RESUMEN
Taxanes, including paclitaxel, are anti-cancer drugs approved for the treatment of prostate cancer but which have limited clinical application due to their hydrophobicity, their low therapeutic index and the emergence of chemoresistance. These side effects may be avoided through the use of new drug delivery systems such as nanoparticles, and paclitaxel-loaded PLGA nanoparticles up to 200 nm in size have shown encouraging results. As it is known that size affects the tissular penetration and distribution of tumors via the enhanced permeability and retention effect, so nanoparticles smaller than 100 nm are potentially interesting vehicles for improving paclitaxel delivery and efficacy. In this work, new paclitaxel-loaded small PLGA nanoparticles, between 49 nm and 95 nm in size and with positive or negative surface charges, were prepared without detergent. They were stable in the presence of serum, and HPLC showed that high paclitaxel loading and stability were achieved. Intracellular uptake of these nanoparticles was studied in PC3 cells by flow cytometry. Confocal studies confirmed a high tubulin destructuration at very low dose with these nanoparticles. This study suggests that both positively and negatively charged paclitaxel-loaded small PLGA nanoparticles deliver this drug into PC3 cells, and that this nanoparticle mode of delivery highly improves paclitaxel efficiency by up to two log-increase. These results also highlight the importance of small nanoparticles for drug delivery in cancer applications and are extremely promising for in vivo studies.
Asunto(s)
Antineoplásicos Fitogénicos/química , Ácido Láctico/química , Nanopartículas/química , Paclitaxel/química , Ácido Poliglicólico/química , Antineoplásicos Fitogénicos/administración & dosificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Endocitosis , Humanos , Ácido Láctico/administración & dosificación , Masculino , Nanopartículas/administración & dosificación , Paclitaxel/administración & dosificación , Tamaño de la Partícula , Ácido Poliglicólico/administración & dosificación , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Propiedades de SuperficieRESUMEN
The fate and phenotype of lesion macrophages is regulated by cellular oxidative stress. Thioredoxin-1 (Trx-1) plays a major role in the regulation of cellular redox balance, with resultant effects on gene expression and cellular responses including cell growth and death. Trx-1 activity is inhibited by interaction with vitamin D-upregulated protein-1 (VDUP-1). Peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed by human monocyte-derived macrophages (HMDM) and PPARgamma agonism has been reported to decrease expression of inflammatory genes and to promote apoptosis of these cells. To determine whether VDUP-1 may be involved in regulating the effects of PPARgamma agonists in macrophages, we investigated the effect of a synthetic PPARgamma agonist (GW929) on the expression of VDUP-1 in HMDM. GW929 concentration-dependently increased HMDM expression of VDUP-1 (mRNA and protein). Transfection of different fragments of the VDUP-1 promoter as well as gel shift analysis revealed the presence of functional PPARgamma response elements (PPRE) in the promoter. Under conditions in which PPAR agonism altered levels of VDUP-1, caspase-3 activity, and macrophage apoptosis were also elevated. The results suggest that PPARgamma activation stimulates apoptosis in human macrophages by altering the cellular redox balance via regulation of VDUP-1.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Macrófagos/efectos de los fármacos , PPAR gamma/agonistas , Aorta/citología , Proteínas Portadoras/genética , Caspasa 3/análisis , Caspasa 3/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ensayo de Cambio de Movilidad Electroforética , Genes Reporteros , Humanos , Etiquetado Corte-Fin in Situ , Luciferasas/metabolismo , Macrófagos/metabolismo , Monocitos/citología , Monocitos/metabolismo , Músculo Liso Vascular/citología , Plásmidos , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Elementos de Respuesta , Transfección , Células U937RESUMEN
Suprachiasmatic nucleus (SCN) neurons generate circadian rhythms, and these neurons normally exhibit loosely-synchronized action potentials. Although electrotonic coupling has long been proposed to mediate this neuronal synchrony, ultrastructural studies have failed to detect gap junctions between SCN neurons. Nevertheless, it has been proposed that neuronal gap junctions exist in the SCN; that they consist of connexin32 or, alternatively, connexin36; and that connexin36 knockout eliminates neuronal coupling between SCN neurons and disrupts circadian rhythms. We used confocal immunofluorescence microscopy and freeze-fracture replica immunogold labeling to examine the distributions of connexin30, connexin32, connexin36, and connexin43 in rat and mouse SCN and used whole-cell recordings to re-assess electrotonic and tracer coupling. Connexin32-immunofluorescent puncta were essentially absent in SCN but connexin36 was relatively abundant. Fifteen neuronal gap junctions were identified ultrastructurally, all of which contained connexin36 but not connexin32, whereas nearby oligodendrocyte gap junctions contained connexin32. In adult SCN, one neuronal gap junction was >600 connexons, whereas 75% were smaller than 50 connexons, which may be below the limit of detectability by fluorescence microscopy and thin-section electron microscopy. Whole-cell recordings in hypothalamic slices revealed tracer coupling with neurobiotin in <5% of SCN neurons, and paired recordings (>40 pairs) did not reveal obvious electrotonic coupling or synchronized action potentials, consistent with few neurons possessing large gap junctions. However, most neurons had partial spikes or spikelets (often <1 mV), which remained after QX-314 [N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide] had blocked sodium-mediated action potentials within the recorded neuron, consistent with spikelet transmission via small gap junctions. Thus, a few "miniature" gap junctions on most SCN neurons appear to mediate weak electrotonic coupling between limited numbers of neuron pairs, thus accounting for frequent detection of partial spikes and hypothetically providing the basis for "loose" electrical or metabolic synchronization of electrical activity commonly observed in SCN neuronal populations during circadian rhythms.
Asunto(s)
Conexinas/fisiología , Uniones Comunicantes/fisiología , Núcleo Supraquiasmático/fisiología , Animales , Conexinas/genética , Detergentes/farmacología , Electrofisiología , Técnica de Fractura por Congelación , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , Microscopía Confocal , Microscopía Electrónica , Neuroglía/fisiología , Neuronas/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Dodecil Sulfato de Sodio/farmacología , Proteína beta1 de Unión Comunicante , Proteína delta-6 de Union ComunicanteRESUMEN
Neuronal gap junctions are abundant in both outer and inner plexiform layers of the mammalian retina. In the inner plexiform layer (IPL), ultrastructurally-identified gap junctions were reported primarily in the functionally-defined and anatomically-distinct ON sublamina, with few reported in the OFF sublamina. We used freeze-fracture replica immunogold labeling and confocal microscopy to quantitatively analyze the morphologies and distributions of neuronal gap junctions in the IPL of adult rat and mouse retina. Under "baseline" conditions (photopic illumination/general anesthesia), 649 neuronal gap junctions immunogold-labeled for connexin36 were identified in rat IPL, of which 375 were photomapped to OFF vs. ON sublaminae. In contrast to previous reports, the volume-density of gap junctions was equally abundant in both sublaminae. Five distinctive morphologies of gap junctions were identified: conventional crystalline and non-crystalline "plaques" (71% and 3%), plus unusual "string" (14%), "ribbon" (7%) and "reticular" (2%) forms. Plaque and reticular gap junctions were distributed throughout the IPL. However, string and ribbon gap junctions were restricted to the OFF sublamina, where they represented 48% of gap junctions in that layer. In string and ribbon junctions, curvilinear strands of connexons were dispersed over 5 to 20 times the area of conventional plaques having equal numbers of connexons. To define morphologies of gap junctions under different light-adaptation conditions, we examined an additional 1150 gap junctions from rats and mice prepared after 30 min of photopic, mesopic and scotopic illumination, with and without general anesthesia. Under these conditions, string and ribbon gap junctions remained abundant in the OFF sublamina and absent in the ON sublamina. Abundant gap junctions in the OFF sublamina of these two rodents with rod-dominant retinas revealed previously-undescribed but extensive pathways for inter-neuronal communication; and the wide dispersion of connexons in string and ribbon gap junctions suggests unique structural features of gap junctional coupling in the OFF vs. ON sublamina.
Asunto(s)
Uniones Comunicantes/ultraestructura , Vías Nerviosas/ultraestructura , Neuronas/ultraestructura , Retina/ultraestructura , Animales , Comunicación Celular/fisiología , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Femenino , Técnica de Fractura por Congelación , Uniones Comunicantes/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Microscopía Inmunoelectrónica , Modelos Neurológicos , Vías Nerviosas/fisiología , Neuronas/fisiología , Estimulación Luminosa , Ratas , Ratas Sprague-Dawley , Retina/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Células Fotorreceptoras Retinianas Bastones/ultraestructura , Visión Ocular/fisiologíaRESUMEN
Each day, approximately 0.5-0.9 l of water diffuses through (primarily) aquaporin-1 (AQP1) channels in the human choroid plexus, into the cerebrospinal fluid of the brain ventricles and spinal cord central canal, through the ependymal cell lining, and into the parenchyma of the CNS. Additional water is also derived from metabolism of glucose within the CNS parenchyma. To maintain osmotic homeostasis, an equivalent amount of water exits the CNS parenchyma by diffusion into interstitial capillaries and into the subarachnoid space that surrounds the brain and spinal cord. Most of that efflux is through AQP4 water channels concentrated in astrocyte endfeet that surround capillaries and form the glia limitans. This report extends the ultrastructural and immunocytochemical characterizations of the crystalline aggregates of intramembrane proteins that comprise the AQP4 "square arrays" of astrocyte and ependymocyte plasma membranes. We elaborate on recent demonstrations in Chinese hamster ovary cells of the effects on AQP4 array assembly resulting from separate vs. combined expression of M1 and M23 AQP4, which are two alternatively spliced variants of the AQP4 gene. Using improved shadowing methods, we demonstrate sub-molecular cross-bridges that link the constituent intramembrane particles (IMPs) into regular square lattices of AQP4 arrays. We show that the AQP4 core particle is 4.5 nm in diameter, which appears to be too small to accommodate four monomeric proteins in a tetrameric IMP. Several structural models are considered that incorporate freeze-fracture data for submolecular "cross-bridges" linking IMPs into the classical square lattices that characterize, in particular, naturally occurring AQP4.
Asunto(s)
Acuaporinas/química , Astrocitos/ultraestructura , Membrana Celular/ultraestructura , Epéndimo/ultraestructura , Técnica de Fractura por Congelación , Empalme Alternativo/genética , Animales , Acuaporina 4 , Acuaporinas/genética , Astrocitos/química , Células CHO , Membrana Celular/química , Cricetinae , Epéndimo/química , Femenino , Aumento de la Imagen , Inmunohistoquímica , Sustancias Macromoleculares/química , Masculino , Modelos Moleculares , Peso Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
OBJECTIVE: Cholesterol accumulation in macrophages is known to alter macrophage biology. In this article we studied the impact of macrophage cholesterol loading on gene expression and identified a novel gene implicated in cell death. METHODS AND RESULTS: The regulated in development and DNA damage response 2 (REDD2) gene was strongly upregulated as THP-1 macrophages are converted to foam cells. These results were confirmed by Northern blot of RNA from human monocyte-derived macrophages (HMDM) treated with oxidized LDL (oxLDL). Human REDD2 shares 86% amino acid sequence identity with murine RTP801-like protein, which is 33% identical to RTP801, a hypoxia-inducible factor 1-responsive gene involved in apoptosis. Treatment of HMDM with desferrioxamine, a molecule that mimics the effect of hypoxia, increased expression of REDD2 in a concentration-dependent fashion. Transfection of U-937 and HMEC cells with a REDD2 expression vector increased the sensitivity of the cells for oxLDL-induced cytotoxicity, by inducing a shift from apoptosis toward necrosis. In contrast, suppression of mRNA expression using siRNA approach resulted in increased resistance to oxLDL treatment. CONCLUSIONS: We showed that stimulation of REDD2 expression in macrophages increases oxLDL-induced cell death, suggesting that REDD2 gene might play an important role in arterial pathology.
Asunto(s)
Muerte Celular/fisiología , Hipoxia/patología , Lipoproteínas LDL/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Proteínas/fisiología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Proteínas Adaptadoras Transductoras de Señales , Arteriosclerosis/genética , Línea Celular , Línea Celular Tumoral , Células Cultivadas , ADN/genética , Deferoxamina/farmacología , Relación Dosis-Respuesta a Droga , Endotelio Vascular/química , Endotelio Vascular/metabolismo , Células Espumosas/fisiología , Humanos , Monocitos/fisiología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transfección/métodos , Células U937/química , Células U937/metabolismoRESUMEN
Uptake of modified low-density lipoproteins (LDLs) by macrophages in the arterial wall is an important event in atherogenesis. Indeed, oxidatively modified LDLs (oxLDLs) are known to affect various cellular processes by modulating oxidation-sensitive signaling pathways. Here we found that the ubiquitous 55 kDa selenoprotein thioredoxin reductase 1 (TrxR1), which is a key enzyme for cellular redox control and antioxidant defense, was upregulated in human atherosclerotic plaques and expressed in foam cells. Using reverse transcription polymerase chain reaction analysis, we also found that oxLDLs, but not native LDLs (nLDLs), dose-dependently increased TrxR1 mRNA in human monocyte-derived macrophages (HMDMs). This stimulating effect was specific for oxLDLs, as pro-inflammatory factors, such as lipopolysaccharides (LPSs), interleukin-1beta (IL-1beta), interleukin-6 (Il-6), and tumor necrosis factor alpha (TNFalpha), under the same conditions, failed to induce TrxR1 mRNA levels to the same extent. Moreover, phorbol ester-differentiated THP-1 cells or HMDMs transiently transfected with TrxR1 promoter fragments linked to a luciferase reporter gene allowed identification of a defined promoter region as specifically responding to the phospholipid component of oxLDLs (p <.05 vs. phospholipid component of nLDLs). Gel mobility shift analyses identified a short 40-nucleotide stretch of the promoter carrying AP-1 and HoxA5 consensus motifs that responded with an altered shift pattern in THP-1 cells treated with oxLDLs, however, without evident involvement of either the Fos, Jun, Nrf2 or HoxA5 transcription factors.
Asunto(s)
Enfermedades de las Arterias Carótidas/enzimología , Regulación Enzimológica de la Expresión Génica , Lipoproteínas LDL/farmacología , Macrófagos/enzimología , Regiones Promotoras Genéticas/genética , Reductasa de Tiorredoxina-Disulfuro/genética , Secuencia de Bases , Enfermedades de las Arterias Carótidas/cirugía , Línea Celular Tumoral , Endarterectomía Carotidea , Humanos , Datos de Secuencia Molecular , Monocitos/fisiología , ARN Mensajero/genética , Tiorredoxina Reductasa 1 , TransfecciónRESUMEN
Combined confocal microscopy and freeze-fracture replica immunogold labeling (FRIL) were used to examine the connexin identity at electrical synapses in goldfish brain and rat retina, and to test for "co-localization" vs. "close proximity" of connexins to other functionally interacting proteins in synapses of goldfish and mouse brain and rat retina. In goldfish brain, confocal microscopy revealed immunofluorescence for connexin35 (Cx35) and NMDA-R1 (NR1) glutamate receptor protein in Mauthner Cell/Club Ending synapses. By FRIL double labeling, NR1 glutamate receptors were found in clusters of intramembrane particles in the postsynaptic membrane extraplasmic leaflets, and these distinctive postsynaptic densities were in close proximity (0.1-0.3 microm) to neuronal gap junctions labeled for Cx35, which is the fish ortholog of connexin36 (Cx36) found at neuronal gap junctions in mammals. Immunogold labeling for Cx36 in adult rat retina revealed abundant gap junctions, including several previously unrecognized morphological types. As in goldfish hindbrain, immunogold double labeling revealed NR1-containing postsynaptic densities localized near Cx36-labeled gap junction in rat inferior olive. Confocal immunofluorescence microscopy revealed widespread co-localization of Cx36 and ZO-1, particularly in the reticular thalamic nucleus and amygdala of mouse brain. By FRIL, ZO-1 immunoreactivity was co-localized with Cx36 at individual gap junction plaques in rat retinal neurons. As cytoplasmic accessory proteins, ZO-1 and possibly related members of the membrane-associated guanylate kinase (MAGUK) family represent scaffolding proteins that may bind to and regulate the activity of many neuronal gap junctions. These data document the power of combining immunofluorescence confocal microscopy with FRIL ultrastructural imaging and immunogold labeling to determine the relative proximities of proteins that are involved in short- vs. intermediate-range molecular interactions in the complex membrane appositions at synapses between neurons.
Asunto(s)
Mapeo Encefálico/métodos , Conexinas/análisis , Proteínas del Ojo/análisis , Proteínas de la Membrana/análisis , Fosfoproteínas/análisis , Proteómica/métodos , Receptores de N-Metil-D-Aspartato/análisis , Animales , Conexinas/biosíntesis , Proteínas del Ojo/biosíntesis , Carpa Dorada , Inmunohistoquímica , Proteínas de la Membrana/biosíntesis , Ratones , Ratones Noqueados , Fosfoproteínas/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/biosíntesis , Proteína de la Zonula Occludens-1 , Proteína delta-6 de Union ComunicanteRESUMEN
Osmotic homeostasis in the brain involves movement of water through aquaporin-4 (AQP4) membrane channels. Perivascular astrocyte end-feet contain distinctive orthogonal lattices (square arrays) assembled from 4- to 6-nm intramembrane particles (IMPs) corresponding to individual AQP4 tetramers. Two isoforms of AQP4 result from translation initiation at methionine residues M1 and M23, but no functional differences are known. In this study, Chinese hamster ovary cells were transfected with M1, M23, or M1+M23 isoforms, and AQP4 expression was confirmed by immunoblotting, immunocytochemistry, and immunogold labeling. Square array organization was examined by freeze-fracture electron microscopy. In astrocyte end-feet, >90% of 4- to 6-nm IMPs were found in square arrays, with 65% in arrays of 13-30 IMPs. In cells transfected with M23, 95% of 4- to 6-nm IMPs were in large assemblies (rafts), 85% of which contained >100 IMPs. However, in M1 cells, >95% of 4- to 6-nm IMPs were present as singlets, with <5% in incipient arrays of 2-12 IMPs. In M1+M23 cells, 4- to 6-nm IMPs were in arrays of intermediate sizes, resembling square arrays in astrocytes. Structural cross-bridges of 1 x 2 nm linked >90% of IMPs in M23 arrays ( approximately 1,000 cross-bridges per microm2) but were rarely seen in M1 cells. These studies show that M23 and M1 isoforms have opposing effects on intramembrane organization of AQP4: M23 forms large square arrays with abundant cross-bridges; M1 restricts square array assembly.
Asunto(s)
Acuaporinas/fisiología , Animales , Acuaporina 4 , Acuaporinas/química , Astrocitos/metabolismo , Células CHO , Cricetinae , Técnica de Fractura por Congelación , Immunoblotting , Inmunohistoquímica , Plásmidos/metabolismo , Isoformas de Proteínas , Ratas , TransfecciónRESUMEN
Metalloproteinases (MMP)-2 and MMP-9 play a role in smooth muscle cell (SMC) migration from the media to the intima following arterial injury. Intravenous administration of adenovirus encoding tissue inhibitor of metalloproteinase-1 (TIMP-1) into balloon-injured rat arteries (3 x 10(11) viral particles/rat; n=7) resulted in a transient expression of TIMP-1 and a significant inhibition of neointima thickening within 16 days ( approximately 40% vs. control; P=0.012). Three days after injury, the number of intimal SMCs was decreased by approximately 98% in TIMP-1-treated rats. However, no alteration was seen in intimal SMC proliferation after 13 days of injury. Therefore, our results show that systemic gene transfer of TIMP-1 is a promising approach in early restenosis treatment.
Asunto(s)
Angioplastia de Balón/efectos adversos , Arterias Carótidas/patología , Estenosis Carotídea/terapia , Terapia Genética , Inhibidor Tisular de Metaloproteinasa-1/genética , Adenoviridae/genética , Animales , Estenosis Carotídea/etiología , Estenosis Carotídea/patología , Movimiento Celular , Vectores Genéticos/administración & dosificación , Humanos , Hiperplasia , Inyecciones Intravenosas , Cinética , Masculino , Metaloproteinasas de la Matriz/metabolismo , Músculo Liso Vascular/fisiopatología , Ratas , Ratas Sprague-Dawley , Inhibidor Tisular de Metaloproteinasa-1/sangreRESUMEN
Endothelin is a potent vasoconstrictor peptide isolated from endothelial cells and it induces smooth muscle cell proliferation. Endothelin-1 secretion is increased in atheroma and induces deleterious effects such as vasospasm and atherosclerosis. Oxidized low-density lipoproteins (LDLs) induce atherosclerosis in the vascular wall, as well as endothelin-1 secretion in endothelial cells and are activators of both peroxisome proliferator-activated receptor-alpha (PPAR-alpha) and PPAR-gamma. PPAR-alpha (fibric acids) and PPAR-gamma (glitazones) activators are used to treat dyslipoproteinemias and type 2 diabetes, respectively. Furthermore, these drugs induce numerous pleiotropic effects, such as inhibiting thrombin-induced endothelin-1 secretion in endothelial cells. This study shows that both PPAR-alpha (Wy 14643) and PPAR-gamma activation (rosiglitazone) partially inhibit oxidized LDL-induced protein kinase C activity and endothelin-1 secretion in endothelial cells at the transcriptional levels and suggests that synthetic PPAR activators are stronger PPAR activators than oxidized LDL. This study also suggests that fibrate and glitazone treatments should have beneficial effects on the vascular wall by reducing endothelin-1 secretion and the resulting vasospasm and atherosclerosis.
Asunto(s)
Endotelina-1/metabolismo , Endotelio Vascular/efectos de los fármacos , Lipoproteínas LDL/farmacología , Proliferadores de Peroxisomas/farmacología , Pirimidinas/farmacología , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Factores de Transcripción/efectos de los fármacos , Animales , Bovinos , Células Cultivadas , Endotelio Vascular/metabolismo , Lipoproteínas LDL/antagonistas & inhibidores , Oxidación-Reducción , Proteína Quinasa C/metabolismoRESUMEN
The aim of this study was to carry out a microbiological evaluation of sites with and without clinical evidence of moderate and severe periodontitis and their correlation with clinical parameters. A total of 52 disease sites and 10 healthy sites were selected according to clinical criteria. The following clinical indexes were measured for all the sites: plaque index, gingival index, blood on probing, depth on probing and insertion level. Samples of subgingival plaque were collected for culture and for differential counts of microbial morphotypes. In disease sites the most frequently isolated were: Prevotella intermedia/nigrescens (65%), Porphyromonas gingivalis (23%), Actinobacillus actinomycetemcomitans (23%), Fusobacterium nucleatum (10%) and Peptostreptococcus sp. (31%). The aerobic gram-positive microflora was predominant in healthy sites. Significant differences were observed in microbial morphotypes between healthy and disease sites: cocci 18.71% and 78.90%, motile rods 46.12% and 16.70%, total spirochetes 26.48% and 2.80%, respectively. The presence of motile rods, spirochetes and P. intermedia/nigrescens were the parameters with most sensitivity to suspect periodontal disease. There were significant differences in the subgingival microflora between healthy and disease sites in patients with moderate and severe periodontitis.
Asunto(s)
Periodontitis/microbiología , Infecciones por Actinobacillus/epidemiología , Infecciones por Actinobacillus/microbiología , Infecciones por Actinobacillus/patología , Adulto , Aggregatibacter actinomycetemcomitans/aislamiento & purificación , Argentina/epidemiología , Infecciones por Bacteroidaceae/epidemiología , Infecciones por Bacteroidaceae/microbiología , Infecciones por Bacteroidaceae/patología , Placa Dental/microbiología , Infecciones por Fusobacterium/epidemiología , Infecciones por Fusobacterium/microbiología , Infecciones por Fusobacterium/patología , Fusobacterium nucleatum/aislamiento & purificación , Infecciones por Bacterias Grampositivas/epidemiología , Infecciones por Bacterias Grampositivas/microbiología , Infecciones por Bacterias Grampositivas/patología , Humanos , Persona de Mediana Edad , Peptostreptococcus/aislamiento & purificación , Índice Periodontal , Periodontitis/epidemiología , Periodontitis/patología , Porphyromonas gingivalis/aislamiento & purificación , Prevotella intermedia/aislamiento & purificación , Índice de Severidad de la EnfermedadRESUMEN
The aim of this study was to carry out a microbiological evaluation of sites with and without clinical evidence of moderate and severe periodontitis and their correlation with clinical parameters. A total of 52 disease sites and 10 healthy sites were selected according to clinical criteria. The following clinical indexes were measured for all the sites: plaque index, gingival index, blood on probing, depth on probing and insertion level. Samples of subgingival plaque were collected for culture and for differential counts of microbial morphotypes. In disease sites the most frequently isolated were: Prevotella intermedia/nigrescens (65), Porphyromonas gingivalis (23), Actinobacillus actinomycetemcomitans (23), Fusobacterium nucleatum (10) and Peptostreptococcus sp. (31). The aerobic gram-positive microflora was predominant in healthy sites. Significant differences were observed in microbial morphotypes between healthy and disease sites: cocci 18.71 and 78.90, motile rods 46.12 and 16.70, total spirochetes 26.48 and 2.80, respectively. The presence of motile rods, spirochetes and P. intermedia/nigrescens were the parameters with most sensitivity to suspect periodontal disease. There were significant differences in the subgingival microflora between healthy and disease sites in patients with moderate and severe periodontitis.
Asunto(s)
Humanos , Adulto , Persona de Mediana Edad , Periodontitis , Aggregatibacter actinomycetemcomitans , Argentina , Fusobacterium nucleatum , Infecciones por Bacterias Grampositivas/epidemiología , Infecciones por Bacterias Grampositivas/microbiología , Infecciones por Bacterias Grampositivas/patología , Infecciones por Actinobacillus/epidemiología , Infecciones por Actinobacillus/microbiología , Infecciones por Actinobacillus/patología , Infecciones por Bacteroidaceae/epidemiología , Infecciones por Bacteroidaceae/microbiología , Infecciones por Bacteroidaceae/patología , Infecciones por Fusobacterium/epidemiología , Infecciones por Fusobacterium/microbiología , Infecciones por Fusobacterium/patología , Peptostreptococcus , Índice Periodontal , Periodontitis , Placa Dental/microbiología , Porphyromonas gingivalis , Prevotella intermedia , Índice de Severidad de la EnfermedadRESUMEN
The aim of this study was to carry out a microbiological evaluation of sites with and without clinical evidence of moderate and severe periodontitis and their correlation with clinical parameters. A total of 52 disease sites and 10 healthy sites were selected according to clinical criteria. The following clinical indexes were measured for all the sites: plaque index, gingival index, blood on probing, depth on probing and insertion level. Samples of subgingival plaque were collected for culture and for differential counts of microbial morphotypes. In disease sites the most frequently isolated were: Prevotella intermedia/nigrescens (65), Porphyromonas gingivalis (23), Actinobacillus actinomycetemcomitans (23), Fusobacterium nucleatum (10) and Peptostreptococcus sp. (31). The aerobic gram-positive microflora was predominant in healthy sites. Significant differences were observed in microbial morphotypes between healthy and disease sites: cocci 18.71 and 78.90, motile rods 46.12 and 16.70, total spirochetes 26.48 and 2.80, respectively. The presence of motile rods, spirochetes and P. intermedia/nigrescens were the parameters with most sensitivity to suspect periodontal disease. There were significant differences in the subgingival microflora between healthy and disease sites in patients with moderate and severe periodontitis.(AU)
Asunto(s)
Humanos , Adulto , Persona de Mediana Edad , Periodontitis/microbiología , Infecciones por Actinobacillus/epidemiología , Infecciones por Actinobacillus/microbiología , Infecciones por Actinobacillus/patología , Aggregatibacter actinomycetemcomitans/aislamiento & purificación , Argentina/epidemiología , Infecciones por Bacteroidaceae/epidemiología , Infecciones por Bacteroidaceae/microbiología , Infecciones por Bacteroidaceae/patología , Placa Dental/microbiología , Infecciones por Fusobacterium/epidemiología , Infecciones por Fusobacterium/microbiología , Infecciones por Fusobacterium/patología , Fusobacterium nucleatum/aislamiento & purificación , Infecciones por Bacterias Grampositivas/epidemiología , Infecciones por Bacterias Grampositivas/microbiología , Infecciones por Bacterias Grampositivas/patología , Peptostreptococcus/aislamiento & purificación , Índice Periodontal , Periodontitis/epidemiología , Periodontitis/patología , Porphyromonas gingivalis/aislamiento & purificación , Prevotella intermedia/aislamiento & purificación , Índice de Severidad de la EnfermedadRESUMEN
High levels of high-density lipoprotein (HDL) cholesterol have been reported to protect against the development of atherosclerosis in humans by increasing reverse cholesterol transport and inhibiting the oxidation of low-density lipoprotein (LDL) due to the paraoxonase content of HDL. The purpose of the present study was to assess if there are any relationships between in vivo increases in serum levels of immunological LDL oxidation markers [autoantibodies against oxidized LDL, autoantibodies against malondialdehyde-modified LDL, LDL immune complexes and anti-cardiolipin autoantibodies], paraoxonase activity and the development of atherosclerosis in control rabbits and in transgenic rabbits expressing human apolipoprotein (apo) A-I. A total of 13 apo A-I transgenic rabbits and 18 non-transgenic littermates were fed on a cholesterol-rich diet (0.4%, w/w) for 14 weeks, and were monitored at weeks 0, 2, 6, 10 and 14. Aortic atherosclerotic lesions were measured at the end of this period. Human apo A-I transgenic rabbits with high HDL cholesterol levels were not protected against the development of atherosclerosis when they were fed on a cholesterol-rich diet which induced dramatic hypercholesterolaemia. Immunological markers of LDL oxidation increased and serum paraoxonase activity decreased similarly in control and transgenic rabbits. In conclusion, the present study demonstrates that high HDL cholesterol levels are ineffective in inhibiting increases in immunological markers of LDL oxidation and the development of atherosclerosis in a mammal with severe hypercholesterolaemia.
Asunto(s)
Apolipoproteína A-I/metabolismo , Arteriosclerosis/inmunología , HDL-Colesterol/fisiología , Hipercolesterolemia/inmunología , Lipoproteínas LDL/inmunología , Animales , Animales Modificados Genéticamente , Anticuerpos Anticardiolipina/sangre , Arteriosclerosis/etiología , Autoanticuerpos/sangre , Biomarcadores , Femenino , Humanos , Hipercolesterolemia/complicaciones , Masculino , Oxidación-Reducción , Conejos , Estadísticas no ParamétricasRESUMEN
The protective effect of di-tert-butylhydroxylated flavonoids (chalcones and arylidenes) against minimally oxidized LDL (mO-LDL)-induced cytotoxicity was studied in cultured bovine aortic endothelial cells. Most of the tested compounds decreased aldehydes formation in medium containing mO-LDL, but their capacity to inhibit LDL oxidation in the cellular medium was not sufficient to totally reduce the cellular toxicity of mO-LDL. Most of the tested flavonoids improved the integrity of cells exposed to mO-LDL, whereas butylated hydroxytoluene was ineffective and quercetin worsened the toxicity of mO-LDL. Moreover these flavonoids induced an increase in GSH cellular levels and their protective effects might be because of their inability to reduce metal ion. Arylidene 6 substituted at position 7 by a hydroxyl group was the most potent compound.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Flavonoides/farmacología , Lipoproteínas LDL/toxicidad , Adenosina Trifosfato/metabolismo , Animales , Bovinos , Células Cultivadas , Medios de Cultivo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Glutatión/metabolismo , Humanos , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
We have identified cells expressing Cx26, Cx30, Cx32, Cx36 and Cx43 in gap junctions of rat central nervous system (CNS) using confocal light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling (FRIL). Confocal microscopy was used to assess general distributions of connexins, whereas the 100-fold higher resolution of FRIL allowed co-localization of several different connexins within individual ultrastructurally-defined gap junction plaques in ultrastructurally and immunologically identified cell types. In >4000 labeled gap junctions found in >370 FRIL replicas of gray matter in adult rats, Cx26, Cx30 and Cx43 were found only in astrocyte gap junctions; Cx32 was only in oligodendrocytes, and Cx36 was only in neurons. Moreover, Cx26, Cx30 and Cx43 were co-localized in most astrocyte gap junctions. Oligodendrocytes shared intercellular gap junctions only with astrocytes, and these heterologous junctions had Cx32 on the oligodendrocyte side and Cx26, Cx30 and Cx43 on the astrocyte side. In 4 and 18 day postnatal rat spinal cord, neuronal gap junctions contained Cx36, whereas Cx26 was present in leptomenigeal gap junctions. Thus, in adult rat CNS, neurons and glia express different connexins, with "permissive" connexin pairing combinations apparently defining separate pathways for neuronal vs. glial gap junctional communication.
Asunto(s)
Encéfalo/metabolismo , Conexina 43/metabolismo , Conexinas/metabolismo , Médula Espinal/metabolismo , Factores de Edad , Animales , Encéfalo/citología , Comunicación Celular/fisiología , Conexina 26 , Conexina 30 , Técnica de Fractura por Congelación/métodos , Uniones Comunicantes/química , Microscopía Confocal , Modelos Anatómicos , Neuroglía/química , Neuroglía/ultraestructura , Neuronas/química , Neuronas/ultraestructura , Ratas , Médula Espinal/citología , Proteína beta1 de Unión Comunicante , Proteína delta-6 de Union ComunicanteRESUMEN
The aim of this study was to carry out a microbiological evaluation of sites with and without clinical evidence of moderate and severe periodontitis and their correlation with clinical parameters. A total of 52 disease sites and 10 healthy sites were selected according to clinical criteria. The following clinical indexes were measured for all the sites: plaque index, gingival index, blood on probing, depth on probing and insertion level. Samples of subgingival plaque were collected for culture and for differential counts of microbial morphotypes. In disease sites the most frequently isolated were: Prevotella intermedia/nigrescens (65
), Porphyromonas gingivalis (23
), Actinobacillus actinomycetemcomitans (23
), Fusobacterium nucleatum (10
) and Peptostreptococcus sp. (31
). The aerobic gram-positive microflora was predominant in healthy sites. Significant differences were observed in microbial morphotypes between healthy and disease sites: cocci 18.71
and 78.90
, motile rods 46.12
and 16.70
, total spirochetes 26.48
and 2.80
, respectively. The presence of motile rods, spirochetes and P. intermedia/nigrescens were the parameters with most sensitivity to suspect periodontal disease. There were significant differences in the subgingival microflora between healthy and disease sites in patients with moderate and severe periodontitis.
RESUMEN
Epidemiological evidence suggests an inverse relationship between dietary intake of flavonoids and cardiovascular risk. The biological activities of flavonoids are related to their antioxidative effects, but they also can be mutagenic, due to the prooxidant activity of the catechol pattern. To prevent these problems, we synthesized new flavonoids where one or two di-tert-butylhydroxyphenyl (DBHP) groups replaced catechol moiety at position 2 of the benzopyrane heterocycle. Two DBHP moieties can also be arranged in an arylidene structure or one DBHP fixed on a chalcone structure. Position 7 on the flavone and arylidene or position 4 on the chalcone was substituted by H, OCH(3), or OH. New structures were compared with quercetin and BHT in an LDL oxidation system induced by Cu(II) ions. Arylidenes and chalcones had the best activities (ED(50) = 0.86 and 0.21) compared with vitamin E, BHT, and quercetin (ED(50) = 10.0, 7. 4, and 2.3 microM). Activity towards stable free radical 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) was measured by log Z and ECR(50) parameters. Synthesized flavones proved to be poor DPPH radical scavengers, the activity increasing with the number of DBHP units. In contrast, arylidenes and chalcones were stronger DPPH radical scavengers (log Z > 3, 0.3 < ECR(50) < 2.12) than BHT (log Z = 0.75, ECR(50) = 12.56) or quercetin (log Z = 2.76, ECR(50) = 0.43). Unlike quercetin, synthesized compounds neither chelated nor reduced copper, proving that these new flavonoids had no prooxidant activity in vitro.