RESUMEN
Botrytized sweet wines are made with berries infected by the fungus Botrytis cinerea. The aim of this study was to identify biomarkers of B. cinerea infection in sweet wines with a focus on laccases which are exocellular oxidase enzymes produced by this fungus during fruit contamination. Total proteins from six commercial sweet wines, including three naturally botrytized wines and three non-botrytized wines were analysed by LC-QTOF-MS. Five laccases, namely laccase-1-BcLCC1, laccase-2-BcLCC2, laccase-3-BcLCC7, laccase-8-BcLCC8 and laccase-12-BcLCC12, were identified in both types of wine. Then, a targeted proteomic approach by LC-MRM was used to semi-quantify laccase-2-BcLCC2 and laccase-3-BcLCC7, in the six samples. LC-MRM targeted analysis of the two enzymes allowed the discrimination of botrytized versus non-botrytized sweet white wines.
Asunto(s)
Biomarcadores/análisis , Botrytis/enzimología , Lacasa/metabolismo , Proteómica , Vino/análisis , Cromatografía Liquida , Frutas/química , Lacasa/química , Vitis/metabolismoRESUMEN
Interactions of anti-inflammatory nimesulide (NM) with different graphene material species were explored employing both ab initio calculations, based on Density Functional Theory (DFT), and a batch adsorption process. The adsorption of NM onto graphene, with and without a vacancy, reduced graphene oxide (rGO) and functionalized graphene nanoribbons was simulated, providing a good understanding of the adsorption process of the NM molecule onto graphene material surfaces. The theoretical results indicate a physisorption interaction between NM and all of the evaluated adsorbents. Based on batch adsorption experiments, it was found that rGO, obtained via a modified Hummers method, is a good nanoadsorbent for the removal of the anti-inflammatory NM from aqueous solutions. The general-order kinetic equation displays the best fit to the experimental data compared with pseudo-first order and pseudo-second order kinetics. The equilibrium data fitted well into the Liu isotherm equation, and the maximum sorption capacity for the adsorption of NM by rGO was 82.4 mg g-1 at 25 °C. Our results of the first principle calculations and the batch adsorption experiments point out that graphene materials are promising nanomaterials for extracting NM from aqueous solutions.
Asunto(s)
Antiinflamatorios no Esteroideos/química , Grafito/química , Modelos Teóricos , Sulfonamidas/química , Adsorción , Semivida , Cinética , Nanoestructuras/química , Óxidos/química , Espectroscopía Infrarroja por Transformada de Fourier , Sulfonamidas/metabolismo , Temperatura , TermogravimetríaRESUMEN
BACKGROUND: The suppressor of cytokine signaling 1 (SOCS1) gene is repressed in prostate cancer (PCa) by epigenetic silencing and microRNA miR30d. Increased expression of the SOCS1-targeting miR30d correlates with higher biochemical recurrence, suggesting a tumor suppressor role of SOCS1 in PCa, but the underlying mechanisms are unclear. We have shown that SOCS1 inhibits MET receptor kinase signaling, a key oncogenic pathway in cancer progression. Here we evaluated the role of SOCS1 in attenuating MET signaling in PCa cells and tumor growth in vivo. METHODS: MET-overexpressing human DU145 and PC3 PCa cell lines were stably transduced with SOCS1, and their growth, migration and invasion of collagen matrix were evaluated in vitro. Cells expressing SOCS1 or the control vector were evaluated for tumor growth in NOD.scid.gamma mice as xenograft or orthotopic tumors. RESULTS: HGF-induced MET signaling was attenuated in SOCS1-expressing DU145 and PC3 cells. Compared with vector control cells, SOCS1-expressing cells showed reduced proliferation and impaired migration following HGF stimulation. DU145 and PC3 cells showed marked ability to invade the collagen matrix following HGF stimulation and this was attenuated by SOCS1. As xenografts, SOCS1-expressing PCa cells showed significantly reduced tumor growth compared with vector control cells. In the orthotopic tumor model, SOCS1 reduced the growth of primary tumors and metastatic spread. Intriguingly, the SOCS1-expressing DU145 and PC3 tumors showed increased collagen deposition, associated with increased frequency of myofibroblasts. CONCLUSIONS: Our findings support the tumor suppressor role of SOCS1 in PCa and suggest that attenuation of MET signaling is one of the underlying mechanisms. SOCS1 in PCa cells also appears to prevent the tumor-promoting functions of cancer-associated fibroblasts.
Asunto(s)
Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Células del Estroma/metabolismo , Proteína 1 Supresora de la Señalización de Citocinas/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Colágeno/metabolismo , Metilación de ADN , Modelos Animales de Enfermedad , Epigénesis Genética , Expresión Génica , Factor de Crecimiento de Hepatocito/metabolismo , Xenoinjertos , Humanos , Masculino , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal , Células del Estroma/patología , Proteína 1 Supresora de la Señalización de Citocinas/genética , Carga Tumoral , Microambiente TumoralRESUMEN
The SOCS1 gene coding for suppressor of cytokine signaling 1 is frequently repressed in hepatocellular carcinoma (HCC), and hence SOCS1 is considered a tumor suppressor in the liver. However, the tumor-suppressor mechanisms of SOCS1 are not yet well understood. SOCS1 is known to inhibit pro-inflammatory cytokine production and signaling and to promote activation of the p53 tumor suppressor. However, we observed that SOCS1-deficient mice developed numerous and large liver tumor nodules following treatment with the hepatocarcinogen diethylnitrosamine (DEN) without showing increased interleukin-6 production or activation of p53. On the other hand, the livers of DEN-treated Socs1-null mice showed elevated levels of p21(CIP1/WAF1) protein (p21). Even though p21 generally functions as a tumor suppressor, paradoxically many cancers, including HCC, are known to express elevated levels of p21 that correlate with poor prognosis. We observed elevated p21 expression also in the regenerating livers of SOCS1-deficient mice and in cisplatin-treated Socs1-null hepatocytes, wherein the p21 protein showed increased stability. We show that SOCS1 interacts with p21 and promotes its ubiquitination and proteasomal degradation. Besides, the DEN-treated livers of Socs1-null mice showed increased nuclear and cytosolic p21 staining, and the latter was associated with growth factor-induced, phosphatidylinositol 3-kinase-dependent phosphorylation of p21 in SOCS1-deficient hepatocytes. Cytosolic p21 is often associated with malignancy and chemo-resistance in many cancers. Accordingly, SOCS1-deficient hepatocytes showed increased resistance to apoptosis that was reversed by shRNA-mediated p21 knockdown. In the regenerating livers of Socs1-null mice, increased p21 expression coincided with elevated cyclinD levels. Correspondingly, SOCS1-deficient hepatocytes showed increased proliferation to growth factor stimulation that was reversed by p21 knockdown. Overall, our findings indicate that the tumor-suppressor functions of SOCS1 in the liver could be mediated, at least partly, via regulation of the expression, stability and subcellular distribution of p21 and its paradoxical oncogenic functions, namely, resistance to apoptosis and increased proliferation.
Asunto(s)
Carcinoma Hepatocelular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , Oncogenes , Proteína 1 Supresora de la Señalización de Citocinas/metabolismo , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , ADN/biosíntesis , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Estabilidad Proteica , Transporte de Proteínas/efectos de los fármacos , Proteína 1 Supresora de la Señalización de Citocinas/deficiencia , Proteína 1 Supresora de la Señalización de Citocinas/genética , Factor de Crecimiento Transformador alfa/farmacologíaRESUMEN
The interactions of sodium diclofenac drug (s-DCF) with different graphene species were investigated using both first principles calculations based on Density Functional Theory (DFT) and adsorption experiments. Through batch adsorption experiments, it was found that rGO was a good adsorbent for removing the s-DCF drug from aqueous solutions. The general-order kinetic model shows the best fit to the experimental data compared with pseudo-first order and pseudo-second order kinetic adsorption models. The equilibrium data (at 25 °C) were fitted to the Liu isotherm model. The maximum sorption capacity for adsorption of the s-DCF drug was 59.67 mg g(-1) for rGO. The s-DCF adsorption onto pristine graphene, graphene with a vacancy, reduced oxide graphene (rGO) and functionalized graphene nanoribbons were simulated providing a good understanding of the adsorption process of this molecule on graphene-family surfaces. The results predict a physisorption regime in all cases. Based on these results, the ab initio calculations and the adsorption experiments point out that the graphene-family are promising materials for extracting s-DCF from wastewater effluents.
Asunto(s)
Diclofenaco/química , Grafito/química , Adsorción , Cinética , Microscopía Electrónica de Rastreo , Espectroscopía Infrarroja por Transformada de Fourier , Termodinámica , TermogravimetríaRESUMEN
Suppressor of cytokine signaling 1 (SOCS1) is considered as a tumor suppressor protein in hepatocellular carcinoma (HCC), but the underlying mechanisms remain unclear. Previously, we have shown that SOCS1-deficient hepatocytes displayed increased responsiveness to hepatocyte growth factor (HGF) due to enhanced signaling via the MET receptor tyrosine kinase. As aberrant MET activation occurs in many tumors including HCC, here we elucidated the mechanisms of SOCS1-mediated regulation. SOCS1 attenuated HGF-induced proliferation of human and mouse HCC cell lines and their growth as tumors in NOD.scid.gamma mice. Tumors formed by SOCS1 expressing HCC cells showed significantly reduced MET expression, indicating that SOCS1 not only attenuates MET signaling but also regulates MET expression. Mechanistically, SOCS1 interacted with MET via the Src homology 2 domain and this interaction was promoted by MET tyrosine kinase activity. The SOCS1-mediated reduction in MET expression does not require the juxtamembrane Y1003 residue implicated in Cbl-mediated downmodulation. Moreover, the proteasome inhibitor MG-132, but not the inhibitors of lysosomal degradation bafilomycin and chloroquine, reversed the SOCS1-mediated reduction in MET expression, indicating that this process is distinct from Cbl-mediated downmodulation. Accordingly, SOCS1 promoted polyubiquitination of MET via K48-dependent but not K63-mediated ubiquitin chain elongation. Furthermore, siRNA-mediated downmodulation of Cbl did not abolish SOCS1-mediated reduction in MET expression in HCC cells. SOCS1-dependent ubiquitination of endogenous MET receptor occurred rapidly following HGF stimulation in HCC cells, leading to proteasomal degradation of phosphorylated MET receptor. These findings indicate that SOCS1 mediates its tumor suppressor functions, at least partly, by binding to MET and interfering with downstream signaling pathways as well as by promoting the turnover of the activated MET receptor. We propose that loss of this control mechanism due to epigenetic repression of SOCS1 could contribute to oncogenic MET signaling in HCC and other cancers, and that MET inhibitors might be useful in treating these patients.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Animales , Células COS , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular , Chlorocebus aethiops , Cloroquina/farmacología , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células Hep G2 , Humanos , Leupeptinas/farmacología , Neoplasias Hepáticas/patología , Ratones , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína 1 Supresora de la Señalización de CitocinasRESUMEN
BACKGROUND: In most patients with breast cancer, radiotherapy induces inflammation that is characterised by an increase of promigratory factors in healthy tissues surrounding the tumour. However, their role in the emergence of the migration phenotype and formation of metastases is still unclear. METHODS: A single mammary gland of BALB/c mice was irradiated with four doses of 6 Gy given at a 24-h interval. After the last session of irradiation, treated and control mammary glands were either collected for quantification of promigratory and proinflammatory factors or were implanted with fluorescent ubiquitination-based cell cycle indicator (FUCCI)-expressing mouse mammary cancer D2A1 cells. The migration of cancer cells in the mammary glands was monitored by optical imaging. On day 21, mammary tumours and lungs were collected for histology analyses and the quantification of metastases. RESULTS: Pre-irradiation of the mammary gland increased by 1.8-fold the migration of cancer cells, by 2-fold the quantity of circulating cancer cells and by 2.4-fold the number of lung metastases. These adverse effects were associated with the induction of interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2). CONCLUSION: The emergence of the metastasis phenotype is believed to be associated with the accumulation of mutations in cancer cells. Our results suggest an alternative mechanism based on promigratory factors from irradiated mammary glands. In clinic, the efficiency of radiotherapy could be improved by anti-inflammatory agents that would prevent the stimulation of cancer cell migration induced by radiation.
Asunto(s)
Movimiento Celular/efectos de la radiación , Neoplasias Pulmonares/secundario , Glándulas Mamarias Animales/efectos de la radiación , Neoplasias Mamarias Experimentales/patología , Células 3T3 , Animales , Línea Celular Tumoral , Ciclooxigenasa 2/biosíntesis , Femenino , Interleucina-6/biosíntesis , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos BALB C , Células Neoplásicas CirculantesRESUMEN
When aberrantly expressed or activated, the Met receptor tyrosine kinase is involved in tumor invasiveness and metastasis. In this study, we have used the Xenopus embryonic system to define the role of various Met proximal-binding partners and downstream signaling pathways in regulating an induced morphogenetic event. We show that expression of an oncogenic derivative of the Met receptor (Tpr-Met) induces ectopic morphogenetic structures during Xenopus embryogenesis. Using variant forms of Tpr-Met that are engineered to recruit a specific signaling molecule of choice, we demonstrate that the sole recruitment of either the Grb2 or the Shc adaptor protein is sufficient to induce ectopic structures and anterior reduction, while the recruitment of PI-3Kinase (PI-3K) is necessary but not sufficient for this effect. In contrast, the recruitment of PLCgamma can initiate the induction, but fails to maintain or elongate supernumerary structures. Finally, evidence indicates that the Ras/Raf/MAPK pathway is necessary, but not sufficient to induce these structures. This study also emphasizes the importance of examining signaling molecules in the regulatory context that is provided by receptor/effector interactions when assessing a role in cell growth and differentiation.
Asunto(s)
Embrión no Mamífero/metabolismo , Inducción Embrionaria/fisiología , Proteínas Proto-Oncogénicas c-met/fisiología , Animales , Femenino , Mutación , Fosforilación , Proteínas Proto-Oncogénicas c-met/genética , XenopusRESUMEN
A detailed structural study of the major acidic triterpene saponins from European oak heartwood, Quercus robur L. and Q. petraea Liebl., revealed the presence of 2 alpha,3 beta,19 alpha-trihydroxyolean-12-ene-24,28-dioic acid (1), 2 alpha,3 beta,19 alpha-23-tetrahydroxyolean-12-ene-24,28-dioic acid (2), and their corresponding glycosides, 28-beta-D-glucopyranosyl-2 alpha,3 beta,19 alpha-trihydroxyolean-12-ene-24,28-dioic acid (3), and 28-beta-D-glucopyranosyl-2 alpha,3 beta,19 alpha,23-tetrahydroxyolean-12-ene-24,28-dioic acid (4). Compounds were isolated and purified by successive flash chromatography and semi-preparative HPLC, and structurally determined by NMR and LC-ESI/MS in the negative ion mode. Compounds 2 and 4 have been identified for the first time in Quercus species and are new compounds.
Asunto(s)
Quercus/química , Saponinas/química , Triterpenos/química , Cromatografía Liquida/métodos , Espectroscopía de Resonancia Magnética/métodos , Estructura Molecular , Extractos Vegetales/química , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
A rapid method that permits separation of grape seed proanthocyanidins according to their polymerization degrees has been developed. This method was based on liquid/liquid extraction and relative solubility of these compounds in different solvents (water, ethyle acetate, methanol, and chloroform). The different fractions obtained were then analyzed by various HPLC techniques (normal phase, reversed phase after thiolysis, and gel permeation) to determine their mean degree of polymerization (DP) and molecular weight profiles. Results show that this method can be used on a preparative scale to separate these polymers according to their sizes.
Asunto(s)
Antocianinas/aislamiento & purificación , Antioxidantes/aislamiento & purificación , Frutas/química , Proantocianidinas , Semillas/química , Cromatografía en Gel/métodos , Cromatografía Líquida de Alta Presión/métodos , Peso MolecularRESUMEN
The antioxidant synergy between (+)-catechin and other wine or biological antioxidants (Trolox, ascorbate, SO(2), uric acid) was measured in vitro using the Folin-Ciocalteu (FC) and metmyoglobin assays. Although the two assays are based on very different reagents (i.e., metal salts versus organic and biochemical reagents), the individual antioxidants showed similar relative activities in both systems. In addition, interaction studies showed simple additive effects in all cases except with the (+)-catechin/SO(2) mixture, which showed a remarkable synergetic effect in both assays.
Asunto(s)
Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Catequina/farmacología , Sinergismo Farmacológico , Modelos Químicos , Sulfitos/farmacología , VinoRESUMEN
We studied the endogenous expression of the serotonin-2A (5-hydroxytryptamine2A, 5-HT2A) 5-HT2C, and a splice-variant of the 5-HT2C receptor in murine Balb/c-3T3 fibroblast cells that is revealed when these cells are maintained in medium containing 5-HT-free serum. RNA editing of the 5-HT2C receptor was exclusively at a single brain-specific site. Addition of 5-HT (EC50 = 23 +/- 2.9 nM) induced an immediate release of calcium from an ionomycin-sensitive intracellular store by coupling to a pertussis toxin-insensitive pathway. The 5-HT-induced calcium mobilization displayed a 5-HT-2-like pharmacology, and ligand binding analyses indicated the presence of specific binding sites (27.5 +/- 2 fmol/mg protein) with a 5-HT2A-like pharmacology. Although the 5-HT2A receptor site was predominant, the smaller component of 5-HT2C receptors alone was sufficient to mediate a maximal calcium response. The 5-HT-induced increase in [Ca2+]i was reversibly inhibited by >75% following a 12-hr pretreatment (T1/2 = 2 hr) with 5-HT (EC50 = 400 nM). Extended treatment (24-96 hr) with 5-HT induced a complete functional desensitization that was associated with a partial (60%) reduction in 5-HT2 receptor number, implicating both receptor down-regulation and post-receptor mechanisms in 5-HT-induced desensitization. Long-term (hours to days) treatment with 5-HT did not modulate DNA synthesis, cell proliferation, or transformation in Balb/c-3T3 cells. These results demonstrate that Balb/c-3T3 cells express endogenous 5-HT2 receptors that are desensitized by the 5-HT present in normal serum, illustrating the importance of growth conditions in the identification of receptor responsiveness. The lack of proliferative response to 5-HT in Balb/c-3T3 suggests a putative role of desensitization as a "safety valve" to prevent abnormal cell growth during sustained 5-HT2 receptor activation.
Asunto(s)
Regulación hacia Abajo , Receptores de Serotonina/metabolismo , Serotonina/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calcio/metabolismo , Medios de Cultivo , Cartilla de ADN , ADN Complementario , Ligandos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Unión Proteica , Ratas , Receptor de Serotonina 5-HT2A , Receptor de Serotonina 5-HT2C , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
NIH-3T3 cells, a nontransformed murine fibroblast cell line previously found to be unresponsive to 5-hydroxytryptamine (5-HT) when cultured in 5-HT-free medium, became responsive to 5-HT, which induced an increase in intracellular calcium concentration. Pharmacological and ligand binding studies showed that NIH-3T3 cells endogenously express a 5-HT2A receptor that, when activated, mobilizes calcium from ionomycin-sensitive intracellular stores via coupling to a pertussis toxin-insensitive pathway. Using reverse transcriptase-PCR cloning and northern blot analysis, the presence of 5-HT2A receptor RNA with a similar nucleotide sequence (99% identity) and molecular size to that of murine brain was detected in NIH-3T3 cells. Responsiveness of the endogenous 5-HT2A receptor in nontransfected cells was completely desensitized after chronic treatment (half-time = 2 h) with 1 microM 5-HT and resensitized on removal of 5-HT. In contrast to NIH-3T3 cells transfected with 5-HT2A receptor cDNA under control of a viral promoter, the long-term agonist-induced functional desensitization in nontransfected NIH-3T3 cells was paralleled by a decrease in both 5-HT2A receptor density and RNA level. These results show that NIH-3T3 cells express an endogenous 5-HT2A receptor that is desensitized by agonist via down-regulation of both receptor number and mRNA. The NIH-3T3 cells provide a novel system for understanding 5-HT2A receptor regulation.
Asunto(s)
Regulación hacia Abajo , ARN Mensajero/metabolismo , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Agonistas de Receptores de Serotonina/farmacología , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calcio/metabolismo , Membranas Intracelulares/metabolismo , Ligandos , Ratones , Datos de Secuencia Molecular , Concentración Osmolar , Reacción en Cadena de la Polimerasa , Serotonina/farmacología , Transcripción GenéticaRESUMEN
In the mouse BP-A31 fibroblasts, mRNAs coding the three isoforms (Ha, Ki, N) of ras are expressed, and there are no activating mutations in the codons 12, 13 or 61. We have produced a subline (Ras2) expressing an oestrogen-inducible v-Ha-ras gene. The contribution of v-Ha-ras to the overall p21ras-GTP content was evaluated by metabolic labelling with 32P. Surprisingly, p21ras-GTP complexes were predominant in the serum-deprived BP-A31 cells as well as in the Ras2 cells. The excess of p21ras-GTP was not due to the lack of the GTPase activating protein. In transient transfection experiments, the serum response element (SRE)-directed CAT was expressed in serum-deprived BP-A31 cells, and insulin caused a further two- to threefold increase in CAT activity. A dominant negative ras mutant (Ha-Ras Asn-17) cancelled both the basal and insulin-induced CAT expression in the BP-A31 but not in the Ras2 cells. Expression of v-Ha-ras in Ras2 cells did not relax their growth factor-dependence and oestradiol had only a minor mitogenic effect. We conclude that p21ras activation does not ensure a complete cell division cycle in these cells, and does not entirely account for the transduction of the mitogenic signal initiated by insulin.
Asunto(s)
Fase G1/fisiología , Genes ras/fisiología , Guanosina Trifosfato/metabolismo , Proteína Oncogénica p21(ras)/metabolismo , Animales , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Medio de Cultivo Libre de Suero , Fibroblastos/citología , Fase G1/genética , Humanos , Insulina/farmacología , Ratones , Proteína Oncogénica p21(ras)/genética , ARN Mensajero/metabolismo , TransfecciónRESUMEN
The hypothesis that antianxiety or antidepressant agents (e.g., 5-HT1A agonists, 5-HT uptake blockers) exert their clinical actions via enhancement of serotonergic neurotransmission due to desensitization of 5-HT1A autoreceptors predicts that regulation of this receptor plays a crucial role in the therapeutic actions of these agents. A multidisciplinary strategy is described for the characterization of the 5-HT1A receptor at the level of cellular signaling mechanisms and genetic regulation, using heterologous expression of the cloned receptor in cell lines, site-directed mutagenesis, isolation of receptor-positive neuronal cell lines, and promoter analysis of the 5-HT1A receptor gene. These analyses will yield new insights into the possible mechanisms down-regulation of 5-HT1A receptor signaling, and may suggest novel sites of inherent defect involved in anxiety syndromes or major depression.
Asunto(s)
Receptores de Serotonina/metabolismo , Transducción de Señal , Transcripción Genética , Antidepresivos/metabolismo , Trastorno Depresivo/metabolismo , Humanos , Serotonina/metabolismoRESUMEN
To evaluate the influence of gestation on peripheral blood T cells, we measured the mRNA production of several cytokines (IL-2, IL-4, IL-10, IFN-gamma) and the p55 subunit of IL-2R at different time points in the blood of pregnant mice and in the placenta. This was made possible by the use of a new PCR technique that is precise and quantitative. Our results show that pregnancy induces profound changes in the expression of these genes in peripheral blood cells. During the first week of gestation, there is an increase in the levels of all the cytokines, followed by a state of immunodepression characterized by levels of cytokines below normal (nonpregnant mice). In the placenta low levels of IL-2 and IL-10 are detected. IFN-gamma mRNA production is higher than the blood IFN-gamma mRNA in the last week of pregnancy. However, the main difference is found for IL-4 mRNA expression where the placenta levels are 5- to 10-fold higher than the blood mRNA expression. We discuss these results in the context of the placenta as a privileged immune site, where IL-4, being the main cytokine, may play a major regulatory role.
Asunto(s)
Citocinas/sangre , Citocinas/metabolismo , Placenta/inmunología , Animales , Secuencia de Bases , Citocinas/genética , Cartilla de ADN/genética , ADN Complementario/genética , Femenino , Expresión Génica , Interferón gamma/sangre , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-10/sangre , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-2/sangre , Interleucina-2/genética , Interleucina-2/metabolismo , Interleucina-4/sangre , Interleucina-4/genética , Interleucina-4/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Placenta/metabolismo , Reacción en Cadena de la Polimerasa , Embarazo , ARN Mensajero/sangre , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
To assess the relationship between insulin-dependent diabetes mellitus (IDDM) self-care management and metabolic control in school-aged children, 21 children ages 10 to 14 years with a duration of IDDM of 5.5 years (range 1 to 13 years) were studied. Self-care was defined as the activities that children with IDDM initiate and perform on their own in the management of their disease. Self-care activity was assessed by a revised Self-Care Questionnaire (SCQ), and metabolic control was determined by HbA1c levels obtained at the same time the SCQ was completed. A higher level of self-care management (higher SCQ) was hypothesized to correlate with better metabolic control (lower HbA1c). Regression analysis of SCQ and HbA1c supported the expected inverse relationship but not at a significant level. The only significant predictors of self-care activity and metabolic control were experience at a specialized camp for children with diabetes and race, with Caucasian children scoring higher in self-care and having lower HbA1c values than African American children.
Asunto(s)
Diabetes Mellitus Tipo 1/prevención & control , Autocuidado/normas , Adolescente , Niño , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Hemoglobina Glucada/análisis , Humanos , Masculino , Análisis de Regresión , Encuestas y CuestionariosRESUMEN
In vitro enzymatic amplification of nucleic acids by PCR or other techniques is a very sensitive method to detect rare DNA segments. We present here a protocol that allows the rapid, sensitive and precise quantification of DNA molecules using PCR amplification run to saturation. The DNA (or cDNA) to be assayed is co-amplified with known amounts of an internal standard DNA. We show that the latter must be almost identical to the assayed DNA, otherwise quantification at the plateau is unreliable. The read-out of the amplification involves one or two additional oligonucleotides. Using fluorescent oligonucleotides as primers in run-off reactions together with an automated DNA sequencer, we could measure the level of expression of several genes, like the murine MHC class I H-2Kd or a specific T cell receptor beta chain transcript in the course of an immunization. mRNA levels were normalized by measuring in a similar manner the number of transcripts encoding the housekeeping gene HPRT. Finally, our procedure might allow the rapid analysis of a large number of samples at the same time, as illustrated by the simultaneous analysis of the mRNAs encoding the CD4 and CD8 murine T cell markers.
Asunto(s)
ADN/análisis , Reacción en Cadena de la Polimerasa/métodos , Animales , Antígenos CD/genética , Secuencia de Bases , Línea Celular , Clonación Molecular , Citometría de Flujo , Antígenos H-2/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Datos de Secuencia Molecular , ARN Mensajero/análisis , Receptores de Antígenos de Linfocitos T/genética , Sensibilidad y Especificidad , VolumetríaRESUMEN
A recombinant baculovirus encoding a single-chain murine major histocompatibility complex class I molecule in which the first three domains of H-2Kd are fused to beta 2-microglobulin (beta 2-m) via a 15-amino acid linker has been isolated and used to infect lepidopteran cells. A soluble, 391-amino acid single-chain H-2Kd (SC-Kd) molecule of 48 kDa was synthesized and glycosylated in insect cells and could be purified in the absence of detergents by affinity chromatography using the anti-H-2Kd monoclonal antibody SF1.1.1.1. We tested the ability of SC-Kd to bind antigenic peptides using a direct binding assay based on photoaffinity labeling. The photoreactive derivative was prepared from the H-2Kd-restricted Plasmodium berghei circumsporozoite protein (P.b. CS) peptide 253-260 (YIPSAEKI), a probe that we had previously shown to be unable to bind to the H-2Kd heavy chain in infected cells in the absence of co-expressed beta 2-microglobulin. SC-Kd expressed in insect cells did not require additional mouse beta 2-m to bind the photoprobe, indicating that the covalently attached beta 2-m could substitute for the free molecule. Similarly, binding of the P.b. CS photoaffinity probe to the purified SC-Kd molecule was unaffected by the addition of exogenous beta 2-m. This is in contrast to H-2KdQ10, a soluble H-2Kd molecule in which beta 2-m is noncovalently bound to the soluble heavy chain, whose ability to bind the photoaffinity probe is greatly enhanced in the presence of an excess of exogenous beta 2-m. The binding of the probe to SC-Kd was allele-specific, since labeling was selectively inhibited only by antigenic peptides known to be presented by the H-2Kd molecule.
Asunto(s)
Baculoviridae/genética , Genes MHC Clase I , Antígenos H-2/aislamiento & purificación , Antígenos H-2/metabolismo , Proteínas Protozoarias , Microglobulina beta-2/aislamiento & purificación , Microglobulina beta-2/metabolismo , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Adenovirus Humanos/genética , Adenovirus Humanos/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/metabolismo , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Línea Celular , Cromatografía de Afinidad , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Genes Virales , Antígenos H-2/genética , Insectos , Cinética , Sustancias Macromoleculares , Ratones , Modelos Estructurales , Datos de Secuencia Molecular , Peso Molecular , Oligodesoxirribonucleótidos , Oligopéptidos/farmacología , Plasmodium berghei/metabolismo , Unión Proteica , Señales de Clasificación de Proteína/genética , Señales de Clasificación de Proteína/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Microglobulina beta-2/genéticaRESUMEN
The expression of the thymidine-thymidylate kinase (HSV1-TK), (ATP: thymidine 5'-phosphotransferase; EC 2.7.1.21) of herpes simplex virus type 1 endows the host cell with a conditional lethal phenotype which depends on the presence of nucleoside analogues metabolized by this enzyme into toxic inhibitors of DNA replication. To generate a recombinant baculovirus that could be selected against by nucleoside analogs, the HSV1-tk coding sequence was placed under the control of the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) immediate early promoterm IE-1(0), and this construction was introduced via homologous recombination into the polyhedrin locus of AcMNPV. Two recombinant baculoviruses harboring this gene construct at the polyhedrin locus were isolated and tested for their ability to replicate in the presence of various concentrations of the nucleoside analog 9-(1,3-Dihydroxy-2-propoxymethyl)guanine (Ganciclovir). Neither Sf9 lepidopteran cell viability nor replication of wild type or beta-Galactosidase-expressing recombinant AcMNPVs were affected by concentrations of Ganciclovir up to 100 microM. In contrast, replication of the recombinant AcMNPV virus harboring the HSV1-tk gene was inhibited by Ganciclovir in a dose-dependent manner. The inhibition was detectable at 2 microM and complete at 100 microM. This property was exploited in model isolations aimed at purifying new recombinant viruses having lost this counter-selectable gene marker as a result of homologous recombination at the polyhedrin locus after cotransfection of the viral DNA with a replacement vector. After being propagated in the presence of Ganciclovir, the progeny of such co-transfections contained over 85% recombinant viruses, demonstrating that counter-selection of parental HSV1-tk-containing viruses by Ganciclovir constitutes a novel approach for recombinant baculovirus isolation.