RESUMO
Cadmium (Cd) accumulation in agricultural systems has caused global environmental and health concerns. Application of phosphate fertiliser to sustain plant production unintentionally accumulated Cd in agricultural soils over time. Rapid and cost-effective Cd monitoring in these soils will help to inform Cd management practices. Compared to total Cd analysis, examining chemical fractions by sequential extraction methods can provide information on the origin, availability, and mobility of soil Cd, and to assess the potential plant Cd uptake. A total of 87 air-dried topsoil (0-15 cm) samples from pastoral farms with a history of long-term application of phosphate fertiliser were analysed using wet chemistry methods for total Cd and Cd forms in exchangeable, acid soluble, metal oxides bound, organic matter bound, and residual fractions. The data acquired using three proximal sensing techniques, visible-near-infrared (vis-NIR), mid-infrared (MIR), and portable X-ray fluorescence (pXRF) spectroscopy were used as input for partial least squares regression to develop models predicting total Cd and Cd fractions. The average total Cd concentration was 0.58 mg Cd/kg soil. For total Cd, cross-validation (cv) results of models using individual vis-NIR, MIR, and pXRF data performed with normalised root mean squared error (nRMSEcv) of 26%, 30%, and 31% and concordance correlation coefficient (CCCcv) of 0.85, 0.77, and 0.75, respectively. For exchangeable Cd, model using MIR data performed with nRMSEcv of 40% and CCCcv of 0.57. For acid soluble and organic matter bound Cd, models using vis-NIR data performed with nRMSEcv of 11% and 33% and CCCcv of 0.97 and 0.84, respectively. Reflectance spectroscopy techniques could potentially be applied as complementary tools to estimate total Cd and plant available and potentially available Cd fractions for effective implementation of Cd monitoring programmes.
Assuntos
Agricultura , Cádmio , Monitoramento Ambiental , Poluentes do Solo , Solo , Cádmio/análise , Poluentes do Solo/análise , Monitoramento Ambiental/métodos , Solo/química , Espectrometria por Raios X/métodosRESUMO
In the early days of the Covid-19 pandemic, images of the virus molecule and 'flatten-the-curve' line charts were inescapable. There is now a vast visual repertoire of vaccines, people wearing face masks in everyday settings, choropleth maps and both bar and line charts. These 'generic visuals' circulate widely in the news media and, however unremarkable, play an important role in representing the crisis in particular ways. We argue that these generic visuals promote banal nationalism, localism and cosmopolitanism in the face of the crisis, and that they do so through the symbolic reiteration of a range of visual resources across news stories. Through an analysis of three major news outlets in the UK, we examine how generic visuals of Covid-19 contribute to these banal visions and versions of belonging and, in doing so, also to foregrounding the role of the state in responding to the crisis.
RESUMO
Arsenic (As) contaminated water is used in South Asian countries to irrigate food crops, but the subsequent uptake of As by vegetables and associated human health risk is poorly understood. We used a pot trial to determine the As uptake of four vegetable species (carrot, radish, spinach and tomato) with As irrigation levels ranging from 50 to 1000 µg L(-1) and two irrigation techniques, non-flooded (70% field capacity for all studied vegetables), and flooded (110% field capacity initially followed by aerobic till next irrigation) for carrot and spinach only. Only the 1000 µg As L(-1) treatment showed a significant increase of As concentration in the vegetables over all other treatments (P < 0.05). The distribution of As in vegetable tissues was species dependent; As was mainly found in the roots of tomato and spinach, but accumulated in the leaves and skin of root crops. There was a higher concentration of As in the vegetables grown under flood irrigation relative to non-flood irrigation. The trend of As bioaccumulation was spinach > tomato > radish > carrot. The As concentration in spinach leaves exceeded the Chinese maximum permissible concentration for inorganic As (0.05 µg g(-1) fresh weight) by a factor of 1.6 to 6.4 times. No other vegetables recorded an As concentration that exceeded this threshold. The USEPA parameters hazard quotient and cancer risk were calculated for adults and adolescents. A hazard quotient value greater than 1 and a cancer risk value above the highest target value of 10(-4) confirms potential risk to humans from ingestion of spinach leaves. In our study, spinach presents a direct risk to human health where flood irrigated with water containing an arsenic concentration greater than 50 µg As L(-1).
Assuntos
Irrigação Agrícola , Arsênio/toxicidade , Água Doce/análise , Verduras/efeitos dos fármacos , Verduras/crescimento & desenvolvimento , Poluentes Químicos da Água/toxicidade , Irrigação Agrícola/métodos , Irrigação Agrícola/normas , Contaminação de Alimentos/análise , Contaminação de Alimentos/prevenção & controle , Humanos , Nova Zelândia , Saúde Pública , Medição de Risco/métodosRESUMO
Wildlife reservoir hosts of bovine tuberculosis (bTB) include Eurasian badgers (Meles meles) and brushtail possum (Trichosurus vulpecula) in the UK and New Zealand, respectively. Similar species warrant further investigation in the northern lower peninsula of Michigan, USA due to the continued presence of bTB on cattle farms. Most research in Michigan, USA has focused on interactions between white-tailed deer (Odocoileus virginianus) and cattle (Bos taurus) for the transmission of the infectious agent of bTB, Mycobacterium bovis, due to high deer densities and feeding practices. However, limited data are available on medium-sized mammals such as Virginia opossum (Didelphis virginiana; hereafter referred to as opossum) and their movements and home range in Michigan near cattle farms. We conducted surveillance of medium-sized mammals on previously depopulated cattle farms for presence of M. bovis infections and equipped opossum with Global Positioning System (GPS) technology to assess potential differences in home range between farms inside and outside the bTB core area that has had cattle test positive for M. bovis. On farms inside the bTB core area, prevalence in opossum was comparable [6%, 95% confidence interval (CI) 2.0-11.0] to prevalence in raccoon (Procyon lotor; 4%, 95% CI 1.0-9.0, P=0.439) whereas only a single opossum tested positive for M. bovis on farms outside the bTB core area. The prevalence in opossum occupying farms that had cattle test positive for M. bovis was higher (6.4%) than for opossum occupying farms that never had cattle test positive for M. bovis (0.9%, P=0.01). Mean size of home range for 50% and 95% estimates were similar by sex (P=0.791) both inside or outside the bTB core area (P=0.218). Although surveillance efforts and home range were not assessed on the same farms, opossum use of farms near structures was apparent as was selection for farms over surrounding forested habitats. The use of farms, stored feed, and structures by opossum, their ability to serve as vectors of M. bovis, and their propensity to ingest contaminated sources of M. bovis requires additional research in Michigan, USA.
Assuntos
Didelphis , Reservatórios de Doenças/veterinária , Mycobacterium bovis , Tuberculose/veterinária , Animais , Bovinos , Feminino , Sistemas de Informação Geográfica , Comportamento de Retorno ao Território Vital , Estimativa de Kaplan-Meier , Modelos Logísticos , Masculino , Michigan/epidemiologia , Mycobacterium bovis/isolamento & purificação , Vigilância da População , Prevalência , Análise Espacial , Tuberculose/diagnóstico , Tuberculose/epidemiologia , Tuberculose/transmissão , Tuberculose Bovina/transmissãoRESUMO
Representative adenoviruses from four of the five major virus subgroups have been shown to interact with the 46-kDa coxsackievirus and adenovirus receptor (CAR) that is widely expressed on many human cell types, suggesting that the ability to bind CAR may be a conserved feature of many of the approximately 50 known adenovirus serotypes. Receptor binding is a function of the distal 'knob' domain of the trimeric viral fiber protein. Here we review recent structural characterizations of knob, CAR and knob-CAR complexes, and we discuss how knob architecture may have evolved to accommodate opposing selective pressures to vary antigenic structure while conserving receptor binding specificity. In contrast to the hypervariability of the solvent-exposed surface of knob, the CAR receptor was found to be non-polymorphic.
Assuntos
Adenovírus Humanos/metabolismo , Receptores Virais/metabolismo , Adenovírus Humanos/química , Adenovírus Humanos/genética , Capsídeo/química , Capsídeo/metabolismo , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Vetores Genéticos/genética , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Receptores Virais/química , Receptores Virais/genéticaRESUMO
We investigated the potential of the South African high-biomass Ni hyperaccumulator Berkheya coddii to phytoextract Co and/or Ni from artificial metalliferous media. Plant accumulation of both metals from single-element substrates indicate that the plant/media metal concentration quotient (bioaccumulation coefficient) increases as total metal concentrations increase. Cobalt was readily taken up by B. coddii with and without the presence of Ni. Nickel uptake was, however, inhibited by the presence of an equal concentration of Co. Bioaccumulation coefficients of Ni and Co for the single element substrates (total metal concentration of 1000 micrograms g-1) were 100 and 50, respectively. Cobalt phytotoxicity was observed above a total Co concentration in plant growth media of 20 micrograms g-1. Elevated Co concentrations significantly decreased the biomass production of B. coddii without affecting the bioaccumulation coefficients. The mixed Ni-Co substrate produced bioaccumulation coefficients of 22 for both Ni and Co. Cobalt phytotoxicity in mixed Ni-Co substrate occurred above a total Co concentration of 15 micrograms g-1. When grown in the presence of both Ni and Co, the bioaccumulation coefficients of each metal were reduced, as compared to single-element substrate. This may indicate competition for binding sites in the root zone. The interference relationship between Ni and Co uptake demonstrated by B. coddii suggests a significant limitation to phytoextraction where both metals are present.
Assuntos
Asteraceae/metabolismo , Cobalto/metabolismo , Metais Pesados/metabolismo , Níquel/metabolismo , Poluentes do Solo/metabolismo , Cobalto/análise , Poluição Ambiental/prevenção & controle , Humanos , Metais Pesados/análise , Níquel/análise , Solo/análise , Poluentes do Solo/análiseRESUMO
Group B coxsackieviruses (CVB) utilize the coxsackievirus-adenovirus receptor (CAR) to recognize host cells. CAR is a membrane protein with two Ig-like extracellular domains (D1 and D2), a transmembrane domain and a cytoplasmic domain. The three-dimensional structure of coxsackievirus B3 (CVB3) in complex with full length human CAR and also with the D1D2 fragment of CAR were determined to approximately 22 A resolution using cryo-electron microscopy (cryo-EM). Pairs of transmembrane domains of CAR associate with each other in a detergent cloud that mimics a cellular plasma membrane. This is the first view of a virus-receptor interaction at this resolution that includes the transmembrane and cytoplasmic portion of the receptor. CAR binds with the distal end of domain D1 in the canyon of CVB3, similar to how other receptor molecules bind to entero- and rhinoviruses. The previously described interface of CAR with the adenovirus knob protein utilizes a side surface of D1.
Assuntos
Adenoviridae/metabolismo , Enterovirus Humano B/metabolismo , Receptores Virais/metabolismo , Adenoviridae/química , Células HeLa , Humanos , Microscopia Eletrônica/métodos , Modelos Moleculares , Receptores Virais/química , Ensaio de Placa ViralRESUMO
Using injections of small molecular weight fluorescein dextran amines, combined with activity-dependent uptake of sulforhodamine 101 (SR101), brainstem circuits presumed to be involved in feeding motor output were investigated. As has been shown previously in other studies, projections to the cerebellar nuclei were identified from the cerebellar cortex, the trigeminal motor nucleus, and the vestibular nuclei. Results presented here suggest an additional pathway from the hypoglossal motor nuclei to the cerebellar nucleus as well as an afferent projection from the peripheral hypoglossal nerve to the Purkinje cell layer of the cerebellar cortex. Injections in the cerebellar cortex combined with retrograde labeling of the peripheral hypoglossal nerve demonstrate anatomical convergence at the level of the medial reticular formation. This suggests a possible integrative region for afferent feedback from the hypoglossal nerve and information through the Purkinje cell layer of the cerebellar cortex. The activity-dependent uptake of SR101 additionally suggests a reciprocal, polysynaptic pathway between this same area of the medial reticular formation and the trigeminal motor nuclei. The trigeminal motor neurons innervate the m adductor mandibulae, the primary mouth-closing muscle. The SR101 uptake clearly labeled the ventrolateral hypoglossal nuclei, the medial reticular formation, and the Purkinje cell layer of the cerebellar cortex. Unlike retrograde labeling of the peripheral hypoglossal nerve, stimulating the hypoglossal nerve while SR101 was bath-applied labeled trigeminal motor neurons. This, combined with the dextran labeling, suggests a reciprocal connection between the trigeminal motor nuclei and the cerebellar nuclei, as well as the medulla. Taken together, these data are important for understanding the neurophysiological pathways used to coordinate the proper timing of an extremely rapid, goal-directed movement and may prove useful for elucidating some of the first principles of sensorimotor integration.
Assuntos
Tronco Encefálico/citologia , Cerebelo/citologia , Comportamento Alimentar/fisiologia , Nervo Hipoglosso/citologia , Movimento/fisiologia , Vias Neurais/citologia , Rana pipiens/anatomia & histologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Tronco Encefálico/efeitos dos fármacos , Tronco Encefálico/fisiologia , Córtex Cerebelar/citologia , Córtex Cerebelar/efeitos dos fármacos , Córtex Cerebelar/fisiologia , Núcleos Cerebelares/citologia , Núcleos Cerebelares/efeitos dos fármacos , Núcleos Cerebelares/fisiologia , Cerebelo/efeitos dos fármacos , Cerebelo/fisiologia , Corantes/farmacocinética , Estimulação Elétrica , Corantes Fluorescentes/farmacologia , Nervo Hipoglosso/efeitos dos fármacos , Nervo Hipoglosso/fisiologia , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/fisiologia , Vias Neurais/efeitos dos fármacos , Vias Neurais/fisiologia , Rana pipiens/fisiologia , Formação Reticular/citologia , Formação Reticular/efeitos dos fármacos , Formação Reticular/fisiologia , Rodaminas/farmacocinética , Núcleos do Trigêmeo/citologia , Núcleos do Trigêmeo/efeitos dos fármacos , Núcleos do Trigêmeo/fisiologiaRESUMO
Anderson, C. W., Dunn, J. J., Freimuth, P. I., Galloway, A. M. and Allalunis-Turner, M. J. Frameshift Mutation in PRKDC, the Gene for DNA-PKcs, in the DNA Repair-Defective, Human, Glioma-Derived Cell Line M059J. Radiat. Res. 156, 2-9 (2001). The glioma-derived cell line M059J is hypersensitive to ionizing radiation, lacks DNA-PK activity, and fails to express protein for the catalytic subunit, DNA-PKcs, while a sister cell line, M059K, derived from the same tumor, has normal DNA-PK activity. Both cell lines are near pentaploid and have multiple copies of chromosome 8, the chromosome on which the DNA-PKcs gene, PRKDC, is located. Sequence analysis of PCR-amplified exons revealed the loss in M059J cells of a single "A" nucleotide in exon 32, corresponding to the first nucleotide of codon 1351 (ACC, Thr) of PRKDC. Loss of the "A" nucleotide would terminate the DNA-PKcs reading frame early in exon 33. DNA from M059K cells had only the wild-type sequence. An analysis of sequences surrounding PRKDC exon 32 from 87 unrelated individuals revealed no polymorphic nucleotides except for a triplet repeat near the 3' end of this exon; no individual had a frameshift mutation in exon 32. No other sequence differences in PRKDC between M059J and M059K cells were observed in approximately 15,000 bp of genomic sequence including the sequences of exons 5 through 38 and surrounding intron sequence, suggesting a possible reduction to homozygosity at this locus prior to acquisition of the mutation leading to the M059J cell line.
Assuntos
Reparo do DNA/genética , Proteínas de Ligação a DNA , Mutação da Fase de Leitura/genética , Glioma/enzimologia , Proteínas Serina-Treonina Quinases/genética , Subunidades Proteicas , Domínio Catalítico/genética , Cromossomos Humanos Par 8/genética , Análise Mutacional de DNA , Proteína Quinase Ativada por DNA , Éxons/genética , Humanos , Masculino , Dados de Sequência Molecular , Proteínas Nucleares , Reação em Cadeia da Polimerase , Poliploidia , Tolerância a Radiação/genética , Análise de Sequência de DNA , Células Tumorais CultivadasRESUMO
In unstressed cells, the tumor suppressor protein p53 is present in a latent state and is maintained at low levels through targeted degradation. A variety of genotoxic stresses initiate signaling pathways that transiently stabilize the p53 protein, cause it to accumulate in the nucleus, and activate it as a transcription factor. Activation leads either to growth arrest at the G1/S or G2/M transitions of the cell cycle or to apoptosis. Recent studies point to roles for multiple post-translational modifications in mediating these events in response to genotoxic stresses through several potentially interacting but distinct pathways. The approximately 100 amino-acid N-terminal and approximately 90 amino-acid C-terminal domains are highly modified by post-translational modifications. The N-terminus is heavily phosphorylated while the C-terminus contains phosphorylated, acetylated and sumoylated residues. Antibodies that recognize p53 only when it has been modified at specific sites have been developed, and studies with these reagents show that most known post-translational modifications are induced when cells are exposed to genotoxic stresses. These recent results, coupled with biochemical and genetic studies, suggest that N-terminal phosphorylations are important for stabilizing p53 and are crucial for acetylation of C-terminal sites, which in combination lead to the full p53-mediated response to genotoxic stresses. Modifications to the C-terminus inhibit the ability of this domain to negatively regulate sequence-specific DNA binding; additionally, they modulate the stability, the oligomerization state, the nuclear import/export process and the degree of ubiquitination of p53.
Assuntos
Processamento de Proteína Pós-Traducional , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Animais , Sítios de Ligação , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Biológicos , Fosforilação , Estrutura Terciária de Proteína , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/químicaRESUMO
Ser-15 of human p53 (corresponding to Ser-18 of mouse p53) is phosphorylated by ataxia-telangiectasia mutated (ATM) family kinases in response to ionizing radiation (IR) and UV light. To determine the effects of phosphorylation of endogenous murine p53 at Ser-18 on biological responses to DNA damage, we introduced a missense mutation (Ser-18 to Ala) by homologous recombination into both alleles of the endogenous p53 gene in mouse embryonic stem (ES) cells. Our analyses showed that phosphorylation of murine p53 at Ser-18 in response to IR or UV radiation was required for a full p53-mediated response to these DNA damage-inducing agents. In contrast, phosphorylation of p53 at Ser-18 was not required for ATM-dependent cellular resistance after exposure to IR. Additionally, efficient acetylation of the C terminus of p53 in response to DNA damage did not require phosphorylation of murine p53 at Ser-18.
Assuntos
Dano ao DNA , Serina/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular , Células Cultivadas , Proteínas de Ligação a DNA , Fase G1 , Humanos , Raios Infravermelhos , Camundongos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/química , Proteínas Supressoras de Tumor , Raios UltravioletaRESUMO
The protein coding segment of the TP53 genes from the glioma-derived cell lines M059J and M059K was sequenced. The sequences from both cell lines were identical over 5039 bp, including the gene segment containing exons 2 through 9, exon 10, and the proximal segment of exon 11. In both cells, the first nucleotide of codon 286 (GAA, Glu) is changed to an A (AAA, Lys). Comparison with the same TP53 segment from the A549 human lung carcinoma cell line revealed several differences in intron sequence.
Assuntos
Substituição de Aminoácidos , Neoplasias Encefálicas/genética , Éxons/genética , Genes p53 , Glioma/genética , Mutação Puntual , Neoplasias Encefálicas/patologia , Carcinoma/genética , Carcinoma/patologia , Códon/genética , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Ciclinas/genética , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Glioma/patologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Células Tumorais CultivadasRESUMO
p53-mediated transcription activity is essential for cell cycle arrest, but its importance for apoptosis remains controversial. To address this question, we employed homologous recombination and LoxP/Cre-mediated deletion to produce mutant murine embryonic stem (ES) cells that express p53 with Gln and Ser in place of Leu25 and Trp26, respectively. p53(Gln25Ser26) was stable but did not accumulate after DNA damage; the expression of p21/Waf1 and PERP was not induced, and p53-dependent repression of MAP4 expression was abolished. Therefore, p53(Gln25Ser26) is completely deficient in transcriptional activation and repression activities. After DNA damage by UV radiation, p53(Gln25Ser26) was phosphorylated at Ser18 but was not acetylated at C-terminal sites, and its DNA binding activity did not increase, further supporting a role for p53 acetylation in the activation of sequence-specific DNA binding activity. Most importantly, p53(Gln25Ser26) mouse thymocytes and ES cells, like p53(-/-) cells, did not undergo DNA damage-induced apoptosis. We conclude that the transcriptional activities of p53 are required for p53-dependent apoptosis.
Assuntos
Apoptose , Dano ao DNA , Genes p53/genética , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Animais , Anexina A5/metabolismo , Sítios de Ligação , Northern Blotting , Western Blotting , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Relação Dose-Resposta à Radiação , Embrião de Mamíferos/metabolismo , Citometria de Fluxo , Glutamina/química , Leucina/química , Proteínas de Membrana/biossíntese , Camundongos , Microscopia de Fluorescência , Modelos Genéticos , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Fosforilação , Recombinação Genética , Serina/química , Células-Tronco/metabolismo , Timo/citologia , Fatores de Tempo , Ativação Transcricional , Triptofano/química , Proteína Supressora de Tumor p53/fisiologia , Raios UltravioletaRESUMO
To characterize the sites in human p53 that become phosphorylated in response to DNA damage, we have developed polyclonal antibodies that recognize p53 only when it is phosphorylated at specific sites. Several attempts to generate an antibody to p53 phosphorylated at Ser(6) using a phosphoserine-containing peptide as an immunogen were unsuccessful; however, phosphorylation-specific antibodies were produced by using the phosphoserine mimetic, l-2-amino-4-phosphono-4, 4-difluorobutanoic acid (F(2)Pab), in place of phosphoserine. Fmoc-F(2)Pab was prepared by an improved synthesis and chemically incorporated using solid phase peptide synthesis. Affinity-purified antibodies elicited by immunizing rabbits with an F(2)Pab peptide coupled to keyhole limpet hemocyanin recognized a p53(1-39) peptide phosphorylated only at Ser(6) but not the unphosphorylated peptide or the same peptide phosphorylated at Ser(9), Ser(15), Ser(20), Ser(33), or Ser(37). Untreated A549 cells exhibited a background of constitutive phosphorylation at Ser(6) that increased approximately 10-fold upon exposure to either ionizing radiation or UV light. Similar results were obtained for Ser(9) using antibodies raised against a conventional phosphopeptide. Ser(9) was phosphorylated by casein kinase 1 in vitro in a phosphoserine 6-dependent manner. Our data identify two additional DNA damage-induced phosphorylations in human p53 and show that F(2)Pab-derivatized peptides can be used to develop phosphorylation site-specific polyclonal antibodies.
Assuntos
Dano ao DNA , Serina/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Doxorrubicina/farmacologia , Humanos , Oligopeptídeos/farmacologia , Fosforilação , Coelhos , Radiação Ionizante , Células Tumorais Cultivadas , Raios UltravioletaRESUMO
In unstressed cells, the tumor suppressor protein p53, a tetrameric transcription factor, is present in a latent state and is maintained at low levels through targeted degradation. A variety of cellular stresses including DNA damage, hypoxia, nucleotide depletion, viral infection, and cytokine-activated signaling pathways that transiently stabilize the p53 protein, cause it to accumulate in the nucleus, and activate it as a transcription factor. Activation leads either to growth arrest at the G1/S or G2/M transitions of the cell cycle or to apoptosis. The molecular mechanisms by which stabilization and activation occur are incompletely understood, but accumulating evidence points to roles for multiple posttranslational modifications in mediating these events through several potentially interacting but distinct pathways. Both the approximately 100 amino acid N-terminal and approximately 90 amino acid C-terminal domains are highly modified by phosphorylation and acetylation, whereas modifications to the central sequence-specific DNA binding domain have not been reported. Seven serines and one threonine in the first 46 residues of the transactivation domain and four to five serines in the carboxyl-terminal domain are now known to be phosphorylated, and Lys320 and Lys382 in the carboxyl-terminal domain (human p53) can be acetylated. Antibodies that recognize p53 only when it has been modified at specific sites have been developed by several laboratories, and studies with these have shown that most of the known posttranslational modifications are induced when cells are exposed to DNA-damaging agents. Exceptions are Ser378, which is reported to be constitutively phosphorylated, and Ser376, which is dephosphorylated in response to DNA damage. These recent results, coupled with biochemical and genetic studies, suggest that several amino-terminal phosphorylations can be important in stabilizing p53 in response to DNA damage and in directing acetylation at C-terminal sites. DNA damage-induced modifications to the C-terminus inhibit the ability of this domain to negatively regulate sequence-specific DNA binding either by inducing a conformational change in the protein or by inhibiting non-sequence-specific DNA binding by the C-terminus. C-terminal modifications also modulate the oligomerization state of p53, and may modulate nuclear import/export. Modifications in response to DNA damage to other components that interact with p53 may also be important. In most cases, clear roles for specific modifications, interactions among individual modifications, and the enzymes responsible for each modification remain to be defined. Nevertheless, the field appears poised for major advances in the understanding of the molecular mechanisms that regulate p53 function.
Assuntos
Processamento de Proteína Pós-Traducional , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Genes p53 , Humanos , Dados de Sequência Molecular , Fosforilação , Proteína Supressora de Tumor p53/químicaRESUMO
In this study, the origins of sensory neurons from the tongue that ascend in the hypoglossal nerve were identified and described in the leopard frog, Rana pipiens. Previous studies have shown that these afferents are used to coordinate the timing of jaw and tongue muscles, and are important in the motor control of feeding. These sensory neurons innervate the tongue bilaterally and appear to originate in the dorsal fungiform papillae of the tongue epithelium.
Assuntos
Nervo Hipoglosso/citologia , Neurônios Aferentes/fisiologia , Papilas Gustativas/anatomia & histologia , Língua/inervação , Animais , Tronco Encefálico/citologia , Células Quimiorreceptoras/fisiologia , Corantes , Dextranos , Rana pipiensRESUMO
The p53 tumor suppressor protein is stabilized in response to ionizing radiation and accumulates in the nucleus. Stabilization is thought to involve disruption of the interaction between the p53 protein and Mdm2, which targets p53 for degradation. Here we show that the direct association between a p53 N-terminal peptide and Mdm2 is disrupted by phosphorylation of the peptide on Thr(18) but not by phosphorylation at other N-terminal sites, including Ser(15) and Ser(37). Thr(18) was phosphorylated in vitro by casein kinase (CK1); this process required the prior phosphorylation of Ser(15). Thr(18) was phosphorylated in vivo in response to DNA damage, and such phosphorylation required Ser(15). Our results suggest that stabilization of p53 after ionizing radiation may result, in part, from an inhibition of Mdm2 binding through a phosphorylation-phosphorylation cascade involving DNA damage-activated phosphorylation of p53 Ser(15) followed by phosphorylation of Thr(18).
Assuntos
Proteínas Nucleares , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Treonina/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Caseína Quinases , Humanos , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2 , Proteína Supressora de Tumor p53/químicaRESUMO
Components of the ras signaling pathway contribute to activation of cellular p53. In MCF-7 cells, p38 kinase activated p53 more effectively than other members of the ras pathway. p53 and p38 kinase exist in the same physical complex, and co-expression of p38 stabilized p53 protein. In vitro, p38 kinase phosphorylated p53 at Ser33 and Ser46, a newly identified site. Mutation of these sites decreased p53-mediated and UV-induced apoptosis, and the reduction correlated with total abrogation of UV-induced phosphorylation on Ser37 and a significant decrease in Ser15 phosphorylation in mutant p53 containing alanine at Ser33 and Ser46. Inhibition of p38 activation after UV irradiation decreased phosphorylation of Ser33, Ser37 and Ser15, and also markedly reduced UV-induced apoptosis in a p53-dependent manner. These results suggest that p38 kinase plays a prominent role in an integrated regulation of N-terminal phosphorylation that regulates p53-mediated apoptosis after UV radiation.
Assuntos
Apoptose/efeitos da radiação , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta , Alanina/metabolismo , Cloranfenicol O-Acetiltransferase/metabolismo , Dano ao DNA , Citometria de Fluxo , Humanos , Proteínas Quinases Ativadas por Mitógeno/genética , Mutagênese Sítio-Dirigida , Fosforilação/efeitos da radiação , Plasmídeos/metabolismo , Serina/metabolismo , Fatores de Tempo , Transcrição Gênica , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
DNA-PK is a nuclear, serine/threonine protein kinase required for repairing DNA double-strand breaks and for V(D)J recombination. To determine the distribution of DNA-PK in human tissues, we assayed paraffin-embedded sections of normal and cancerous tissues for DNA-PKcs and Ku80 by immunohistochemistry. We also assayed for Brca2, a human tumor suppressor gene that is implicated in the repair of DNA strand-breaks. Brca2 was strongly expressed in epithelial cells of the breast, endometrium, and thymus, in tingible body macrophages of follicular germinal centers of lymphoid tissue, and in reticuloendothelial cells in the spleen. DNA-PKcs and Ku80 expression was usually parallel, but both were expressed in a highly cell- and tissue-specific manner. The highest levels were observed in spermatogenic cells (but not in spermatozoa), and in neurons and glial cells of the central and autonomic nervous system. Neither protein was consistently expressed in liver nor in resting mammary epithelium, but lactating breast epithelium was strongly positive for DNA-PKcs and Ku80. In contrast to established human cell cultures, expression between cells in the same tissue was highly selective in the epidermis, exocrine pancreas, renal glomeruli, the red pulp of the spleen, and within cellular compartments of tonsils, lymph nodes, and thymus. Most cancerous tissues were consistently positive for DNA-PKcs and Ku80, except invasive carcinoma of the breast. DNA-PKcs, Ku80, and Ku70 mRNAs were expressed in all normal tissues with relatively little variation in levels. Our results suggest that the apparent absence of DNA-PKcs and Ku80 from some cells or tissues is a consequence of post-transcriptional mechanisms that regulate protein levels.