RESUMEN
OBJECTIVE: Chlamydia trachomatis is the most prevalent bacteria causing sexually transmitted infections. In women, this infection can cause cervicitis and urethritis, although it's usually asymptomatic. The aim of this study was to investigate the prevalence of C. trachomatis in women attending the lab Instituto de Previsión Social and detect the genotypes. METHODS: Endocervical samples from 505 symptomatic and asymptomatic women were assayed. It was determined the presence of C. trachomatis by PCR through amplification of a fragment of the cryptic plasmid. Positive samples were genotyped by the partial amplification of the ompA gene and analyzed phylogenetically. RESULTS: Forty-three positive samples were detected to infection with C. trachomatis, obtaining a prevalence of 8.5% (IC 95%: 6.4-11.3%). The prevalence of C. trachomatis was higher in women with vaginal symptoms [11.3% (30/265) vs. 5.4% (13/240)] (p = 0.018), as well as in women under 26 year-old [11.5% (28/244) vs. 6.2% (15/246)] (p = 0.021). Based on phylogenetic analysis, it was observed that 62% of the samples were genotype E, 15% genotype J, 15% genotype D, and 8% genotype F. CONCLUSIONS: This work is the first contribution on the molecular epidemiology of C. trachomatis in the Misiones province, Argentina, which shows the rate of prevalence of this bacterium and offers information on circulating genotypes.
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Infecciones por Chlamydia/epidemiología , Chlamydia trachomatis/genética , Adolescente , Adulto , Argentina , Femenino , Genotipo , Humanos , Persona de Mediana Edad , Epidemiología Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Prevalencia , Enfermedades Vaginales/microbiología , Adulto JovenRESUMEN
Thiol groups of cysteine residues represent redox centers involved in multiple biological functions. It has been postulated that changes in the redox status of mammalian epididymal spermatozoa contribute to the sperm maturation process. The present work shows the thiol-disulfide protein profile of stallion epididymal spermatozoa achieved by two-dimension electrophoresis and MALDI-TOF/TOF mass spectrometry of proteins labeled with a thiol-reactive fluorescent tag, monobromobimane. Our results have shown the formation of disulfide bonds in several sperm protein fractions during the epididymal maturation process. The majority of the oxidized thiol sperm proteins identified correspond to structural molecules of the flagellum (as the outer dense fiber-1 protein - ODF1), followed by glycolytic enzymes (as glyceraldehyde-3-phosphate dehydrogenase spermatogenic), antioxidant protectors (as glutathione S-transferase and phospholipid hydroperoxide glutathione peroxidase - PHGPx). The magnitude of the thiol oxidation differs between proteins, and was more drastic in polypeptides with molecular weights of up to 33kDa, identified as ODF1 and PHGPx. A kinase anchor protein, a voltage-dependent anion channel protein and a zona pellucida-binding protein were also found in the polypeptide samples that contained oxidized SH groups. These proteins may be modified or controlled by the mechanisms involved in the cysteine-redox changes, corroborating the belief that a correct degree of protein oxidation is required for the stabilization of sperm structure, protection against oxidative damage, induction of progressive sperm motility and fertilization.
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Disulfuros/análisis , Caballos , Proteínas de Plasma Seminal/análisis , Espermatozoides/química , Compuestos de Sulfhidrilo/análisis , Animales , Electroforesis en Gel Bidimensional , Caballos/metabolismo , Masculino , Proteoma/análisis , Proteoma/metabolismo , Proteínas de Plasma Seminal/química , Proteínas de Plasma Seminal/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Recuperación de la Esperma/veterinaria , Espermatozoides/metabolismoRESUMEN
Tumor-associated immune cells often lack immune effector activities, and instead they present protumoral functions. To understand how tumors promote this immunological switch, invasive and noninvasive breast cancer cell (BRC) lines were cocultured with a promonocytic cell line in a Matrigel-based 3D system. We hypothesized that if communication exists between tumor and immune cells, coculturing would result in augmented expression of genes associated with tumor malignancy. Upregulation of proteases MMP1 and MMP9 and inflammatory COX2 genes was found likely in response to soluble factors. Interestingly, changes were more apparent in promonocytes and correlated with the aggressiveness of the BRC line. Increased gene expression was confirmed by collagen degradation assays and immunocytochemistry of prostaglandin 2, a product of COX2 activity. Untransformed MCF-10A cells were then used as a sensor of soluble factors with transformation-like capabilities, finding that acini formed in the presence of supernatants of the highly aggressive BRC/promonocyte cocultures often exhibited total loss of the normal architecture. These data support that tumor cells can modify immune cell gene expression and tumor aggressiveness may importantly reside in this capacity. Modeling interactions in the tumor stroma will allow the identification of genes useful as cancer prognostic markers and therapy targets.
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Células Acinares/patología , Neoplasias de la Mama/patología , Colágeno/metabolismo , Ciclooxigenasa 2/metabolismo , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Células Precursoras de Monocitos y Macrófagos/enzimología , Células Acinares/metabolismo , Neoplasias de la Mama/genética , Comunicación Celular/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Técnicas de Cocultivo , Dinoprostona/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Matriz Extracelular/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Modelos Biológicos , Células Precursoras de Monocitos y Macrófagos/patología , Invasividad Neoplásica , Fenotipo , Proteolisis , Solubilidad , Regulación hacia ArribaRESUMEN
The expression of α-D-mannosidase activity was fluorometrically and electrophoretically assessed in spermatozoa, epididymal fluid and homogenates of stallion epididymal tissue. Enzyme activity had regional differences; it was higher (P<0.05) in samples from the cauda epididymal region than in samples from the proximal caput region (largely composed of efferent ducts). Based on enzyme activity, as a function of pH of the assay substrate, electrophoretic analysis in native and native/SDS-PAGE conditions, and the effect of inhibitors or activators, we inferred the presence of at least two catalytically active forms of α-D-mannosidase. The neutral form of the enzyme (α-mannosidase II) was activated by Co2+, whereas the acid form (optimum pH 3.5 to 4.0) was sensitive to swainsonine (an inhibitor of α-mannosidase I), stabilized or stimulated by Zn2+, and not activated by Co2+ (activator of the neutral form). The activity of the acid form of the enzyme was highest in the epididymal fluid, where it seemed to be mainly in a secretory form. This form of the enzyme may have a role in plasma membrane remodeling associated with sperm maturation. In contrast, the activity of α-mannosidase II was higher in mature spermatozoa. It has been postulated that α-mannosidase II may act as a receptor in the recognition and binding of the complementary carbohydrate moieties present on the zona pellucida. With non-denaturing electrophoresis, α-D-mannosidase had an electrophoretic mobility of 0.35 and 0.24. When resolved by 1D and 2D SDS-PAGE (under denaturing conditions) the enzyme had a major protein band of molecular weight 154 kDa in spermatozoa and epididymal samples. Based on its properties under native conditions, we inferred that this enzyme might interact with other proteins and form transitory aggregates.
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Epidídimo/fisiología , Caballos/fisiología , Semen/enzimología , Espermatozoides/enzimología , alfa-Manosidasa/metabolismo , Animales , Cloruros/farmacología , Cobalto/farmacología , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Epidídimo/enzimología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Concentración de Iones de Hidrógeno , Masculino , Swainsonina/farmacología , Compuestos de Zinc/farmacología , alfa-Manosidasa/antagonistas & inhibidores , alfa-Manosidasa/genéticaRESUMEN
OBJECTIVE: To study the association of HLA-B27 with IgG antibodies to different enterobacterial HSP60s in patients with ankylosing spondylitis (AS). METHODS: IgG antibodies to 60 kDa enterobacterial HSPs were determined by ELISA in paired samples of sera and synovial fluid from 21 HLA-B27+ ankylosing spondylitis (AS) patients; and in sera from 32 HLA-B27+ AS patients, 35 HLA-B27+ healthy relatives of AS patients, and 60 HLA-B27- healthy individuals with no family members with AS. RESULTS: HLA-B27+ patients and healthy individuals showed significantly higher IgG antibody levels to recombinant enterobacterial HSP60s than HLA-B27- healthy controls. The levels of anti-HSP60Sf and anti-HSP60Ec antibodies correlated with disease activity and anti-HSP60Ec antibodies with male gender. No association between enterobacterial HSP60 antibody levels and disease duration was observed. All groups had lower levels of IgG antibodies to rHSP60 from Streptococcus pyogenes (rHSP60 Spy). In paired samples of sera and synovial fluid from B27+ patients, IgG antibodies to enterobacterial HSP60s were detected, but in significantly higher levels in sera than in synovial fluid. The anti-rHSPSpy IgG response in these samples was lower and similar in the three groups. CONCLUSIONS: A correlation was found between HLA-B27 and the response to recombinat enterobacterial HSP60s. This response could be associated with disease activitir and gender in some proteins and the presence eof IgG antibodies to these proteins in synovial fluid could be associated with the inflammatory process and initiation of AS.
Asunto(s)
Chaperonina 60/inmunología , Infecciones por Enterobacteriaceae/inmunología , Antígeno HLA-B27/inmunología , Espondilitis Anquilosante/inmunología , Líquido Sinovial/inmunología , Adolescente , Adulto , Anticuerpos Antibacterianos/sangre , Chaperonina 60/biosíntesis , Enterobacteriaceae/inmunología , Infecciones por Enterobacteriaceae/sangre , Infecciones por Enterobacteriaceae/complicaciones , Femenino , Antígeno HLA-B27/genética , Humanos , Indígenas Norteamericanos/genética , Masculino , México , Persona de Mediana Edad , Proteínas Recombinantes/inmunología , Espondilitis Anquilosante/sangre , Espondilitis Anquilosante/microbiología , Líquido Sinovial/microbiología , Adulto JovenRESUMEN
'The highly packed chromatin of mature spermatozoa results from replacement of somatic-like histones by highly basic arginine- and cysteine-rich protamines during spermatogenesis, with additional conformational changes in chromatin structure during epididymal transit. The objective of the present study was to compare the nuclear characteristics of immature and mature epididymal stallion spermatozoa, using a variety of experimental approaches. Resistance to in vitro decondensation of chromatin, following exposure to SDS-DTT and alkaline thioglycolate, increased significantly in mature spermatozoa. Evaluation of the thiol-disulfide status (monobromobimane labeling) demonstrated that immature cells obtained from ductulli efferentes contained mostly thiol groups, whereas these groups were oxidized in mature cells collected from the cauda epididymidis. Based on atomic absorption spectrophotometry, maturation of stallion spermatozoa was accompanied by a 60% reduction in the Zn(2+) content of sperm cells, concomitant with increased concentrations of this ion in epididymal fluid. Furthermore, the degree of disulfide bonding was inversely correlated with susceptibility of chromatin to acid denaturation (SCSA). Collectively, these data were consistent with the hypothesis that maturation of stallion spermatozoa involves oxidation of sulphydryl groups to form intra- and intermolecular disulfide links between adjacent protamines, with loss of zinc as an integral feature. These changes endow mechanical and chemical resistance to the nucleus, ensuring efficient transmission of the paternal genome at fertilization.
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Cromatina/metabolismo , Caballos/fisiología , Maduración del Esperma/fisiología , Espermatogénesis/fisiología , Espermatozoides/fisiología , Zinc/metabolismo , Animales , Cromatina/ultraestructura , Eyaculación/fisiología , Epidídimo/fisiología , Masculino , Oxidación-Reducción , Espermatozoides/metabolismo , Compuestos de Sulfhidrilo/análisis , Zinc/análisisRESUMEN
OBJECTIVE: Apoptosis appears to be the mode of cell death by which damaged cells are removed from the lesional tissue. The aim of this study was to examine keratinocyte apoptosis and caspase-3 (CPP32) expression in oral lichen planus (OLP). MATERIALS AND METHODS: Paraffin-embedded samples of OLP (n = 30) and normal oral mucosa (NOM; n = 5) were prepared for haematoxylin-eosin (H & E), immunohistochemistry and electron microscopy. The number of apoptotic cells and the proportion of total cells that were either apoptotic (apoptotic index; AI) or mitotic (mitotic index; MI) were assessed in H & E stained sections. An immunostaining-intensity-distribution index (IIDI; proportion of stained cells x staining intensity) was used to assess CPP32 immunoreactivity. RESULTS: Results showed a significant increase in the number of apoptotic cells in OLP (P < 0.001). In OLP, all apoptotic bodies were found in the basal and prickle epithelial layers. Compared with NOM, the AI was significantly greater in atrophic (P < 0.05), reticular (P < 0.001) and plaque-like (P < 0.01) OLP. The MI was significantly greater in plaque-like OLP (P < 0.01). The proportion of CPP32-positive cells and the IIDI were significantly greater in all forms of OLP compared with NOM (P < 0.05). No difference in CPP32 expression was evident between clinical forms of OLP. Electron microscopy confirmed the light microscopic finding of apoptosis. CONCLUSION: Keratinocyte apoptosis and caspase-3 expression co-localized to the basal and parabasal epithelial layers, suggesting that proliferating epithelial cells may be targeted for destruction in OLP. Differences in epithelial AI and MI may underlie the various clinical and histological appearances of OLP.
Asunto(s)
Apoptosis , Caspasas/análisis , Precursores Enzimáticos/análisis , Queratinocitos/enzimología , Liquen Plano Oral/enzimología , Adulto , Anciano , Atrofia , Caspasa 3 , Núcleo Celular/enzimología , Núcleo Celular/ultraestructura , Cromatina/enzimología , Cromatina/ultraestructura , Colorantes , Citoplasma/enzimología , Citoplasma/ultraestructura , Células Epiteliales/enzimología , Células Epiteliales/patología , Colorantes Fluorescentes , Humanos , Queratinocitos/patología , Liquen Plano Oral/patología , Linfocitos/enzimología , Linfocitos/patología , Microscopía Electrónica , Persona de Mediana Edad , Mitosis , Mucosa Bucal/enzimología , Mucosa Bucal/patologíaRESUMEN
AIMS: To determine the levels of IgG class antibodies to recombinant heat shock protein 60 kDa of Yersinia enterocolitica (rHSP60Ye), Klebsiella pneumoniae (rHSP60Kp), Escherichia coli (rHSP60Ec), Shigella flexneri (rHSP60Sf), and Streptococcus pyogenes (rHSP60Sp) in the serum of patients with HLA-B27 associated acute anterior uveitis (HLA-B27 associated AAU), idiopathic acute anterior uveitis (idiopathic AAU), pars planitis, Vogt-Koyanagi-Harada (VKH), and healthy subjects. METHODS: The genes that code for HSP60Ye, HSP60Kp, HSP60Ec, HSP60Sf, and HSP60Sp were cloned by PCR from genomic DNA. The rHSPs were purified by affinity using a Ni-NTA resin. The serum levels of IgG class antibodies to rHSP60s were determined by ELISA in patients with uveitis (n = 42) and in healthy subjects (n = 25). RESULTS: The majority of patients with uveitis had higher levels of IgG class antibodies to rHSP60Ye compared with levels of healthy subjects (p = 0.01), although these differences were only observed in the HLA-B27 associated AAU (p = 0.005) and in pars planitis patients (p = 0.001). The levels of IgG antibodies to the rHSP60Kp, rHSP60Sf, rHSP60Ec, and rHSP60Sp were similar in patients with uveitis and in healthy subjects (p>0.05). CONCLUSION: The results suggest that HSP60Ye could be involved in the aetiology of HLA-B27 associated AAU and pars planitis.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Chaperonina 60/inmunología , Inmunoglobulina G/sangre , Pars Planitis/microbiología , Uveítis Anterior/microbiología , Yersinia enterocolitica/inmunología , Enfermedad Aguda , Adolescente , Adulto , Femenino , Predisposición Genética a la Enfermedad , Antígeno HLA-B27/sangre , Humanos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Pars Planitis/inmunología , Proteínas Recombinantes/inmunología , Recurrencia , Uveítis Anterior/inmunología , Síndrome Uveomeningoencefálico/inmunología , Síndrome Uveomeningoencefálico/microbiologíaRESUMEN
This study demonstrated that when the regeneration of the axotomized sciatic nerve is induced through tubulization with chitosan, this biomaterial does not induce immunostimulation or immunodepression in the dog. Canine females were distributed among three groups: an intact control group which was only isolated, an axotomized control group, and an axotomized group which was tubulized with 3% chitosan prostheses. In vitro culture and phagocytosis tests, as well as IgG and IgM serum concentrations, were determined in peripheral blood on days 0, 15, 30 and 60. The results showed that chitosan implants did not importantly affect the immune response.
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Axotomía , Quitina/análogos & derivados , Quitina/inmunología , Regeneración Nerviosa , Prótesis e Implantes , Nervio Ciático/fisiología , Animales , Quitosano , Perros , Femenino , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Activación de Linfocitos , Fagocitosis , Nervio Ciático/inmunologíaRESUMEN
1. Eggs from wild and captive populations of Greater Rhea (Rhea americana) were studied to determine their physical and chemical characteristics. 2. Significant differences were found among populations in almost all chemical parameters studied, whereas within physical parameters only shell weight (as a proportion of the entire egg) and density showed differences. 3. Eggs from wild populations had the highest protein and linolenic acid and the lowest total lipid contents, while cholesterol levels of these eggs and of those from the largest captive area were the lowest.
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Huevos/análisis , Reiformes/fisiología , Animales , Animales Domésticos , Animales Salvajes , Colesterol/análisis , Proteínas del Huevo/análisis , Cáscara de Huevo/química , Huevos/normas , Lípidos/análisisAsunto(s)
Arteriolas/embriología , Riñón/irrigación sanguínea , Arteria Renal/embriología , Venas Renales/embriología , Animales , Arteriolas/crecimiento & desarrollo , Diferenciación Celular , Sustancias de Crecimiento/fisiología , Riñón/embriología , Ratones , Neovascularización Fisiológica , Arteria Renal/crecimiento & desarrollo , Venas Renales/crecimiento & desarrollo , Células Madre/citologíaAsunto(s)
Animales , Conejos , Arteriolas/embriología , Arteria Renal/embriología , Venas Renales/embriología , Riñón/irrigación sanguínea , Arteriolas/crecimiento & desarrollo , Arteria Renal/crecimiento & desarrollo , Venas Renales/crecimiento & desarrollo , Células Madre/citología , Diferenciación Celular , Sustancias de Crecimiento/fisiología , Neovascularización Fisiológica , Riñón/embriologíaRESUMEN
The occurrence, localization and ultrastructural characteristics of a blood-tissue barrier throughout the stallion proximal seminal excurrent duct system were studied by the exclusion of electron-dense tracers and freeze-fracture techniques. Striking differences were observed in the distribution of lanthanum tracer and in the geometrical organization of the zonulae occludentes along the ductus efferentes, epididymides and vas deferens. The zonulae occludentes domain, the principal structural component of the blood-epididymis barrier, differed in permeability, width and strand numbers along the ductus. The flow of tracer was not impeded by the vascular endothelium, the peritubular myoid layer or other surface membrane specialization. The tight junctions of the ductuli efferentes are poorly developed but unlike those of rats, guinea pigs or man they are not associated with gap junctions. The result of the tracer experiments and the low number of tight junctional strands in the ductuli efferentes suggests that the barrier of the ductuli efferentes corresponds to the 'leaky type'. In the epididymis the zonulae occludentes are well developed throughout the duct. The greatest number of strands, especially in the cauda epididymidis regions, correlates well with a decreased junctional permeability in this area. Another evidence for the existence of the stallion blood-epididymis barrier are the differences in the proteins electrophoretic profiles between blood plasma as compared with the fluid inside the seminal ductus. This junctional complexes contribute to create a highly defined luminal fluid microenvironment that ensures the sperm maturation and survival.
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Barrera Hematotesticular , Capilares/citología , Epidídimo/citología , Animales , Capilares/ultraestructura , Permeabilidad Capilar , Epidídimo/irrigación sanguínea , Técnica de Fractura por Congelación , Caballos , Masculino , Microscopía Electrónica , Ratas , Uniones Estrechas/ultraestructuraRESUMEN
Different venlafaxine doses (1, 5, and 10 mg/kg) and saline solution were administered to ten male Wistar rats (Latin-Square design). Compared with saline, venlafaxine produced a dose-related suppression of REM sleep and an increase in wake time while slow wave sleep was reduced. This effect is similar to the one that has been reported with some tricyclic antidepressants.
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Antidepresivos de Segunda Generación/farmacología , Ciclohexanoles/farmacología , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Sueño REM/efectos de los fármacos , Animales , Electroencefalografía/efectos de los fármacos , Masculino , Ratas , Ratas Wistar , Sueño REM/fisiología , Clorhidrato de VenlafaxinaRESUMEN
En este trabajo los efectos hepatotóxicos del anticonvulsionantes 4-hidroxi,, 4-etil, 4-fenil butiramida (HEPB) y sus homólogos HEPA y HEPP fueron evaluados y comparados con los efectos del valproato de sodio y del fenobarbital en cultivos de hepatocitos de larga sobrevivencia. Los hepatocitos se sembraron sobre una monocapa alimentadora de células 3T3 letalmente tratadas con mitomicina C y fueron expuestos por una o dos semanas a los anticonvulsionantes (50-500 mg/ml). Se seterminó en el medio de cultivo la liberación de las enzimas citoplásticas transaminasa glutámico oxalacética (GOT). Transaminasa glutámico pirúvica (GPT) y deshidrogenasa láctica (LDH), se determinó también el contenido de triclicéridos (TG) mediante tinción con rojo oleoso O. HEPB produjo gotas intracitoplásmicas de lípidos, ensanchamiento de los espacios intercelulares y retracción de las células. HEPA y HEPP produjeron efectos similares pero en menor grado. El valproato de sodio causó retracción de las células, ensanchamiento de los espacios intracelulares y también vacuolización de las células. Después de una exposición de una semana el valproato de sodio produjo la liberación más alta de TGO, TGP y LDH. Los cultivos tratados con los otros anticonvulsionantes mostraron poca diferencia con los cultivos control. Después de dos semanas de exposición los efectos fueron menores. El mayor contenido de TG lo produjo el HEPB, mientras que el valpoatro de sodio produjo una disminución en el contenido de TG. Nuestros resultados muestran que el rango de toxicidad de los anticonvulsionantes probados es valproato de sodio >HEPB>HEPA>HEPP>fenobarbital
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Ratas , Animales , Masculino , Ácido Valproico/farmacocinética , Anticonvulsivantes , Butiratos , Butiratos/toxicidad , Hepatopatías/inducido químicamente , Fenobarbital/farmacocinética , Ratas Wistar/cirugíaRESUMEN
In the search for the pathogenic consequences of the molecular mimicry between the Klebsiella pneumoniae nitrogenase and the HLA-B27 antigen, sera from individuals belonging to 16 kindreds with juvenile-onset ankylosing spondylitis cases, were analyzed for antibodies against nitrogenase-positive and -negative K. pneumoniae whole bacterial extracts. An initial screening for nitrogenase producing K. pneumoniae strains was performed in 31 clinical isolates. The best nitrogenase producing strain was selected as well as a non producing one for immunoblot analysis using sera from 82 subjects, 55 HLA-B27 positive, of which 26 had some clinical manifestations. Even though electrophoretic patterns were different in both strains, there was no distinctive differential recognition of the 30-40 kDa proteins where the nitrogenase subcomponent which shares the sequence QTDRED with the HLA-B27 molecule is located. On the other hand, strong recognition of a protein of 60 kDa (p60Kp) was detected in 75% of HLA-B27 positive tested subjects independently of their clinical status. Studies on the nature of this protein and its participation in the pathogenesis of ankylosing spondylitis are now in progress.
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Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Proteínas Bacterianas/inmunología , Klebsiella pneumoniae/inmunología , Imitación Molecular , Nitrogenasa/inmunología , Espondilitis Anquilosante/inmunología , Especificidad de Anticuerpos , Enfermedades Autoinmunes/genética , Reacciones Cruzadas , Antígeno HLA-B27/análisis , Antígeno HLA-B27/genética , Humanos , Klebsiella pneumoniae/enzimología , Espondilitis Anquilosante/genéticaRESUMEN
Porphyrin production and excretion and the effects of lead exposure were studied in long-term cultures of adult rat hepatocytes cultured on a feeder layer of 3T3 cells after addition of 5-aminolevulinic acid. Porphyrin excretion into the culture medium showed an irregular profile during the first 10 days, with a maximum increase of 50% at day 4 and at day 10 a value similar to that of day 1. Thereafter, porphyrin excretion decreased progressively to 18% of the initial value after 4 weeks. The cellular porphyrin content, after 7 and 28 days in culture, reached values 3.8 and 2.4-fold higher than the corresponding day 1 value. The exposure to 0.5 and 2.4 microM Pb2+ for up to 28 days produced a biphasic effect on porphyrin excretion. Firstly, there was a progressive decrease up to 81% during the first 6 days of lead exposure and, secondly, this effect was followed by an increase reaching control values at day 15 and of up to 6.7-fold after 22 days of exposure to 2.4 microM Pb2+. Similar changes were observed in cellular porphyrin content. The exposure to 0.5 and 2.4 microM Pb2+ for 2 and 4 weeks also produced morphological alterations and release of cytoplasmic enzymes. Our results show that hepatocytes cultured on 3T3 cells produce and excrete porphyrins for 28 days and that exposure for 4 weeks to micromolar lead concentrations alters these functions and cell morphology and produces cytotoxic effects which are better evaluated by monitoring alterations in porphyrin excretion than by enzyme leakage. They also suggest that this culture system is a useful model for assessing the toxic effects of xenobiotics on the biosynthesis of heme by liver cells.
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Hígado/efectos de los fármacos , Compuestos Organometálicos/toxicidad , Porfirinas/biosíntesis , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Hígado/citología , Hígado/metabolismo , Masculino , Porfirinas/metabolismo , Ratas , Ratas WistarRESUMEN
To study the functionality of the urea cycle in long-term cultures of adult rat hepatocytes, urea production and the activity of two urea cycle enzymes were measured in hepatocytes cultured on 3T3 cells for 15 days. Urea production was also measured in cultures maintained with medium containing either 0.4 mm arginine or 0.4 mm ornithine and in cultures exposed to different concentrations of NH(4)Cl, an in vivo inducer of urea production. In hepatocytes seeded on 3T3 cells, urea production decreased gradually to 50% of the initial value after 15 days. Urea production was similar in 3T3-hepatocyte cultures maintained for 11 days with medium containing ornithine or arginine. Hepatocytes exposed for 24 hr to 1, 3 and 5 mm NH(4)Cl showed an average increase in urea production of 25, 50 and 69%, respectively, above that of unexposed cultures over 15 days. Ornithine transcarbamylase (OTC) activity decreased by 84% after 5 days in culture and remained constant thereafter, while arginase activity remained constant over 15 days. In contrast, in hepatocytes seeded on plastic substratum, urea production decreased to 24% of the initial value after 8 days in culture. OTC and arginase activities also decreased to 13 and 10% of their initial values after 8 days in culture. These results show that 3T3-hepatocyte cultures from adult rats produce urea from ornithine and/or arginine for at least 15 days and respond to an inducer of urea production as in vivo. They also show that these cultures have decreasing and constant levels of OTC and arginase activities, respectively, owing probably to an adaptative response dependent on substrate concentrations and hormonal regulation. These findings also suggest that 3T3-hepatocyte cultures are a suitable in vitro system to study urea production, its regulation by substrates and hormones and its alteration by drugs and toxic chemicals.