RESUMEN
Quantum dots (QDs), an important class of emerging nanomaterial, are widely anticipated to find application in many consumer and clinical products in the near future. Premarket regulatory scrutiny is, thus, an issue gaining considerable attention. Previous review papers have focused primarily on the toxicity of QDs. From the point of view of product regulation, however, parameters that determine exposure (e.g., dosage, transformation, transportation, and persistence) are just as important as inherent toxicity. We have structured our review paper according to regulatory risk assessment practices, in order to improve the utility of existing knowledge in a regulatory context. Herein, we summarize the state of academic knowledge on QDs pertaining not only to toxicity, but also their physicochemical properties, and their biological and environmental fate. We conclude this review with recommendations on how to tailor future research efforts to address the specific needs of regulators.
Asunto(s)
Puntos Cuánticos , Pruebas de Toxicidad , Animales , Humanos , Medición de RiesgoRESUMEN
OBJECTIVE: To characterize a novel splice variant of the alpha subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMRalpha), which we discovered in human neutrophils. METHODS: We used reverse transcriptase polymerase chain reaction to identify, characterize, and examine the expression of a novel splice variant of the GMRalpha transcript. At the protein level, surface plasmon resonance was used to measure the affinity of a recombinant soluble form of the novel GMRalpha protein for GM-CSF ligand. The full-length novel GMRalpha protein was expressed in a recombinant cell culture system, and its expression and localization were examined using Western blotting, I(125) GM-CSF binding assays, flow cytometry, and a soluble GMRalpha enzyme-linked immunosorbent assay. RESULTS: The novel GMRalpha transcript identified herein contains a previously undescribed exon of the GMRalpha gene; this exon derives from an Alu DNA repeat element, and is alternatively spliced in the novel GMRalpha transcript. Inclusion of this 102 nucleotide exon results in translation of a protein product, which we have named Alu-GMRalpha. Alu-GMRalpha is identical to cell surface GMRalpha, but additionally contains a 34 amino-acid insert in the juxtamembrane region of the extracellular domain of GMRalpha. Functionally, the Alu-GMRalpha-specific epitope does not modify the ability of the protein to bind GM-CSF, but rather appears to be preferentially targeted by ectodomain proteases to mediate the release of a third soluble GM-CSF receptor into the extracellular space. CONCLUSIONS: This study provides the first example of a cytokine receptor system in which soluble receptors are produced by three distinct mechanisms. Our results highlight the importance of soluble GMRalpha proteins in regulation of GM-CSF signaling.
Asunto(s)
Empalme Alternativo/fisiología , Elementos Alu/fisiología , Exones/fisiología , Neutrófilos/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Secuencia de Aminoácidos/genética , Animales , Clonación Molecular , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Ligandos , Ratones , Mutagénesis Insercional/fisiología , Células 3T3 NIH , Neutrófilos/citología , Péptido Hidrolasas/metabolismo , Estructura Terciaria de Proteína/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal/fisiología , Resonancia por Plasmón de SuperficieRESUMEN
The integrity of the human genome is preserved by signal transduction pathways called checkpoints, which delay progression through the cell cycle when DNA damage is present. Three checkpoint proteins, hRad9, hRad1, and hHus1, form a proliferating cell nuclear antigen-like, heterotrimeric complex that has been proposed to function in the initial detection of DNA structural abnormalities. hRad9 is highly modified by phosphorylation, in a constitutive manner and in response to both DNA damage and cell cycle position. Here we present evidence that Thr292 of hRad9 is subject to Cdc2-dependent phosphorylation in mitosis. Furthermore, our data are also consistent with four other hRad9 phosphorylation sites (Ser277, Ser328, Ser336, and Thr355) being regulated in part by Cdc2. We also identify Ser387 as a novel site of hRad9 constitutive phosphorylation and show that phosphorylation at Ser387 is a prerequisite for one form of DNA damage-induced hyperphosphorylation of hRad9. Characterization of nonphosphorylatable mutants has revealed that hRad9 phosphorylation plays a critical role in checkpoint signaling. Overexpression of these mutants blocks the interaction between hRad9 and the DNA damage-responsive protein TopBP1 and impairs the cellular response to DNA damage during S phase.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Proteína Quinasa CDC2/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiología , Ciclo Celular/efectos de la radiación , Proteínas de Ciclo Celular/genética , Línea Celular , Daño del ADN , Células HeLa , Humanos , Mitosis , Mutagénesis Sitio-Dirigida , Fosforilación , ARN Interferente Pequeño/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Treonina/química , TransfecciónRESUMEN
Soluble GM-CSF receptor alpha subunit (sGMRalpha) is a soluble isoform of the GMRalpha that is believed to arise exclusively through alternative splicing of the GMRalpha gene product. The sGMRalpha mRNA is expressed in a variety of tissues, but it is not clear which cells are capable of secreting the protein. We show here that normal human monocytes, but not lymphocytes, constitutively secrete sGMRalpha. Stimulation of monocytes with GM-CSF, LPS, PMA, or A23187 rapidly up-regulates the secretion of sGMRalpha in a dose-dependent manner, demonstrating that secretion is also regulated. To determine whether sGMRalpha arose exclusively through alternative splicing of the GMRalpha gene product, or whether it could also be generated through ectodomain shedding of GMRalpha, we engineered a murine pro-B cell line (Ba/F3) to express exclusively the cDNA for cell surface GMRalpha (Ba/F3.GMRalpha). The Ba/F3.GMRalpha cell line, but not the parental Ba/F3 cell line, constitutively shed a sGMRalpha-like protein that bound specifically to GM-CSF, was equivalent in size to recombinant alternatively spliced sGMRalpha (60 kDa), and was recognized specifically by a mAb raised against the ectodomain of GMRalpha. Furthermore, a broad-spectrum metalloprotease inhibitor (BB94) reduced constitutive and PMA-, A23187-, and LPS-induced secretion of sGMRalpha by monocytes, suggesting that shedding of GMRalpha by monocytes may be mediated in part through the activity of metalloproteases. Taken together, these observations demonstrate that sGMRalpha is constitutively secreted by monocytes, that GM-CSF and inflammatory mediators up-regulate sGMRalpha secretion, and that sGMRalpha arises not only through alternative splicing but also through ectodomain shedding of cell surface GMRalpha.