排序方式: 共有22条查询结果,搜索用时 31 毫秒
1.
R. Ashton Lavoie Jeffrey T. Zugates Andrew T. Cheeseman Matt A. Teten Srivatsan Ramesh Julia M. Freeman Summer Swango Jeremy Fitzpatrick Amod Joshi Bradley Hollers Zufan Debebe Tyler K. Lindgren Amber N. Kozak Vinay K. Kondeti Mary K. Bright Eric J. Yearley Alexander Tracy Jacob A. Irwin Michael Guerrero 《Biotechnology and bioengineering》2023,120(10):2953-2968
Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or “empty” capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%. 相似文献
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Neal R. Rasmussen Tricia M. Wright Samira A. Brooks Kathryn E. Hacker Zufan Debebe Adam B. Sendor Matthew P. Walker Michael Ben Major Jennifer Green Geoffrey M. Wahl W. Kimryn Rathmell 《The Journal of biological chemistry》2013,288(36):26301-26310
Expression of the receptor tyrosine kinase-like orphan receptor 2 (Ror2) has been identified in an increasing array of tumor types and is known to play a role as an important mediator of Wnt signaling cascades. In this study, we aimed to clarify Ror2 interactions with the Wnt pathways within the context of renal cell carcinoma (RCC). An examination of Ror2 expression in primary human RCC tumors showed a significant correlation with several Wnt signaling genes, including the classical feedback target gene Axin2. We provide evidence that Ror2 expression results in a partially activated state for canonical Wnt signaling through an increased signaling pool of β-catenin, leading to an enhancement of downstream target genes following Wnt3a stimulation in both renal and renal carcinoma-derived cells. Additionally, inhibition of low-density lipoprotein receptor-related protein 6 (LRP6) with either siRNA or dickkopf decreased the response to Wnt3a stimulation, but no change was seen in the increased β-catenin pool associated with Ror2 expression, suggesting that LRP6 cofactor recruitment is necessary for a Wnt3a-induced signal but that it does not participate in the Ror2 effect on β-catenin signaling. These results highlight a new role for Ror2 in conveying a tonic signal to stabilize soluble β-catenin and create a poised state of enhanced responsiveness to Wnt3a exogenous signals in RCC. 相似文献
3.
Improved bio-energy yields via sequential ethanol fermentation and biogas digestion of steam exploded oat straw 总被引:2,自引:0,他引:2
Dererie DY Trobro S Momeni MH Hansson H Blomqvist J Passoth V Schnürer A Sandgren M Ståhlberg J 《Bioresource technology》2011,102(6):4449-4455
Using standard laboratory equipment, thermochemically pretreated oat straw was enzymatically saccharified and fermented to ethanol, and after removal of ethanol the remaining material was subjected to biogas digestion. A detailed mass balance calculation shows that, for steam explosion pretreatment, this combined ethanol fermentation and biogas digestion converts 85-87% of the higher heating value (HHV) of holocellulose (cellulose and hemicellulose) in the oat straw into biofuel energy. The energy (HHV) yield of the produced ethanol and methane was 9.5-9.8 MJ/(kg dry oat straw), which is 28-34% higher than direct biogas digestion that yielded 7.3-7.4 MJ/(kg dry oat straw). The rate of biogas formation from the fermentation residues was also higher than from the corresponding pretreated but unfermented oat straw, indicating that the biogas digestion could be terminated after only 24 days. This suggests that the ethanol process acts as an additional pretreatment for the biogas process. 相似文献
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Harder DR Narayanan J Gebremedhin D 《American journal of physiology. Heart and circulatory physiology》2011,300(5):H1557-H1565
While myogenic force in response to a changing arterial pressure has been described early in the 20th century, it was not until 1984 that the effect of a sequential increase in intraluminal pressure on cannulated cerebral arterial preparations was found to result in pressure-dependent membrane depolarization associated with spike generation and reduction in lumen diameter. Despite a great deal of effort by different laboratories and investigators, the identification of the existence of a mediator of the pressure-induced myogenic constriction in arterial muscle remained a challenge. It was the original finding by our laboratory that demonstrated the capacity of cerebral arterial muscle cells to express the cytochrome P-450 4A enzyme that catalyzes the formation of the potent vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid, the production of which in cerebral arterial muscle cells increases with the elevation in intravascular pressure. 20-HETE activates protein kinase C and causes the inhibition of Ca(2+)-activated K(+) channels, depolarizes arterial muscle cell membrane, and activates L-type Ca(2+) channel to increase intracellular Ca(2+) levels and evoke vasoconstriction. The inhibition of 20-HETE formation attenuates pressure-induced arterial myogenic constriction in vitro and blunts the autoregulation of cerebral blood flow in vivo. We suggest that the formation and action of cytochrome P-450-derived 20-HETE in cerebral arterial muscle could play a critically important role in the control of cerebral arterial tone and the autoregulation of cerebral blood flow under physiological conditions. 相似文献
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The metabolism of 25-hydroxyvitamin D(3) was studied with a crude mitochondrial cytochrome P450 extract from pig kidney and with recombinant human CYP27A1 (mitochondrial vitamin D(3) 25-hydroxylase) and porcine CYP2D25 (microsomal vitamin D(3) 25-hydroxylase). The kidney mitochondrial cytochrome P450 catalyzed the formation of 1alpha,25-dihydroxyvitamin D(3), 24,25-dihydroxyvitamin D(3) and 25,27-dihydroxyvitamin D(3). An additional metabolite that was separated from the other hydroxylated products on HPLC was also formed. The formation of this 25-hydroxyvitamin D(3) metabolite was dependent on NADPH and the mitochondrial electron transferring protein components. A monoclonal antibody directed against purified pig liver CYP27A1 immunoprecipitated the 1alpha- and 27-hydroxylase activities towards 25-hydroxyvitamin D(3) as well as the formation of the unknown metabolite. These results together with substrate inhibition experiments indicate that CYP27A1 is responsible for the formation of the unknown 25-hydroxyvitamin D(3) metabolite in kidney. Recombinant human CYP27A1 was found to convert 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3), 25,27-dihydroxyvitamin D(3) and a major metabolite with the same retention time on HPLC as that formed by kidney mitochondrial cytochrome P450. Gas chromatography-mass spectrometry (GC-MS) analysis of the unknown enzymatic product revealed it to be a triol different from other known hydroxylated 25-hydroxyvitamin D(3) metabolites such as 1alpha,25-, 23,25-, 24,25-, 25,26- or 25,27-dihydroxyvitamin D(3). The product had the mass spectrometic properties expected for 4beta,25-dihydroxyvitamin D(3). Recombinant porcine CYP2D25 converted 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3) and 25,26-dihydroxyvitamin D(3). It can be concluded that both CYP27A1 and CYP2D25 are able to carry out multiple hydroxylations of 25-hydroxyvitamin D(3). 相似文献
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René Thieme Susanne Kurz Marlen Kolb Tewodros Debebe Susanne Holtze Michaela Morhart Klaus Huse Karol Szafranski Matthias Platzer Thomas B. Hildebrandt Gerd Birkenmeier 《PloS one》2015,10(6)
Background
The naked mole-rat (NMR) is a long-lived and cancer resistant species. Identification of potential anti-cancer and age related mechanisms is of great interest and makes this species eminent to investigate anti-cancer strategies and understand aging mechanisms. Since it is known that the NMR expresses higher liver mRNA-levels of alpha 2-macroglobulin than mice, nothing is known about its structure, functionality or expression level in the NMR compared to the human A2M.Results
Here we show a comprehensive analysis of NMR- and human plasma-A2M, showing a different prediction in glycosylation of NMR-A2M, which results in a higher molecular weight compared to human A2M. Additionally, we found a higher concentration of A2M (8.3±0.44 mg/mL vs. and 4.4±0.20 mg/mL) and a lower total plasma protein content (38.7±1.79 mg/mL vs. 61.7±3.20 mg/mL) in NMR compared to human. NMR-A2M can be transformed by methylamine and trypsin resulting in a conformational change similar to human A2M. NMR-A2M is detectable by a polyclonal antibody against human A2M. Determination of tryptic and anti-tryptic activity of NMR and human plasma revealed a higher anti-tryptic activity of the NMR plasma. On the other hand, less proteolytic activity was found in NMR plasma compared to human plasma.Conclusion
We found transformed NMR-A2M binding to its specific receptor LRP1. We could demonstrate lower protein expression of LRP1 in the NMR liver tissue compared to human but higher expression of A2M. This was accompanied by a higher EpCAM protein expression as central adhesion molecule in cancer progression. NMR-plasma was capable to increase the adhesion in human fibroblast in vitro most probably by increasing CD29 protein expression. This is the first report, demonstrating similarities as well as distinct differences between A2M in NMR and human plasma. This might be directly linked to the intriguing phenotype of the NMR and suggests that A2M might probably play an important role in anti-cancer and the anti-aging mechanisms in the NMR. 相似文献8.
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Raymond C Koehler Debebe Gebremedhin David R Harder 《Journal of applied physiology》2006,100(1):307-317
Astrocytes send processes to synapses and blood vessels, communicate with other astrocytes through gap junctions and by release of ATP, and thus are an integral component of the neurovascular unit. Electrical field stimulations in brain slices demonstrate an increase in intracellular calcium in astrocyte cell bodies transmitted to perivascular end-feet, followed by a decrease in vascular smooth muscle calcium oscillations and arteriolar dilation. The increase in astrocyte calcium after neuronal activation is mediated, in part, by activation of metabotropic glutamate receptors. Calcium signaling in vitro can also be influenced by adenosine acting on A2B receptors and by epoxyeicosatrienoic acids (EETs) shown to be synthesized in astrocytes. Prostaglandins, EETs, arachidonic acid, and potassium ions are candidate mediators of communication between astrocyte end-feet and vascular smooth muscle. In vivo evidence supports a role for cyclooxygenase-2 metabolites, EETs, adenosine, and neuronally derived nitric oxide in the coupling of increased blood flow to increased neuronal activity. Combined inhibition of the EETs, nitric oxide, and adenosine pathways indicates that signaling is not by parallel, independent pathways. Indirect pharmacological results are consistent with astrocytes acting as intermediaries in neurovascular signaling within the neurovascular unit. For specific stimuli, astrocytes are also capable of transmitting signals to pial arterioles on the brain surface for ensuring adequate inflow pressure to parenchymal feeding arterioles. Therefore, evidence from brain slices and indirect evidence in vivo with pharmacological approaches suggest that astrocytes play a pivotal role in regulating the fundamental physiological response coupling dynamic changes in cerebral blood flow to neuronal synaptic activity. Future work using in vivo imaging and genetic manipulation will be required to provide more direct evidence for a role of astrocytes in neurovascular coupling. 相似文献
10.
Qian W Chen JJ Human J Aya T Zhu J Biswas K Peterkin T Hungate RW Arik L Johnson E Kumar G Joseph S Jona J Guo HX Wu Z 《Bioorganic & medicinal chemistry letters》2012,22(2):1061-1067
In a series of bradykinin B1 antagonists, we discovered that replacement of oxopiperazine acetamides with dehydro-oxopiperazine acetamides provided compounds with enhanced activity against the B1 receptor. The synthesis and SAR leading to potent analogs with reduced molecular weight will be discussed. 相似文献