisiología , Espacio Extracelular/fisiología , Membranas Intracelulares/enzimología , Neuronas Motoras/fisiología , Transducción de Señal/fisiología , Trombina/fisiología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Línea Celular , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Fragmentación del ADN , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Ratones , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/enzimología , Trombina/farmacología , Inhibidores de Topoisomerasa I , Inhibidores de Topoisomerasa IIRESUMEN
Effects of two lectins, wheat germ agglutinin (WGA) and concanavalin A (Con A), on platelet functional reactions and interaction of lectins with the platelet membrane glycoproteins (GPs) have been studied. Both lectins stimulated platelet aggregation and secretion of serotonin from platelet dense granules. The effects of WGA and Con A were blocked by specific sugars, N-acetyl-D-glucosamine and alpha-methyl-D-mannopyranoside, respectively, by adenylate cyclase activator prostaglandin E1, and by anti-GP IIb-IIIa monoclonal antibody (monAB), CRC64, that inhibits platelet interaction with fibrinogen. The data indicate that both lectins interacting with the carbohydrate moiety on the platelet surface stimulated not passive agglutination but fibrinogen--GP IIb-IIIa-dependent platelet aggregation which is coupled with the secretion from granules and activation of the intracellular systems of signal transduction. However, there were significant differences between the stimulatory effects of WGA and Con A. WGA induced more pronounced and quick platelet aggregation and stimulated several times higher serotonin secretion than Con A. In addition, adhesion studies showed that plastic-adsorbed WGA appeared to be a nonadhesive substrate, whereas Con A effectively stimulated platelet adhesion. Unlike Con A-induced platelet aggregation, adhesion to Con A substrate was not inhibited by monAB CRC64, i.e., was not dependent on GP IIb-IIIa--fibrinogen interaction. Binding of lectins with major platelet GPs was studied using immobilized WGA and Con A and platelet lysate as a source of GPs. Platelet lysate was incubated with immobilized lectins and then binding of individual GPs was evaluated using specific mono- and polyclonal antibodies. WGA binds with GP Ib and P-selectin but not with other GPs tested. Interaction of Con A with platelet GPs was less specific. This lectin binds with GP IIb-IIIa, GP Ib, GP IV, and P-selectin. Although GP Ib appeared to be the main protein which bound WGA on platelet surface, anti-GP Ib antibodies failed to affect WGA-induced platelet aggregation, but inhibited WGA-induced agglutination of fixed platelets. Thus, interaction of the WGA with GP Ib could not be considered as a major stimulus initiating WGA-dependent platelet activation and aggregation.
Asunto(s)
Plaquetas/efectos de los fármacos , Concanavalina A/farmacología , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/metabolismo , Aglutininas del Germen de Trigo/farmacología , Plaquetas/citología , Plaquetas/metabolismo , Adhesión Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Humanos , Glicoproteínas de Membrana Plaquetaria/inmunología , Unión Proteica , Serotonina/metabolismoRESUMEN
Thrombin, the ultimate enzyme in the blood coagulation cascade, has prominent actions on various cells, including neurons. As in platelets, thrombin increases [Ca2+]i mobilization in neurons, and also retracts neurites. Both these effects are mediated through a G protein-coupled, proteolytically activated receptor for thrombin (PAR-1). Prolonged exposure to thrombin kills neurons via apoptosis, that may also involve PAR-1 activation. Increased [Ca2+]i has been a unifying mechanism proposed for cell death in several neurodegenerative diseases. Thrombin-elevated calcium levels may activate intracellular cascades in neurons leading to cell death. Since thrombin mediates its diverse effects on cells through both heterotrimeric and monomeric G proteins, we also explored what effect altering differential G protein coupling would have on the neuronal response to thrombin. We studied calcium mobilization by thrombin in a model motor neuronal cell line, NSC19, using fluorescence image analysis. Confirming effects in other neuronal types, thrombin caused dramatic increases in [Ca2+]i levels, both transiently and after prolonged exposure, which involved activation and cleavage of the PAR-1 receptor. Using enzyme linked immunosorbent assay (ELISA) and dot-blot analysis, we found that the N-terminal fragment of PAR-1 was released into the medium after exposure to thrombin. We confirmed that PAR-1 protein and mRNA expression occurred in motor neurons. We found that cholera toxin inhibited thrombin-mediated Ca2+ influx, pertussis toxin did not significantly alter thrombin action, and lovastatin, a small 21-kDa Ras GTPase (Rho) modulator, showed a tendency to reduce the thrombin effect. These data indicate that thrombin-increased [Ca2+]i, sufficient to trigger cell death in motor neurons, might be approached in vivo by modulating thrombin signaling through PAR-1.
Asunto(s)
Calcio/metabolismo , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Receptores de Trombina/metabolismo , Trombina/farmacología , Animales , Línea Celular , Toxina del Cólera/farmacología , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células Híbridas , Interfase/efectos de los fármacos , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Lovastatina/farmacología , Ratones , Toxina del Pertussis , Receptor PAR-1 , Receptores de Trombina/efectos de los fármacos , Trombina/antagonistas & inhibidores , Trombina/efectos de los fármacos , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
STUDY DESIGN: Serum withdrawal was introduced to a spinal cord motor neuron cell line to investigate the mode of cell death. OBJECTIVES: To characterize the death of motor neurons in culture, to gain insight into mechanisms that could be important in spinal cord diseases. SUMMARY OF BACKGROUND DATA: Normal reduction of cell number during central nervous system development is brought about by programmed cell death. These same apoptotic processes probably play a role in a variety of central nervous system disorders, including traumatic injury. Although certain proteolytic processes are involved, the molecular details involved in the apoptotic induction have not been fully elucidated. METHODS: To identify apoptosis, several criteria were used, including analysis of chromatin condensation with DNA-specific stains (propidium iodide and Hoechst 33342); in situ end-labeling of DNA fragments in apoptotic nuclei with terminal deoxynucleotidyl transferase; fragmentation of DNA separated on agarose gel electrophoresis; and cleavage of a characteristic substrate for apoptotic proteases, alpha-fodrin, into signature cleavage fragments. RESULTS: The NSC19 cell line exhibited motor neuron characteristics morphologically, with typical cellular structure, and biochemically, by synthesizing choline acetyl transferase. Under various treatments including serum withdrawal (loss of trophic factors), cell loss occurred through an apoptotic cell death pathway. CONCLUSIONS: A murine motor neuron cell line, NSC19, has been used to investigate apoptosis in this in vitro system. Cell death occurs by apoptosis, suggesting that this cell line may provide a useful model for studying apoptotic mechanisms in spinal cord degeneration and injury.
Asunto(s)
Apoptosis/fisiología , Neuronas Motoras/fisiología , Animales , Azacitidina/farmacología , Proteínas Portadoras/metabolismo , Línea Celular , Inhibidores Enzimáticos/farmacología , Ratones , Proteínas de Microfilamentos/metabolismo , Mitosis/efectos de los fármacos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Inhibidores de Proteínas QuinasasRESUMEN
Precise determination of mRNA levels is an essential element in any investigation of complex regulatory systems. Classical methodologies such as Northern hybridization suffer from requirements for significant samples of material and also a degree of nonspecificity. Recently, quantitative techniques involving PCR amplification have been devised. We have developed and applied such procedures to the determination of prothrombin messages in skeletal muscle cells during development. In addition to its role in the blood coagulation cascade, the serine protease thrombin has been shown to participate in several signaling events in the neuromuscular system. The inactive precursor, prothrombin, primarily produced in the liver, has also been shown to be synthesized and developmentally-regulated in the brain. In skeletal muscle, thrombin is a mediator of activity-dependent polyneuronal synapse elimination (ADPSE) which occurs in early postnatal development. Recent experiments showing that thrombin is released from myotubes in culture under the influence of acetylcholine suggest that locally-synthesized prothrombin may be the source of this Hebbian synaptic interaction. We have determined that prothrombin is expressed in skeletal muscle, as the likely source of thrombin involved in ADPSE, and the current results show the quantitative expression of muscle prothrombin during this time of intense synapse remodeling.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Reacción en Cadena de la Polimerasa , Protrombina/genética , ARN Mensajero/biosíntesis , Animales , Células Cultivadas , Clonación Molecular , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Desarrollo de Músculos , Proteínas Musculares/biosíntesis , Músculo Esquelético/embriología , Músculo Esquelético/crecimiento & desarrollo , Especificidad de Órganos , Protrombina/biosíntesis , ARN Mensajero/análisis , ARN Mensajero/genética , Sinapsis/metabolismoRESUMEN
In order to develop in vitro models of CNS injury, astrocytes have been mechanically injured in culture to study reactive astrocytosis. However, scratch injury models of pure neuronal cultures have not yet been exploited to study programmed cell death (PCD). For this study, we examined model motor neurons (NSC19 cells) in culture and found time-dependent cell death in proximity (within 2.5 mm) to a physical scratch injury. Injury-induced cell death was apoptotic verified by positively-stained nuclei using both the in situ end-labeling (ISEL) procedure and Hoechst 33342. Unexpectedly, cells proximal to the injury site were not affected by the injury until 3 days later suggesting that adjacent motor neuron loss was dependent on a 'death signal' produced by direct injury to sister neurons. 'Executioners' in apoptosis include free radicals, cell cycle kinases and cysteine proteases (caspases). Extracellular serine proteases, such as thrombin and granzyme B, may activate such intracellular pathways and several inhibitors (serpins), such as CrmA, are effective in blocking apoptosis. Since protease nexin I (PNI), a serpin homologous with CrmA, prevents apoptosis of lumbar motor neurons and is increased after nerve injury, we examined mRNA by RT-PCR for PNI expression. Of interest, although we were unable to find significant levels of PNI message in NSC19 cells, we did detect it in the parent neuroblastoma.
Asunto(s)
Apoptosis , Azacitidina/farmacología , Neuronas Motoras/patología , Precursor de Proteína beta-Amiloide , Animales , Bencimidazoles , Proteínas Portadoras/análisis , Proteínas Portadoras/biosíntesis , Línea Celular , Núcleo Celular/ultraestructura , Colina O-Acetiltransferasa/análisis , Cromatina/ultraestructura , Ratones , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiología , Reacción en Cadena de la Polimerasa , Nexinas de Proteasas , ARN Mensajero/biosíntesis , Receptores de Superficie Celular , Inhibidores de Serina Proteinasa/biosíntesis , Transcripción GenéticaRESUMEN
The blood coagulation cascade proteolytic enzyme, thrombin, affects many cell types, including neurons and astrocytes, in which it prevents process outgrowth and induces significant morphological degeneration and even cell death. Since thrombin may contribute significantly to pathological conditions in the central nervous system (CNS), where it is synthesized locally, we measured the levels of thrombin and its precursor, prothrombin, in the cerebrospinal fluid (CSF) of 67 individuals from 6 groups: non-neurologic controls (NNC); spinal degenerative disease (SDD); peripheral nerve disease (PND); cerebrovascular, neuroimmune and seizure disorders and tumor (CNSD); traumatic brain injury (TBI) and neurodegenerative disorders (NDD). We employed a sensitive chromogenic assay utilizing the thrombin specific tripeptide substrate, S-2238, to evaluate CSF levels of thrombin and prothrombin. The latter estimated after its conversion to active enzyme by the snake venom prothrombinase, ecarin. No measurable active thrombin was detected in these CSF samples. However, activatable prothrombin was measured in all groups. The mean activatable prothrombin concentrations (in nM) were 7.26 +/- 3.39 (NNC); 8.85 +/- 3.09 (SDD); 6.78 +/- 2.58 (PND); 6.33 +/- 3.87 (CNSD); 5.10 +/- 1.86 (TBI), and 7.80 +/- 3.27 (NDD). Duncan's multiple comparison test showed significant reduction (p <0.05) in prothrombin levels of the TBI group. Our data suggests that the prothrombin zymogen gains access to the CSF, likely across either an intact or compromised blood-brain barrier (BBB), in increased amounts with age. Reduced levels in TBI patients may have diagnostic and/or prognostic value.