RESUMEN

The manganese salt bath is considered a primary standard for determining the absolute emission rate of radionuclide neutron sources. The National Research Council of Canada has recently revived its manganese salt bath and a full description of the system is given here. The physical characteristics of the bath, as well as the methods for determining the efficiency of the bath system and the induced activity in the bath, are described. An in-depth analysis of the fraction of neutrons captured in the manganese and the correction factor for neutron losses is also provided. Finally, the results of emission rate measurements of four different sources, complete with an uncertainty budget, are given. The emission rates of three americium-beryllium neutron sources and one californium-252 neutron source were found to agree with the known values, within a standard uncertainty of 1.7%.

Asunto(s)

Californio , Manganeso , Dosis de Radiación , Calibración , Californio/análisis , Neutrones , Americio/análisis , Berilio/análisis , Canadá , Radiometría/métodos

RESUMEN

Non-melanoma skin cancers are the most prevalent form of cancer, with cutaneous squamous cell carcinoma (cscc) being the 2nd most common type. Patients presenting with high-risk lesions associated with locally advanced or metastatic cscc face high rates of recurrence and mortality. Accurate staging and risk stratification for patients can be challenging because no system is universally accepted, and no Canadian guidelines currently exist. Patients with advanced cscc are often deemed ineligible for either or both of curative surgery and radiation therapy (rt) and, until recently, were limited to off-label systemic cisplatin-fluorouracil or cetuximab therapy, which offers modest clinical benefits and potentially severe toxicity. A new systemic therapy, cemiplimab, has been approved for the treatment of locally advanced and metastatic cscc. In the present review, we provide recommendations for patient classification and staging based on current guidelines, direction for determining patient eligibility for surgery and rt, and an overview of the available systemic treatment options for advanced cscc and of the benefits of a multidisciplinary approach to patient management.

Asunto(s)

Carcinoma de Células Escamosas/terapia , Neoplasias Cutáneas/terapia , Carcinoma de Células Escamosas/patología , Humanos , Metástasis de la Neoplasia , Neoplasias Cutáneas/patología

RESUMEN

We previously reported that STAT1 expression is frequently abrogated in human estrogen receptor-α-positive (ERα(+)) breast cancers and mice lacking STAT1 spontaneously develop ERα(+) mammary tumors. However, the precise mechanism by which STAT1 suppresses mammary gland tumorigenesis has not been fully elucidated. Here we show that STAT1-deficient mammary epithelial cells (MECs) display persistent prolactin receptor (PrlR) signaling, resulting in activation of JAK2, STAT3 and STAT5A/5B, expansion of CD61(+) luminal progenitor cells and development of ERα(+) mammary tumors. A failure to upregulate SOCS1, a STAT1-induced inhibitor of JAK2, leads to unopposed oncogenic PrlR signaling in STAT1(-/-) MECs. Prophylactic use of a pharmacological JAK2 inhibitor restrains the proportion of luminal progenitors and prevents disease induction. Systemic inhibition of activated JAK2 induces tumor cell death and produces therapeutic regression of pre-existing endocrine-sensitive and refractory mammary tumors. Thus, STAT1 suppresses tumor formation in mammary glands by preventing the natural developmental function of a growth factor signaling pathway from becoming pro-oncogenic. In addition, targeted inhibition of JAK2 may have significant therapeutic potential in controlling ERα(+) breast cancer in humans.

Asunto(s)

Receptor alfa de Estrógeno/metabolismo , Janus Quinasa 2/metabolismo , Neoplasias Mamarias Animales/metabolismo , Células Madre Neoplásicas/metabolismo , Factor de Transcripción STAT1/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Femenino , Compuestos Heterocíclicos con 3 Anillos/farmacología , Janus Quinasa 2/antagonistas & inhibidores , Neoplasias Mamarias Animales/tratamiento farmacológico , Neoplasias Mamarias Animales/genética , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Factor de Transcripción STAT1/deficiencia , Transducción de Señal/efectos de los fármacos , Proteína 1 Supresora de la Señalización de Citocinas

RESUMEN

The design, simulation results and measurements of a new neutron energy spectrometer are presented. The device, which may be called NNS, for Nested Neutron Spectrometer, works under the same principles as a Bonner Sphere Spectrometer (BSS) System, i.e. whereby a thermal neutron detector is surrounded by a polyethylene moderator. However, the moderator is cylindrical in shape. The different thicknesses of moderator are created by inserting one cylinder into another, much like nested Russian dolls. This design results in a much lighter instrument that is also easier to use in the field. Simulations and measurements show that, despite its shape, the device can be made to offer a near angular isotropic response to neutrons and that unfolded neutron spectra are in agreement with those obtained with a more traditional BSS.

Asunto(s)

Neutrones , Polietileno/química , Protección Radiológica/instrumentación , Análisis Espectral/instrumentación , Diseño de Equipo , Dosis de Radiación , Protección Radiológica/métodos , Análisis Espectral/métodos

RESUMEN

Human papillomaviruses (HPV) have now been identified as a necessary cause of benign and malignant lesions of the differentiating epithelium, particularly cervical cancer, the second most prevalent cancer in women worldwide. While two prophylactic HPV vaccines and screening programs are available, there is currently no antiviral drug for the treatment of HPV infections and associated diseases. The recent progress toward the identification and characterization of specific molecular targets for small molecule-based approaches provides prospect for the development of effective HPV antiviral compounds. Traditionally, antiviral therapies target viral enzymes. HPV encode for few proteins, however, and rely extensively on the infected cell for completion of their life cycle. This article will review the functions of the viral E1 helicase, which encodes the only enzymatic function of the virus, of the E2 regulatory protein, and of the viral E6 and E7 oncogenes in viral replication and pathogenesis. Particular emphasis will be placed on the recent progress made towards the development of novel small molecule inhibitors that specifically target and inhibit the functions of these viral proteins, as well as their interactions with other viral and/or cellular proteins.

RESUMEN

The goals of this study were to investigate the response of the rat supraspinatus tendon to overuse at the molecular level using transcriptional profiling, and to identify potential markers of tendinopathy. Adult rats were subjected to an overuse protocol that consists of downhill running (10% grade) at 17 m/min for 1 h/day, 5 days/week, for a total of either 1, 2, or 4 weeks. Another group of rats served as nonrunning time 0 controls. Transcriptional profiling was performed on the supraspinatus and patellar tendons using an Affymetrix rat genome array. A gene was considered to be differentially expressed if the p value from an ANOVA test was less than 0.01 and the difference between runners and controls was at least twofold at any time point. The supraspinatus tendon had increased expression of well-known cartilage genes such as col2a1, aggrecan, and sox9. These genes were not regulated in the patellar tendon, an internal comparator. Few genes associated with inflammation, or angiogenesis, were differentially expressed, and no significant change in the regulation of matrix metalloproteinases was detected. The results of this study suggest that by expressing more cartilage genes, the tendon is converting toward a fibrocartilage phenotype as a result of the repetitive loading and repeated compression of the tendon as it passes through the acromial arch.

Asunto(s)

Trastornos de Traumas Acumulados/genética , Trastornos de Traumas Acumulados/fisiopatología , Perfilación de la Expresión Génica , Lesiones del Manguito de los Rotadores , Manguito de los Rotadores/fisiopatología , Animales , Modelos Animales de Enfermedad , Fibrocartílago/lesiones , Fibrocartílago/fisiopatología , Marcadores Genéticos , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Ratas , Ratas Sprague-Dawley , Transcripción Genética , Soporte de Peso

RESUMEN

The E1 helicase of papillomaviruses is required for replication of the viral double-stranded DNA genome, in conjunction with cellular factors. DNA replication is initiated at the viral origin by the assembly of E1 monomers into oligomeric complexes that have unwinding activity. In vivo, this process is catalyzed by the viral E2 protein, which recruits E1 specifically at the origin. For bovine papillomavirus (BPV) E1 a minimal DNA-binding domain (DBD) has been identified N-terminal to the enzymatic domain. In this study, we characterized the DBD of human papillomavirus 11 (HPV11), HPV18, and BPV E1 using a quantitative DNA binding assay based on fluorescence anisotropy. We found that the HPV11 DBD binds DNA with an affinity and sequence requirement comparable to those of the analogous domain of BPV but that the HPV18 DBD has a higher affinity for nonspecific DNA. By comparing the DNA-binding properties of a dimerization-defective protein to those of the wild type, we provide evidence that dimerization of the HPV11 DBD occurs only on two appropriately positioned E1 binding-sites and contributes approximately a 10-fold increase in binding affinity. In contrast, the HPV11 E1 helicase purified as preformed hexamers binds DNA with little sequence specificity, similarly to a dimerization-defective DBD. Finally, we show that the amino acid substitution that prevents dimerization reduces the ability of a longer E1 protein to bind to the origin in vitro and to support transient HPV DNA replication in vivo, but has little effect on its ATPase activity or ability to oligomerize into hexamers. These results are discussed in light of a model of the assembly of replication-competent double hexameric E1 complexes at the origin.

Asunto(s)

ADN Helicasas/química , Proteínas de Unión al ADN/química , Papillomaviridae/enzimología , Proteínas Virales/química , Secuencia de Bases , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dimerización , Polarización de Fluorescencia , Humanos , Datos de Secuencia Molecular , Mutación , Papillomaviridae/química , Papillomaviridae/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo

RESUMEN

The affinity of the origin-binding domain (OBD) of simian virus 40 large T antigen for its cognate origin was measured at equilibrium using a DNA binding assay based on fluorescence anisotropy. At a near-physiological concentration of salt, the affinities of the OBD for site II and the core origin were 31 and 50 nM, respectively. Binding to any of the four 5'-GAGGC-3' binding sites in site II was only slightly weaker, between 57 and 150 nM. Although the OBD was shown previously to assemble as a dimer on two binding sites spaced by 7 bp, we found that increasing the distance between both binding sites by 1 to 3 bp had little effect on affinity. Similar results were obtained for full-length T antigen in absence of nucleotide. Addition of ADP-Mg, which promotes hexamerization of T antigen, greatly increased the affinity of full-length T antigen for the core origin and for nonspecific DNA. The implications of these findings for the assembly of T antigen at the origin and its transition to a non-specific DNA helicase are discussed.

Asunto(s)

Antígenos Transformadores de Poliomavirus/química , Antígenos Transformadores de Poliomavirus/metabolismo , Proteínas de Unión al ADN/metabolismo , Origen de Réplica/fisiología , Animales , Antígenos Transformadores de Poliomavirus/genética , Secuencia de Bases , Sitios de Unión , Replicación del ADN , ADN Viral/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Dimerización , Polarización de Fluorescencia , Datos de Secuencia Molecular

RESUMEN

Overuse injuries and trauma in tendon often involve acute or chronic pain and eventual matrix destruction. Anti-inflammatory drugs have been used as a treatment, however, the cellular and molecular mechanisms of the destructive processes in tendon are not clearly understood. It is thought that an inflammatory event may be involved as an initiating factor. Mediators of the inflammatory response include cytokines released from macrophages and monocytes. Interleukin-1 beta (IL-1 beta) is a candidate proinflammatory cytokine that is active in connective tissues such as bone and cartilage. We hypothesized that tendon cells would express receptors and respond to IL-1 beta in an initial "molecular inflammation" cascade, that is, connective tissue cell expression of cytokines that induce matrix destructive enzymes. This cascade results in expression of matrix metalloproteinases (MMPs) and aggrecanases that may lead to matrix destruction. Normal human tendon cells from six patients were isolated, grown to quiescence and treated with human recombinant IL-1 beta in serum-free medium for 16 h. Total RNA was isolated and mRNA expression assessed by semiquantitative RT-PCR. IL-1 beta (1 nM) induced mRNAs for cyclooxygenase 2 (COX2), MMP-1, -3, -13 and aggrecanase-1 as well as IL-1 beta and IL-6, whereas mRNAs for COX1 and MMP-2 were expressed constitutively. The IL-1 beta-treated tendon cells released prostaglandin E(2) (PGE(2)) in the medium, suggesting that the inducible COX2 catalyzed this synthesis. Induction of PGE(2) was detectable at 10 pM IL-1 beta. IL-1 beta also stimulated MMP-1 and -3 protein secretion. Induction of MMP-1 and -3 was detectable at 10 pM IL-1 beta. Post-injury or after some other inciting events, exogenous IL-1 beta released upon bleeding or as leakage of local capillaries may drive a proinflammatory response at the connective tissue cell level. The resulting induction of COX2, MMP-1 and -3 may underscore a potential for nonlymphocyte-mediated cytokine production of MMPs that causes matrix destruction and a loss of tendon biomechanical properties. Endogenous IL-1 beta might contribute to the process through a positive feedback loop by stimulating expression and accumulation of MMPs in the tendon matrix.

Asunto(s)

Interleucina-1/biosíntesis , Interleucina-1/farmacología , Interleucina-6/biosíntesis , Isoenzimas/biosíntesis , Metaloendopeptidasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Tendones/efectos de los fármacos , Proteínas ADAM , Proteína ADAMTS4 , Adulto , Anciano , Células Cultivadas , Ciclooxigenasa 2 , Cartilla de ADN/química , Dinoprostona/metabolismo , Relación Dosis-Respuesta a Droga , Inducción Enzimática , Retroalimentación Fisiológica/fisiología , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Humanos , Interleucina-1/genética , Interleucina-6/genética , Isoenzimas/genética , Masculino , Proteínas de la Membrana , Metaloendopeptidasas/genética , Persona de Mediana Edad , Procolágeno N-Endopeptidasa , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tendones/citología , Tendones/enzimología

RESUMEN

The objective of this work was to assess the response of tendon to chronic repetitive loading. Controlled muscle stimulation was used to load the rabbit Achilles tendon at a frequency of 1.25 Hz for two hours per day, three days per week for a period of 11 weeks. Average peak tendon force was 26 N during the protocol. The loading protocol did not modify the gross morphology of the tissue, nor its water content or cellularity. Increases in mRNA expression of collagen Type III and MMPs were observed, but no signs of injury were detected by histologic examination of tendon and paratenon structures. The lack of a detectable injury response suggests that the tendons were not loaded beyond their capacity for repair. Factors additional to mechanical loading such as aging, illness or stress may be necessary to produce pathology.

Asunto(s)

Tendón Calcáneo/metabolismo , Tendón Calcáneo/patología , Animales , Colágeno/genética , Femenino , Factor II del Crecimiento Similar a la Insulina/genética , Interleucina-1/genética , Metaloproteinasa 3 de la Matriz/genética , ARN Mensajero/metabolismo , Conejos , Estrés Mecánico , Factor de Necrosis Tumoral alfa/genética

RESUMEN

To better characterize the enzymatic activities required for human papillomavirus (HPV) DNA replication, the E1 helicases of HPV types 6 and 11 were produced using a baculovirus expression system. The purified wild type proteins and a version of HPV11 E1 lacking the N-terminal 71 amino acids, which was better expressed, were found to be hexameric over a wide range of concentrations and to have helicase and ATPase activities with relatively low values for K(m)(ATP) of 12 microm for HPV6 E1 and 6 microm for HPV11 E1. Interestingly, the value of K(m)(ATP) was increased 7-fold in the presence of the E2 transactivation domain. In turn, ATP was found to perturb the co-operative binding of E1 and E2 to DNA. Mutant and truncated versions of in vitro translated E1 were used to identify a minimal ATPase domain composed of the C-terminal 297 amino acids. This fragment was expressed, purified, and found to be fully active in ATP hydrolysis, single-stranded DNA binding, and unwinding assays, despite lacking the minimal origin-binding domain.

Asunto(s)

Adenosina Trifosfato/metabolismo , ADN Helicasas/metabolismo , Papillomaviridae/enzimología , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Biopolímeros , Catálisis , ADN Helicasas/química , Cartilla de ADN , Datos de Secuencia Molecular , Papillomaviridae/aislamiento & purificación , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato

Asunto(s)

Derecho Penal/métodos , Enfermería de Urgencia/métodos , Medicina Legal/métodos , Examen Físico/métodos , Violación/diagnóstico , California , Vestuario , Colorantes , Dermatoglifia del ADN , Documentación , Femenino , Control de Formularios y Registros , Humanos , Relaciones Interpersonales , Registros Médicos , Evaluación en Enfermería/métodos , Examen Físico/enfermería , Violación/psicología

RESUMEN

Transcription by RNA polymerase II is accompanied by cyclic phosphorylation and dephosphorylation of the carboxy-terminal heptapeptide repeat domain (CTD) of its largest subunit. We have used deletion and point mutations in Fcp1p, a TFIIF-interacting CTD phosphatase, to show that the integrity of its BRCT domain, like that of its catalytic domain, is important for cell viability, mRNA synthesis, and CTD dephosphorylation in vivo. Although regions of Fcp1p carboxy terminal to its BRCT domain and at its amino terminus were not essential for viability, deletion of either of these regions affected the phosphorylation state of the CTD. Two portions of this carboxy-terminal region of Fcp1p bound directly to the first cyclin-like repeat in the core domain of the general transcription factor TFIIB, as well as to the RAP74 subunit of TFIIF. These regulatory interactions with Fcp1p involved closely related amino acid sequence motifs in TFIIB and RAP74. Mutating the Fcp1p-binding motif KEFGK in the RAP74 (Tfg1p) subunit of TFIIF to EEFGE led to both synthetic phenotypes in certain fcp1 tfg1 double mutants and a reduced ability of Fcp1p to activate transcription when it is artificially tethered to a promoter. These results suggest strongly that this KEFGK motif in RAP74 mediates its interaction with Fcp1p in vivo.

Asunto(s)

Fosfoproteínas Fosfatasas/metabolismo , Saccharomyces cerevisiae/enzimología , Factores de Transcripción TFII , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Regulación Fúngica de la Expresión Génica , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Espectroscopía de Resonancia Magnética , Metilmetanosulfonato/farmacología , Mutación , Fenotipo , Fosfoproteínas Fosfatasas/genética , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína/genética , ARN Polimerasa II/química , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factor de Transcripción TFIIB , Factores de Transcripción/genética , Activación Transcripcional

RESUMEN

The E1 helicase of papillomavirus is required, in addition to host cell DNA replication factors, during the initiation and elongation phases of viral episome replication. During initiation, the viral E2 protein promotes the assembly of enzymatically active multimeric E1 complexes at the viral origin of DNA replication. In this study we used the two-hybrid system and chemical cross-linking to demonstrate that human papillomavirus type 11 (HPV11) E1 can self-associate in yeast and form hexamers in vitro in a reaction stimulated by single-stranded DNA. Self-association in yeast was most readily detected using constructs spanning the E1 C-terminal domain (amino acids 353 to 649) and was dependent on a minimal E1-E1 interaction region located between amino acids 353 and 431. The E1 C-terminal domain was also able to oligomerize in vitro but, in contrast to wild-type E1, did so efficiently in the absence of single-stranded DNA. Sequences located between amino acids 191 and 353 were necessary for single-stranded DNA to modulate oligomerization of E1 and were also required, together with the rest of the C terminus, for binding of E1 to the origin. Two regions within the C-terminal domain were identified as important for oligomerization: the ATP-binding domain and region A, which is located within the minimal E1-E1 interaction domain and is one of four regions of E1 that is highly conserved with the large T antigens of simian virus 40 and polyomavirus. Amino acid substitutions of highly conserved residues within the ATP-binding domain and region A were identified that reduced the ability of E1 to oligomerize and bind to the origin in vitro and to support transient DNA replication in vivo. These results support the notion that oligomerization of E1 occurs primarily through the C-terminal domain of the protein and is allosterically regulated by DNA and ATP. The bipartite organization of the E1 C-terminal domain is reminiscent of that found in other hexameric proteins and suggests that these proteins may oligomerize by a similar mechanism.

Asunto(s)

ADN Viral/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Papillomaviridae/química , Origen de Réplica , Proteínas Virales/química , Proteínas Virales/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia Conservada , Replicación del ADN , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Papillomaviridae/genética , Papillomaviridae/metabolismo , Estructura Terciaria de Proteína , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/genética

RESUMEN

The HPV E1 and E2 proteins along with cellular factors, are required for replication of the viral genome. In this study we show that in vitro synthesized HPV11 E1 can support DNA replication in a cell-free system and is able to cooperate with E2 to recruit the host polymerase alpha primase to the HPV origin in vitro. Deletion analysis revealed that the N-terminal 166 amino acids of E1, which encompass a nuclear localization signal and a cyclin E-binding motif, are dispensable for E1-dependent DNA replication and for recruitment of pol alpha primase to the origin in vitro. A shorter E1 protein lacking the N-terminal 190 amino acids supported cell-free DNA replication at less than 25% the efficiency of wild-type E1 and was active in the pol alpha primase recruitment assay. An even shorter E1 protein lacking a functional DNA-binding domain due to a truncation of the N-terminal 352 amino acids was inactive in both assays despite the fact that it retains the ability to associate with E2 or pol alpha primase in the absence of ori DNA. We provide additional functional evidence that E1 interacts with pol alpha primase through the p70 subunit of the complex by showing that p70 can be recruited to the HPV origin by E1 and E2 in vitro, that the domain of E1 (amino acids 353-649) that binds to pol alpha primase in vitro is the same as that needed for interaction with p70 in the yeast two-hybrid system, and that exogenously added p70 competes with the interaction between E1 and pol alpha primase and inhibits E1-dependent cell-free DNA replication. On the basis of these results and the observation that pol alpha primase competes with the interaction between E1 and E2 in solution, we propose that these three proteins assemble at the origin in a stepwise process during which E1, following its interaction with E2, must bind to DNA prior to interacting with pol alpha primase.

Asunto(s)

Replicación del ADN , ADN Viral/biosíntesis , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Papillomaviridae/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Replicación Viral , Animales , Unión Competitiva , Línea Celular , Ciclina E/metabolismo , ADN Polimerasa I/química , ADN Polimerasa I/metabolismo , ADN Primasa/química , ADN Primasa/metabolismo , ADN Viral/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/fisiología , Papillomaviridae/química , Papillomaviridae/genética , Papillomaviridae/fisiología , Unión Proteica , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , Origen de Réplica/genética , Eliminación de Secuencia/genética , Transcripción Genética , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/genética

RESUMEN

The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II is phosphorylated soon after transcriptional initiation. We show here that the essential FCP1 gene of S. cerevisiae is linked genetically to RNA polymerase II and encodes a CTD phosphatase essential for dephosphorylation of RNA polymerase II in vivo. Fcp1p contains a phosphatase motif, psi psi psi DXDX(T/V)psi psi, which is novel for eukaryotic protein phosphatases and essential for Fcp1p to function in vivo. This motif is also required for recombinant Fcp1p to dephosphorylate the RNA polymerase II CTD or the artificial substrate p-nitrophenylphosphate in vitro. The effects of fcp1 mutations in global run-on and genome-wide expression studies show that transcription by RNA polymerase II in S. cerevisiae generally requires CTD phosphatase.

Asunto(s)

Fosfoproteínas Fosfatasas/genética , ARN Polimerasa II/genética , Saccharomyces cerevisiae/enzimología , Mutación , Nitrofenoles/metabolismo , Compuestos Organofosforados/metabolismo , Fosforilación , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Temperatura , Transcripción Genética/genética

RESUMEN

OBJECTIVE: Our experiments were designed to test the hypothesis that tendon cells might respond differently to applied strain in vitro than in vivo. DESIGN: We tested cells in whole tendons from exercised chickens and from isolated surface (TSC) and internal tendon (TIF) in vitro that were subjected to mechanical strain. We hypothesized that tendon cells differentially express genes in response to mechanical loading in vivo and in vitro. METHODS: We utilized an in-vivo exercise model in which chickens were run on a treadmill in an acute loading regime for 1 h 45 min with the balance of time at rest to 6 h total time. Gene expression was analyzed by a differential display technique. In addition, isolated avian flexor digitorum profundus TSC and TIF cells were subjected to cyclic stretching at 1 Hz, 5% average elongation for 6 h, +/- PDGF-BB, IGF-I, TGF-beta 1, PTH, estrogen, PGE2, or no drug and/or no load. mRNA was then collected and samples were subjected to differential display analysis. CONCLUSIONS: Load with or without growth factor and hormone treatments induced expression of novel genes as well as some known genes that were novel to tendon cells. We conclude that the study of gene expression in mechanically loaded cells in vivo and in vitro will lead to the discovery of novel and important marker proteins that may yield clues to positive and negative cell strain responses that are protective under one set of conditions and destructive under another.

Asunto(s)

Regulación de la Expresión Génica/fisiología , Tendones/citología , Animales , Northern Blotting , Técnicas de Cultivo de Célula , Pollos , Sustancias de Crecimiento/farmacología , Hormonas/farmacología , Condicionamiento Físico Animal/fisiología , Biosíntesis de Proteínas , ARN Mensajero/genética , Estrés Mecánico , Tendones/efectos de los fármacos , Tendones/metabolismo

RESUMEN

Replication of the genome of human papillomaviruses (HPV) is initiated by the recruitment of the viral E1 helicase to the origin of DNA replication by the viral E2 protein, which binds specifically to the origin. We determined, for HPV type 11 (HPV-11), that the C-terminal 296 amino acids of E1 are sufficient for interaction with the transactivation domain of E2 in the yeast two-hybrid system and in vitro. This region of E1 encompasses the ATP-binding domain. Here we have examined the role of this ATP-binding domain, and of ATP, on E2-dependent binding of E1 to the origin. Several amino acid substitutions in the phosphate-binding loop (P loop), which is implicated in binding the triphosphate moiety of ATP, abolished E2 binding, indicating that the structural integrity of this domain is essential for the interaction. The structural constraints imposed on the E1 P loop may differ between HPV-11 and bovine papillomavirus type 1 (BPV-1), since the P479S substitution that inactivates BPV-1 E1 is tolerated in the HPV-11 enzyme. Other substitutions in the E1 P loop, or in two other conserved motifs of the ATP-binding domain, were tolerated, indicating that ATP binding is not essential for interaction with E2. Nevertheless, ATP-Mg stimulated the E2-dependent binding of E1 to the origin in vitro. This stimulation was maximal at the physiological temperature (37 degrees C) and did not require ATP hydrolysis. In contrast, ATP-Mg did not stimulate the E2-dependent binding to the origin of an E1 protein containing only the C-terminal domain (353 to 649) or that of mutant E1 proteins with alterations in the DNA-binding domain. These results are discussed in light of a model in which the E1 ATP-binding domain is required for formation of the E2-binding surface and can, upon the binding of ATP, facilitate and/or stabilize the interaction of E1 with the origin.

Asunto(s)

Adenosina Trifosfato/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Papillomaviridae/metabolismo , Origen de Réplica , Proteínas Virales/metabolismo , Sustitución de Aminoácidos , Animales , Sitios de Unión , Bovinos , ADN Helicasas/genética , Replicación del ADN , Proteínas de Unión al ADN/genética , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Glutamina/genética , Glutamina/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Magnesio , Papillomaviridae/genética , Papillomaviridae/fisiología , Prolina/genética , Prolina/metabolismo , Saccharomyces cerevisiae , Serina/genética , Serina/metabolismo , Temperatura , Proteínas Virales/genética , Replicación Viral

RESUMEN

This work addresses the symbiotic culture of the arbuscular mycorrhizal (AM) fungus Glomus intraradices with Daucus carota hairy roots transformed by Agrobacterium rhizogenes, in two submerged culture systems: Petri dish and airlift bioreactor. AM fungi play an active role in plant nutrition and protection against plant pathogens. These fungi are obligate biotrophs as they depend on a host plant for their needs in carbohydrates. The effect of the mycorrhizal roots inoculum-to-medium volume ratio on the growth of both symbionts was studied. A critical inoculating condition was observed at approximately 0.6 g dry biomass (DW). L-1 medium, above which root growth was significantly reduced when using a low-salt minimal (M) liquid medium previously developed for hairy root-AM fungi co-culture. Below critical inoculum conditions the maximum specific root growth and specific G. intraradices spore production rates of 0.021 and 0.035 d-1, respectively, were observed for Petri dish cultures. Maximum spore production in the airlift bioreactor was ten times lower than that of Petri dish cultures and obtained with the lowest inoculum assessed (0.13 g DW. L-1 medium) with 1.82 x 10(5) +/- 4.05 x 10(4) (SEM) spores (g DW inoculum)-1 (L medium)-1 in 107 d. This work proposes a second-generation bioprocess for AM fungi propagule production in bioreactors. Copyright 1999 John Wiley & Sons, Inc.