RESUMEN
To evaluate physiological roles of the large, second cytoplasmic loops (C2) situated between the M3 and M4 transmembrane domains of nicotinic acetylcholine receptor (nAChR) subunits. We have constructed chimeric ß2 (ß2χ) and ß4 (ß4χ) subunits in which the "nested" C2 domains (but not the "proximal" sequences of â¼14 residues immediately adjacent to the M3 or M4 domains) of these ß subunits were replaced by the corresponding sequence from the serotonin 5-HT3A receptor subunit. We previously reported that heterologously expressed nAChR containing α4 and ß2χ subunits displayed a faster whole-cell current decay in its agonist response compared to responses of all-wild-type α4ß2-nAChR. This suggests an unexpected, functional role for the C2 domain of the ß2 subunit in α4ß2-nAChR acute desensitization. Here we report that there also is faster desensitization of α4ß4χ-nAChR relative to α4ß4-nAChR stably and heterologously expressed in the human SH-EP1 cell-line. In addition, cell-attached, single-channel recording shows that both acetylcholine-activated α4ß2χ- and α4ß4χ-nAChR have a significantly lower mean open probability, shorter mean open-time, and a longer mean closed-time than their fully wild-type counterparts while not having different conductance amplitudes. These findings reveal microscopic bases for the faster desensitization of α4(∗)-nAChR containing chimeric instead of wild-type ß subunits. Our findings also remain consistent with novel and unexpected roles of ß subunit-nested C2 domains in modulation of α4(∗)-nAChR function.
Asunto(s)
Receptores Nicotínicos/metabolismo , Acetilcolina/farmacología , Secuencia de Aminoácidos , Animales , Atropina/farmacología , Línea Celular , Agonistas Colinérgicos/farmacología , Citoplasma , Humanos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Antagonistas Muscarínicos , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Técnicas de Placa-Clamp , Receptores Nicotínicos/genética , Receptores de Serotonina 5-HT3/genética , Receptores de Serotonina 5-HT3/metabolismo , Alineación de SecuenciaRESUMEN
BACKGROUND AND OBJECTIVE: Hyperglycemia and advanced glycation end-products (AGEs) have been hypothesized as the etiologic factors of diabetic periodontitis. The aim of this study was to clarify in greater detail the patterns of AGE-mediated periodontal inflammation under various physiological conditions. MATERIAL AND METHODS: The deposition of AGEs and expression of the receptor for AGEs (RAGE) were identified by immunohistochemistry in Sprague-Dawley rats with experimentally induced periodontitis or diabetes. Human periodontal ligament cells (PDLCs) and mesenchymal stem cells (MSCs) were cultured under simulated conditions of hyperglycemia, Porphyromonas gingivalis lipopolysaccharide (LPS) stimulation and matrix glycation. Cell viability and expression of toll-like receptors (TLRs), Rage, an inflammatory signaling initiator (nuclear factor kappa light chain enhancer of activator ß cells), an oxidative stressor (heme oxygenase-1) and collagen synthesis (type I and type IV) genes were evaluated. RESULTS: The deposition of AGEs and the expression of Rage were evident in the inflamed periodontal tissues in all rats and appeared to be enhanced in rats with diabetes. Matrix glycation augmented cytotoxicity, up-regulated RAGE and TLRs in both PDLCs and MSCs, and significantly activated downstream inflammatory signaling in MSCs. Oxidative stress was significantly increased under matrix glycation in both PDLCs and MSCs and was significantly increased at a high-glucose concentration in MSCs. A consistent decrease in expression of type I and type IV collagens was observed in MSCs, but a delayed reduction was noted in PDLCs. CONCLUSIONS: Matrix glycation modulated cell behavior to induce inflammation equivalent to that produced by incubation with P. gingivalis LPS. Periodontal inflammation also led to matrix glycation, thus demonstrating a definite interaction between diabetes and periodontitis.
Asunto(s)
Productos Finales de Glicación Avanzada/inmunología , Lipopolisacáridos/inmunología , Ligamento Periodontal/inmunología , Porphyromonas gingivalis/inmunología , Transducción de Señal/inmunología , Pérdida de Hueso Alveolar/inmunología , Pérdida de Hueso Alveolar/patología , Animales , Supervivencia Celular/inmunología , Células Cultivadas , Colágeno Tipo I/análisis , Colágeno Tipo II/análisis , Diabetes Mellitus Experimental/inmunología , Hemo-Oxigenasa 1/análisis , Humanos , Hiperglucemia/inmunología , Masculino , Células Madre Mesenquimatosas/inmunología , Subunidad p50 de NF-kappa B/análisis , Estrés Oxidativo/fisiología , Ligamento Periodontal/citología , Periodontitis/inmunología , Periodontitis/patología , Ratas , Ratas Sprague-Dawley , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/análisis , Receptores Inmunológicos/inmunología , Estreptozocina , Receptores Toll-Like/análisis , Regulación hacia Arriba/inmunologíaRESUMEN
Heat generated during implant osteotomy might lead to osteonecrosis and delayed bone repair, thus resulting in impaired early osseointegration and fixation of bone-anchoring devices. In this study, we proposed to overcome heat-induced injury to bone by fabricating core-shell polymeric biodegradable microspheres encapsulating a mitogenic factor, platelet-derived growth factor (PDGF), and a differentiation factor, simvastatin, in a simultaneous or sequential release profile. Microspheres encapsulating bovine serum albumin (BSA), PDGF, simvastatin, PDGF-in-core with simvastatin-in-shell, and simvastatin-in-core with PDGF-in-shell were delivered to fill standardized osteotomy sites on edentulous ridges of rat maxillae under irrigated or non-irrigated conditions. In the absence of irrigation, significant reduction of cell viability and increase in inflammation and sequestrum formation without evidence of osteogenesis were observed. Both PDGF and simvastatin deliveries facilitated cell viability and reduced osteonecrosis. Localized osteogenesis was seen under simvastatin treatment, while generalized but primitive osteogenesis was noted in PDGF-treated osteotomy sites. In addition, sequential PDGF-simvastatin delivery further augmented osteogenesis and promoted bone maturation. The results suggested that sequential PDGF-simvastatin delivery was an effective modality to prevent heat-induced complications and facilitate bone apposition after implant osteotomy, potentially favoring the early fixation of bone-anchoring devices and oral implant osseointegration.
Asunto(s)
Sistemas de Liberación de Medicamentos , Calor/efectos adversos , Osteogénesis/efectos de los fármacos , Osteonecrosis/prevención & control , Osteotomía/efectos adversos , Factor de Crecimiento Derivado de Plaquetas/administración & dosificación , Simvastatina/administración & dosificación , Implantes Absorbibles , Animales , Masculino , Maxilar/lesiones , Maxilar/cirugía , Microesferas , Osteonecrosis/etiología , Polietilenglicoles , Poliglactina 910 , Ratas , Ratas Sprague-Dawley , Microtomografía por Rayos XRESUMEN
BACKGROUND AND OBJECTIVES: TGF-ß1 exerts important physiological functions in osteogenesis and chondrogenesis and may be of therapeutic interest. The aim of this work was to develop a scalable purification process of TGF-ß1 from virally inactivated human platelets. STUDY DESIGN AND METHODS: Apheresis platelet concentrates (N=12) were solvent/detergent (S/D) treated (1% TnBP/1% Triton X-45; 31°C) and the resulting platelet lysates were clarified by oil extraction and centrifugation, then chromatographed on an anion-exchange DEAE-Sepharose Fast-Flow column equilibrated in a PBS buffer, pH 7.5. The column was washed to eliminate unbound proteins and the S/D agents. Bound proteins were eluted using a 1 M NaCl-PBS buffer pH 7.5 (DEAE-eluate). The content in TGF-ß1, PDGF-AB, VEGF, IGF-1, EGF, and b-FGF was measured by ELISA. Proteins, lipids, and S/D agents were assessed. Protein profile was determined by SDS-PAGE under reduced or non-reduced conditions. RESULTS: Most proteins, including albumin and immunoglobulins G, A, and M did not bind to the DEAE column as evidenced also by SDS-PAGE. Essentially all PDGF, VEGF, and IGF were in the breakthrough. The DEAE-eluate contained close to 60% of the TGF-ß1 at a mean concentration of about 102 ng/ml, whereas EGF, b-FGF were at about 0.72 and 0.18 ng/ml, respectively. The content in TnBP and Triton X-45 was <2 ppm. CONCLUSION: A fraction enriched in TGF-ß1 can be prepared from virally inactivated human platelet lysates using an easily scale process. Its interest in regenerative medicine and cell therapy will be evaluated in further studies.
Asunto(s)
Plaquetas/química , Factor de Crecimiento Transformador beta1/química , Factor de Crecimiento Transformador beta1/aislamiento & purificación , Inactivación de Virus , Plaquetas/virología , Cromatografía por Intercambio Iónico/métodos , Citocinas/química , Humanos , Inmunoglobulinas/química , Octoxinol/químicaRESUMEN
BACKGROUND: Human platelet growth factors (HPGF) are essential for tissue regeneration and may replace fetal bovine serum (FBS) in cell therapy. No method for the manufacture of standardized virally inactivated HPGF has been developed yet. STUDY DESIGN AND METHODS: Platelet concentrates (PC) were subjected to solvent/detergent (S/D) treatment (1% TnBP/1% Triton X-45), oil extraction, hydrophobic interaction chromatography and sterile filtration. Platelet-derived growth factor (PDGF)-AB, -BB and -AA, transforming growth factor-beta1 (TGF-beta1), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1) and vascular endothelium growth factor (VEGF) were measured by ELISA. Composition in proteins and lipids was determined, protein profiles were obtained by SDS-PAGE, and TnBP and Triton X-45 were assessed by gas chromatography and high-performance liquid chromatography, respectively. Cell growth promoting activity of HPGF was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay using human embryonic kidney (HEK293A) fibroblast and Statens Seruminstitute rabbit corneal (SIRC) epithelial cell lines. RESULTS: The GF preparation contained a mean of 16.66, 2.04, 1.53, 72.19, 0.33, 48.59 and 0.44 ng/ml of PDGF-AB, -BB, -AA, TGF-beta1, EGF, IGF-1 and VEGF, respectively. The protein profile was typical of platelet releasates and had less than 2 p.p.m. of residual S/D agents. MTS assay of HEK293A and SIRC cultures showed that the GF preparation at 10% and 0.1% (v/v), respectively, could successfully replace 10% FBS for cell proliferation. Cell-stimulating activity of HPGF on HEK293A was over twice that of PC releasates. CONCLUSION: STANDARDIZED and functional virally inactivated HPGF can be prepared from human PC for possible applications in cell therapy and regenerative medicine.
Asunto(s)
Plaquetas/química , Péptidos y Proteínas de Señalización Intercelular/aislamiento & purificación , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Trasplante de Células/métodos , Fraccionamiento Químico/métodos , Ensayo de Inmunoadsorción Enzimática , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Conejos , Medicina Regenerativa/métodosRESUMEN
BACKGROUND: Helicobacter pylori is a bacterial pathogen strongly associated with ulcer diseases and gastric cancer. The bacterial-induced alteration of cell-cycle control in host cells may play a role in the pathogenetic mechanisms. The aims of this study were to define the effect of H. pylori on the G2/M to G1 transition in a gastric cell line. METHODS: Cultured gastric cells, AGS, were synchronized in the S/early G2 phase and treated with intact H. pylori. The cell-cycle distribution of AGS cells was determined by flow cytometry. The activity of cdc2 kinase, as well as of some parameters that affect the kinase activity, was also examined. RESULTS: H. pylori delays cell-cycle progression at the G2/M phase in AGS cells. The G2/M delay was associated with reduced activity of cdc2 kinase. Both down-regulation of cell-cycle regulators (p34cdc2, cyclin B1 and cdc25C) and decreased association between p34cdc2 and cyclin B1 were found to be associated with the activity of cdc2 kinase abated after the H. pylori infection. In addition, the H. pylori-induced G2/M delay required direct contact between the bacteria and host cells. CONCLUSIONS: H. pylori inhibits G2/M to G1 progression and causes a reduction of cell division in gastric epithelial cells.
Asunto(s)
Adenocarcinoma/fisiopatología , Proteína Quinasa CDC2/metabolismo , Ciclo Celular , Helicobacter pylori/fisiología , Neoplasias Gástricas/fisiopatología , Adenocarcinoma/metabolismo , Adenocarcinoma/microbiología , Western Blotting , Ciclina B/metabolismo , Ciclina B1 , Regulación hacia Abajo , Fase G1/fisiología , Fase G2/fisiología , Humanos , Factor Promotor de Maduración/metabolismo , Pruebas de Precipitina , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/microbiología , Células Tumorales CultivadasRESUMEN
Effects of cytisine (cy), 3-bromocytisine (3-Br-cy), 5-bromocytisine (5-Br-cy) and 3,5-dibromocytisine (3,5-diBr-cy) on human (h) alpha7-, alpha4beta2- and alpha4beta4 nicotinic acetylcholine (nACh) receptors, expressed in Xenopus oocytes and cell lines, have been investigated. Cy and its bromo-isosteres fully inhibited binding of both [alpha-(125)I]bungarotoxin ([alpha-(125)I]BgTx) to halpha7- and [(3)H]cy to halpha4beta2- or halpha4beta4-nACh receptors. 3-Br-cy was the most potent inhibitor of both [alpha-(125)I]BgTx and [(3)H]cy binding. Cy was less potent than 3-Br-cy, but 5-Br-cy and 3,5-diBr-cy were the least potent inhibitors. Cy and 3-Br-cy were potent full agonists at halpha7-nACh receptors but behaved as partial agonists at halpha4beta2- and halpha4beta4-nACh receptors. 5-Br-cy and 3,5-diBr-cy had low potency and were partial agonists at halpha7- and halpha4beta4-nACh receptors, but they elicited no responses on halpha4beta2-nACh receptors. Cy and 3-Br-cy produced dual dose-response curves (DRC) at both halpha4beta2- and halpha4beta4-nACh receptors, but ACh produced dual DRC only at halpha4beta2-nACh receptors. Low concentrations of cy, 3-Br-cy and 5-Br-cy enhanced ACh responses of oocytes expressing halpha4beta2-nACh receptors, but at high concentrations they inhibited the responses. In contrast, 3,5-diBr-cy only inhibited, in a competitive manner, ACh responses of halpha4beta2-nACh receptors. It is concluded that bromination of the pyridone ring of cy produces marked changes in effects of cy that are manifest as nACh receptor subtype-specific differences in binding affinities and in functional potencies and efficacies.
Asunto(s)
Citosina/análogos & derivados , Citosina/farmacología , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Acetilcolina/metabolismo , Acetilcolina/farmacología , Animales , Unión Competitiva/efectos de los fármacos , Unión Competitiva/fisiología , Bungarotoxinas/metabolismo , Bungarotoxinas/farmacología , Citosina/química , Electrofisiología , Expresión Génica/fisiología , Humanos , Radioisótopos de Yodo , Oocitos/fisiología , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tritio , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
The expression of two heat-responsive cct (chaperonin-containing Tcp-1) genes from the archaeon Haloferax volcanii was investigated at the transcription level. The cct1 and cct2 genes, which encode proteins of 560 and 557 amino acids, respectively, were identified on cosmid clones of an H. volcanii genomic library and subsequently sequenced. The deduced amino acid sequences of these genes exhibited a high degree of similarity to other archaeal and eucaryal cct family members. Expression of the cct genes was characterized in detail for the purpose of developing a model for studying transcription regulation in the domain Archaea. Northern (RNA) analysis demonstrated that the cct mRNAs were maximally induced after heat shock from 37 to 55 degrees C and showed significant heat inducibility after 30 min at 60 degrees C. Transcription of cct mRNAs was also stimulated in response to dilute salt concentrations. Transcriptional analysis of cct promoter regions coupled to a yeast tRNA reporter gene demonstrated that 5' flanking sequences up to position -233 (cct1) and position -170 (cct2) were sufficient for promoting heat-induced transcription. Transcript analysis indicated that both basal transcription and stress-induced transcription of the H. volcanii cct genes were directed by a conserved archaeal consensus TATA motif (5'-TTTATA-3') centered at -25 relative to the mapped initiation site. Comparison of the cct promoter regions also revealed a striking degree of sequence conservation immediately 5' and 3' of the TATA element.
Asunto(s)
Proteínas Arqueales , Regulación de la Expresión Génica Arqueal , Haloferax/genética , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/genética , Transcripción Genética , Secuencia de Aminoácidos , Secuencia de Bases , Chaperonina con TCP-1 , Chaperoninas/química , Chaperoninas/genética , Clonación Molecular , Secuencia Conservada , Genes Arqueales , Genes Reporteros , Haloferax/química , Proteínas de Choque Térmico/química , Calor , Humanos , Chaperonas Moleculares/química , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , TATA BoxRESUMEN
Poisoning is a well known cause of morbidity and mortality in children. In Taiwan, little information has been published regarding the status of pediatric poisoning exposures. To provide more information on pediatric poisoning exposures for the purpose of poison prevention, a retrospective study was designed and conducted to analyse the data of National Poison Centre (NPC), Taiwan. All telephone inquiries concerning poisoning exposures in those under 19 years of age, received by NPC-Taiwan from July 1985 through December 1993, were included in this study. The age, sex, reason for exposure, route of exposure, substances involved and clinical outcome of those telephone calls were then analyzed. A total of 5,812 inquiries concerning poisoning exposures in children were recorded. Male exposures were more prevalent than females (59%) Vs. 41%) Accidental exposures accounted for 77.7% of the cases and most were exposed by the oral route. Substances most frequently ingested were household products, benzodiazepines and pesticides. The data revealed a mortality rate of 1.4%. Accidental poisoning exposures from household products and drugs remain a significant problem for those younger than 6 years of age. Further education of parents and care takers and the employment of child-resistant containers are needed to prevent cases of pediatric poisoning. Reduction of amphetamine abuse in adolescents is also of major concern and deserves more attention.
Asunto(s)
Intoxicación/epidemiología , Intoxicación/etiología , Adolescente , Distribución por Edad , Benzodiazepinas/envenenamiento , Niño , Preescolar , Femenino , Productos Domésticos/envenenamiento , Humanos , Incidencia , Lactante , Masculino , Plaguicidas/envenenamiento , Centros de Control de Intoxicaciones/estadística & datos numéricos , Estudios Retrospectivos , Distribución por Sexo , Taiwán/epidemiologíaRESUMEN
UNLABELLED: The Taiwan National Poison Center has received more than 30,000 telephone calls since its establishment in July 1985. OBJECTIVE: To obtain more information about poisoning exposures in Taiwan, a retrospective analysis was conducted of all telephone calls to the center concerning human poisoning exposures July 1985 through December 1993. METHODS: The following data were tabulated: age, sex, intent of exposure, route of exposure, substances ingested and clinical severity. RESULTS: During the eight years (1985-1993), 23,436 telephone calls concerning human poisoning exposure were recorded. Adults accounted for most cases (75.2%) and exposures involving males (54.2%) were somewhat more prevalent than female poisoning exposures (44.7%). Intentional poisonings (54.6%) were more common than unintentional poisonings (40.1%), with an inverse relationship in pediatric poisoning exposures. After amphetamines, the most frequently ingested poisons were pesticides, benzodiazepines, and cleaning products. Fatalities occurred most frequently following ingestion of pesticides. The mortality rate was 5.7% for all exposures. CONCLUSIONS: Human poisoning is a serious problem in Taiwan. The reduction of suicide attempts is a major objective. Childhood poisonings are underreported and of high mortality.
Asunto(s)
Centros de Control de Intoxicaciones/estadística & datos numéricos , Intoxicación/epidemiología , Adolescente , Adulto , Anciano , Anfetaminas/efectos adversos , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Mortalidad , Exposición Profesional/estadística & datos numéricos , Plaguicidas/efectos adversos , Estudios Retrospectivos , Caracteres Sexuales , Suicidio/estadística & datos numéricos , Taiwán/epidemiologíaRESUMEN
Taiwan cobra (Naja naja atra) snake venom was separated into 19 fractions by means of CM-Sephadex C-50 column chromatography. Anticoagulant Fractions V-VII were refractionated by gel filtration on Sephadex G-50 and the purified component possessed phospholipase A2 activity and an inhibitory effect on collagen-induced platelet aggregation. The anticoagulant action could be antagonized by phospholipid or platelet factor 3. Anticoagulant Fraction XVII was also further refractionated by gel filtration on Sephadex G-50 and the purified component was shown to be cardiotoxin. It was a weak anticoagulant, caused direct hemolysis and potentiated collagen-induced platelet aggregation. Thromboelastographic studies showed that the anticoagulant action of cobra venom is due to the synergistic effects of phospholipase A2 and cardiotoxin.
Asunto(s)
Anticoagulantes/aislamiento & purificación , Venenos Elapídicos/aislamiento & purificación , Animales , Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Cromatografía en Gel , Proteínas Cardiotóxicas de Elápidos/aislamiento & purificación , Proteínas Cardiotóxicas de Elápidos/farmacología , Venenos Elapídicos/farmacología , Hemólisis/efectos de los fármacos , Técnicas In Vitro , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A/farmacología , Fosfolipasas A2 , Agregación Plaquetaria/efectos de los fármacos , ConejosRESUMEN
Cobra venom phospholipase A2 induced a biphasic effect on washed rabbit platelets. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The aggregation and thromboxane B2 formation were inhibited by indomethacin, mepacrine, tetracaine and imipramine, while PGE1 and sodium nitroprusside inhibited only the aggregation, but not the thromboxane B2 formation. The second phase was an inhibitory effect on platelet aggregation induced by arachidonic acid, PAF, ADP or collagen but not that by thrombin or ionophore A23187. The longer the incubation time of cobra venom phospholipase A2 with platelets, the more the inhibitory effect. The aggregating and anti-aggregating effects could be overcome by bovine serum albumin. Lysophosphatidylcholine (Lyso-PC) and arachidonic acid showed synergistic inhibition in platelet aggregation. Lyso-PC decreased thromboxane B2 formation in platelets formed by collagen. The inhibitory effect of Lyso-PC on platelet aggregation was more marked at lower calcium concentrations. It is concluded that the aggregating effect of exogenous addition of venom phospholipase A2 is due to thromboxane formation and the antiplatelet effect is similar to those produced by arachidonic acid and lysophosphatidylcholine.