Enhancement of transient gene expression by fed-batch culture of HEK 293 EBNA1 cells in suspension

Enhanced green fluorescence protein (GFP) and erythropoietin (EPO) were used as reporters to assess and improve transient gene expression in HEK 293 EBNA1 cells. The production of EPO only lasted 3 days and reached 18.1 mg/l in suspension cultures in 1 l batch bioreactors. However, GFP expression examined in well-plate experiments persisted for 12 days in transfected cells but decreased rapidly within the next 15 days. These results suggest that the retaining of a plasmid in cells may not be a limiting factor for protein expression in large-scale transient transfection. To improve cell maintenance and protein expression, a fed-batch culture was performed using an enriched medium, a mixture of equal volumes of 293 SFM II medium and a 5 × amino acid solution prepared based on DMEM/F12 medium formula. EPO reached 33.6 mg/l, representing 86% increase over that of the batch culture. Moreover, the total amount of EPO produced was increased by 165% in view of the volume increase in the fed-batch culture. The serum-free medium used in this work enables cells growing well and transfection without medium change. Thus, the process reported here is simple and easy to scale up.     

Resurrecting abandoned proteins with pure water: CD and NMR studies of protein fragments solubilized in salt-free water

Many proteins expressed in Escherichia coli cells form inclusion bodies that are neither refoldable nor soluble in buffers. Very surprisingly, we recently discovered that all 11 buffer-insoluble protein fragments/domains we have, with a great diversity of cellular function, location, and molecular size, could be easily solubilized in salt-free water. The circular dichroism (CD) and NMR characterization led to classification of these proteins into three groups: group 1, with no secondary structure by CD and with narrowly-dispersed but sharp (1)H-(15)N heteronuclear single quantum correlation (HSQC) peaks; group 2, with secondary structure by CD but with HSQC peaks broadened and, consequently, only a small set of peaks detectable; and group 3, with secondary structure by CD and also well-separated HSQC peaks. Intriguingly, we failed to find any protein with a tight tertiary packing. Therefore, we propose that buffer-insoluble proteins may lack intrinsic ability to reach or/and to maintain a well-packed conformation, and thus are trapped in partially-folded states with many hydrophobic side chains exposed to the bulk solvent. As such, a very low ionic strength is sufficient to screen out intrinsic repulsive interactions and, consequently, allow the hydrophobic clustering/aggregation to occur. Marvelously enough, it appears that in pure water, proteins have the potential to manifest their full spectrum of structural states by utilizing intrinsic repulsive interactions to suppress the attractive hydrophobic clustering. Our discovery not only gives a novel insight into the properties of insoluble proteins, but also sheds the first light that we know of on previously unknown regimes associated with proteins.     

Expression of glucose-6-phosphatase system genes in murine cortex and hypothalamus.

The glucose-6-phosphatase (G6Pase) system participates in the regulation of glucose homeostasis by converting glucose-6-phosphate (G6P) into glucose and inorganic phosphates. We have used an RT-PCR-based cloning and sequencing approach to study the expression of components of the G6Pase system in the hypothalamus and cortex tissues of the ob/ob mouse. We observed the expression of hepatic G6Pase catalytic subunit, G6PC, in both tissues, although increased template inputs were required for its detection. Conversely, expression of both the mouse homologue of the previously-described brain-specific G6P translocase T1 (G6PT1) variant and of the hepatic G6PT1 isoform was easily detectable in hypothalamus and cortex tissues. Of the proposed G6Pase catalytic subunit homologues, the expression of murine ubiquitous G6Pase catalytic subunit-related protein (UGRP, G6PC3) was also easily detectable in both tissues. However, islet-specific G6Pase catalytic subunit-related protein (IGRP, G6PC2) was expressed in a tissue-specific manner, and was detectable only in hypothalamus tissue at increased template inputs. We conclude that cells within ob/ob mouse hypothalamus and cortex tissues express genes with either established or proposed roles in G6P hydrolysis.     

Ena/VASP proteins regulate cortical neuronal positioning

Development of the multilayered cerebral cortex involves extensive regulated migration of neurons arising from the deeper germinative layers of the mammalian brain. The anatomy and formation of the cortical layers has been well characterized; however, the underlying molecular mechanisms that control the migration and the final positioning of neurons within the cortex remain poorly understood. Here, we report evidence for a key role of Ena/VASP proteins, a protein family implicated in the spatial control of actin assembly and previously shown to negatively regulate fibroblast cell speeds, in cortical development. Ena/VASP proteins are highly expressed in the developing cortical plate in cells bordering Reelin-expressing Cajal-Retzius cells and in the intermediate zone through which newly born cells migrate. Inhibition of Ena/VASP function through retroviral injections in utero led to aberrant placement of early-born pyramidal neurons in the superficial layers of both the embryonic and the postnatal cortex in a cell-autonomous fashion. The abnormally placed pyramidal neurons exhibited grossly normal morphology and polarity. Our results are consistent with a model in which Ena/VASP proteins function in vivo to control the position of neurons in the mouse neocortex.     

Large scale international replication and meta-analysis study confirms association of the 15q14 locus with myopia. The CREAM consortium

Myopia is a complex genetic disorder and a common cause of visual impairment among working age adults. Genome-wide association studies have identified susceptibility loci on chromosomes 15q14 and 15q25 in Caucasian populations of European ancestry. Here, we present a confirmation and meta-analysis study in which we assessed whether these two loci are also associated with myopia in other populations. The study population comprised 31 cohorts from the Consortium of Refractive Error and Myopia (CREAM) representing 4 different continents with 55,177 individuals; 42,845 Caucasians and 12,332 Asians. We performed a meta-analysis of 14 single nucleotide polymorphisms (SNPs) on 15q14 and 5 SNPs on 15q25 using linear regression analysis with spherical equivalent as a quantitative outcome, adjusted for age and sex. We calculated the odds ratio (OR) of myopia versus hyperopia for carriers of the top-SNP alleles using a fixed effects meta-analysis. At locus 15q14, all SNPs were significantly replicated, with the lowest P value 3.87?×?10(-12) for SNP rs634990 in Caucasians, and 9.65?×?10(-4) for rs8032019 in Asians. The overall meta-analysis provided P value 9.20?×?10(-23) for the top SNP rs634990. The risk of myopia versus hyperopia was OR 1.88 (95?% CI 1.64, 2.16, P?相似文献   

Ndfip1 regulates nuclear Pten import in vivo to promote neuronal survival following cerebral ischemia

PTEN (phosphatase and tensin homologue deleted on chromosome TEN) is the major negative regulator of phosphatidylinositol 3-kinase signaling and has cell-specific functions including tumor suppression. Nuclear localization of PTEN is vital for tumor suppression; however, outside of cancer, the molecular and physiological events driving PTEN nuclear entry are unknown. In this paper, we demonstrate that cytoplasmic Pten was translocated into the nuclei of neurons after cerebral ischemia in mice. Critically, this transport event was dependent on a surge in the Nedd4 family-interacting protein 1 (Ndfip1), as neurons in Ndfip1-deficient mice failed to import Pten. Ndfip1 binds to Pten, resulting in enhanced ubiquitination by Nedd4 E3 ubiquitin ligases. In vitro, Ndfip1 overexpression increased the rate of Pten nuclear import detected by photobleaching experiments, whereas Ndfip1(-/-) fibroblasts showed negligible transport rates. In vivo, Ndfip1 mutant mice suffered larger infarct sizes associated with suppressed phosphorylated Akt activation. Our findings provide the first physiological example of when and why transient shuttling of nuclear Pten occurs and how this process is critical for neuron survival.     

Phosphorylation of the gastric tumor suppressor RUNX3 following H. pylori infection results in its localization to the cytoplasm

As H. pylori infection progresses, intestinal metaplasia (IM), a key event in gastric carcinogenesis, develops in the stomach. The mechanism by which H. pylori infection causes the trans-differentiation of gastric cells to intestinal-type cells remains an important question. In the current study, we found that RUNX3 is deregulated in all human IM specimens examined by either down regulation or mislocalization; Aberrant localization of a gastric tumor suppressor RUNX3 is observed in most human cases of IM with concurrent H. pylori infection, and RUNX3 is down-regulated in most cases of IM without H. pylori-infection. The cytoplasmic mislocalization of a RUNX3 was associated with H. pylori-induced c-Src activation and RUNX tyrosine phosphorylation. Moreover, gastric epithelial cells of Runx3(-/-) mice expressed the intestinal markers Muc2 and Li-Cadherin, which suggests that the deregulation of Runx3 is a key event in the intestinalization of the gastric epithelium. Collectively, the results of the current study suggest that RUNX3 deregulation is associated with H. pylori-induced pathogenesis and the development of IM.     

Bimodal collagen fibril diameter distributions direct age-related variations in tendon resilience and resistance to rupture

Scaling relationships have been formulated to investigate the influence of collagen fibril diameter (D) on age-related variations in the strain energy density of tendon. Transmission electron microscopy was used to quantify D in tail tendon from 1.7- to 35.3-mo-old (C57BL/6) male mice. Frequency histograms of D for all age groups were modeled as two normally distributed subpopulations with smaller (D(D1)) and larger (D(D2)) mean Ds, respectively. Both D(D1) and D(D2) increase from 1.6 to 4.0 mo but decrease thereafter. From tensile tests to rupture, two strain energy densities were calculated: 1) u(E) [from initial loading until the yield stress (σ(Y))], which contributes primarily to tendon resilience, and 2) u(F) [from σ(Y) through the maximum stress (σ(U)) until rupture], which relates primarily to resistance of the tendons to rupture. As measured by the normalized strain energy densities u(E)/σ(Y) and u(F)/σ(U), both the resilience and resistance to rupture increase with increasing age and peak at 23.0 and 4.0 mo, respectively, before decreasing thereafter. Multiple regression analysis reveals that increases in u(E)/σ(Y) (resilience energy) are associated with decreases in D(D1) and increases in D(D2), whereas u(F)/σ(U) (rupture energy) is associated with increases in D(D1) alone. These findings support a model where age-related variations in tendon resilience and resistance to rupture can be directed by subtle changes in the bimodal distribution of Ds.     

Identification of the protein kinases that activate the E3 ubiquitin ligase Pellino 1 in the innate immune system

The E3 ubiquitin ligase Pellino 1 can be interconverted between inactive and active forms by a reversible phosphorylation mechanism. In vitro, phosphorylation and activation can be catalysed by either the IRAKs [IL (interleukin)-1-receptor-associated kinases] IRAK1 and IRAK4, or the IKK {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase}-related kinases [IKK? and TBK1 (TANK {TRAF [TNF (tumour-necrosis-factor)-receptor-associated factor]-associated NF-κB activator}-binding kinase 1)]. In the present study we establish that IRAK1 is the major protein kinase that mediates the IL-1-stimulated activation of Pellino 1 in MEFs (mouse embryonic fibroblasts) or HEK (human embryonic kidney)-293 cells, whereas the IKK-related kinases activate Pellino 1 in TNFα-stimulated MEFs. The IKK-related kinases are also the major protein kinases that activate Pellino 1 in response to TLR (Toll-like receptor) ligands that signal via the adaptors MyD88 (myeloid differentiation primary response gene 88) and/or TRIF [TIR (Toll/IL-1 receptor) domain-containing adaptor protein inducing interferon β]. The present studies demonstrate that, surprisingly, the ligands that signal via MyD88 do not always employ the same protein kinase to activate Pellino 1. Our results also establish that neither the catalytic activity of IRAK1 nor the activation of Pellino 1 is required for the initial transient activation of NF-κB and MAPKs (mitogen-activated protein kinases) that is triggered by IL-1 or TNFα in MEFs, or by TLR ligands in macrophages. The activation of Pellino 1 provides the first direct readout for IRAK1 catalytic activity in cells.     

Identification of Regulators of Polyploidization Presents Therapeutic Targets for Treatment of AMKL

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our?study implicates five networks of kinases that?regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in?vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.