RESUMEN
The temporal effect of discharge and limnology on fish composition and species diversity in a floodplain lake at the confluence of the Amazon and Negro Rivers was evaluated. Species richness, abundance and assemblage composition were strongly influenced by seasonal discharge of the Amazon and Negro Rivers, which affects lateral connectivity, water conductivity and temperature. As a consequence, temporal ß-diversity was high in the lake and the assemblage was dominated by seasonally transient species. Relatively large species known to feed on resources within the floodplain were captured almost exclusively during the flood period. During the dry season, the assemblage was dominated by fishes adapted to harsh conditions of high temperature and low dissolved oxygen concentrations. An open system with high spatial and temporal heterogeneity created by the meeting of two large rivers with different water chemistry, Lago Catalão has a dynamic fish assemblage. Given its high temporal ß-diversity and abundance of fishes, many of great importance in local fisheries, Lago Catalão and other floodplain lakes in this region merit special attention for conservation.
Asunto(s)
Ecosistema , Peces , Estaciones del Año , Animales , Brasil , Inundaciones , Lagos , RíosRESUMEN
Pothomorphe umbellata, a native Brazilian plant, is popularly known to be effective in the treatment of skin lesions. This benefit is attributed to 4-nerolidylcatechol (4-NC), a compound extracted from P. umbellata. Since melanomas show prominent resistance to apoptosis and exhibit extreme chemoresistance to multiple forms of therapy, novel compounds addressing induction of cell death are worth investigating. Here, we evaluated effects on cell cycle progression and possible cytotoxic activity of 4-NC in melanoma cell lines as well as human dermal fibroblasts. Inhibitory effects on cell invasion and MMP activity were also investigated. 4-NC showed cytotoxic activity for all melanoma cell lines tested (IC50=20-40 microM, 24h for tumoral cell lines; IC50=50 microM for fibroblast cell line) associated with its capacity to induce apoptosis. Furthermore, this is the first time that 4-NC is described as an inhibitor of cell invasiveness, due mainly to a G1 cell cycle arrest and inhibition of MMP-2 activity in melanoma cell lines.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Catecoles/farmacología , Melanoma/tratamiento farmacológico , Piperaceae/química , Neoplasias Cutáneas/tratamiento farmacológico , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Melanoma/secundario , Invasividad Neoplásica/patología , Invasividad Neoplásica/prevención & control , Extractos Vegetales/química , Extractos Vegetales/farmacología , Neoplasias Cutáneas/patologíaRESUMEN
The in vitro ability of Pothomorphe umbellata ethanolic crude extract to inhibit matrix metalloproteinase (MMP) in normal cornea and in cornea after alkali injury was demonstrated. Corneas of albino rabbits were injured with 1 N NaOH for 20 s. After 48 h the corneas were excised, homogenized and analyzed for MMP-9 (92 kDa), pro-MMP-2 (72 kDa) and MMP-2 (67 kDa) activity by gelatin zymography. The activity was also measured in untreated corneas. After electrophoresis of 20 microg protein, gels were incubated with 50, 100, or 250 microg/mL lyophilized hydroethanolic (1:1) root crude extract of P. umbellata standardized for 4-nerolidylcatechol (7.09%). The activity of the enzymes was compared with that of untreated gel. At 48 h after injury, the activity of all MMPs was increased compared with untreated eyes. When the gels were incubated with P. umbellata extract the activity of MMP-2, pro-MMP-2 and MMP-9 decreased in a dose-dependent manner. MMP-9 activity decreased by approximately 50% after incubation with 50 microg/mL and was completely abolished at 100 and 250 microg/mL of the extract. After incubation with 50 microg/mL the activity of pro-MMP-2 and MMP-2 also decreased by 50%. The activity of pro-MMP-2 was almost completely abolished after incubation with 250 microg/mL of the extract. For MMP-2 the incubation with 100 or 250 microg/mL of the extract of P. umbellata promoted a 10-fold decrease in activity. In conclusion, P. umbellata root crude extract can be useful as an alternative therapy to control MMP activity after corneal injury.
Asunto(s)
Quemaduras Químicas/enzimología , Lesiones de la Cornea , Inhibidores Enzimáticos/farmacología , Quemaduras Oculares/inducido químicamente , Inhibidores de la Metaloproteinasa de la Matriz , Piperaceae/química , Animales , Córnea/enzimología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/aislamiento & purificación , Quemaduras Oculares/enzimología , Metaloproteinasas de la Matriz/metabolismo , Fitoterapia , Extractos Vegetales/farmacología , ConejosRESUMEN
The in vitro ability of Pothomorphe umbellata ethanolic crude extract to inhibit matrix metalloproteinase (MMP) in normal cornea and in cornea after alkali injury was demonstrated. Corneas of albino rabbits were injured with 1 N NaOH for 20 s. After 48 h the corneas were excised, homogenized and analyzed for MMP-9 (92 kDa), pro-MMP-2 (72 kDa) and MMP-2 (67 kDa) activity by gelatin zymography. The activity was also measured in untreated corneas. After electrophoresis of 20 æg protein, gels were incubated with 50, 100, or 250 µg/mL lyophilized hydroethanolic (1:1) root crude extract of P. umbellata standardized for 4-nerolidylcatechol (7.09 percent). The activity of the enzymes was compared with that of untreated gel. At 48 h after injury, the activity of all MMPs was increased compared with untreated eyes. When the gels were incubated with P. umbellata extract the activity of MMP-2, pro-MMP-2 and MMP-9 decreased in a dose-dependent manner. MMP-9 activity decreased by approximately 50 percent after incubation with 50 µg/mL and was completely abolished at 100 and 250 µg/mL of the extract. After incubation with 50 µg/mL the activity of pro-MMP-2 and MMP-2 also decreased by 50 percent. The activity of pro-MMP-2 was almost completely abolished after incubation with 250 µg/mL of the extract. For MMP-2 the incubation with 100 or 250 µg/mL of the extract of P. umbellata promoted a 10-fold decrease in activity. In conclusion, P. umbellata root crude extract can be useful as an alternative therapy to control MMP activity after corneal injury.
Asunto(s)
Animales , Conejos , Quemaduras Químicas/enzimología , Córnea/lesiones , Inhibidores Enzimáticos/farmacología , Quemaduras Oculares/inducido químicamente , Metaloproteinasas de la Matriz/antagonistas & inhibidores , Piperaceae/química , Córnea/enzimología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/aislamiento & purificación , Quemaduras Oculares/enzimología , Metaloproteinasas de la Matriz/metabolismo , Fitoterapia , Extractos Vegetales/farmacologíaRESUMEN
In this study we evaluated the activity of Pothomorphe umbellata root extract on hairless mice chronically exposed to UVB radiation (76.5 mJ/cm(2), 4 days per week for 22 weeks). Mouse dorsal surfaces were treated topically with 20 mg/cm(2) of a carbomer 940 gel (vehicle) with or without P. umbellata root extract to a final concentration of 0.1%, for 2 h before irradiation. Another irradiated group received no topical treatment. A fourth group received no treatment and was not irradiated. Visible skin wrinkling was evaluated using a scale ranging from 0 to 4, where 0 corresponds to no skin modification, and 4 to the maximum visual skin alteration observed in our experiments. Histological measurements were carried out on standard haematoxylin & eosin stained sections. The mean distances between the outermost surface of the epidermis (excluding the stratum corneum) and the dermal-epidermal junction were determined by morphometric analysis. These distances were statistically increased in the irradiated control groups when compared to the nonirradiated control group and to the irradiated group using P. umbellata root extract. These data demonstrate that P. umbellata may be successfully used as a topical skin-protecting agent against the deleterious effects of UV radiation.
Asunto(s)
Fitoterapia/métodos , Piperaceae , Traumatismos Experimentales por Radiación/prevención & control , Protectores contra Radiación/uso terapéutico , Envejecimiento de la Piel/efectos de la radiación , Administración Cutánea , Animales , Ratones , Ratones Pelados , Extractos Vegetales/uso terapéutico , Raíces de Plantas , Traumatismos Experimentales por Radiación/patología , Índice de Severidad de la Enfermedad , Envejecimiento de la Piel/patología , Rayos Ultravioleta/efectos adversosRESUMEN
A characteristic feature of neoplastic transformation is a perpetual activation of oncogenic proteins. Here, we studied signal transducers and activators of transcription (STAT) in patients with mycosis fungoides (MF)/cutaneous T-cell lymphoma (CTCL). Malignant lymphocytes in dermal infiltrates of CTCL tumors showed frequent and intense nuclear staining with anti-PY-STAT3 antibody, indicating a constitutive activation of STAT3 in vivo in tumor stages. In contrast, only sporadic and faint staining was observed in indolent lesions of patch and plaque stages of MF. Moreover, neoplastic lymphocytes in the epidermal Pautrier abscesses associated with early stages of MF did not express activated STAT3. To address the role of STAT3 in survival/apoptosis, CTCL tumor cells from an advanced skin tumor were transfected with either wild-type STAT3 (STAT3wt) or dominant-negative STAT3 (STAT3D). Forced inducible expression of STAT3D triggered a significant increase in tumor cells undergoing apoptosis, whereas forced expression of STAT3wt or empty vector had no effect. In conclusion, a profound in vivo activation of STAT3 is observed in MF tumors but not in the early stages of MF. Moreover, STAT3 protects tumor cells from apoptosis in vitro. Taken together, these findings suggest that STAT3 is a malignancy factor in CTCL.
Asunto(s)
Apoptosis , Proteínas de Unión al ADN/metabolismo , Linfoma Cutáneo de Células T/química , Transactivadores/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/fisiología , Femenino , Humanos , Inmunohistoquímica , Linfocitos/química , Linfocitos/patología , Linfoma Cutáneo de Células T/etiología , Linfoma Cutáneo de Células T/patología , Masculino , Persona de Mediana Edad , Micosis Fungoide/química , Micosis Fungoide/patología , Invasividad Neoplásica/patología , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiología , Factor de Transcripción STAT3 , Neoplasias Cutáneas/química , Neoplasias Cutáneas/patología , Transactivadores/análisis , Transactivadores/fisiologíaRESUMEN
To study cell motility in different phases of the cell cycle, time-lapse recording by computer-assisted microscopy of unsynchronised cells from three mammalian cell lines (L929, BT4Cn, HeLa) was used for the determination of the displacements of individual cells. The displacements were used for calculation of three key parameters describing cell motility: speed, persistence time and rate of diffusion. All investigated cell lines demonstrated a lower cell displacement in the G2 phase than in the G1/S phases. This was caused by a decrease in speed and/or persistence time. The decrease in motility was accompanied by changes in morphology reflecting the larger volume of cells in G2 than in G1. Furthermore, L-cells and HeLa-cells appeared to be less adherent in the G2 phase. Transfection of L-cells with constitutively active Rac1 led to a general increase in the speed and rate of diffusion in G2 to levels comparable to those of control cells in G1. In contrast, transfection with dominant-negative Rac1 reduced cell speed and resulted in cellular displacements, which were identical in G1 and G2. These observations indicate that migration of cultured cells is regulated in a cell-cycle-dependent manner, and that an enhancement of Rac1 activity is sufficient for a delay of the reduced cell displacement otherwise seen in G2.
Asunto(s)
Ciclo Celular , Movimiento Celular/fisiología , Proteína de Unión al GTP rac1/fisiología , Animales , Western Blotting , División Celular , Línea Celular Tumoral , Tamaño de la Célula , Citometría de Flujo , Fase G2 , Glioma/patología , Células HeLa , Humanos , Procesamiento de Imagen Asistido por Computador , Cinética , Células L , Ratones , Microscopía Fluorescente , Ratas , Transfección , Grabación en Video , Proteína de Unión al GTP rac1/genéticaRESUMEN
Antigen (Ag)-specific CD8+ T cells are a major host defence against viral infections. In the present study, we generated human CD8+ T-cell lines specific towards influenza matrix peptide (IMP)-pulsed Ag-presenting cells. We compared the effect of interleukin-2 (IL-2) and IL-15 on the proliferation and cytotoxic activity of primary and secondary IMP-specific cytotoxic T lymphocyte (CTL) culture. In primary CTL cultures, IL-15-induced cell expansion was considerably reduced as compared with IL-2-induced cell expansion, and IL-15 favoured the outgrowth of CTLs without peptide specificity in these cultures. Secondary IMP-specific CD8+ T cells were generated by the addition of IL-2 during two cycles of restimulation. From the third restimulation, identical CTL cultures were expanded with either IL-2 or IL-15 in parallel. Cell expansion as well as Ag specificity was considerably reduced after a 5 day culture period in the presence of IL-15. No or low CD69 expression was observed in IL-15-cultured CTLs, whereas IL-2-cultured CTLs contained high fractions of CD69+ cells. Furthermore, a high fraction of these latter cells coexpressed the cytotoxic marker CD56. However, IL-15-cultured CTLs exhibited cytotoxic activity without detectable expression of CD56, suggesting that CD56 is not essential for cytotoxic activity. Thus, the results presented suggest that IL-15 favours the outgrowth of unspecific cytotoxic effector T cells.
Asunto(s)
Citotoxicidad Inmunológica , Interleucina-15/farmacología , Fragmentos de Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas de la Matriz Viral/inmunología , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Antígenos Virales/inmunología , Apoptosis , Antígeno CD56/metabolismo , División Celular , Línea Celular , Células Cultivadas , Citocinas/biosíntesis , Pruebas Inmunológicas de Citotoxicidad , Humanos , Interleucina-2/farmacología , Lectinas Tipo C , Activación de Linfocitos , Receptores de Interleucina-15 , Receptores de Interleucina-2/metabolismo , Linfocitos T Citotóxicos/efectos de los fármacosRESUMEN
Human retinal pigment epithelial (RPE) cells are capable of presenting bacterial superantigens (SAg) to T cells in vitro by ligation of MHC class II molecules on RPE cells with the T cell receptor. The purpose of this study was to evaluate the involvement of adhesion molecules in presentation of SAg. Cultured human fetal and adult RPE cells were treated with interferon-gamma (IFN-gamma, 500 U ml(-1) for 72 hr) and afterwards pulsed with the SAg staphylococcal enterotoxin A (SEA, 500 ng ml(-1) for 2 hr) followed by coculture with freshly obtained T cells isolated from peripheral blood. Proliferation was measured by (3)H-thymidine incorporation assay. In selected experiments, either RPE or T cells were pre-treated with blocking antibodies specific for cell surface molecules. For comparison, dendritic cells were used as superantigen presenting cells for T cells. This study showed that presentation of SEA by RPE cells to resting T cells was dependent on the presence of the molecules CD2, CD58 and CD18, CD54. The cycling status of T cells was decisive, thus resting T cells but not activated T cells were capable to proliferate in response to SEA presentation. Proliferation of T cells induced by adult RPE cells was comparable to the proliferation induced by dendritic cells at concentrations of SAg above 100 ng ml(-1), but at concentrations of SAg below 10 ng ml(-1) the response was significantly lower for SAg presented by RPE cells compared to dendritic cells. The results demonstrate that CD2-CD58 and CD18-CD54 interactions are critical for SAg presentation by RPE cells to T cells. The findings thus suggest that also presentation of peptides to resting T cells by RPE cells may be dependent upon these interactions.
Asunto(s)
Presentación de Antígeno/fisiología , Antígenos CD/fisiología , Epitelio Pigmentado Ocular/inmunología , Superantígenos/inmunología , Linfocitos T/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos CD18/fisiología , Antígenos CD2/fisiología , Antígenos CD58/fisiología , División Celular/fisiología , Separación Celular , Células Cultivadas , Citometría de Flujo , Humanos , Molécula 1 de Adhesión Intercelular/fisiología , Interferón gamma/fisiología , Estadísticas no Paramétricas , Linfocitos T/citologíaRESUMEN
CD8(+) CD56(+) cells isolated from human peripheral blood lymphocytes have been shown recently to represent a population of cytotoxic active T cells. However, it is not known if these cells are intrathymically or extrathymically developed or how these cells are influenced by growth factors. In the present study, we investigated the effects of interleukin-2 (IL-2) and IL-15 on human thymocytes with respect to development of CD8(+) CD56(+) T cells. Freshly isolated thymocytes contain few CD8(+) CD56(+) cells, but the number of these cells increases significantly when thymocytes are grown in the presence of IL-15 or IL-2. However, IL-15 induced a significantly higher fraction of CD8(+) CD56(+) cells compared with IL-2. Thus, although IL-2 and IL-15 are known to have a number of redundant functions, we here demonstrate that IL-15 is superior to IL-2 in inducing CD8(+) CD56(+) T cells from cultures of thymocytes. The majority of the IL-15-grown CD8(+) CD56(+) cells were CD45R0(+), representing a memory phenotype, and showed high expression of the IL-15R-complex and high numbers of CD69(+) cells. Moreover, cytotoxic activity was confined to this cell population.
Asunto(s)
Antígeno CD56/análisis , Interleucina-15/farmacología , Linfocitos T Citotóxicos/inmunología , Timo/inmunología , Sangre/inmunología , Células Cultivadas , Pruebas Inmunológicas de Citotoxicidad , Humanos , Memoria Inmunológica , Inmunofenotipificación , Interleucina-12/farmacología , Receptores de Interleucina-15 , Receptores de Interleucina-2/biosíntesis , Subgrupos de Linfocitos T/clasificación , Linfocitos T Citotóxicos/efectos de los fármacosRESUMEN
Cutaneous T cell lymphomas (CTCLs) often show abnormal interleukin-2 (IL-2) receptor signaling. In this study, we investigated the role of Gab2, a recently identified adaptor molecule involved in IL-2 receptor signaling in CTCLs. We show that Gab2 was transiently phosphorylated by tyrosine in human mycosis fungoides (MF) tumor T cells upon IL-2 stimulation and that SHP2 as well as Stat5a associated inducibly with Gab2. IL-15, but not IL-4, also induced tyrosine phosphorylation of Gab2, suggesting that the IL-2 receptor beta-chain is important for IL-2-induced Gab2 phosphorylation. Preincubation of cells with the Src family kinase inhibitor, PP1, surprisingly increased the IL-2- and IL-15-induced tyrosine phosphorylation of Gab2, indicating that an Src family kinase member negatively regulates IL-2 receptor signaling in MF T cells. Thus, although Gab2 seems to function normally in MF T cells compared to normal T cells, Gab2 itself might be abnormally regulated by an Src family kinase.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Interleucina-15/metabolismo , Interleucina-2/metabolismo , Proteínas de la Leche , Micosis Fungoide/inmunología , Fosfoproteínas/metabolismo , Proteínas Tirosina Fosfatasas/biosíntesis , Neoplasias Cutáneas/inmunología , Linfocitos T/metabolismo , Transactivadores/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Sitios de Unión , Inducción Enzimática , Humanos , Interleucina-15/farmacología , Interleucina-2/farmacología , Péptidos y Proteínas de Señalización Intracelular , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas/farmacología , Factor de Transcripción STAT5 , Linfocitos T/efectos de los fármacos , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor , Tirosina/metabolismo , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
The suppressors of cytokine signalling (SOCS) proteins comprise a newly identified family of negative feedback regulators of cytokine signalling. SOCS expression is differentially induced upon cytokine stimulation in different cell types. Here we show that interferon-alpha (IFNalpha) is a potent inducer of SOCS expression in human T cells, as high expression of CIS, SOCS-1, SOCS-2, and SOCS-3 was detectable after IFNalpha stimulation. After 4 h of stimulation, CIS, SOCS-1, and SOCS-3 expression had returned to baseline levels, whereas SOCS-2 expression had not declined. In contrast, after IL-2 induction neither CIS, SOCS-1, nor SOCS-2 expression levels declined after 6 h. In conclusion, we provide the first evidence that IFNalpha induces SOCS expression in human T cells. Moreover, we show that IFNalpha and IL-2 induce distinct patterns of expression kinetics, suggesting that dynamic changes in cytokine sensitivity might be mediated via induction of SOCS expression with different kinetics in T cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Proteínas Portadoras/genética , Proteínas de Unión al ADN , Expresión Génica/efectos de los fármacos , Interferón-alfa/inmunología , Péptidos y Proteínas de Señalización Intracelular , Proteínas/genética , Proteínas Represoras , Transducción de Señal , Transactivadores , Factores de Transcripción , Linfocitos T CD4-Positivos/efectos de los fármacos , Línea Celular , Humanos , Interferón-alfa/farmacología , Interleucina-2/farmacología , Proteína 1 Supresora de la Señalización de Citocinas , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de CitocinasRESUMEN
Interleukin-2 (IL-2) is a growth factor which upon binding to high-affinity receptors (IL-2Ralphabetagamma) triggers mitogenesis in T cells. IL-2Ralpha expression is restricted to T cells which have recently encountered antigen, and in healthy individuals the majority (>95%) of peripheral T cells are IL-2Ralpha negative. An aberrant expression of IL-2Ralpha has recently been described in cutaneous T-cell lymphoma (CTCL). Here, we study the regulation of IL-2Ralpha expression and STATs in a tumor cell line obtained from peripheral blood from a patient with Sezary syndrome (SS), a leukemic variant of CTCL. We show that (1) STAT3 (a transcription factor known to regulate IL-2Ralpha transcription) is constitutively tyrosine-phosphorylated in SS tumor cells, but not in non-malignant T cells; (2) STAT3 binds constitutively to a STAT-binding sequence in the promotor of the IL-2Ralpha gene; (3) the Janus kinase inhibitor, tyrphostine AG490, inhibits STAT3 activation, STAT3 DNA binding, and IL-2Ralpha mRNA and protein expression in parallel; and (4) tyrphostine AG490 inhibits IL-2 driven mitogenesis and triggers apoptosis in SS tumor cells. In conclusion, we provide the first example of a constitutive STAT3 activation in SS tumor cells. Moreover, our findings suggest that STAT3 activation might play an important role in the constitutive IL-2Ralpha expression, survival, and growth of malignant SS cells.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de Unión al ADN/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Síndrome de Sézary/metabolismo , Neoplasias Cutáneas/metabolismo , Transactivadores/metabolismo , Tirfostinos/farmacología , Apoptosis/efectos de los fármacos , Humanos , Janus Quinasa 3 , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Receptores de Interleucina-2/análisis , Factor de Transcripción STAT3 , Síndrome de Sézary/tratamiento farmacológico , Síndrome de Sézary/patología , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Células Tumorales CultivadasRESUMEN
Retinal pigment epithelial (RPE) cells have been proposed to play a part in maintaining the eye as an immune privileged organ. However, our knowledge of the implicated mechanism is still sparse. Fas ligand (FasL) expression of RPE cells is generally recognized to be essential for the immune privilege of the eye, but due to contradictory published results, it is unclear whether RPE cells express this molecule. The purpose of this study was to investigate the expression of FasL in RPE cells in vitro and in vivo. Cultured human fetal and adult RPE cells were examined by flow cytometry, Western blotting, RT-PCR and RNase Protection assay for FasL expression. Additionally, sections of ocular tissue were stained for FasL by immunohistochemistry. None of the used methods indicated FasL expression in cultured fetal or adult RPE cells of various passages. However, RPE cells in vivo, as judged from tissue sections, were positive for FasL, indicating a discrepancy between RPE cells in vitro and in vivo with regard to this molecule.
Asunto(s)
Glicoproteínas de Membrana/biosíntesis , Epitelio Pigmentado Ocular/inmunología , Epitelio Pigmentado Ocular/metabolismo , Receptor fas/metabolismo , Adulto , Western Blotting , Línea Celular , Proteína Ligando Fas , Feto , Citometría de Flujo , Humanos , Sueros Inmunes/metabolismo , Inmunohistoquímica , Ligandos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Epitelio Pigmentado Ocular/citología , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleasas/metabolismoRESUMEN
Signal transducer and activator of transcription 6 (STAT6) is essential for the biological activities of interleukin-4 (IL-4) and the development of allergic responses in mice. Here we report on a sensitive and specific assay for STAT6 activation in response to IL-4. We took advantage of double-stranded oligonucleotide probes containing a STAT6-binding gene-sequence from the promotor of the immunoglobulin heavy chain germline epsilon transcript to study the IL-4-induced DNA binding of STAT6. Using these probes, we show that repeated adjacent STAT6-binding sites result in enhanced STAT6-DNA binding. Moreover, the distance between the binding sites is critical for STAT-DNA binding, i.e. STAT6 binding is decreased at distances above 20 nucleotides between neighbouring binding sites. Using this assay to study cross-talk between IL-4 and chemokines, we provide evidence that MIP-1beta and MIG inhibit IL-4-induced STAT6 activation, whereas other chemokines and cytokines do not. In conclusion, our data show that oligonucleotide fishing is a supplementary tool for studying cytokine cross-talk at a genomic level.
Asunto(s)
Quimiocinas/metabolismo , Interleucina-4/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Citocinas/metabolismo , ADN/genética , ADN/metabolismo , Humanos , Interleucina-4/farmacología , Ratones , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Fosforilación , Factor de Transcripción STAT6 , Transactivadores/química , Tirosina/metabolismoRESUMEN
Glucagon-like peptide-2 (GLP-2) induces intestinal growth in mice; but in normal rats, it seems less potent, possibly because of degradation of GLP-2 by the enzyme dipeptidyl peptidase IV (DPP-IV). The purpose of this study was to investigate the survival and effect of GLP-2 in rats and mice after s.c. injection of GLP-2 with or without the specific DPP-IV inhibitor, valine-pyrrolidide (VP). Rats were injected s.c. with 40 microg GLP-2 or 40 microg GLP-2+15 mg VP. Plasma was collected at different time points and analyzed, by RIA, for intact GLP-2. Rats were treated for 14 days with: saline; 15 mg VP; 40 microg GLP-2, 40 microg GLP-2+15 mg VP; 40 microg GLP-2 (3-33). Mice were treated for 10 days with: saline; 5 microg GLP-2; 5 microg GLP-2+1.5 mg VP; 25 microg GLP-2; 25 microg GLP-2 (3-33). In both cases, body weight, intestinal weight, length, and morphometric data were measured. After s.c. injection, the plasma concentration of GLP-2 reached a maximum after 15 min, and elevated concentrations persisted for 4-8 h. With VP, the concentration of intact GLP-2 was about 2-fold higher for at least the initial 60 min. Rats treated with GLP-2+VP had increased (P < 0.01) small-bowel weight (4.68 +/- 0.11%, relative to body weight), compared with the two control groups, [3.01 +/- 0.06% (VP) and 2.94 +/- 0.07% (NaCl)] and GLP-2 alone (3.52 +/- 0.10%). In mice, the growth effect of 5 microg GLP-2+VP was comparable with that of 25 microg GLP-2. GLP-2 (3-33) had no effect in rats, but it had a weak effect on intestinal growth in mice. The extensive GLP-2 degradation in rats can be reduced by VP, and DPP-IV inhibition markedly enhances the intestinotrophic effect of GLP-2 in both rats and mice. We propose that DPP-IV inhibition may be considered to enhance the efficacy of GLP-2 as a therapeutic agent.
Asunto(s)
Dipeptidil Peptidasa 4/metabolismo , Inhibidores Enzimáticos/farmacología , Intestinos/efectos de los fármacos , Intestinos/crecimiento & desarrollo , Péptidos/farmacología , Animales , Peso Corporal , Femenino , Péptido 2 Similar al Glucagón , Péptidos Similares al Glucagón , Humanos , Ratones , Ratones Endogámicos C57BL , Tamaño de los Órganos , Péptidos/metabolismo , Pirroles/farmacología , Ratas , Ratas Wistar , Proteínas Recombinantes/farmacología , Valina/farmacologíaRESUMEN
The Huntington disease gone encodes the protein huntington, which is widely expressed during embryonic development and in mature tissues. In order to elucidate the physiological function of huntington, which so far is unknown, we intend to study the effect of antisense down-regulated huntington expression. We have found an inhibiting effect of a phosphorothioated oligodeoxynucleotide (PS-ODN) added to the culture medium of embryonic teratocarcinoma cells (NT2) and postmitotic neurons (NT2N neurons) differentiated from the NT2 cells. Specific inhibition of expression of endogenous huntington was achieved in NT2N neurons in the concentration range of 1-5 microM PS-ODN, whereas no inhibition was obtained in NT2 cells. We describe in detail the selection of the target sequence for the antisense oligo and the uptake, intracellular distribution, and stability of the antisense PS-ODN in the two cell types. Antisense down-regulation of huntington in this model of human neurons represents a suitable approach to study its normal function.
Asunto(s)
Proteínas del Tejido Nervioso/genética , Neuronas/fisiología , Proteínas Nucleares/genética , Oligonucleótidos Antisentido/farmacocinética , Animales , Anticuerpos , Exones , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Expresión Génica/fisiología , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Técnicas In Vitro , Mitosis , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/inmunología , Neuronas/química , Neuronas/citología , Proteínas Nucleares/análisis , Proteínas Nucleares/inmunología , Oligonucleótidos Antisentido/análisis , Biosíntesis de Proteínas , ARN Mensajero/análisis , Conejos , Teratocarcinoma , Células Tumorales CultivadasRESUMEN
The role of Lck in IL-2-induced proliferation and cell survival is still controversial. Here, we show that the Src family kinase inhibitor, PP1, reduced the IL-2-induced proliferation of human T cells significantly without inhibiting the anti-apoptotic effect of IL-2. As Lck is the only Src family kinase activated upon IL-2 stimulation in T cells, this indicates that Lck is involved in IL-2-induced proliferation but not survival. IL-2-induced MAP kinase activation was only slightly inhibited by PP1, suggesting that Lck is not essential for IL-2-induced MAP kinase activation in human T cells. We found that an IL-2-sensitive, human mycosis fungoides-derived tumor T cell line is Lck negative, and that the IL-2-induced MAP kinase activation is comparable to non-cancerous T cells, although a little delayed in kinetics. An Lck expressing clone was established by transfecting Lck into mycosis fungoides tumor T cells, but Lck had no influence on the delayed kinetics of MAP kinase activation, indicating that Lck is not essential for MAP kinase activation in mycosis fungoides tumor T cells or in non-cancerous T cells. Taken together, this indicates that Lck is involved in IL-2-induced proliferation, but not cell survival, through a pathway not involving MAP kinase.
Asunto(s)
Interleucina-2/farmacología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Linfocitos T/citología , Linfocitos T/enzimología , Secuencia de Bases , División Celular/efectos de los fármacos , División Celular/fisiología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Cartilla de ADN/genética , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Micosis Fungoide/enzimología , Micosis Fungoide/genética , Pirazoles/farmacología , Pirimidinas/farmacología , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Linfocitos T/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
The effects of IL-15, as compared to IL-2, on growth of human thymocytes has been evaluated. Expression of comparable amounts of receptor chains was found on IL-2 and IL-15 cultured thymocytes, as well as comparable receptor signalling. However, IL-15 was superior to IL-2 in promoting CD8(+)thymocyte growth, whereas CD4(+)cells proliferated to a higher extent in IL-2 cultures. CD4(+)8(+)and CD4(-)8(-)thymocytes expanded equally well in both culture types.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Interleucina-15/farmacología , Linfocitos T/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica/inmunología , Humanos , Interleucina-2/farmacología , Receptores de Interleucina-15 , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/citología , Linfocitos T/efectos de los fármacosRESUMEN
PURPOSE: The aim of this study was to determine the role of Bcl-2, Bcl-X L, Bax, and c-Fos in regulation of apoptosis, induced by ultraviolet-light A (UV-A) and daunorubicin (DNR), in retinal pigment epithelium (RPE) cells grown on bovine extracellular matrix (ECM)-coated or uncoated plastic dishes. METHODS: Apoptosis in confluent RPE cells cultured on ECM-coated or uncoated dishes was induced by UV-A or DNR. Apoptosis was detected by 7-amino-actinomycin D labeling followed by flow cytometry and by terminal deoxy-transferase mediated X-dUTP nick end labeling (TUNEL). Cellular expression of Bcl-2, Bcl-X L, Bax, and c-Fos was determined by the use of antibodies and flow cytometry, Western blot analysis, and immunocytochemical staining. RESULTS: Both UV-A and DNR induce apoptosis in human RPE cells in vitro. Human fetal RPE cells grown on ECM-coated dishes were significantly more resistant to UV-A or DNR induced apoptosis than cells grown on uncoated dishes. RPE cells grown on ECM-coated dishes expressed higher Bcl-2 levels and lower Bax levels compared to cells grown on uncoated dishes. However, Bcl-X L and c-Fos levels were comparable in the two cultures. After UV-A or DNR treatment, Bcl-2, Bcl-X L, Bax, and c-Fos levels were differently regulated in cells grown on ECM-coated dishes compared to cells grown on uncoated dishes. CONCLUSION: A significant protection against apoptosis of RPE cells grown on ECM compared to cells grown on uncoated plastic dishes was found after exposure to UV-A or DNR. This protection was found to be proportionally correlated to the anti-apoptotic protein Bcl-2 and inversely correlated to the expression of Bax. Furthermore a sustained induction and expression of c-Fos was found to correlate to a higher percentage of apoptotic cells of RPE cells grown on plastic. These findings demonstrate that ECM is of great importance for RPE cell survival during noxious stimuli and points out the essential role for a healthy Bruch's Membrane (BM) for RPE survival.