RESUMO
Wheat is the staple food crop in temperate countries and increasingly consumed in developing countries, displacing traditional foods. However, wheat products are typically low in bioavailable iron and zinc, contributing to deficiencies in these micronutrients in countries where wheat is consumed as a staple food. Two factors contribute to the low contents of bioavailable iron and zinc in wheat: the low concentrations of these minerals in white flour, which is most widely consumed, and the presence of phytates in mineral-rich bran fractions. Although high zinc types of wheat have been developed by conventional plant breeding (biofortification), this approach has failed for iron. However, studies in wheat and other cereals have shown that transgenic (also known as genetically modified; GM) strategies can be used to increase the contents of iron and zinc in white flour, by converting the starchy endosperm tissue into a 'sink' for minerals. Although such strategies currently have low acceptability, greater understanding of the mechanisms which control the transport and deposition of iron and zinc in the developing grain should allow similar effects to be achieved by exploiting naturally induced genetic variation. When combined with conventional biofortification and innovative processing, this approach should provide increased mineral bioavailability in a range of wheat products, from white flour to wholemeal.
RESUMO
Inadequate intake of essential minerals such as iron and zinc is a public health concern in the UK, particularly for girls and young women. Approximately 30% and 50% of the zinc and iron, respectively, in the UK diet is provided by cereals. In wheat, most of the iron and zinc is contained within the aleurone cell layer; however, aleurone is removed during processing of wheat into white flour. While elemental iron powder is added back into white flour at the milling stage, there is no restoration of zinc. Elemental iron powder has very low bioavailability, and therefore, in our current Biotechnology and Biological Sciences Research Council Diet and Health Research Industry Club-funded project, we are investigating the potential use of aleurone as a bioavailable source of minerals that could be added to wheat-based foods. This work has relevance for the food industry and may establish the use of aleurone as a functional food ingredient for fortification of a range of cereal-based food products.
RESUMO
Despite the rapid pace of biomedical innovation, research and development (R&D) productivity in the pharmaceutical industry has not improved broadly. Increasingly, firms need to leverage new approaches to product development and commercial execution, while maintaining adaptability to rapid changes in the marketplace and in biomedical science. Firms are also seeking ways to capture some of the talent, infrastructure, and innovation that depends on federal R&D investment. As a result, a major transition to external innovation is taking place across the industry. One example of these external innovation initiatives is the Sanofi-MIT Partnership, which provided seed funding to MIT investigators to develop novel solutions and approaches in areas of interest to Sanofi. These projects were highly collaborative, with information and materials flowing both ways. The relatively small amount of funding and short time frame of the awards built an adaptable and flexible process to advance translational science.
Assuntos
Pesquisa Biomédica/organização & administração , Difusão de Inovações , Indústria Farmacêutica/organização & administração , Universidades/organização & administração , Comportamento Cooperativo , Eficiência Organizacional , Humanos , Pesquisa Translacional Biomédica/organização & administraçãoRESUMO
Cereals constitute important sources of iron in human diet; however, much of the iron in wheat is lost during processing for the production of white flour. This study employed novel food processing techniques to increase the bioaccessibility of naturally occurring iron in wheat. Iron was localized in wheat by Perl's Prussian blue staining. Soluble iron from digested wheat flour was measured by a ferrozine spectrophotometric assay. Iron bioaccessibility was determined using an in vitro simulated peptic-pancreatic digestion, followed by measurement of ferritin (a surrogate marker for iron absorption) in Caco-2 cells. Light microscopy revealed that iron in wheat was encapsulated in cells of the aleurone layer and remained intact after in vivo digestion and passage through the gastrointestinal tract. The solubility of iron in wholegrain wheat and in purified wheat aleurone increased significantly after enzymatic digestion with Driselase, and following mechanical disruption using micromilling. Furthermore, following in vitro simulated peptic-pancreatic digestion, iron bioaccessibility, measured as ferritin formation in Caco-2 cells, from micromilled aleurone flour was significantly higher (52%) than from whole aleurone flour. Taken together our data show that disruption of aleurone cell walls could increase iron bioaccessibility. Micromilled aleurone could provide an alternative strategy for iron fortification of cereal products.
Assuntos
Manipulação de Alimentos/métodos , Mucosa Intestinal/metabolismo , Ferro/metabolismo , Triticum/química , Disponibilidade Biológica , Células CACO-2 , Digestão , Farinha/análise , Humanos , Modelos Biológicos , Sementes/química , Sementes/metabolismo , Triticum/metabolismoRESUMO
Kras-induced non-small-cell lung adenocarcinoma is the major subtype of lung cancers and is associated with poor prognosis. Using a lung cancer mouse model that expresses a cre-mediated KrasG12D mutant, we identified a critical role for the cell surface molecule CD44 in mediating cell proliferation downstream of oncogenic Kras signaling. The deletion of CD44 attenuates lung adenocarcinoma formation and prolongs the survival of these mice. Mechanistically, CD44 is required for the activation of Kras-mediated signaling through the mitogen-activated protein kinase (MAPK) pathway and thus promotes tumor cell proliferation. Together, these results reveal an unrecognized role for CD44 in oncogenic Kras-induced lung adenocarcinoma and suggest that targeting CD44 could be an effective strategy for halting Kras-dependent carcinomas.
Assuntos
Adenocarcinoma/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Receptores de Hialuronatos/metabolismo , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Receptores de Hialuronatos/genética , Neoplasias Pulmonares/patologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Terapia de Alvo Molecular , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de SinaisRESUMO
Octamer transcription factor-1 (Oct-1) has recently been shown to function as a stress sensor that promotes cell survival subsequent to DNA damage. Here, we show that the survival signal imparted by Oct-1 following exposure to ionizing radiation (IR) is dependent upon DNA-dependent protein kinase (DNA-PK)-dependent phosphorylation of a cluster of 13 specific ser/thr residues within the N-terminal transcriptional regulatory domain of Oct-1. Although IR treatment did not affect the recruitment of Oct-1 to the histone H2B promoter, the recruitment of RNA polymerase II, TATA-binding protein and histone H4 acetylation were strongly reduced, consistent with a decrease in Oct-1 transcriptional regulatory potential following IR exposure. Ser/Thr-Ala substitution of 13 sites present in Oct-1 transcriptional regulatory domain eliminated Oct-1 phosphorylation subsequent to IR exposure. Further, these substitutions prevented Oct-1 from rescuing the survival of IR-treated Oct-1-/- murine embryonic fibroblasts, providing a direct link between DNA-PK-dependent phosphorylation and the contribution of Oct-1 to cell survival. These results implicate Oct-1 as a primary effector in a DNA-PK-dependent cell survival pathway that is activated by double-stranded DNA breaks.
Assuntos
Substituição de Aminoácidos , Dano ao DNA , Proteína Quinase Ativada por DNA/metabolismo , Fator 1 de Transcrição de Octâmero/genética , Células 3T3 , Sequência de Aminoácidos , Animais , Sítios de Ligação , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Relação Dose-Resposta à Radiação , Histonas/genética , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fator 1 de Transcrição de Octâmero/metabolismo , Fosforilação , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos da radiação , Serina/genética , Serina/metabolismo , Treonina/genética , Treonina/metabolismo , TransfecçãoRESUMO
BACKGROUND: Only 10% of dietary iron is absorbed in the duodenum which implies that 90% (approximately 9 mg day(-1)) reaches the lower small intestine and colon. Therefore the purpose of this study was to assess the iron transport capacity of the rat proximal colon and to determine whether iron absorption is regulated by changes in dietary iron content. MATERIALS AND METHODS: Rats were fed for 14 days on either iron adequate (44 mg Fe kg(-1) diet) or iron-deficient (< 0.5 mg Fe kg(-1) diet) diets. The 59Fe transport across the colonic epithelium and its subsequent appearance in the blood were measured in vivo. In separate studies the colon was excised and used to measure divalent metal transporter expression. RESULTS: Divalent metal transporter (DMT1) was expressed at the apical membrane of the surface epithelium in rat proximal colon. In animals fed an iron-deficient diet, DMT1 mRNA and protein expression were increased. This was accompanied by a significant increase in tissue 59Fe uptake. CONCLUSIONS: The proximal colon can absorb non-haem iron from the intestinal lumen. The purpose of this mechanism remains to be elucidated.
Assuntos
Colo/metabolismo , Ferro da Dieta/farmacocinética , Animais , Transporte Biológico , Western Blotting , Proteínas de Transporte de Cátions/metabolismo , Duodeno/metabolismo , Heme , Absorção Intestinal , Mucosa Intestinal/metabolismo , Ferro/sangue , Deficiências de Ferro , Ferro da Dieta/administração & dosagem , Proteínas de Ligação ao Ferro/metabolismo , Masculino , Ratos , Ratos WistarRESUMO
microRNAs (miRNAs) represent a large set of master regulators of gene expression. They constitute 1-4% of human genes and probably regulate 30% of protein-encoding genes. These small regulatory RNAs act at a posttranscriptional level-mediating translational repression and/or mRNA degradation-through their association with Argonaute protein and target mRNAs. In this paper, we discuss various mechanisms by which miRNAs regulate posttranscriptionally, including their subcellular localization. Recent results indicate that the majority of miRNA-targeted and thus translationally repressed mRNA is probably distributed in the diffuse cytoplasm, even though a small fraction is concentrated in subcellular compartments, such as processing bodies or stress granules; notably, the stress granule localization of Argonaute depends on the presence of miRNAs. Here we discuss the structural requirement of these subcellular compartments in light of their potential miRNA functions.
Assuntos
MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Humanos , Mamíferos , Modelos Biológicos , Biossíntese de Proteínas , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Frações Subcelulares/metabolismoAssuntos
Inativação Gênica , RNA/metabolismo , Animais , Evolução Biológica , Códon sem Sentido , Metilação de DNA , Elementos de DNA Transponíveis , RNA/biossíntese , RNA/genética , RNA Helicases/metabolismo , RNA de Cadeia Dupla/biossíntese , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Polimerase Dependente de RNA/metabolismoRESUMO
In vitro selection was used to sample SnRNA-related sequences for ribozyme activities, and several 2',5'-branch-forming ribozymes were isolated. One such ribozyme is highly dependent upon an 11-nt motif that contains a conserved U6 snRNA sequence (ACAGAGA-box) known to be important for pre-mRNA splicing. The ribozyme reaction is similar to the first step of splicing in that an internal 2'-hydroxyl of an unpaired adenosine attacks at the 5'-phosphate of a guanosine. It differs in that the leaving group is diphosphate rather than a 5' exon. The finding that lariat formation can be accomplished by a small RNA with sequences related to U6 snRNA indicates that the RNA available in the spliceosome may be involved in RNA-catalyzed branch formation.
Assuntos
Variação Genética , Precursores de RNA/genética , RNA Catalítico/metabolismo , RNA Nuclear Pequeno/genética , Animais , Sequência de Bases , Sequência Conservada , Éxons , Biblioteca Gênica , Íntrons , Cinética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos , Filogenia , Precursores de RNA/química , Precursores de RNA/metabolismo , Splicing de RNA , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade por Substrato , Trypanosomatina/genéticaRESUMO
The CDK9-cyclin T kinase complex, positive transcription elongation factor b (P-TEFb), stimulates the process of elongation of RNA polymerase (Pol) II during transcription of human immunodeficiency virus. P-TEFb associates with the human immunodeficiency virus Tat protein and with the transactivation response element to form a specific complex, thereby mediating efficient elongation. Here, we show that P-TEFb preferentially phosphorylates hSPT5 as compared with the carboxyl-terminal domain of RNA Pol II in vitro. Phosphorylation of hSPT5 by P-TEFb occurred on threonine and serine residues in its carboxyl-terminal repeat domains. In addition, we provide several lines of evidence that P-TEFb is a CDK-activating kinase (CAK)-independent kinase. For example, CDK9 was not phosphorylated by CAK, whereas CDK2-cyclin A kinase activity was dramatically enhanced by CAK. Therefore, it is likely that P-TEFb participates in regulation of elongation by RNA Pol II by phosphorylation of its substrates, hSPT5 and the CTD of RNA Pol II, in a CAK-independent manner.
Assuntos
Proteínas Cromossômicas não Histona , Proteínas Fúngicas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição , Sequência de Bases , Primers do DNA , Fosforilação , Fator B de Elongação Transcricional Positiva , RNA Polimerase II/químicaRESUMO
Lactate and ammonia are the two major waste products formed during mammalian cell growth. Accumulation of these side products can have a negative effect on cell growth, and has drawn recent attention because of their inhibitory effects on the specific product synthesis rate. Our aim is to reduce lactate formation in the cell culture by genetically manipulating of the pathway of lactate synthesis with an aim to achieve high monoclonal antibody production. We have partially disrupted the LDH-A gene by homologous recombination in hybridoma cells (ATCC-CRL-1606). The cells that received the newly introduced DNA were selected by G418, and an LDH-deficient cell was identified by a screening method based on medium color changing in 96-well plates. A variant cell, LDH-neo21, was identified through this screening method and was characterized. The specific productivity of lactate by LDH-neo21 cells was 50% lower than that of parental cells. Intracellular LDH enzyme activity was significantly reduced. The cell growth was improved both in terms of cell density and cell viability. Total cell density potentially reached 5 x 10(6) cells/mL while the parental hybridoma cells had a cell density of 3.5 x 10(6) cells/mL, which represented a 30% increase. The antibody production of LDH-neo21 cells was threefold greater than that of parental cells during 5-day batch culture. Polymerase chain reaction (PCR) results showed that at least one copy of the LDH-A gene was disrupted in the LDH-neo21 cells. The variant of the hybridoma cell exhibited a significant advantage of reduced lactate formation in the cell culture with a high concentration of glucose, which led to a higher production of monoclonal antibody. 2001 John Wiley & Sons, Inc.
Assuntos
Anticorpos Monoclonais/genética , Engenharia Genética/métodos , L-Lactato Desidrogenase/genética , Lactatos/metabolismo , Amônia/metabolismo , Animais , Anticorpos Monoclonais/biossíntese , Divisão Celular , DNA/genética , Vetores Genéticos , Hibridomas , L-Lactato Desidrogenase/metabolismo , Mamíferos , Camundongos , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes/biossíntese , Transfecção/métodosRESUMO
Microscopy studies have shown that XIST RNA colocalizes with the inactive X chromosome (Xi). However, the molecular basis for this colocalization is unknown. Here we provide two lines of evidence from chromatin immunoprecipitation experiments that XIST RNA physically associates with the Xi chromatin. First, XIST RNA can be co-precipitated by antiserum against macroH2A, a histone H2A variant enriched in the Xi. Second, XIST RNA can be co-precipitated by antisera that recognize unacetylated, but not acetylated, isoforms of histones H3 and H4. The specificity of XIST RNA association with hypoacetylated chromatin, together with the previous finding that hypoacetylated histone H4 is enriched at promoters of X-inactivated genes, raises the possibility that XIST RNA may contribute to the hypoacetylation of specific regions of the Xi so as to alter the expression of X-linked genes.
Assuntos
RNA não Traduzido/genética , Fatores de Transcrição/genética , Cromossomo X , Animais , Especificidade de Anticorpos , Células Cultivadas , Cromatina/química , Mecanismo Genético de Compensação de Dose , Fibronectinas/genética , Histonas/genética , Soros Imunes , Camundongos , Regiões Promotoras Genéticas , RNA Longo não Codificante , RNA não Traduzido/imunologia , Fatores de Transcrição/imunologiaRESUMO
Double-stranded RNA (dsRNA) directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). Using a recently developed Drosophila in vitro system, we examined the molecular mechanism underlying RNAi. We find that RNAi is ATP dependent yet uncoupled from mRNA translation. During the RNAi reaction, both strands of the dsRNA are processed to RNA segments 21-23 nucleotides in length. Processing of the dsRNA to the small RNA fragments does not require the targeted mRNA. The mRNA is cleaved only within the region of identity with the dsRNA. Cleavage occurs at sites 21-23 nucleotides apart, the same interval observed for the dsRNA itself, suggesting that the 21-23 nucleotide fragments from the dsRNA are guiding mRNA cleavage.
Assuntos
Trifosfato de Adenosina/metabolismo , RNA Antissenso/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Drosophila/embriologia , Drosophila/genética , Dados de Sequência Molecular , Nucleotídeos , Biossíntese de Proteínas , Processamento Pós-Transcricional do RNA , RNA Interferente PequenoAssuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/genética , Elementos de DNA Transponíveis , Regulação da Expressão Gênica , Inativação Gênica , RNA de Cadeia Dupla/genética , RNA de Helmintos/genética , RNA Mensageiro/genética , Animais , Feminino , Genes de Helmintos , Proteínas de Helminto/genética , Proteínas de Helminto/fisiologia , Masculino , Mutação , RNA de Helmintos/metabolismo , RNA Mensageiro/metabolismoRESUMO
Host cell factor 1 (HCF-1; also called C1) is a 230-kDa protein which is cleaved posttranslationally into separate but associated N- and C-terminal polypeptides. These polypeptides are components of the C1 complex, along with Oct-1 and the viral protein VP16. The C1 complex is formed when herpes simplex virus (HSV) infects a cell and is responsible for transcription of the HSV immediate-early genes. A temperature-sensitive mutation in the N-terminal kelch domain of HCF-1 reversibly arrests cells in a G(0)-like state when grown at the nonpermissive temperature, and the same domain interacts with VP16 in the formation of the C1 complex. The form of HCF-1 in primary G(0) cells was investigated by using peripheral blood mononucleocytes and serum-arrested human primary fibroblasts. A novel 50-kDa N-terminal fragment of HCF-1 encompassing the kelch domain was identified in the cytoplasm of these cells. This fragment arises by proteolysis of the full-length HCF-1 protein and is able to associate with VP16.
Assuntos
Proteína Vmw65 do Vírus do Herpes Simples/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas/metabolismo , Fase de Repouso do Ciclo Celular/fisiologia , Proteínas de Transporte , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fator C1 de Célula Hospedeira , Humanos , Leucócitos Mononucleares/citologia , Fator 1 de Transcrição de Octâmero , Ligação Proteica , Estrutura Terciária de Proteína , Simplexvirus , Fatores de Transcrição/metabolismo , Latência ViralRESUMO
The SRm160/300 splicing coactivator, which consists of the serine/arginine (SR)-related nuclear matrix protein of 160 kDa and a 300-kDa nuclear matrix antigen, functions in splicing by promoting critical interactions between splicing factors bound to pre-mRNA, including snRNPs and SR family proteins. In this article we report the isolation of a cDNA encoding the 300-kDa antigen and investigate the activity of it and SRm160 in splicing. Like SRm160, the 300-kDa antigen contains domains rich in alternating S and R residues but lacks an RNA recognition motif; the protein is accordingly named "SRm300." SRm300 also contains a novel and highly conserved N-terminal domain, several unique repeated motifs rich in S, R, and proline residues, and two very long polyserine tracts. Surprisingly, specific depletion of SRm300 does not prevent the splicing of pre-mRNAs shown previously to require SRm160/300. Addition of recombinant SRm160 alone to SRm160/300-depleted reactions specifically activates splicing. The results indicate that SRm160 may be the more critical component of the SRm160/300 coactivator in the splicing of SRm160/300-dependent pre-mRNAs.
Assuntos
Antígenos Nucleares , Proteínas Associadas à Matriz Nuclear , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Splicing de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais , Núcleo Celular/metabolismo , Células Cultivadas , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , DNA Complementar/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/imunologia , Testes de Precipitina , Splicing de RNA/fisiologia , RNA Mensageiro/metabolismo , Spliceossomos/metabolismoRESUMO
As we contemplate the nature of life sciences in the 21st century, we should briefly consider the changes that have occurred in the past century. Surely, the sources of progress of this science in the next century are the advances emerging now. Furthermore, the likely pace of discovery and change in life sciences in the next century can best be estimated by a reflection on its history.
Assuntos
Disciplinas das Ciências Biológicas/tendências , Animais , Previsões , HumanosRESUMO
We have identified a new pyrimidine-tract binding factor, PUF, that is required, together with U2AF, for efficient reconstitution of RNA splicing in vitro. The activity has been purified and consists of two proteins, PUF60 and the previously described splicing factor p54. p54 and PUF60 form a stable complex in vitro when cotranslated in a reaction mixture. PUF activity, in conjunction with U2AF, facilitates the association of U2 snRNP with the pre-mRNA. This reaction is dependent upon the presence of the large subunit of U2AF, U2AF65, but not the small subunit U2AF35. PUF60 is homologous to both U2AF65 and the yeast splicing factor Mud2p. The C-terminal domain of PUF60, the PUMP domain, is distantly related to the RNA-recognition motif domain, and is probably important in protein-protein interactions.