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71.
Todd J Menkhaus Sara U Eriksson Paul B Whitson Charles E Glatz 《Biotechnology and bioengineering》2002,77(2):148-154
Host selection can be a strategy to simplify downstream processing for protein recovery. Advancing capabilities for using plants as hosts offers new host opportunities that have received only limited attention from a downstream processing perspective. Here, we investigated the potential of using a polycationic precipitating agent (polyethylenimine; PEI) to precipitate an acidic model protein (beta-glucuronidase; GUS) from aqueous plant extracts. To assess the potential of host selection to enhance the ease of recovery, the same procedure was applied to oilseed extracts of canola, corn (germ), and soy. For comparison, PEI precipitation of GUS was also evaluated from a crude bacterial fermentation broth. Two versions of the target protein were investigated--the wild-type enzyme (WTGUS) and a genetically engineered version containing 10 additional aspartates on each of the enzyme's four homologous subunits (GUSD10). It was found that canola was the most compatible expression host for use with this purification technique. GUS was completely precipitated from canola with the lowest dosage of PEI (30 mg PEI/g total protein), and over 80% of the initial WTGUS activity was recovered with 18-fold purification. Precipitation from soy gave yields over 90% for WTGUS but only 1.3-fold enrichment. Corn, although requiring the most PEI relative to total protein to precipitate (210 mg PEI/g total protein for 100% precipitation), gave intermediate results, with 81% recovery of WTGUS activity and a purification factor of 2.6. The addition of aspartate residues to the target protein did not enhance the selectivity of PEI precipitation in any of the systems tested. In fact, the additional charge reduced the ability to recover GUSD10 from the precipitate, resulting in lower yields and enrichment ratios compared to WTGUS. Compared to the bacterial host, plant systems provided lower polymer dosage requirements, higher yields of recoverable activity and greater purification factors. 相似文献
72.
Mafalda C. O. Figueiredo Susana A. L. Lobo Sara H. Sousa Fábio P. Pereira Judy D. Wall Lígia S. Nobre Lígia M. Saraiva 《Journal of bacteriology》2013,195(11):2684-2690
Desulfovibrio species are Gram-negative anaerobic sulfate-reducing bacteria that colonize the human gut. Recently, Desulfovibrio spp. have been implicated in gastrointestinal diseases and shown to stimulate the epithelial immune response, leading to increased production of inflammatory cytokines by macrophages. Activated macrophages are key cells of the immune system that impose nitrosative stress during phagocytosis. Hence, we have analyzed the in vitro and in vivo responses of Desulfovibrio vulgaris Hildenborough to nitric oxide (NO) and the role of the hybrid cluster proteins (HCP1 and HCP2) and rubredoxin oxygen oxidoreductases (ROO1 and ROO2) in NO protection. Among the four genes, hcp2 was the gene most highly induced by NO, and the hcp2 transposon mutant exhibited the lowest viability under conditions of NO stress. Studies in murine macrophages revealed that D. vulgaris survives incubation with these phagocytes and triggers NO production at levels similar to those stimulated by the cytokine gamma interferon (IFN-γ). Furthermore, D. vulgaris
hcp and roo mutants exhibited reduced viability when incubated with macrophages, revealing that these gene products contribute to the survival of D. vulgaris during macrophage infection. 相似文献
73.
In vitro improvement of quail primordial germ cell expansion through activation of TGF‐beta signaling pathway
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74.
75.
Giovanni Corso Joana Figueiredo Simone Pietro De Angelis Federica Corso Antonia Girardi Joana Pereira Raquel Seruca Bernardo Bonanni Patricia Carneiro Gabriella Pravettoni Elena Guerini Rocco Paolo Veronesi Giacomo Montagna Virgilio Sacchini Sara Gandini 《Journal of cellular and molecular medicine》2020,24(11):5930-5936
E-cadherin protein (CDH1 gene) integrity is fundamental to the process of epithelial polarization and differentiation. Deregulation of the E-cadherin function plays a crucial role in breast cancer metastases, with worse prognosis and shorter overall survival. In this narrative review, we describe the inactivating mechanisms underlying CDH1 gene activity and its possible translation to clinical practice as a prognostic biomarker and as a potential targeted therapy. 相似文献
76.
Jacopo Burrello Sara Bolis Carolina Balbi Alessio Burrello Elena Provasi Elena Caporali Lorenzo Grazioli Gauthier Andrea Peirone Fabrizio D'Ascenzo Silvia Monticone Lucio Barile Giuseppe Vassalli 《Journal of cellular and molecular medicine》2020,24(17):9945-9957
The current standard biomarker for myocardial infarction (MI) is high‐sensitive troponin. Although powerful in clinical setting, search for new markers is warranted as early diagnosis of MI is associated with improved outcomes. Extracellular vesicles (EVs) attracted considerable interest as new blood biomarkers. A training cohort used for diagnostic modelling included 30 patients with STEMI, 38 with stable angina (SA) and 30 matched‐controls. Extracellular vesicle concentration was assessed by nanoparticle tracking analysis. Extracellular vesicle surface‐epitopes were measured by flow cytometry. Diagnostic models were developed using machine learning algorithms and validated on an independent cohort of 80 patients. Serum EV concentration from STEMI patients was increased as compared to controls and SA. EV levels of CD62P, CD42a, CD41b, CD31 and CD40 increased in STEMI, and to a lesser extent in SA patients. An aggregate marker including EV concentration and CD62P/CD42a levels achieved non‐inferiority to troponin, discriminating STEMI from controls (AUC = 0.969). A random forest model based on EV biomarkers discriminated the two groups with 100% accuracy. EV markers and RF model confirmed high diagnostic performance at validation. In conclusion, patients with acute MI or SA exhibit characteristic EV biomarker profiles. EV biomarkers hold great potential as early markers for the management of patients with MI. 相似文献
77.
Pithecellobium dulce has been used in traditional medicine to treat various ailments owing to its restorative properties. The biological activities and chemical profiles of the lipophilic fraction of P. dulce bark and leaves were assessed herein. Fatty acid methyl esters (FAME) and unsaponifiable matter (USM) were prepared and analyzed by GC/MS. A total of 40 compounds were identified in the bark saponifiable fraction, whereas 9 compounds were annotated in the leaves. Palmitic acid methyl ester was the major compound identified accounting for 41.48 % of the bark and 19.03 % of the leaves composition. Besides, linolenic acid methyl ester (22.40 %) and linoleic acid (12.69 %) were annotated in the leaves saponifiable fraction. A total of 63 compounds were detected in the bark USM and 4 compounds were identified in the leaves. Phytol represented the major component in the leaves (52.57 %) followed by lupeol (20.68 %) and lupenone (8.60 %). Meanwhile, n‐dodecane dominated in the bark USM accounting for 24.69 % of the total composition. The leaves and bark lipophilic fractions revealed moderate antioxidant and antibacterial activities. Both extracts showed no antifungal activity. No cytotoxicity was observed for both lipophilic fractions. P. dulce offers a good source of antioxidant compounds that can be introduced to food and pharmaceutical industry. 相似文献
78.
Mono‐ and multimetallic nanoparticles (NPs) have diverse and tunable physicochemical properties that arise from their compositions as well as crystallite size and shape. The ability to control precisely the composition and structure of NPs through synthesis is central to achieving state‐of‐the‐art designer metal NPs for use as catalysts and electrocatalysts. However, a major limitation to the use of designer metal NPs as catalysts is the ability to scale their syntheses while maintaining structural precision. To address this challenge, continuous flow routes to metal NPs involving the use of droplet microreactors are being developed, providing the synthetic versatility necessary to achieve known and completely new nanostructures. This progress report outlines how the chemistry and process parameters of droplet microreactors can be used to achieve high performing nanocatalysts through control of NP composition, size, shape, and architecture and outlines directions toward previously unimaginable nanostructures. 相似文献
79.
In this work a new highly fluorescent N,N‐dimethyl benzylamine–palladium(II) yu complex was synthesized by the reaction of [Pd2{(C,N–C6H4CH2N(CH3)2}2(μ‐OAc)]2] with curcumin. The structure of the synthesized complex was characterized using Fourier transform infra‐red (FT‐IR) spectroscopy, 1H nuclear magnetic resonance spectroscopy, and elemental analysis. Fluorescence quantum yield (ΦF) values of the synthesized complex in dimethyl sulfoxide (DMSO), acetonitrile, ethanol, and methanol were 0.160, 0.104, 0.068, and 0.061, respectively. The fluorescence signal of the complex in the organic solvents was very sensitive to the water content of the organic solvent. The quenching effect of water was used to determine trace amounts of water in the heteroatom‐containing organic solvents (ethanol, methanol, acetonitrile) and redox‐active solvents (DMSO). The linear ranges for determination of water (v/v %) in ethanol, DMSO and acetonitrile were found to be 0.03–14.5, 0.08–13.8, and 0.07–18.8, respectively. Two linear ranges were found for determination of water (v/v %) in methanol (0.1–1.2 and 4.7–25.0). Detection limit (DL) values were calculated to be 0.001, 0.05, 0.004, and 0.01 (v/v %) in ethanol, methanol, acetonitrile, and DMSO, respectively. The proposed method overcomes the problems of the standard Karl Fischer method for determination of water in DMSO. In addition, it gave the best DL value for determination of water in ethanol compared with all published papers to date. 相似文献
80.
Rossana Saracino Chiara Capponi Sara Di Persio Carla Boitani Silvia Masciarelli Francesco Fazi Stefania Fera Elena Vicini 《Molecular reproduction and development》2020,87(4):419-429
Glial cell line‐derived neurotrophic factor (GDNF) and retinoic acid (RA) are two molecules crucial for the regulation of the spermatogonial compartment of the testis. During the cycle of the seminiferous epithelium, their relative concentration oscillates with lower GDNF levels in stages where RA levels are high. It has been recently shown that RA negatively regulates Gdnf expression but the mechanisms behind are so far unknown. Here, we show that RA directly downregulates Gdnf mRNA levels in primary murine Sertoli cells through binding of RARα to a novel DR5‐RARE on Gdnf promoter. Pharmacological inhibition and chromatin immunoprecipitation–quantitative polymerase chain reaction analysis suggested that the underlying mechanism involved histone deacetylase activity and epigenetic repression of Gdnf promoter upon RA treatment. 相似文献