RESUMEN
The vertebrate retina is said to be inverted because the photoreceptors are oriented in the posterior direction and are thus unable to maximize photodetection under conditions of low illumination. The tapetum lucidum is a photoreflective structure located posterior to the photoreceptors in the eyes of some fish and terrestrial animals. The tapetum reflects light forward, giving incident photons a "second chance" to collide with a photoreceptor, substantially enhancing retinal photosensitivity in dim light. Across vertebrates (and arthropods), there are a wide variety of tapeta that vary in structure, chemical composition, and even tissue architecture, indicating repeated convergent evolution. To date, the tapetum has not been observed in any cephalopod, however, which also possess a camera-like eye, but with the retinal photoreceptors oriented in the anterior direction. We therefore hypothesize that the tapetum lucidum is a compensatory adaptation for the suboptimal design of the inverted retina of vertebrates.
Asunto(s)
Coroides , Retina , Animales , Células Fotorreceptoras , Células Fotorreceptoras de Vertebrados , VertebradosRESUMEN
Plant residue is currently an underutilized resource in forensic investigations despite the fact that many crime scenes, as well as suspects and victims, harbor plant-derived residue that could be recovered and analyzed. Notwithstanding the considerable skill of forensic botanists, current methods of species determination could benefit from tools for DNA-based species identification. However, DNA barcoding in plants has been hampered by sequence complications in the plant genome. Following a database search for usable barcodes, broad-spectrum primers were designed and utilized to amplify and sequence the rbcL, trnL-F, and rrn18 genetic loci from a variety of household plants. Once obtained, these DNA sequences were used to design species-targeted primers that could successfully discriminate the source of plant residue from among the 21 species tested.
Asunto(s)
Código de Barras del ADN Taxonómico , ADN de Plantas/genética , Sitios Genéticos , Botánica , Cartilla de ADN , Ciencias Forenses , Genoma de Plastidios/genética , Humanos , Mitocondrias/genética , Reacción en Cadena de la Polimerasa , ARN Ribosómico 18S/genética , Análisis de Secuencia de ADNRESUMEN
Research on the human microbiome, the microbiota that live in, on, and around the human person, has revolutionized our understanding of the complex interactions between microbial life and human health and disease. The microbiome may also provide a valuable tool in forensic death investigations by helping to reveal the postmortem interval (PMI) of a decedent that is discovered after an unknown amount of time since death. Current methods of estimating PMI for cadavers discovered in uncontrolled, unstudied environments have substantial limitations, some of which may be overcome through the use of microbial indicators. In this project, we sampled the microbiomes of decomposing human cadavers, focusing on the skin microbiota found in the nasal and ear canals. We then developed several models of statistical regression to establish an algorithm for predicting the PMI of microbial samples. We found that the complete data set, rather than a curated list of indicator species, was preferred for training the regressor. We further found that genus and family, rather than species, are the most informative taxonomic levels. Finally, we developed a k-nearest- neighbor regressor, tuned with the entire data set from all nasal and ear samples, that predicts the PMI of unknown samples with an average error of ±55 accumulated degree days (ADD). This study outlines a machine learning approach for the use of necrobiome data in the prediction of the PMI and thereby provides a successful proof-of- concept that skin microbiota is a promising tool in forensic death investigations.
Asunto(s)
Microbiota , Piel/microbiología , Bacterias/genética , Bacterias/aislamiento & purificación , Cadáver , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Patologia Forense , Humanos , Aprendizaje Automático , Cambios Post Mortem , Factores de TiempoRESUMEN
The CCN family is composed of six cysteine-rich, modular, and conserved proteins whose functions span a variety of tissues and include cell proliferation, adhesion, angiogenesis, and wound healing. Roles for the CCN proteins throughout the entire body including the skin, kidney, brain, blood vessels, hematopoietic compartment and others, are continuously being elucidated. Likewise, an understanding of the regulation of this important gene family is constantly becoming clearer, through identification of transcription factors that directly activate, repress, or respond to upstream cell signaling pathways, as well as other forms of gene expression control. Vitamin D (1,25-dihydroxyvitamin D3 or calcitriol), a vitamin essential for numerous biological processes, acts as a potent gene expression modulator. The regulation of the CCN gene family members by calcitriol has been described in many contexts. Here, we provide a concise and thorough overview of what is known about calcitriol and its regulation of the CCN genes, and argue that its regulation is of physiological importance in a wide breadth of tissues in which CCN genes function. In addition, we highlight the effects of vitamin D on CCN gene expression in the setting of two common pathologic conditions, fibrosis and cancer, and propose that the therapeutic effects of vitamin D3 described in these disease states may in part be attributable to CCN gene modulation. As vitamin D is perfectly safe in a wide range of doses and already showing promise as an adjuvant therapeutic agent, a deeper understanding of its control of CCN gene expression may have profound implications in clinical management of disease.
Asunto(s)
Proteínas CCN de Señalización Intercelular/genética , Regulación de la Expresión Génica/efectos de los fármacos , Terapia Neoadyuvante , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Vitamina D/uso terapéutico , Animales , Fibrosis , Humanos , Modelos Biológicos , Vitamina D/farmacologíaRESUMEN
Connective Tissue Growth Factor (CCN2/CTGF) and Nephroblastoma Overexpressed (CCN3/NOV) execute key functions within the hematopoietic compartment. Both are abundant in the bone marrow stroma, which is a niche for hematopoiesis and supports marrow function. Roles for 1,25-dihydroxyvitamin D3 (calcitriol) and all-trans retinoic acid in the bone marrow have also been elucidated. Interestingly, some of the annotated roles of these vitamins overlap with established functions of CCN2 and CCN3. Yet, no factor has been identified that unifies these observations. In this study, we report the regulation of the CTGF and NOV genes by Myeloid Zinc Finger-1 (MZF-1), a hematopoietic transcription factor. We show the interaction of MZF-1 with the CTGF and NOV promoters in several cell types. Up-regulation of MZF-1 via calcitriol and vitamin A induces expression of CTGF and NOV, implicating a role for these vitamins in the functions of these two genes. Lastly, knockdown of MZF1 reduces levels of CTGF and NOV. Collectively, our results argue that MZF-1 regulates the CTGF and NOV genes in the hematopoietic compartment, and may be involved in their respective functions in the stroma.
Asunto(s)
Factor de Crecimiento del Tejido Conjuntivo/biosíntesis , Hematopoyesis/genética , Factores de Transcripción de Tipo Kruppel/genética , Proteína Hiperexpresada del Nefroblastoma/biosíntesis , Médula Ósea/metabolismo , Calcitriol/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/genética , Regulación de la Expresión Génica , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteína Hiperexpresada del Nefroblastoma/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Tretinoina/metabolismoRESUMEN
Recently, new tissue-specific functions for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been discovered, aside from its archetypal function in glycolysis. This casts doubt on the legitimacy of using GAPDH as a normalization control for gene expression analysis. We report the binding of the myeloid zinc finger-1 (MZF-1) transcription factor to the human GAPDH promoter. Furthermore, we show that up-regulation of MZF-1 by 1,25-dihydroxyvitamin D3 (calcitriol) induces GAPDH in HS-5 stromal fibroblasts, while knockdown of MZF1 by shRNA leads to a concomitant reduction in GAPDH expression. This argues that MZF-1 regulates GAPDH, indicating a role for GAPDH in calcitriol-mediated signaling.
Asunto(s)
Calcitriol/farmacología , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Secuencia de Bases , Calcitriol/metabolismo , Línea Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Interferente Pequeño , Transducción de Señal , Tretinoina/metabolismo , Tretinoina/farmacologíaRESUMEN
Federal workplace drug testing was initiated during the late 1980s. Since then, numerous methods have been employed to subvert these drug tests, adulteration of urine samples being the most common. A wide variety of adulterants has been reported to date along with suitable methods of their detection. Recently, websites have claimed that zinc sulfate can be an effective adulterant to bypass drug testing. Herein, these claims are investigated using standard drug detection kits and urine samples adulterated with zinc. Drug-free urine samples were fortified with different amounts methamphetamines and benzoylecgonine, to which zinc sulfate was added to study its effect. Urine samples from acute marijuana smokers were also obtained in order to study the effects of zinc supplements on THC drug testing. All urine drug testing was performed using ELISA detection kits manufactured by Immunalysis. Both zinc sulfate and zinc supplements are effective in interfering with the detection of all three drugs by Immunalysis drug detection kits. Also, no suitable method could be established to detect zinc in urine samples. Zinc can be an effective adulterant in urine for some illicit drugs that are commonly screened under routine drug testing.
Asunto(s)
Cocaína/orina , Dronabinol/orina , Drogas Ilícitas/orina , Metanfetamina/orina , Detección de Abuso de Sustancias/métodos , Zinc/química , Cocaína/química , Dronabinol/química , Interacciones Farmacológicas , Ensayo de Inmunoadsorción Enzimática , Humanos , Drogas Ilícitas/química , Metanfetamina/químicaRESUMEN
BACKGROUND: Nevoid basal cell carcinoma syndrome (NBCCS) is a rare genetic disease which causes a variety of dermatological lesions, especially basal cell carcinomas (BCCs), often on the face, neck, and head. METHODS: Persons attending a national NBCCS support group meeting were asked to participate in survey-based assessments of quality of life and depressive symptoms. Inclusion criteria required a self-reported NBCCS diagnosis, voluntary agreement to participate, and age over 18 years. Exclusion criteria included cognitive impairment. Skin-related quality of life was assessed with Skindex-29, completed by 32 participants. Depressive symptomatology was determined with the Center for Epidemiological Studies Depression Scale (CES-D), completed by 18 participants. Sociodemographic, medical, and social variables were also analyzed. RESULTS: Median Skindex-29 scores for the emotions, symptoms, and functioning scales were 42.50, 32.14, and 28.13, respectively (means: 41.17, 37.05, and 29.30, respectively). These scores were slightly higher than those observed in patients with neurofibromatosis type 1, a similar genetic disease with skin symptoms. The CES-D scores (median = 15.50, mean = 17.50) suggested that 50% of participants had significant depressive symptomatology. Variables showing moderate associations with the scores included diet, number of affected family members, and treatment type. Interestingly, the number of BCCs had no effect. CONCLUSIONS: Nevoid basal cell carcinoma syndrome impacts the quality of life of its subjects in a similar manner to other genodermatoses. Depressive symptoms are particularly prevalent. Several demographic, medical, and social characteristics affect these outcomes. Thus, the psychological impact of this disorder should be evaluated in the course of considering the care of persons with NBCCS.
Asunto(s)
Síndrome del Nevo Basocelular/epidemiología , Síndrome del Nevo Basocelular/psicología , Depresión/epidemiología , Depresión/psicología , Calidad de Vida , Adulto , Trastornos del Conocimiento/epidemiología , Trastornos del Conocimiento/psicología , Salud de la Familia , Femenino , Encuestas de Atención de la Salud , Humanos , Masculino , Persona de Mediana Edad , Prevalencia , Índice de Severidad de la Enfermedad , Conducta SocialRESUMEN
Three approaches to molecular phylogenetics are demonstrated to biology students as they explore molecular data from Homo sapiens and four related primates. By analyzing DNA sequences, protein sequences, and chromosomal maps, students are repeatedly challenged to develop hypotheses regarding the ancestry of the five species. Although these exercises were designed to supplement and enhance classroom instruction on phylogeny, cladistics, and systematics in the context of a postsecondary majors-level introductory biology course, the activities themselves require very little prior student exposure to these topics. Thus, they are well suited for students in a wide range of educational levels, including a biology class at the secondary level. In implementing this exercise, we have observed measurable gains, both in student comprehension of molecular phylogeny and in their acceptance of modern evolutionary theory. By engaging students in modern phylogenetic activities, these students better understood how biologists are currently using molecular data to develop a more complete picture of the shared ancestry of all living things.
Asunto(s)
Biología/educación , Filogenia , Enseñanza/métodos , Evolución Biológica , Curriculum , Humanos , EstudiantesRESUMEN
Virus infection of macrophages stimulates the expression of proinflammatory and antiviral genes interleukin-1 (IL-1), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In this study, we show that phosphatidylinositol 3-kinase (PI3K) is required for the inflammatory response of macrophages to virus infection. When macrophages are infected with encephalomyocarditis virus (EMCV) there is a rapid and transient activation of PI3K and phosphorylation of its downstream target Akt. Inhibitors of PI3K attenuate EMCV- and double-stranded RNA-induced iNOS, COX-2 and IL-1 beta expression in RAW264.7 cells and mouse peritoneal macrophages. The attenuation of inflammatory gene expression in response to PI3K inhibition correlates with the induction of macrophage apoptosis. The morphology of macrophages shifts from activation in response to EMCV infection to apoptosis in the cells treated with PI3K inhibitors and EMCV. These morphological changes are accompanied by the activation of caspase-3. These findings suggest that PI3K plays a central role in the regulation of macrophage responses to EMCV infection. When PI3K is activated, it participates in the regulation of inflammatory gene expression; however, if PI3K is inhibited macrophages are unable to mount an inflammatory antiviral response and die by apoptosis.
Asunto(s)
Infecciones por Cardiovirus/inmunología , Virus de la Encefalomiocarditis/inmunología , Activación de Macrófagos/inmunología , Fosfatidilinositol 3-Quinasas/inmunología , ARN Bicatenario/inmunología , Animales , Infecciones por Cardiovirus/enzimología , Caspasa 3/inmunología , Línea Celular , Ciclooxigenasa 2/inmunología , Virus de la Encefalomiocarditis/enzimología , Interleucina-1beta/inmunología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/virología , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/inmunología , Proteínas Proto-Oncogénicas c-akt/inmunologíaRESUMEN
Intracellular levels of zinc have shown a strong inverse correlation to growth and malignancy of prostate cancer. To date, studies of zinc supplementation in prostate cancer have been equivocal and have not accounted for bioavailability of zinc. Therefore, we hypothesized that direct intra-tumoral injection of zinc could impact prostate cancer growth. In this study, we evaluated the cytotoxic properties of the pH neutral salt zinc acetate on the prostate cancer cell lines PC3, DU145 and LNCaP. Zinc acetate killed prostate cancer cell lines in vitro, independent of androgen sensitivity, in a dose-dependent manner in a range between 200 and 600 microM. Cell death occurred rapidly with 50% cell death by six hours and maximal cell death by 18 hours. We next established a xenograft model of prostate cancer and tested an experimental treatment protocol of direct intra-tumoral injection of zinc acetate. We found that zinc treatments halted the growth of the prostate cancer tumors and substantially extended the survival of the animals, whilst causing no detectable cytoxicity to other tissues. Thus, our studies form a solid proof-of-concept that direct intra-tumoral injection of zinc acetate could be a safe and effective treatment strategy for prostate cancer.
Asunto(s)
Neoplasias Hormono-Dependientes/tratamiento farmacológico , Neoplasias Hormono-Dependientes/patología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Acetato de Zinc/administración & dosificación , Animales , Proliferación Celular , Humanos , Inyecciones Intralesiones , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
alpha-thrombin is a potent mitogen for fibroblasts and initiates a rapid signal transduction pathway leading to the activation of Ras and the stimulation of cell cycle progression. While the signaling events downstream of Ras have been studied in significant detail and appear well conserved across many species and cell types, the precise molecular events beginning with thrombin receptor activation and leading to the activation of Ras are not as well understood. In this study, we examined the immediate events in the rapid response to alpha-thrombin, in a single cell type, and found that an unexpected degree of specificity exists in the pathway linking alpha-thrombin to Ras activation. Specifically, although IIC9 cells express all three Ras isoforms, only N-Ras is rapidly activated by alpha-thrombin. Further, although several Galpha subunits associate with PAR1 and are released following stimulation, only Galpha(i2) couples to the rapid activation of Ras. Similarly, although IIC9 cells express many Gbeta and Ggamma subunits, only a subset associates with Galpha(i2), and of those, only a single Gbetagamma dimer, Gbeta(1)gamma(5), participates in the rapid activation of N-Ras. We then hypothesized that co-localization into membrane microdomains called lipid rafts, or caveolae, is at least partially responsible for this degree of specificity. Accordingly, we found that all components localize to lipid rafts and that disruption of caveolae abolishes the rapid activation of N-Ras by alpha-thrombin. We thus report the molecular elucidation of an extremely specific and rapid signal transduction pathway linking alpha-thrombin stimulation to the activation of Ras.
Asunto(s)
Fibroblastos/metabolismo , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Microdominios de Membrana/metabolismo , Trombina/farmacología , Proteínas ras/metabolismo , Animales , Células CHO , Colesterol/metabolismo , Cricetinae , Cricetulus , Fibroblastos/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Humanos , Microdominios de Membrana/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Mdm2 is the most important regulator of p53, the chief responder of various modes of cellular stress, including DNA damage and oncogenic insult. Many alternative and aberrant splice products of the Mdm2 gene product have been described, but relatively little is known about the origin, function, or consequence of these variants. Recently, a novel splice form of mdm2 was discovered which incorporates 108bp of intronic sequence into the mature Mdm2 mRNA. The additional sequence encodes in-frame stop codons, resulting in severely truncated mdm2 protein. Most intriguingly, this alternative splice form, termed Mdm2(+108), is acutely induced by the chemotherapeutic agents Adriamycin and Actinomycin D, but not other DNA damaging agents. The effect of Mdm2(+108) induction is a rapid and robust accumulation of p53, arguing that the function of this alternative splice event is to engage the p53 tumor surveillance pathway and restrain proliferation of cells damaged with these potently genotoxic compounds.
Asunto(s)
Daño del ADN , Proteínas Proto-Oncogénicas c-mdm2/genética , Empalme del ARN , Proteína p53 Supresora de Tumor/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Apoptosis , Secuencia de Bases , Línea Celular Tumoral , Dactinomicina/farmacología , Doxorrubicina/farmacología , Humanos , Intrones , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The retinoblastoma protein pRb plays a pivotal role in G(1)- to S-phase cell cycle progression and is among the most frequently mutated gene products in human cancer. Although much focus has been placed on understanding how the A/B pocket and COOH-terminal domain of pRb cooperate to relieve transcriptional repression of E2F-responsive genes, comparatively little emphasis has been placed on the function of the NH(2)-terminal region of pRb and the interaction of the multiple domains of pRb in the full-length context. Using "reverse mutational analysis" of Rb(DeltaCDK) (a dominantly active repressive allele of Rb), we have previously shown that restoration of Thr-373 is sufficient to render Rb(DeltaCDK) sensitive to inactivation via cyclin-CDK phosphorylation. This suggests that the NH(2)-terminal region plays a more critical role in pRb regulation than previously thought. In the present study, we have expanded this analysis to include additional residues in the NH(2)-terminal region of pRb and further establish that the mechanism of pRb inactivation by Thr-373 phosphorylation is through the dissociation of E2F. Most surprisingly, we further have found that removal of the COOH-terminal domain of either RbDeltaCDK(+T373) or wild-type pRb yields a functional allele that cannot be inactivated by phosphorylation and is repressive of E2F activation and S-phase entry. Our data demonstrate a novel function for the NH(2)-terminal domain of pRb and the necessity for cooperation of multiple domains for proper pRb regulation.
Asunto(s)
Proliferación Celular , Factores de Transcripción E2F/metabolismo , Proteína de Retinoblastoma/metabolismo , Fase S , Sitios de Unión , Línea Celular Tumoral , Ciclina E/genética , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Factores de Transcripción E2F/genética , Factor de Transcripción E2F1/metabolismo , Genes Reporteros , Humanos , Mutación , Fosforilación , Regiones Promotoras Genéticas , Unión Proteica , Estructura Terciaria de Proteína , Proteína de Retinoblastoma/genética , Treonina , Transcripción Genética , TransfecciónRESUMEN
Progression through the mammalian cell division cycle is regulated by the sequential activation of cyclin-dependent kinases, CDKs, at specific phases of the cell cycle. Cyclin A-CDK2 and cyclin A-CDK1 phosphorylate nuclear substrates during S and G(2) phases, respectfully. However, the DNA helicase complex, MCM2-7, is loaded onto the origin of replications in G(1), prior to the normally scheduled induction of cyclin A. It has previously been shown that cyclin A-CDKs phosphorylate MCM2 and MCM4 in vitro, thereby diminishing helicase activity. Thus, in this study we hypothesize that, in vivo, cyclin A-CDK activity during G(1) would result in an inhibition of progression into the S phase. To test this, we establish an in vivo method of inducing cyclin A-CDK activity in G(1) phase and observe that activation of cyclin A-CDK, but not cyclin E-CDK complexes, inhibit DNA synthesis without affecting other G(1) events such as cyclin D synthesis, E2F activation and cdc6 loading onto chromatin. We further report that the mechanism of this S phase inhibition occurs, at least in part, through impaired loading of MCM onto chromatin, presumably due to decreased levels of cdt1 and premature phosphorylation of MCM by cyclin A-CDK. In addition to providing in vivo confirmation of in vitro predictions regarding cyclin A-CDK phosphorylation of the MCM complex, our results provide insight into the cellular effects of unscheduled cyclin A-CDK activity in mammalian cells.
Asunto(s)
Cromatina/metabolismo , Ciclina A/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/biosíntesis , Fase G1 , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Señales de Localización Nuclear , Transporte de Proteínas , Fase SRESUMEN
Mdm2, as the most important negative regulator of p53, plays an important homeostatic role in regulating cell division and the cellular response to DNA damage, oncogenic insult and other forms of cellular stress. We discovered that the DNA damaging agent adriamycin (doxorubicin) induces a novel aberrantly spliced Mdm2 mRNA which incorporates 108 bp of intronic sequence not normally found in the Mdm2 mature mRNA. Accordingly, we term this Mdm2 splice variant Mdm2(+108). Importantly, this insertion introduces in-frame nonsense codons, thus encoding a profoundly truncated mdm2 protein lacking the C-terminal RING finger domain and the E3 ubiquitin ligase activity. A wide range of pharmacological testing revealed that Mdm2(+108) is induced, in mouse and rat cells, in specific response to Adriamycin and actinomycin D, but not other modes of DNA damage. Meanwhile, antibodies against the N-terminal region of mdm2 reveal a marked reduction in detectable mdm2 protein upon Adriamycin treatment, while p53 accumulates to strikingly high levels. We thus conclude that this alternative spicing of Mdm2 may be an important mechanism to facilitate massive accumulation of p53 in response to genotoxic agents.
Asunto(s)
Empalme Alternativo/efectos de los fármacos , Daño del ADN , Dactinomicina/farmacología , Doxorrubicina/farmacología , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Secuencia de Bases , Western Blotting , Células Cultivadas , Ratones , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Using genome-wide approaches, we have elucidated the regulatory circuitry governed by the XBP1 transcription factor, a key effector of the mammalian unfolded protein response (UPR), in skeletal muscle and secretory cells. We identified a core group of genes involved in constitutive maintenance of ER function in all cell types and tissue- and condition-specific targets. In addition, we identified a cadre of unexpected targets that link XBP1 to neurodegenerative and myodegenerative diseases, as well as to DNA damage and repair pathways. Remarkably, we found that XBP1 regulates functionally distinct targets through different sequence motifs. Further, we identified Mist1, a critical regulator of differentiation, as an important target of XBP1, providing an explanation for developmental defects associated with XBP1 loss of function. Our results provide a detailed picture of the regulatory roadmap governed by XBP1 in distinct cell types as well as insight into unexplored functions of XBP1.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Redes Reguladoras de Genes/genética , Proteínas Nucleares/metabolismo , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sitios de Unión , Inmunoprecipitación de Cromatina , Biología Computacional , Proteínas de Unión al ADN/genética , Retículo Endoplásmico/metabolismo , Metabolismo Energético , Regulación de la Expresión Génica , Genoma/genética , Ratones , Datos de Secuencia Molecular , Desarrollo de Músculos , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Enfermedades Musculares/genética , Enfermedades Neurodegenerativas/genética , Proteínas Nucleares/genética , Unión Proteica , Pliegue de Proteína , Ratas , Factores de Transcripción del Factor Regulador X , Reproducibilidad de los Resultados , Especificidad por Sustrato , Factores de Transcripción , Proteína 1 de Unión a la X-BoxRESUMEN
Previous studies in our laboratory have shown that constitutive cyclin E expression can alleviate the requirement for cyclin D-CDK activity in the inactivation of the retinoblastoma protein (pRb). Rb(DeltaCDK), a mutant construct of pRb with 15 of the 16 CDK phosphorylation sites mutated to alanine represses activation of E2F by mitogen, despite cyclin E overexpression. However, restoration of the four cyclin E-CDK2 phosphorylation sites to Rb(DeltaCDK) renders this construct sensitive to inactivation by CDK phosphorylation. In the present study, we engage a "reverse mutational analysis" by restoring cyclin E-CDK2 phosphorylation sites to Rb(DeltaCDK) individually and in combinations in an attempt to discover phosphorylation sites on Rb that are critical for inactivation. Surprisingly, we report that, in both rodent and human cells, restoration of threonine-373 to Rb(DeltaCDK), alone or in combination with other phospho-restorations, results in a loss of the constitutively repressive effect of this construct on E2F activation. Further, induction of endogenous cyclin A protein is blocked by Rb(DeltaCDK), but not by mutants of Rb(DeltaCDK) containing a restored threonine-373. Finally, while S phase entry is blocked by expression of Rb(DeltaCDK), restoration of threonine-373 largely attenuates this effect. These findings reveal that phosphorylation of threonine-373 by CDK2-cyclin E represent a potentially crucial event in the inactivation of the pRb protein.
Asunto(s)
Fosfotreonina/metabolismo , Proteína de Retinoblastoma/metabolismo , Animales , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Ciclina A/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Análisis Mutacional de ADN , Factores de Transcripción E2F/metabolismo , Humanos , Proteínas Mutantes/metabolismo , Mutación/genética , Fosforilación , Fase SRESUMEN
UNLABELLED: In a new study, a systematic screen for genes necessary for normal cell cycle progression has been completed in Drosophila S2 cells. THE RESULTS: some familiar faces and some new faces add to our appreciation of the staggering complexity of cellular growth and proliferation. The apparent utility of genome-wide RNA interference screens is validated once again.