RESUMEN

In March 2007, mung bean (Vigna radiata) sprouts produced in an indoor sprouting facility in northern California developed brown lesions beginning 5 days after germination. Dark brown-to-reddish brown lesions with distinct margins developed on the stem, hypocotyl, and first true leaves of affected sprouts. Although all seed is routinely soaked in 20,000 mg Ca(OCl)2/liter of water for 15 min before germination, approximately 5 to 10% of the bean sprouts in several growing baskets (1.5 × 1.5 m) were affected and had to be discarded. Each basket contained approximately 1 t of sprouts. To isolate the causal organism, symptomatic stems were surface disinfested for 1 min in 0.5% NaOCl and incubated on acidified potato dextrose agar (PDA) at 25°C. Cultures were identified as Rhizoctonia solani on the basis of morphological features including right-angled branching of brown hyphae and the presence of sclerotia. PCR amplification of the internal transcribed spacer region was performed with primers RS1 and RS4 (2). Sequences were identical to R. solani AG4-HG-II in GenBank (Accession No. AF354074). To conduct pathogenicity tests, a 5-mm2-diameter disk from the margin of a culture of the fungus on PDA was placed in the center of 25 5-day-old germinated sprouts placed in a plastic box (15 × 10 × 5 cm) held at 25°C. Two isolates of R. solani cultured from different lots of sprouts were included in the assays. Controls received noncolonized agar. Treatments were replicated four times and each experiment was repeated three times. A moist paper towel was included in each box to maintain humidity. After 3 days, symptoms developed in the inoculated boxes but not in the noninoculated boxes. The fungus was reisolated from lesions, completing Koch's postulates. To our knowledge, this is the first report of R. solani on mung bean sprouts in a commercial sprouting facility. However, R. solani has been associated with root rot of mung bean plants in the field (1). References: (1) T. R. Anderson. Can. Plant Dis. Surv. 65:1, 1985. (2) C. Guillemaut et al. Can. J. Microbiol. 49:556, 2003.

RESUMEN

Glucocorticoid administration to mice results in a rapid loss of bone mineral density due to an imbalance in osteoblast and osteoclast numbers. Whereas excess glucocorticoids reduce both osteoblast and osteoclast precursors, cancellous osteoclast number surprisingly does not decrease as does osteoblast number, presumably due to the ability of glucocorticoids to promote osteoclast life span. Whether glucocorticoids act directly on osteoclasts in vivo to promote their life span and whether this contributes to the rapid loss of bone with glucocorticoid excess remains unknown. To determine the direct effects of glucocorticoids on osteoclasts in vivo, we expressed 11beta-hydroxysteroid dehydrogenase type 2, an enzyme that inactivates glucocorticoids, specifically in the osteoclasts of transgenic mice using the tartrate-resistant acid phosphatase promoter. Bone mass, geometry, and histomorphometry were similar in untreated wild-type and transgenic animals. Glucocorticoid administration for 7 d caused equivalent increases in cancellous osteoblast apoptosis, and equivalent decreases in osteoblasts, osteoid, and bone formation, in wild-type and transgenic mice. In contrast, glucocorticoids stimulated expression of the mRNA for calcitonin receptor, an osteoclast product, in wild-type but not transgenic mice. Consistent with the previous finding that glucocorticoids decrease osteoclast precursors and prolong osteoclast life span, glucocorticoids decreased cancellous osteoclast number in the transgenic mice but not wild-type mice. In accord with this decrease in osteoclast number, the loss of bone density observed in wild-type mice was strikingly prevented in transgenic mice. These results demonstrate for the first time that the early, rapid loss of bone caused by glucocorticoid excess results from direct actions on osteoclasts.

Asunto(s)

Densidad Ósea/efectos de los fármacos , Glucocorticoides/farmacología , Osteoclastos/efectos de los fármacos , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/metabolismo , Animales , Desarrollo Óseo/genética , Huesos/metabolismo , Dexametasona/efectos adversos , Dexametasona/farmacología , Femenino , Glucocorticoides/efectos adversos , Masculino , Ratones , Ratones Transgénicos , Especificidad de Órganos , Osteoclastos/metabolismo , Prednisolona/farmacología , Columna Vertebral/citología , Columna Vertebral/efectos de los fármacos , Columna Vertebral/crecimiento & desarrollo , Transgenes

RESUMEN

Elucidation of kinase-initiated routes by which the estrogen receptors alpha and beta (ERalpha and ERbeta) control gene transcription, along with evidence of distinct biologic outcomes in response to ligands that can selectively activate nongenotropic signaling of the ERs or the androgen receptor, suggest that the ERs control a range of genes wider than that regulated by their direct association with DNA. To ascertain the extent and significance of nongenotropic ER-mediated transcription, we employed transduced HeLa cells expressing wild-type ERalpha or the ligand binding domain of ERalpha localized to the cell membrane (E-Mem), the OB-6 osteoblastic cell line, MCF-7 breast carcinoma cells and uteri from mice treated with 17beta-estradiol (E(2)), or the nongenotropic signaling activator 4-estren-3alpha,17beta-diol (estren). E(2) and estren induced ERK1/2 and Akt phosphorylation in ERalpha or E-Mem stably transfected HeLa cells; however, the phosphorylation kinetics differed between the two cell lines. In all four models, nongenotropic ER actions regulated a population of genes distinct from those regulated by genotropic ER actions. Specifically, the expression of Wnt2, Frizzled10, Egr-1, and c-Fos was strongly up-regulated in E-Mem-containing HeLa cells treated with E(2) or estren, or in ERalpha-containing HeLa cells treated with estren. Up-regulation of Frizzled10 by estren was reproduced in MCF-7 cells. Egr-1 was up-regulated by both estren and E(2); but complement 3, only by E(2) in the uteri. Estren had no effect on complement 3, cathepsin D, progesterone receptor, bcl-2, and cyclin D1 in MCF-7 cells, whereas E(2) up-regulated all these estrogen response element or activating protein-1-containing genes. These results support an extensive divergence in gene expression depending on the mode of ER activation.

Asunto(s)

Receptor alfa de Estrógeno/fisiología , Regulación de la Expresión Génica , Transcripción Genética , Animales , Sitios de Unión , Huesos/metabolismo , Estradiol/farmacología , Estrenos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Receptores Frizzled , Células HeLa , Humanos , Ratones , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Neurotransmisores/genética , Proteína wnt2/genética

RESUMEN

Since 1994; the emphasis in the provision of health services in South Africa has shifted from hospital-based care to a community-based comprehensive primary health care system; especially important in the management of chronic diseases; such as Diabetes Mellitus (DM). The incidence of DM is rapidly increasing on a global basis according to the World Health Organization; resulting in the development of evidence-based guidelines for control and management of diabetes mellitus in many countries around the world. The aim of these guidelines is to provide optimal care for patients as efficiently and cost-effectively as possible; facilitate the early detection of the condition and to provide a framework for patient education; essential for self-management and self-monitoring of the condition. Registered nurses play an extremely important role in the management of patients with DM. In this study; the views of registered nurses on the national guidelines for the control and management of DM (Type 2) were explored and described and methods of facilitating the implementation of the national guidelines in practice were identified. A qualitative; exploratory; descriptive and contextual approach was used. Registered nurses who participated in this study had definite positive views on the guidelines; were satisfied with the content of the guidelines and viewed them as an effective contribution to the management of DM; if implemented correctly. However; the participants identified several factors hindering the effective implementation of the guidelines. Recommendations to assist registered nurses in the implementation and utilisation of the national guidelines for the management of DM were constructed

Asunto(s)

Diabetes Mellitus , Manejo de la Enfermedad , Programas Nacionales de Salud , Enfermería , Pacientes Ambulatorios , Atención Primaria de Salud

RESUMEN

Both chronic excess of PTH, as in hyperparathyroidism, and intermittent elevation of PTH (by daily injections) increase the number of osteoblasts; albeit, the former is associated with bone catabolism and the later with bone anabolism. Intermittent PTH increases osteoblast number by attenuating osteoblast apoptosis, an effect that requires the transcription factor Runx2. However, chronic elevation of PTH does not affect osteoblast apoptosis because it stimulates the proteasomal degradation of Runx2. Here, we studied the effects of PTH on Sost, a Runx2 target gene expressed in osteocytes (former osteoblasts embedded in the bone matrix), which antagonizes the pro-osteoblastogenic actions of bone morphogenetic proteins and Wnts. We report that continuous infusion of PTH to mice for 4 d decreased Sost mRNA expression in vertebral bone by 80-90%. This effect was accompanied by a comparable reduction of sclerostin, the product of Sost, in osteocytes, as determined by quantitative immunoblot analysis of bone extracts and by immunostaining. In contrast, a single injection of PTH caused a transient 50% reduction in Sost mRNA at 2 h, but four daily injections had no effect on Sost mRNA or sclerostin. PTH strongly decreased Sost expression in osteocytes formed in primary cultures of neonatal murine calvaria cells as well as in osteocytic MLO-A5 cells, demonstrating a direct effect of PTH on this cell type. These results, together with evidence that sclerostin antagonizes bone morphogenetic proteins and Wnts, strongly suggest that suppression of Sost by PTH represents a novel mechanism for hormonal control of osteoblastogenesis mediated by osteocytes.

Asunto(s)

Proteínas Morfogenéticas Óseas/metabolismo , Osteoblastos/citología , Osteocitos/metabolismo , Hormona Paratiroidea/farmacología , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proteínas Morfogenéticas Óseas/genética , División Celular/efectos de los fármacos , Células Cultivadas , Esquema de Medicación , Femenino , Marcadores Genéticos/genética , Glicoproteínas , Humanos , Inyecciones , Péptidos y Proteínas de Señalización Intercelular , Vértebras Lumbares/metabolismo , Ratones , Hormona Paratiroidea/administración & dosificación , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , Cráneo/citología

RESUMEN

We show that sex steroids protect the adult murine skeleton through a mechanism that is distinct from that used to preserve the mass and function of reproductive organs. The classical genotropic actions of sex steroid receptors are dispensable for their bone protective effects, but essential for their effects on reproductive tissues. A synthetic ligand (4-estren-3alpha,17beta-diol) that reproduces the nongenotropic effects of sex steroids, without affecting classical transcription, increases bone mass and strength in ovariectomized females above the level of the estrogen-replete state and is at least as effective as dihydrotestosterone in orchidectomized males, without affecting reproductive organs. Such ligands merit investigation as potential therapeutic alternatives to hormone replacement for osteoporosis in both women and men [corrected].

Asunto(s)

Densidad Ósea/efectos de los fármacos , Huesos/efectos de los fármacos , Estrenos/farmacología , Osteoblastos/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Huesos/fisiología , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Células Cultivadas , Fuerza Compresiva/efectos de los fármacos , Dihidrotestosterona/farmacología , Estradiol/farmacología , Estrenos/metabolismo , Femenino , Humanos , Masculino , Ratones , Orquiectomía , Tamaño de los Órganos/efectos de los fármacos , Osteoblastos/fisiología , Osteocalcina/sangre , Osteoclastos/fisiología , Osteogénesis/efectos de los fármacos , Osteoporosis/tratamiento farmacológico , Ovariectomía , Pirazoles/farmacología , Receptores de Estrógenos/metabolismo , Vesículas Seminales/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas , Útero/efectos de los fármacos , Útero/metabolismo

RESUMEN

The rates of osteoblast and osteoclast formation are tightly balanced, possibly due to the requirement of mesenchymal osteoblast progenitors for osteoclastogenesis. Osteoblast differentiation requires the transcription factor Cbfa1, whereas osteoclastogenesis results from the interaction between receptor activator of NF kappa B ligand (RANKL), expressed on stromal/osteoblastic cells, and RANK, a surface receptor on hematopoietic precursors. A striking decrease in the number of osteoclasts in Cbfa1-deficient mice suggested that Cbfa1 might be involved in RANKL expression. To investigate this possibility and to elucidate the mechanisms regulating RANKL expression, we isolated the 5'-flanking region of the murine RANKL gene and found that it contains two potential binding sites for Cbfa1 (OSE2-like sites). Cbfa1 bound to either of these sites in gel shift assays and stimulated the activity of a chimeric promoter consisting of multimerized RANKL OSE2-like sites inserted upstream from a minimal thymidine kinase (tk) promoter in transient transfections. However, Cbfa1 cotransfection did not stimulate murine RANKL promoter-luciferase constructs. Further analysis revealed that removal of these sites from the RANKL promoter by either site-directed mutagenesis or 5'-deletion did not alter the basal activity of promoter-reporter constructs. Conditional expression of Cbfa1 in a stromal/osteoblastic cell line stimulated osteocalcin mRNA by fivefold, but had no significant effect on RANKL mRNA levels. Conversely, conditional expression of a dominant-negative form of Cbfa1 in the same cell line inhibited osteocalcin mRNA by threefold, but had no effect on RANKL mRNA. Although these results cannot rule out a novel function for Cbfa1 in RANKL expression, they demonstrate that Cbfa1 does not regulate RANKL gene activity in the same manner as known targets of this transcription factor, such as osteocalcin.

Asunto(s)

Proteínas Portadoras/biosíntesis , Glicoproteínas de Membrana/biosíntesis , Proteínas de Neoplasias , Osteoblastos/fisiología , Células del Estroma/fisiología , Factores de Transcripción/fisiología , Secuencia de Aminoácidos/fisiología , Animales , Secuencia de Bases/fisiología , Línea Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Datos de Secuencia Molecular , Ligando RANK , ARN Mensajero/biosíntesis , Receptor Activador del Factor Nuclear kappa-B

RESUMEN

The relationship of the classical receptors and their transcriptional activity to nongenotropic effects of steroid hormones is unknown. We demonstrate herein a novel paradigm of sex steroid action on osteoblasts, osteocytes, embryonic fibroblasts, and HeLa cells involving activation of a Src/Shc/ERK signaling pathway and attenuating apoptosis. This action is mediated by the ligand binding domain and eliminated by nuclear targeting of the receptor protein; ERalpha, ERbeta, or AR can transmit it with similar efficiency irrespective of whether the ligand is an estrogen or an androgen. This antiapoptotic action can be dissociated from the transcriptional activity of the receptor with synthetic ligands, providing proof of principle for the development of function-specific-as opposed to tissue-selective-and gender-neutral pharmacotherapeutics.

Asunto(s)

Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Transducción de Señal/fisiología , Andrógenos/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Sitios de Unión/fisiología , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Estrógenos/farmacología , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Células HeLa , Humanos , Técnicas In Vitro , Masculino , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/fisiología , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteoclastos/fisiología , Fragmentos de Péptidos/farmacología , Receptores Androgénicos/química , Receptores de Estrógenos/química , Factores Sexuales , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/fisiología , Dominios Homologos src/fisiología , Familia-src Quinasas/metabolismo

RESUMEN

Currently, lifelong immunosuppression is required for organ transplant recipients. The majority of transplant recipients will eventually develop chronic rejection with resultant graft loss, despite treatment with powerful immunosuppressive agents. These agents are also associated with numerous toxicities including reduced immunity against infection and malignancy. Therefore, the central goal in transplant science is to devise tolerance strategies in an attempt to establish a state of prolonged non-reactivity against the allograft, accompanied with preservation of an intact immune system. Although predictable tolerance induction has been elusive, we found that short course of the novel immunomodulatory agent, anti-CD45RB monoclonal antibody, leads to indefinite acceptance of renal allografts in mice, and has been shown to markedly prolong allograft survival in primates. We review the current state of development of this antibody, and the progress made in defining its mechanism of action.

Asunto(s)

Anticuerpos Monoclonales/uso terapéutico , Terapia de Inmunosupresión/métodos , Antígenos Comunes de Leucocito , Inmunología del Trasplante , Traslado Adoptivo , Animales , Anticuerpos Monoclonales/administración & dosificación , Autoinmunidad , Humanos , Tolerancia Inmunológica , Inmunosupresores/administración & dosificación , Técnicas In Vitro , Ratones , Modelos Biológicos , Primates , Trasplante Heterólogo

RESUMEN

Interleukin-6 (IL-6)-type cytokines stimulate osteoclast formation by activating the glycoprotein 130 (gp130) receptor subunit on stromal/osteoblastic cells, which in turn leads to signal transducer and activator of transcription 3 (STAT3)-mediated expression of receptor activator of NF-kappaB ligand (RANKL). Based on evidence that gp130 expression is regulated by a variety of cytokines and hormones, we have determined here whether changes in gp130 levels directly contribute to the magnitude of the osteoclastogenic stimulus delivered by IL-6-type cytokines. To accomplish this, gp130 protein levels were modulated using a tetracycline-regulated expression system in a stromal/osteoblastic cell line, UAMS-32, which supports osteoclast formation. Removal of doxycycline from the culture medium elevated gp130 expression and increased the responsiveness of a STAT-responsive promoter-luciferase construct to IL-6 complexed with its soluble receptor (IL-6+sIL-6R), but diminished the responsiveness to oncostatin M (OSM). IL-6+sIL-6R-stimulated osteoclast formation was greater when osteoclast precursors were cocultured with the cells expressing elevated gp130 levels than when cells expressing low gp130 levels were used. However, increased gp130 levels reduced OSM-stimulated osteoclast formation. These results establish that the level of gp130 in stromal/osteoblastic cells directly modulates the magnitude of the osteoclastogenic response to IL-6-type cytokines such that an increase in gp130 increases the cellular responsiveness to IL-6+sIL-6R but reduces responsiveness to OSM.

Asunto(s)

Antígenos CD/metabolismo , Células de la Médula Ósea/metabolismo , Interleucina-5/farmacología , Glicoproteínas de Membrana/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Células 3T3 , Animales , Antígenos CD/genética , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Línea Celular , Células Cultivadas , Receptor gp130 de Citocinas , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Glicoproteínas de Membrana/genética , Ratones , Oncostatina M , Osteoclastos/citología , Péptidos/farmacología , Receptores de Interleucina-6/metabolismo , Factor de Transcripción STAT3 , Transducción de Señal , Transactivadores/metabolismo

RESUMEN

Bone morphogenetic proteins (BMPs) have been heretofore implicated in the induction of osteoblast differentiation from uncommitted progenitors during embryonic skeletogenesis and fracture healing. We have tested the hypothesis that BMPs are also involved in the osteoblastogenesis that takes place in the bone marrow in postnatal life. To do this, we took advantage of the properties of noggin, a recently discovered protein that binds BMP-2 and -4 and blocks their action. Addition of human recombinant noggin to bone marrow cell cultures from normal adult mice inhibited both osteoblast and osteoclast formation; these effects were reversed by exogenous BMP-2. Consistent with these findings, BMP-2 and -4 and BMP-2/4 receptor transcripts and proteins were detected in these primary cultures, in a bone marrow-derived stromal/osteoblastic cell line, as well as in murine adult whole bone; noggin expression was also documented in all these preparations. Moreover, addition of antinoggin antibody caused an increase in osteoblast progenitor formation. These findings suggest that BMP-2 and -4 are expressed in the bone marrow in postnatal life and serve to maintain the continuous supply of osteoblasts and osteoclasts; and that, in fact, BMP-2/4-induced commitment to the osteoblastic lineage is a prerequisite for osteoclast development. Hence, BMPs, perhaps in balance with noggin and possibly other antagonists, may provide the tonic baseline control of the rate of bone remodeling on which other inputs (e.g., hormonal, biomechanical, etc.) operate.

Asunto(s)

Células de la Médula Ósea/citología , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Osteoblastos/citología , Osteoclastos/citología , Proteínas/farmacología , Factor de Crecimiento Transformador beta , Animales , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 4 , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1 , Receptores de Proteínas Morfogenéticas Óseas de Tipo II , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Portadoras , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Ratones , Pruebas de Neutralización , Proteínas Serina-Treonina Quinasas/genética , Receptores de Factores de Crecimiento/genética , Proteínas Recombinantes/farmacología

RESUMEN

Interleukin (IL)-6-type cytokines stimulate osteoclastogenesis by activating gp130 in stromal/osteoblastic cells and may mediate some of the osteoclastogenic effects of other cytokines and hormones. To determine whether STAT3 is a downstream effector of gp130 in the osteoclast support function of stromal/osteoblastic cells and whether the gp130/STAT3 pathway is utilized by other osteoclastogenic agents, we conditionally expressed dominant negative (dn)-STAT3 or dn-gp130 in a stromal/osteoblastic cell line (UAMS-32) that supports osteoclast formation. Expression of either dominant negative protein abolished osteoclast formation stimulated by IL-6 + soluble IL-6 receptor, oncostatin M, or IL-1 but not by parathyroid hormone or 1,25-dihydroxyvitamin D3. Because previous studies suggested that IL-6-type cytokines may stimulate osteoclastogenesis by inducing expression of the tumor necrosis factor-related protein, receptor activator of NF-kappaB ligand (RANKL), we conditionally expressed RANKL in UAMS-32 cells and found that this was sufficient to stimulate osteoclastogenesis. Moreover, dn-STAT3 blocked the ability of either IL-6 + soluble IL-6 receptor or oncostatin M to induce RANKL. These results establish that STAT3 is essential for gp130-mediated osteoclast formation and that the target of STAT3 during this process is induction of RANKL. In addition, this study demonstrates that activation of the gp130-STAT3 pathway in stromal/osteoblastic cells mediates the osteoclastogenic effects of IL-1, but not parathyroid hormone or 1, 25-dihydroxyvitamin D3.

Asunto(s)

Antígenos CD/metabolismo , Calcitriol/metabolismo , Proteínas de Unión al ADN/metabolismo , Interleucina-1/metabolismo , Glicoproteínas de Membrana/metabolismo , FN-kappa B/metabolismo , Osteoblastos/enzimología , Hormona Paratiroidea/metabolismo , Células del Estroma/enzimología , Transactivadores/metabolismo , Animales , Proteínas Portadoras/metabolismo , Receptor gp130 de Citocinas , Activación Enzimática , Ligandos , Ratones , Ratones Endogámicos C57BL , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Factor de Transcripción STAT3 , Transducción de Señal , Transfección

RESUMEN

The cyclin-dependent kinase inhibitor p21(WAF1,CIP1,SDI1) plays a critical role in cell differentiation, and it has been shown to confer resistance to apoptosis. Based on this, and on evidence that activation of the gp130/signal transducer and activator of transcription (STAT) signal transduction pathway by interleukin (IL)-6 type cytokines promotes differentiation and prevents apoptosis in osteoblastic cells, we have investigated the possibility that p21 is a downstream effector of this signaling pathway in osteoblasts. We report that either oncostatin M (OSM) or IL-6 plus soluble IL-6 receptor increased the levels of p21 mRNA and protein in the osteoblast-like human osteosarcoma cell line MG63 and stimulated the activity of a 2.4-kilobase pair segment of the human p21 gene promoter. Further, nuclear extracts from cytokine-stimulated MG63 cells formed protein-DNA complexes with a 19-base pair nucleotide fragment of the p21 promoter containing a single STAT response element. The identity of the binding proteins as Stat3 and Stat1 was demonstrated with specific antibodies. In addition, and in support of a mediating role of STATs in the activation of the p21 promoter, overexpression of Stat3 potentiated the cytokine effect on the p21 promoter; whereas a dominant negative Stat3, or a mutation of the STAT response element on the promoter, significantly reduced the cytokine effect. Finally, antisense oligonucleotides complementary to p21 mRNA inhibited OSM-induced stimulation of alkaline phosphatase expression and antagonized the protective effect of OSM on anti-Fas-induced apoptosis. These results demonstrate that p21 is a downstream effector of gp130/Stat3 activation and a critical mediator of the pro-differentiating and anti-apoptotic effects of IL-6 type cytokines on human osteoblastic cells.

Asunto(s)

Ciclinas/genética , Interleucina-6/fisiología , Osteoblastos/citología , Regulación hacia Arriba/fisiología , Antígenos CD/metabolismo , Apoptosis/fisiología , Secuencia de Bases , Diferenciación Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Receptor gp130 de Citocinas , Humanos , Glicoproteínas de Membrana/metabolismo , Oligonucleótidos Antisentido , Osteoblastos/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/fisiología

RESUMEN

CONTEXT: Studies of high-risk families with multiple early-onset cases of breast cancer have been useful for assessing the type and spectrum of germline mutations on the BRCA1 gene, but do not provide guidance to women with modest family history profiles. Thus, studies of women from the general population are needed to determine the BRCA1 mutation frequency in women perceived to be at high risk, and to develop profiles of those most likely to be carriers. OBJECTIVE: To characterize frequency and spectrum of germline BRCA1 mutations in 2 categories of women identified via population-based studies hypothesized to be at increased risk of carrying such mutations: those diagnosed as having breast cancer before age 35 years and those diagnosed before age 45 years who have first-degree breast cancer family history. DESIGN: Study subjects were drawn from 2 population-based case-control studies of breast cancer in young women on the basis of their family history or their age of diagnosis. Cases were younger than 35 years or were younger than 45 years with first-degree family history at the time of breast cancer diagnosis and were ascertained via a population-based cancer registry, and controls (women without breast cancer) were identified via random-digit dialing. SETTING: Three counties in western Washington State. MAIN OUTCOME MEASURE: BRCA1 germline mutations in study subjects identified in DNA from peripheral blood lymphocytes by single-strand conformation polymorphism analysis using primer pairs that span the BRCA1 coding region and intron-exon boundaries. RESULTS: Of 193 women diagnosed as having breast cancer before age 35 years, none of whom were selected on the basis of family history status, 12 (6.2%, 95% confidence interval [CI], 3.2%-10.6%) had germline BRCA1 mutations. In 208 women diagnosed before age 45 years who had first-degree breast cancer family history, 15 (7.2%, 95% CI,4.1%-11.6%) had germline mutations in BRCA1. In both groups, there were variations in mutation frequency noted by age and by family history. Mutation frequency decreased with increasing age of diagnosis. Higher proportions of mutations were seen in cases with at least 1 relative diagnosed as having breast cancer before age 45 years, in cases with greater numbers of affected relatives, and those with ovarian cancer family history. Mutation frequency did not vary by bilateral breast cancer family history. No frameshift or nonsense mutations were observed in 71 control women with a first-degree family history, although missense changes of unknown significance were seen in cases and controls. CONCLUSIONS: Women with BRCA1 germline mutations lacked a common family history profile. Also, a large proportion of the women with a first-degree breast cancer family history and women diagnosed as having breast cancer before age 35 years did not carry germline BRCA1 mutations. Hence, while early-onset disease and a strong breast cancer family history may be useful guidelines for checking BRCA1 status, these findings on women drawn from the general population suggest that it may be difficult to develop BRCA1 mutation screening criteria among women with modest family history profiles.

Asunto(s)

Neoplasias de la Mama/genética , Genes BRCA1 , Adulto , Factores de Edad , Neoplasias de la Mama/epidemiología , Análisis Mutacional de ADN , Femenino , Mutación de Línea Germinal , Haplotipos , Heterocigoto , Humanos , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Factores de Riesgo

RESUMEN

Both estrogen and androgen exert their antiosteoporotic effects, at least in part, by inhibiting IL-6 production, thereby suppressing osteoclastogenesis. Several observations, however, suggest that besides increased IL-6 production, sensitivity of the osteoclastogenic process to this cytokine is altered after ovariectomy. Based on this and evidence that the ligand-binding subunit of the IL-6 receptor (gp80) is a limiting factor for the actions of IL-6 on bone, we hypothesized that sex steroids regulate expression of the IL-6 receptor as well. We report that 17beta-estradiol or dihydrotestosterone in vitro decreased the abundance of the gp80 mRNA as well as the mRNA of the signal-transducing subunit of the IL-6 receptor (gp130) in cells of the bone marrow stromal/osteoblastic lineage, and also decreased gp130 protein levels. These effects did not require new protein synthesis. In contrast to sex steroids, parathyroid hormone stimulated gp130 expression; this effect was opposed by sex steroids. Consistent with these findings, ovariectomy in mice caused an increase in expression of gp80, gp130, and IL-6 mRNAs in ex vivo bone marrow cell cultures as determined by quantitative reverse transcription (RT)-PCR, and confirmed on an individual cell basis using in situ RT-PCR. The demonstration of increased expression of the IL-6 receptor after loss of sex steroids provides an explanation for why IL-6 is important for skeletal homeostasis in the sex steroid-deficient, but not replete, state.

Asunto(s)

Médula Ósea/efectos de los fármacos , Células del Tejido Conectivo/efectos de los fármacos , Dihidrotestosterona/farmacología , Estradiol/farmacología , Receptores de Interleucina-6/biosíntesis , Animales , Células de la Médula Ósea/efectos de los fármacos , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Interleucina-6/biosíntesis , Interleucina-6/genética , Ratones , Osteoblastos/efectos de los fármacos , Ovariectomía , Receptores de Interleucina-6/genética , Células del Estroma/efectos de los fármacos

RESUMEN

Glycoprotein 130 (gp130), a shared component of all the receptors for the interleukin-6 cytokine family, transduces cytokine signals in part by activating latent cytoplasmic signal transducers and activators of transcription (STATs). STATs subsequently translocate into the nucleus and stimulate gene expression. In the studies reported here, the 5'-flanking region of the human gp130 gene was isolated and the transcription initiation sites were mapped. To demonstrate that the isolated DNA fragment contained a functional promoter, a plasmid construct containing 2433 base pairs of the gp130 5'-flanking region, inserted upstream from the firefly luciferase gene, was transiently transfected into HepG2 hepatoma cells. The construct exhibited constitutive promoter activity. In addition, a 5-h treatment with interleukin-6 or oncostatin M stimulated the activity of this promoter severalfold. Localization of the cytokine response element by 5'-deletion analysis and site-directed mutagenesis revealed a cis-acting binding site for activated STAT complexes. Furthermore, DNA binding analysis demonstrated that this element binds activated STAT1 and STAT3 homo- and heterodimers. This STAT-binding element was sufficient to confer cytokine stimulation to a minimal herpesvirus thymidine kinase promoter. These results establish that the DNA fragment we have isolated contains the human gp130 promoter and that interleukin-6 type cytokines may influence the activity of this promoter via activated STATs.

Asunto(s)

Antígenos CD/biosíntesis , Antígenos CD/genética , Proteínas de Unión al ADN/metabolismo , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Regiones Promotoras Genéticas , Receptores de Citocinas/biosíntesis , Receptores de Citocinas/genética , Transactivadores/metabolismo , Secuencia de Bases , Carcinoma Hepatocelular , Núcleo Celular/metabolismo , Receptor gp130 de Citocinas , Regulación de la Expresión Génica , Genes Reporteros , Biblioteca Genómica , Humanos , Cinética , Neoplasias Hepáticas , Luciferasas/biosíntesis , Datos de Secuencia Molecular , Plásmidos , Proteínas Recombinantes de Fusión/biosíntesis , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Transducción de Señal , Transfección , Células Tumorales Cultivadas

RESUMEN

In both vertebrate and invertebrate cells, the 60-kDa Ro autoantigen is bound to small cytoplasmic RNAs known as Y RNAs. In Xenopus oocytes, the 60-kDa Ro protein is also complexed with a class of 5S rRNA precursors that contain internal mutations. Because these 5S rRNA precursors are processed inefficiently and degraded eventually, the Ro protein may function in a quality control pathway for 5S rRNA biosynthesis. We have investigated the sequence and secondary structure determinants in the mutant 5S rRNAs that confer binding by the 60-kDa Ro protein. The mutant 5S rRNAs fold to form an alternative helix that is required for recognition by the 60-kDa Ro protein. Mutations that disrupt the alternative helix eliminate Ro protein binding, whereas compensatory changes that restore the helix are bound efficiently by the Ro protein. When the structure of the mutant RNA was probed using dimethylsulfate and oligonucleotide-directed RNase H cleavage, the results were consistent with the formation of the alternative structure. The La protein, which is also complexed with the mutant 5S rRNA precursors, protects similar sequences from nuclease digestion as does the 60-kDa Ro protein. Thus, the binding sites for these two proteins are either nearby on the RNA, or the two proteins may be complexed through protein-protein interactions. When the human Ro protein is expressed in the yeast Saccharomyces cerevisiae, the protein binds wild-type 5S rRNA precursors, suggesting that a population of wild-type precursors also folds into the alternative structure.

Asunto(s)

Autoantígenos/metabolismo , Conformación de Ácido Nucleico , ARN Ribosómico 5S/química , ARN Ribosómico 5S/metabolismo , ARN Citoplasmático Pequeño , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Animales , Autoantígenos/genética , Secuencia de Bases , Drosophila melanogaster , Humanos , Datos de Secuencia Molecular , Precursores del ARN/química , Precursores del ARN/metabolismo , Ribonucleasa T1/metabolismo , Ribonucleoproteínas/genética , Saccharomyces cerevisiae , Relación Estructura-Actividad , Xenopus laevis , Antígeno SS-B

RESUMEN

Anti-Mi-2 autoantibody is strongly associated with dermatomyositis and found in sera of 20% of patients. Mi-2 antigen contains at least eight components and previous evidence suggested that the 240-kD protein was the antigenic component for at least some sera. In this study, anti-M-2 patient sera were used to screen human thymocyte and HeLa cell lambda gt11 expression libraries, and two clones from each had plaques specifically reactive with anti-Mi-2 sera. Studies with affinity-purified antibody supported the identification of the clones. All of 44 anti-Mi-2 sera reacted with the plaques, but none of 44 control sera reacted significantly. The cDNAs were identical, and full sequencing of one revealed an open reading frame spanning a 1,054-bp insert. Rescreening the library with the cDNA yielded a 1,589-bp cDNA that continued the open reading frame. The Mi-2 cDNA hybridized to a single 7.5-8.0 kb mRNA of HeLa cells, by Northern blot. Rabbit antiserum directed at a portion of the cDNA product reacted with HeLa 240-kD Mi-2 protein. The sequence was notable for four potential zinc-fingers and several charged regions. The protein encoded by the cDNA produced in vitro reacted with only one of five of the Mi-2 sera. These findings indicate that the Mi-2 240 kD is a novel protein that is antigenic for all Mi-2 sera, and strongly suggests that a major common epitope is conformational in nature.

Asunto(s)

Adenosina Trifosfatasas , Autoantígenos/genética , ADN Helicasas , Dermatomiositis/inmunología , Secuencia de Aminoácidos , Animales , Autoanticuerpos/inmunología , Secuencia de Bases , ADN Complementario/análisis , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Epítopos , Células HeLa , Humanos , Masculino , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2 , Datos de Secuencia Molecular , Peso Molecular , Conejos , Dedos de Zinc

RESUMEN

In virtually all vertebrate cells, Ro RNPs consist of the 60-kDa Ro autoantigen bound to one of several small cytoplasmic RNA molecules known as Y RNAs. Because the 60-kDa Ro autoantigen is also found complexed with defective precursors of 5S rRNA in Xenopus oocytes, we have proposed that this protein functions in a quality control, or discard pathway, for 5S RNA biosynthesis (O'Brien CA, Wolin SL, 1994, Genes & Dev 8:2891-2903). The role of the Y RNAs in this pathway is unknown. To begin a genetic analysis of Ro RNP function, we have characterized these particles in the nematode Caenorhabditis elegans. The C. elegans Ro protein is 12 kDa larger than the vertebrate protein; the larger size is due in part to an N-terminal extension and to two insertions in the RNA recognition motif. In contrast to all previously described vertebrate species, the Ro protein appears bound to a single Y RNA in C. elegans. Similar to vertebrate Y RNAs, the C. elegans Y RNA can be folded to form a pyrimidine-rich internal loop and a long stem in which the 5' and 3' ends are base paired. Within the stem is a conserved bulged helix that is proposed to be the binding site of the Ro protein. Interestingly, although the human protein can bind the nematode Y RNA, the C. elegans protein does not bind human Y RNAs. This is the first description of Ro RNPs in an invertebrate species.

Asunto(s)

Autoantígenos/análisis , Caenorhabditis elegans/química , ARN/análisis , Ribonucleoproteínas/análisis , Secuencia de Aminoácidos , Animales , Autoantígenos/metabolismo , Secuencia de Bases , Caenorhabditis elegans/embriología , Humanos , Immunoblotting , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Pruebas de Precipitina , Unión Proteica , ARN/genética , ARN/metabolismo , ARN Citoplasmático Pequeño , Ribonucleoproteínas/metabolismo , Análisis de Secuencia de ARN , Homología de Secuencia de Aminoácido

RESUMEN

YRNAs are small cytoplasmic RNAs that are components of the Ro ribonucleoprotein complex. This complex, which also includes the 60-kDa Ro protein, is a human autoantigen which is conserved among vertebrates, and is of unknown function. Multiple sequences with YRNA homology, known as YRNA-like sequences, have been detected in rabbit, mouse, duck, iguana and frog genomes with human Y cDNA probes. As judged by Northern blots of total RNA, however, not all of these genomic YRNA-like sequences are expressed. Complementary DNA and putative gene sequences for iguana Y3 (iY3) and iguana Y4 (iY4) Ro RNAs have been determined and used, along with previously sequenced human and frog Ro YRNA sequences, to construct the most likely Y3 and Y4 RNA secondary structures. The data presented indicate that Y3 is the most conserved Ro RNA, not only by its more consistent presence in other species, but also at the levels of sequence divergence and secondary structure similarity. The differences observed between the secondary structure solutions for the Y3 and Y4 Ro RNAs are consistent with the possibility that these RNAs perform different cellular functions.

Asunto(s)

ARN/genética , Ribonucleoproteínas/metabolismo , Vertebrados/genética , Animales , Autoantígenos/química , Autoantígenos/genética , Secuencia de Bases , Secuencia Conservada , Sondas de ADN , Humanos , Iguanas/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN/química , ARN Citoplasmático Pequeño , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Homología de Secuencia de Ácido Nucleico , Xenopus/genética