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Sample records for culture filtrate protein

  1. Early secretory antigenic target protein-6/culture filtrate protein-10 fusion protein-specific Th1 and Th2 response and its diagnostic value in tuberculous pleural effusion

    Institute of Scientific and Technical Information of China (English)

    戈启萍

    2013-01-01

    Objective To detect the Th1 and Th2 cell percentage in pleural effusion mononuclear cells (PEMCs) stimulated by early secretory antigenic target protein-6 (ESAT-6) /culture filtrate protein-10 (CFP-10) fusion protein (E/C) with flow cytometry (FCM) ,and to explore the local antigen specific Th1 and Th2 response and

  2. Identification and characterization of a 29-kilodalton protein from Mycobacterium tuberculosis culture filtrate recognized by mouse memory effector cells

    DEFF Research Database (Denmark)

    Rosenkrands, I; Rasmussen, P.B.; Carnio, M;

    1998-01-01

    in two-dimensional electrophoresis gels as three distinct spots just below the antigen 85 complex component MPT59, CFP29 is present in both culture filtrate and the membrane fraction from M. tuberculosis, suggesting that this antigen is released from the envelope to culture filtrate during growth...

  3. Prolactin modulates cytokine production induced by culture filtrate proteins of M. bovis through different signaling mechanisms in THP1 cells.

    Science.gov (United States)

    Martínez-Neri, Priscila A; López-Rincón, Gonzalo; Mancilla-Jiménez, Raúl; del Toro-Arreola, Susana; Muñoz-Valle, José Francisco; Fafutis-Morris, Mary; Bueno-Topete, Miriam Ruth; Estrada-Chávez, Ciro; Pereira-Suárez, Ana Laura

    2015-01-01

    The immunomodulatory functions of prolactin (PRL) are well recognized. Augmented PRL plasma levels were observed in patients with advanced tuberculosis (TB). Recently, we have reported that LPS and Mycobacterium bovis (M. bovis) induced differential expression of PRL receptor (PRLR) isoforms in THP-1 cells and bovine macrophages, respectively. The aim of this work was to determine whether PRL should be considered as a potential modulator of the signaling pathways and cytokine synthesis, induced by culture filtrate protein (CFP) from M. bovis in THP-1 monocytes. The THP-1 cells were stimulated with PRL (20ng/mL), M. bovis CFP (50μg/mL). PRLR as well as phosphorylated STAT3, STAT5, Akt1/2/3, ERK1/2 and p38 expression were evaluated by Western blot. IL1-β, TNF-α, IL-6, IL-12, IL-8, and IL-10 concentrations were measured by ELISA. Our results demonstrated that the expression pattern of PRLR short isoforms is induced by M. bovis CFP. M bovis CFP induced phosphorylation of Akt2, ERK1/2, p38, STAT3, and STAT5 pathways. In turn, PRL only activated the JAK2/STAT3-5 signaling pathway. However, when combined both stimuli, PRL significantly increased STAT3-5 phosphorylation and downregulated Akt2, ERK1/2, and p38 phosphorylation. As expected, M. bovis CFP induced substantial amounts of IL1-β, IL-6, TNF-α, IL-8, IL-12, and IL-10. However, the PRL costimulation considerably decreased IL1-β, TNF-α, and IL-12 secretion, and increased IL-10 production. This results suggest that up-regulation of IL-10 by PRL might be modulating the pro-inflammatory response against mycobacterial antigens through the MAPK pathway.

  4. Improving basic and membrane protein MS detection of the culture filtrate proteins from Mycobacterium tuberculosis H37Rv by biomimetic affinity pre-fractionation.

    Science.gov (United States)

    Ma, Guorong; Zhou, Fuqiang; Gao, Lina; Sun, Zhanqiang; Deng, Li; Zhang, Shulin; Li, Rongxiu

    2017-04-09

    The culture filtrate proteins (CFPs) from Mycobacterium tuberculosis (MTB) have been shown to induce protective immune responses in human and animal models, making them a promising source of candidate targets for tuberculosis drugs, vaccines and diagnostics. The constituents of the MTB CFP proteome are complex and vary with growth conditions. To effectively profile CFPs, gel-based pre-fractionation is usually performed before MS analysis. In this study, we describe a novel pre-fractionation approach by which the proteome is divided into seven partially overlapping fractions by biomimetic affinity chromatography (BiAC) using a six-column cascade. The LC-MS/MS analysis of individual fractions identified a total of 541 CFPs, including 61 first-time identifications. Notably, ∼1/3 (20/61) of these novel CFPs are membrane proteins, among which 9 proteins have 2-14 transmembrane domains (TMD). In addition, ∼1/4 (14/61) of the CFPs are basic proteins with pI values greater than 9.0. Our data demonstrate that BiAC pre-fractionation markedly improves protein detection by LC-MS/MS, and the coverage of basic and hydrophobic proteins in particular is remarkably increased. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  5. Role of alpha-crystallin, early-secreted antigenic target 6-kDa protein and culture filtrate protein 10 as novel diagnostic markers in osteoarticular tuberculosis

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    Nazia Rizvi

    2016-07-01

    Full Text Available Osteoarticular tuberculosis constitutes about 3% of all tuberculosis cases. Early and accurate diagnosis of tuberculosis is a challenging problem especially in the case of osteoarticular tuberculosis owing to the lower number of bacilli. However, an accurate and timely diagnosis of the disease results in an improved efficacy of the given treatment. Besides the limitations of conventional methods, nowadays molecular diagnostic techniques have emerged as a major breakthrough for the early diagnosis of tuberculosis with high sensitivity and specificity. Alpha-crystallin is a dominantly expressed protein responsible for the long viability of the pathogen during the latent phase under certain stress conditions such as hypoxia and nitric oxide stress. Two other proteins—early secreted antigenic target-6 and culture filtrate protein-10—show high expression in the active infective phase of Mycobacterium tuberculosis. In this article, we focus on the different proteins expressed dominantly in latent/active tuberculosis, and which may be further used as prognostic biomarkers for diagnosing tuberculosis, both in latent and active phases.

  6. Effects of broth culture filtrate protein of VacA+ Helicobacter pylori on the proliferation and apoptosis of gastric epithelial cells.

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    Zhao, Yu-qing; Guo, Tao; Qian, Jia-ming

    2013-01-01

    Infection with Helicobacter pylori (H. pylori) may lead to chronic inflammation of the stomach epithelium, mucosal atrophy, imbalance of proliferation and apoptosis of epithelial cells; resulting in chronic gastritis, peptic ulcer, gastric cancer, and many other clinical outcomes. Why and how H. pylorus leads to gastric cancer is not clear yet. Through in vitro experiments, this study evaluated the effects of broth culture filtrate protein (BCF-P) from the supernatant of liquid culture media of H. pylori on proliferation and apoptosis of immortalized human gastric epithelial cell lines (GES-1) and gastric cancer cell lines (AGS). For the study, GES-1 and AGS cell lines mix with BCF-P and epidermal growth factor (EGF). MTT assay and flow cytometry (FCM) determined the levels of proliferation and apoptosis. Detected expression levels of cyclooxygenase-2 (COX-2) and Fas mRNA by reverse transcription (RT)-PCR. Also did analysis of the effects of BCF-P on epidermal growth factor receptor (EGFR) tyrosine kinase activity of GES-1 and AGS cells by non-radioactive enzyme-linked assay. The Student's t test and one-way analysis of variance (ANOVA) were used for statistical analysis. BCF-P inhibited proliferation of GES-1 and AGS cells in a concentration-dependent manner. The inhibition rates are respectively 68.7% in AGS and 61.4% in GES-1. With the same dose and time for inhibiting the proliferation, BCF-P failed to induce apoptosis of GES-1 and AGS cells. Effects of BCF-P reduced the expression of Fas mRNA of GES-1 and AGS cells (P cells (P cells. BCF-P inhibited the proliferation of AGS and GES-1 cells in vitro, unrelated to apoptosis. Effects of BCF-P on gastric epithelial cells in vitro are not equivalent to that of EGF.

  7. Effects of broth culture filtrate protein of VacA+ Helicobacter pylori on the proliferation and apoptosis of gastric epithelial cells

    Institute of Scientific and Technical Information of China (English)

    ZHAO Yu-qing; GUO Tao; QIAN Jia-ming

    2013-01-01

    Background Infection with Helicobacterpylori (H.pylori) may lead to chronic inflammation of the stomach epithelium,mucosal atrophy,imbalance of proliferation and apoptosis of epithelial cells; resulting in chronic gastritis,peptic ulcer,gastric cancer,and many other clinical outcomes.Why and how H.pylorus leads to gastric cancer is not clear yet.Through in vitro experiments,this study evaluated the effects of broth culture filtrate protein (BCF-P) from the supernatant of liquid culture media of H.pylori on proliferation and apoptosis of immortalized human gastric epithelial cell lines (GES-1) and gastric cancer cell lines (AGS).Methods For the study,GES-1 and AGS cell lines mix with BCF-P and epidermal growth factor (EGF).MTT assay and flow cytometry (FCM) determined the levels of proliferation and apoptosis.Detected expression levels of cyclooxygenase-2 (COX-2) and Fas mRNA by reverse transcription (RT)-PCR.Also did analysis of the effects of BCF-P on epidermal growth factor receptor (EGFR) tyrosine kinase activity of GES-1 and AGS cells by non-radioactive enzyme-linked assay.The Student's t test and one-way analysis of variance (ANOVA) were used for statistical analysis.Results BCF-P inhibited proliferation of GES-1 and AGS cells in a concentration-dependent manner.The inhibition rates are respectively 68.7% in AGS and 61.4% in GES-1.With the same dose and time for inhibiting the proliferation,BCF-P failed to induce apoptosis of GES-1 and AGS cells.Effects of BCF-P reduced the expression of Fas mRNA of GES-1 and AGS cells (P <0.05).This is consistent with the effects of EGF.BCF-P reduced the expression of COX-2mRNA of AGS cells (P <0.05).This is opposite to the effects of EGF (P <0.05).Effects of BCF-P improved more than three times the EGFR tyrosine kinase activity of GES-1 and AGS cells.Conclusions BCF-P inhibited the proliferation of AGS and GES-1 cells in vitro,unrelated to apoptosis.Effects of BCF-P on gastric epithelial cells in vitro are not

  8. Screening culture filtrates of fungi for activity against Tylenchulus semipenetrans

    Energy Technology Data Exchange (ETDEWEB)

    Verdejo-Lucas, S.; Viera, A.; Stchigel, A. M.; Sorribas, F. J.

    2009-07-01

    Culture filtrates of 20 fungi isolated from citrus soils were screened for their activity against Tylenchulus semipenetrans in both in vitro and greenhouse tests. The filtrates of Talaromyces cyanescens (isolates 2-4 and 2-5), Paecilomyces lilacinus, Chaetomium robustum, Acremonium strictum, Engyodontium album, Myrothecium verrucaria, Emericella rugulosa, and Tarracomyces gigaspora consistently inhibited the motility of second-stage juveniles at various concentrations of the filtrate. Dose-response models were used to determine the filtrate concentration required to inhibit the motility of 50% of the juveniles (CI50). The culture filtrate of P. lilacinus showed the highest activity with a CI50 value of 58% that differed from that of C. robustum (CI50 = 68%), and A. strictum CI50 = 82%. The culture filtrates of P. lilacinus, E. album, and T. cyanescens 2-5 maintained their activity when autoclaved at 120 degree centigrade for 20 min. The autoclaved filtrate of T. cyanescens 2-4 was more effective at inhibiting juvenile motility (CI50 = 28%) than that of T. cyanescens 2-5 (CI50 = 80%), C. robustum (CI50 = 72 %) and P. lilacinus (CI50 = 72%). The culture filtrate of T. cyanescens 2-4 also inhibited egg hatching. Nematode reproduction on Cleopatra mandarin and Carrizo citrange were respectively reduced by the culture filtrate of P. lilacinus and the autoclaved filtrate of T. cyanescens 2-4. These results support the hypothesis that soil fungi may contribute to regulate nematode densities by the production of secondary metabolites with nematicidal activity. (Author) 30 refs.

  9. Oviposition Attractancy of Bacterial Culture Filtrates: response of Culex quinquefasciatus

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    S Poonam

    2002-04-01

    Full Text Available Oviposition attractants could be used for monitoring as well as controlling mosquitoes by attracting them to lay eggs at chosen sites. In the present study, culture filtrates of seven bacterial species were tested for their attractancy against gravid females of Culex quinquefasciatus. When their oviposition active indices (OAI were studied, the culture filtrates of Bacillus cereus and Pseudomonas fluorescens exhibited oviposition attractancy (OAI = >0.3 at 100 ppm and the OAI were respectively 0.70 and 0.47. Culture filtrates of B. thuringiensis var. israelensis (wild type, B. t. var. israelensis (mutant and B. sphaericus showed attractancy at 2000 ppm with OAI of respectively 0.71, 0.59 and 0.68. However, the OAI of B. megaterium as well as Azospirillum brasilense was 0.13 (at 2000 ppm, which was less than 0.3 required to be considered them as attractants. When the oviposition attractancy of the bacterial culture filtrates were compared with that of a known oviposition attractant, p-cresol (at 10 ppm, the culture filtrates of B. t. var. israelensis (wild type and B. cereus were found to be more active than p-cresol, respectively with 64.2 and 54.3% oviposition.

  10. Biological Control of Aphid Using Fungal Culture and Culture Filtrates of Beauveria bassiana.

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    Kim, Jeong Jun; Jeong, Gayoung; Han, Ji Hee; Lee, Sangyeob

    2013-12-01

    Aphids are one of the most destructive pests in crop production such as pepper, cucumber, and eggplants. The importance of entomopathogenic fungi as alternative pest control agents is increasing. Conidia of entomopathogenic fungi are influenced by environmental conditions, such as temperature and relative humidity, and cause slow and fluctuating mortality. These factors have prevented wider application and use of biocontrol agents. For investigation of means of mitigation of such problems, we conducted bioassays with 47 fungal culture filtrates in order to evaluate the potential of secondary metabolites produced by entomopathogenic fungi for use in aphid control. Among 47 culture filtrates cultured potato dextrose broth, filtrate of Beauveria bassiana Bb08 showed the highest mortality (78%) against green peach aphid three days after treatments. Filtrate of Bb08 cultured in Adamek's medium showed higher toxicity as 100% to third instar nymphs of the aphid compared with seven other filtrates cultured in different broths amended with colloidal chitin or oil. The culture filtrates and fungal cultures from media amended with colloidal chitin or oil had lower control efficacies than filtrates without these additives in three different media. These results indicate that the fungal culture fluid or culture filtrate of B. bassiana Bb08 cultured in Adamek's medium has potential for development as a mycopesticide for aphid control.

  11. Culture: A Filtration Process during Communication in Education.

    Science.gov (United States)

    de Lange, Rudi

    This paper focuses on the filtration process of culture during communication in education with reference to visual elements. An introduction provides a review of some communication models--graphic representations of theories that attempt to predict and explain the process of communication. These simple models are discussed: Aristotle's model of…

  12. Expression of 87 kD protein in the broth culture filtrate of Helicobacter pylori and its association with the vacuolating effect

    Institute of Scientific and Technical Information of China (English)

    SHI Li; YIE Gui-an; NAN Qing-zhen; SUN Yong; ZHANG Ya-li; ZHANG Zhen-shu; ZHOU Dian-yuan

    2001-01-01

    To study the vacuolating effect of Helicobacter pylori(H.pylori). Method: The vacuolating effect and its relationship with vacuolating cytotoxin antigen (an 87 kD protein) were investigated by the method of cytotoxic test, SDS-PAGE and scanning. Result: Of the 62 clinical isolates, 43 strains were H.pylori (Toxin+) with vacuolating effect, while the others were H.pylori (Toxin-) without vacuolating effect. Altogether 78.26%(36/46) patients with peptic ulcer were infected with H.pylori (Toxin+) strains, and only 42.86%(6/14) who had gastritis were infected with H.pylori (Toxin+) strains, with significant difference between them(χ2=4.83,P<0.05). A protein with relativemolecular mass of 87 kD was identified in the broth culture filter(BCF) of 30.23% H. Pylori (Toxin+) strains (13/43) but in none of that of H.pylori (Toxin-) strains, and the difference was statistically significant(P<0.05). There was a significant and concordant relationship between the OD value of the protein band and the titer of vacuolating activity of H.pylori (Toxin+) (r=0.67 and P<0.05 by linear regression analysis). Conclusion: H.pylori (Toxin+) were more often associated with peptic ulcerous diseases than with gastritis diseases. The vacuolating effect of H.pylori (Toxin+) may be caused by the 87 kD protein.

  13. DESTRUCTION OF XENOBIOTICS BY CULTURE FILTRATE FROM XYLOTROPHIC BASIDIOMYCETES

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    Fedotov O. V.

    2015-12-01

    Full Text Available The article deals with the efficiency of pollutants biodegradation by xylotrophic basidiomycetes submerged cultures grown on standard glucose-peptone medium (GPM. The efficiency of pollutants biodegradation was determined by the model compound – dye Methyl Orange. The purpose of the work is screening of 19 species 81 strains xylotrophic basidiomycetes cultures on the indicator of the dye oxidative degradation efficiency and exploring the possibility of induction of this indicator by modifying the culture medium. The biodegradation efficiency was determined by following method. Assigned amount of culture filtrate (experiment or medium (control was added to the 0.001% solution of Methyl Orange in sodium acetate buffer. pH of the reaction mixture was 4.4 units. Samples were incubated at +40°C for 48 hours. Then pH of the reaction mixture was set up at 3.1 units using sodium acetate buffer and the optical density of solutions at a wavelength of 506 nm was measured. The efficiency of biodegradation was calculated by the difference of the optical density of control and experiment as a percentage. The most promising strains – F. velutipes F-1105, P. eryngii P-er, T. hirsuta Th-11 and D. quercina Dq-08 were selected. The composition of the glucose-peptone medium was modified for these strains by the introduction in the medium lignosulfonate, Tween 80, Kirk’s minerals solution and selecting the concentration of these components. According to the study for the purpose of pollutants degradation it is advisable to cultivate F. velutipes F-1105 strain on modified GPM, which further comprises at 1 l: lignosulfonate – 3.5 g; Tween 80 – 1.0 g, Kirk’s minerals solution – 70 ml; P. eryngii P-er strain – 5.0 g, 1.0 g, 70 ml; T. hirsuta Th-11 strain – 5.0 g, 1.0 g, 105 ml; and D. quercina Dq-08 strain – 6.5 g, 1.0 g, 105 ml, respectively. This allowed to increase the model compound degradation efficiency by the culture filtrate of strain F

  14. Sterile Filtration of Highly Concentrated Protein Formulations: Impact of Protein Concentration, Formulation Composition, and Filter Material.

    Science.gov (United States)

    Allmendinger, Andrea; Mueller, Robert; Huwyler, Joerg; Mahler, Hanns-Christian; Fischer, Stefan

    2015-10-01

    Differences in filtration behavior of concentrated protein formulations were observed during aseptic drug product manufacturing of biologics dependent on formulation composition. The present study investigates filtration forces of monoclonal antibody formulations in a small-scale set-up using polyvinylidene difluoride (PVDF) or polyethersulfone (PES) filters. Different factors like formulation composition and protein concentration related to differences in viscosity, as well as different filtration rates were evaluated. The present study showed that filtration behavior was influenced by the presence or absence of a surfactant in the formulation, which defines the interaction between filter membrane and surface active formulation components. This can lead to a change in filter resistance (PES filter) independent on the buffer system used. Filtration behavior was additionally defined by rheological non-Newtonian flow behavior. The data showed that high shear rates resulting from small pore sizes and filtration pressure up to 1.0 bar led to shear-thinning behavior for highly concentrated protein formulations. Differences in non-Newtonian behavior were attributed to ionic strength related to differences in repulsive and attractive interactions. The present study showed that the interplay of formulation composition, filter material, and filtration rate can explain differences in filtration behavior/filtration flux observed for highly concentrated protein formulations thus guiding filter selection.

  15. Effects of different Helicobacter pylori culture filtrates on growth of gastric epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Yan-Guo Yan; Gang Zhao; Jin-Ping Ma; Shi-Rong Cai; Wen-Hua Zhan

    2008-01-01

    AIM: To study the effects of different Helicobacter pylori (H py/orl) culture filtrates on growth of gastric epithelial cells.METHODS: Broth culture filtrates of H pylori were prepared. Gastric epithelial cells were treated with the filtrates, and cell growth was determined by growth curve and flow cytometry. DNA damage of gastric epithelial cells was measured by single-cell microgel electrophoresis.RESULTS: Gastric epithelial cells proliferated actively when treated by CagA-gene-positive broth culture filtrates, and colony formation reached 40%. The number of cells in S phase increased compared to controls. Comet assay showed 41.2% comet cells in GES-1 cells treated with CagA-positive filtrates (P<0.05).CONCLUSION: CagA-positive filtrates enhance the changes in morphology and growth characteristics of human gastric epithelial tumor cells. DNA damage maybe one of the mechanisms involved in the growth changes.

  16. In Vitro Selection of Peanut Somatic Embryos on Medium Containing Culture Filtrate of Sclerotium rolfsii and Plantlet Regeneration

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    YUSNITA

    2005-06-01

    Full Text Available Attempts to identify somaclonal variants of peanut with resistance to Sclerotium stem rot disease due to infection of S. rolfsii were conducted. The objectives of this study were to develop in vitro selection method using culture filtrates of S. rolfsii, identify culture filtrate-insensitive somatic embryo (SE of peanut after in vitro selection and regenerate peanut R0 lines originated from culture filtrate-insensitive SE. To achieve these objectives, peanut embryogenic tissues were cultured on selective medium containing various concentrations of S. rolfsii culture filtrates and sublethal concentration of the filtrates. Medium containing sublethal level of S. rolfsii culture filtrates was used to identify culture filtrate-insensitive SE of peanut. Subsequently, the selected SEs were germinated, plantlets were regenerated and preliminary tested against S. rolfsii. Results of the experiments showed that addition of S. rolfsii culture filtrates into medium for inducing peanut somatic embryos drastically reduced their growth and proliferation. S. rolfsii culture filtrates at 10% concentration has significantly reduced the number of proliferated SE per explant. However, sublethal level was achieved at 30% of culture filtrates concentration. Responses of five peanut cultivars against 30% of culture filtrates were similar, indicating they were similar in their susceptibility against S. rolfsii. A number of culture filtrate-insensitive SE were identified after culturing 1500 clumps of embryogenic tissue of peanut cv. Kelinci for three consecutive passages on medium containing 30% of culture filtrates. Germination of selected SE and regeneration of plantlet from culture filtrate-insensitive SE resulted in 50 peanut R0 lines. These lines have been grown in the plastic house and produced normal seeds for further evaluation. Results of S. rolfsii inoculation indicated the existence of chimera for insensitivity against S. rolfsii.

  17. Response of Vitis vinifera cell cultures to Eutypa lata and Trichoderma atroviride culture filtrates: expression of defence-related genes and phenotypes.

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    Mutawila, C; Stander, C; Halleen, F; Vivier, M A; Mostert, L

    2017-03-01

    Cell suspension cultures of Vitis vinifera cv. Dauphine berries were used to study the response to the vascular pathogen, Eutypa lata, in comparison with a biological control agent, Trichoderma atroviride, that was previously shown to be effective in pruning wound protection. The expression of genes coding for enzymes of the phenylpropanoid pathway and pathogenesis-related (PR) proteins was profiled over a 48-h period using quantitative reverse transcriptase PCR. The cell cultures responded to elicitors of both fungi with a hypersensitive-like response that lead to a decrease in cell viability. Similar genes were triggered by both the pathogen and biocontrol agent, but the timing patterns and magnitude of expression was dependent on the specific fungal elicitor. Culture filtrates of both fungi caused upregulation of phenylalanine ammonia-lyase (PAL), 4-coumaroyl Co-A ligase (CCo-A) and stilbene synthase (STS), and a downregulation of chalcone synthase (CHS) genes. The pathogen filtrate caused a biphasic pattern in the upregulation of PAL and STS genes which was not observed in cells treated with filtrates of the biocontrol agent. Analytical assays showed significantly higher total phenolic content and chitinolytic enzyme activity in the cell cultures treated with the T. atroviride filtrate compared to the pathogen filtrate. These results corresponded well to the higher expression of PAL and chitinase class IV genes. The response of the cell cultures to T. atroviride filtrate provides support for the notion that the wound protection by the biocontrol agent at least partially relies on the induction of grapevine resistance mechanisms.

  18. Effect of temperature, pH and detergents on the antifungal activity of bacterial culture filtrates against Mycosphaerella fijiensis

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    Eilyn Mena

    2014-01-01

    Full Text Available The bacteria associated to crops have been studied as potential biocontrol agents. However, few investigations on the interaction Musa spp. - Mycosphaerella fijiensis-Musa associated bacteria have been developed. Consequently, bacterial metabolites involved and the effect on them of physical and chemical factors remain unknown. Therefore, this study aimed to determine the effect of temperature, pH and detergents on bacterial culture filtrates with antifungal activity in vitro against Mycosphaerella fijiensis. The pathogen growth inhibition was assessed by absorbance reading at OD 565nm. It was found that the antifungal activity of the bacterial culture filtrates against M. fijiensis, varied in the presence of different values of temperature, pH, and types of detergents and this was related to the bacterial strain. The results suggested the possible protein nature of the metabolites with antifungal activity. Keywords: bacteria, biological control, antifungal metabolites

  19. A Laboratory Exercise for Visible Gel Filtration Chromatography Using Fluorescent Proteins

    Science.gov (United States)

    Zhang, Wenqiang; Cao, Yibin; Xu, Lishan; Gong, Jufang; Sun, Meihao

    2015-01-01

    Gel filtration chromatography (GFC) separates molecules according to size and is one of the most widely used methods for protein purification. Here, red fluorescent protein (RFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and/or their fusion proteins were prokaryotically expressed, purified,…

  20. A Laboratory Exercise for Visible Gel Filtration Chromatography Using Fluorescent Proteins

    Science.gov (United States)

    Zhang, Wenqiang; Cao, Yibin; Xu, Lishan; Gong, Jufang; Sun, Meihao

    2015-01-01

    Gel filtration chromatography (GFC) separates molecules according to size and is one of the most widely used methods for protein purification. Here, red fluorescent protein (RFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and/or their fusion proteins were prokaryotically expressed, purified,…

  1. Characterization of Proteins in Filtrate from Biodegradation of Crop Residue

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    Horton, Wileatha; Trotman, A. A.

    1997-01-01

    Biodegradation of plant biomass is a feasible path for transformation of crop residue and recycling of nutrients for crop growth. The need to model the effects of factors associated with recycling of plant biomass resulting from hydroponic sweet potato production has led to investigation of natural soil isolates with the capacity for starch hydrolysis. This study sought to use nondenaturing gel electrophoresis to characterize the proteins present in filtered effluent from bioreactors seeded with starch hydrolyzing bacterial culture used in the biodegradation of senesced sweet potato biomass. The study determined the relative molecular weight of proteins in sampled effluent and the protein banding pattern was characterized. The protein profiles of effluent were similar for samples taken from independent runs under similar conditions of starch hydrolysis. The method can be used as a quality control tool for confirmation of starch hydrolysis of crop biomass. In addition, this method will allow monitoring for presence of contaminants within the system-protein profiles indicative of new enzymes in the bioreactors.

  2. Culture filtrate antigens and allergens of Epicoccum nigrum cultivated in modified semi-synthetic medium.

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    Bisht, Vandana; Singh, Bhanu Pratap; Kumar, Raj; Arora, Naveen; Sridhara, Susheela

    2002-05-01

    Epicoccum nigrum (EN) is an important fungal allergen for nasobronchial allergy. Fungal extracts should contain all the relevant allergen components from spores, mycelium and culture medium for the purpose of allergy diagnosis and therapy. EN extract from spore-mycelial mass has been standardized, but the culture filtrate (CF) allergens of EN have not been studied as EN grows poorly in synthetic medium. The objective of the present study was to obtain a standard CF extract of EN by cultivating the source material in a modified semi-synthetic medium and to compare this with the EN cellular extract. Sabouraud's medium containing yeast extract (50 mg/l) was filtered using 10-kDa cut-off membrane and the lower molecular mass media components were used to cultivate EN. The CF obtained after removing the spore-mycelia was dialyzed to remove media components. The CF extract was characterized by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot. It was compared with EN spore-mycelial extract by enzyme-linked immunosorbent assay (ELISA), ELISA inhibition and by intradermal testing on allergy patients. The CF extract of EN resolved into 30 protein bands on SDS-PAGE. About 27 IgG bands were detected using anti-EN rabbit antibodies and 12 IgE bands by EN-sensitive pooled patients' sera. Periodate modification of CF proteins showed that the carbohydrate moieties are not important for IgE binding. Protein components of 26, 34 and 43 kDa were recognized as the major CF allergens. Three different batches of CF extract required 7.5-9 ng of self protein for 50% inhibition of binding to anti-EN rabbit antibodies in ELISA. Intradermal testing with CF extract showed comparable allergenic potency to standardized EN spore-mycelial extract, although it contained some allergenic proteins in higher amounts as compared to the spore-mycelial extract. In summary, the semi-synthetic medium has been suitably modified for obtaining EN CF antigens. This medium can

  3. Effects of varied cultures and inductors on production of proteins and toxicity to nematodes or insects in the broth filtrate of Duddingtonia flagrans%不同培养基及诱导物对捕食性真菌Duddingtonia flagrans发酵液中蛋白质的产生及杀虫作用的影响

    Institute of Scientific and Technical Information of China (English)

    赵晓慧; 王瑞; 杨晓野; 杨莲茹; 苏佳; 李军燕; 李文生

    2012-01-01

    To study the production of proteins and toxicity to nematodes or insects in the broth filtrate of the nematode-trapping fungus,different liquid media with varied nutritional ingredients and inductors were used to culture Duddingtonia flagrans.The fermentation broth filtrations were collected,and the protein content,protease activity,alkaline phosphatase activity and toxicity of the broth filtration to nematodes or insects were evaluated to determine the best medium and induction agent for producing exoproteins and toxic substance to nematodes or insects.The results showed that different media and induction agents could significantly influence the metabolic process of D.flagrans,and it led to vary the types,concentration,activity of proteins and the phosphatase activity.Moreover,we also found the fermentation broth of D.flagrans not only had nematicidal activity,but also had a role to kill the fly larvae.The results above provided an important information for further studying the mechanism of nematode-trapping fungus to killing its prey.%为研究捕食性真菌Duddingtonia flagrans发酵液中蛋白质的产生及杀虫作用,使用不同营养成分组成的液体培养基及诱导物,对Duddingtonia flagrans进行培养和诱导,测定其发酵液中的蛋白质含量、蛋白酶活性、磷酸酶活性和杀虫作用,进而确定Duddingtonia flagrans产生胞外蛋白质和杀虫作用最适的培养基与诱导物。结果显示,不同培养基及诱导物对Duddingtonia flagrans的代谢过程会产生明显影响,导致其分泌的蛋白质种类、浓度以及蛋白酶和磷酸酶的活性显著不同;Duddingtonia flagrans发酵液不仅具有杀线虫活性,而且有杀蝇蛆作用。上述结果为进一步研究捕食线虫性真菌的杀虫机理提供了重要参考依据。

  4. Understanding and modeling alternating tangential flow filtration for perfusion cell culture.

    Science.gov (United States)

    Kelly, William; Scully, Jennifer; Zhang, Di; Feng, Gang; Lavengood, Mathew; Condon, Jason; Knighton, John; Bhatia, Ravinder

    2014-01-01

    Alternating tangential flow (ATF) filtration has been used with success in the Biopharmaceutical industry as a lower shear technology for cell retention with perfusion cultures. The ATF system is different than tangential flow filtration; however, in that reverse flow is used once per cycle as a means to minimize fouling. Few studies have been reported in the literature that evaluates ATF and how key system variables affect the rate at which ATF filters foul. In this study, an experimental setup was devised that allowed for determination of the time it took for fouling to occur for given mammalian (PER.C6) cell culture cell densities and viabilities as permeate flow rate and antifoam concentration was varied. The experimental results indicate, in accordance with D'Arcy's law, that the average resistance to permeate flow (across a cycle of operation) increases as biological material deposits on the membrane. Scanning electron microscope images of the post-run filtration surface indicated that both cells and antifoam micelles deposit on the membrane. A unique mathematical model, based on the assumption that fouling was due to pore blockage from the cells and micelles in combination, was devised that allowed for estimation of sticking factors for the cells and the micelles on the membrane. This model was then used to accurately predict the increase in transmembane pressure during constant flux operation for an ATF cartridge used for perfusion cell culture.

  5. Controlling the rejection of protein during membrane filtration by adding selected polyelectrolytes

    DEFF Research Database (Denmark)

    Pinelo, Manuel; Ferrer Roca, Carme; Meyer, Anne S.

    2012-01-01

    Electrostatic interactions among the charged groups on proteins and/or between proteins and other solutes significantly affect the aggregation/deposition phenomena that induce fouling and decrease permeate flux during membrane purification of proteins. Such interactions can be turned into an adva...... help enhance the performance of membrane filtration for fractionation/purification of a target protein by significantly reducing fouling and modifying rejection/selectivity....

  6. Structural Revision of Isovalertatins D03 and D23 Isolated from the Culture Filtrate of Streptomyces luteogriseus

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Two aminooligosaccharides, isovalertatins D03 (1) and D23 (2), were isolated from the culture filtrate of Streptomyces luteogriseus. Their structures were reinvestigated and revised by spectroscopic evidences including ESI multistage mass spectrometry and 2-dimensional NMR techniques.

  7. Effect of Lactobacillus paracasei Culture Filtrates and Artichoke Polyphenols on Cytokine Production by Dendritic Cells

    Science.gov (United States)

    Sisto, Angelo; Luongo, Diomira; Treppiccione, Lucia; De Bellis, Palmira; Di Venere, Donato; Lavermicocca, Paola; Rossi, Mauro

    2016-01-01

    The most recent trend in research on probiotic bacteria aims at the exploitation of bioactive bacterial compounds that are responsible for health-promoting effects and suitable for medical applications. Therefore, the main purpose of this study was to ascertain if the immunomodulatory effects of L. paracasei strains on dendritic cells (DCs) were caused by bacterial metabolites released in the culture medium. For that reason, bacterial strains were grown in two media generally used for the culture of DCs, and the effects of culture filtrates on the maturation of DCs and cytokine production were evaluated. Moreover, to reveal potential synergistic effects on the immunomodulation of DCs, an artichoke phenolic extract (APE) was added to the media before bacterial growth. The experiments pointed out an interesting anti-inflammatory activity of a culture filtrate obtained after growing a probiotic L. paracasei strain in one of the media supplemented with APE. Therefore, this culture filtrate—which combines the anti-inflammatory activity and the other well-known health-promoting properties of artichoke phenolic compounds—could represent the basis for future particular exploitations. PMID:27754398

  8. Simplification of vacuole structure during plant cell death triggered by culture filtrates of Erwinia carotovora

    Institute of Scientific and Technical Information of China (English)

    Yumi Hirakawa; Toshihisa Nomura; Seiichiro Hasezawa; Takumi Higaki

    2015-01-01

    Vacuoles are suggested to play crucial roles in plant defense-related cel death. During programmed cel death, previous live cel imaging studies have observed vacuoles to become simpler in structure and have implicated this simplification as a prelude to the vacuole’s rupture and consequent lysis of the plasma membrane. Here, we examined dynamics of the vacuole in cel cycle-synchronized tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yel ow 2) cel s during cel death induced by application of culture filtrates of Erwinia carotovora. The filtrate induced death in about 90%of the cel s by 24 h. Prior to cel death, vacuole shape simplified and endoplasmic actin filaments disassembled;however, the vacuoles did not rupture until after plasma membrane integrity was lost. Instead of facilitating rupture, the simplification of vacuole structure might play a role in the retrieval of membrane components needed for defense-related cel death.

  9. Fast Filtration of Bacterial or Mammalian Suspension Cell Cultures for Optimal Metabolomics Results.

    Science.gov (United States)

    Bordag, Natalie; Janakiraman, Vijay; Nachtigall, Jonny; González Maldonado, Sandra; Bethan, Bianca; Laine, Jean-Philippe; Fux, Elie

    2016-01-01

    The metabolome offers real time detection of the adaptive, multi-parametric response of the organisms to environmental changes, pathophysiological stimuli or genetic modifications and thus rationalizes the optimization of cell cultures in bioprocessing. In bioprocessing the measurement of physiological intracellular metabolite levels is imperative for successful applications. However, a sampling method applicable to all cell types with little to no validation effort which simultaneously offers high recovery rates, high metabolite coverage and sufficient removal of extracellular contaminations is still missing. Here, quenching, centrifugation and fast filtration were compared and fast filtration in combination with a stabilizing washing solution was identified as the most promising sampling method. Different influencing factors such as filter type, vacuum pressure, washing solutions were comprehensively tested. The improved fast filtration method (MxP® FastQuench) followed by routine lipid/polar extraction delivers a broad metabolite coverage and recovery reflecting well physiological intracellular metabolite levels for different cell types, such as bacteria (Escherichia coli) as well as mammalian cells chinese hamster ovary (CHO) and mouse myeloma cells (NS0).The proposed MxP® FastQuench allows sampling, i.e. separation of cells from medium with washing and quenching, in less than 30 seconds and is robustly designed to be applicable to all cell types. The washing solution contains the carbon source respectively the 13C-labeled carbon source to avoid nutritional stress during sampling. This method is also compatible with automation which would further reduce sampling times and the variability of metabolite profiling data.

  10. FILTRATION OF NUCLEOCAPSID PROTEIN OF NEWCASTLE DISEASE VIRUS FOR IMMUNODIAGNOSTIC KIT

    Directory of Open Access Journals (Sweden)

    R. Ahmad Raus

    2011-10-01

    Full Text Available The aim of the present study is to purify recombinant nucleocapsid (NP protein of Newcastle disease virus (NDV using microfitration fitration system and improve the processing conditions as well. A microporous membrane screening using two different sizes of membrane which are 0.1µm and 0.45µm was performed before further improvement on NP protein filtration was carried out. Several conditions of the independent variables that affect the microfiltration process such as the temperature, transmembrane pressure (TMP, and viscosity were observed. It turned out to be that 0.45µm membrane gave higher yield of NP protein than that of 0.1µm membrane. Thus, 0.45µm membrane was used to improve the filtration process for NP protein. Based on the Full Factorial Design (STATISTICA 8.0, Statsoft, Inc., eight sets of experiment were designed to identify the best conditions for the NP protein filtration. From these experiments, the optimal conditions that gave the highest yield of NP protein are; TMP 4.5psi, viscosity 2.39cP at 4ºC. Based on ANOVA (analysis of variance, all the independent variables studied do not significantly affect the purification of NP protein. However, based on the lowest p value for each variable corresponding to the response, temperature has the largest effect for the NP protein yield.

  11. Production of a ruminant protein supplement by anaerobic fermentation of feedlot waste filtrate

    Energy Technology Data Exchange (ETDEWEB)

    Reddy, C.A.; Erdman, M.D.

    1977-01-01

    In studies initiated to develop simple and efficient procedures for the production of feed supplements, it was shown that the filtrate from feedlot wastes diluted with water and filtered could be fermented under anaerobic conditions by mixed rumen bacteria, Lactobacilli, or natural microflora from the feedlot wastes to produce a protein-rich feed supplement. The filtrate is low in carbohydrate and therefore supplemental carbohydrate in the form of whey, molasses, starch from potato processing wastes, or corn starch is necessary. Rigid anaerobic conditions need not be maintained nor must aseptic conditions be observed. (JSR)

  12. Fast response filter module with plug flow of filtrate for on-line sampling from submerged cultures of filamentous fungi

    NARCIS (Netherlands)

    Poulsen, B.R.; Ruijter, G.J.G.; Panneman, H.; Iversen, J.J.L.; Visser, J.

    2004-01-01

    Automatic and accurate sampling is both convenient and sometimes necessary to obtain detailed information about cell cultures. We developed an autoclavable sampling system in which culture broth was pumped through an ultrafiltration cross-flow module with a novel filtrate collecting principle and a

  13. [Culture-filtrate producing condition and biological activity of Fusarium solani].

    Science.gov (United States)

    Ding, Wenjiao; Li, Jinhua; Chai, Zhaoxiang

    2009-10-01

    To study the culture-filtrate producing condition of Fusarium Solani isolated from Astragalus root and explore the mechanism Astragalus root rot disease caused by, in order to find theoretical support for screening resistant germ plasma via mycotoxin. The method of germinating seeds in petri dish with filter paper and inhibition method for embryo growth were used to study the biological activity and the specialty of cultural filtrate of 10 F. solani isolates. The toxin produced by F. solani had strong inhibition effect in the different nutrient media, at different temperatures and under different light conditions. With extension of culturing time, embryo inhibition rate went up gradually with the strongest inhibition at the 12th day and the inhibition ratio between 92.0% -52.0%. The toxin produced at 5 degrees C to 35 degrees C inhibited embryo germination of Astragalus differently with the strongest at 25 degrees C, and next to it at 20,30 degrees C. The impact of light on bioactive substances of the toxin was not statistically distinctive, but the 24-hour darkness was benefit to toxin production. PSC had a stronger inhibition rate than the other nutrient media, next to it was PDB. After autoclaving, the toxin still kept toxic to embryo of Astragalus, which indicated that the toxin was tolerant to high temperatures. The toxin produced by F. solani at different growing condition had strong biological activity, was tolerant to high temperature. The best condition for F. solani to produce toxin was that it was cultured in PSC liquid medium, in dark, at 25 degrees C for 12 d. The toxin produced by isolate HQM40 was non-host specific toxin.

  14. Fast Filtration of Bacterial or Mammalian Suspension Cell Cultures for Optimal Metabolomics Results.

    Directory of Open Access Journals (Sweden)

    Natalie Bordag

    Full Text Available The metabolome offers real time detection of the adaptive, multi-parametric response of the organisms to environmental changes, pathophysiological stimuli or genetic modifications and thus rationalizes the optimization of cell cultures in bioprocessing. In bioprocessing the measurement of physiological intracellular metabolite levels is imperative for successful applications. However, a sampling method applicable to all cell types with little to no validation effort which simultaneously offers high recovery rates, high metabolite coverage and sufficient removal of extracellular contaminations is still missing. Here, quenching, centrifugation and fast filtration were compared and fast filtration in combination with a stabilizing washing solution was identified as the most promising sampling method. Different influencing factors such as filter type, vacuum pressure, washing solutions were comprehensively tested. The improved fast filtration method (MxP® FastQuench followed by routine lipid/polar extraction delivers a broad metabolite coverage and recovery reflecting well physiological intracellular metabolite levels for different cell types, such as bacteria (Escherichia coli as well as mammalian cells chinese hamster ovary (CHO and mouse myeloma cells (NS0.The proposed MxP® FastQuench allows sampling, i.e. separation of cells from medium with washing and quenching, in less than 30 seconds and is robustly designed to be applicable to all cell types. The washing solution contains the carbon source respectively the 13C-labeled carbon source to avoid nutritional stress during sampling. This method is also compatible with automation which would further reduce sampling times and the variability of metabolite profiling data.

  15. Microspectroscopic investigation of the membrane clogging during the sterile filtration of the growth media for mammalian cell culture.

    Science.gov (United States)

    Cao, Xiaolin; Loussaert, James A; Wen, Zai-qing

    2016-02-01

    Growth media for mammalian cell culture are very complex mixtures of several dozens of ingredients, and thus the preparation of qualified media is critical to viable cell density and final product titers. For liquid media prepared from powdered ingredients, sterile filtration is required prior to use to safeguard the cell culture process. Recently one batch of our prepared media failed to pass through the sterile filtration due to the membrane clogging. In this study, we report the root cause analysis of the failed sterile filtration based on the investigations of both the fouling media and the clogged membranes with multiple microspectroscopic techniques. Cellular particles or fragments were identified in the fouling media and on the surfaces of the clogged membranes, which were presumably introduced to the media from the bacterial contamination. This study demonstrated that microspectroscopic techniques may be used to rapidly identify both microbial particles and inorganic precipitates in the cell culture media.

  16. Affinity filtration coupled with capillary-based affinity purification for the isolation of protein complexes.

    Science.gov (United States)

    Qureshi, M S; Sheikh, Q I; Hill, R; Brown, P E; Dickman, M J; Tzokov, S B; Rice, D W; Gjerde, D T; Hornby, D P

    2013-08-01

    The isolation of complex macromolecular assemblies at the concentrations required for structural analysis represents a major experimental challenge. Here we present a method that combines the genetic power of site-specific recombination in order to selectively "tag" one or more components of a protein complex with affinity-based rapid filtration and a final step of capillary-based enrichment. This modified form of tandem affinity purification produces highly purified protein complexes at high concentrations in a highly efficient manner. The application of the method is demonstrated for the yeast Arp2/3 heptameric protein complex involved in mediating reorganization of the actin cytoskeleton.

  17. Effect of sampling time on the culturability of airborne fungi and bacteria sampled by filtration.

    Science.gov (United States)

    Durand, Kate T H; Muilenberg, Michael L; Burge, Harriet A; Seixas, Noah S

    2002-01-01

    Air sampling of bioaerosols by filtration may be preferable for many epidemiological studies because the methods can be used to collect personal samples for a full work-shift. There is some concern, however, that the viability of fungal spores and bacterial cells might be compromised by sampling for as long as a full shift. This study was designed to determine the effect of sampling up to 6 h on the viability (measured by culture) of airborne fungi and bacteria at composting facilities. Six side-by-side samples were collected in two locations at each of three composting facilities for 1 h at 2 l/m on polycarbonate filters. Two samples in each set were then capped while clean, HEPA-filtered air was drawn across two others for an additional 2 h and across the last two for an additional 5 h. Filters were washed and the samples were analyzed for culturable bacteria and fungi, and for total bacteria and fungi by microscopic counting. Concentrations ranged from 1.7 x 10(3) to 6.2 x 10(7) c.f.u./m3 of culturable fungi and 1.17 x 10(4) to 1.0 x 10(6) c.f.u./m3 of culturable bacteria. In linear models that included duration of sampling, location, and the interaction of location and sample duration, neither sample duration nor the interaction term were significant predictors of the logs of the concentrations of culturable fungi or bacteria or of the ratio of the logs of the culturable concentrations to total concentrations for fungi or bacteria. This suggests that increased sampling time does not affect the viability of the organisms commonly found in the air at composting facilities.

  18. Insecticidal Activity of Ethyl Acetate Extracts from Culture Filtrates of Mangrove Fungal Endophytes

    Science.gov (United States)

    Abraham, Silva; Basukriadi, Adi; Pawiroharsono, Suyanto

    2015-01-01

    In the search for novel potent fungi-derived bioactive compounds for bioinsecticide applications, crude ethyl acetate culture filtrate extracts from 110 mangrove fungal endophytes were screened for their toxicity. Toxicity tests of all extracts against brine shrimp (Artemia salina) larvae were performed. The extracts with the highest toxicity were further examined for insecticidal activity against Spodoptera litura larvae and acetylcholinesterase (AChE) inhibition activity. The results showed that the extracts of five isolates exhibited the highest toxicity to brine shrimp at 50% lethal concentration (LC50) values of 7.45 to 10.24 ppm. These five fungal isolates that obtained from Rhizophora mucronata were identified based on sequence data analysis of the internal transcribed spacer region of rDNA as Aspergillus oryzae (strain BPPTCC 6036), Emericella nidulans (strains BPPTCC 6035 and BPPTCC 6038), A. tamarii (strain BPPTCC 6037), and A. versicolor (strain BPPTCC 6039). The mean percentage of S. litura larval mortality following topical application of the five extracts ranged from 16.7% to 43.3%. In the AChE inhibition assay, the inhibition rates of the five extracts ranged from 40.7% to 48.9%, while eserine (positive control) had an inhibition rate of 96.8%, at a concentration of 100 ppm. The extracts used were crude extracts, so their potential as sources of AChE inhibition compounds makes them likely candidates as neurotoxins. The high-performance liquid chromatography profiles of the five extracts differed, indicating variations in their chemical constituents. This study highlights the potential of culture filtrate ethyl acetate extracts of mangrove fungal endophytes as a source of new potential bioactive compounds for bioinsecticide applications. PMID:26190921

  19. Insecticidal Activity of Ethyl Acetate Extracts from Culture Filtrates of Mangrove Fungal Endophytes.

    Science.gov (United States)

    Abraham, Silva; Basukriadi, Adi; Pawiroharsono, Suyanto; Sjamsuridzal, Wellyzar

    2015-06-01

    In the search for novel potent fungi-derived bioactive compounds for bioinsecticide applications, crude ethyl acetate culture filtrate extracts from 110 mangrove fungal endophytes were screened for their toxicity. Toxicity tests of all extracts against brine shrimp (Artemia salina) larvae were performed. The extracts with the highest toxicity were further examined for insecticidal activity against Spodoptera litura larvae and acetylcholinesterase (AChE) inhibition activity. The results showed that the extracts of five isolates exhibited the highest toxicity to brine shrimp at 50% lethal concentration (LC50) values of 7.45 to 10.24 ppm. These five fungal isolates that obtained from Rhizophora mucronata were identified based on sequence data analysis of the internal transcribed spacer region of rDNA as Aspergillus oryzae (strain BPPTCC 6036), Emericella nidulans (strains BPPTCC 6035 and BPPTCC 6038), A. tamarii (strain BPPTCC 6037), and A. versicolor (strain BPPTCC 6039). The mean percentage of S. litura larval mortality following topical application of the five extracts ranged from 16.7% to 43.3%. In the AChE inhibition assay, the inhibition rates of the five extracts ranged from 40.7% to 48.9%, while eserine (positive control) had an inhibition rate of 96.8%, at a concentration of 100 ppm. The extracts used were crude extracts, so their potential as sources of AChE inhibition compounds makes them likely candidates as neurotoxins. The high-performance liquid chromatography profiles of the five extracts differed, indicating variations in their chemical constituents. This study highlights the potential of culture filtrate ethyl acetate extracts of mangrove fungal endophytes as a source of new potential bioactive compounds for bioinsecticide applications.

  20. Filtration as the main mechanism of increased protein extravasation in liver cirrhosis

    DEFF Research Database (Denmark)

    Henriksen, J H; Parving, H H; Lassen, N A;

    1980-01-01

    albumin and 125I-labelled human IgG in eight patients with cirrhosis of the liver. The mean wedged hepatic venous pressure was 22 mmHg (range 13-34). TERalb and and TERIgG/TERalb ratio was on average 8.4 +/- 0.8%/h (SD), and 7.4 +/- 1.9%/h (SD), respectively and these values are significantly increased...... filtration (bulk flow) is the dominant process of the increase microvascular protein escape in cirrhosis, due most likely to increased hepatic, but also to increased extrahepatic splanchnic transcapillary protein flux....

  1. Simultaneous clarification of Escherichia coli culture and purification of extracellularly produced penicillin G acylase using tangential flow filtration and anion-exchange membrane chromatography (TFF-AEMC).

    Science.gov (United States)

    Orr, Valerie; Scharer, Jeno; Moo-Young, Murray; Honeyman, C Howie; Fenner, Drew; Crossley, Lisa; Suen, Shing-Yi; Chou, C Perry

    2012-07-01

    Downstream purification often represents the most cost-intensive step in the manufacturing of recombinant proteins since conventional purification processes are lengthy, technically complicated, and time-consuming. To address this issue, herein we demonstrated the simultaneous clarification and purification of the extracellularly produced recombinant protein by Escherichia coli using an integrated system of tangential flow filtration and anion exchange membrane chromatography (TFF-AEMC). After cultivation in a bench-top bioreactor with 1L working volume using the developed host/vector system for high-level expression and effective secretion of recombinant penicillin G acylase (PAC), the whole culture broth was applied directly to the established system. One-step purification of recombinant PAC was achieved based on the dual nature of membrane chromatography (i.e. microfiltration-sized pores and anion-exchange chemistry) and cross-flow operations. Most contaminant proteins in the extracellular medium were captured by the anion-exchange membrane and cells remained in the retentate, whereas extracellular PAC was purified and collected in the filtrate. The batch time for both cultivation and purification was less than 24h and recombinant PAC with high purity (19 U/mg), yield (72% recovery), and productivity (41 mg of purified PAC per liter of culture) was obtained. Due to the nature of the non-selective protein secretion system and the versatility of ion-exchange membrane chromatography, the developed system can be widely applied for effective production and purification of recombinant proteins.

  2. Aspergillus culture filtrates and sputum sols from patients with pulmonary aspergillosis cause damage to human respiratory ciliated epithelium in vitro.

    Science.gov (United States)

    Amitani, R; Murayama, T; Nawada, R; Lee, W J; Niimi, A; Suzuki, K; Tanaka, E; Kuze, F

    1995-10-01

    Aspergillus species frequently colonize lower respiratory tracts and lungs with localized underlying conditions (healed tuberculous cavity, cystic fibrosis, bronchiectasis, etc.) even in subjects without systemic predisposing factors. We investigated the in vitro effects of culture filtrates of Aspergillus species and sputum sols from patients with pulmonary aspergillosis on ciliary beat frequency (CBF) and epithelial integrity of human respiratory ciliated epithelium. Culture filtrates of 25 clinically isolated fungi (16 Aspergillus fumigatus, three Aspergillus niger, one Aspergillus flavus, three Candida albicans, and two Cryptococcus neoformans) were obtained by culturing the fungi in Medium-199 at 37 degrees C for 7 days, and five sputum sols were obtained from patients with pulmonary aspergillosis infected by A. fumigatus. During 6 h experiments using a photometric technique, 14 out of 16 A. fumigatus culture filtrates caused progressive and significant reduction in CBF associated with marked epithelial disruption, whilst the culture filtrates of A. niger and A. flavus caused minor epithelial damage without slowing of CBF, and Medium-199 alone (Control) showed neither epithelial damage nor slowing of CBF. All of the sputum sols also caused significant slowing of CBF as well as epithelial disruption. Culture filtrates of C. albicans and Cr. neoformans had no effects on human respiratory epithelium. We conclude that Aspergillus species, especially A. fumigatus release a factor (or factors) which causes damage to respiratory epithelium and slows CBF, and that these factors may contribute to the colonization of the lower respiratory tracts by the Aspergillus species and may possibly contribute to the further proliferation and spread of the lesions in pulmonary aspergillosis.

  3. Four New Isovalertatins M33, D13, D33, and D43, from the Culture Filtrate of Streptomyces luteogriseus

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Four new aminooligosaccharides, isovalertatins M33 1, D13 2, D33 3, and D43 4, were isolated from the culture filtrate of Streptomyces luteogriseus. Their structures were established by chemical and spectroscopic evidences including ESI multistage mass spectrometry and 2-dimensional NMR techniques, and were found in full consistencies with the previous profiling results proposed by LC/ESI/MS analysis.

  4. Immunotoxic effect of thiamethoxam in immunized mice with Brucella abortus cultural filtrate antigen

    Science.gov (United States)

    Salema, L. H.; Alwan, M. J.; Yousif, Afaf Abdulrahman

    2016-01-01

    Aim: This study was planned for determination the toxic effect of thiamethoxam (TMX) in immunized mice with Brucella abortus culture filtrate antigen (CFBAgs) (as a vaccine) and its role of TMX on decrease activity of B. abortus antigen on eliciting of humoral and cellular immunity. Materials and Methods: To achieve these goals 60 female mice were used, 7-8 weeks age, they were divided equally into three groups (20 in each group) and treated as follows: 1st group: Mice were immunized with CFBAgs intraperitoneally in two doses, 2 weeks intervals with (protein concentration 2 mg\\ml), 2nd group: Mice immunized as in the 1st group and was administrated orally with 1/10 lethal dose 50% of TMX (83.7 mg/kg B.W.) for 4 weeks daily, 3rd group was administrated orally with 0.3 ml normal saline served as a control group. At day 28 post immunization (PI) delayed type hypersensitivity (skin test) was done, and serum samples were collected at day 30 (PI) for detection of passive hemagglutination test (PHA); interferon gamma (IFN-γ) which was done by enzyme-linked immunosorbent assay test in addition to phagocytes assay. Results: The results of skin test post injection with soluble antigen of B. abortus intradermally showed a high significantly mean values at p≤0.05 of footpad skin thickness in the 1st group of mice which recorded (0.51±0.002 mm) as compared with the 2nd group of mice which showed (0.08±0.002 mm) after 24 h; the mean values of skin thickness were declined in the 1st mice (0.46±0.002) and 2nd mice (0.070±0.001) at 48 h; control group showed a negative results. These results were agreed with results of serum levels of IFN-γ (pg/ml) that showed that a significant increase the vaccinated 1st group (406.36±1.52), than those values in the 2nd group (151.61±0.89) and negative result in 3rd group (46.47±0.60), in addition to results of PHA test which showed a significant increase in antibody titer in the 1st group (139±12.16) with low level of serum antibody

  5. Immunotoxic effect of thiamethoxam in immunized mice with Brucella abortus cultural filtrate antigen

    Directory of Open Access Journals (Sweden)

    L. H. Salema

    2016-12-01

    Full Text Available Aim: This study was planned for determination the toxic effect of thiamethoxam (TMX in immunized mice with Brucella abortus culture filtrate antigen (CFBAgs (as a vaccine and its role of TMX on decrease activity of B. abortus antigen on eliciting of humoral and cellular immunity. Materials and Methods: To achieve these goals 60 female mice were used, 7-8 weeks age, they were divided equally into three groups (20 in each group and treated as follows: 1st group: Mice were immunized with CFBAgs intraperitoneally in two doses, 2 weeks intervals with (protein concentration 2 mg\\ml, 2nd group: Mice immunized as in the 1st group and was administrated orally with 1/10 lethal dose 50% of TMX (83.7 mg/kg B.W. for 4 weeks daily, 3rd group was administrated orally with 0.3 ml normal saline served as a control group. At day 28 post immunization (PI delayed type hypersensitivity (skin test was done, and serum samples were collected at day 30 (PI for detection of passive hemagglutination test (PHA; interferon gamma (IFN-γ which was done by enzyme-linked immunosorbent assay test in addition to phagocytes assay. Results: The results of skin test post injection with soluble antigen of B. abortus intradermally showed a high significantly mean values at p≤0.05 of footpad skin thickness in the 1st group of mice which recorded (0.51±0.002 mm as compared with the 2nd group of mice which showed (0.08±0.002 mm after 24 h; the mean values of skin thickness were declined in the 1st mice (0.46±0.002 and 2nd mice (0.070±0.001 at 48 h; control group showed a negative results. These results were agreed with results of serum levels of IFN-γ (pg/ml that showed that a significant increase the vaccinated 1st group (406.36±1.52, than those values in the 2nd group (151.61±0.89 and negative result in 3rd group (46.47±0.60, in addition to results of PHA test which showed a significant increase in antibody titer in the 1st group (139±12.16 with low level of serum antibody

  6. Proteomic profile of culture filtrate from the Brazilian vaccine strain Mycobacterium bovis BCG Moreau compared to M. bovis BCG Pasteur

    Directory of Open Access Journals (Sweden)

    Degrave Wim M

    2011-04-01

    Full Text Available Abstract Background Bacille Calmette-Guerin (BCG is currently the only available vaccine against tuberculosis (TB and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs from M. bovis BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection. Results The 2DE proteomic map of M. bovis BCG Moreau CFPs in the pH range 3 - 8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa, Rv1926c (BCG1965c, Mpb63 and Rv1886c (BCG1923c, Ag85B in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2 and Rv0350 (BCG0389, DnaK, show the opposite pattern. Conclusions Here we report the detailed 2DE profile of CFPs from M. bovis BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB.

  7. Pengaruh Cairan Cultur Filtrate Fibroblast (CFF Terhadap Penyembuhan Luka; Penelitian eksperimental pada Rattus Norvegicus Galur Wistar

    Directory of Open Access Journals (Sweden)

    Oky Masir

    2012-11-01

    Full Text Available AbstrakLatar belakang:Metode penyembuhan luka telah mengalami perkembangan, baik berupa suatu produk atau stimulan terhadap proses biologis tubuh dalam menkompensasi luka. Fibroblas merupakan salah satu komponen penyembuhan yang berperan penting dalam proses fibroplasia. Culture Filtrate Fibroblast (CFF merupakan hasil kultur fibroblas yang akan dibuktikan efeknya terhadap proses percepatan penyembuhan luka pada penelitian ini. Metode. Penelitian ini menggunakan desain eksperimental dengan metode post test only control group design dan rancangan acak kelompok (RAK dengan menggunakan tikus putih wistar. Hewan coba dibagi menjadi 4 kelompok, yaitu 2 kelompok perlakuan yang diberikan CFF ke area eksisi luka dan kelompok kontrol yang diberikan larutan NaCl 0,9% ke area eksisi luka. Data diolah dengan SPSS 16.0. Data Kategori dianalisa dengan Chi-squared dan data numerik dengan Independent T-test. Hasil. Dari tingkat penyembuhan tidak ditemukan perbedaan pada kedua kelompok, namun perubahan restriksi jaringan lebih besar pada kelompok perlakuan. Pada skor pembentukan kolagen, derajat epitelisasi serta jumlah pembentukan pembuluh darah baru pada hari ke-3 tidak ditemukan perbedaan antara kedua kelompok. Namun pada pengamatan hari ke-7 memperlihatkan pembentukan kolagen, derajat epitelisasi serta jumlah pembentukan pembuluh darah baru lebih banyak pada kelompok perlakuan. Pada fibrosis hari ke-3 dan hari ke-7 memperlihatkan terjadinya fibrosis lebih banyak pada kelompok perlakuan dibanding kontrol. Pada pengamatan terjadinya infeksi hari ke-3 memperlihatkan infeksi lebih sedikit pada kelompok perlakuan dan terjadinya infeksi sama pada hari ke-7. Kesimpulan. CFF memberikan tingkat penyembuhan luka yang lebih baik dibanding NaCl.Kata kunci: CFF, NaCl 0,9 %, tingkat penyembuhan luka.Abstract Background: Wound healing methods have been developed, either a product or a stimulant to the body's biological processes in wound compensation. Fibroblasts is one

  8. Biological control of post-harvest diseases on apple by using plant essential oils and Trichoderma culture filtrates

    OpenAIRE

    2013-01-01

    Pure essential oils have been purified and chemically characterized from cinnamon, clove and black pepper. All these oils have antifungal properties and when applied to apple fruits in post harvest can efficiently control Botrytis cinerea, Alternaria alternata or Penicillium expansum. In addition, application of these compounds or Trichoderma culture filtrate can induce systemic resistance mechanisms in apple fruit. This has been confirmed through phytoalexins and proteomic analysis. The accu...

  9. Influence of different species of Penicillium and their culture filtrates on seed germination and seedling growth of sorghum

    Directory of Open Access Journals (Sweden)

    Koteswara Rao Vankudoth

    2014-12-01

    Full Text Available Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 The present investigations influence of Penicillium species and their culture filtrates on the seed germination and seedling growth of sorghum was studied. Culture filtrate of P. nordicum, P. verrucosum, P. chrysogenum, P. commune, P. citrinum, P.camemberti and P. digitatum were comparatively more toxic and significantly inhibited the seed germination and seedling growth varied with the species. However, pathogenicity of different inoculum of Penicillium species also exhibited varying seedling growth inhibition with P. citrinum, P. chrysogenum, P. commune, P. italicum, P. verrucosum and P. expansum assayed by water agar method. A significant phytotoxicity of these species inhibited coleoptile 32-70%, radicle 10-87% and leaf growth 20-86% with correlation coefficient 0.65, 0.67 and 0.79%, and were observed respectively. In-vitro mycotoxin production was assayed by culture filtrates of major mycotoxigenic strains revealed production of ochratoxin A (OTA, cyclopiazonic acid (CPA, rubratoxin B (RTB, griseofulvin (GRI, citrinin (CIT, patulin (PAT, penitrem A (PENA and mycophenolic acid (MPA screened by TLC/HPLC. Toxicity of species of Penicillium on seed germination, coleoptile, radicle, and leaf elongation inhibition may be attributed to the toxin-chemotypes produced by the species of Penicillium. However, even non-toxigenic strains of Penicillium also caused mild inhibition which may be attributed to the presence of other toxin-chemotypes.

  10. Filtrate of Phellinus linteus Broth Culture Reduces Infarct Size Significantly in a Rat Model of Permanent Focal Cerebral Ischemia.

    Science.gov (United States)

    Suzuki, Sakiko; Kawamata, Takakazu; Okada, Yoshikazu; Kobayashi, Tomonori; Nakamura, Tomoyuki; Hori, Tomokatsu

    2011-01-01

    Phellinus linteus, a natural growing mushroom, has been known to exhibit anti-tumor, anti-inflammatory, anti-allergic and anti-oxidant effects. Aiming to exploit the neuroprotective effects of P. linteus, we evaluated its effects on infarct volume reduction in a rat model of focal cerebral ischemia. Male Sprague-Dawley rats were subjected to right middle cerebral artery occlusion. Filtrate of P. linteus broth culture (various doses), fractionated filtrate (based on molecular weight) or control medium was administered intraperitoneally to rats before or after ischemia induction. Rats were killed at 24 h after the stroke surgery. Cortical and caudoputaminal infarct volumes were determined separately using an image analysis program following staining with 2,3,5-triphenyltetrazolium chloride. Significant cortical infarct volume reductions were found in the pre-treatment groups (30 and 60 minutes before onset of cerebral ischemia) compared with the control group, showing dose dependence. Posttreatment (30 minutes after ischemic onset) also significantly reduced cortical infarct volume. Furthermore, the higher molecular weight (≥12 000) fraction of the culture filtrate was more effective compared with the lower molecular weight fraction. The present findings suggest that P. linteus may be a new promising approach for the treatment of focal cerebral ischemia, with the additional benefit of a wide therapeutic time window since significant infarct volume reduction is obtained by administration even after the ischemic event. Our finding that the higher molecular weight fraction of the P. linteus culture filtrate demonstrated more prominent effect may provide a clue to identify the neuroprotective substances and mechanisms.

  11. Filtrate of Phellinus linteus Broth Culture Reduces Infarct Size Significantly in a Rat Model of Permanent Focal Cerebral Ischemia

    Directory of Open Access Journals (Sweden)

    Sakiko Suzuki

    2011-01-01

    Full Text Available Phellinus linteus, a natural growing mushroom, has been known to exhibit anti-tumor, anti-inflammatory, anti-allergic and anti-oxidant effects. Aiming to exploit the neuroprotective effects of P. linteus, we evaluated its effects on infarct volume reduction in a rat model of focal cerebral ischemia. Male Sprague-Dawley rats were subjected to right middle cerebral artery occlusion. Filtrate of P. linteus broth culture (various doses, fractionated filtrate (based on molecular weight or control medium was administered intraperitoneally to rats before or after ischemia induction. Rats were killed at 24 h after the stroke surgery. Cortical and caudoputaminal infarct volumes were determined separately using an image analysis program following staining with 2,3,5-triphenyltetrazolium chloride. Significant cortical infarct volume reductions were found in the pre-treatment groups (30 and 60 minutes before onset of cerebral ischemia compared with the control group, showing dose dependence. Posttreatment (30 minutes after ischemic onset also significantly reduced cortical infarct volume. Furthermore, the higher molecular weight (≥12 000 fraction of the culture filtrate was more effective compared with the lower molecular weight fraction. The present findings suggest that P. linteus may be a new promising approach for the treatment of focal cerebral ischemia, with the additional benefit of a wide therapeutic time window since significant infarct volume reduction is obtained by administration even after the ischemic event. Our finding that the higher molecular weight fraction of the P. linteus culture filtrate demonstrated more prominent effect may provide a clue to identify the neuroprotective substances and mechanisms.

  12. Influence of incubation time of Trichoderma harzianum Rifai and antifungal activity of culture filtrate against Bipolaris oryzae

    Directory of Open Access Journals (Sweden)

    Ernesto Juniors Pérez Torres

    2016-03-01

    Full Text Available This work was developed at Agriculture Microbiology laboratory in the Agriculture Faculty at Universidad Central “Martha Abreu” de Las Villas, with the aim to evaluate the influence of incubation time of Trichoderma harzianum Rifai on antifungal activity of its culture filtrates against Bipolaris oryzae. Flask with Czapek broth was used for growing T. harzianum, then one disc of mycelium were incubated during 20 and 30 days at 28±1ºC and dark. Trichoderma harzianum (strain A-34 was submited to filtration process, and comparison were done among concentration of 0%, 25%, 50%, 75%, 100% (v/v. Major inhibition porcentaje were observed in concentrations of 75% y 100% both at 20 and 30 days of incubation. At 20 day the inhibition porcentaje was greater in comparsison with 30 days.

  13. Development of supported biomimetic membranes for insertion of aquaporin protein water channels for novel water filtration applications

    DEFF Research Database (Denmark)

    Hansen, Jesper Søndergaard

    Aquaporins represent a class of membrane protein channels found in all living organisms that selectively transport water molecules across biological membranes. The work presented in this thesis was motivated by the conceptual idea of incorporating aquaporin water channels into biomimetic membranes......). This constitutes a new methodology to correctly and functionally reconstitute membrane proteins in controllable amounts into giant vesicles. The method for formation of giant protein vesicles subsequently led to the first functional prototype of an aquaporin-membrane water filtration device....

  14. Inhibition of Fusarium solani Infection in Murine Keratocytes by Lactobacillus salivarius ssp. salivarius JCM1231 Culture Filtrate In Vitro.

    Science.gov (United States)

    Hu, Jianzhang; Chen, Fang; Kan, Tong; Zhuang, Hua; Zhang, Jingjin; Han, Xiaoli

    2017-06-21

    To explore the inhibitory activity of Lactobacillus salivarius ssp. salivarius JCM1231 (L. salivarius JCM1231) culture filtrate against Fusarium solani (F. solani) and its effects on murine keratocytes (MKs) infected with F. solani. L. salivarius JCM1231 was cultured in an anaerobic incubator for 24 h, and the L. salivarius culture filtrate (LSCF) was prepared .The antifungal activity of L. salivarius JCM1231 against F. solani was determined with a plate overlay assay, agar diffusion assay, and conidial germination inhibition test. The effects of temperature, pH, and proteolytic enzymes on the antifungal activity of LSCF were detected with microtiter plate-well assay and conidial germination inhibition assay. Furthermore, the effects of LSCF on MKs infected with F. solani were detected. Cell activity and apoptosis were measured using methylthiazoletetrazolium assays and flow cytometry analysis, respectively. The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) cytokines were measured using real-time polymerase chain reactions and enzyme-linked immunosorbent assays (ELISA), and mycotoxin production was detected with high-performance liquid chromatography tandem mass spectrometry. Conidial germination and mycelia growth of F. solani were significantly inhibited by LSCF. The antifungal substances produced by L. salivarius JCM1231 were heat unstable, proteinaceous, and sensitive to proteolytic enzymes and were active within a narrow acidic pH range between 2.0 and 4.0. In the presence of 15 µg/ml of LSCF, cell activity was significantly increased, and cell apoptosis, the level of IL-6 and TNF-α expressions, and mycotoxin (zearalenone and fumonisin B1) productions were decreased significantly in MKs infected with F. solani. L. salivarius JCM1231 culture filtrate can effectively inhibit F. solani growth and protect MKs against F. solani infection.

  15. Partial calcium depletion during membrane filtration affects gelation of reconstituted milk protein concentrates.

    Science.gov (United States)

    Eshpari, H; Jimenez-Flores, R; Tong, P S; Corredig, M

    2015-12-01

    Milk protein concentrate powders (MPC) with improved rehydration properties are often manufactured using processing steps, such as acidification and high-pressure processing, and with addition of other ingredients, such as sodium chloride, during their production. These steps are known to increase the amount of serum caseins or modify the mineral equilibrium, hence improving solubility of the retentates. The processing functionality of the micelles may be affected. The aim of this study was to investigate the effects of partial acidification by adding glucono-δ-lactone (GDL) to skim milk during membrane filtration on the structural changes of the casein micelles by observing their chymosin-induced coagulation behavior, as such coagulation is affected by both the supramolecular structure of the caseins and calcium equilibrium. Milk protein concentrates were prepared by preacidification with GDL to pH 6 using ultrafiltration (UF) and diafiltration (DF) followed by spray-drying. Reconstituted UF and DF samples (3.2% protein) treated with GDL showed significantly increased amounts of soluble calcium and nonsedimentable caseins compared with their respective controls, as measured by ion chromatography and sodium dodecyl sulfate-PAGE electrophoresis, respectively. The primary phase of chymosin-induced gelation was not significantly different between treatments as measured by the amount of caseino-macropeptide released. The rheological properties of the reconstituted MPC powders were determined immediately after addition of chymosin, both before and after dialysis against skim milk, to ensure similar serum composition for all samples. Reconstituted samples before dialysis showed no gelation (defined as tan δ=1), and after re-equilibration only control UF and DF samples showed gelation. The gelation properties of reconstituted MPC powders were negatively affected by the presence of soluble casein, and positively affected by the amount of both soluble and insoluble

  16. T-cell recognition of Mycobacterium tuberculosis culture filtrate fractions in tuberculosis patients and their household contacts

    DEFF Research Database (Denmark)

    Demissie, A; Ravn, P; Olobo, J

    1999-01-01

    We examined the immune responses of patients with active pulmonary tuberculosis (TB) and their healthy household contacts to short-term culture filtrate (ST-CF) of Mycobacterium tuberculosis or molecular mass fractions derived from it. Our goal was to identify fractions strongly recognized......, to secreted mycobacterial antigens is suggestive of an early stage of infection by M. tuberculosis, which could in time result in overt disease or containment of the infection. This possibility is currently being investigated by follow-up studies of the household contacts....

  17. T-cell recognition of Mycobacterium tuberculosis culture filtrate fractions in tuberculosis patients and their household contacts

    DEFF Research Database (Denmark)

    Demissie, A; Ravn, P; Olobo, J

    1999-01-01

    We examined the immune responses of patients with active pulmonary tuberculosis (TB) and their healthy household contacts to short-term culture filtrate (ST-CF) of Mycobacterium tuberculosis or molecular mass fractions derived from it. Our goal was to identify fractions strongly recognized......, to secreted mycobacterial antigens is suggestive of an early stage of infection by M. tuberculosis, which could in time result in overt disease or containment of the infection. This possibility is currently being investigated by follow-up studies of the household contacts....

  18. Isolation and purification of bovine immunoglobulins: use of Sephacryl S-300 filtration avoids protein precipitation steps.

    Science.gov (United States)

    Collard, A; Pivont, P; Portetelle, D; Gregoire, R; Burny, A; Antoine, H

    1984-01-01

    A simplified purification method for bovine IgG1, IgG2, IgM, IgA and S-IgA is presented. It takes advantage of Sephacryl S-300 gel filtration. When applied to this column, colostral whey can be separated into IgM, S-IgA and IgG rich fractions. Filtration of blood serum leads to IgM, IgA and IgG rich fractions. IgM, IgG1, IgG2, S-IgA and IgA are then further purified by anion exchange chromatography.

  19. Filtration Fundamentals.

    Science.gov (United States)

    Ward, Ken; Hunsaker, Scot

    1997-01-01

    Examines how choice of commercial swimming-pool filtration systems is driven by the project-specific needs of the pools. Also highlighted are definitions of specific terms used when discussing filtration systems. Questions that pool designers can answer to make filtration-system purchasing decisions are listed. (GR)

  20. Cell recycled culture of succinic acid-producing Anaerobiospirillum succiniciproducens using an internal membrane filtration system.

    Science.gov (United States)

    Lee, Pyung-Cheon; Lee, Sang-Yup; Chang, Ho-Nam

    2008-07-01

    Cell recycled culture of succinic acid-producing Anaerobiospirillum succiniciproducens was anaerobically carried out using an internal membrane filter module in order to examine the physiological response of A. succiniciproducens to a high-cell-density environment. The optimal growth of A. succiniciproducens and its enhanced succinic acid productivity were observed under CO2-rich conditions, established by adding NaHCO3 and Na2CO3, in the cell recycled system. A. succiniciproducens grew up to 6.50 g-DCW/l, the highest cell concentration obtained so far, in cell recycled cultures. The cells did not change their morphology, which is known to be easily changed in unfavorable or stress environments. The maximum productivity of succinic acid was about 3.3 g/l/h, which is 3.3 times higher than those obtained in batch cultures. These results can serve as a guide for designing highly efficient cell recycled systems for succinic acid at a commercial level.

  1. Water filtration rate and infiltration/accumulation of low density lipoproteins in 3 different modes of endothelial/smooth muscle cell co-cultures.

    Science.gov (United States)

    Ding, ZuFeng; Fan, YuBo; Deng, XiaoYan

    2009-11-01

    Using different endothelial/smooth muscle cell co-culture modes to simulate the intimal structure of blood vessels, the water filtration rate and the infiltration/accumulation of LDL of the cultured cell layers were studied. The three cell culture modes of the study were: (i) The endothelial cell monolayer (EC/Phi); (ii) endothelial cells directly co-cultured on the smooth muscle cell monolayer (EC-SMC); (iii) endothelial cells and smooth muscle cells cultured on different sides of a Millicell-CM membrane (EC/SMC). It was found that under the same condition, the water filtration rate was the lowest for the EC/SMC mode and the highest for the EC/Phi mode, while the infiltration/accumulation of DiI-LDLs was the lowest in the EC/Phi mode and the highest in the EC-SMC mode. It was also found that DiI-LDL infiltration/accumulation in the cultured cell layers increased with the increasing water filtration rate. The results from the in vitro model study therefore suggest that the infiltration/accumulation of the lipids within the arterial wall is positively correlated with concentration polarization of atherogenic lipids, and the integrity of the endothelium plays an important role in the penetration and accumulation of atherogenic lipids in blood vessel walls.

  2. Water filtration rate and infiltration/accumulation of low density lipoproteins in 3 different modes of endothelial/smooth muscle cell co-cultures

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Using different endothelial/smooth muscle cell co-culture modes to simulate the intimal structure of blood vessels, the water filtration rate and the infiltration/accumulation of LDL of the cultured cell layers were studied. The three cell culture modes of the study were: (i) The endothelial cell monolayer (EC/Φ); (ii) endothelial cells directly co-cultured on the smooth muscle cell monolayer (EC-SMC); (iii) endothelial cells and smooth muscle cells cultured on different sides of a Millicell-CM membrane (EC/SMC). It was found that under the same condition, the water filtration rate was the lowest for the EC/SMC mode and the highest for the EC/Φ mode, while the infiltration/accumulation of DiI-LDLs was the lowest in the EC/Φ mode and the highest in the EC-SMC mode. It was also found that DiI-LDL infiltration/accumulation in the cultured cell layers increased with the increasing water filtration rate. The results from the in vitro model study therefore suggest that the infiltration/accumulation of the lipids within the arterial wall is positively correlated with concentration polarization of atherogenic lipids, and the integrity of the endothelium plays an important role in the penetration and accumulation of atherogenic lipids in blood vessel walls.

  3. Optimization of protein fractionation by skim milk microfiltration: Choice of ceramic membrane pore size and filtration temperature.

    Science.gov (United States)

    Jørgensen, Camilla Elise; Abrahamsen, Roger K; Rukke, Elling-Olav; Johansen, Anne-Grethe; Schüller, Reidar B; Skeie, Siv B

    2016-08-01

    The objective of this study was to investigate how ceramic membrane pore size and filtration temperature influence the protein fractionation of skim milk by cross flow microfiltration (MF). Microfiltration was performed at a uniform transmembrane pressure with constant permeate flux to a volume concentration factor of 2.5. Three different membrane pore sizes, 0.05, 0.10, and 0.20µm, were used at a filtration temperature of 50°C. Furthermore, at pore size 0.10µm, 2 different filtration temperatures were investigated: 50 and 60°C. The transmission of proteins increased with increasing pore size, giving the permeate from MF with the 0.20-µm membrane a significantly higher concentration of native whey proteins compared with the permeates from the 0.05- and 0.10-µm membranes (0.50, 0.24, and 0.39%, respectively). Significant amounts of caseins permeated the 0.20-µm membrane (1.4%), giving a permeate with a whitish appearance and a casein distribution (αS2-CN: αS1-CN: κ-CN: β-CN) similar to that of skim milk. The 0.05- and 0.10-µm membranes were able to retain all caseins (only negligible amounts were detected). A permeate free from casein is beneficial in the production of native whey protein concentrates and in applications where transparency is an important functional characteristic. Microfiltration of skim milk at 50°C with the 0.10-µm membrane resulted in a permeate containing significantly more native whey proteins than the permeate from MF at 60°C. The more rapid increase in transmembrane pressure and the significantly lower concentration of caseins in the retentate at 60°C indicated that a higher concentration of caseins deposited on the membrane, and consequently reduced the native whey protein transmission. Optimal protein fractionation of skim milk into a casein-rich retentate and a permeate with native whey proteins were obtained by 0.10-µm MF at 50°C.

  4. Modeling of Filtration Processes—Microfiltration and Depth Filtration for Harvest of a Therapeutic Protein Expressed in Pichia pastoris at Constant Pressure

    Directory of Open Access Journals (Sweden)

    Muthukumar Sampath

    2014-12-01

    Full Text Available Filtration steps are ubiquitous in biotech processes due to the simplicity of operation, ease of scalability and the myriad of operations that they can be used for. Microfiltration, depth filtration, ultrafiltration and diafiltration are some of the most commonly used biotech unit operations. For clean feed streams, when fouling is minimal, scaling of these unit operations is performed linearly based on the filter area per unit volume of feed stream. However, for cases when considerable fouling occurs, such as the case of harvesting a therapeutic product expressed in Pichia pastoris, linear scaling may not be possible and current industrial practices involve use of 20–30% excess filter area over and above the calculated filter area to account for the uncertainty in scaling. In view of the fact that filters used for harvest are likely to have a very limited lifetime, this oversizing of the filters can add considerable cost of goods for the manufacturer. Modeling offers a way out of this conundrum. In this paper, we examine feasibility of using the various proposed models for filtration of a therapeutic product expressed in Pichia pastoris at constant pressure. It is observed that none of the individual models yield a satisfactory fit of the data, thus indicating that more than one fouling mechanism is at work. Filters with smaller pores were found to undergo fouling via complete pore blocking followed by cake filtration. On the other hand, filters with larger pores were found to undergo fouling via intermediate pore blocking followed by cake filtration. The proposed approach can be used for more accurate sizing of microfilters and depth filters.

  5. Identification of three elicitins and a galactan-based complex polysaccharide from a concentrated culture filtrate of Phytophthora infestans efficient against Pectobacterium atrosepticum.

    Science.gov (United States)

    Saubeau, Guillaume; Gaillard, Fanny; Legentil, Laurent; Nugier-Chauvin, Caroline; Ferrières, Vincent; Andrivon, Didier; Val, Florence

    2014-09-26

    The induction of plant immunity by Pathogen Associated Molecular Patterns (PAMPs) constitutes a powerful strategy for crop protection. PAMPs indeed induce general defense responses in plants and thus increase plant resistance to pathogens. Phytophthora infestans culture filtrates (CCFs) are known to induce defense responses and decrease the severity of soft rot due to Pectobacterium atrosepticum in potato tubers. The aim of this study was to identify and characterize the active compounds from P. infestans filtrate. The filtrate was fractionated by gel filtration, and the protection effects against P. atrosepticum and the ability to induce PAL activity were tested for each fraction. The fraction active in protection (F1) also induced PAL activity, as did the whole filtrate. Three elicitins (INF1, INF4 and INF5) were identified in F1b, subfraction of F1, by MALDI-TOF-MS and MS/MS analyses. However, deproteinized F1b still showed biological activity against the bacterium, revealing the presence of an additional active compound. GC-MS analyses of the deproteinized fraction highlighted the presence of a galactan-based complex polysaccharide. These experiments demonstrate that the biological activity of the CCF against P. atrosepticum results from a combined action of three elicitins and a complex polysaccharide, probably through the activation of general defense responses.

  6. Identification of Three Elicitins and a Galactan-Based Complex Polysaccharide from a Concentrated Culture Filtrate of Phytophthora infestans Efficient against Pectobacterium atrosepticum

    Directory of Open Access Journals (Sweden)

    Guillaume Saubeau

    2014-09-01

    Full Text Available The induction of plant immunity by Pathogen Associated Molecular Patterns (PAMPs constitutes a powerful strategy for crop protection. PAMPs indeed induce general defense responses in plants and thus increase plant resistance to pathogens. Phytophthora infestans culture filtrates (CCFs are known to induce defense responses and decrease the severity of soft rot due to Pectobacterium atrosepticum in potato tubers. The aim of this study was to identify and characterize the active compounds from P. infestans filtrate. The filtrate was fractionated by gel filtration, and the protection effects against P. atrosepticum and the ability to induce PAL activity were tested for each fraction. The fraction active in protection (F1 also induced PAL activity, as did the whole filtrate. Three elicitins (INF1, INF4 and INF5 were identified in F1b, subfraction of F1, by MALDI-TOF-MS and MS/MS analyses. However, deproteinized F1b still showed biological activity against the bacterium, revealing the presence of an additional active compound. GC-MS analyses of the deproteinized fraction highlighted the presence of a galactan-based complex polysaccharide. These experiments demonstrate that the biological activity of the CCF against P. atrosepticum results from a combined action of three elicitins and a complex polysaccharide, probably through the activation of general defense responses.

  7. Addi-Chek filtration, BACTEC, and 10-ml culture methods for recovery of microorganisms from dialysis effluent during episodes of peritonitis.

    Science.gov (United States)

    Males, B M; Walshe, J J; Garringer, L; Koscinski, D; Amsterdam, D

    1986-02-01

    The Addi-Chek (filtration; Millipore Corp., Bedford, Mass.) and BACTEC (radiometric detection of growth in culture media; Johnston Laboratories, Inc., Towson, Md.) systems were compared with the 10-ml culture (centrifugation) method for the recovery of microorganisms from peritoneal dialysate collected from patients with clinical evidence of peritonitis and containing greater than or equal to 200 leukocytes per mm3. Both alternate methods were comparable, and results were not significantly different from those of the conventional 10-ml culture method. All systems were adversely affected in their capacity to recover organisms when dialysates had been collected during periods of antimicrobial therapy.

  8. Fungal protein and ethanol from lignocelluloses using Rhizopus pellets under simultaneous saccharification, filtration and fermentation (SSFF

    Directory of Open Access Journals (Sweden)

    Somayeh FazeliNejad

    2016-03-01

    Full Text Available The economic viability of the 2nd generation bioethanol production process cannot rely on a single product but on a biorefinery built around it. In this work, ethanol and fungal biomass (animal feed were produced from acid-pretreated wheat straw slurry under an innovative simultaneous saccharification, fermentation, and filtration (SSFF strategy. A membrane unit separated the solids from the liquid and the latter was converted to biomass or to both biomass and ethanol in the fermentation reactor containing Rhizopus sp. pellets. Biomass yields of up to 0.34 g/g based on the consumed monomeric sugars and acetic acid were achieved. A surplus of glucose in the feed resulted in ethanol production and reduced the biomass yield, whereas limiting glucose concentrations resulted in higher consumption of xylose and acetic acid. The specific growth rate, in the range of 0.013-0.015/h, did not appear to be influenced by the composition of the carbon source. Under anaerobic conditions, an ethanol yield of 0.40 g/g was obtained. The present strategy benefits from the easier separation of the biomass from the medium and the fungus ability to assimilate carbon residuals in comparison with when yeast is used. More specifically, it allows in-situ separation of insoluble solids leading to the production of pure fungal biomass as a value-added product.

  9. Shortening filtrations

    Institute of Scientific and Technical Information of China (English)

    ENOCHS Edgar E.

    2012-01-01

    Let C be a set of modules.We argue that there is an ordinal κ such that if a module has a filtration by modules in C,then it has a filtration of length κ by direct sums of modules in C.As an application we give another way to prove a result of Saorín and (S)(t)oví(c)ek and of (S)(t)oví(c)ek.

  10. Monitoring the fractionation of a whey protein isolate during dead-end membrane filtration using fluorescence and chemometric methods.

    Science.gov (United States)

    Elshereef, Rand; Budman, Hector; Moresoli, Christine; Legge, Raymond L

    2010-01-01

    During membrane-based separation of proteins, changes in protein concentration of the permeate and retentate streams occurs over time. The current work proposes a new approach for monitoring the changes in concentrations of proteins in both permeate and retentate by making use of data collected using fluorescence spectroscopy and intrinsic protein fluorescence analyzed by multivariate statistical techniques. Whey protein isolate consists mainly of alpha-lactalbumin (alpha-LA), beta-lactoglobulin (beta-LG), and small proportion of bovine serum albumin (BSA) and was used as a model system in this study. A fiber optic probe (FOP) was used to acquire multiwavelength fluorescence spectra for permeate and retentate streams at different times during UF-based separation of the components from a multicomponent solution. Multivariate regression models were developed for predicting the concentrations of alpha-LA, beta-LG, and BSA by establishing a calibration model between data acquired using the FOP and the corresponding protein concentration levels measured by size-exclusion chromatography. The model was validated using FOP data that were not previously used for calibration of the regression models. This comparison showed that concentrations of alpha-LA, beta-LG, and BSA could be predicted directly from FOP data within reasonable accuracy by making use of multivariate calibration tools. This approach has several attractive features including that it is nondestructive, fast, and relatively simple to perform. This technique has potential practical applications as it could offer the opportunity for in situ monitoring of membrane filtration processes by tracking individual protein transmission and selectivity of fractionation.

  11. Herbicidal Potential of Drechslera spp. Culture Filtrates Against Parthenium hysterophorus L. Potencial Herbicida de Filtrados de Cultivo de Drechslera spp. Contra Parthenium hysterophorus L.

    Directory of Open Access Journals (Sweden)

    Arshad Javaid

    2011-12-01

    Full Text Available Herbicidal activity of culture filtrates of four Drechslera spp., namely D. australiensis (Bugnic. Subram. & B.L. Jain, D. biseptata (Sacc. & Roum. M.J. Richardson & E.M. Fraser, D. hawaiiensis Bugnic. ex M.B. Ellis, and D. holmii (Luttr. Subram. & P.C. Jain, prepared in malt extract broth was investigated against parthenium weed (Parthenium hysterophorus L. in both laboratory bioassays and pots. In laboratory bioassays, the effect of original (100% and diluted (50% culture filtrates of the four Drechslera spp. was studied on parthenium germination and seedling growth in 90 mm diameter Petri plates. Original culture filtrate of all the four Drechslera species significantly reduced germination, shoot length, shoot fresh biomass, root length, and root fresh biomass of parthenium seedlings by 43 to 77%, 77 to 82%, 69 to 82%, 90 to 92%, and 67 to 83%, respectively, as compared to the control. In pot trials, foliar application of original fungal culture filtrates was carried out on 1-wk and 2-wk old parthenium seedlings. Culture filtrates of all the four Drechslera spp., except D. holmii, markedly reduced parthenium shoot dry weight. Two-week-old plants were more susceptible to foliar spray than the 1-wk old plants. There was a 13 to 55% and 28 to 65% reduction in shoot dry weight of 1-wk and 2-wk old parthenium plants, respectively, due to culture filtrates of various Drechslera spp. The present study concludes that Drechslera spp. culture filtrates can be used as alternative herbicides for parthenium weed management.Se investigó la actividad herbicida de filtrados de cultivos de cuatro especies de Drechslera spp., i.e. D. australiensis (Bugnic. Subram. & B.L. Jain, D. biseptata (Sacc. & Roum. M.J. Richardson & E.M. Fraser, D. hawaiiensis Bugnic. ex M.B. Ellis, and D. holmii (Luttr. Subram. & PC. Jain, preparados en caldo de extracto de malta contra la maleza Parthenium hysterophorus L. en bioensayos de laboratorio y en macetas. En los bioensayos

  12. PATHOGENIC BEHAVIOUR OF ALTERNARIA ALTERNATA AND PHYTOTOXICITY OF ITS CULTURE FILTRATES ON LEPIDIUM SATIVUM: A MEDICINAL HERB OF IMMENSE PHARMACOLOGICAL POTENTIAL

    Directory of Open Access Journals (Sweden)

    U. K. KHARE

    2013-01-01

    Full Text Available Alternaria alternata causing leaf spot in Lepidium sativum was isolated and purified from diseased leaf tissuescollected from collected from the Medicinal and Aromatic plant garden, Department of Crop and HerbalPhysiology, Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur (M.P.; isolated and purified on Potato DextroseAgar media. Microscopic examination of a seven days old culture revealed hyaline, septate and branched mycelia,conidiophores with 30.0-80.2 µ length and 3-6 µ width and obclavate to obpyriform conidia (23-30 x 9.2-12.7µ with short conical beak arranged in acropetal fashion. The isolated culture and its culture filtrates wereinoculated to germinated seedlings of chandrasur and also incubated with healthy leaves in a growth chamber.Typical symptoms of Alternaria leaf spot was observed both in in vivo and in vitro inoculated plantlets anddetached leaves respectively. Chlorosis on the hypocotyls and leaves were observed. A. alternata was consistentlyreisolated from symptomatic leaf tissues on PDA. Thus, an efficient and reliable screening method wherein theeffect of the selection agent (pathogen culture, culture filtrate/phytotoxin was demonstrated providing soundpharmacological rational in terms of micro propagation and development of Alternaria resistant L. sativum, animportant medicinal herb.

  13. Effect of nitrogen sources and vitamins on ligninolytic enzyme production by some white-rot fungi. Dye decolorization by selected culture filtrates.

    Science.gov (United States)

    Levin, Laura; Melignani, Eliana; Ramos, Araceli Marcela

    2010-06-01

    The effect of amino acids, complex nitrogen sources and vitamin addition on Trametes trogii, Trametes villosa and Coriolus versicolor var. antarcticus ligninolytic enzyme production, was evaluated. Dye decolorization by their culture filtrates was compared. Glutamic acid followed by peptone, were the best N sources for laccase and manganese peroxidase production. The three fungi produced two laccase isoenzymes (molecular weights from 38 up to 150 kDa); their pattern of production was not affected by medium composition. Although the response was not uniform, vitamin addition sometimes stimulated ligninolytic enzyme production, but never inhibited it. Thiamine induced manganese peroxidase production. T. trogii grown in glutamic acid produced culture filtrates with the highest laccase (188.3 U/ml) and manganese peroxidase activities (4.5 U/ml), rendering the best results in decolorization. These crude filtrates were able to decolorize in half hour (at pH 4.5, 30 degrees C): 13%, 23%, 40%, 46%, 82%, 94% and 95% of Gentian Violet, Xylidine, Congo Red, Malachite Green, Remazol Brilliant Blue R, Indigo Carmine and Anthraquinone Blue, respectively. (c) 2010 Elsevier Ltd. All rights reserved.

  14. Biorefinery of microalgal soluble proteins by sequential processing and membrane filtration

    NARCIS (Netherlands)

    Safi, C.; Olivieri, G.; Pina Campos, Rui; Engelen-Smit, N.; Mulder, W.J.; Broek, van den L.A.M.; Sijtsma, L.

    2017-01-01

    A mild biorefinery process was investigated on the microalga Nannochloropsis gaditana, to obtain an enriched fraction of water soluble proteins free from chlorophyll. After harvesting, a 100 g.L−1 solution of cells was first subjected to cell disruption by either high-pressure

  15. Biorefinery of microalgal soluble proteins by sequential processing and membrane filtration.

    Science.gov (United States)

    Safi, C; Olivieri, G; Campos, R P; Engelen-Smit, N; Mulder, W J; van den Broek, L A M; Sijtsma, L

    2017-02-01

    A mild biorefinery process was investigated on the microalga Nannochloropsis gaditana, to obtain an enriched fraction of water soluble proteins free from chlorophyll. After harvesting, a 100g.L(-1) solution of cells was first subjected to cell disruption by either high-pressure homogenization (HPH) or enzymatic treatment (ENZ). HPH resulted in a larger release of proteins (49%) in the aqueous phase compared to the Alcalase incubation (35%). In both cases, an ultrafiltration/diafiltration (UF/DF) was then performed on the supernatant obtained from cell disruption by testing different membrane cut-off (1000kDa, 500kDa and 300kDa). After optimising the process conditions, the combination of ENZ→UF/DF ended in a larger overall yield of water soluble proteins (24.8%) in the permeate compared to the combination of HPH→UF/DF (17.4%). A gel polarization model was implemented to assess the maximum achievable concentration factor during ultrafiltration and the mass transfer coefficient related to the theoretical permeation flux rate. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. Influence of the charge of low molecular weight proteins on their efficacy of filtration and/or adsorption on dialysis membranes with different intrinsic properties.

    Science.gov (United States)

    Moachon, N; Boullange, C; Fraud, S; Vial, E; Thomas, M; Quash, G

    2002-02-01

    Hemodialysis membranes eliminate by filtration low-molecular-weight toxic metabolites (urea and creatinine) with minimum interactions between blood components and the membrane itself. However, the ability of a membrane to adsorb specific proteins could be beneficial if the accumulation of these same proteins is implicated in the genesis of a pathological condition. Beta-amyloidosis which accompanies the elevation of beta2-microglobulin (11.8 kDa) in the plasma of dialysed patients is one such condition (Biochem. Biophys. Res. Commun. 129 (3) (1985) 701-706: Lancet 1 (1986) 1240-1311). To determine whether increases in plasma beta2-microglobulin levels were due to differences in filtration efficacy of the membrane used and/or to certain characteristics of this protein, e.g. its charge (pI 5.7) the adsorption and filtration of [3H] beta2-microglobulin and [3H] lysozyme of similar MW 14.5 kDa, but pI: 10.8 were compared on different membranes. It was found that, neither [3H] beta2-microglobulin nor [3H] lysozyme are removed by cuprophan, whereas over 75% of beta2-microglobulin is removed by filtration on polyacrylonitrile, polyacrylonitrile-polyethyleneimine, polysulfone and >95% by adsorption to polymethylmethacrylate-BK. For lysozyme, removal by adsorption is >95% on polyacrylonitrile and polyacrylonitrile-polyethyleneimine, 72% on polymethylmethacrylate-BK and by filtration is 95% on polysulfone. Hemodialysis membranes must therefore not simply be considered as filters of low-molecular-weight metabolites but should be equally assessed for their capacity to eliminate potentially deleterious low-molecular-weight plasma proteins.

  17. Fast Swinnex Filtration (FSF): A fast and robust sampling and extraction method suitable for metabolomics analysis of cultures grown in complex media

    DEFF Research Database (Denmark)

    McCloskey, Douglas; Utrilla, Jose; Naviaux, Robert K.;

    2015-01-01

    of a suitable method for anaerobic cultures grown in complex media. Given that a vast majority of bacteria are facultative or obligate anaerobes that grow to low biomass density and need to be cultured in complex media, a suitable sampling and extraction strategy for anaerobic cultures is needed. In this work......Liquid chromatography tandem mass spectrometry (LC–MS/MS) provides a powerful means to analyze intracellular metabolism. A prerequisite to accurate metabolomics analysis using LC–MS/MS is a robust sampling and extraction protocol. One unaddressed area in sampling is a detailed examination......, we develop a fast-filtration method using pressuredriven Swinnex filters. We show that the method is fast enough to provide an accurate snapshot of intracellular metabolism, reduces matrix interference from the media to improve the number of compounds that can be detected, and is applicable...

  18. [Molecular-genetic polymorphism of cellular lines of wheat resistant to cultural filtrate Gaeumannomyces graminis var. tritici and plant-regenerants from them].

    Science.gov (United States)

    Bavol, A V; Dubrovna, O V

    2009-01-01

    DNA polymorphism of wheat cellular lines resistant to culture filtrate of G. graminis var. tritici and regenerated plants has been investigated using ISSR-analysis. Specific changes in DNA sequence were revealed in resistant calluses. It was established, that all resistant cellular lines differed by the presence of specific amplicons of 2347 bp (primer 5'-TCTCTCTCTCTCTCTCG-3') and 1745 bp (primer 5'-AGAGAGAGAGAGAGAGTC-3'), as well as the absence of the amplicon of 1108 bp (primer 5'-ACACACACACACACACC-3') from the initial callus and the callus not exposed to the action of selective factor. These changes have been also revealed in R0 and R1 plants.

  19. GSPEL - Air Filtration Laboratory

    Data.gov (United States)

    Federal Laboratory Consortium — Evaluation capabilities for air filtration devices The Air Filtration Lab provides testing of air filtration devices to demonstrate and validate new or legacy system...

  20. Estimated glomerular filtration rate in sickle cell anemia is associated with polymorphisms of bone morphogenetic protein receptor 1B.

    Science.gov (United States)

    Nolan, Vikki G; Ma, Qianli; Cohen, Herbert T; Adewoye, Adeboye; Rybicki, Anne C; Baldwin, Clinton; Mahabir, Rhea N; Homan, Erica P; Wyszynski, Diego F; Fabry, Mary E; Nagel, Ronald L; Farrer, Lindsay A; Steinberg, Martin H

    2007-03-01

    Renal disease is common in sickle cell anemia. In this exploratory work, we used data from a longitudinal study of the natural history of sickle cell disease to examine the hypothesis that polymorphisms (SNPs) in selected candidate genes are associated with glomerular filtration rate (GFR). DNA samples and clinical and laboratory data were available for 1,140 patients with sickle cell anemia. GFR was estimated using the Cockcroft-Gault and Schwartz formulas for adults and children, respectively. We examined approximately 175 haplotype tagging (ht) SNPs in about 70 genes of the TGFbeta/BMP pathway for their association with GFR using linear regression. Four SNPs in BMPR1B, a bone morphogenetic protein (BMP) receptor gene, yielded statistically significant associations (P values ranging from 0.015 to 0.046). Three haplotypes in this gene were also associated with GFR. The TGF-beta/BMP pathway has been associated with the development of diabetic nephropathy, which has some features in common with sickle cell nephropathy. Our results suggest that, as with other subphenotypes of sickle cell disease, renal function may be genetically modulated.

  1. Hydrophilic compounds in culture filtrates of Fusarium oxysporum f. sp. cubense GCV [01210] induce protection to banana leave toward a main pathogen phytotoxic component

    Directory of Open Access Journals (Sweden)

    Nayanci Portal González

    2014-07-01

    Full Text Available Panama disease caused by Fusarium oxysporum f. sp. cubense (Foc, is among the most important diseases in Musa spp. Foc is a necrotrophic fungus, their phytotoxins play a role in disease development. Previously culture filtrate (FCC 15 days incubation with differential phytotoxic activity against two Musa cultivars was obtained. From this, the main fraction with nonspecific phytotoxic activity against both cultivars was purified. In this study, the biological activity of the aqueous phase and the main phytotoxic fraction purified from organic extract of Fusarium oxysporum f. sp. cubense VCG [01210] Race 1 FCC was determined on banana leaves of cv. `Gros Michel' (susceptible and `FHIA-01' (resistant. Foc FCC phytotoxic effect was confirmed. The aqueous phase showed no phytotoxic activity on both cultivars, while the simultaneous application of the aqueous phase with the main phytotoxic fraction induced a differential response of tissues in susceptible and resistant cultivars evaluated. The results indicated that the compounds present in the aqueous phase are required to induce the protection of leaf tissue against phytotoxic main component of the pathogen. Key words: culture filtrate, Panama disease, resistant, susceptible

  2. Scale-down of continuous filtration for rapid bioprocess design: Recovery and dewatering of protein precipitate suspensions.

    Science.gov (United States)

    Reynolds, T; Boychyn, M; Sanderson, T; Bulmer, M; More, J; Hoare, M

    2003-08-20

    The early specification of bioprocesses often has to be achieved with small (tens of millilitres) quantities of process material. If extensive process discovery is to be avoided at pilot or industrial scale, it is necessary that scale-down methods be created that not only examine the conditions of process stages but also allows production of realistic output streams (i.e., streams truly representative of the large scale). These output streams can then be used in the development of subsequent purification operations. The traditional approach to predicting filtration operations is via a bench-scale pressure filter using constant pressure tests to examine the effect of pressure on the filtrate flux rate and filter cake dewatering. Interpretation of the results into cake resistance at unit applied pressure (alpha) and compressibility (n) is used to predict the pressure profile required to maintain the filtrate flux rate at a constant predetermined value. This article reports on the operation of a continuous mode laboratory filter in such a way as to prepare filter cakes and filtrate similar to what may be achieved at the industrial scale. Analysis of the filtration rate profile indicated the filter cake to have changing properties (compressibility) with time. Using the insight gained from the new scale-down methodology gave predictions of the flux profile in a pilot-scale candle filter superior to those obtained from the traditional batch filter used for laboratory development.

  3. MALDI-TOF identification of Gram-negative bacteria directly from blood culture bottles containing charcoal: Sepsityper® kits versus centrifugation-filtration method.

    Science.gov (United States)

    Riederer, Kathleen; Cruz, Kristian; Shemes, Stephen; Szpunar, Susan; Fishbain, Joel T

    2015-06-01

    Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry has dramatically altered the way microbiology laboratories identify clinical isolates. Direct blood culture (BC) detection may be hampered, however, by the presence of charcoal in BC bottles currently in clinical use. This study evaluates an in-house process for extraction and MALDI-TOF identification of Gram-negative bacteria directly from BC bottles containing charcoal. Three hundred BC aliquots were extracted by a centrifugation-filtration method developed in our research laboratory with the first 96 samples processed in parallel using Sepsityper® kits. Controls were colonies from solid media with standard phenotypic and MALDI-TOF identification. The identification of Gram-negative bacteria was successful more often via the in-house method compared to Sepsityper® kits (94.7% versus 78.1%, P≤0.0001). Our in-house centrifugation-filtration method was further validated for isolation and identification of Gram-negative bacteria (95%; n=300) directly from BC bottles containing charcoal.

  4. Atividade alelopática do filtrado de cultura produzido por Fusarium solani Allelopathic activity of culture filtrate produced by Fusarium solani

    Directory of Open Access Journals (Sweden)

    A.P.S. Souza Filho

    2007-03-01

    Full Text Available As plantas daninhas se constituem no principal problema a impor limitação à exploração da agropecuária nas áreas tropicais. Entretanto, o controle químico dessas plantas tem gerado insatisfações de ordem social, quer porque contaminam as fontes de recursos naturais ou por comprometerem a qualidade dos alimentos da dieta dos animais, em geral, e dos humanos, em particular. Os objetivos deste trabalho foram identificar e caracterizar a atividade alelopática do filtrado de cultura produzido pelo fungo Fusarium solani f. sp. pipers. Foram avaliados os efeitos das toxinas, nas concentrações de 1,0 e 4,0%, sobre a germinação de sementes e o desenvolvimento da radícula e do hipocótilo das plantas daninhas malícia (Mimosa pudica e mata-pasto (Senna obtusifolia. Os resultados mostraram presença de atividade alelopática inibitória, com variações de acordo com a concentração e a planta receptora. A intensidade dos efeitos inibitórios induzidos pelo extrato esteve positivamente associada à concentração, com efeitos mais intensos verificados a 4,0%. Independentemente da concentração e do bioensaio, a espécie malícia se mostrou mais sensível aos efeitos do filtrado da cultura. O desenvolvimento da radícula foi o fator da planta mais intensamente inibido. Os resultados indicam a existência de potencial de utilização da toxina produzida pelo fungo, como fonte alternativa no controle de plantas daninhas, o que justifica estudos mais avançados.Weeds are a major problem limiting agriculture and cattle raising activities in the tropics. Current chemical control measures have raised environmental concerns due to their potential of contaminating natural resources and compromising the quality of animal feed. The objective of this paper was to identify and characterize the potential allelopathic activity of Fusarium solani f. sp. pipers culture filtrate. The effects of the toxin were analyzed at 1% and 4% concentration, on seed

  5. 枯萎病菌毒素培养滤液对唐菖蒲幼苗毒性的初步研究%Poisoning effects of toxin culture filtrate of Fusarium wilt pathogen on seedling of gladiolus

    Institute of Scientific and Technical Information of China (English)

    胡颖慧; 龚束芳; 李彩华; 冯晓晓; 尚芬; 杨涛; 车代弟

    2012-01-01

    对唐菖蒲枯萎病菌进行了产毒培养基的筛选,研究毒素培养滤液对胚根生长的抑制作用、对幼苗根系活力的影响及对幼苗的致萎作用.结果表明:唐菖蒲枯萎病菌在Czapek培养基中培养15 d所产生的培养滤液对唐菖蒲胚根的抑制作用最强,胚根生长抑制率高达99.79%;而菌丝干重在PD培养基中培养13 d达到最高值l 242.27 mg;毒素培养滤液经121℃灭菌20 min后对唐菖蒲胚根仍有很强的抑制作用,表现出较高的热稳定性;与对照相比,毒素培养滤液能够显著降低唐菖蒲幼苗根系活力,随处理时间的延长和处理浓度的增加,萎蔫级数逐渐增加.说明枯萎病菌毒素培养滤液是唐菖蒲枯萎病的致病因子,可以利用其筛选唐菖蒲抗枯萎病品种.%Four kinds of culture media for gladiolus Fusarium wilt pathogen were used to produce culture filtrate. Poisoning effects of culture filtrate of gladiolus Fusarium wilt on radicles growth, root activity and seedling of gladiolus were studied. The results showed that the culture filtrate had the strongest inhibition effect on the radicle of gladiolus at the 15th day cultured in Czapek liquid medium and the inhibition ratio was 99. 79% . The optimum medium for hyphae growth was PD medium. The hyphae growth reached to maximum at the 13 th day. The culture filtrate possessed the capacity of thermal stability, which could inhibit the growth of radical after sterilized 20 min under 1211. The culture filtrate significantly inhibited activity of gladiolus root and caused wilting of seedling. With the extension of culturing time and concentration of culture filtrate,the wilting degree of seedlings increased. The above indicated the culture filtrate of gladiolus Fusarium wilt pathogen was pathogenic factor of gladiolus wilt, and it could be used to screen disease-resistance gladiolus variety.

  6. Relationship of serum RBP, NEFA and FKN content with urinary protein excretion and glomerular filtration function damage in patients with early diabetic nephropathy

    Institute of Scientific and Technical Information of China (English)

    Zhi Yang; Ji Zhang; Li-Ping Tang

    2016-01-01

    Objective:To analyze the relationship of serum RBP, NEFA and FKN content with urinary protein excretion and glomerular filtration function damage in patients with early diabetic nephropathy.Methods: A total of 76 patients with early diabetic nephropathy were included in observation group and 80 healthy subjects were included in control group. Differences in retinol-binding protein (RBP), non-esterified fatty acid (NEFA) and chemokine Fractalkine (FKN) content in serum as well as urinary protein excretion indexes and glomerular filtration function indexes were compared between the two groups immediately after admission, and the correlation of RBP, NEFA and FKN content with the disease was further analyzed.Results:Serum RBP, NEFA and FKN content of observation group were higher than those of control group; 24 h total urine volume, 24 h urinary albumin and urinary albumin excretion rate as well as urine-specific proteins Kim-1, NGAL, L-FABP and CysC content of observation group were higher than those of control group and positively correlated with serum RBP, NEFA and FKN content; GFR level of observation group was lower than that of control group and negatively correlated with serum RBP, NEFA and FKN content while serum Cr, BUN, VF, LP-a,β2-MG and HA content were higher than those of control group and positively correlated with serum RBP, NEFA and FKN content.Conclusions:Serum RBP, NEFA and FKN content significantly increase in patients with early diabetic nephropathy and are correlated with urinary protein excretion and glomerular filtration function damage.

  7. GSPEL - Air Filtration Laboratory

    Data.gov (United States)

    Federal Laboratory Consortium — Evaluation capabilities for air filtration devicesThe Air Filtration Lab provides testing of air filtration devices to demonstrate and validate new or legacy system...

  8. Wtip- and gadd45a-interacting protein dendrin is not crucial for the development or maintenance of the glomerular filtration barrier.

    Directory of Open Access Journals (Sweden)

    Zhijie Xiao

    Full Text Available Glomerular podocyte cells are critical for the function of the renal ultrafiltration barrier. Especially, the highly specialized cell-cell junction of podocytes, the slit diaphragm, has a central role in the filtration barrier. This is highlighted by the fact that mutations in molecular components of the slit diaphragm, including nephrin and Cd2-associated protein (Cd2ap, result in proteinuric diseases in man. Dendrin is a poorly characterized cytosolic component of the slit diaphragm in where it interacts with nephrin and Cd2ap. Dendrin is highly specific for the podocyte slit diaphragm, suggesting that it has a dedicated role in the glomerular filtration barrier. In this study, we have generated a dendrin knockout mouse line and explored the molecular interactions of dendrin. Dendrin-deficient mice were viable, fertile, and had a normal life span. Morphologically, the glomerulogenesis proceeded normally and adult dendrin-deficient mice showed normal glomerular histology. No significant proteinuria was observed. Following glomerular injury, lack of dendrin did not affect the severity of the damage or the recovery process. Yeast two-hybrid screen and co-immunoprecipitation experiments showed that dendrin binds to Wt1-interacting protein (Wtip and growth arrest and DNA-damage-inducible 45 alpha (Gadd45a. Wtip and Gadd45a mediate gene transcription in the nucleus, suggesting that dendrin may have similar functions in podocytes. In line with this, we observed the relocation of dendrin to nucleus in adriamycin nephropathy model. Our results indicate that dendrin is dispensable for the function of the normal glomerular filtration barrier and that dendrin interacts with Wtip and Gadd45a.

  9. Proteomic analysis of formalin-fixed paraffin-embedded glomeruli suggests depletion of glomerular filtration barrier proteins in two-kidney, one-clip hypertensive rats.

    Science.gov (United States)

    Finne, Kenneth; Vethe, Heidrun; Skogstrand, Trude; Leh, Sabine; Dahl, Tone D; Tenstad, Olav; Berven, Frode S; Reed, Rolf K; Vikse, Bjørn Egil

    2014-12-01

    It is well known that hypertension may cause glomerular damage, but the molecular mechanisms involved are still incompletely understood. In the present study, we used formalin-fixed paraffin-embedded (FFPE) tissue to investigate changes in the glomerular proteome in the non-clipped kidney of two-kidney one-clip (2K1C) hypertensive rats, with special emphasis on the glomerular filtration barrier. 2K1C hypertension was induced in 6-week-old Wistar Hannover rats (n = 6) that were sacrificed 23 weeks later and compared with age-matched sham-operated controls (n = 6). Tissue was stored in FFPE tissue blocks and later prepared on tissue slides for laser microdissection. Glomeruli without severe morphological damage were isolated, and the proteomes were analysed using liquid chromatography-tandem mass spectrometry. 2K1C glomeruli showed reduced abundance of proteins important for slit diaphragm complex, such as nephrin, podocin and neph1. The podocyte foot process had a pattern of reduced abundance of transmembrane proteins but unchanged abundances of the podocyte cytoskeletal proteins synaptopodin and α-actinin-4. Lower abundance of important glomerular basement membrane proteins was seen. Possible glomerular markers of damage with increased abundance in 2K1C were transgelin, desmin and acyl-coenzyme A thioesterase 1. Microdissection and tandem mass spectrometry could be used to investigate the proteome of isolated glomeruli from FFPE tissue. Glomerular filtration barrier proteins had reduced abundance in the non-clipped kidney of 2K1C hypertensive rats. © The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA.

  10. Effects of cell suspension and cell·free culture filtrate of Pseudomonas aeruginosa in the control of root rot-root kont disease complex of tomato (Lycopersicon esculentum Mill.

    Directory of Open Access Journals (Sweden)

    I. A. Siddiqui

    2013-12-01

    Full Text Available The plant growth-promoting rhizobacterium Pseudomonas aeruginosa strain IE-6 was tested for antagonistic activity towards Meloidogyne javanica, the root-knot nematode and soilbome root-infecting fungi viz., Macrophomina phaseolina, Fusarium solani and Rhizoctonia solani under laboratory and greenhouse conditions. Cell-free culture filtrate of the bacterium caused significant reduction in egg hatching of M.javanica and inhibited radial growth of fungi in vitro. Cell-free culture filtrate also caused lyses in mycelium of F.solani. Under greenhouse conditions, soil drenches with the aqueous cell suspension or cell-free culture resulted in a considerable reduction in nematode population densities in soil and subsequent root-knot development due to M.javanica. In addition to nematode control, rhizobacterium application also inhibited root-infection caused by soilborne root~infecting fungi with significant enhancement of growth of tomato seedlings.

  11. Accelerating Smith-Waterman Alignment for Protein Database Search Using Frequency Distance Filtration Scheme Based on CPU-GPU Collaborative System.

    Science.gov (United States)

    Liu, Yu; Hong, Yang; Lin, Chun-Yuan; Hung, Che-Lun

    2015-01-01

    The Smith-Waterman (SW) algorithm has been widely utilized for searching biological sequence databases in bioinformatics. Recently, several works have adopted the graphic card with Graphic Processing Units (GPUs) and their associated CUDA model to enhance the performance of SW computations. However, these works mainly focused on the protein database search by using the intertask parallelization technique, and only using the GPU capability to do the SW computations one by one. Hence, in this paper, we will propose an efficient SW alignment method, called CUDA-SWfr, for the protein database search by using the intratask parallelization technique based on a CPU-GPU collaborative system. Before doing the SW computations on GPU, a procedure is applied on CPU by using the frequency distance filtration scheme (FDFS) to eliminate the unnecessary alignments. The experimental results indicate that CUDA-SWfr runs 9.6 times and 96 times faster than the CPU-based SW method without and with FDFS, respectively.

  12. Glomerular Filtration Barrier Assembly: An insight

    OpenAIRE

    Arif, Ehtesham; Nihalani, Deepak

    2013-01-01

    A glomerulus is the network of capillaries that resides in the Bowman’s capsule that functions as a filtration unit of kidney. The glomerular function ensures that essential plasma proteins are retained in blood and the filtrate is passed on as urine. The glomerular filtration assembly is composed of three main cellular barriers that are critical for the ultrafiltration process, the fenestrated endothelium, glomerular basement membrane and highly specialized podocytes. The podocytes along wit...

  13. 橡胶树黑团孢叶斑病菌培养滤液对薇甘菊的除草活性%Herbicidal Effect of Periconia heveae Culture Filtrate on Mikania micrantha

    Institute of Scientific and Technical Information of China (English)

    古鑫; 范志伟; 张国良; 黄乔乔; 程汉亭; 付卫东; 沈奕德; 刘丽珍

    2012-01-01

    [Objective] The paper was to explore the herbicidal effect of Periconia heveae culture filtrate on Mikania micrantha. [ Method ] Through indoor bioassay method, the inhibitory effects of culture filtrates of seven kinds of plant disease fungi on seed germination and seedling growth of M. Micrantha were studied, and the security of P. Heveae culture filtrate on pepper ( Capsicum annuum) and tomato ( Solarium lycop-ersicum ) was also evaluated. [ Result ] The culture filtrates of P. Heveae had strong inhibitory effects on seed germination and the growth of root, shoot and seedling of M. Micrantha, and the inhibition rates were 72.7% , 93% , 32.4% , and 54.6% (fresh weight), respectively. Security tests showed that culture filtrates of P. Heveae were safe to seed germination and root and shoot growth of tomato, but they inhibited seed germination of pepper. [Conclusion] As potential biological herbicide, the culture filtrate and metabolites of P. Heveae is worthy of further development.%[目的]探讨橡胶树黑团孢叶斑病菌培养滤液对薇甘菊(Mikania micrantha H.B.K.)的除草活性.[方法]采用室内生物活性 测定方法,研究了7种植物病害真菌培养滤液对恶性入侵杂草薇甘菊种子萌发和幼苗生长的抑制作用,并评价了橡胶树黑团孢叶斑病真菌培养滤液对辣椒和番茄的安全性.[结果]橡胶树黑团孢叶斑病病原真菌培养滤液对薇甘菊种子萌发以及根、茎、幼苗生长均有较强的抑制作用,抑制率分别达72.7%、93.0%、32.4%和54.6%(鲜重).橡胶树黑团孢叶斑病病原真菌培养滤液对番茄种子萌发和根茎生长均表现安全,而对辣椒种子萌发有抑制作用.[结论]橡胶树黑团孢叶斑病病原真菌毒素培养滤液及其代谢物值得作为生物除草剂进一步开发.

  14. Cheese production using kefir culture entrapped in milk proteins.

    Science.gov (United States)

    Dimitrellou, Dimitra; Kandylis, Panagiotis; Kourkoutas, Yiannis; Koutinas, Athanasios A; Kanellaki, Maria

    2015-05-01

    The aim of the present study was to evaluate the use of kefir culture entrapped in casein and in whey protein as starter cultures for the production of Feta-type cheese. Microbiological analysis showed that counts of enterobacteria, coliforms, and staphylococci were significantly reduced due to kefir culture. In addition, the effect of kefir culture on the formation of volatile compounds, such as esters, organic acids, alcohols, carbonyl compounds, and lactones, was also investigated using the SPME GC/MS technique. Cheese samples produced with kefir culture entrapped in milk proteins presented improved profile of aroma-related compounds. Principal component analysis of the results indicated that the volatile composition of the different cheese types was dependent on the nature of the starter culture. Finally, the sensory evaluation showed that the products produced with kefir culture had a soft, fine taste, and were of improved quality.

  15. PURIFICATION OF WATER SOLUBLE PROTEINS (2S ALBUMINS EXTRACTED FROM PEANUT DEFATTED FLOUR AND ISOLATION OF THEIR ISOFORMS BY GEL FILTRATION AND ANION EXCHANGE CHROMATOGRAPHY

    Directory of Open Access Journals (Sweden)

    IMÈNE BOUALEG

    2017-06-01

    Full Text Available 2S albumins are water-soluble seed storage proteins present in dicotyledonous plants, including legumes. In peanuts, 2S albumins have been identified as major allergens. In this work, we aimed to study these water soluble allergenic proteins. They were extracted in water from peanut defatted flour (oilcake. It was quantified by Bradford method. The total and insoluble proteins content was determined by Kjeldahl method (% P = N x 6.25. The crude 2S albumins were purified using gel-filtration chromatography. Anion exchange chromatography analysis was applied to isolate their isoforms. The recorded values for total and insoluble proteins are 45.49 % and 36.65 % consecutively. A value of 9.99 % was determined for water soluble proteins content which correspond to 20 % compared to the total proteins. Analysis by Sephadex G-75 chromatography of soluble extract gave two majors peaks in which, the Mr ~ 25 kDa peak was predicted to be pure 2S albumin fraction. Using DAEA-cellulose chromatography, two peaks were appeared from pure 2S albumins, it were predicted that 2S albumin isoforms theoretically represent the peanut major allergens Ara h2 and Ara h6. These approaches are the basis for further studies may involve immunological analysis to understand the impact of these biomolecules on peanut allergenicity.

  16. Production and Purification of Remazol Brilliant Blue R Decolorizing Peroxidase from the Culture Filtrate of Pleurotus ostreatus

    Science.gov (United States)

    Shin, K.; Oh, I.; Kim, C.

    1997-01-01

    An extracellular H(inf2)O(inf2)-requiring Remazol brilliant blue R (RBBR) decolorizing enzymatic activity was found in the culture medium of Pleurotus ostreatus. The enzymatic activity was maximally obtained in idiophase, and the optimum C/N ratio was 24. High C/N ratios repressed the enzymatic activity, and addition of veratryl alcohol had no effect on the production of enzyme. The enzyme was purified by ammonium sulfate fractionation, Sephacryl S-200 HR chromatography, DEAE Sepharose CL-6B chromatography, and Mono Q chromatography. The purification of RBBR decolorizing peroxidase, as judged by the final specific activity of 6.00 U/mg, was 54.5-fold, with a yield of 9.9%. The molecular mass of the native enzyme determined by gel permeation chromatography was found to be about 73 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme was a monomer with a molecular mass of 71 kDa. The enzyme was optimally active at pH 3.0 to 3.5 and at 25(deg)C. Under standard assay conditions, the apparent K(infm) values of the enzyme toward RBBR and H(inf2)O(inf2) were 10.99 and 32.97 (mu)M, respectively. The enzyme had affinity toward various phenolic compounds and artificial dyes, and it was inhibited by Na(inf2)S(inf2)O(inf5), potassium cyanide, NaN(inf3), and cysteine. The absorption spectrum of the enzyme exhibited maxima at 407, 510, and 640 nm. The addition of H(inf2)O(inf2) to the enzyme resulted in an absorbance decrease at 407 and 510 nm. PMID:16535590

  17. Direct identification of microorganisms from positive blood cultures using the lysis-filtration technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): a multicentre study.

    Science.gov (United States)

    Farina, Claudio; Arena, Fabio; Casprini, Patrizia; Cichero, Paola; Clementi, Massimo; Cosentino, Marina; Degl'Innocenti, Roberto; Giani, Tommaso; Luzzaro, Francesco; Mattei, Romano; Mauri, Carola; Nardone, Maria; Rossolini, Gian Maria; Serna Ortega, Paula Andrea; Vailati, Francesca

    2015-04-01

    Microbial identification from blood cultures is essential to institute optimal antibiotic therapy and improve survival possibilities. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been successfully applied to identify bacteria and yeasts from positive blood cultures broths. The aim of this multicentre study was to evaluate the reliability of the lysis-filtration technique associated with MALDI-TOF MS to directly identify microorganisms from 765 positive blood cultures collected in six Italian hospitals. Overall, 675/765 (78.1%) blood isolates were correctly identified at the species level, with significant differences between Gram-negative and Gram-positive bacteria (92.6%, and 69.8%, respectively). Some difficulties arise in identifying Streptococcus pneumoniae, Staphylococcus aureus, yeasts and anaerobes. The lysis-filtration protocol is a suitable procedure in terms of performance in identifying microorganisms, but it is quite expensive and technically time-consuming since the time of filtration is not regular for all the samples. The application of the MALDI-TOF MS technique to the direct microbial identification from positive blood cultures is a very promising approach, even if more experience must be gained to minimize errors and costs.

  18. Development of an in process control filtration-assisted chemiluminometric immunoassay to quantify foot and mouth disease virus (FMDV) non-capsid proteins in vaccine-antigen batches.

    Science.gov (United States)

    Capozzo, Alejandra Victoria; Martínez, Manuel Rosendo; Schielen, Wilhelmus Joseph Gerardus

    2010-09-14

    In many countries, foot and mouth disease (FMD) is controlled by vaccination and surveillance against non-capsid proteins (NCP); therefore vaccines are required not to induce antibodies against NCP. Vaccine purity is evaluated by repeated inoculation of naïve cattle, an expensive and time consuming protocol that raises several animal welfare concerns. We have developed an in process control filtration-assisted chemiluminometric immunoassay (FAL-ELISA), to detect and quantify NCP in vaccine-antigen batches regardless of its volume and composition. Samples are filtered through PVDF-filter microplates pre-coated with a monoclonal antibody against NCP. Filtration removes all unbound components in the sample and captured NCP are detected by anti-NCP conjugate followed by incubation with the substrate, luminol/peroxide. Analytical detection limit was 2 ng for purified NCP and 4 ng for vaccine-antigen batches spiked with NCP, which makes this assay sensitive enough to be applied to purity control of FMD vaccines. Vaccine components did not interfere with the antibody and substrate reactions in the assay. FAL-ELISA is an alternative for the in vivo tests, observing the objective to Replace, Reduce and Refine the use of animals for quality control of immunobiologicals.

  19. Production of recombinant proteins in suspension-cultured plant cells.

    Science.gov (United States)

    Plasson, Carole; Michel, Rémy; Lienard, David; Saint-Jore-Dupas, Claude; Sourrouille, Christophe; de March, Ghislaine Grenier; Gomord, Véronique

    2009-01-01

    Plants have emerged in the past decade as a suitable alternative to the current production systems for recombinant pharmaceutical proteins and, today their potential for low-cost production of high quality, much safer and biologically active mammalian proteins is largely documented. Among various plant expression systems being explored, genetically modified suspension-cultured plant cells offer a promising system for production of biopharmaceuticals. Indeed, when compared to other plant-based production platforms that have been explored, suspension-cultured plant cells have the advantage of being totally devoid of problems associated with the vagaries of weather, pest, soil and gene flow in the environment. Because of short growth cycles, the timescale needed for the production of recombinant proteins in plant cell culture can be counted in days or weeks after transformation compared to months needed for the production in transgenic plants. Moreover, recovery and purification of recombinant proteins from plant biomass is an expensive and technically challenging business that may amount to 80-94% of the final product cost. One additional advantage of plant cell culture is that the recombinant protein fused with a signal sequence can be expressed and secreted into the culture medium, and therefore recovered and purified in the absence of large quantities of contaminating proteins. Consequently, the downstream processing of proteins extracted from plant cell culture medium is less expensive, which may/does balance the higher costs of fermentation. When needed for clinical use, recombinant proteins are easily produced in suspension-cultured plant cells under certified, controllable and sterile conditions that offer improved safety and provide advantages for good manufacturing practices and regulatory compliance. In this chapter, we present basic protocols for rapid generation of transgenic suspension-cultured cells of Nicotiana tabacum, Oriza sativa and Arabidopis

  20. Ethanol production by high cell-density culture with periodic reversion of circulating flow in cross-flow filtration. Ryudo hoko henkan cross flow rokaho wo mochiita komitsudo baiyoho ni okeru ethanol seisan

    Energy Technology Data Exchange (ETDEWEB)

    Asakura, T.; Toda, K. (The University of Tokyo, Tokyo (Japan). Institute of Applied Microbiology)

    1991-07-25

    For the purpose of effective production of ethanol, high cell-density culture was examined using a bioreactor with a flat-membrane filtlation module for three kinds of ethanol production microorganism, and the productivity of ethanol fermentation was investigated. The flow direction of the culture broth circulating along a flat membrane in the filtration module was changed periodically, at intervals of 4 minutes and 1 minute, as a result of which, the filtration flux was stabilized to some degree and increased five-fold over that of the control experiment. When the cell concentration in the reactor was maintained at a constant by draining broth at a proper rate, the filtration flux was stabilized further and increased twenty-fold over that of the control experiment. The productivity and exit concentration of ethanol in this cell recycle culture when the cell concentration was 200 gl{sup {minus}1} of Bakers yeast, were 83.7 gl{sup {minus}1}h{sup {minus}1} and 30.2 gl{sup {minus}1}, respectively. For the other two kinds of yeast, high ethanol concentration and productivity were confirmed, concluding that this production method was effective. 25 refs., 9 figs., 1 tab.

  1. Glomerular filtration rate

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/007305.htm Glomerular filtration rate To use the sharing features on this page, please enable JavaScript. Glomerular filtration rate (GFR) is a test used to check ...

  2. Therapeutically important proteins from in vitro plant tissue culture systems.

    Science.gov (United States)

    Doran, Pauline M

    2013-01-01

    Plant cells cultured in liquid medium in bioreactors are now being used commercially to produce biopharmaceutical proteins. The emergence of in vitro plant cell culture as a production vehicle reflects the importance of key biosafety and biocontainment concerns affecting the competitiveness of alternative systems such as mammalian cell culture and agriculture. Food plant species are particularly attractive as hosts for in vitro protein production: the risk of transgene escape and food chain contamination is eliminated using containment facilities, while regulatory approval for oral delivery of drugs may be easier than if non-edible species were used. As in whole plants, proteolysis in cultured plant cells can lead to significant degradation of foreign proteins after synthesis; however, substantial progress has been made to counter the destructive effects of proteases in plant systems. Although protein secretion into the culture medium is advantageous for product recovery and purification, measures are often required to minimise extracellular protease activity and product losses due to irreversible surface adsorption. Disposable plastic bioreactors, which are being used increasingly in mammalian cell bioprocessing, are also being adopted for plant cell culture to allow rapid scale-up and generation of saleable product. This review examines a range of technical and regulatory issues affecting the choice of industrial production platform for foreign proteins, and assesses progress in the development of in vitro plant systems for biopharmaceutical production.

  3. Peritoneal culture alters Streptococcus pneumoniae protein profiles and virulence properties

    Science.gov (United States)

    Orihuela, C. J.; Janssen, R.; Robb, C. W.; Watson, D. A.; Niesel, D. W.

    2000-01-01

    We have examined the properties of Streptococcus pneumoniae cultured in the murine peritoneal cavity and compared its virulence-associated characteristics to those of cultures grown in vitro. Analysis of mRNA levels for specific virulence factors demonstrated a 2.8-fold increase in ply expression and a 2.2-fold increase in capA3 expression during murine peritoneal culture (MPC). Two-dimensional gels and immunoblots using convalescent-phase patient sera and murine sera revealed distinct differences in protein production in vivo (MPC). MPC-grown pneumococci adhered to A549 epithelial cell lines at levels 10-fold greater than those cultured in vitro.

  4. Association between serum insulin-like growth factor I or IGF-binding protein 3 and estimated glomerular filtration rate: results of a population-based sample

    Directory of Open Access Journals (Sweden)

    Dittmann Kathleen

    2012-12-01

    Full Text Available Abstract Background Insulin-like growth factor I (IGF-I, which is mostly carried in blood by IGF-binding protein 3 (IGFBP-3, was associated to the glomerular filtration rate and chronic kidney disease in a multiethnic study among US adults. The aim of the present study was to investigate whether serum IGF-I or IGFBP-3 are associated with estimated glomerular filtration rate (eGFR in a population-based study of Caucasian adults. Methods Data from 4028 subjects (2048 women aged 20 to 81 years from the Study of Health in Pomerania (SHIP were analyzed. Total serum IGF-I and IGFBP-3 concentrations were determined by chemiluminescence immunoassays and categorized into sex- and age-specific quartiles. Results After adjusting for age, waist circumference and type 2 diabetes mellitus, analysis of variance (ANOVA revealed inverse associations between serum IGF-I concentrations and eGFR in men as well as between serum IGFBP-3 concentrations and eGFR in men and women. Logistic regression analyses confirmed these findings and showed that high IGF-I or IGFBP-3 concentrations were associated with an increased risk of decreased eGFR (2 in men or women. These relations became stronger when lower eGFR cut-offs were used for the analyses. Conclusion Our data revealed associations of increased serum IGF-I concentrations and decreased eGFR in men but not in women and an association of increased serum IGFBP-3 concentrations and decreased eGFR in both sexes.

  5. Rotary filtration system

    Science.gov (United States)

    Herman, David T.; Maxwell, David N.

    2011-04-19

    A rotary filtration apparatus for filtering a feed fluid into permeate is provided. The rotary filtration apparatus includes a container that has a feed fluid inlet. A shaft is at least partially disposed in the container and has a passageway for the transport of permeate. A disk stack made of a plurality of filtration disks is mounted onto the shaft so that rotation of the shaft causes rotation of the filtration disks. The filtration disks may be made of steel components and may be welded together. The shaft may penetrate a filtering section of the container at a single location. The rotary filtration apparatus may also incorporate a bellows seal to prevent leakage along the shaft, and an around the shaft union rotary joint to allow for removal of permeate. Various components of the rotary filtration apparatus may be removed as a single assembly.

  6. Origins of the poor filtration characteristics of wheat starch hydrolysates

    NARCIS (Netherlands)

    Matser, A.M.; Steeneken, P.A.M.

    1998-01-01

    The effects of wheat starch components on the filtration characteristics of wheat starch hydrolysates were investigated with a model-based approach. The filtration rate was not affected by the removal of the pentosans or by altering the conformation of the protein. On the other hand, the filtration

  7. Enrichment and purification of casein glycomacropeptide from whey protein isolate using supercritical carbon dioxide processing and membrane filtration

    Science.gov (United States)

    Whey protein concentrates (WPC) and isolates (WPI), which are dried, concentrated forms of cheese whey, are comprised mainly of beta–lactoglobulin (beta-LG), a–lactalbumin (a-LA), and glycomacropeptide (GLY), and are added to foods to boost their nutritional and functional properties. In previous st...

  8. Rapid identification of bacteria and yeasts from positive-blood-culture bottles by using a lysis-filtration method and matrix-assisted laser desorption ionization-time of flight mass spectrum analysis with the SARAMIS database.

    Science.gov (United States)

    Fothergill, Amy; Kasinathan, Vyjayanti; Hyman, Jay; Walsh, John; Drake, Tim; Wang, Yun F Wayne

    2013-03-01

    Rapid identification of microorganisms causing bloodstream infections directly from a positive blood culture would decrease the time to directed antimicrobial therapy and greatly improve patient care. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a fast and reliable method for identifying microorganisms from positive culture. This study evaluates the performance of a novel filtration-based method for processing positive-blood-culture broth for immediate identification of microorganisms by MALDI-TOF with a Vitek MS research-use-only system (VMS). BacT/Alert non-charcoal-based blood culture bottles that were flagged positive by the BacT/Alert 3D system were included. An aliquot of positive-blood-culture broth was incubated with lysis buffer for 2 to 4 min at room temperature, the resulting lysate was filtered through a membrane, and harvested microorganisms were identified by VMS. Of the 259 bottles included in the study, VMS identified the organisms in 189 (73%) cultures to the species level and 51 (19.7%) gave no identification (ID), while 6 (2.3%) gave identifications that were considered incorrect. Among 131 monomicrobic isolates from positive-blood-culture bottles with one spot having a score of 99.9%, the IDs for 131 (100%) were correct to the species level. In 202 bottles where VMS was able to generate an ID, the IDs for 189 (93.6%) were correct to the species level, whereas the IDs provided for 7 isolates (3.5%) were incorrect. In conclusion, this method does not require centrifugation and produces a clean spectrum for VMS analysis in less than 15 min. This study demonstrates the effectiveness of the new lysis-filtration method for identifying microorganisms directly from positive-blood-culture bottles in a clinical setting.

  9. Differential effects of DEAE negative mode chromatography and gel-filtration chromatography on the charge status of Helicobacter pylori neutrophil-activating protein.

    Science.gov (United States)

    Hong, Zhi-Wei; Yang, Yu-Chi; Pan, Timothy; Tzeng, Huey-Fen; Fu, Hua-Wen

    2017-01-01

    Helicobacter pylori neutrophil-activating protein (HP-NAP) is involved in H. pylori-associated gastric inflammation. HP-NAP is also a vaccine candidate, a possible drug target, and a potential diagnostic marker for H. pylori-associated diseases. Previously, we purified recombinant HP-NAP by one-step diethylaminoethyl (DEAE) negative mode chromatography by collecting the unbound fraction at pH 8.0 at 4°C. It remains unclear why HP-NAP does not bind to DEAE resins at the pH above its isoelectric point during the purification. To investigate how pH affects the surface net charge of HP-NAP and its binding to DEAE resins during the purification, recombinant HP-NAP expressed in Escherichia coli was subjected to DEAE negative mode chromatography at pH ranging from 7.0 to 9.0 at 25°C and the surface charge of purified HP-NAP was determined by capillary electrophoresis. A minimal amount of HP-NAP was detected in the elution fraction of DEAE Sepharose resin at pH 8.5, whereas recombinant HP-NAP was detected in the elution fraction of DEAE Sephadex resin only at pH 7.0 and 8.0. The purified recombinant HP-NAP obtained from the unbound fractions was not able to bind to DEAE resins at pH 7.0 to 9.0. In addition, the surface charge of the purified HP-NAP was neutral at pH 7.0 to 8.0 and was either neutral or slightly negative at pH 8.5 and 9.0. However, recombinant HP-NAP purified from gel-filtration chromatography was able to bind to DEAE Sepharose resin at pH 7.0 to 9.0 and DEAE Sephadex resin at pH 7.0. At pH 8.5 and 9.0, only the negatively charged species of HP-NAP were found. Thus, recombinant HP-NAP with different charge status can be differentially purified by DEAE negative mode chromatography and gel-filtration chromatography. Furthermore, the charge distribution on the surface of HP-NAP, the presence of impure proteins, and the overall net charge of the resins all affect the binding of HP-NAP to DEAE resins during the negative purification.

  10. Filtration as the main transport mechanism of protein exchange between plasma and the peritoneal cavity in hepatic cirrhosis

    DEFF Research Database (Denmark)

    Henriksen, Jens Henrik Sahl; Lassen, N A; Parving, H H;

    1980-01-01

    with carcinomatous ascites. Trans-vascular escape rates of albumin (TERa) and IgG (TERg) were determined in the cirrhotic patients from the disappearance of simultaneously intravenously injected 131I-labelled serum albumin and 124I-labelled IgG. Peritoneal space to plasma appearance times ranged 0.1-3.3 h......, but significantly higher (P TERa and TERg were on average 9.6 and 8.6% of intravascular protein masses per hour, mean TERg/TERa ratio was 0.95. Peritoneal space...

  11. Effect of membrane length, membrane resistance, and filtration conditions on the fractionation of milk proteins by microfiltration.

    Science.gov (United States)

    Piry, A; Heino, A; Kühnl, W; Grein, T; Ripperger, S; Kulozik, U

    2012-04-01

    We investigated the fractionation of casein micelles and the whey protein β-lactoglobulin (β-LG) of skim milk by crossflow microfiltration (0.1 μm) for the first time by a novel approach as a function of membrane length and membrane resistance. A special module was constructed with 4 sections and used to assess the effects of membrane length by measuring flux and β-LG permeation (or transmission) as a function of transmembrane pressure and membrane length. Depending on the position, the membranes were partly controlled by a deposit layer. A maximum for β-LG mass flow through the various membrane sections was found, depending on the position along the membrane. To study the effect of convective flow toward the membrane, membranes with 4 different intrinsic permeation resistances were assessed in terms of the permeation and fouling effects along the flow channel. From these findings, we derived a ratio between transmembrane pressure and membrane resistance, which was useful in reducing the effect of deposit formation and, thus, to optimize the protein permeation. In addition, the fouling effect was investigated in terms of reversible and irreversible fouling and, in addition, by differentiation between pressure-induced fouling and adsorption-induced (pressure-independent) fouling, again as a function of membrane length.

  12. Tamm-Horsfall protein regulates circulating and renal cytokines by affecting glomerular filtration rate and acting as a urinary cytokine trap.

    Science.gov (United States)

    Liu, Yan; El-Achkar, Tarek M; Wu, Xue-Ru

    2012-05-11

    Although few organ systems play a more important role than the kidneys in cytokine catabolism, the mechanism(s) regulating this pivotal physiological function and how its deficiency affects systemic cytokine homeostasis remain unclear. Here we show that elimination of Tamm-Horsfall protein (THP) expression from mouse kidneys caused a marked elevation of circulating IFN-γ, IL1α, TNF-α, IL6, CXCL1, and IL13. Accompanying this were enlarged spleens with prominent white-pulp macrophage infiltration. Lipopolysaccharide (LPS) exacerbated the increase of serum cytokines without a corresponding increase in their urinary excretion in THP knock-out (KO) mice. This, along with the rise of serum cystatin C and the reduced inulin and creatinine clearance from the circulation, suggested that diminished glomerular filtration may contribute to reduced cytokine clearance in THP KO mice both at the baseline and under stress. Unlike wild-type mice where renal and urinary cytokines formed specific in vivo complexes with THP, this "trapping" effect was absent in THP KO mice, thus explaining why cytokine signaling pathways were activated in renal epithelial cells in such mice. Our study provides new evidence implicating an important role of THP in influencing cytokine clearance and acting as a decoy receptor for urinary cytokines. Based on these and other data, we present a unifying model that underscores the role of THP as a major regulator of renal and systemic immunity.

  13. Use of Protein Hydrolysates in Industrial Starter Culture Fermentations

    Science.gov (United States)

    Ummadi, Madhavi (Soni); Curic-Bawden, Mirjana

    Lactic acid bacteria (LAB) have been used as starter cultures for fermenting foods long before the importance of microorganisms were recognized. The most important group of LAB are the lactococci, lactobacilli, streptococci, and pediococci. Additionally, bifidobacteria have been included as a probiotic, providing added value to the product. Since the genera involved are so diverse, the nutritional requirements (energy, carbon and nitrogen sources) differ significantly between and within species. Designing an optimum fermentation medium for production of active and vigorous LAB starter cultures and probiotics requires selecting the right raw ingredients, especially protein hydrolysates that can provide adequate nutrients for growth and viability. This chapter attempts to describe the application of various commercial protein hydrolysates used for production of dairy and meat starter cultures, with special emphasis on meeting the nitrogen requirements of industrially important LAB species.

  14. Towards an Understanding of How Protein Hydrolysates Stimulate More Efficient Biosynthesis in Cultured Cells

    Science.gov (United States)

    Siemensma, André; Babcock, James; Wilcox, Chris; Huttinga, Hans

    In the light of the growing demand for high quality plant-derived hydrolysates (i.e., HyPep™ and UltraPep™ series), Sheffield Bio-Science has developed a new hydrolysate platform that addresses the need for animal-free cell culture medium supplements while also minimizing variability concerns. The platform is based upon a novel approach to enzymatic digestion and more refined processing. At the heart of the platform is a rationally designed animal component-free (ACF) enzyme cocktail that includes both proteases and non-proteolytic enzymes (hydrolases) whose activities can also liberate primary components of the polymerized non-protein portion of the raw material. This enzyme system is added during a highly optimized process step that targets specific enzyme-substrate reactions to expand the range of beneficial nutritional factors made available to cells in culture. Such factors are fundamental to improving the bio-performance of the culture system, as they provide not merely growth-promoting peptides and amino acids, but also key carbohydrates, lipids, minerals, and vitamins that improve both rate and quality of protein expression, and serve to improve culture life due to osmo-protectant and anti-apoptotic properties. Also of significant note is that, compared to typical hydrolysates, the production process is greatly reduced and requires fewer steps, intrinsically yielding a better-controlled and therefore more reproducible product. Finally, the more sophisticated approach to enzymatic digestion renders hydrolysates more amenable to sterile filtration, allowing hydrolysate end users to experience streamlined media preparation and bioreactor supplementation activities. Current and future development activities will evolve from a better understanding of the complex interactions within a handful of key biochemical pathways that impact the growth and productivity of industrially relevant organisms. Presented in this chapter are some examples of the efforts that

  15. Extraction of endo-pectinase activity from the culture filtrate of Polyporus squamosus by aqueous two-phase systems composed of low molecular mass polyethylene glycol and phosphate salt

    Directory of Open Access Journals (Sweden)

    Peričin Draginja M.

    2004-01-01

    Full Text Available Separation of endo-pectinase activity from the culture filtrate of Polyporus squamosus - strain MMOL76, by aqueous two-phase partitioning technique in polyethylene glycol/sodium dihydrogen phosphate system was investigated as the first operation in the downstream processing of enzyme. The best results concerning the partitioning coefficient and the top phase yield were achieved in the polyethylene glycol 400/sodium dihydrogen phosphate system at the tie-line length 78.9 % at pH 3.8 (K = 8, with a 90% yield.

  16. HEPES inhibits the conversion of prion protein in cell culture.

    Science.gov (United States)

    Delmouly, Karine; Belondrade, Maxime; Casanova, Danielle; Milhavet, Ollivier; Lehmann, Sylvain

    2011-05-01

    HEPES is a well-known buffering reagent used in cell-culture medium. Interestingly, this compound is also responsible for significant modifications of biological parameters such as uptake of organic molecules, alteration of oxidative stress mechanisms or inhibition of ion channels. While using cell-culture medium supplemented with HEPES on prion-infected cells, it was noticed that there was a significant concentration-dependent inhibition of accumulation of the abnormal isoform of the prion protein (PrP(Sc)). This effect was present only in live cells and was thought to be related to modification of the PrP environment or biology. These results could modify the interpretation of cell-culture assays of prion therapeutic agents, as well as of previous cell biology results obtained in the field using HEPES buffers. This inhibitory effect of HEPES could also be exploited to prevent contamination or propagation of prions in cell culture.

  17. Benchtop isolation and characterization of functional exosomes by sequential filtration.

    Science.gov (United States)

    Heinemann, Mitja L; Ilmer, Matthias; Silva, Leslie P; Hawke, David H; Recio, Alejandro; Vorontsova, Maria A; Alt, Eckhard; Vykoukal, Jody

    2014-12-05

    Early and minimally invasive detection of malignant events or other pathologies is of utmost importance in the pursuit of improved patient care and outcomes. Recent evidence indicates that exosomes and extracellular vesicles in serum and body fluids can contain nucleic acid, protein, and other biomarkers. Accordingly, there is great interest in applying these clinically as prognostic, predictive, pharmacodynamic, and early detection indicators. Nevertheless, existing exosome isolation methods can be time-consuming, require specialized equipment, and/or present other inefficiencies regarding purity, reproducibility and assay cost. We have developed a straightforward, three-step protocol for exosome isolation of cell culture supernatants or large volumes of biofluid based on sequential steps of dead-end pre-filtration, tangential flow filtration (TFF), and low-pressure track-etched membrane filtration that we introduce here. Our approach yields exosome preparations of high purity and defined size distribution and facilitates depletion of free protein and other low-molecular-weight species, extracellular vesicles larger than 100nm, and cell debris. Samples of exosomes prepared using the approach were verified morphologically by nanoparticle tracking analysis and electron microscopy, and mass spectrometry analyses confirmed the presence of previously reported exosome-associated proteins. In addition to being easy-to-implement, sequential filtration yields exosomes of high purity and, importantly, functional integrity as a result of the relatively low-magnitude manipulation forces employed during isolation. This answers an unmet need for preparation of minimally manipulated exosomes for investigations into exosome function and basic biology. Further, the strategy is amenable to translation for clinical exosome isolations because of its speed, automatability, scalability, and specificity for isolating exosomes from complex biological samples.

  18. Magnetic-seeding filtration

    Energy Technology Data Exchange (ETDEWEB)

    Ying, T.Y.; Chin, C.J.; Lu, S.C.; Yiacoumi, S. [Georgia Institute of Technology, Atlanta, GA (United States). School of Civil and Environmental Engineering] [and others

    1997-10-01

    Magnetic-seeding filtration consists of two steps: heterogeneous particle flocculation of magnetic and nonmagnetic particles in a stirred tank and high-gradient magnetic filtration (HGMF). The effects of various parameters affecting magnetic-seeding filtration (HGMF). The effects of various parameters affecting magnetic seeding filtration are theoretically and experimentally investigated. A trajectory model that includes hydrodynamic resistance, van der Waals, and electrostatic forces is developed to calculate the flocculation frequency in a turbulent-shear regime. Fractal dimension is introduced to simulate the open structure of aggregates. A magnetic-filtration model that consists of trajectory analysis, a particle build-up model, a breakthrough model, and a bivariate population-balance model is developed to predict the breakthrough curve of magnetic-seeding filtration. A good agreement between modeling results and experimental data is obtained. The results show that the model developed in this study can be used to predict the performance of magnetic-seeding filtration without using empirical coefficients or fitting parameters. 35 refs., 7 figs., 1 tab.

  19. Same day identification and full panel antimicrobial susceptibility testing of bacteria from positive blood culture bottles made possible by a combined lysis-filtration method with MALDI-TOF VITEK mass spectrometry and the VITEK2 system.

    Science.gov (United States)

    Machen, Alexandra; Drake, Tim; Wang, Yun F Wayne

    2014-01-01

    Rapid identification and antimicrobial susceptibility testing of microorganisms causing bloodstream infections or sepsis have the potential to improve patient care. This proof-of-principle study evaluates the Lysis-Filtration Method for identification as well as antimicrobial susceptibility testing of bacteria directly from positive blood culture bottles in a clinical setting. A total of 100 non-duplicated positive blood cultures were tested and 1012 microorganism-antimicrobial combinations were assessed. An aliquot of non-charcoal blood culture broth was incubated with lysis buffer briefly before being filtered and washed. Microorganisms recovered from the filter membrane were first identified by using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight VITEK® Mass Spectrometry (VITEK MS). After quick identification from VITEK MS, filtered microorganisms were inoculated to VITEK®2 system for full panel antimicrobial susceptibility testing analysis. Of 100 bottles tested, the VITEK MS resulted in 94.0% correct organism identification to the species level. Compared to the conventional antimicrobial susceptibility testing methods, direct antimicrobial susceptibility testing from VITEK®2 resulted in 93.5% (946/1012) category agreement of antimicrobials tested, with 3.6% (36/1012) minor error, 1.7% (7/1012) major error, and 1.3% (13/1012) very major error of antimicrobials. The average time to identification and antimicrobial susceptibility testing was 11.4 hours by using the Lysis-Filtration method for both VITEK MS and VITEK®2 compared to 56.3 hours by using conventional methods (pblood culture can be achieved and can be used for appropriate antibiotic therapy and antibiotic stewardship.

  20. Same day identification and full panel antimicrobial susceptibility testing of bacteria from positive blood culture bottles made possible by a combined lysis-filtration method with MALDI-TOF VITEK mass spectrometry and the VITEK2 system.

    Directory of Open Access Journals (Sweden)

    Alexandra Machen

    Full Text Available Rapid identification and antimicrobial susceptibility testing of microorganisms causing bloodstream infections or sepsis have the potential to improve patient care. This proof-of-principle study evaluates the Lysis-Filtration Method for identification as well as antimicrobial susceptibility testing of bacteria directly from positive blood culture bottles in a clinical setting. A total of 100 non-duplicated positive blood cultures were tested and 1012 microorganism-antimicrobial combinations were assessed. An aliquot of non-charcoal blood culture broth was incubated with lysis buffer briefly before being filtered and washed. Microorganisms recovered from the filter membrane were first identified by using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight VITEK® Mass Spectrometry (VITEK MS. After quick identification from VITEK MS, filtered microorganisms were inoculated to VITEK®2 system for full panel antimicrobial susceptibility testing analysis. Of 100 bottles tested, the VITEK MS resulted in 94.0% correct organism identification to the species level. Compared to the conventional antimicrobial susceptibility testing methods, direct antimicrobial susceptibility testing from VITEK®2 resulted in 93.5% (946/1012 category agreement of antimicrobials tested, with 3.6% (36/1012 minor error, 1.7% (7/1012 major error, and 1.3% (13/1012 very major error of antimicrobials. The average time to identification and antimicrobial susceptibility testing was 11.4 hours by using the Lysis-Filtration method for both VITEK MS and VITEK®2 compared to 56.3 hours by using conventional methods (p<0.00001. Thus, the same-day results of microorganism identification and antimicrobial susceptibility testing directly from positive blood culture can be achieved and can be used for appropriate antibiotic therapy and antibiotic stewardship.

  1. Same Day Identification and Full Panel Antimicrobial Susceptibility Testing of Bacteria from Positive Blood Culture Bottles Made Possible by a Combined Lysis-Filtration Method with MALDI-TOF VITEK Mass Spectrometry and the VITEK2 System

    Science.gov (United States)

    Machen, Alexandra; Drake, Tim; Wang, Yun F. (Wayne)

    2014-01-01

    Rapid identification and antimicrobial susceptibility testing of microorganisms causing bloodstream infections or sepsis have the potential to improve patient care. This proof-of-principle study evaluates the Lysis-Filtration Method for identification as well as antimicrobial susceptibility testing of bacteria directly from positive blood culture bottles in a clinical setting. A total of 100 non-duplicated positive blood cultures were tested and 1012 microorganism-antimicrobial combinations were assessed. An aliquot of non-charcoal blood culture broth was incubated with lysis buffer briefly before being filtered and washed. Microorganisms recovered from the filter membrane were first identified by using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight VITEK® Mass Spectrometry (VITEK MS). After quick identification from VITEK MS, filtered microorganisms were inoculated to VITEK®2 system for full panel antimicrobial susceptibility testing analysis. Of 100 bottles tested, the VITEK MS resulted in 94.0% correct organism identification to the species level. Compared to the conventional antimicrobial susceptibility testing methods, direct antimicrobial susceptibility testing from VITEK®2 resulted in 93.5% (946/1012) category agreement of antimicrobials tested, with 3.6% (36/1012) minor error, 1.7% (7/1012) major error, and 1.3% (13/1012) very major error of antimicrobials. The average time to identification and antimicrobial susceptibility testing was 11.4 hours by using the Lysis-Filtration method for both VITEK MS and VITEK®2 compared to 56.3 hours by using conventional methods (p<0.00001). Thus, the same-day results of microorganism identification and antimicrobial susceptibility testing directly from positive blood culture can be achieved and can be used for appropriate antibiotic therapy and antibiotic stewardship. PMID:24551067

  2. Establishment of sorghum cell suspension culture system for ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-03-18

    Mar 18, 2008 ... Total soluble proteins (TSP) and culture filtrate (CF) proteins were extracted from the cell culture ... Even though. Arabidopsis provides for an excellent model system for .... stained, destained and imaged using a Molecular Imager PharosFX ... system, are dynamic and heterogeneous, being com- posed of a ...

  3. Filtration in Porous Media

    DEFF Research Database (Denmark)

    Yuan, Hao; Shapiro, Alexander

    There is a considerable and ongoing effort aimed at understanding the transport and the deposition of suspended particles in porous media, especially non-Fickian transport and non-exponential deposition of particles. In this work, the influential parameters in filtration models are studied...... to understand their effects on the non-Fickian transport and the non-exponential deposition. The filtration models are validated by the comparisons between the modelling results and the experimental data.The elliptic equation with distributed filtration coefficients may be applied to model non-Fickian transport...... and hyperexponential deposition. The filtration model accounting for the migration of surface associated particles may be applied for non-monotonic deposition....

  4. Water Filtration Products

    Science.gov (United States)

    1986-01-01

    American Water Corporation manufactures water filtration products which incorporate technology originally developed for manned space operations. The formula involves granular activated charcoal and other ingredients, and removes substances by catalytic reactions, mechanical filtration, and absorption. Details are proprietary. A NASA literature search contributed to development of the compound. The technology is being extended to a deodorizing compound called Biofresh which traps gas and moisture inside the unit. Further applications are anticipated.

  5. Manufacture of biopharmaceutical proteins by mammalian cell culture systems.

    Science.gov (United States)

    Tolbert, W R

    1990-01-01

    In the last several years, dramatic advances have been in the development of new biopharmaceuticals including monoclonal antibodies for diagnosis and treatment and such genetically engineered proteins as tPA, Factor VIIIc, erythropoietin and soluble CD4, an anti-AIDS protein. Currently, there are several hundred such candidate drugs in human clinical trials. In most cases, these protein-based drugs will require manufacture by mammalian cell culture due to the inability of lower organisms to properly glycosylate, fold, make correct disulfide bonds and secrete active biomolecular forms. The need for large scale production from cell culture will greatly increase as more of the products in clinical trials are approved for commercial production. This will require significant reduction in manufacturing costs per gram, concomitant with increased capacity to hundreds or perhaps even thousands of kilograms annually. As an example, Invitron's multi-reactor manufacturing facility has operated at greater than one-half million liters per year and has experience with more than 250 mammalian cell lines for producing protein drug products.

  6. Identification and characterization of a 29-kilodalton protein from Mycobacterium tuberculosis culture filtrate recognized by mouse memory effector cells

    DEFF Research Database (Denmark)

    Rosenkrands, I; Rasmussen, P.B.; Carnio, M

    1998-01-01

    , Determination of the N-terminal amino acid sequence allowed cloning and sequencing of the cfp29 gene. The nucleotide sequence showed 62% identity to the bacteriocin Linocin from Brevibacterium linens, Purified recombinant histidine-tagged CFP29 and native CFP29 had similar T-cell stimulatory properties...

  7. Nitration of plant apoplastic proteins from cell suspension cultures.

    Science.gov (United States)

    Szuba, Agnieszka; Kasprowicz-Maluśki, Anna; Wojtaszek, Przemysław

    2015-04-29

    Nitric oxide causes numerous protein modifications including nitration of tyrosine residues. This modification, though one of the greatest biological importance, is poorly recognized in plants and is usually associated with stress conditions. In this study we analyzed nitrotyrosines from suspension cultures of Arabidopsis thaliana and Nicotiana tabacum, treated with NO modulators and exposed to osmotic stress, as well as of BY2 cells long-term adapted to osmotic stress conditions. Using confocal microscopy, we showed that the cell wall area is one of the compartments most enriched in nitrotyrosines within a plant cell. Subsequently, we analyzed nitration of ionically-bound cell-wall proteins and identified selected proteins with MALDI-TOF spectrometry. Proteomic analysis indicated that there was no significant increase in the amount of nitrated proteins under the influence of NO modulators, among them 3-morpholinosydnonimine (SIN-1), considered a donor of nitrating agent, peroxynitrite. Moreover, osmotic stress conditions did not increase the level of nitration in cell wall proteins isolated from suspension cells, and in cultures long-term adapted to stress conditions; that level was even reduced in comparison with control samples. Among identified nitrotyrosine-containing proteins dominated the ones associated with carbon circulation as well as the numerous proteins responding to stress conditions, mainly peroxidases. High concentrations of nitric oxide found in the cell wall and the ability to produce large amounts of ROS make the apoplast a site highly enriched in nitrotyrosines, as presented in this paper. Analysis of ionically bound fraction of the cell wall proteins indicating generally unchanged amounts of nitrotyrosines under influence of NO modulators and osmotic stress, is noticeably different from literature data concerning, however, the total plant proteins analysis. This observation is supplemented by further nitroproteome analysis, for cells long

  8. Silver nanoparticle protein corona composition in cell culture media.

    Directory of Open Access Journals (Sweden)

    Jonathan H Shannahan

    Full Text Available The potential applications of nanomaterials as drug delivery systems and in other products continue to expand. Upon introduction into physiological environments and driven by energetics, nanomaterials readily associate proteins forming a protein corona (PC on their surface. This PC influences the nanomaterial's surface characteristics and may impact their interaction with cells. To determine the biological impact of nanomaterial exposure as well as nanotherapeutic applications, it is necessary to understand PC formation. Utilizing a label-free mass spectrometry-based proteomics approach, we examined the composition of the PC for a set of four silver nanoparticles (AgNPs including citrate-stabilized and polyvinlypyrrolidone-stabilized (PVP colloidal silver (20 or 110 nm diameter. To simulate cell culture conditions, AgNPs were incubated for 1 h in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, washed, coronal proteins solubilized, and proteins identified and quantified by label-free LC-MS/MS. To determine which attributes influence PC formation, the AgNPs were characterized in both water and cell culture media with 10% FBS. All AgNPs associated a common subset of 11 proteins including albumin, apolipoproteins, keratins, and other serum proteins. 110 nm citrate- and PVP-stabilized AgNPs were found to bind the greatest number of proteins (79 and 85 respectively compared to 20 nm citrate- and PVP-stabilized AgNPs (45 and 48 respectively, suggesting a difference in PC formation based on surface curvature. While no relationships were found for other protein parameters (isoelectric point or aliphatic index, the PC on 20 nm AgNPs (PVP and citrate consisted of more hydrophobic proteins compared to 110 nm AgNPs implying that this class of proteins are more receptive to curvature-induced folding and crowding in exchange for an increased hydration in the aqueous environment. These observations demonstrate the significance of

  9. Culture and the Immune System: Cultural Consonance in Social Support and C-reactive Protein in Urban Brazil.

    Science.gov (United States)

    Dressler, William W; Balieiro, Mauro C; Ribeiro, Rosane P; Dos Santos, José Ernesto

    2016-06-01

    In this article, we examine the distribution of a marker of immune system stimulation-C-reactive protein-in urban Brazil. Social relationships are associated with immunostimulation, and we argue that cultural dimensions of social support, assessed by cultural consonance, are important in this process. Cultural consonance is the degree to which individuals, in their own beliefs and behaviors, approximate shared cultural models. A measure of cultural consonance in social support, based on a cultural consensus analysis regarding sources and patterns of social support in Brazil, was developed. In a survey of 258 persons, the association of cultural consonance in social support and C-reactive protein was examined, controlling for age, sex, body mass index, low-density lipoprotein cholesterol, depressive symptoms, and a social network index. Lower cultural consonance in social support was associated with higher C-reactive protein. Implications of these results for future research are discussed.

  10. Visualization of water flow during filtration using flat filtration materials

    Directory of Open Access Journals (Sweden)

    Hrůza Jakub

    2012-04-01

    Full Text Available Filtration materials are very important elements of some industrial appliances. Water filtration is a separation of solid materials from fluid. Solid particles are captured on the frontal area of the filtration textile and only liquid passes through it. It is important to know the filtration process in a detailed way to be able to develop filtration materials. Visualization of filtration process enables a better view of the filtration. This method also enables to determine efficiency and homogeneity of filtration using image analysis. For this purpose, a new waterfiltration measuring setup was proposed and constructed. Filtration material is mounted into the optically transparent place in the setup. Laser sheet is directed into this place as in the case of Particle Image Velocimetry measuring method. Monochrome and sensitive camera records the light scattered by seeding particles in water. The seeding particles passing through the filter serve for measuring filtration efficiency, and also for visualization of filtration process. Filtration setup enables to measure also the pressure drop and a flow. The signals are processed by National Instruments compactDAQ system and UMA software. Microfibrous and nanofibrous filtration materials are tested by this measuring method. In the case of nanofibrous filtration, appropriate size of seeding particles is needed to be used to perform a process of filtration.

  11. Changes in glomerular filtration rate, lithium clearance and plasma protein clearances in the early phase after unilateral nephrectomy in living healthy renal transplant donors

    DEFF Research Database (Denmark)

    Strandgaard, S; Kamper, A; Skaarup, P;

    1988-01-01

    1. Glomerular and tubular function was studied before and 2 months after unilateral nephrectomy in 14 healthy kidney donors by measurement of the clearances of 51Cr-labelled ethylenediaminetetra-acetate, lithium, beta 2-microglobulin, albumin and immunoglobulin G. 2. The glomerular filtration rate...

  12. Staphylococcus carnosus: from starter culture to protein engineering platform.

    Science.gov (United States)

    Löfblom, John; Rosenstein, Ralf; Nguyen, Minh-Thu; Ståhl, Stefan; Götz, Friedrich

    2017-10-02

    Since the 1950s, Staphylococcus carnosus is used as a starter culture for sausage fermentation where it contributes to food safety, flavor, and a controlled fermentation process. The long experience with S. carnosus has shown that it is a harmless and "food grade" species. This was confirmed by the genome sequence of S. carnosus TM300 that lacks genes involved in pathogenicity. Since the development of a cloning system in TM300, numerous genes have been cloned, expressed, and characterized and in particular, virulence genes that could be functionally validated in this non-pathogenic strain. A secretion system was developed for production and secretion of industrially important proteins and later modified to also enable display of heterologous proteins on the surface. The display system has been employed for various purposes, such as development of live bacterial delivery vehicles as well as microbial biocatalysts or bioadsorbents for potential environmental or biosensor applications. Recently, this surface display system has been utilized for display of peptide and protein libraries for profiling of protease substrates and for generation of various affinity proteins, e.g., Affibody molecules and scFv antibodies. In addition, by display of fragmented antigen-encoding genes, the surface expression system has been successfully used for epitope mapping of antibodies. Reviews on specific applications of S. carnosus have been published earlier, but here we provide a more extensive overview, covering a broad range of areas from food fermentation to sophisticated methods for protein-based drug discovery, which are all based on S. carnosus.

  13. The filtration properties of a dimorphic yeast

    OpenAIRE

    McCarthy, Anthony A

    2001-01-01

    A dimorphic yeast Kluyveromyces marxianu var. marxianus NRRLy2415 which exhibits a wide range of mean morphological forms was used as a model organism to investigate the role of cell morphology on the dead-end and crossflow filtration behaviour. Varying the culturing conditions produced cell suspensions of different mean morphology. Batch fermentations were used to produce yeast-like morphologies and continuous cultures produced cells more mycelial in nature. Semi-automated image analysis was...

  14. Microfluidic colloid filtration

    Science.gov (United States)

    Linkhorst, John; Beckmann, Torsten; Go, Dennis; Kuehne, Alexander J. C.; Wessling, Matthias

    2016-03-01

    Filtration of natural and colloidal matter is an essential process in today’s water treatment processes. The colloidal matter is retained with the help of micro- and nanoporous synthetic membranes. Colloids are retained in a “cake layer” – often coined fouling layer. Membrane fouling is the most substantial problem in membrane filtration: colloidal and natural matter build-up leads to an increasing resistance and thus decreasing water transport rate through the membrane. Theoretical models exist to describe macroscopically the hydrodynamic resistance of such transport and rejection phenomena; however, visualization of the various phenomena occurring during colloid retention is extremely demanding. Here we present a microfluidics based methodology to follow filter cake build up as well as transport phenomena occuring inside of the fouling layer. The microfluidic colloidal filtration methodology enables the study of complex colloidal jamming, crystallization and melting processes as well as translocation at the single particle level.

  15. CENTRIFUGAL MEMBRANE FILTRATION

    Energy Technology Data Exchange (ETDEWEB)

    Daniel J. Stepan; Bradley G. Stevens; Melanie D. Hetland

    1999-10-01

    The overall project consists of several integrated research phases related to the applicability, continued development, demonstration, and commercialization of the SpinTek centrifugal membrane filtration process. Work performed during this reporting period consisted of Phase 2 evaluation of the SpinTek centrifugal membrane filtration technology and Phase 3, Technology Partnering. During Phase 1 testing conducted at the EERC using the SpinTek ST-IIL unit operating on a surrogate tank waste, a solids cake developed on the membrane surface. The solids cake was observed where linear membrane velocities were less than 17.5 ft/s and reduced the unobstructed membrane surface area up to 25%, reducing overall filtration performance. The primary goal of the Phase 2 research effort was to enhance filtration performance through the development and testing of alternative turbulence promoter designs. The turbulence promoters were designed to generate a shear force across the entire membrane surface sufficient to maintain a self-cleaning membrane capability and improve filtration efficiency and long-term performance. Specific Phase 2 research activities included the following: System modifications to accommodate an 11-in.-diameter, two-disk rotating membrane assembly; Development and fabrication of alternative turbulence promoter designs; Testing and evaluation of the existing and alternative turbulence promoters under selected operating conditions using a statistically designed test matrix; and Data reduction and analysis; The objective of Phase 3 research was to demonstrate the effectiveness of SpinTek's centrifugal membrane filtration as a pretreatment to remove suspended solids from a liquid waste upstream of 3M's WWL cartridge technology for the selective removal of technetium (Tc).

  16. Magnetic-seeding filtration

    Energy Technology Data Exchange (ETDEWEB)

    Depaoli, D. [Oak Ridge National Lab., TN (United States)

    1996-10-01

    This task will investigate the capabilities of magnetic-seeding filtration for the enhanced removal of magnetic and nonmagnetic particulates from liquids. This technology appies to a wide range of liquid wastes, including groundwater, process waters, and tank supernatant. Magnetic-seeding filtration can be used in several aspects of treatment, such as (1) removal of solids, particularly those in the colloidal-size range that are difficult to remove by conventional means; (2) removal of contaminants by precipitation processes; and (3) removal of contaminants by sorption processes.

  17. Filtration by eyelashes

    Science.gov (United States)

    Vistarakula, Krishna; Bergin, Mike; Hu, David

    2010-11-01

    Nearly every mammalian and avian eye is rimmed with lashes. We investigate experimentally the ability of lashes to reduce airborne particle deposition in the eye. We hypothesize that there is an optimum eyelash length that maximizes both filtration ability and extent of peripheral vision. This hypothesis is tested using a dual approach. Using preserved heads from 36 species of animals at the American Museum of Natural History, we determine the relationship between eye size and eyelash geometry (length and spacing). We test the filtration efficacy of these geometries by deploying outdoor manikins and measuring particle deposition rate as a function of eyelash length.

  18. Optimization of Separation and Purification of Lactic Acid Bacterial Protein with Gel-filtrationChromatography%凝胶层析分离纯化乳酸菌菌体蛋白条件的优化

    Institute of Scientific and Technical Information of China (English)

    赵春燕; 康素花; 荣向华; 张旋

    2016-01-01

    The lactic acid bacterial protein were separated and purified by gel-filtration chromatography. Se-lecting different gel-filtration chromatography media,column height,flow rate and sample weight to elute lactic acid bacterial protein of Sephadex G-25 desalting. The elution peak was observed. The result showed that all theabove four factors could affect the separation and purification performance of the lactic acid bacterial protein ingel-filtration chromatography by various degrees. In this test,the best separation and purification performancewas achieved using Sephadex G-75 with 45 cm column height,0.75 mL/min flow rate and 5%CV sample weight.%采用凝胶过滤层析分离纯化乳酸菌菌体蛋白,选择不同的凝胶过滤介质、过滤柱高度、洗脱流速、样品上样量对经过SephadexG-25脱盐处理的乳酸菌菌体蛋白进行洗脱,观察洗脱峰.结果表明,凝胶过滤介质、过滤柱过度、洗脱流速和样品上样量均不同程度的影响菌体蛋白的纯化效果.在本试验中,分离纯化乳酸菌菌体蛋白的凝胶过滤层析参数为:SephadexG-75的层析介质以45 cm的柱床高度,洗脱流速0.75 mL/min,样品上样量为5%CV时,对乳酸菌菌体蛋白可达到最佳的分离纯化效果.

  19. Enlargements of filtrations and applications

    CERN Document Server

    Corcuera, J M

    2012-01-01

    In this paper we review some old and new results about the enlargement of filtrations problem, as well as their applications to credit risk and insider trading problems. The enlargement of filtrations problem consists in the study of conditions under which a semimartingale remains a semimartingale when the filtration is enlarged, and, in such a case, how to find the Doob-Meyer decomposition. Filtrations may be enlarged in different ways. In this paper we consider initial and progressive filtration enlargements made by random variables and processes. Keywords: Credit Risk, Insider Trading, Enlargement of Filtrations

  20. Cellulose filtration of blood from malaria patients for improving ex vivo growth of Plasmodium falciparum parasites

    DEFF Research Database (Denmark)

    Mkumbaye, Sixbert I; Minja, Daniel T R; Jespersen, Jakob S;

    2017-01-01

    BACKGROUND: Establishing in vitro Plasmodium falciparum culture lines from patient parasite isolates can offer deeper understanding of geographic variations of drug sensitivity and mechanisms of malaria pathogenesis and immunity. Cellulose column filtration of blood is an inexpensive, rapid...... and effective method for the removal of host factors, such as leucocytes and platelets, significantly improving the purification of parasite DNA in a blood sample. METHODS: In this study, the effect of cellulose column filtration of venous blood on the initial in vitro growth of P. falciparum parasite isolates....... falciparum merozoite surface protein 2 genotyping was performed using nested PCR on extracted genomic DNA, and the var gene transcript levels were investigated, using quantitative PCR on extracted RNA, at admission and 4 days of culture. RESULTS: The cellulose-filtered parasites grew to higher parasitaemia...

  1. Water Treatment Technology - Filtration.

    Science.gov (United States)

    Ross-Harrington, Melinda; Kincaid, G. David

    One of twelve water treatment technology units, this student manual on filtration provides instructional materials for six competencies. (The twelve units are designed for a continuing education training course for public water supply operators.) The competencies focus on the following areas: purposes of sedimentation basins and flocculation…

  2. The Glomerular Filtration Barrier: Components and Crosstalk

    Directory of Open Access Journals (Sweden)

    Madhav C. Menon

    2012-01-01

    Full Text Available The glomerular filtration barrier is a highly specialized blood filtration interface that displays a high conductance to small and midsized solutes in plasma but retains relative impermeability to macromolecules. Its integrity is maintained by physicochemical and signalling interplay among its three core constituents—the glomerular endothelial cell, the basement membrane and visceral epithelial cell (podocyte. Understanding the pathomechanisms of inherited and acquired human diseases as well as experimental injury models of this barrier have helped to unravel this interdependence. Key among the consequences of interference with the integrity of the glomerular filtration barrier is the appearance of significant amounts of proteins in the urine. Proteinuria correlates with kidney disease progression and cardiovascular mortality. With specific reference to proteinuria in human and animal disease phenotypes, the following review explores the roles of the endothelial cell, glomerular basement membrane, and the podocyte and attempts to highlight examples of essential crosstalk within this barrier.

  3. Dynamic optical filtration

    Science.gov (United States)

    Chretien, Jean-Loup (Inventor); Lu, Edward T. (Inventor)

    2005-01-01

    A dynamic optical filtration system and method effectively blocks bright light sources without impairing view of the remainder of the scene. A sensor measures light intensity and position so that selected cells of a shading matrix may interrupt the view of the bright light source by a receptor. A beamsplitter may be used so that the sensor may be located away from the receptor. The shading matrix may also be replaced by a digital micromirror device, which selectively sends image data to the receptor.

  4. Magnetic flocculation and filtration

    Energy Technology Data Exchange (ETDEWEB)

    Yiacoumi, Sotira; Chin, Ching-Ju; Yin, Tung-Yu [Georgia Inst. of Tech., Atlanta, GA (United States). School of Civil and Environmental Engineering; Tsouris, C., DePaoli, D.W.; Chattin, M.R.; Spurrier, M. [Oak Ridge National Lab., TN (United States)

    1996-10-01

    A model is available in predicting flocculation frequencies between particles of various properties under the influence of a magnetic field. This model provides a basic understanding of fundamental phenomena, such as particle-particle and particle-collector interactions, occurring in HGMF (high gradient magnetic field), and will be extended to describe experimental data of particle flocculation and filtration and predict the performance of high- gradient magnetic filters. It is also expected that this model will eventually lead to a tool for design and optimization of magnetic filters for environmental, metallurgical, biochemical, and other applications.

  5. Approaches to Optimizing Animal Cell Culture Process: Substrate Metabolism Regulation and Protein Expression Improvement

    Science.gov (United States)

    Zhang, Yuanxing

    Some high value proteins and vaccines for medical and veterinary applications by animal cell culture have an increasing market in China. In order to meet the demands of large-scale productions of proteins and vaccines, animal cell culture technology has been widely developed. In general, an animal cell culture process can be divided into two stages in a batch culture. In cell growth stage a high specific growth rate is expected to achieve a high cell density. In production stage a high specific production rate is stressed for the expression and secretion of qualified protein or replication of virus. It is always critical to maintain high cell viability in fed-batch and perfusion cultures. More concern has been focused on two points by the researchers in China. First, the cell metabolism of substrates is analyzed and the accumulation of toxic by-products is decreased through regulating cell metabolism in the culture process. Second, some important factors effecting protein expression are understood at the molecular level and the production ability of protein is improved. In pace with the rapid development of large-scale cell culture for the production of vaccines, antibodies and other recombinant proteins in China, the medium design and process optimization based on cell metabolism regulation and protein expression improvement will play an important role. The chapter outlines the main advances in metabolic regulation of cell and expression improvement of protein in animal cell culture in recent years.

  6. Approaches to optimizing animal cell culture process: substrate metabolism regulation and protein expression improvement.

    Science.gov (United States)

    Zhang, Yuanxing

    2009-01-01

    Some high value proteins and vaccines for medical and veterinary applications by animal cell culture have an increasing market in China. In order to meet the demands of large-scale productions of proteins and vaccines, animal cell culture technology has been widely developed. In general, an animal cell culture process can be divided into two stages in a batch culture. In cell growth stage a high specific growth rate is expected to achieve a high cell density. In production stage a high specific production rate is stressed for the expression and secretion of qualified protein or replication of virus. It is always critical to maintain high cell viability in fed-batch and perfusion cultures. More concern has been focused on two points by the researchers in China. First, the cell metabolism of substrates is analyzed and the accumulation of toxic by-products is decreased through regulating cell metabolism in the culture process. Second, some important factors effecting protein expression are understood at the molecular level and the production ability of protein is improved. In pace with the rapid development of large-scale cell culture for the production of vaccines, antibodies and other recombinant proteins in China, the medium design and process optimization based on cell metabolism regulation and protein expression improvement will play an important role. The chapter outlines the main advances in metabolic regulation of cell and expression improvement of protein in animal cell culture in recent years.

  7. Magnetic-seeding filtration

    Energy Technology Data Exchange (ETDEWEB)

    DePaoli, D.W.; Tsouris, C. [Oak Ridge National Lab., TN (United States); Yiacoumi, Sotira

    1997-10-01

    Magnetic-seeding filtration is a technology under development for the enhanced removal of magnetic and non-magnetic particulates from liquids. This process involves the addition of a small amount of magnetic seed particles (such as naturally occurring iron oxide) to a waste suspension, followed by treatment with a magnetic filter. Non-magnetic and weakly magnetic particles are made to undergo nonhomogeneous flocculation with the seed particles, forming flocs of high magnetic susceptibility that are readily removed by a conventional high-gradient magnetic filter. This technology is applicable to a wide range of liquid wastes, including groundwater, process waters, and tank supernatants. Magnetic-seeding filtration may be used in several aspects of treatment, such as (1) removal of solids, particularly those in the colloidal size range that are difficult to remove by conventional means; (2) removal of contaminants by precipitation processes; and (3) removal of contaminants by sorption processes. Waste stream characteristics for which the technology may be applicable include (1) particle sizes ranging from relatively coarse (several microns) to colloidal particles, (2) high or low radiation levels, (3) broad-ranging flow rates, (4) low to moderate solids concentration, (5) cases requiring high decontamination factors, and (6) aqueous or non-aqueous liquids. At this point, the technology is at the bench-scale stage of development; laboratory studies and fundamental modeling are currently being employed to determine the capabilities of the process.

  8. Microcystin-Bound Protein Patterns in Different Cultures of Microcystis aeruginosa and Field Samples

    Science.gov (United States)

    Wei, Nian; Hu, Lili; Song, Lirong; Gan, Nanqin

    2016-01-01

    Micocystin (MC) exists in Microcystis cells in two different forms, free and protein-bound. We examined the dynamic change in extracellular free MCs, intracellular free MCs and protein-bound MCs in both batch cultures and semi-continuous cultures, using high performance liquid chromatography and Western blot. The results showed that the free MC per cell remained constant, while the quantity of protein-bound MCs increased with the growth of Microcystis cells in both kinds of culture. Significant changes in the dominant MC-bound proteins occurred in the late exponential growth phase of batch cultures, while the dominant MC-bound proteins in semi-continuous cultures remained the same. In field samples collected at different months in Lake Taihu, the dominant MC-bound proteins were shown to be similar, but the amount of protein-bound MC varied and correlated with the intracellular MC content. We identified MC-bound proteins by two-dimensional electrophoresis immunoblots and mass spectrometry. The 60 kDa chaperonin GroEL was a prominent MC-bound protein. Three essential glycolytic enzymes and ATP synthase alpha subunit were also major targets of MC-binding, which might contribute to sustained growth in semi-continuous culture. Our results indicate that protein-bound MC may be important for sustaining growth and adaptation of Microcystis sp. PMID:27754336

  9. [Clinical experience of automated double filtration plasmapheresis].

    Science.gov (United States)

    Lee, C T; Chuang, F R; Hsu, K T; Lam, K K; Liao, S C; Liu, C C; Chen, J B; Jang, S W; Chien, Y S; Pan, H H

    1996-12-01

    Double filtration plasmapheresis, one kind of fractionation plasmapheresis, was developed from membrane type plasmapheresis to remove only the pathogen and return the normal protein back to the patient. We started our automated double filtration plasmapheresis since December 1993. There were 13 patients who received one hundred treatments totally during one year period. And they are myasthenia gravis (8 patients); acute inflammatory demyelinating polyneuropathy (1 patient), multiple myeloma (1 patient); acquired factor VIII inhibitor (1 patient); autoimmune hemolytic anemia (1 patient); systemic lupus erythematous (1 patient). Technically double filtration plasmapheresis is easy to perform and time-saving. It also makes necessity of replacement fluid less frequent. Incidence of complication is rare, and this includes hypotension 2%, palpitation 1%, headache 1%, hemolysis 4%, air emboli 1%, high secondary pressure 2%, and no motality during our treatment. Clinical response is documented in cases of myasthenia gravis; acute inflammatory demyelinating polyneuropathy and acquired factor VIII inhibitor in our study. In conclusion, double filtration plasmapheresis is a time-saving, convenient, and safe therapeutic modality with rare complication. Because its effectiveness on limited kinds of diseases and costs relatively high price, thus plasmapheresis should be used in selected cases and treat aggressively if indicated.

  10. Recombinant Protein Production and Insect Cell Culture and Process

    Science.gov (United States)

    Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor); OConnor, Kim C. (Inventor); Francis, Karen M. (Inventor); Andrews, Angela D. (Inventor); Prewett, Tracey L. (Inventor)

    1997-01-01

    A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production.

  11. Modus of filtration.

    Science.gov (United States)

    Meltzer, Theodore H

    2006-01-01

    Experience teaches that particles larger than the pores of a filter cannot negotiate its passage. Other retention mechanisms are less obvious than sieve retention or size exclusion. They are electrical in nature, and find expression in the bonding alliances that mutually attract (or repel) filters and particles. The influence of hydrogen bonds, of van der Waals forces, of hydrophobic adsorptions, and of transient polarities on particle retentions are set forth in terms of the double electrical layer concept that also governs colloidal destabilizations. The origins of differences in membrane porosities is explained, as also the importance of the filtration conditions. The singularity of the particle-fluid-filter relationship on organism and/or pore size alteration is stressed.

  12. Filtration properties of nonwovens.

    Science.gov (United States)

    Gador, W; Jankowska, E

    1999-01-01

    This paper presents the results and conclusions from experimental investigations concerning filtration properties of nonwovens. The needled nonwovens were made from polyester fibres (PTE) with average fibre diameter 12 micrometres and polypropylene fibres (PP) with average fibre diameter 32 micrometres. Nonwovens were produced out of each of those fibres or out of a mixture of polyester and polypropylene fibres. This paper also presents investigations of nonwoven fabric made of polypropylene fibres (PP) with average fibre diameter 2.6 micrometres, which was formed according to melt-blown technology. Oil mist, as challenge aerosol, was used to evaluate the performance of filter media at various aerosol velocities. The average oil mist test aerosol particle diameter was 0.3 micrometre. Filter penetration was measured at oil mist concentration 0.24 g/m(3).

  13. Characterization of cell envelope proteins of Staphylococcus epidermidis cultured in human peritoneal dialysate.

    OpenAIRE

    Smith, D G; Wilcox, M. H.; Williams, P.; Finch, R G; Denyer, Stephen Paul

    1991-01-01

    The cell envelope protein profiles of Staphylococcus epidermidis cultured in used human peritoneal dialysate (HPD) differed markedly from those of cells cultured in nutrient broth. Compared with broth-grown cells, many cell wall proteins were repressed in HPD, although three proteins of 42, 48, and 54 kDa predominated and an iron-repressible 130-kDa protein was induced. Growth in HPD also resulted in expression of two cell membrane proteins of 32 and 36 kDa which were iron repressible. Sodium...

  14. Air filtration in HVAC systems

    CERN Document Server

    Ginestet, Alain; Tronville, Paolo; Hyttinen, Marko

    2010-01-01

    Air filtration Guidebook will help the designer and user to understand the background and criteria for air filtration, how to select air filters and avoid problems associated with hygienic and other conditions at operation of air filters. The selection of air filters is based on external conditions such as levels of existing pollutants, indoor air quality and energy efficiency requirements.

  15. Selective release from cultured mammalian cells of heat-shock (stress) proteins that resemble glia-axon transfer proteins.

    Science.gov (United States)

    Hightower, L E; Guidon, P T

    1989-02-01

    Cultured rat embryo cells were stimulated to rapidly release a small group of proteins that included several heat-shock proteins (hsp110, hsp71, hscp73) and nonmuscle actin. The extracellular proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. Heat-shocked cells released the same set of proteins as control cells with the addition of the stress-inducible hsp110 and hsp71. Release of these proteins was not blocked by either monensin or colchicine, inhibitors of the common secretory pathway. A small amount of the glucose-regulated protein grp78 was externalized by this pathway. The extracellular accumulation of these proteins was inhibited after they were synthesized in the presence of the lysine analogue aminoethyl cysteine. It is likely that the analogue-substituted proteins were misfolded and could not be released from cells, supporting our conclusion that a selective release mechanism is involved. Remarkably, actin and the squid heat-shock proteins homologous to rat hsp71 and hsp110 are also among a select group of proteins transferred from glial cells to the squid giant axon, where they have been implicated in neuronal stress responses (Tytell et al.: Brain Res., 363:161-164, 1986). Based in part on the similarities between these two sets of proteins, we hypothesized that these proteins were released from labile cortical regions of animal cells in response to perturbations of homeostasis in cells as evolutionarily distinct as cultured rat embryo cells and squid glial cells.

  16. Influence of anabolic agents on protein synthesis and degradation in muscle cells grown in culture

    Energy Technology Data Exchange (ETDEWEB)

    Roeder, R.A.; Thorpe, S.D.; Byers, F.M.; Schelling, G.T.; Gunn, J.M.

    Muscle cell culture (L/sub 6/) studies were conducted to determine whether anabolic agents have a direct effect on the muscle cell. The effect of zeranol, testosterone propionate, estradiol benzoate, progesterone, dexamethasone and anabolic agent-dexamethasone combinations on protein synthesis and degradation were measured. Myoblast and myotube cultures were pretreated with 1 ..mu..M compounds for 12, 24 and 48 h before a 6-h synthesis or degradation measuring period. Protein synthesis was determined as cpm of (/sup 3/H) leucine incorporated per mg cell protein. Protein degradation was measured by a pulse-chase procedure using (/sup 3/H) leucine and expressed as the percentage labeled protein degraded in 6 h. Progesterone slightly increased protein synthesis in myoblast cultures. Testosterone propionate had no effect on synthesis. Protein synthesis was decreased by estradiol benzoate in myotube cultures. Protein degradation was not altered appreciably by anabolic agents. Protein synthesis was initially inhibited in myotubes by dexamethasone, but increased in myoblasts and myotubes in the extended incubation time. Dexamethasone also consistently increased protein degradation, but this required several hours to be expressed. Anabolic agents did not interfere with dexamethasone-induced increases in protein synthesis and degradation. The magnitude of response and sensitivity were similar for both the myoblast and the more fully differentiated myotube for all compounds tested. These results indicate that anabolic agents at the 1 ..mu..M level do not have a direct anabolic effect on muscle or alter glucocorticoid-induced catabolic response in muscle.

  17. Production of therapeutic proteins through plant tissue and cell culture

    Directory of Open Access Journals (Sweden)

    Reza S. Gharelo

    2016-04-01

    Full Text Available Nowadays, pharmaceutical recombinant protein is increasingly used in treatment of many diseases such as hepatitis, anemia, diabetes and cancer. Different protein expression systems have been used for the expression of recombinant proteins in which each of them face obstacles that make utilizing them as comprehensive expression system in order to express wide variety of proteins difficult. Plant cell as a eukaryotic expression system have many advantages compared to other hosts. They are very "safe" and significantly decrease concerns about the contamination of recombinant proteins with human pathogens. In addition to this, plants as eukaryotic expression system perform proper post-translational modification, in case of eukaryotic proteins, and appropriate folding resulting in right function in biological environments. Therefore, the production of pharmaceutical protein through plant cells can be absolutely promising approach. In this review, the production of pharmaceutical protein in plant cells, advantages and disadvantages, offered methods and techniques for developing recombinant protein yields, and affective factors on the whole process of pharmaceutical protein expression in the molecular level will be reviewed.

  18. A Sandwiched-Culture Technique for Evaluation of Heterologous Protein Production in a Filamentous Fungus

    Science.gov (United States)

    Ásgeirsdóttir, Sigrídur A.; Scholtmeijer, Karin; Wessels, Joseph G. H.

    1999-01-01

    Aspergillus niger is known for its efficient excretion machinery. However, problems have often arisen in obtaining high amounts of heterologous proteins in the culture medium. Here we present a quick method using sandwiched colonies to evaluate transgenic strains for secretion of heterologous proteins. Expressing the ABH1 hydrophobin of Agaricus bisporus in A. niger, we showed that low production levels of the heterologous protein are probably due to extracellular proteolytic degradation of the protein. PMID:10224030

  19. Embryo culture in teratological surveillance and serum proteins in development. Progress report, 1979-1980

    Energy Technology Data Exchange (ETDEWEB)

    Klein, N.W.

    1980-07-01

    Research progress for the period 1979-1980 is reported. The feasibility of using rat embryo cultures to test the teratogenic activity of serum was studied. The mechanisms regulating the synthesis of serum proteins were investigated. (ACR)

  20. Problems of multiphase fluid filtration

    CERN Document Server

    Konovalov, AN

    1994-01-01

    This book deals with a spectrum of problems related to the mathematical modeling of multiphase filtration. Emphasis is placed on an inseparable triad: model - algorithm - computer code. An analysis of new and traditional filtration problems from the point of view of both their numerical implementation and the reproduction of one or another technological characteristics of the processes under consideration is given. The basic principles which underlie the construction of efficient numerical methods taking into account the filtration problems are discussed: non-evolutionary nature, degeneration,

  1. Induction of Bone Matrix Protein Expression by Native Bone Matrix Proteins in C2C12 Culture

    Institute of Scientific and Technical Information of China (English)

    ZHEN-MING HU; SEAN A. F. PEEL; STEPHEN K. C. HO; GEORGE K. B. SANDOR; CAMERON M. L. CLOKIE

    2009-01-01

    Objective To study the expression of bone matrix protein (BMP) induced by bovine bone morphogenetic proteins (BMPs) in vitro. Methods Type I collagen, osteopontin (OPN), osteonectin (ON), osteocalcin (OC), and bone sialoprotein (BSP) were detected by immunohistochemistry in C2C12 cultured from day 1 to day 28. Results The signaling of bone matrix protein expression became weaker except for type I collagen, OC and BSP after 5 days. Fourteen days after culture, the positive signaling of type I collagen, OPN, ON, OC, and BSP was gradually declined, and could be detected significantly as compared with that of the negative control on day 28. BMP assay showed that the Ikaline phosphatase (ALP) activity was higher in C2C12 culture than in the control during the 14-day culture. Also, total protein and DNA significantly increased during the 14-day culture. High levels of ALP were seen in preosteoblasts and osteoblsts in vivo and in differentiating ostcoblasts in vitro. ALP was well recognized as a marker reflecting osteoblastic activity. Conclusion Native bovine BMP induces conversion of myoblasts into osteoblasts, produces type 1 collagen, and plays significantly role in osteoinduction and bone matrix mineralization of C2C12 in vitro.

  2. Molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures.

    Science.gov (United States)

    Schillberg, Stefan; Raven, Nicole; Fischer, Rainer; Twyman, Richard M; Schiermeyer, Andreas

    2013-01-01

    Plants have been used for more than 20 years to produce recombinant proteins but only recently has the focus shifted away from proof-of-principle studies (i.e. is my protein expressed and is it functional?) to a serious consideration of the requirements for sustainable productivity and the regulatory approval of pharmaceutical products (i.e. is my protein safe, is it efficacious, and does the product and process comply with regulatory guidelines?). In this context, plant tissue and cell suspension cultures are ideal production platforms whose potential has been demonstrated using diverse pharmaceutical proteins. Typically, cell/tissue cultures are grown in containment under defined conditions, allowing process controls to regulate growth and product formation, thus ensuring regulatory compliance. Recombinant proteins can also be secreted to the culture medium, facilitating recovery and subsequent purification because cells contain most of the contaminating proteins and can be removed from the culture broth. Downstream processing costs are therefore lower compared to whole plant systems, balancing the higher costs of the fermentation equipment. In this article, we compare different approaches for the production of valuable proteins in plant cell suspension and tissue cultures, describing the advantages and disadvantages as well as challenges that must be overcome to make this platform commercially viable. We also present novel strategies for system and process optimization, helping to increase yields and scalability.

  3. Process validation for cell culture-derived pharmaceutical proteins.

    Science.gov (United States)

    Lubiniecki, A S; Wiebe, M E; Builder, S E

    1990-01-01

    Principles of process validation are extremely powerful tools in assurance of product quality. They are especially useful for reducing those risks not easily measured routinely during production. When combined with effective process and facility design principles, characterization of cell banks and products, appropriate lot release tests, and adherence to cGMP, safe cell culture biologicals can be prepared in a reliable manner.

  4. Mitochondrial trifunctional protein deficiency in human cultured fibroblasts

    DEFF Research Database (Denmark)

    Djouadi, Fatima; Habarou, Florence; Le Bachelier, Carole

    2016-01-01

    Mitochondrial trifunctional protein (MTP) deficiency caused by HADHA or HADHB gene mutations exhibits substantial molecular, biochemical, and clinical heterogeneity and ranks among the more severe fatty acid oxidation (FAO) disorders, without pharmacological treatment. Since bezafibrate has been...

  5. Latest aspects of mechanical filtration

    Directory of Open Access Journals (Sweden)

    Stanislav Koláček

    2013-01-01

    Full Text Available The aim of this study was to describe and unify all knowledge about mechanic filtration. The first part deals with the parameters and properties of filtration. Here some important basic concepts are explained such as pressure gradient, filter life, etc. There’s also a description of convenient filtration technology for coarse and fine materials, such as sand, smoke or soot. The second part primarily focuses on the real use and application of filters for liquid and gaseous media. The differences in construction between different types of filters for filtration of fuels, oils, hydraulic fluids, air and cabin filters are described. The last section is focused mainly on new materials for the production of filters. These materials are ceramic or nanomaterials, which can actually be enriched for example with antibacterial silver or some fungicides.

  6. Ribosome-inhibiting proteins from in vitro cultures of Phytolacca dodecandra

    DEFF Research Database (Denmark)

    Thomsen, S.; Hansen, Harald S.; Nyman, U.

    1991-01-01

    Phytolacca dodecandra (L'Herit) grown in cell cultures was investigated for content of ribosome-inhibiting proteins, which was evaluated hy measuring inhibition of protein synthesis in a cell-free rat liver extract. Calli initiated from leaf, cotyledon, radicle, and hypocotyl and suspension cells...

  7. Protein markers for identification of Yersinia pestis and their variation related to culture

    Energy Technology Data Exchange (ETDEWEB)

    Wunschel, David S. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Engelmann, Heather E. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Victry, Kristin D. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Clowers, Brian H. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Sorensen, Christina M. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Valentine, Nancy B. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Mahoney Fahey, Christine M. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Wietsma, Thomas W. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Wahl, Karen L. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2013-12-11

    The detection of high consequence pathogens, such as Yersinia pestis, is well established in biodefense laboratories for bioterror situations. Laboratory protocols are well established using specified culture media and a growth temperature of 37 °C for expression of specific antigens. Direct detection of Y. pestis protein markers, without prior culture, depends on their expression. Unfortunately protein expression can be impacted by the culture medium which cannot be predicted ahead of time. Furthermore, higher biomass yields are obtained at the optimal growth temperature (i.e. 28 °C–30 °C) and therefore are more likely to be used for bulk production. Analysis of Y. pestis grown on several types of media at 30 °C showed that several protein markers were found to be differentially detected in different media. Analysis of the identified proteins against a comprehensive database provided an additional level of organism identification. Peptides corresponding to variable regions of some proteins could separate large groups of strains and aid in organism identification. This work illustrates the need to understand variability of protein expression for detection targets. The potential for relating expression changes of known proteins to specific media factors, even in nutrient rich and chemically complex culture medium, may provide the opportunity to draw forensic information from protein profiles.

  8. Health benefits of particle filtration.

    Science.gov (United States)

    Fisk, W J

    2013-10-01

    The evidence of health benefits of particle filtration in homes and commercial buildings is reviewed. Prior reviews of papers published before 2000 are summarized. The results of 16 more recent intervention studies are compiled and analyzed. Also, reviewed are four studies that modeled health benefits of using filtration to reduce indoor exposures to particles from outdoors. Prior reviews generally concluded that particle filtration is, at best, a source of small improvements in allergy and asthma health effects; however, many early studies had weak designs. A majority of recent intervention studies employed strong designs and more of these studies report statistically significant improvements in health symptoms or objective health outcomes, particularly for subjects with allergies or asthma. The percentage improvement in health outcomes is typically modest, for example, 7% to 25%. Delivery of filtered air to the breathing zone of sleeping allergic or asthmatic persons may be more consistently effective in improving health than room air filtration. Notable are two studies that report statistically significant improvements, with filtration, in markers that predict future adverse coronary events. From modeling, the largest potential benefits of indoor particle filtration may be reductions in morbidity and mortality from reducing indoor exposures to particles from outdoor air.

  9. Health Benefits of Particle Filtration

    Energy Technology Data Exchange (ETDEWEB)

    Fisk, William J.

    2013-10-01

    The evidence of health benefits of particle filtration in homes and commercial buildings is reviewed. Prior reviews of papers published before 2000 are summarized. The results of 16 more recent intervention studies are compiled and analyzed. Also reviewed are four studies that modeled health benefits of using filtration to reduce indoor exposures to particles from outdoors. Prior reviews generally concluded that particle filtration is, at best, a source of small improvements in allergy and asthma health effects; however, many early studies had weak designs. A majority of recent intervention studies employed strong designs and more of these studies report statistically significant improvements in health symptoms or objective health outcomes, particularly for subjects with allergies or asthma. The percentage improvement in health outcomes is typically modest, e.g., 7percent to 25percent. Delivery of filtered air to the breathing zone of sleeping allergic or asthmatic persons may be more consistently effective in improving health than room air filtration. Notable are two studies that report statistically significant improvements, with filtration, in markers that predict future adverse coronary events. From modeling, the largest potential benefits of indoor particle filtration may be reductions in morbidity and mortality from reducing indoor exposures to particles from outdoor air.

  10. Health Benefits of Particle Filtration

    Energy Technology Data Exchange (ETDEWEB)

    Fisk, William J.

    2013-10-01

    The evidence of health benefits of particle filtration in homes and commercial buildings is reviewed. Prior reviews of papers published before 2000 are summarized. The results of 16 more recent intervention studies are compiled and analyzed. Also, reviewed are four studies that modeled health benefits of using filtration to reduce indoor exposures to particles from outdoors. Prior reviews generally concluded that particle filtration is, at best, a source of small improvements in allergy and asthma health effects; however, many early studies had weak designs. A majority of recent intervention studies employed strong designs and more of these studies report statistically significant improvements in health symptoms or objective health outcomes, particularly for subjects with allergies or asthma. The percent age improvement in health outcomes is typically modest, for example, 7percent to 25percent. Delivery of filtered air to the breathing zone of sleeping allergic or asthmatic persons may be more consistently effective in improving health than room air filtration. Notable are two studies that report statistically significant improvements, with filtration, in markers that predict future adverse coronary events. From modeling, the largest potential benefits of indoor particle filtration may be reductions in morbidity and mortality from reducing indoor exposures to particles from outdoor air.

  11. Filtration and compression of organic materials

    DEFF Research Database (Denmark)

    Christensen, Morten Lykkegaard; Keiding, Kristian

    The conventional filtration theory has been based on filtrations of incompressible particles such as anatase, kaolin and clay. The filtration models have later been used for organic slurries but can often not explain the observed experimental data. At constant pressure, the filtrate volume does...

  12. 40 CFR 141.73 - Filtration.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 22 2010-07-01 2010-07-01 false Filtration. 141.73 Section 141.73... PRIMARY DRINKING WATER REGULATIONS Filtration and Disinfection § 141.73 Filtration. A public water system..., and does not meet all of the criteria in § 141.71 (a) and (b) for avoiding filtration, must...

  13. Hepatitis C virus proteins do not directly trigger fibrogenic events in cultured human liver myofibroblasts.

    Science.gov (United States)

    Tan, K; Guibert, C; Neaud, V; Rosenbaum, J

    2003-11-01

    Although liver fibrosis is the major complication of hepatitis C virus (HCV) infection, the mechanisms of fibrogenesis in this setting are not completely understood. The aim of this study was to test the direct effect of HCV proteins on signalling- and fibrosis-related events in cultured human liver myofibroblasts, the effector cells of liver fibrogenesis. Cultured myofibroblasts were exposed to recombinant HCV core, a structural protein, and nonstructural proteins (NS) 3, NS 4 and NS 5. HCV proteins did not significantly increase DNA synthesis in myofibroblasts. We then examined if these proteins affected early signalling events. None of the HCV proteins affected the phosphorylation of the mitogen activated protein kinases/extracellular regulated kinases 1 and 2, or of the phosphatidylinositol 3-kinase target, Akt. HCV proteins had also no effect on intracellular calcium concentration. In other experiments, fibrogenesis-related parameters were measured. None of the HCV proteins had any effect on the secretion of type I collagen, tissue inhibitor of matrix metalloproteinases type 1, gelatinase or urokinase. Alpha-smooth muscle actin expression was also not modified. In summary, our experiments do not support a direct effect of these HCV proteins on fibrogenic cells.

  14. 转KAP6.1-GFP-蜘蛛拖丝蛋白基因核心序列4S绵羊成纤维细胞株的筛选%Filtration of Transgenic Sheep Skin Fibroblasts with KAP6.1-GFP-polymerized Spider Dragline Silk Protein Gene(4S)

    Institute of Scientific and Technical Information of China (English)

    王春生; 原璐; 宁方勇; 吴治昊; 朴善花; 安铁洙

    2011-01-01

    with normal fibroblast, cell growth curve was "S" shape, population doubling time (PDT) shorten gradually along with culture time increase, plating efficiency was gradually increasing in 24h. These biological analyses showed the transgenic cells were similar with normal sheep fibroblast. Biological characters of the transgenic cells after freezing-thawing were similar with fresh sheep fibroblast. The PCR result showed that the vector constructed was integrated into sheep genome. [Conclusion] The transgenic cells with polymerized spider dragline silk protein gene were filtrated, and thus laid a foundation for the transgenic sheep with expression spider dragline silk protein in hair follicle.

  15. Cyclodextrins inhibit replication of scrapie prion protein in cell culture.

    Science.gov (United States)

    Prior, Marguerite; Lehmann, Sylvain; Sy, Man-Sun; Molloy, Brendan; McMahon, Hilary E M

    2007-10-01

    Prion diseases are fatal neurodegenerative disorders that are caused by the conversion of a normal host-encoded protein, PrP(C), to an abnormal, disease-causing form, PrP(Sc). This paper reports that cyclodextrins have the ability to reduce the pathogenic isoform of the prion protein PrP(Sc) to undetectable levels in scrapie-infected neuroblastoma cells. Beta-cyclodextrin removed PrP(Sc) from the cells at a concentration of 500 microM following 2 weeks of treatment. Structure activity studies revealed that antiprion activity was dependent on the size of the cyclodextrin. The half-maximal inhibitory concentration (IC(50)) for beta-cyclodextrin was 75 microM, whereas alpha-cyclodextrin, which possessed less antiprion activity, had an IC(50) of 750 microM. This report presents cyclodextrins as a new class of antiprion compound. For decades, the pharmaceutical industry has successfully used cyclodextrins for their complex-forming ability; this ability is due to the structural orientation of the glucopyranose units, which generate a hydrophobic cavity that can facilitate the encapsulation of hydrophobic moieties. Consequently, cyclodextrins could be ideal candidates for the treatment of prion diseases.

  16. Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures.

    Science.gov (United States)

    Xu, Jianfeng; Ge, Xumeng; Dolan, Maureen C

    2011-01-01

    "Molecular farming" in plants with significant advantages in cost and safety is touted as a promising platform for the production of complex pharmaceutical proteins. While whole-plant produced biopharmaceuticals account for a significant portion of the preclinical and clinical pipeline, plant cell suspension culture, which integrates the merits of whole-plant systems with those of microbial fermentation, is emerging as a more compliant alternative "factory". However, low protein productivity remains a major obstacle that limits extensive commercialization of plant cell bioproduction platform. This review highlights the advantages and recent progress in plant cell culture technology and outlines viable strategies at both the biological and process engineering levels for advancing the economic feasibility of plant cell-based protein production. Approaches to overcome and solve the associated challenges of this culture system that include non-mammalian glycosylation and genetic instability will also be discussed.

  17. Microtubule Associated Protein 1b (MAP1B Is a Marker of the Microtubular Cytoskeleton in Podocytes but Is Not Essential for the Function of the Kidney Filtration Barrier in Mice.

    Directory of Open Access Journals (Sweden)

    Markus Gödel

    Full Text Available Podocytes are essential for the function of the kidney glomerular filter. A highly differentiated cytoskeleton is requisite for their integrity. Although much knowledge has been gained on the organization of cortical actin networks in podocyte's foot processes, less is known about the molecular organization of the microtubular cytoskeleton in primary processes and the cell body. To gain an insight into the organization of the microtubular cytoskeleton of the podocyte, we systematically analyzed the expression of microtubule associated proteins (Maps, a family of microtubules interacting proteins with known functions as regulator, scaffold and guidance proteins. We identified microtubule associated protein 1b (MAP1B to be specifically enriched in podocytes in human and rodent kidney. Using immunogold labeling in electron microscopy, we were able to demonstrate an enrichment of MAP1B in primary processes. A similar association of MAP1B with the microtubule cytoskeleton was detected in cultured podocytes. Subcellular distribution of MAP1B HC and LC1 was analyzed using a double fluorescent reporter MAP1B fusion protein. Subsequently we analyzed mice constitutively depleted of MAP1B. Interestingly, MAP1B KO was not associated with any functional or structural alterations pointing towards a redundancy of MAP proteins in podocytes. In summary, we established MAP1B as a specific marker protein of the podocyte microtubular cytoskeleton.

  18. Increased adsorption of histidine-tagged proteins onto tissue culture polystyrene

    DEFF Research Database (Denmark)

    Holmberg, Maria; Hansen, Thomas Steen; Lind, Johan Ulrik

    2012-01-01

    In this study we compare histidine-tagged and native proteins with regards to adsorption properties. We observe significantly increased adsorption of proteins with an incorporated polyhistidine amino acid motif (HIS-tag) onto tissue culture polystyrene (TCPS) compared to similar proteins without...... a HIS-tag. The effect is not observed on polystyrene (PS). Adsorption experiments have been performed at physiological pH (7.4) and the effect was only observed for the investigated proteins that have pI values below or around 7.4. Competitive adsorption experiments with imidazole...

  19. Surface proteins in normal and transformed rat liver epithelial cells in culture.

    Science.gov (United States)

    Bannikov, G. A.; Saint Vincent, L.; Montesano, R.

    1980-01-01

    The pattern of surface proteins of different types of normal and transformed rat liver cells have been studied in culture by means of lactoperoxidase-catalysed iodination procedures, followed by SDS-gel electrophoresis. The cells examined were primary cultures of epithelial liver cells, long-term cultures of epithelial liver cells, in vitro transformed epithelial liver cell lines and liver tumour-cell lines; mesenchymal cells from liver and skin were also examined. The principal surface proteins of primary cultures of epithelial cells from adult or neonatal rats had components with mol. wts of 140,000-160,000, 100,000 and 40,000-70,000. A band that had the same position as fibronectin from mesenchymal cells was also present and this band, as well as other iodinated components, were less sensitive to trypsin than fibroblastic fibronectin. A similar pattern of iodinated proteins was seen in long-term cultures of epithelial liver cells, with a great reduction in the number and intensity of the bands in the mol. wt region below 100,000. Almost all the in vitro transformed and tumour epithelial cell lines contain a protein with a mol. wt 135,000 as one of the major iodinated bands, and in contrast to the observation in transformed fibroblasts, the fibronectin was retained by most of these transformed cell lines. Images Fig. 1 Fig. 2 Fig. 3 PMID:7053205

  20. Cake creep during filtration of flocculated manure

    DEFF Research Database (Denmark)

    Christensen, Morten Lykkegaard; Keiding, Kristian

    the distribution of N and P on the fields. Filtration is a useful method for such a separation. Furthermore, chemicals can be added to flocculate the solids and thereby increase the filterability i.e. the specific filter-cake resistance can be reduced from 1015 m/kg to 1011 m/kg. Both the amount of added chemicals......, and the mixing procedure affect the result, and lab-scale experiments are often used to study how these pre-treatments influence the filtration process. However, the existing mathematical filtration models are based on filtration of inorganic particles and cannot simulate the filtration data obtained when manure...... that the discrepancy between the filtration theory and the observed filtration behaviour is due to a time-dependent collapse of the formed cake (creep). This can also explain the observed behaviour when flocculated manure is filtered. The filtration data can be simulated if cake creep is adopted in the filtration...

  1. Effects of Proteins from Culture Medium on Surface Property of Silanes- Functionalized Magnetic Nanoparticles

    Directory of Open Access Journals (Sweden)

    Chen ZP

    2008-01-01

    Full Text Available Abstract Monodisperse magnetic nanoparticles (MNPs were synthesized by thermal decomposition of iron-oleate and functionalized with silanes bearing various functional groups such as amino group (NH2, short-chain poly(ethylene glycol (PEG, and carboxylic group (COOH. Then, silanes-functionalized magnetic nanoparticles (silanes-MNPs were incubated in cell culture medium plus fetal calf serum to investigate the effects of proteins from culture medium on surface property of MNPs. Zeta potential measurements showed that although surface charges of silanes-MNPs were different, they exhibited negative charges at neutral pH and approximate isoelectric points after they were incubated in cell culture medium. The reason was that silanes-MNPs could easily adsorb proteins from culture medium via non-covalent binding, resulting in the formation of protein-silanes-MNPs conjugates. Moreover, silanes-MNPs with various functional groups had different adsorption capacity to proteins, as confirmed by Coomassie blue fast staining method. The in vitro cell experiments showed that protein-silanes-MNPs had higher cellular uptake by cancer cells than silanes-MNPs.

  2. Emulsions for interfacial filtration.

    Energy Technology Data Exchange (ETDEWEB)

    Grillet, Anne Mary; Bourdon, Christopher Jay; Souza, Caroline Ann; Welk, Margaret Ellen; Hartenberger, Joel David; Brooks, Carlton, F.

    2006-11-01

    We have investigated a novel emulsion interfacial filter that is applicable for a wide range of materials, from nano-particles to cells and bacteria. This technology uses the interface between the two immiscible phases as the active surface area for adsorption of targeted materials. We showed that emulsion interfaces can effectively collect and trap materials from aqueous solution. We tested two aqueous systems, a bovine serum albumin (BSA) solution and coal bed methane produced water (CBMPW). Using a pendant drop technique to monitor the interfacial tension, we demonstrated that materials in both samples were adsorbed to the liquid-liquid interface, and did not readily desorb. A prototype system was built to test the emulsion interfacial filter concept. For the BSA system, a protein assay showed a progressive decrease in the residual BSA concentration as the sample was processed. Based on the initial prototype operation, we propose an improved system design.

  3. Improving IAQ Via Air Filtration.

    Science.gov (United States)

    Monk, Brian

    1999-01-01

    Provides tips on using air filtration to control indoor air quality in educational facilities, including dedicated spaces with unique air quality conditions such as in libraries, museums and archival storage areas, kitchens and dining areas, and laboratories. The control of particulate contaminants, gaseous contaminants, and moisture buildup are…

  4. Filtration combustion: Smoldering and SHS

    Science.gov (United States)

    Matkowsky, Bernard J.

    1995-01-01

    Smolder waves and SHS (self-propagating high-temperature synthesis) waves are both examples of combustion waves propagating in porous media. When delivery of reactants through the pores to the reaction site is an important aspect of the process, it is referred to as filtration combustion. The two types of filtration combustion have a similar mathematical formulation, describing the ignition, propagation and extinction of combustion waves in porous media. The goal in each case, however, is different. In smoldering the desired goal is to prevent propagation, whereas in SHS the goal is to insure propagation of the combustion wave, leading to the synthesis of desired products. In addition, the scales in the two areas of application may well differ. For example, smoldering generally occurs at a relatively low temperature and with a smaller propagation velocity than SHS filtration combustion waves. Nevertheless, the two areas of application have much in common, so that mechanisms learned about in one application can be used to advantage in the other. In this paper we discuss recent results in the areas of filtration combustion.

  5. Demonstration of creep during filtration

    DEFF Research Database (Denmark)

    Christensen, Morten Lykkegaard; Bugge, Thomas Vistisen; Kirchheiner, Anders Løvenbalk

    The classical filtration theory assumes a unique relationship between the local filter cake porosity and the local effective pressure. For a number of compressible materials, it has however been observed that during the consolidation stage this may not be the case. It has been found that the prod......The classical filtration theory assumes a unique relationship between the local filter cake porosity and the local effective pressure. For a number of compressible materials, it has however been observed that during the consolidation stage this may not be the case. It has been found...... that the production of filtrate also depends on the characteristic time for the filter cake solids to deform. This is formulated in the Terzaghi-Voigt model in which a secondary consolidation is introduced. The secondary consolidation may be visualized by plots of the relative cake deformation (U) v.s. the square...... magnitude as the primary consolidation (defined by the hydraulic retardation), the creep phenomenon may occur during filtration. This will lead to Ruth's plots characterized by a concave with two (more or less) distinct slopes. The slopes are defined by the relationship between the porosity...

  6. 凝胶过滤色谱和Bradford法测定发酵类抗生素中蛋白残留量%Gel Filtration Chromatography Combined with Bradford Method for Determination of Total Residual Protein in Ferment Antibiotics

    Institute of Scientific and Technical Information of China (English)

    许明哲; 马仕洪; 胡昌勤

    2004-01-01

    目的以发酵类抗生素盐酸林可霉素为例,建立一个快速、简单、灵敏度高的可用于发酵类抗生素中蛋白残留量控制的方法.方法利用凝胶过滤色谱(GFC)首先将蛋白质与抗生素分离,收集蛋白组分,再利用蛋白检测方法Bradford法(考马氏亮兰染色法)对残留的蛋白进行定量;色谱柱为SuperdexTM Peptide HR 10/30;检测波长为214 nm;流速为1 mL·min-1;流动相为0.01 mol·L-1磷酸盐缓冲液;进样量为500 μL.结果本方法蛋白(BSA)的平均回收率大于90%,蛋白浓度在0-12 μg·mL-1之间符合曲线方程y=-0.002 4x2 + 0.064 2x + 0.002 9,r2=0.999 9; 检测限为3 ng·mL-1(相当于盐酸林可霉素中蛋白的残留量为7×10-7).结论本方法简便,快速,灵敏,可以用于控制发酵类抗生素中蛋白的残留量.%Aim A novel method has been developed for evaluation of the levels of total residual protein in antibiotics produced by fermentation using gel filtration chromatography (GFC) combined with Bradford assay based on determination of residual protein in lincomycin hydrochloride. Methods The chromatographic conditions were SuperdexTM peptide column, 0.01 mol*L-1 phosphate buffer solution as mobile phase, and flow rate of 1 mL·min-1. Five hundred microliters of lincomycin hydrochloride solution (3 g of lincomycin hydrochloride dissolved in 10 mL of mobile phase) was injected into the chromatograph and the eluted solution was collected between 6 min and 14.5 min (protein eluted from column within this period), and the residual content of total protein in the eluted solution was assayed using Bradford assay method. Results The average recovery was more than 90% for bovine serum albumin, the calibration equation for the range of 0-12 μg·mL-1 of protein was y=-0.002 4x2+0.064 2x+0.002 9, r2=0.999 9, RSD=0.1%-0.9%, and the LOD and LOQ were 3 and 10 ng·mL-1 of protein, respectively. Conclusion The novel method for determining the residual protein in ferment antibio

  7. The Membrane Protein LasM Promotes the Culturability of Legionella pneumophila in Water

    Science.gov (United States)

    Li, Laam; Faucher, Sébastien P.

    2016-01-01

    The water-borne pathogen Legionella pneumophila (Lp) strongly expresses the lpg1659 gene in water. This gene encodes a hypothetical protein predicted to be a membrane protein using in silico analysis. While no conserved domains were identified in Lpg1659, similar proteins are found in many Legionella species and other aquatic bacteria. RT-qPCR showed that lpg1659 is positively regulated by the alternative sigma factor RpoS, which is essential for Lp to survive in water. These observations suggest an important role of this novel protein in the survival of Lp in water. Deletion of lpg1659 did not affect cell morphology, membrane integrity or tolerance to high temperature. Moreover, lpg1659 was dispensable for growth of Lp in rich medium, and during infection of the amoeba Acanthamoeba castellanii and of THP-1 human macrophages. However, deletion of lpg1659 resulted in an early loss of culturability in water, while over-expression of this gene promoted the culturability of Lp. Therefore, these results suggest that lpg1659 is required for Lp to maintain culturability, and possibly long-term survival, in water. Since the loss of culturability observed in the absence of Lpg1659 was complemented by the addition of trace metals into water, this membrane protein is likely a transporter for acquiring essential trace metal for maintaining culturability in water and potentially in other metal-deprived conditions. Given its role in the survival of Lp in water, Lpg1659 was named LasM for Legionella aquatic survival membrane protein. PMID:27734007

  8. The membrane protein LasM Promotes the Culturability of Legionella pneumophila in Water

    Directory of Open Access Journals (Sweden)

    Laam Li

    2016-09-01

    Full Text Available The water-borne pathogen Legionella pneumophila (Lp strongly expresses the lpg1659 gene in water. This gene encodes a hypothetical protein predicted to be a membrane protein using in silico analysis. While no conserved domains were identified in Lpg1659, similar proteins are found in many Legionella species and other aquatic bacteria. RT-qPCR showed that lpg1659 is positively regulated by the alternative sigma factor RpoS, which is essential for Lp to survive in water. These observations suggest an important role of this novel protein in the survival of Lp in water. Deletion of lpg1659 did not affect cell morphology, membrane integrity or tolerance to high temperature. Moreover, lpg1659 was dispensable for growth of Lp in rich medium, and during infection of the amoeba Acanthamoeba castellanii and of THP-1 human macrophages. However, deletion of lpg1659 resulted in an early loss of culturability in water, while over-expression of this gene promoted the culturability of Lp. Therefore, these results suggest that lpg1659 is required for Lp to maintain culturability, and possibly long-term survival, in water. Since the loss of culturability observed in the absence of Lpg1659 was complemented by the addition of trace metals into water, this membrane protein is likely a transporter for acquiring essential trace metal for maintaining culturability in water and potentially in other metal-deprived conditions. Given its role in the survival of Lp in water, Lpg1659 was named LasM for Legionella aquatic survival membrane protein.

  9. Targeted Protein Degradation by Salmonella under Phagosome-Mimicking Culture Conditions Investigated Using Comparative Peptidomics

    Energy Technology Data Exchange (ETDEWEB)

    Manes, Nathan P.; Gustin, Jean K.; Rue, Joanne; Mottaz, Heather M.; Purvine, Samuel O.; Norbeck, Angela D.; Monroe, Matthew E.; Zimmer, Jennifer S.; Metz, Thomas O.; Adkins, Joshua N.; Smith, Richard D.; Heffron, Fred

    2007-04-01

    The pathogen Salmonella enterica is known to cause both food poisoning and typhoid fever. Due to the emergence of antibiotic-resistant isolates and the threat of bioterrorism (e.g., contamination of the food supply), there is a growing need to study this bacterium. In this investigation, comparative peptidomics was used to study Salmonella enterica serovar Typhimurium cultured in either a rich medium or in an acidic, low magnesium, and minimal nutrient medium designed to roughly mimic the macrophage phagosomal compartment (within which Salmonella are known to survive). Native peptides from cleared cell lysates were enriched by using isopropanol extraction and analyzed by using both LC-MS/MS and LC-FTICR-MS. We identified 5,163 distinct peptides originating from 682 proteins and the data clearly indicated that compared to cells cultured in the rich medium, Salmonella cultured in the phagosome-mimicking medium had dramatically higher abundances of a wide variety of protein degradation products, especially from ribosomal proteins. Salmonella from the same cultures were also analyzed by using bottom-up proteomics, and when the peptidomic and proteomic data were analyzed together, two clusters of proteins targeted for proteolysis were tentatively identified. Possible roles of targeted proteolysis by phagocytosed Salmonella are discussed.

  10. A sandwiched-culture technique for evaluation of heterologous protein production in a filamentous fungus

    NARCIS (Netherlands)

    Asgeirsdottir, SA; Scholtmeijer, K; Wessels, JGH

    1999-01-01

    Aspergillus niger is known for its efficient excretion machinery. However, problems have often arisen in obtaining high amounts of heterologous proteins in the culture medium. Here we present a quick method using sandwiched colonies to evaluate transgenic strains for secretion of heterologous protei

  11. Fibronectin in cultured rat keratinocytes: distribution, synthesis, and relationship to cytoskeletal proteins

    DEFF Research Database (Denmark)

    Gibson, W T; Couchman, J R; Badley, R A;

    1983-01-01

    immunofluorescence staining of cultures grown in the absence of a feeder layer using an antiserum which had been cross-adsorbed with foetal calf serum proteins to remove antibodies which recognised serum fibronectin. The distribution of fibronectin in areas of cell-cell and cell-substratum contact...

  12. Correlating composition and functionality of soy protein hydrolysates used in animal cell cultures

    NARCIS (Netherlands)

    Gupta, A.J.

    2015-01-01

    Abstract Soy protein hydrolysates are often supplemented to chemically defined (CD) media in cell cultures, but there is little understanding of the effect of their composition on their functionality (viable cell density, total immunoglobulin (IgG), and specific IgG production). To

  13. COMPARATIVE PATHOGENESIS OF HALOACETIC ACID AND PROTEIN KINASE INHIBITOR EMBRYOTOXICITY IN MOUSE WHOLE EMBRYO CULTURE

    Science.gov (United States)

    Comparative pathogenesis of haloacetic acid and protein kinase inhibitor embryotoxicity in mouse whole embryo culture.Ward KW, Rogers EH, Hunter ES 3rd.Curriculum in Toxicology, University of North Carolina at Chapel Hill, 27599-7270, USA.Haloacetic acids ...

  14. SELECCIÓN Y REGENERACIÓN in vitro DE SOMACLONES DE TOMATE DE ÁRBOL (Solanum betacea cav. Sendt UTILIZANDO FILTRADOS DE CULTIVO DE Colletotrichum acutatum CON ACTIVIDAD PECTINASA SELECTION AND in vitro REGENERATION OF SOMACLONES OF TREE TOMATO (Solanum betacea cav. Sendt USING CULTURE FILTRATES OF Colletotrichum acutatum WITH PECTINASA ACTIVITY

    Directory of Open Access Journals (Sweden)

    Carlos Patiño Torres

    2007-12-01

    Full Text Available Se utilizaron filtrados de cultivo de Colletotrichum acutatum, agente causal de la antracnosis del tomate de árbol, para la selección in vitro de variantes somaclonales de ésta planta con resistencia potencial a la enfermedad. El filtrado, utilizado a varias concentraciones, demostró ser un agente de selección efectivo cuando se integró al medio de cultivo, pues causó niveles elevados de mortalidad de los explantes comparado con los tratamientos sin filtrado. Igualmente, se estudió el efecto de la benziladenina (BA ó BAP sobre la respuesta organogénica de explantes de tejido foliar de tomate de árbol.Culture filtrates of Colletotrichum acutatum, causal agent of the anthracnose disease in tree tomato, were used for the in vitro selection of somaclonal variants with potential resistance to this disease. Culture filtrates used at different concentrations, showed to be an effective agent of selection when they were mixed with the growth media. The findings indicated a high level of mortality for the explants growing on the ammended media as compared with these growing on non ammended one. The effect of the benziladenina (BA ó BAP on the organogenic response of leaf explants of tomato tree was also studied.

  15. A fluorescence anisotropy method for measuring protein concentration in complex cell culture media.

    Science.gov (United States)

    Groza, Radu Constantin; Calvet, Amandine; Ryder, Alan G

    2014-04-22

    The rapid, quantitative analysis of the complex cell culture media used in biopharmaceutical manufacturing is of critical importance. Requirements for cell culture media composition profiling, or changes in specific analyte concentrations (e.g. amino acids in the media or product protein in the bioprocess broth) often necessitate the use of complicated analytical methods and extensive sample handling. Rapid spectroscopic methods like multi-dimensional fluorescence (MDF) spectroscopy have been successfully applied for the routine determination of compositional changes in cell culture media and bioprocess broths. Quantifying macromolecules in cell culture media is a specific challenge as there is a need to implement measurements rapidly on the prepared media. However, the use of standard fluorescence spectroscopy is complicated by the emission overlap from many media components. Here, we demonstrate how combining anisotropy measurements with standard total synchronous fluorescence spectroscopy (TSFS) provides a rapid, accurate quantitation method for cell culture media. Anisotropy provides emission resolution between large and small fluorophores while TSFS provides a robust measurement space. Model cell culture media was prepared using yeastolate (2.5 mg mL(-1)) spiked with bovine serum albumin (0 to 5 mg mL(-1)). Using this method, protein emission is clearly discriminated from background yeastolate emission, allowing for accurate bovine serum albumin (BSA) quantification over a 0.1 to 4.0 mg mL(-1) range with a limit of detection (LOD) of 13.8 μg mL(-1). Copyright © 2014. Published by Elsevier B.V.

  16. BDNF downregulates 5-HT(2A) receptor protein levels in hippocampal cultures

    DEFF Research Database (Denmark)

    Trajkovska, V; Santini, M A; Marcussen, Anders Bue;

    2009-01-01

    Both brain-derived neurotrophic factor (BDNF) and the serotonin receptor 2A (5-HT(2A)) have been related to depression pathology. Specific 5-HT(2A) receptor changes seen in BDNF conditional mutant mice suggest that BDNF regulates the 5-HT(2A) receptor level. Here we show a direct effect of BDNF...... on 5-HT(2A) receptor protein levels in primary hippocampal neuronal and mature hippocampal organotypic cultures exposed to different BDNF concentrations for either 1, 3, 5 or 7 days. In vivo effects of BDNF on hippocampal 5-HT(2A) receptor levels were further corroborated in (BDNF +/-) mice...... with reduced BDNF levels. In primary neuronal cultures, 7 days exposure to 25 and 50ng/mL BDNF resulted in downregulation of 5-HT(2A), but not of 5-HT(1A), receptor protein levels. The BDNF-associated downregulation of 5-HT(2A) receptor levels was also observed in mature hippocampal organotypic cultures...

  17. Characterization of cytoskeletal and junctional proteins expressed by cells cultured from human arachnoid granulation tissue

    Directory of Open Access Journals (Sweden)

    Mehta Bhavya C

    2005-10-01

    Full Text Available Abstract Background The arachnoid granulations (AGs are projections of the arachnoid membrane into the dural venous sinuses. They function, along with the extracranial lymphatics, to circulate the cerebrospinal fluid (CSF to the systemic venous circulation. Disruption of normal CSF dynamics may result in increased intracranial pressures causing many problems including headaches and visual loss, as in idiopathic intracranial hypertension and hydrocephalus. To study the role of AGs in CSF egress, we have grown cells from human AG tissue in vitro and have characterized their expression of those cytoskeletal and junctional proteins that may function in the regulation of CSF outflow. Methods Human AG tissue was obtained at autopsy, and explanted to cell culture dishes coated with fibronectin. Typically, cells migrated from the explanted tissue after 7–10 days in vitro. Second or third passage cells were seeded onto fibronectin-coated coverslips at confluent densities and grown to confluency for 7–10 days. Arachnoidal cells were tested using immunocytochemical methods for the expression of several common cytoskeletal and junctional proteins. Second and third passage cultures were also labeled with the common endothelial markers CD-31 or VE-cadherin (CD144 and their expression was quantified using flow cytometry analysis. Results Confluent cultures of arachnoidal cells expressed the intermediate filament protein vimentin. Cytokeratin intermediate filaments were expressed variably in a subpopulation of cells. The cultures also expressed the junctional proteins connexin43, desmoplakin 1 and 2, E-cadherin, and zonula occludens-1. Flow cytometry analysis indicated that second and third passage cultures failed to express the endothelial cell markers CD31 or VE-cadherin in significant quantities, thereby showing that these cultures did not consist of endothelial cells from the venous sinus wall. Conclusion To our knowledge, this is the first report of

  18. Fractionnement de protéines du lait par filtration dynamique.

    OpenAIRE

    Espina, V.

    2009-01-01

    This thesis focuses on milk proteins fractionation by dynamic filtration. A two-stage process has been proposed in order to separate milk proteins into three main fractions: casein micelles, α-Lactalbumin and β-Lactoglobulin. The first stage of the process consists in microfiltration of skim milk for separating casein micelles from whey proteins. The performances of two dynamic filtration modules: the Multi Shaft Disk (MSD) module and the rotating disk module have been compared. The results h...

  19. Filtration Understanding: FY10 Testing Results and Filtration Model Update

    Energy Technology Data Exchange (ETDEWEB)

    Daniel, Richard C.; Billing, Justin M.; Burns, Carolyn A.; Peterson, Reid A.; Russell, Renee L.; Schonewill, Philip P.; Shimskey, Rick W.

    2011-04-04

    This document completes the requirements of Milestone 2-4, Final Report of FY10 Testing, discussed in the scope of work outlined in the EM31 task plan WP-2.3.6-2010-1. The focus of task WP 2.3.6 is to improve the U.S. Department of Energy’s (DOE’s) understanding of filtration operations for high-level waste (HLW) to improve filtration and cleaning efficiencies, thereby increasing process throughput and reducing the Na demand (through acid neutralization). Developing the cleaning/backpulsing requirements will produce much more efficient operations for both the Hanford Tank Waste Treatment and Immobilization Plant (WTP) and the Savannah River Site (SRS), thereby significantly increasing throughput by limiting cleaning cycles. The scope of this work is to develop the understanding of filter fouling to allow developing this cleaning/backpulsing strategy.

  20. Effect of culture conditions on the growth of biomass Yarrowia lipolytica - producing protein feed

    Directory of Open Access Journals (Sweden)

    O. S. Korneeva

    2016-01-01

    Full Text Available Fodder yeast is highly valuable protein-vitamin products. Protein digestibility by yeast and amino acid content, superior proteins of animal origin. Fodder yeast protein digested in animals by 95 %. The biological value of yeast protein is determined by the presence of a significant amount of essential amino acids. Moreover, yeast cells contain many vitamins microelement and a significant amount of fat, in which the predominant unsaturated fatty acid. Currently, fodder yeast successfully used in livestock and poultry, so the demand for them is increasing every year. For the production of fodder yeast using a yeast having the necessary technological properties: the ability of rapid growth in aerobic conditions to form protein, amino acids and vitamins, resistant crop production, the development of resistance to foreign microorganisms. Intensive education yeast biomass contributes to a number of conditions, including pH, temperature and aeration of the culture occupy an important place. The main criterion for comparison and selection of a culture medium for this is the speed of its growth and ability to assimilate all of the nutrients with high economic factor. It depends on the performance of the enterprise, energy consumption and other technical - economic performance. The effect of pH of the medium on the biomass accumulation of yeast Yarrowia lipolytica. Found that at pH 5,2 - 5,5 observed maximum growth rate of the yeast cells. The effect of temperature on the accumulation of yeast biomass. The temperature of the culture medium determines the intensity of metabolism in cells. It was found that the optimal growth temperature of the culture Yarrowia lipolytica is 33 0C. The effect of aeration on the growth rate of yeast cells. Tro-established that the maximum increase of biomass was obtained with the aeration of 70 cm3 /cm3hrs.

  1. Modulation of neurite branching by protein phosphorylation in cultured rat hippocampal neurons.

    Science.gov (United States)

    Audesirk, G; Cabell, L; Kern, M

    1997-09-20

    The control of branching of axons and dendrites is poorly understood. It has been hypothesized that branching may be produced by changes in the cytoskeleton [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture, NeuroReport 4 (1993) 412-419; P. Friedrich, A. Aszodi, MAP2: a sensitive cross-linker and adjustable spacer in dendritic architecture, FEBS Lett. 295 (1991) 5-9]. The assembly and stability of microtubules, which are prominent cytoskeletal elements in both axons and dendrites, are regulated by microtubule-associated proteins, including tau (predominantly found in axons) and MAP2 (predominantly found in dendrites). The phosphorylation state of tau and MAP2 modulates their interactions with microtubules. In their low-phosphorylation states, tau and MAP2 bind to microtubules and increase microtubule assembly and/or stability. Increased phosphorylation decreases these effects. Diez-Guerra and Avila [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture, NeuroReport 4 (1993) 412-419] found that protein phosphorylation correlates with neurite branching in cultured rat hippocampal neurons, and hypothesized that increased protein phosphorylation stimulates neurite branching. To test this hypothesis, we cultured rat hippocampal neurons in the presence of specific modulators of serine-threonine protein kinases and phosphatases. Inhibitors of several protein kinases, which would be expected to decrease protein phosphorylation, reduced branching. KT5720, an inhibitor of cyclic AMP-dependent protein kinase, and KN62, an inhibitor of Ca(2+)-calmodulin-dependent protein kinases, inhibited branching of both axons and dendrites. Calphostin C and chelerythrine, inhibitors of protein kinase C, inhibited branching of axons but not dendrites. Treatments that would be expected to increase protein phosphorylation, including inhibitors of protein

  2. 40 CFR 141.173 - Filtration.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 22 2010-07-01 2010-07-01 false Filtration. 141.173 Section 141.173... PRIMARY DRINKING WATER REGULATIONS Enhanced Filtration and Disinfection-Systems Serving 10,000 or More People § 141.173 Filtration. A public water system subject to the requirements of this subpart that...

  3. Transforming growth factor-beta and the glomerular filtration barrier

    Directory of Open Access Journals (Sweden)

    Ayesha Ghayur

    2013-03-01

    Full Text Available The increasing burden of chronic kidney disease worldwide and recent advancements in the understanding of pathologic events leading to kidney injury have opened up new potential avenues for therapies to further diminish progression of kidney disease by targeting the glomerular filtration barrier and reducing proteinuria. The glomerular filtration barrier is affected by many different metabolic and immune-mediated injuries. Glomerular endothelial cells, the glomerular basement membrane, and podocytes—the three components of the filtration barrier—work together to prevent the loss of protein and at the same time allow passage of water and smaller molecules. Damage to any of the components of the filtration barrier can initiate proteinuria and renal fibrosis. Transforming growth factor-beta (TGF-β is a pleiotropic cytokine strongly associated with the fibrogenic response. It has a known role in tubulointerstitial fibrosis. In this review we will highlight what is known about TGF-β and how it interacts with the components of glomerular filtration barrier and causes loss of function and proteinuria.

  4. Culture phases, cytotoxicity and protein expressions of agarose hydrogel induced Sp2/0, A549, MCF-7 cell line 3D cultures.

    Science.gov (United States)

    Ravi, Maddaly; Kaviya, S R; Paramesh, V

    2016-05-01

    Advancements in cell cultures are occurring at a rapid pace, an important direction is culturing cells in 3D conditions. We demonstrate the usefulness of agarose hydrogels in obtaining 3 dimensional aggregates of three cell lines, A549, MCF-7 and Sp2/0. The differences in culture phases, susceptibility to cisplatin-induced cytotoxicity are studied. Also, the 3D aggregates of the three cell lines were reverted into 2D cultures and the protein profile differences among the 2D, 3D and revert cultures were studied. The analysis of protein profile differences using UniProt data base further augment the usefulness of agarose hydrogels for obtaining 3D cell cultures.

  5. High-yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development.

    Science.gov (United States)

    Zhang, Ningning; Gonzalez, Maria; Savary, Brett; Xu, Jianfeng

    2016-03-01

    Low-yield protein production remains the most significant economic hurdle with plant cell culture technology. Fusions of recombinant proteins with hydroxyproline-O-glycosylated designer glycopeptide tags have consistently boosted secreted protein yields. This prompted us to study the process development of this technology aiming to achieve productivity levels necessary for commercial viability. We used a tobacco BY-2 cell culture expressing EGFP as fusion with a glycopeptide tag comprised of 32 repeat of "Ser-Pro" dipeptide, or (SP)32 , to study cell growth and protein secretion, culture scale-up, and establishment of perfusion cultures for continuous production. The BY-2 cells accumulated low levels of cell biomass (~7.5 g DW/L) in Schenk & Hildebrandt medium, but secreted high yields of (SP)32 -tagged EGFP (125 mg/L). Protein productivity of the cell culture has been stable for 6.0 years. The BY-2 cells cultured in a 5-L bioreactor similarly produced high secreted protein yield at 131 mg/L. Successful operation of a cell perfusion culture for 30 days was achieved under the perfusion rate of 0.25 and 0.5 day(-1) , generating a protein volumetric productivity of 17.6 and 28.9 mg/day/L, respectively. This research demonstrates the great potential of the designer glycopeptide technology for use in commercial production of valuable proteins with plant cell cultures. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Mobile surface water filtration system

    Directory of Open Access Journals (Sweden)

    Aashish Vatsyayan

    2012-09-01

    Full Text Available To design a mobile system for surface water filtrationMethodology: the filtration of surface impurities begins with their retraction to concentrated thickness using non ionising surfactants, then isolation using surface tension property and sedimentation of impurities in process chamber using electrocoagulation. Result:following studies done to determine the rate of spreading of crude oil on water a method for retraction of spread crude oil to concentrated volumes is developed involving addition of non -ionising surfactants in contrast to use of dispersants. Electrocoagulation process involves multiple processes taking place to lead to depositionof impurities such as oil, grease, metals. Studies of experiments conducted reveals parameters necessary for design of electrocoagulation process chamber though a holistic approach towards system designing is still required. Propeller theory is used in determining the required design of propeller and the desired thrust, the overall structure will finally contribute in deciding the choice of propeller.

  7. Cell culture and animal infection with distinct Trypanosoma cruzi strains expressing red and green fluorescent proteins.

    Science.gov (United States)

    Pires, S F; DaRocha, W D; Freitas, J M; Oliveira, L A; Kitten, G T; Machado, C R; Pena, S D J; Chiari, E; Macedo, A M; Teixeira, S M R

    2008-03-01

    Different strains of Trypanosoma cruzi were transfected with an expression vector that allows the integration of green fluorescent protein (GFP) and red fluorescent protein (RFP) genes into the beta-tubulin locus by homologous recombination. The sites of integration of the GFP and RFP markers were determined by pulse-field gel electrophoresis and Southern blot analyses. Cloned cell lines selected from transfected epimastigote populations maintained high levels of fluorescent protein expression even after 6 months of in vitro culture of epimastigotes in the absence of drug selection. Fluorescent trypomastigotes and amastigotes were observed within Vero cells in culture as well as in hearts and diaphragms of infected mice. The infectivity of the GFP- and RFP-expressing parasites in tissue culture cells was comparable to wild type populations. Furthermore, GFP- and RFP-expressing parasites were able to produce similar levels of parasitemia in mice compared with wild type parasites. Cell cultures infected simultaneously with two cloned cell lines from the same parasite strain, each one expressing a distinct fluorescent marker, showed that at least two different parasites are able to infect the same cell. Double-infected cells were also detected when GFP- and RFP-expressing parasites were derived from strains belonging to two distinct T. cruzi lineages. These results show the usefulness of parasites expressing GFP and RFP for the study of various aspects of T. cruzi infection including the mechanisms of cell invasion, genetic exchange among parasites and the differential tissue distribution in animal models of Chagas disease.

  8. Serum-free culture alters the quantity and protein composition of neuroblastoma-derived extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Jinghuan Li

    2015-05-01

    Full Text Available Extracellular vesicles (EVs play a significant role in cell–cell communication in numerous physiological processes and pathological conditions, and offer promise as novel biomarkers and therapeutic agents for genetic diseases. Many recent studies have described different molecular mechanisms that contribute to EV biogenesis and release from cells. However, little is known about how external stimuli such as cell culture conditions can affect the quantity and content of EVs. While N2a neuroblastoma cells cultured in serum-free (OptiMEM conditions did not result in EVs with significant biophysical or size differences compared with cells cultured in serum-containing (pre-spun conditions, the quantity of isolated EVs was greatly increased. Moreover, the expression levels of certain vesicular proteins (e.g. small GTPases, G-protein complexes, mRNA processing proteins and splicing factors, some of which were previously reported to be involved in EV biogenesis, were found to be differentially expressed in EVs under different culture conditions. These data, therefore, contribute to the understanding of how extracellular factors and intracellular molecular pathways affect the composition and release of EVs.

  9. Centrifugal membrane filtration -- Task 9

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-08-01

    The Energy and Environmental Research Center (EERC) has teamed with SpinTek Membrane Systems, Inc., the developer of a centrifugal membrane filtration technology, to demonstrate applications for the SpinTek technology within the US Department of Energy (DOE) Environmental management (EM) Program. The technology uses supported microporous membranes rotating at high rpm, under pressure, to separate suspended and colloidal solids from liquid streams, yielding a solids-free permeate stream and a highly concentrated solids stream. This is a crosscutting technology that falls under the Efficient Separations and Processing Crosscutting Program, with potential application to tank wastes, contaminated groundwater, landfill leachate, and secondary liquid waste streams from other remediation processes, including decontamination and decommissioning systems. Membrane-screening tests were performed with the SpinTek STC-X4 static test cell filtration unit, using five ceramic membranes with different pore size and composition. Based on permeate flux, a 0.25-{micro}m TiO{sub 2}/Al{sub 2}O{sub 3} membrane was selected for detailed performance evaluation using the SpinTek ST-IIL centrifugal membrane filtration unit with a surrogate tank waste solution. An extended test run of 100 hr performed on a surrogate tank waste solution showed some deterioration in filtration performance, based on flux, apparently due to the buildup of solids near the inner portion of the membrane where relative membrane velocities were low. Continued testing of the system will focus on modifications to the shear pattern across the entire membrane surface to affect improved long-term performance.

  10. Effect of JP-8 jet fuel exposure on protein expression in human keratinocyte cells in culture.

    Science.gov (United States)

    Witzmann, F A; Monteiro-Riviere, N A; Inman, A O; Kimpel, M A; Pedrick, N M; Ringham, H N; Riviere, J E

    2005-12-30

    Dermal exposure to jet fuel is a significant occupational hazard. Previous studies have investigated its absorption and disposition in skin, and the systemic biochemical and immunotoxicological sequelae to exposure. Despite studies of JP-8 jet fuel components in murine, porcine or human keratinocyte cell cultures, proteomic analysis of JP-8 exposure has not been investigated. This study was conducted to examine the effect of JP-8 administration on the human epidermal keratinocyte (HEK) proteome. Using a two-dimensional electrophoretic approach combined with mass spectrometric-based protein identification, we analyzed protein expression in HEK exposed to 0.1% JP-8 in culture medium for 24 h. JP-8 exposure resulted in significant expression differences (p<0.02) in 35 of the 929 proteins matched and analyzed. Approximately, a third of these alterations were increased in protein expression, two-thirds declined with JP-8 exposure. Peptide mass fingerprint identification of effected proteins revealed a variety of functional implications. In general, altered proteins involved endocytotic/exocytotic mechanisms and their cytoskeletal components, cell stress, and those involved in vesicular function.

  11. Enhancing Protein Expression in HEK-293 Cells by Lowering Culture Temperature

    Science.gov (United States)

    Lin, Chi-Yen; Huang, Zhen; Wen, Wei; Wu, Andrew; Wang, Congzhou; Niu, Li

    2015-01-01

    Animal cells and cell lines, such as HEK-293 cells, are commonly cultured at 37°C. These cells are often used to express recombinant proteins. Having a higher expression level or a higher protein yield is generally desirable. As we demonstrate in this study, dropping culture temperature to 33°C, but not lower, 24 hours after transient transfection in HEK-293S cells will give rise to ~1.5-fold higher expression of green fluorescent protein (GFP) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. By following the time course of the GFP-expressing cells growing at 37°C and 33°C from 24 hours after transfection (including 19 hours recovery at 37°C in the normal growth medium), we found that a mild hypothermia (i.e., 33°C) reduces the growth rate of HEK-293S cells, while increasing cellular productivity of recombinant proteins. As a result, green cells remain undivided in a longer period of time. Not surprisingly, the property of a recombinant protein expressed in the cells grown at 33°C is unaffected, as shown by the use of AMPA receptors. We further demonstrate with the use of PC12 cells that this method may be especially useful when a recombinant protein is difficult to express using a chemical-based, transient transfection method. PMID:25893827

  12. The effect of starter culture and annatto on the flavor and functionality of whey protein concentrate.

    Science.gov (United States)

    Campbell, R E; Miracle, R E; Drake, M A

    2011-03-01

    The flavor of whey protein can carry over into ingredient applications and negatively influence consumer acceptance. Understanding sources of flavors in whey protein is crucial to minimize flavor. The objective of this study was to evaluate the effect of annatto color and starter culture on the flavor and functionality of whey protein concentrate (WPC). Cheddar cheese whey with and without annatto (15 mL of annatto/454 kg of milk, annatto with 3% wt/vol norbixin content) was manufactured using a mesophilic lactic starter culture or by addition of lactic acid and rennet (rennet set). Pasteurized fat-separated whey was then ultrafiltered and spray dried into WPC. The experiment was replicated 4 times. Flavor of liquid wheys and WPC were evaluated by sensory and instrumental volatile analyses. In addition to flavor evaluations on WPC, color analysis (Hunter Lab and norbixin extraction) and functionality tests (solubility and heat stability) also were performed. Both main effects (annatto, starter) and interactions were investigated. No differences in sensory properties or functionality were observed among WPC. Lipid oxidation compounds were higher in WPC manufactured from whey with starter culture compared with WPC from rennet-set whey. The WPC with annatto had higher concentrations of p-xylene, diacetyl, pentanal, and decanal compared with WPC without annatto. Interactions were observed between starter and annatto for hexanal, suggesting that annatto may have an antioxidant effect when present in whey made with starter culture. Results suggest that annatto has a no effect on whey protein flavor, but that the starter culture has a large influence on the oxidative stability of whey.

  13. Although it is rapidly metabolized in cultured rat hepatocytes, lauric acid is used for protein acylation

    OpenAIRE

    Rioux, Vincent; Daval, Stéphanie; Guillou, Hervé; Jan, Sophie; Legrand, Philippe

    2003-01-01

    International audience; This study was designed to examine the metabolic fate of exogenous lauric acid in cultured rat hepatocytes, in terms of both lipid metabolism and acylation of proteins. Radiolabeled [ 1-$^{14}$C] -lauric acid at 0.1 mM in the culture medium was rapidly taken up by the cells ($94.8 \\pm 2.2\\%$ of the initial radioactivity was cleared from the medium after a 4 h incubation) but its incorporation into cellular lipids was low ($24.6 \\pm 4.2\\%$ of initial radioactivity after...

  14. Protein and Carbohydrate Accumulation in Normal and High-Lysine Barley in Spike Culture

    DEFF Research Database (Denmark)

    Mather, D.E; Giese, Nanna Henriette

    1984-01-01

    -soluble, hordein, and non-protein N fractions appeared to be normal. Endosperm dry weight and starch were lower than in intact plants and declined at higher N levels. A linear relationship was observed between starch content and the concentration of sucrose in the endosperm water. Uptake of culture medium......Spikes of barley cv. Bomi and high-lysine mutants Riso 1508 and Riso 56 were cultured on liquid media at varying N and sucrose levels. Bomi accumulated N in response to increasing N levels in the medium and a higher level was reached than in spikes of intact plants. The distribution of N in salt...

  15. Although it is rapidly metabolized in cultured rat hepatocytes, lauric acid is used for protein acylation.

    Science.gov (United States)

    Rioux, Vincent; Daval, Stéphanie; Guillou, Hervé; Jan, Sophie; Legrand, Philippe

    2003-01-01

    This study was designed to examine the metabolic fate of exogenous lauric acid in cultured rat hepatocytes, in terms of both lipid metabolism and acylation of proteins. Radiolabeled [14C]-lauric acid at 0.1 mM in the culture medium was rapidly taken up by the cells (94.8 +/- 2.2% of the initial radioactivity was cleared from the medium after a 4 h incubation) but its incorporation into cellular lipids was low (24.6 +/- 4.2% of initial radioactivity after 4 h), due to the high beta-oxidation of lauric acid in hepatocytes (38.7 +/- 4.4% after the same time). Among cellular lipids, lauric acid was preferentially incorporated into triglycerides (10.6 +/- 4.6% of initial radioactivity after 4 h). Lauric acid was also rapidly converted to palmitic acid by two successive elongations. Protein acylation was detected after metabolic labeling of the cells with [11,12-3H]-lauric acid. Two-dimensional electrophoresis separation of the cellular proteins and autoradiography evidenced the incorporation of radioactivity into 35 well-resolved proteins. Radiolabeling of several proteins resulted from covalent linkage to the precursor [11,12-3H]-lauric acid or to its elongation product, myristic acid. The covalent linkages between these proteins and lauric acid were broken by base hydrolysis, indicating that the linkage was of the thioester or ester-type. Endogenous myristic acid produced by lauric acid elongation was used for both protein N-myristoylation and protein S-acylation. Therefore, these results show for the first time that, although it is rapidly metabolized in hepatocytes, exogenous lauric acid is a substrate for the acylation of liver proteins.

  16. Cake Filtration in Viscoelastic Polymer Solutions

    Science.gov (United States)

    Surý, Alexander; Machač, Ivan

    2009-07-01

    In this contribution, the filtration equations for a cake filtration in viscoelastic fluids are presented. They are based on a capillary hybrid model for the flow of a power law fluid. In order to express the elastic pressure drop excess in the flow of viscoelastic filtrate through the filter cake and filter screen, modified Deborah number correction functions are included into these equations. Their validity was examined experimentally. Filtration experiments with suspensions of hardened polystyrene particles (Krasten) in viscoelastic aqueous solutions of polyacryl amides (0.4% and 0.6%wt. Kerafloc) were carried out at a constant pressure on a cylindrical filtration unit using filter screens of different resistance.

  17. Insulin receptors mediate growth effects in cultured fetal neurons. I. Rapid stimulation of protein synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Heidenreich, K.A.; Toledo, S.P. (Univ. of California-San Diego, La Jolla (USA))

    1989-09-01

    In this study we have examined the effects of insulin on protein synthesis in cultured fetal chick neurons. Protein synthesis was monitored by measuring the incorporation of (3H)leucine (3H-leu) into trichloroacetic acid (TCA)-precipitable protein. Upon addition of 3H-leu, there was a 5-min lag before radioactivity occurred in protein. During this period cell-associated radioactivity reached equilibrium and was totally recovered in the TCA-soluble fraction. After 5 min, the incorporation of 3H-leu into protein was linear for 2 h and was inhibited (98%) by the inclusion of 10 micrograms/ml cycloheximide. After 24 h of serum deprivation, insulin increased 3H-leu incorporation into protein by approximately 2-fold. The stimulation of protein synthesis by insulin was dose dependent (ED50 = 70 pM) and seen within 30 min. Proinsulin was approximately 10-fold less potent than insulin on a molar basis in stimulating neuronal protein synthesis. Insulin had no effect on the TCA-soluble fraction of 3H-leu at any time and did not influence the uptake of (3H)aminoisobutyric acid into neurons. The isotope ratio of 3H-leu/14C-leu in the leucyl tRNA pool was the same in control and insulin-treated neurons. Analysis of newly synthesized proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that insulin uniformly increased the incorporation of 14C-leu into all of the resolved neuronal proteins. We conclude from these data that (1) insulin rapidly stimulates overall protein synthesis in fetal neurons independent of amino acid uptake and aminoacyl tRNA precursor pools; (2) stimulation of protein synthesis is mediated by the brain subtype of insulin receptor; and (3) insulin is potentially an important in vivo growth factor for fetal central nervous system neurons.

  18. Dynamical Systems, Cytokine Storms, and Blood Filtration

    Science.gov (United States)

    Foster, Glenn; Hubler, Alfred

    2008-03-01

    Various infections and non-infectious diseases can trigger immune cells and the proteins (cytokines) the cells use to communicate with each other to be caught in a positive feedback loop; this ``cytokine storm'' is frequently fatal. By examining the network of cytokine-immune cell interactions we will illustrate why anti-mediator drugs have been generally ineffective in stopping this feedback. A more effective approach may be to try and reduce interactions by dampening many signals at once by filtering the cytokines out of the blood directly (think dialysis). We will argue that feedback on an out of control nonlinear dynamical system is easier to understand than its normal healthy state and apply filtration to a toy model of immune response.

  19. Teratogens induce a subset of small heat shock proteins in Drosophila primary embryonic cell cultures.

    Science.gov (United States)

    Buzin, C H; Bournias-Vardiabasis, N

    1984-01-01

    Drosophila embryonic cells placed into culture just after gastrulation differentiate in vitro over the next 24 hr. A number of drugs that are teratogenic in mammalian systems have been found to inhibit muscle or neuron differentiation (or both) in these developing cultures. We have examined, by two-dimensional gel electrophoresis, the effects of these drugs on protein synthesis in embryonic cells. For nine teratogens tested, cells treated for 20 hr with the drug show a dramatic induction of three proteins of about 20 kilodaltons, in addition to the normal proteins synthesized by untreated cells. Three teratogens as well as all eight nonteratogens tested did not show this induction. The induced proteins appear to be identical to three of the heat shock proteins (hsp 23, 22a, and 22b), as shown by electrophoretic mobilities and peptide mapping by partial proteolysis. A 37 degrees C heat shock of the embryonic cells produces the full complement of heat shock proteins, whereas drug-treated cells induce only the subset hsp 23, 22a, and 22b but not hsp 26 or 27. beta-Ecdysterone, the Drosophila molting hormone, also inhibits embryonic differentiation and induces hsp 23, 22a, and 22b, a partial subset of the heat shock proteins (hsp 22, 23, 26, and 27) induced by the hormone in imaginal discs and some Drosophila continuous cell lines. Dose-response studies of several drugs show a correlation between the degree of inhibition of differentiation and the level of induction of hsp 23, 22a, and 22b. The induction of heat shock proteins by drugs may reflect specific types of stress that can also give rise to teratogenesis. Images PMID:6588379

  20. Effects of Shuanghuangbu on the total protein content and ultrastructure in cultured human periodontal ligament cells

    Institute of Scientific and Technical Information of China (English)

    许彦枝; 邹慧儒; 王小玲; 刘世正; 王永军

    2004-01-01

    Background Successful periodontal regeneration depends on the migration, proliferation and differentiation of periodontal ligament cells in periodontal defects. The total protein content and the ultrastructure demonstrate the metabolizability and activity of periodontal ligament cells. This study was conducted to observe the effects of Shuanghuangbu, a mixture of medicinal herbs, on the total protein content and the ultrastructure of human periodontal ligament cells.Methods Periodontal ligament cells were grown to confluence and then cultured in Dulbecco's modified eagle medium (DMEM) supplemented with Shuanghuangbu over the concentration range of 0 to 1000 μg/ml. The total protein content in cultured cells was determined by using Coommasie brilliant blue technique. Periodontal ligament cells were incubated in 0 and 100 μg/ml Shuanghuangbu decoction for 5 days, then observed through transmission electron microscope.Results The total protein content of human periodontal ligament cells increased in each experiment group added 10-1000 μg/ml Shuanghuangbu respectively, and the effect of 100 μg/ml was excellent. Under the transmission electron microscope, there were more rough endoplasmic reticulums and mitochodrias in the experiment group than those in the control group. Conclusion Shuanghuangbu stimulates the protein synthesis of human periodontal ligament cells and improves human periodontal ligament cells' metabolizability and activity.

  1. Evaluating the Role of Viral Proteins in HIV-Mediated Neurotoxicity Using Primary Human Neuronal Cultures.

    Science.gov (United States)

    Rao, Vasudev R; Eugenin, Eliseo A; Prasad, Vinayaka R

    2016-01-01

    Despite the inability of HIV-1 to infect neurons, over half of the HIV-1-infected population in the USA suffers from neurocognitive dysfunction. HIV-infected immune cells in the periphery enter the central nervous system by causing a breach in the blood-brain barrier. The damage to the neurons is mediated by viral and host toxic products released by activated and infected immune and glial cells. To evaluate the toxicity of any viral isolate, viral protein, or host inflammatory protein, we describe a protocol to assess the neuronal apoptosis and synaptic compromise in primary cultures of human neurons and astrocytes.

  2. Fast filtration for metabolome sampling of suspended animal cells

    OpenAIRE

    Volmer, Martin; Northoff, Stefan; Scholz, Sebastian; Thüte, Tobias; Büntemeyer, Heino; Noll, Thomas

    2010-01-01

    Abstract A new method for sampling suspended animal cells by fast filtration is presented that allows rapid quenching of cellular metabolism and efficient separation of the cells from culture medium. Compared to sampling with a microstructure heat exchanger or centrifugation without prior quenching, the adenylate energy charge and the measured concentrations especially of metabolites with a high turnover rate or of metabolites early in metabolic pathways were substantially higher. ...

  3. Fast filtration for metabolome sampling of suspended animal cells

    OpenAIRE

    Volmer, Martin; Northoff, Stefan; Scholz, Sebastian; Thüte, Tobias; Büntemeyer, Heino; Noll, Thomas

    2010-01-01

    Abstract A new method for sampling suspended animal cells by fast filtration is presented that allows rapid quenching of cellular metabolism and efficient separation of the cells from culture medium. Compared to sampling with a microstructure heat exchanger or centrifugation without prior quenching, the adenylate energy charge and the measured concentrations especially of metabolites with a high turnover rate or of metabolites early in metabolic pathways were substantially higher. ...

  4. Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes.

    Science.gov (United States)

    Storey, Stephen M; McIntosh, Avery L; Huang, Huan; Landrock, Kerstin K; Martin, Gregory G; Landrock, Danilo; Payne, H Ross; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

    2012-04-15

    A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2/SCP-x/L-FABP null mice and hepatocytes. Taken together

  5. Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes

    Science.gov (United States)

    Storey, Stephen M.; McIntosh, Avery L.; Huang, Huan; Landrock, Kerstin K.; Martin, Gregory G.; Landrock, Danilo; Payne, H. Ross; Atshaves, Barbara P.; Kier, Ann B.

    2012-01-01

    A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2/SCP-x/L-FABP null mice and hepatocytes. Taken together

  6. Engineer medium and feed for modulating N-glycosylation of recombinant protein production in CHO cell culture

    DEFF Research Database (Denmark)

    Fan, Yuzhou; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    2016-01-01

    Chinese hamster ovary (CHO) cells have become the primary expression system for the production of complex recombinant proteins due to their long-term success in industrial scale production and generating appropriate protein N-glycans similar to that of humans. Control and optimization of protein N......-glycosylation is crucial, as the structure of N-glycans can largely influence both biological and physicochemical properties of recombinant proteins. Protein N-glycosylation in CHO cell culture can be controlled and tuned by engineering medium, feed, culture process, as well as genetic elements of the cell...

  7. Protein and Glycoprotein Patterns Related to Morphogenesis in Mammillaria gracillis Pfeiff. Tissue Culture

    Directory of Open Access Journals (Sweden)

    Biljana Balen

    2002-01-01

    Full Text Available As plants with Crassulacean Acid Metabolism (CAM, cacti are highly affected by artificial environmental conditions in tissue culture. Plants of Mammillaria gracillis Pfeiff. (Cactaceae propagated in vitro produced callus spontaneously. This habituated callus regenerated normal and hyperhydric shoots without the addition of growth regulators. In order to compare habituated callus with the tumorous one, cactus cells were transformed with two strains of Agrobacterium tumefaciens: the wild strain B6S3 (tumour line TW and the rooty mutant GV3101 (tumour line TR. Gene expression in cactus plants, habituated callus, regenerated shoots and two tumour lines was analysed at the level of cellular and extracellular protein and glycoprotein profiles. Proteins were separated by SDS-polyacrylamide gel electrophoresis and 2-D PAGE electrophoresis and silver stained. Concavalin A-peroxidase staining detected glycoproteins with D-manose in their glycan component on protein blots. Developmentally specific protein patterns of Mammillaria gracillis tissue lines were detected. The 2-D PAGE electrophoresis revealed some tissue specific protein groups. The cellular glycoprotein of 42 kDa detected by ConA was highly expressed in undifferentiated tissues (habituated callus, TW and TR tumours and in hyperhydric regenerants. Tumours produced extracellular proteins of 33, 23 and 22 kDa. The N glycosylation of cellular and extracellular proteins was related to specific developmental stage of cactus tissue.

  8. The Susd2 protein regulates neurite growth and excitatory synaptic density in hippocampal cultures.

    Science.gov (United States)

    Nadjar, Yann; Triller, Antoine; Bessereau, Jean-Louis; Dumoulin, Andrea

    2015-03-01

    Complement control protein (CCP) domains have adhesion properties and are commonly found in proteins that control the complement immune system. However, an increasing number of proteins containing CCP domains have been reported to display neuronal functions. Susd2 is a transmembrane protein containing one CCP domain. It was previously identified as a tumor-reversing protein, but has no characterized function in the CNS. The present study investigates the expression and function of Susd2 in the rat hippocampus. Characterization of Susd2 during development showed a peak in mRNA expression two weeks after birth. In hippocampal neuronal cultures, the same expression profile was observed at 15days in vitro for both mRNA and protein, a time consistent with synaptogenesis in our model. At the subcellular level, Susd2 was located on the soma, axons and dendrites, and appeared to associate preferentially with excitatory synapses. Inhibition of Susd2 by shRNAs led to decreased numbers of excitatory synaptic profiles, exclusively. Also, morphological parameters were studied on young (5DIV) developing neurons. After Susd2 inhibition, an increase in dendritic tree length but a decrease in axon elongation were observed, suggesting changes in adhesion properties. Our results demonstrate a dual role for Susd2 at different developmental stages, and raise the question whether Susd2 and other CCP-containing proteins expressed in the CNS could be function-related.

  9. Genetically engineered binding proteins as biosensors for fermentation and cell culture.

    Science.gov (United States)

    Ge, Xudong; Tolosa, Leah; Simpson, Jen; Rao, Govind

    2003-12-20

    The signal-transduction properties and the potential applications of two engineered binding proteins from E. coli were extensively studied. Both proteins have a single cysteine mutation in their polypeptide chains, which allow the introduction of an environmentally sensitive fluorophore: ANS for glucose-binding protein (GBP) and acrylodan for glutamine-binding protein (QBP). Both proteins respond to their ligands in the micromolar range. The proteins can be stored at 4 degrees C for at least 5 months. Apparent binding constant, protein concentration, and fluorophore are three major factors that affect the biosensor's responsive ranges. The binding of the ligand is quick and reversible in solution, but the unfavorable dissociation equilibrium and mass-transfer resistance for encapsulated proteins can delay the response to several minutes and the recovery to hours. Simulated results show that using dialysis tubing with a diameter of 1 mm or less is possible to reduce the recovery time to less than 30 minutes. The potential applications of GBP were studied in yeast fermentation and E. coli fermentations in three different scales: 150 mL, 5 mL, and 100 microL. The results were compared with an YSI 2700 Chemistry Analyzer. Although the latter could not give reliable results for the E. coli fermentations as the glucose concentration in LB medium is close to its lower detection limit, the glucose biosensor presented here was successfully applied to each situation. Glutamine-binding protein was tested in cell cultures of two different scales (100 mL and 100 microL) and the results were also compared with those obtained with YSI. Both QBP and YSI gave good results for the 100-mL cell culture, but the relatively large sample volume requirement of YSI (at least 5 microL) prevented it from being used in the 100-microL cell culture. Because of their small sample volume requirements (less than 1 microL) and high sensitivity, the assays described here might find wide applications

  10. Influence of Selenium Content in the Culture Medium on Protein Profile of Yeast Cells Candida utilis ATCC 9950

    Directory of Open Access Journals (Sweden)

    Marek Kieliszek

    2015-01-01

    Full Text Available Selenium is an essential trace element for human health and it has been recognized as a component of several selenoproteins with crucial biological functions. It has been identified as a component of active centers of many enzymes, as well as integral part of biologically active complexes. The aim of the study was to evaluate the protein content and amino acid profile of the protein of fodder yeast Candida utilis ATCC 9950 cultured in media control and experimental enriched selenium. Protein analysis was performed using SDS-PAGE method consisting of polyacrylamide gel electrophoresis in the presence of SDS. The highest contents of soluble protein (49,5 mg/g were found in yeast cells after 24-hour culture conducted in control (YPD medium. In the presence of selenium there were determined small amounts of protein content. With increasing time of yeast culture (to 72 hours the control and experimental media were reported to reduce soluble protein content. In electropherogram proteins from control cultures was observed the presence of 10 protein fractions, but in all the experimental cultures (containing 20, 30, and 40 mg/L selenium of 14 protein fractions. On the basis of the molecular weights of proteins, it can be concluded that they were among others: selenoprotein 15 kDa and selenoprotein 18 kDa.

  11. 利用辣椒疫霉培养滤液体外筛选胡椒抗瘟病无性系研究%In vitro Selection of Black Pepper (Piper nigrum Linn.) Somaclones Resistant to Foot Rot Using Culture Filtrate of Phytophthora capsici

    Institute of Scientific and Technical Information of China (English)

    刘进平; 郑成木

    2004-01-01

    Based on shoot-tip multiplication technique, the application of in vitro selection of black pepper somaclones resistant to foot rot disease caused by fungus Phytophthora capsici was carried out using a large-leaf variety Daye (Lampong Type) which is widely cultivated in Hainan but highly susceptible to P. capsici as explant resources. The results demonstrated that sterilization methods significantly influenced on the toxicity of the culture filtrate of P. capsici. The fungal filtrate toxicity could be maintained using filtrate sterilization by addition of the fungal filtrate into selective medium instead of autoclave sterilization. The survival rate of shoot tips and multiple shoots decreased with the increasing concentrations of the fungal culture filtrate. Some cultures blackened and died finally, and some formed calli or remained dormant. After 2 subcultures onto the same selective medium,microshoots were rooted in vitro and transferred to the greenhouse conditions for screening the resistance to fungus P. capsici. The plants exhibited no external symptoms of the disease during three successive times of resistance assay once two weeks were considered to be resistant. As the concentrations of fungal culture filtrate increased, the total number of regenerated plants obtained declined but the frequencies of plants resistant to P. capsici increased. 1 (1.54%), 4 (20.00%) and 3 (42.86%) disease resistant somaclonal variants were obtained at concentration of 25%, 50% and 75% fungal culture filtrate as selective agents, respectively, with the total number of 8 plants resistant to P. capsici.%在胡椒(Piper nigrum Linn.)茎尖丛生增殖技术的基础上,以印尼大叶种"Lampong Type"无菌实生苗作外植体源,利用辣椒疫霉(Phytophthora capsici)培养滤液对胡椒茎尖及其增殖形成的丛生芽进行体外选择.辣椒疫霉培养滤液的不同灭菌方法对辣椒疫霉培养滤液的毒性影响显著,过滤灭菌方式可以保持辣椒疫霉

  12. Water Filtration Using Plant Xylem

    CERN Document Server

    Lee, Jongho; Chambers, Valerie; Venkatesh, Varsha; Karnik, Rohit

    2013-01-01

    Effective point-of-use devices for providing safe drinking water are urgently needed to reduce the global burden of waterborne disease. Here we show that plant xylem from the sapwood of coniferous trees - a readily available, inexpensive, biodegradable, and disposable material - can remove bacteria from water by simple pressure-driven filtration. Approximately 3 cm3 of sapwood can filter water at the rate of several liters per day, sufficient to meet the clean drinking water needs of one person. The results demonstrate the potential of plant xylem to address the need for pathogen-free drinking water in developing countries and resource-limited settings.

  13. Mechanism of reduced glomerular filtration rate in chronic malnutrition.

    Science.gov (United States)

    Ichikawa, I; Purkerson, M L; Klahr, S; Troy, J L; Martinez-Maldonado, M; Brenner, B M

    1980-05-01

    To determine the physiological basis for the low glomerular filtration rate in chronic malnutrition, micropuncture studies were performed in Munich-Wistar rats chronically pair-fed isocaloric diets of either low (group 1, nine rats) or high protein content (group 2, nine rats). Despite the absence of hypoalbuminemia, average values for single nephron and total kidney glomerular filtration rate were nearly 35% lower in group 1 than in group 2. Mean values for glomerular capillary and Bowman's space hydraulic pressures were essentially identical in the two groups, thereby excluding glomerular transcapillary hydraulic pressure difference as the cause for the low filtration rates in group 1 animals. On the other hand, average glomerular capillary plasma flow rate and glomerular capillary ultrafiltration coefficient were significantly lower (by approximately 25 and approximately 50%, respectively) in group 1 than in group 2. The fall in glomerular capillary plasma flow rate was the consequence of increased afferent and efferent arteriolar resistances. Plasma and erythrocyte volumes were found to be equal in five additional pairs of group 1 and group 2 rats. Thus, the substantial alterations in the ultrafiltration coefficient, glomerular capillary plasma flow rate, and renal arteriolar resistances responsible for the low filtration rate in group 1 animals were not merely a consequence of decreased circulating blood or plasma volumes. Mean values for glomerular cross sectional area were significantly lower in group 1 than in group 2 despite similar values for kidney weight in the two groups. This reduction in glomerular cross sectional area in group 1 rats is presumed to reflect a decrease in effective filtration surface area and therefore likely accounts, at least in part, for the decline in ultrafiltration coefficient observed in this group.Finally, since the daily caloric intake of group 2 animals was restricted because of pair feeding requirements tied to the group 1

  14. Morphological and protein profile comparison of large vessel and microvascular endothelial cells in culture

    Energy Technology Data Exchange (ETDEWEB)

    Beer, D.M.; Kim, J.S.; Carson, M.P.; Haudeuschild, C.C.; Patton, W.F.; Jacobson, B.S.

    1986-05-01

    Bovine adrenal medulla (AmMEC) and brain (BrMEC) microvessel endothelial cells, and bovine aortic (BAE) endothelial cells were isolated and cultured under identical conditions using a modification of a technique previously described for BrMEC. The cells were isolated and passaged under conditions minimizing cell surface alterations. Primary cultures were confluent in 4-6 days at a plating density in the region of 10/sup 4/ cells/cm/sup 2/. BAEs maintained a cobblestone morphology and a denser monolayer than MECs in primary and passaged cells whether the cells were passaged using Pancreatin, Trypsin-EDTA, or Collagenase-EDTA. MECs were initially elongate and became more like BAEs with passaging. BAEs and AmMECs were examined for differences in whole cell, Triton extracted cytoskeleton and plasma membrane (PM) protein profiles by two-dimensional gel electrophoresis. Cells were labeled with /sup 35/S-methionine and PM by lactoperoxidase catalyzed iodination. Though for the most part protein patterns were similar, several proteins in the PM and cytoskeletal preparations differed. A significant difference in the isoelectric forms of proteins with the same molecular weight was observed in the PM.

  15. Purification and Characterization of an Antibacterial Protein from the Cultured Mycelia of Cordyceps sinensis

    Institute of Scientific and Technical Information of China (English)

    HU Zheng; YE Maoqing; XIA Liqiu; TU Wenjuan; LI Liang; ZOU Guolin

    2006-01-01

    An antibacterial protein was isolated from the cultured mycelia of Cordyceps sinensis, and was designated as Cordyceps sinensis Antibacterial Protein (CSAP). CSAP was single-chained, with an apparent molecular mass of 35 × 103 revealed by SDS-PAGE and a novel hydrophobic N-terminal sequence N-ALATQHGAP. The antimicrobial assays showed CSAP could inhibit the growth of Gram-positive and Gram-negative bacteria but no significant inhibition against fungi or yeasts. Furthermore, the antibacterial activity of CSAP was not bactericidal but bacteriostatic. It was the first time that an antibacterial protein was described in the Cordyceps species, which might involve in the chemical defense mechanism of the hosts.

  16. Pig manure treatment and purification by filtration.

    Science.gov (United States)

    Makara, A; Kowalski, Z

    2015-09-15

    This study aimed to develop a new, complex pig manure treatment and filtration process. The final scheme, called the AMAK process, comprised the following successive steps: mineralization with mineral acids, alkalization with lime milk, superphosphate addition, a second alkalization, thermal treatment, and pressure filtration. The proposed method produced a filtrate with 95%, 80%, and 96% reductions in chemical oxygen demand, nitrogen content, and phosphorus content, respectively. An advantage of the proposed method was that it incorporated a crystalline phase into the solid organic part of the manure, which enabled high filtration rates (>1000 kg m(-2) h(-1)) and efficient separation. The process also eliminated odor emissions from the filtrate and sediment. The treated filtrate could be used to irrigate crops or it could be further treated in conventional biological wastewater treatment plants. The sediment could be used for producing mineral-organic fertilizer. The AMAK process is inexpensive, and it requires low investment costs.

  17. Isolation of an 18,000-dalton hypusine-containing protein from cultured mouse neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Dou, Q.P.; Chen, K.Y.

    1987-05-01

    An 18,000-dalton protein can be metabolically labeled by (TH)putrescine or spermidine in mammalian cells. The labeling is due to a post-translational conversion of a lysine residue to hypusine residue. Previous studies indicated that the labeling is growth-dependent and is greatly diminished in mouse neuroblastoma cells after differentiation. To further study the physiological functions of this protein in the differentiation of mouse neuroblastoma cells, they have developed a simple procedure to purify this protein from cultured NB-15 mouse neuroblastoma cells. The 4-steps procedure included a Cibacron-Blue column, an omega-diaminooctyl-agarose column, a Sephadex G-50 column, and a Mono Q column. The procedure resulted in a 500-fold purification and the preparation appeared to be homogenous as judged by SDS-PAGE. Peptide map analysis using V-8 protease digestion method indicated that the 18,000-dalton hypusine-containing protein from NB-15 cells was identical to eukaryotic initiation factor 4D isolated from rabbit reticulocytes. This purification scheme also enabled them to detect a very faintly labeled protein in NB-15 cells. This weakly labeled protein had an apparent molecular weight of 22,000-dalton and pI of 5.0.

  18. Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

    Directory of Open Access Journals (Sweden)

    Jun Nakanishi, Hidekazu Nakayama, Kazuo Yamaguchi, Andres J Garcia and Yasuhiro Horiike

    2011-01-01

    Full Text Available The development of methods for the off–on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs of three disulfide compounds containing (i a photocleavable poly(ethylene glycol (PEG, (ii nitrilotriacetic acid (NTA and (iii hepta(ethylene glycol (EG7. Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag sequences in its Ni2+-ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG7 underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII7–10 to the irradiated regions. In contrast, when bovine serum albumin—a major serum protein—was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII7-10 was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

  19. Dewatering of Chlorella pyrenoidosa using diatomite dynamic membrane: filtration performance, membrane fouling and cake behavior.

    Science.gov (United States)

    Zhang, Yalei; Zhao, Yangying; Chu, Huaqiang; Zhou, Xuefei; Dong, Bingzhi

    2014-01-01

    The diatomite dynamic membrane (DDM) was utilized to dewater Chlorella pyrenoidosa of 2 g dry weight/L under continuous-flow mode, whose ultimate algae concentration ranged from 43 g to 22 g dry weight/L of different culture time. The stable flux of DDM could reach 30 L/m(2) h over a 24 h operation time without backwash. Influences of extracellular organic matters (EOM) on filtration behavior and membrane fouling were studied. The DDM was divided into three sub-layers, the slime layer, the algae layer and the diatomite layer from the outside to the inside of the cake layer based on components and morphologies. It was found that EOM caused membrane fouling by accumulating in the slime and algae layers. The DDM intercepted polysaccharides, protein-like substances, humic-like substances and some low-MW organics. Proteins were indicated the major membrane foulants with increased protein/polysaccharide ratio from the slime layer to the diatomite layer as culture time increased. This method could be applied to subsequent treatment of microalgae coupling technology of wastewater treatment or microalgae harvesting for producing biofuel. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Silver nanoparticles inhibit fish gill cell proliferation in protein-free culture medium.

    Science.gov (United States)

    Yue, Yang; Behra, Renata; Sigg, Laura; Schirmer, Kristin

    2016-10-01

    While short-term exposures of vertebrate cells, such as from fish, can be performed in defined, serum-free media, long-term cultures generally require addition of growth factors and proteins, normally supplied with a serum supplement. However, proteins are known to alter nanoparticle properties by binding to nanoparticles. Therefore, in order to be able to study nanoparticle-cell interactions for extended periods, the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, was adapted to proliferate in a commercial, serum-free medium, InVitrus VP-6. The newly adapted cell strain was named RTgill-W1-pf (protein free). These cells proliferate at a speed similar to the RTgill-W1 cells cultured in a fully supplemented medium containing 5% fetal bovine serum. As well, they were successfully cryopreserved in liquid nitrogen and fully recovered after thawing. Yet, senescence set in after about 10 passages in InVitrus VP-6 medium, revealing that this medium cannot fully support long-term culture of the RTgill-W1 strain. The RTgill-W1-pf cell line was subsequently applied to investigate the effect of silver nanoparticles (AgNP) on cell proliferation over a period of 12 days. Indeed, cell proliferation was inhibited by 10 μM AgNP. This effect correlated with high levels of silver being associated with the cells. The new cell line, RTgill-W1-pf, can serve as a unique representation of the gill cell-environment interface, offering novel opportunities to study nanoparticle-cell interactions without serum protein interference.

  1. Side Stream Filtration for Cooling Towers

    Energy Technology Data Exchange (ETDEWEB)

    None

    2012-10-20

    This technology evaluation assesses side stream filtration options for cooling towers, with an objective to assess key attributes that optimize energy and water savings along with providing information on specific technology and implementation options. This information can be used to assist Federal sites to determine which options may be most appropriate for their applications. This evaluation provides an overview of the characterization of side stream filtration technology, describes typical applications, and details specific types of filtration technology.

  2. Secretory expression of human protein in the Yeast Pichia pastoris by controlled fermentor culture.

    Science.gov (United States)

    Murasugi, Akira

    2010-06-01

    The heterologous protein expression system of Pichia pastoris is now widely used for expression of many human proteins, because the efficiently expressed proteins will be correctly folded in Pichia pastoris cells and also efficiently secreted from the cells. Recombinant human serum albumin (rHSA) is efficiently secreted from Pichia pastoris. Nowadays, the expression of rHSA exceeds 10g in 1 L fermentor culture broth, and the protein is completely purified. Recombinant HSA expressed in Pichia pastoris was approved as a medicine by the authorities in 2007, and launched in 2008 in Japan. One of the insulin precursors (IP) was also successfully expressed in Pichia pastoris, and secreted up to 3.6g in 1 L medium using a multi-copy transformant. The insulin precursor could be efficiently converted to insulin, the final product, in vitro. Human growth hormone was also expressed in Pichia pastoris, and secreted up to 49 mg in 1 L medium. These proteins are also important for clinical applications. Midkine and pleiotrophin may be two of the candidates for clinical applications. Secretion signals, the copy number of an expression cassette in transformants, and culture conditions for fermentation were examined for efficient expression of these proteins in Pichia pastoris. The best signal was selected, and other factors were optimized. The amounts of native midkine and native pleiotrophin expressed were approximately 0.36g and 0.26g in 1 L medium, respectively. Expression of bile-salt stimulated lipase (BSSL) had been extremely low in the beginning of a fermentor culture experiment. However, approximately 1 g rBSSL in 1 L medium was finally expressed in a fermentor by unlimited feeding of glycerol for cell growth and optimization of other factors. BSSL from human milk and rBSSL from Pichia cells are glycosylated. The structure differences between these glycans are obvious. When humanization of Pichia glycans is established by genetic engineering, the Pichia pastoris expression

  3. Persistent homology in graph power filtrations.

    Science.gov (United States)

    Parks, Allen D; Marchette, David J

    2016-10-01

    The persistence of homological features in simplicial complex representations of big datasets in R (n) resulting from Vietoris-Rips or Čech filtrations is commonly used to probe the topological structure of such datasets. In this paper, the notion of homological persistence in simplicial complexes obtained from power filtrations of graphs is introduced. Specifically, the rth complex, r ≥ 1, in such a power filtration is the clique complex of the rth power G(r) of a simple graph G. Because the graph distance in G is the relevant proximity parameter, unlike a Euclidean filtration of a dataset where regional scale differences can be an issue, persistence in power filtrations provides a scale-free insight into the topology of G. It is shown that for a power filtration of G, the girth of G defines an r range over which the homology of the complexes in the filtration are guaranteed to persist in all dimensions. The role of chordal graphs as trivial homology delimiters in power filtrations is also discussed and the related notions of 'persistent triviality', 'transient noise' and 'persistent periodicity' in power filtrations are introduced.

  4. Air filtration and indoor air quality

    DEFF Research Database (Denmark)

    Bekö, Gabriel

    2006-01-01

    decent ventilation and air cleaning/air filtration, high indoor air quality cannot be accomplished. The need for effective air filtration has increased with increasing evidence on the hazardous effects of fine particles. Moreover, the air contains gaseous pollutants, removal of which requires various air....... These contradictions should motivate manufacturers and researchers to develop new efficient filtration techniques and/or improve the existing ones. Development of low polluting filtration techniques, which are at the same time easy and inexpensive to maintain is the way forward in the future....

  5. Filtration and compression of organic materials

    DEFF Research Database (Denmark)

    Christensen, Morten Lykkegaard; Keiding, Kristian

    The conventional filtration theory has been based on filtrations of incompressible particles such as anatase, kaolin and clay. The filtration models have later been used for organic slurries but can often not explain the observed experimental data. At constant pressure, the filtrate volume does...... not increase in proportion with square root time when e.g. sewage sludge and manure are filtered. Different explanations have been suggested. However, organic slurries are complex mixtures and it is therefore difficult to evaluate the suggested explanations and develops new models. One possible solution...

  6. Ultrastructural and immunocytochemical detection of keratins and extracellular matrix proteins in lizard skin cultured in vitro.

    Science.gov (United States)

    Alibardi, Lorenzo; Polazzi, Elisabetta

    2012-04-01

    The present study shows the localization of epidermal and dermal proteins produced in lizard skin cultivated in vitro. Cells from the skin have been cultured for up to one month to detect the expression of keratins, actin, vimentin and extracellular matrix proteins (fibronectin, chondroitin sulphate proteoglycan, elastin and collagen I). Keratinocytes and dermal cells weakly immunoreact for Pan-Cytokeratin but not with the K17-antibody at the beginning of the cell culture when numerous keratin bundles are present in keratinocyte cytoplasm. The dense keratin network disappears after 7-12 days in culture, and K17 becomes detectable in both keratinocytes and mesenchymal cells isolated from the dermis. While most epidermal cells are lost after 2 weeks of in vitro cultivation dermal cells proliferate and form a pellicle of variable thickness made of 3-8 cell layers. The fibroblasts of this dermal equivalent produces an extracellular matrix containing chondroitin sulphate proteoglycan, collagen I, elastic fibers and fibronectin, explaining the attachment of the pellicle to the substratum. The study indicates that after improving keratinocyte survival a skin equivalent for lizard epidermis would be feasible as a useful tool to analyze the influence of the dermis on the process of epidermal differentiation and the control of the shedding cycle in squamates.

  7. Synthesis of Salt Soluble Proteins in Barley. Pulse-Labeling Study of Grain Filling in Liquid-Cultured Detached Spikes

    DEFF Research Database (Denmark)

    Giese, Nanna Henriette; Hejgaard, Jørn

    1984-01-01

    The accumulation of salt-soluble proteins in the endosperm of developing barley (Hordeum vulgare L.) grains was examined. Detached spikes of barley were cultured at different levels of nitrogen nutrition and pulse-labeled with [14C] sucrose at specific times after anthesis. Proteins were extracted...... to increased nitrogen nutrition. Two major components, β-amylase and protein Z in particular, had a synthesis profile almost identical to that of the endosperm storage protein, hordein....

  8. Theoretical comparison of filtration by the renal glomerulus and artificial membranes.

    Science.gov (United States)

    Tsuji, M; Sakai, K

    1999-01-01

    Improvement in filtration performance of artificial membranes will be possible if their structure mimics the renal glomerulus. Blood filtration with glomerular capillary and artificial membranes was, therefore, modeled to clarify the effects of their structure on filtration rates. Filtration rates were obtained by dividing membrane modules axially into a number of sections and using a calculus of finite differences. The modules were assumed to be composed of straight hollow fibers arranged in parallel, with a membrane surface area of 1.5 m2. The mean transmembrane pressure (TMP) was assumed to be too low for a protein gel layer to form on the membrane surface. A decrease in the inner diameter of membrane hollow fibers led to an increase in filtration rate because of an increased film mass transfer coefficient. A decrease in hollow fiber length also produced an increase in filtration rate because of decreased axial TMP drop. The glomerular capillary has a higher filtration rate than artificial membranes because of the low TMP drop and the low osmotic pressure at the membrane surface. Decreasing both the inner diameter and the length of the hollow fibers is effective in increasing the filtration rate at constant TMP.

  9. Membrane filters and membrane-filtration processes for health care.

    Science.gov (United States)

    Eudailey, W A

    1983-11-01

    The development of membrane-filtration processes is reviewed, and current types and uses of membrane filtration in health care is discussed. Development of adequate support structures for filters and of disposable filtration devices has facilitated development of filtration processes for pharmaceutical industry, manufacturing in hospital pharmacies, and direct patient care. Hydrophobic filters have also been developed; aqueous solutions cannot wet the pore structures of these filters and therefore cannot pass. Sterility-testing systems have also been developed. There are two types of filters: depth (constructed of compacted fibers) and membrane (which have a homogeneous internal structure). Depth filters retain only a portion of particles in a particular size range and are generally not acceptable for use in health care. Membrane filters retain all particles of a given size. Types of membrane filters are selected for specific uses based on needed flow rates, particulate load, and retention capability. Membrane filters may be validated using bacterial-passage, bubble-point, and diffusion tests. Most membrane filters used in health care are microporous filters that retain particles in the 0.1-10-micron size range. Applications are currently being developed for ultrafilters, which retain both particles and substances with large molecular structures such as proteins, and reverse-osmosis filter membranes, which allow only water or water-miscible solvents of very low molecular weights to pass. Experience in engineering designs, quality assurance, and test procedures has led to the development of many safe, reliable, and effective membrane products for health care.

  10. Evidence for sequential appearance of cartilage matrix proteins in developing mouse limbs and in cultures of mouse mesenchymal cells.

    Science.gov (United States)

    Franzen, A; Heinegard, D; Solursh, M

    1987-01-01

    The initiation of synthesis and the accumulation of four cartilage matrix proteins (type II collagen and three noncollagenous proteins, one of Mr 148, one of Mr 59, and an oligometric protein of Mr above 500 with 100-kDa subunits, respectively) were studied in developing mouse limbs and in cultures of limb bud mesenchyme by means of immunolocalization. On day 13 of gestation, type II collagen was observed throughout the entire humerus, whereas the 148-kDa protein was localized only in the central portion. Neither the 100-kDa-subunit protein nor the 59-kDa protein could be demonstrated in the humerus at that stage. On day 14 1/2, type II collagen and the 148-kDa protein were codistributed throughout the humerus. The 100-kDa-subunit protein was detectable in the periphery of the humerus, whereas little 59-kDa protein could yet be demonstrated. On day 18, all four proteins being studied could be detected immunologically in the developing mouse humerus. They differed in immunolocalization. Type Ii collagen, the 148-kDa protein, and the 100-kDa-subunit protein were codistributed throughout the distal and proximal parts of the cartilage. However, the 148-kDa protein could no longer be detected immunochemically in the outermost part of the cartilage in the proximal shoulder joint. The 148-kDa protein codistributed with type II collagen and the 100-kDa-subunit protein in the distal cartilaginous region, where joint development was less advanced. On the other hand, the 59-kDa protein was not demonstrated directly within the hyaline cartilaginous structures, but surrounded the entire structure. This protein was also present in the same part of the proximal joint region as that in which the 148-kDa protein was not detected. To develop an in vitro model for studies of skeletogenesis, mesenchymal cells prepared from mouse limb buds were cultured as micromass cultures at high initial cell density to favor chondrogenesis. On day 3 of culture, type II collagen was the only protein

  11. Identification of immunoreactive proteins of Streptococcus agalactiae isolated from cultured tilapia in China.

    Science.gov (United States)

    Liu, Guangjin; Zhang, Wei; Lu, Chengping

    2013-12-01

    Streptococcus agalactiae (Group B streptococcus, GBS) is an important zoonotic pathogen that can cause lethal infections in humans and animals, including aquatic species. Immunoreactive proteins of the S. agalactiae strain, GD201008-001, isolated from cultured tilapia in China, were screened by immunoproteomics using hyperimmune sera, convalescent guinea pig sera and GD201008-001-infected tilapia antisera as primary detection antibodies. A total of 16 different proteins were identified including 13 novel immunoreactive proteins of S. agalactiae. Four proteins, serine-rich repeat glycoprotein 1, branched-chain alpha-keto acid dehydrogenase (BKD) subunit E2, 5'-nucleotidase family protein and ornithine carbamoyltransferase, were shown to react with the three types of sera and thus were considered to represent novel S. agalactiae vaccine candidate antigens. Our findings represent the basis for vaccine development for piscine S. agalactiae and are necessary for understanding virulence factors and immunogenicity of S. agalactiae with different hosts.

  12. Expression of hepatitis C virus envelope protein 2 induces apoptosis in cultured mammalian cells

    Institute of Scientific and Technical Information of China (English)

    Li-Xin Zhu; Jing Liu; You-Hua Xie; Yu-Ying Kong; Ye Ye; Chun-Lin Wang; Guang-Di Li; Yuan Wang

    2004-01-01

    AIM: To explore the role of hepatitis C virus (HCV) envelope protein 2 (E2) in the induction of apoptosis.METHODS: A carboxyterminal truncated E2 (E2-661) was transiently expressed in several cultured mammalian cell lines or stably expressed in Chinese hamster ovary (CHO)cell line. Cell proliferation was assessed by 3H thymidine uptake. Apoptosis was examined by Hoechst 33258staining, flow cytometry and DNA fragmentation analysis.RESULTS: Reduced proliferation was readily observed in the E2-661 expressing cells. These cells manifested the typical features of apoptosis, including cell shrinkage,chromatin condensation and hypodiploid genomic DNA content. Similar apoptotic cell death was observed in an E2-661 stably expressing cell line.CONCLUSION: HCV E2 can induce apoptosis in cultured mammalian cells.

  13. Non-Mulberry and Mulberry Silk Protein Sericins as Potential Media Supplement for Animal Cell Culture.

    Science.gov (United States)

    Sahu, Neety; Pal, Shilpa; Sapru, Sunaina; Kundu, Joydip; Talukdar, Sarmistha; Singh, N Ibotambi; Yao, Juming; Kundu, Subhas C

    2016-01-01

    Silk protein sericins, in the recent years, find application in cosmetics and pharmaceuticals and as biomaterials. We investigate the potential of sericin, extracted from both mulberry Bombyx mori and different non-mulberry sources, namely, tropical tasar, Antheraea mylitta; muga, Antheraea assama; and eri, Samia ricini, as growth supplement in serum-free culture medium. Sericin supplemented media containing different concentrations of sericins from the different species are examined for attachment, growth, proliferation, and morphology of fibrosarcoma cells. The optimum sericin supplementation seems to vary with the source of sericins. The results indicate that all the sericins promote the growth of L929 cells in serum-free culture media; however, S. ricini sericin seems to promote better growth of cells amongst other non-mulberry sericins.

  14. Genotypic analysis of meningococcal factor h-binding protein from non-culture clinical specimens.

    Directory of Open Access Journals (Sweden)

    Stephen A Clark

    Full Text Available Factor H-Binding Protein (fHbp is an outer membrane protein antigen included in two novel meningococcal group B vaccines and, as such, is an important typing target. Approximately 50% of meningococcal disease cases in England and Wales are confirmed using real-time PCR on non-culture clinical specimens only. Protocols for typing fHbp from this subset of cases have not yet been established. Here we present a nested PCR-based assay designed to amplify and sequence fHbp from non-culture clinical specimens. From analytical sensitivity experiments carried out using diluted DNA extracts, an estimated analytical sensitivity limit of 6 fg/µL of DNA (<3 genome copies/µL was calculated. The sensitivity of the assay was shown to be comparable to the ctrA-directed real-time PCR assay currently used to confirm invasive disease diagnoses from submitted clinical specimens. A panel of 96 diverse, patient-matched clinical specimen/isolate pairs from invasive disease cases was used to illustrate the breadth of strain coverage for the assay. All fHbp alleles sequenced from the isolates matched those derived from previous whole genome analyses. The first-round PCR primer binding sites are highly conserved, however an exceptional second-round PCR primer site mismatch in one validation isolate prevented amplification. In this case, amplification from the corresponding clinical specimen was achieved, suggesting that the use of a nested PCR procedure may compensate for any minor mismatches in round-two primer sites. The assay was successful at typing 91/96 (94.8% of the non-culture clinical specimens in this study and exhibits sufficient sensitivity to type fHbp from the vast majority of non-culture clinical specimens received by the Meningococcal Reference Unit, Public Health England.

  15. Tangential flow filtration of hemoglobin.

    Science.gov (United States)

    Palmer, Andre F; Sun, Guoyong; Harris, David R

    2009-01-01

    Bovine and human hemoglobin (bHb and hHb, respectively) was purified from bovine and human red blood cells via tangential flow filtration (TFF) in four successive stages. TFF is a fast and simple method to purify Hb from RBCs using filtration through hollow fiber (HF) membranes. Most of the Hb was retained in stage III (100 kDa HF membrane) and displayed methemoglobin levels less than 1%, yielding final concentrations of 318 and 300 mg/mL for bHb and hHb, respectively. Purified Hb exhibited much lower endotoxin levels than their respective RBCs. The purity of Hb was initially assessed via SDS-PAGE, and showed tiny impurity bands for the stage III retentate. The oxygen affinity (P(50)) and cooperativity coefficient (n) were regressed from the measured oxygen-RBC/Hb equilibrium curves of RBCs and purified Hb. These results suggest that TFF yielded oxygen affinities of bHb and hHb that are comparable to values in the literature. LC-MS was used to measure the molecular weight of the alpha (alpha) and beta (beta) globin chains of purified Hb. No impurity peaks were present in the HPLC chromatograms of purified Hb. The mass of the molecular ions corresponding to the alpha and beta globin chains agreed well with the calculated theoretical mass of the alpha- and beta- globin chains. Taken together, our results demonstrate that HPLC-grade Hb can be generated via TFF. In general, this method can be more broadly applied to purify Hb from any source of RBCs. This work is significant, since it outlines a simple method for generating Hb for synthesis and/or formulation of Hb-based oxygen carriers.

  16. Novel Nuclear Protein Complexes of Dystrophin 71 Isoforms in Rat Cultured Hippocampal GABAergic and Glutamatergic Neurons.

    Directory of Open Access Journals (Sweden)

    Rafael Rodríguez-Muñoz

    Full Text Available The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f, during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV. By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (≥60% and multipolar Glutamatergic (≤40% neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC: dystrophin 71d or dystrophin 71f bound to β-dystroglycan, α1-, β-, α2-dystrobrevins, α-syntrophin, and syntrophin-associated protein nNOS (Dp71d-DAPC or Dp71f-DAPC, respectively, in the hippocampal neurons. Furthermore, both complexes were localized in interchromatin granule cluster structures (nuclear speckles of neuronal nucleoskeleton preparations. The present study evinces that each Dp71's complexes differ slightly in dystrobrevins composition. The results demonstrated that Dp71d-DAPC was mainly localized in bipolar GABAergic and Dp71f-DAPC in multipolar Glutamatergic hippocampal neurons. Taken together, our results show that dystrophin 71d, dystrophin 71f and DAP integrate protein complexes, and both complexes were associated to nuclear speckles structures.

  17. Novel Nuclear Protein Complexes of Dystrophin 71 Isoforms in Rat Cultured Hippocampal GABAergic and Glutamatergic Neurons.

    Science.gov (United States)

    Rodríguez-Muñoz, Rafael; Cárdenas-Aguayo, María Del Carmen; Alemán, Víctor; Osorio, Beatriz; Chávez-González, Oscar; Rendon, Alvaro; Martínez-Rojas, Dalila; Meraz-Ríos, Marco Antonio

    2015-01-01

    The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f), during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV). By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (≥60%) and multipolar Glutamatergic (≤40%) neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC): dystrophin 71d or dystrophin 71f bound to β-dystroglycan, α1-, β-, α2-dystrobrevins, α-syntrophin, and syntrophin-associated protein nNOS (Dp71d-DAPC or Dp71f-DAPC, respectively), in the hippocampal neurons. Furthermore, both complexes were localized in interchromatin granule cluster structures (nuclear speckles) of neuronal nucleoskeleton preparations. The present study evinces that each Dp71's complexes differ slightly in dystrobrevins composition. The results demonstrated that Dp71d-DAPC was mainly localized in bipolar GABAergic and Dp71f-DAPC in multipolar Glutamatergic hippocampal neurons. Taken together, our results show that dystrophin 71d, dystrophin 71f and DAP integrate protein complexes, and both complexes were associated to nuclear speckles structures.

  18. Acute guttate psoriasis patients have positive streptococcus hemolyticus throat cultures and elevated antistreptococcal M6 protein titers.

    Science.gov (United States)

    Zhao, Guang; Feng, Xiaoling; Na, Aihua; Yongqiang, Jiang; Cai, Qing; Kong, Jian; Ma, Huijun

    2005-02-01

    To further study the role of Streptococci hemolyticus infection and streptococcal M6 protein in the pathogenesis of acute guttate psoriasis, streptococcal cultures were taken from the throats of 68 patients with acute guttate psoriasis. PCR technique was applied to detect M6 protein encoding DNA from those cultured streptococci. Pure M6 protein was obtained by Sephacry/S-200HR and Mono-Q chromatography from proliferated Streptococcus hemolyticus. Antistreptococcal M6 protein titers were measured in the serum of patients with acute guttate psoriasis, plaque psoriasis and healthy controls by ELISA. A high incidence of Streptococcus hemolyticus culture was observed in the guttate psoriatic group compared with the plaque psoriasis and control groups. Fourteen strains of Streptococcus hemolyticus were cultured from the throats of 68 acute guttate psoriasis patients. Of these, 5 strains contain DNA encoding the M6 protein gene as confirmed by PCR technique. More than 85% purification of M6 protein was obtained from Streptococcus pyogenes. Applying our pure M6 protein with the ELISA methods, we found that the titer of antistreptococcal M6 protein was significantly higher in the serum of guttate psoriasis patients than in the control or plaque psoriasis groups (P psoriasis have a high incidence of Streptococcus hemolyticus in their throats and raised titers of antistreptococcal M6 protein in their sera.

  19. Characterization of the secretome of suspension cultures of Medicago species reveals proteins important for defense and development.

    Science.gov (United States)

    Kusumawati, Lucia; Imin, Nijat; Djordjevic, Michael A

    2008-10-01

    Molecular events occurring in the plant apoplast contribute to important developmental and defense responses. To define the secretome of Medicago, we used suspension cultures to isolate and identify secreted proteins as a first step to determining their functions. Proteins in the extracellular medium of the suspension cultures were examined using SDS-PAGE, tandem mass spectrometry (MALDI-TOF/TOF) and bioinformatics tools. There were 39 proteins identified in the cultures derived from M. sativa, M. truncatula 2HA (an embryogenic line), and M. truncatula sickle (an ethylene-insensitive mutant). N-Terminal secretion signals were detected in 34 proteins and five other proteins were predicted to be secreted via a nonclassical (ER-independent) route. All samples possessed defense related proteins including pathogenesis related (PR) proteins. The glycoprotein, SIEP1L, was found only in M. sativa. Three secreted proteinases were identified in M. truncatula, including a serine carboxypeptidase detected only in 2HA. Some proteins were unique to a cell culture line. Quantitative real time RT-PCR was used to determine mRNA expression of selected genes corresponding to proteins found only in 2HA or sickle or in both. The results correlate well with the proteomic data. For instance, a GDSL-lipase gene known to be regulated by ethylene was found only in 2HA but not in the ethylene insensitive mutant. Similarly, the PR1a protein, expressed from a well recognized ethylene-regulated gene, was found in 2HA but not sickle. These experiments indicate that the suspension culture systems established here are useful to avoid contamination from cytoplasmic proteins and to identify secreted proteins in Medicago, and should have application in other plant systems.

  20. Production of functional killer protein in batch cultures upon a shift from aerobic to anaerobic conditions

    Directory of Open Access Journals (Sweden)

    Gildo Almeida da Silva

    2011-06-01

    Full Text Available The aim of this work was to study the production of functional protein in yeast culture. The cells of Saccharomyces cerevisiae Embrapa 1B (K+R+ killed a strain of Saccharomyces cerevisiae Embrapa 26B (K-R-in grape must and YEPD media. The lethal effect of toxin-containing supernatant and the effect of aeration upon functional killer production and the correlation between the products of anaerobic metabolism and the functional toxin formation were evaluated. The results showed that at low sugar concentration, the toxin of the killer strain of Sacch. cerevisiae was only produced under anaerobic conditions . The system of killer protein production showed to be regulated by Pasteur and Crabtree effects. As soon as the ethanol was formed, the functional killer toxin was produced. The synthesis of the active killer toxin seemed to be somewhat associated with the switch to fermentation process and with concomitant alcohol dehydrogenase (ADH activity.

  1. A novel domain regulating degradation of the glomerular slit diaphragm protein podocin in cell culture systems.

    Directory of Open Access Journals (Sweden)

    Markus Gödel

    Full Text Available Mutations in the gene NPHS2 are the most common cause of hereditary steroid-resistant nephrotic syndrome. Its gene product, the stomatin family member protein podocin represents a core component of the slit diaphragm, a unique structure that bridges the space between adjacent podocyte foot processes in the kidney glomerulus. Dislocation and misexpression of slit diaphragm components have been described in the pathogenesis of acquired and hereditary nephrotic syndrome. However, little is known about mechanisms regulating cellular trafficking and turnover of podocin. Here, we discover a three amino acids-comprising motif regulating intracellular localization of podocin in cell culture systems. Mutations of this motif led to markedly reduced degradation of podocin. These findings give novel insight into the molecular biology of the slit diaphragm protein podocin, enabling future research to establish the biological relevance of podocin turnover and localization.

  2. Activation of protein kinase C inhibits potassium currents in cultured endothelial cells.

    Science.gov (United States)

    Zhang, H; Weir, B; Daniel, E E

    1995-04-01

    The effect of protein kinase C on potassium channels in cultured endothelial cells was investigated by using whole-cell patch-clamp techniques. Activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu), but not phorbol 12-monomyristate (PMM), an inactive analogue of phorbol esters, depressed an outward calcium-dependent potassium current. The inhibitory actions of PMA and PDBu could be reversed by the kinase inhibitor H-7. Cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum calcium pump, and LP-805, a novel vasodilator which also releases endothelium-derived relaxing factors, activated the outward calcium-dependent potassium conductance. PMA and PDBu, but not PMM, reduced the outward conductance induced by cyclopiazonic acid and LP-805. These effects of PMA and PDBu on potassium currents may be mediated either by phosphorylation of ion channels, or by decreasing intracellular calcium concentration.

  3. Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens

    DEFF Research Database (Denmark)

    Skjøt, R L; Oettinger, T; Rosenkrands, I;

    2000-01-01

    Culture filtrate from Mycobacterium tuberculosis contains protective antigens of relevance for the generation of a new antituberculosis vaccine. We have identified two previously uncharacterized M. tuberculosis proteins (TB7.3 and TB10.4) from the highly active low-mass fraction of culture filtrate...... in cultures of peripheral blood mononuclear cells from human tuberculosis (TB) patients, Mycobacterium bovis BCG-vaccinated donors, and nonvaccinated donors. The two ESAT-6 family members, TB10.4 and CFP10, were very strongly recognized and induced gamma interferon release at the same level (CFP10...

  4. Efficacy of Syringe Filtration for the Selective Isolation of Campylobacter from Chicken Carcass Rinse.

    Science.gov (United States)

    Chon, Jung-Whan; Kim, Hong-Seok; Kim, Dong-Hyeon; Kim, Young-Ji; Sung, Kidon; Kim, Hyunsook; Seo, Kun-Ho

    2017-06-01

    We investigated the efficacy of syringe filtration for selective isolation of Campylobacter from chicken carcass rinse by combining syringe filtration with the conventional culture method. Whole chicken carcass rinses were incubated in Bolton enrichment broth, set aside or subjected to syringe filtration, and streaked on Campy-Cefex agar with or without cefoperazone antibiotic supplement. Compared with the conventional method without filtration, 0.65-μm-pore-size syringe filtration resulted in a significantly higher number of Campylobacter-positive samples (23.8 to 37.5% versus 70.0 to 72.5%; P Campylobacter (93.8% versus 6.3 to 26.3%), and a lower growth index (1 = growth of a few colonies; 2 = growth of colonies on about half of the plate; and 3 = growth on most of the plate) for competing microbiota (2.9 to 3.0 versus 1.2 to 1.4). When syringe filtration was applied, agar plates containing the antibiotic had significantly less contamination (6.3% versus 26.3%; P Campylobacter isolation rate was similar (P > 0.05). Syringe filtration combined with conventional enrichment improved the rate and selectivity of Campylobacter isolation from chicken carcasses.

  5. Morphological Variability and Distinct Protein Profiles of Cultured and Endosymbiotic Symbiodinium cells Isolated from Exaiptasia pulchella.

    Science.gov (United States)

    Pasaribu, Buntora; Weng, Li-Chi; Lin, I-Ping; Camargo, Eddie; Tzen, Jason T C; Tsai, Ching-Hsiu; Ho, Shin-Lon; Lin, Mong-Rong; Wang, Li-Hsueh; Chen, Chii-Shiarng; Jiang, Pei-Luen

    2015-10-20

    Symbiodinium is a dinoflagellate that plays an important role in the physiology of the symbiotic relationships of Cnidarians such as corals and sea anemones. However, it is very difficult to cultivate free-living dinoflagellates after being isolated from the host, as they are very sensitive to environmental changes. How these symbiont cells are supported by the host tissue is still unclear. This study investigated the characteristics of Symbiodinium cells, particularly with respect to the morphological variability and distinct protein profiles of both cultured and endosymbiotic Symbiodinium which were freshly isolated from Exaiptasia pulchella. The response of the cellular morphology of freshly isolated Symbiodinium cells kept under a 12 h L:12 h D cycle to different temperatures was measured. Cellular proliferation was investigated by measuring the growth pattern of Symbiodinium cells, the results of which indicated that the growth was significantly reduced in response to the extreme temperatures. Proteomic analysis of freshly isolated Symbiodinium cells revealed twelve novel proteins that putatively included transcription translation factors, photosystem proteins, and proteins associated with energy and lipid metabolism, as well as defense response. The results of this study will bring more understandings to the mechanisms governing the endosymbiotic relationship between the cnidarians and dinoflagellates.

  6. Morphological Variability and Distinct Protein Profiles of Cultured and Endosymbiotic Symbiodinium cells Isolated from Exaiptasia pulchella

    Science.gov (United States)

    Pasaribu, Buntora; Weng, Li-Chi; Lin, I.-Ping; Camargo, Eddie; Tzen, Jason T. C.; Tsai, Ching-Hsiu; Ho, Shin-Lon; Lin, Mong-Rong; Wang, Li-Hsueh; Chen, Chii-Shiarng; Jiang, Pei-Luen

    2015-10-01

    Symbiodinium is a dinoflagellate that plays an important role in the physiology of the symbiotic relationships of Cnidarians such as corals and sea anemones. However, it is very difficult to cultivate free-living dinoflagellates after being isolated from the host, as they are very sensitive to environmental changes. How these symbiont cells are supported by the host tissue is still unclear. This study investigated the characteristics of Symbiodinium cells, particularly with respect to the morphological variability and distinct protein profiles of both cultured and endosymbiotic Symbiodinium which were freshly isolated from Exaiptasia pulchella. The response of the cellular morphology of freshly isolated Symbiodinium cells kept under a 12 h L:12 h D cycle to different temperatures was measured. Cellular proliferation was investigated by measuring the growth pattern of Symbiodinium cells, the results of which indicated that the growth was significantly reduced in response to the extreme temperatures. Proteomic analysis of freshly isolated Symbiodinium cells revealed twelve novel proteins that putatively included transcription translation factors, photosystem proteins, and proteins associated with energy and lipid metabolism, as well as defense response. The results of this study will bring more understandings to the mechanisms governing the endosymbiotic relationship between the cnidarians and dinoflagellates.

  7. Fluorescence Microspectroscopy for Testing the Dimerization Hypothesis of BACE1 Protein in Cultured HEK293 Cells

    Science.gov (United States)

    Gardeen, Spencer; Johnson, Joseph L.; Heikal, Ahmed A.

    2016-06-01

    Alzheimer's Disease (AD) is a neurodegenerative disorder that results from the formation of beta-amyloid plaques in the brain that trigger the known symptoms of memory loss in AD patients. The beta-amyloid plaques are formed by the proteolytic cleavage of the amyloid precursor protein (APP) by the proteases BACE1 and gamma-secretase. These enzyme-facilitated cleavages lead to the production of beta-amyloid fragments that aggregate to form plaques, which ultimately lead to neuronal cell death. Recent detergent protein extraction studies suggest that BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. In this contribution, we examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using complementary fluorescence spectroscopy and microscopy methods. Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal, and differential interference contrast to monitor the localization and distribution of intracellular BACE1. Complementary fluorescence lifetime and anisotropy measurements enabled us to examine the conformational and environmental changes of BACE1 as a function of substrate binding. Using fluorescence correlation spectroscopy, we also quantified the diffusion coefficient of BACE1-EGFP on the plasma membrane as a means to test the dimerization hypothesis as a fucntion of substrate-analog inhibitition. Our results represent an important first towards examining the substrate-mediated dimerization hypothesis of BACE1 in live cells.

  8. Nitration of soluble proteins in organotypic culture models of Parkinson's disease.

    Science.gov (United States)

    Larsen, Trine R; Söderling, Ann-Sofi; Caidahl, Kenneth; Roepstorff, Peter; Gramsbergen, Jan Bert

    2008-02-01

    Protein nitration due to oxidative and nitrative stress has been linked to the pathogenesis of Parkinson's disease (PD), but its relationship to the loss of dopamine (DA) or tyrosine hydroxylase (TH) activity is not clear. Here we quantified protein-bound 3-nitrotyrosine (3-NT) by a novel gas chromatography/negative chemical ionization tandem mass spectrometry technique and DA and 3,4-dihydroxyphenylalanine (DOPA) by HPLC in tissues or medium of organotypic, mouse mesencephalon cultures after acute or chronic treatments with the peroxynitrite donor 3-morpholino-sydnonimine (SIN-1), the dopaminergic toxin 1-methyl-4-phenylpyridinium (MPP(+)) or the lipophilic complex I inhibitor rotenone. Incubation with SIN-1 (24 h) or MPP(+) treatments (48 h) caused dose-dependent protein nitration reaching a maximum of eightfold increase by 10 mM SIN-1 or twofold by 10 microM MPP(+), but significant DA depletions occurred at much lower concentrations of MPP(+) (1 microM). Chronic MPP(+) or rotenone treatments (3 weeks) caused maximum protein nitration by 1 microM (twofold) or 10nM (fourfold), respectively. Co-treatment with the nitric oxide synthase inhibitor l-NAME (300 microM) prevented protein nitration by MPP(+), but did not protect against MPP(+)-induced DA depletion or inhibition of TH activity. Acute incubation with 100 microM SIN-1 inhibited TH activity, which could be blocked by co-treatment with the tetrahydrobiopterin precursor l-sepiapterin, but tissue DA depletions required higher doses of SIN-1 (>1 mM, 24 h) and longer survival. In conclusion, protein nitration and TH activity or DA depletion are not directly related in these models.

  9. Subcellular compartmentalization in protoplasts from Artemisia annua cell cultures: engineering attempts using a modified SNARE protein.

    Science.gov (United States)

    Di Sansebastiano, Gian Pietro; Rizzello, Francesca; Durante, Miriana; Caretto, Sofia; Nisi, Rossella; De Paolis, Angelo; Faraco, Marianna; Montefusco, Anna; Piro, Gabriella; Mita, Giovanni

    2015-05-20

    Plants are ideal bioreactors for the production of macromolecules but transport mechanisms are not fully understood and cannot be easily manipulated. Several attempts to overproduce recombinant proteins or secondary metabolites failed. Because of an independent regulation of the storage compartment, the product may be rapidly degraded or cause self-intoxication. The case of the anti-malarial compound artemisinin produced by Artemisia annua plants is emblematic. The accumulation of artemisinin naturally occurs in the apoplast of glandular trichomes probably involving autophagy and unconventional secretion thus its production by undifferentiated tissues such as cell suspension cultures can be challenging. Here we characterize the subcellular compartmentalization of several known fluorescent markers in protoplasts derived from Artemisia suspension cultures and explore the possibility to modify compartmentalization using a modified SNARE protein as molecular tool to be used in future biotechnological applications. We focused on the observation of the vacuolar organization in vivo and the truncated form of AtSYP51, 51H3, was used to induce a compartment generated by the contribution of membrane from endocytosis and from endoplasmic reticulum to vacuole trafficking. The artificial compartment crossing exocytosis and endocytosis may trap artemisinin stabilizing it until extraction; indeed, it is able to increase total enzymatic activity of a vacuolar marker (RGUSChi), probably increasing its stability. Exploring the 51H3-induced compartment we gained new insights on the function of the SNARE SYP51, recently shown to be an interfering-SNARE, and new hints to engineer eukaryote endomembranes for future biotechnological applications.

  10. Microanalysis characterization of bioactive protein-bound polysaccharides produced by Amanita ponderosa cultures.

    Science.gov (United States)

    Salvador, Cátia; Martins, M Rosário; Caldeira, A Teresa

    2015-02-01

    Different compounds of edible mushrooms are responsible for their bioactivity. The ability to synthesize polysaccharides, namely protein-polysaccharide (PPS) complexes, is related to the antioxidant capacity of these compounds and present great interest in preventing a number of diseases, including cancer, cardiovascular and auto-immune diseases, and accelerated aging. Amanita ponderosa are wild edible mushrooms that grow in Mediterranean "montado" areas [Portuguese name given to cork oak (Quercus suber) and holm oak (Quercus ilex) forests]. The aim of this study was to evaluate the production of PPS complexes obtained from A. ponderosa cultures using a new microanalytical approach to quickly and easily monitor the production process. Microanalysis using Fourier-transform infrared using attenuated total reflection and Raman spectroscopy of PPS samples showed spectra compatible with identification of this type of compound in culture extracts. PPS separated by size-exclusion chromatography showed seven main complexes. Molecular weights of the main PPS complexes isolated from cultures ranged between 1.5 and 20 kDa and did not present toxicity against Artemia salina, demonstrating the potential of A. ponderosa as a source of biologically active compounds with nutraceutical value. Application of this microanalytical approach to monitoring the production of PPS compounds can be successfully applied in biotechnological processes.

  11. The effect of protein-protein and protein-membrane interactions on membrane fouling in ultrafiltration

    NARCIS (Netherlands)

    Huisman, I.H.; Prádanos, P.; Hernández, A.

    2000-01-01

    It was studied how protein-protein and protein-membrane interactions influence the filtration performance during the ultrafiltration of protein solutions over polymeric membranes. This was done by measuring flux, streaming potential, and protein transmission during filtration of bovine serum albumin

  12. The effect of protein-protein and protein-membrane interactions on membrane fouling in ultrafiltration

    NARCIS (Netherlands)

    Huisman, I.H.; Prádanos, P.; Hernández, A.

    2000-01-01

    It was studied how protein-protein and protein-membrane interactions influence the filtration performance during the ultrafiltration of protein solutions over polymeric membranes. This was done by measuring flux, streaming potential, and protein transmission during filtration of bovine serum albumin

  13. EFFECT OF MATRINE ON EXPRESSION OF HCCR1 AND HCCR2 PROTEINS IN CULTURAL HUMAN HEPATOCELLULAR CARCINOMAS CELLS

    Institute of Scientific and Technical Information of China (English)

    GENG Dong-jin; CHEN Jun-hao; CHEN Lei-lei; GU Xiang-fang; ZHANG Le; HAN Li; LIU Yong

    2005-01-01

    Objective: To explore the effects of matrine on HCCR1 and HCCR2 expression in cultural human hepatocellular carcinomas (HCC) cells at the level of gene and protein. Methods: Three methods, representational difference analysis(RDA) of cDNA, microarrays and fluorescence in situ hybridization (FISH) were used to detect levels of mRNA and protein expression of HCCR1 and HCCR-2 before and after treatment of matrine. Results: Matrine had inhibitory effect on the mRNA and protein expression of HCCR1 and HCCR2 in cultural HCC cells. Conclusion: Matrine has inhibitory effect on gene transcription, protein expression of HCCR1 and HCCR2 in cultural HCC cells.

  14. Review series: The cell biology of renal filtration.

    Science.gov (United States)

    Scott, Rizaldy P; Quaggin, Susan E

    2015-04-27

    The function of the kidney, filtering blood and concentrating metabolic waste into urine, takes place in an intricate and functionally elegant structure called the renal glomerulus. Normal glomerular function retains circulating cells and valuable macromolecular components of plasma in blood, resulting in urine with just trace amounts of proteins. Endothelial cells of glomerular capillaries, the podocytes wrapped around them, and the fused extracellular matrix these cells form altogether comprise the glomerular filtration barrier, a dynamic and highly selective filter that sieves on the basis of molecular size and electrical charge. Current understanding of the structural organization and the cellular and molecular basis of renal filtration draws from studies of human glomerular diseases and animal models of glomerular dysfunction.

  15. Induction of inflammatory responses from THP-1 cells by cell-free filtrates from clinical isolates of Alloiococcus otitidis.

    Science.gov (United States)

    Ashhurst-Smith, Christopher; Hall, Sharron T; Burns, Christine J; Stuart, John; Blackwell, C Caroline

    2014-04-01

    In our model system using the THP-1 monocytic cell line, whole heat-killed cells of Alloiococcus otitidis elicited several pro-inflammatory cytokines identified in ear effusions of children with otitis media (OM). Levels of these cytokines were equivalent to or greater than those elicited by a standard Gram-positive otopathogen, Streptococcus pneumoniae. The current study examined the hypothesis that extracellular material produced by A. otitidis might also contribute to the inflammatory responses in OM. Cell-free culture filtrates of recent A. otitidis isolates (n = 39) were tested for induction of pro-inflammatory cytokines from THP-1 cells primed with IFN-γ. The highest responses were from IL-8 followed by IL-1β, and the lowest from IL-6. Filtrates from nine isolates were treated with lysozyme or proteinase K to assess the nature of the extracellular stimulants. Peptidoglycan was not a major component eliciting the responses. There was no correlation between colony type or β-haemolysin production. Proteinase K treatment indicated extracellular proteins might induce the inflammatory responses, particularly the 70-75 ku band. Further studies on the role of the extracellular proteins of A. otitidis and cytokine responses in pathogenesis of ear infections are needed.

  16. Filtrating forms of soil bacteria

    Science.gov (United States)

    Van'kova, A. A.; Ivanov, P. I.; Emtsev, V. T.

    2013-03-01

    Filtrating (ultramicroscopic) forms (FF) of bacteria were studied in a soddy-podzolic soil and the root zone of alfalfa plants as part of populations of the most widespread physiological groups of soil bacteria. FF were obtained by filtering soil solutions through membrane filters with a pore diameter of 0.22 μm. It was established that the greater part of the bacteria in the soil and in the root zone of the plants has an ultramicroscopic size: the average diameter of the cells is 0.3 μm, and their length is 0.6 μm, which is significantly less than the cell size of banal bacteria. The number of FF varies within a wide range depending on the physicochemical conditions of the habitat. The FF number's dynamics in the soil is of a seasonal nature; i.e., the number of bacteria found increases in the summer and fall and decreases in the winter-spring period. In the rhizosphere of the alfalfa, over the vegetation period, the number of FF and their fraction in the total mass of the bacteria increase. A reverse tendency is observed in the rhizoplane. The morphological particularities (identified by an electron microscopy) and the nature of the FF indicate their physiological activity.

  17. Fluorescence fluctuation analysis of BACE1-GFP fusion protein in cultured HEK293 cells

    Science.gov (United States)

    Gardeen, Spencer; Johnson, Joseph L.; Heikal, Ahmed A.

    2016-10-01

    Beta-site APP cleaving enzyme 1 (BACE1) is a type I transmembrane aspartyl protease. In the amyloidogenic pathway, BACE1 provides β-secretase activity that cleaves the amyloid precursor protein (APP) that leads to amyloid beta (Aβ) peptides. The aggregation of these Aβ will ultimately results in amyloid plaque formation, a hallmark of Alzheimer's disease (AD). Amyloid aggregation leads to progressive memory impairment and neural loss. Recent detergent protein extraction studies suggest that the untreated BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. Here, we examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using fluorescence correlation spectroscopy (FCS). Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal and DIC microscopy to monitor labeled BACE1 localization and distribution within the cell. Our one-photon fluorescence fluctuation autocorrelation of BACE1- EGFP on the plasma membrane of HEK cells is modeled using two diffusing species on the plasma membrane with estimated diffusion coefficients of 1.39 x 10-7 cm2/sec and 2.8 x 10-8 cm2/sec under resting conditions and STA-200 inhibition, respectively. Anomalous diffusion model also provided adequate description of the observed autocorrelation function of BACE1- EGFP on the plasma membrane with an estimate exponent (α) of 0.8 and 0.5 for resting and STA-200 treated cells, respectively. The corresponding hydrodynamic radius of this transmembrane fusion protein was estimated using the measured diffusion coefficients assuming both Stokes-Einstein and Saffman-Delbruck models. Our results suggest a complex diffusion pattern of BACE1-EGFP on the plasma membrane of HEK cells with the possibility for dimer formation, especially under STA-200 inhibition.

  18. PDF-based heterogeneous multiscale filtration model.

    Science.gov (United States)

    Gong, Jian; Rutland, Christopher J

    2015-04-21

    Motivated by modeling of gasoline particulate filters (GPFs), a probability density function (PDF) based heterogeneous multiscale filtration (HMF) model is developed to calculate filtration efficiency of clean particulate filters. A new methodology based on statistical theory and classic filtration theory is developed in the HMF model. Based on the analysis of experimental porosimetry data, a pore size probability density function is introduced to represent heterogeneity and multiscale characteristics of the porous wall. The filtration efficiency of a filter can be calculated as the sum of the contributions of individual collectors. The resulting HMF model overcomes the limitations of classic mean filtration models which rely on tuning of the mean collector size. Sensitivity analysis shows that the HMF model recovers the classical mean model when the pore size variance is very small. The HMF model is validated by fundamental filtration experimental data from different scales of filter samples. The model shows a good agreement with experimental data at various operating conditions. The effects of the microstructure of filters on filtration efficiency as well as the most penetrating particle size are correctly predicted by the model.

  19. Removal of benzocaine from water by filtration with activated carbon

    Science.gov (United States)

    Howe, G.E.; Bills, T.D.; Marking, L.L.

    1990-01-01

    Benzocaine is a promising candidate for registration with the U.S. Food and Drug Administration for use as an anesthetic in fish culture, management, and research. A method for the removal of benzocaine from hatchery effluents could speed registration of this drug by eliminating requirements for data on its residues, tolerances, detoxification, and environmental hazards. Carbon filtration effectively removes many organic compounds from water. This study tested the effectiveness of three types of activated carbon for removing benzocaine from water by column filtration under controlled laboratory conditions. An adsorptive capacity was calculated for each type of activated carbon. Filtrasorb 400 (12 x 40 mesh; U.S. standard sieve series) showed the greatest capacity for benzocaine adsorption (76.12 mg benzocaine/g carbon); Filtrasorb 300 (8 x 30 mesh) ranked next (31.93 mg/g); and Filtrasorb 816 (8 x 16 mesh) absorbed the least (1.0 mg/g). Increased adsorptive capacity was associated with smaller carbon particle size; however, smaller particle size also impeded column flow. Carbon filtration is a practical means for removing benzocaine from treated water.

  20. Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein.

    Science.gov (United States)

    Hashimoto, Takuma; Horikawa, Daiki D; Saito, Yuki; Kuwahara, Hirokazu; Kozuka-Hata, Hiroko; Shin-I, Tadasu; Minakuchi, Yohei; Ohishi, Kazuko; Motoyama, Ayuko; Aizu, Tomoyuki; Enomoto, Atsushi; Kondo, Koyuki; Tanaka, Sae; Hara, Yuichiro; Koshikawa, Shigeyuki; Sagara, Hiroshi; Miura, Toru; Yokobori, Shin-Ichi; Miyagawa, Kiyoshi; Suzuki, Yutaka; Kubo, Takeo; Oyama, Masaaki; Kohara, Yuji; Fujiyama, Asao; Arakawa, Kazuharu; Katayama, Toshiaki; Toyoda, Atsushi; Kunieda, Takekazu

    2016-09-20

    Tardigrades, also known as water bears, are small aquatic animals. Some tardigrade species tolerate almost complete dehydration and exhibit extraordinary tolerance to various physical extremes in the dehydrated state. Here we determine a high-quality genome sequence of Ramazzottius varieornatus, one of the most stress-tolerant tardigrade species. Precise gene repertoire analyses reveal the presence of a small proportion (1.2% or less) of putative foreign genes, loss of gene pathways that promote stress damage, expansion of gene families related to ameliorating damage, and evolution and high expression of novel tardigrade-unique proteins. Minor changes in the gene expression profiles during dehydration and rehydration suggest constitutive expression of tolerance-related genes. Using human cultured cells, we demonstrate that a tardigrade-unique DNA-associating protein suppresses X-ray-induced DNA damage by ∼40% and improves radiotolerance. These findings indicate the relevance of tardigrade-unique proteins to tolerability and tardigrades could be a bountiful source of new protection genes and mechanisms.

  1. Growth-promoting effect on iron-sulfur proteins on axenic cultures of Entamoeba dispar

    Directory of Open Access Journals (Sweden)

    Khalifa S.A.M.

    2006-03-01

    Full Text Available A growth-promoting factor (GPF that promotes the growth of Entamoeba dispar under axenic culture conditions was found in fractions of mitochondria (Mt, hydrogenosomes (Hg and chloroplasts (Cp obtained from cells of six different protozoan, mammalian and plant species. We were able to extract the GPF from the Cp-rich leaf cells of a plant (spiderwort: Commelina communis L. in an acetone-soluble fraction as a complex of chlorophyll with low molecular weight proteins (molecular weight [MW] approximately 4,600. We also found that on treatment with 0.6 % complexes of 2-mercapthoethanol (2ME, complexes of chlorophyll-a with iron-sulphur (Fe-S proteins (e.g., ferredoxins [Fd] from spinach and Clostridium pasteurianum and noncomplex rubredoxin (Rd from C. pasteurianum have a growth-promoting effect on E. dispar. These findings suggest that E. dispar may lack a sufficient quantity of some essential components of Fe-S proteins, such as Fe-S center.

  2. Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein

    Science.gov (United States)

    Hashimoto, Takuma; Horikawa, Daiki D.; Saito, Yuki; Kuwahara, Hirokazu; Kozuka-Hata, Hiroko; Shin-I, Tadasu; Minakuchi, Yohei; Ohishi, Kazuko; Motoyama, Ayuko; Aizu, Tomoyuki; Enomoto, Atsushi; Kondo, Koyuki; Tanaka, Sae; Hara, Yuichiro; Koshikawa, Shigeyuki; Sagara, Hiroshi; Miura, Toru; Yokobori, Shin-ichi; Miyagawa, Kiyoshi; Suzuki, Yutaka; Kubo, Takeo; Oyama, Masaaki; Kohara, Yuji; Fujiyama, Asao; Arakawa, Kazuharu; Katayama, Toshiaki; Toyoda, Atsushi; Kunieda, Takekazu

    2016-01-01

    Tardigrades, also known as water bears, are small aquatic animals. Some tardigrade species tolerate almost complete dehydration and exhibit extraordinary tolerance to various physical extremes in the dehydrated state. Here we determine a high-quality genome sequence of Ramazzottius varieornatus, one of the most stress-tolerant tardigrade species. Precise gene repertoire analyses reveal the presence of a small proportion (1.2% or less) of putative foreign genes, loss of gene pathways that promote stress damage, expansion of gene families related to ameliorating damage, and evolution and high expression of novel tardigrade-unique proteins. Minor changes in the gene expression profiles during dehydration and rehydration suggest constitutive expression of tolerance-related genes. Using human cultured cells, we demonstrate that a tardigrade-unique DNA-associating protein suppresses X-ray-induced DNA damage by ∼40% and improves radiotolerance. These findings indicate the relevance of tardigrade-unique proteins to tolerability and tardigrades could be a bountiful source of new protection genes and mechanisms. PMID:27649274

  3. Particle contamination of parenteralia and in-line filtration of proteinaceous drugs.

    Science.gov (United States)

    Werner, Benjamin Patrick; Winter, Gerhard

    2015-12-30

    Protein drug products play an important role in the treatment of severe diseases. However, due to the instability of these complex molecules, protein aggregates can form which can compromise drug safety and efficacy including immunogenic reactions. In-line filtration during the administration of these drugs can serve as a final safeguarding step to protect patients from risks associated with proteinaceous particles. A unique analysis of more than 300 marketed protein drug products revealed that already around 16% of all these products are filtered during preparation or administration. Further, the research revealed that no standardized filtration practice exists. Broad variances regarding filter membrane or pore size are found and sometimes no specifications are mentioned at all. The benefits as well as possible negative impacts of filtration like filter shedding, extractables or drug adsorption are critically assessed. Several proposals to improve the current filtration practice and to expand the number of in-line filtered protein drug products are made. The suggestions include the demand for the specific usage of one filter membrane type, the establishment of a filtration routine for unfiltered protein drugs and a statistical analysis between filtered and non-filtered products with similar formulations to identify possible differences in the immunogenicity rate.

  4. Engineer medium and feed for modulating N-glycosylation of recombinant protein production in CHO cell culture

    DEFF Research Database (Denmark)

    Fan, Yuzhou; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    2017-01-01

    -glycosylation is crucial, as the structure of N-glycans can largely influence both biological and physicochemical properties of recombinant proteins. Protein N-glycosylation in CHO cell culture can be controlled and tuned by engineering medium, feed, culture process, as well as genetic elements of the cell......Chinese hamster ovary (CHO) cells have become the primary expression system for the production of complex recombinant proteins due to their long-term success in industrial scale production and generating appropriate protein N-glycans similar to that of humans. Control and optimization of protein N....... In this chapter, we will focus on how to carry out experiments for N-glycosylation modulation through medium and feed optimization. The workflow and typical methods involved in the experiment process will be presented....

  5. Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture.

    Science.gov (United States)

    Imsoonthornruksa, Sumeth; Pruksananonda, Kamthorn; Parnpai, Rangsun; Rungsiwiwut, Ruttachuk; Ketudat-Cairns, Mariena

    2015-01-01

    To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins.

  6. Glutathione, glutathione disulfide, and S-glutathionylated proteins in cell cultures.

    Science.gov (United States)

    Giustarini, Daniela; Galvagni, Federico; Tesei, Anna; Farolfi, Alberto; Zanoni, Michele; Pignatta, Sara; Milzani, Aldo; Marone, Ilaria M; Dalle-Donne, Isabella; Nassini, Romina; Rossi, Ranieri

    2015-12-01

    The analysis of the global thiol-disulfide redox status in tissues and cells is a challenging task since thiols and disulfides can undergo artificial oxido-reductions during sample manipulation. Because of this, the measured values, in particular for disulfides, can have a significant bias. Whereas this methodological problem has already been addressed in samples of red blood cells and solid tissues, a reliable method to measure thiols and disulfides in cell cultures has not been previously reported. Here, we demonstrate that the major artifact occurring during thiol and disulfide analysis in cultured cells is represented by glutathione disulfide (GSSG) and S-glutathionylated proteins (PSSG) overestimation, due to artificial oxidation of glutathione (GSH) during sample manipulation, and that this methodological problem can be solved by the addition of N-ethylmaleimide (NEM) immediately after culture medium removal. Basal levels of GSSG and PSSG in different lines of cultured cells were 3-5 and 10-20 folds higher, respectively, when the cells were processed without NEM. NEM pre-treatment also prevented the artificial reduction of disulfides that occurs during the pre-analytical phase when cells are exposed to an oxidant stimulus. In fact, in the absence of NEM, after medium removal, GSH, GSSG and PSSG levels restored their initial values within 15-30 min, due to the activity of reductases and the lack of the oxidant. The newly developed protocol was used to measure the thiol-disulfide redox status in 16 different line cells routinely used for biomedical research both under basal conditions and after treatment with disulfiram, a thiol-specific oxidant (0-200 μM concentration range). Our data indicate that, in most cell lines, treatment with disulfiram affected the levels of GSH and GSSG only at the highest concentration. On the other hand, PSSG levels increased significantly also at the lower concentrations of the drug, and the rise was remarkable (from 100 to 1000

  7. Lifecycle management for recombinant protein production using mammalian cell culture technology.

    Science.gov (United States)

    Moran, Enda; Gammell, Patrick

    2014-01-01

    Product lifecycle management refers to the oversight process and activities carried out to fully realize the commercial potential and value of a product in the marketplace. It is typical for many changes to be introduced to the production processes and testing methods for biopharmaceutical drugs over their lifetime in the commercial marketplace. Technology lifecycle management, as discussed here, refers to the management of the different phases or generations of processes and methods used to make and test the active biopharmaceutical ingredient or drug product, and the adoption of different devices used to present the drug product to patients. The factors to consider when making changes to a commercial biopharmaceutical manufacturing process as part of a technology lifecycle management program are discussed. A case study outlines one approach taken in bringing forward a major process change to a cell culture process for the production of a therapeutic recombinant protein.

  8. Metal reduction at bulk chemical filtration

    Science.gov (United States)

    Umeda, Toru; Daikoku, Shusaku; Tsuzuki, Shuichi; Murakami, Tetsuya

    2017-03-01

    OK73 thinner and cyclohexanone, both of which were spiked with metals were passed through Nylon 6,6 filter, varying flow rate, which include the conditions of both point-of-use and bulk filtrations. The influent and effluent metal concentrations were measured using ICP-MS for metal removal efficiency of the filtration. As a result, removal efficiency for some metals descended depending on the flow rate, while others maintained. Slower flow rate is recommended to maintain low metal concentration in bulk filtration based on the result. Metals in cyclohexanone were reduced at higher efficiency than in OK73 thinner, agrees with a metal removal model of hydrophilic adsorbent in organic solvent, evidenced in our previous paper. Further, metal reduction on 300 mm φ Si wafer after coating organic solvents with Nylon 6,6 filtration was evidenced with TREX analysis.

  9. Filtration in coal liquefaction - Influence of filtration conditions in non-hydrogenated systems

    Science.gov (United States)

    Clarke, J. W.; Rantell, T. D.

    1980-01-01

    A series of experiments has been carried out to study the effects of filtration conditions upon the rate of filtration of non-hydrogenated coal digests. The results show the dependence of cake resistivity on both the filtration temperature and pressure. Filter cakes were found to be compressible, resulting in smaller increases in rate with increasing pressure than with incompressible cakes. The filtration temperature determines the packing of residual solids in the cake which in turn affects the cake resistivity. An empirical relation has been derived between filtration temperature and resistivity. With increasing temperature there is an increase in filtration rate due to the reduced viscosity, but a reduction owing to a higher packing density of solids in the filter cake.

  10. Bone morphogenetic protein-5 (BMP-5 promotes dendritic growth in cultured sympathetic neurons

    Directory of Open Access Journals (Sweden)

    Higgins Dennis

    2001-09-01

    Full Text Available Abstract Background BMP-5 is expressed in the nervous system throughout development and into adulthood. However its effects on neural tissues are not well defined. BMP-5 is a member of the 60A subgroup of BMPs, other members of which have been shown to stimulate dendritic growth in central and peripheral neurons. We therefore examined the possibility that BMP-5 similarly enhances dendritic growth in cultured sympathetic neurons. Results Sympathetic neurons cultured in the absence of serum or glial cells do not form dendrites; however, addition of BMP-5 causes these neurons to extend multiple dendritic processes, which is preceded by an increase in phosphorylation of the Smad-1 transcription factor. The dendrite-promoting activity of BMP-5 is significantly inhibited by the BMP antagonists noggin and follistatin and by a BMPR-IA-Fc chimeric protein. RT-PCR and immunocytochemical analyses indicate that BMP-5 mRNA and protein are expressed in the superior cervical ganglia (SCG during times of initial growth and rapid expansion of the dendritic arbor. Conclusions These data suggest a role for BMP-5 in regulating dendritic growth in sympathetic neurons. The signaling pathway that mediates the dendrite-promoting activity of BMP-5 may involve binding to BMPR-IA and activation of Smad-1, and relative levels of BMP antagonists such as noggin and follistatin may modulate BMP-5 signaling. Since BMP-5 is expressed at relatively high levels not only in the developing but also the adult nervous system, these findings suggest the possibility that BMP-5 regulates dendritic morphology not only in the developing, but also the adult nervous system.

  11. Dynamic Effects of Cerium on Syntheses of Soluble Protein and Taxol in Suspension Culture of Taxus Chinensis Var. Mairei Cells

    Institute of Scientific and Technical Information of China (English)

    李景川; 马忠海; 元英进; 孙安慈; 胡昌序

    2001-01-01

    The dynamic effects of Ce4+ on the syntheses of soluble protein and taxol in suspension cultures of Taxus chinensis var. mairei cells were studied. The phenomena of “partition” and “bifurcation” were observed in studying the dynamic effect of Ce4+ on soluble protein synthesis and cell activity. That is, Ce4+ of low concentration improves the soluble protein synthetic strength and cell activity, while Ce4+of high concentration is harmful to protein synthesis and cell activity. In addition, Ce4+ of appropriate concentration enhances taxol synthesis.

  12. Indoor particles affect vascular function in the aged - An air filtration-based intervention study

    DEFF Research Database (Denmark)

    Brauner, E.V.; Forchhammer, L.; Moller, P.

    2008-01-01

    factors, P-selectin, plasma amyloid A, C-reactive protein, fibrinogen, IL-6, tumor necrosis factor-alpha, protein oxidation measured as 2-aminoadipic semialdehyde in plasma, urinary 8-iso-prostaglandin F-2 alpha, and blood pressure. Indoor air filtration significantly improved MVF by 8.1% (95% confidence...

  13. Low molecular weight peptides derived from sarcoplasmic proteins produced by an autochthonous starter culture in a beaker sausage model

    Directory of Open Access Journals (Sweden)

    Constanza M. López

    2015-06-01

    Significance: The selection of a specific autochthonous starter culture guarantees the hygiene and typicity of fermented sausages. The identification of new peptides as well as new target proteins by means of peptidomics represents a significant step toward the elucidation of the role of microorganisms in meat proteolysis. Moreover, these peptides may be further used as biomarkers capable to certify the use of the applied autochthonous starter culture described here.

  14. Complications and Management of the Filtration Bleb

    Institute of Scientific and Technical Information of China (English)

    Clement; W; N; Chan

    1992-01-01

    Filtration surgery is the commonest operation performed for closed angle or open angle glaucoma when medical treatment or laser trabeculoplasty has failed to control the intraocular pressure. It is characterized by the formation of an artificial drainage fistula between the anterior chamber and subconjunctival space. The successful operation is evidenced by the appearance of a subconjunctival filtration bleb which in turn depends on the patency of this pathway. Despite numerous modification, the procedu...

  15. The Glomerular Filtration Barrier: Components and Crosstalk

    OpenAIRE

    Madhav C. Menon; Chuang, Peter Y.; Cijiang John He

    2012-01-01

    The glomerular filtration barrier is a highly specialized blood filtration interface that displays a high conductance to small and midsized solutes in plasma but retains relative impermeability to macromolecules. Its integrity is maintained by physicochemical and signalling interplay among its three core constituents—the glomerular endothelial cell, the basement membrane and visceral epithelial cell (podocyte). Understanding the pathomechanisms of inherited and acquired human diseases as well...

  16. In vitro culture of feline embryos increases stress-induced heat shock protein 70 and apoptotic related genes.

    Science.gov (United States)

    Sananmuang, Thanida; Phutikanit, Nawapen; Nguyen, Catherine; Manee-In, Sukanya; Techakumphu, Mongkol; Tharasanit, Theerawat

    2013-01-01

    Developmental competence and quality of in vitro produced embryos has been demonstrated to be lower than in vivo derived embryos. This study aimed specifically to determine the effects of in vitro culture of feline embryos using various culture densities on developmental competence and expression of stress- and apoptotic-related genes in terms of heat shock protein 70 (HSP70) and apoptotic-related (BAX and BCL-2) gene expressions. In experiment 1, we characterized the inducible form of a feline-specific HSP70 mRNA sequence, as it has not been previously reported. The primers for feline HSP70 mRNA were synthesized and tested on heat-treated cat fibroblasts. In experiment 2, feline embryos were cultured at different culture densities (embryo:culture volume; 1:1.25, 1:5 and 1:20). The developmental competence was determined along with HSP70, BAX and BCL-2 transcript abundances using quantitative RT-PCR. In vivo derived embryos were used as a control group. A partial cat HSP70 mRNA sequence (190 bp) was characterized and exhibited high nucleotide identity (93 to 96%) with other species. Cleaved embryos cultured at high density (1:1.25) developed to blastocysts at a lower rate than those generated from lower densities. Irrespective of the culture densities used, in vitro cultured blastocysts showed increased levels of HSP70 and BAX transcripts compared with in vivo counterparts. Blastocysts derived from the highest culture density (1:1.25) showed higher levels of upregulation of HSP70 and BAX transcripts than those cultured at lower culture densities (1:5 and 1:20). In conclusion, increased levels of pro-apoptotic (BAX) and stress-response (HSP70) transcripts correlated with developmental incompetence of embryos cultured at high embryonic density, indicating that stress accumulated during in vitro embryo culture affected the fate for embryo development and quality.

  17. cultural

    Directory of Open Access Journals (Sweden)

    Irene Kreutz

    2006-01-01

    Full Text Available Es un estudio cualitativo que adoptó como referencial teorico-motodológico la antropología y la etnografía. Presenta las experiencias vivenciadas por mujeres de una comunidad en el proceso salud-enfermedad, con el objetivo de comprender los determinantes sócio-culturales e históricos de las prácticas de prevención y tratamiento adoptados por el grupo cultural por medio de la entrevista semi-estructurada. Los temas que emergieron fueron: la relación entre la alimentación y lo proceso salud-enfermedad, las relaciones con el sistema de salud oficial y el proceso salud-enfermedad y lo sobrenatural. Los dados revelaron que los moradores de la comunidad investigada tienen un modo particular de explicar sus procedimientos terapéuticos. Consideramos que es papel de los profesionales de la salud en sus prácticas, la adopción de abordajes o enfoques que consideren al individuo en su dimensión sócio-cultural e histórica, considerando la enorme diversidad cultural en nuestro país.

  18. Properties of the filtrate from treatment of pig manure by filtration method

    Directory of Open Access Journals (Sweden)

    Makara Agnieszka

    2017-02-01

    Full Text Available This paper presents properties of filtrate obtained from pig manure using the AMAK treatment process, which includes the mineralization of macro- and microfertilizer components by the hydrolyzing of organic matter into forms that are bioavailable to plants. Filtration produced two products, sediment and filtrate. The quality of the filtrate allowed for its use as a substitute of water to irrigate crops. Concentrations of heavy metals are very low and therefore the quality of the filtrate fully complies with European standard concerning fertilizers. The used mineralization process practically eliminated odors from the filtrate. The reduction of specific odor emission by 99.1-99.5% in samples taken from above the filtrate, respectively compared to the odor concentration found in samples taken from above raw pig manure. Sediment could be used as raw material for production of mineral-organic fertilizer. Filtrate and filtration sediments analyses show that the majority of nitrogen and other fertilizing compounds included in raw pig manure remains in sediment.

  19. Optimization of biomolecule separation by combining microscale filtration and design-of-experiment methods.

    Science.gov (United States)

    Kazemi, Amir S; Kawka, Karina; Latulippe, David R

    2016-10-01

    There is considerable interest in developing microscale (i.e., high-throughput) methods that enable multiple filtration experiments to be run in parallel with smaller sample amounts and thus reduce the overall required time and associated cost to run the filtration tests. Previous studies to date have focused on simply evaluating the filtration capacity, not the separation performance. In this work, the stirred-well filtration (SWF) method was used in combination with design-of-experiment (DOE) methods to optimize the separation performance for three binary mixtures of bio-molecules: protein-protein, protein-polysaccharide, and protein-DNA. Using the parallel based format of the SWF method, eight constant-flux ultrafiltration experiments were conducted at once to study the effects of stirring conditions, permeate flux, and/or solution conditions (pH, ionic strength). Four separate filtration tests were conducted for each combination of process variables; in total, over 100 separate tests were conducted. The sieving coefficient and selectivity results are presented to match the DOE design format and enable a greater understanding of the effects of the different process variables that were studied. The method described herein can be used to rapidly determine the optimal combination of process factors that give the best separation performance for a range of membrane-based separations applications and thus obviate the need to run a large number of traditional lab-scale tests. Biotechnol. Bioeng. 2016;113: 2131-2139. © 2016 Wiley Periodicals, Inc.

  20. [The creatine effect on RNA and protein synthesis in growing culture of chick embryo myoblasts].

    Science.gov (United States)

    Zilber, M L; Litvinova, V N; Morozov, V I; Pliskin, A V; Pshendin, A I; Rogozkin, V A

    1975-01-01

    4hr incubation of the growing culture of chick embrio myoblasts in the presence of 5 mM creatine resulted, regardless of a well-defined lowering of cell membrane permeability to labelled precursors, in: (1) the 1.5-fold induction of 14C-orotic acid incorporation into total cellular RNA; (2) the 1.9-fold stimulation of DNA-dependent RNA-polymerase activity and (3) the preferable, in comparison with total proteins, 14C-leucine incorporation in the myosin heavy chain. The stimulating effect of creatine on RNA-poly-merase deals with a definite enzyme fraction and it is caused by the augmentation of the transcriptional complex enzyme activity. Polyacrilamide gel electrophoresis of total 14C-RNA revealed a relative increase of radioactivity after creatine treatment in both high-molecular pike and the fraction corresponding to 26S RNA, the relative proportion of mRNA containing Poly-A in the composition of total cellular RNA being invariable. The data obtained show that, though creatine as a positive regulator manifestates its effect at different stages of the sucessive chain of reactions leading to muscular protein synthesis, the direct realization of creatine regulatory function is connected with nucleus and is fullfilled at the transcription level.

  1. 21 CFR 177.2910 - Ultra-filtration membranes.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Ultra-filtration membranes. 177.2910 Section 177... Components of Articles Intended for Repeated Use § 177.2910 Ultra-filtration membranes. Ultra-filtration... the processing of food, under the following prescribed conditions; (a)(1) Ultra-filtration...

  2. Controlled shear filtration: A novel technique for animal cell separation.

    Science.gov (United States)

    Vogel, J H; Kroner, K H

    1999-06-20

    A novel rotary microfiltration technique specifically suited for the separation of animal cells has been developed. The concept allows the independent adjustment of wall shear stress, transmembrane pressure, and residence time, allowing straightforward optimization of the microfiltration process. By using a smooth, conically shaped rotor, it is possible to establish a controlled shear field in which animal cells experience a significant hydrodynamic lift away from the membrane surface. It is shown in preliminary experiments that shear-induced cell-rupture speeds up membrane clogging and that cell debris poses the most significant problem in harvesting of BHK cell cultures by dynamic microfiltration. However, a threshold value of shear stability exists which depends on the frequency of passing the shear field, the residence time in the shear field, as well as on cell status. By operating close to this threshold value, cell viability can be maintained while concentration polarization is efficiently minimized. By applying this concept, it is possible to attain flux rates several times higher compared to conventional crossflow filtration. Controlled shear filtration (CSF) can be used for batch harvesting as well as for cell retention in high cell density systems. In batch harvesting of hIL-2 from rBHK cell culture, a constant flux rate of 290 L h-1 m-2 has been adjusted without indication of membrane clogging or fouling.

  3. Rate of filtration by Perna viridis pre-exposed to heavy metals

    OpenAIRE

    Prabhudeva, K.N.; Menon, N. R.

    1985-01-01

    Perna viridis exposed to different concentrations of copper and zinc for varying periods and then the rate of filtration estimated under metal free culture conditions. The concentrations under which the animals were maintained before the experiments were 0.025 to 0.150 p.p.m. of zinc and 0.005 to 0.08 p.p.m. of copper.

  4. Description of the surface water filtration and ozone treatment system at the Northeast Fishery Center

    Science.gov (United States)

    A water filtration and ozone disinfection system was installed at the U.S. Fish and Wildlife Service's Northeast Fishery Center in Lamar, Pennsylvania to treat a surface water supply that is used to culture sensitive and endangered fish. The treatment system first passes the surface water through dr...

  5. Overexpression of citrus polygalacturonase-inhibiting protein in citrus black rot pathogen Alternaria citri.

    Science.gov (United States)

    Katoh, Hiroshi; Nalumpang, Sarunya; Yamamoto, Hiroyuki; Akimitsu, Kazuya

    2007-05-01

    The rough lemon (Citrus jambhiri) gene encoding polygalacturonase-inhibiting protein (RlemPGIPA) was overexpressed in the pathogenic fungus Alternaria citri. The overexpression mutant AcOPI6 retained the ability to utilize pectin as a sole carbon source, and the overexpression of polygalacturonase-inhibiting protein did not have any effect on the growth of AcOPI6 in potato dextrose and pectin medium. The pathogenicity of AcOPI6 to cause a black rot symptom in citrus fruits was also unchanged. Polygalacturonase-inhibiting protein was secreted together with endopolygalacturonase into culture filtrates of AcOPI6, and oligogalacturonides were digested from polygalacturonic acid by both proteins in the culture filtrates. The reaction mixture containing oligogalacturonides possessed activity for induction of defense-related gene, RlemLOX, in rough lemon leaves.

  6. A continuous-exchange cell-free protein synthesis system based on extracts from cultured insect cells.

    Directory of Open Access Journals (Sweden)

    Marlitt Stech

    Full Text Available In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds.

  7. Effects of bone morphogenetic protein-7 stimulation on osteoblasts cultured on different biomaterials.

    Science.gov (United States)

    Açil, Yahya; Springer, Ingo N G; Broek, Vanessa; Terheyden, Hendrik; Jepsen, Søren

    2002-01-01

    The objective of the present study was to investigate the effects of an in vitro stimulation of human osteoblasts by recombinant human bone morphogenetic protein-7 (rhBMP-7) on the collagen types and the quantity of the collagen cross-links synthesized in a three-dimensional culture on various biomaterials for bone replacement. Trabecular bone chips were harvested from human iliac crests, and cell cultures were established at standard conditions. One hundred and fifty nanograms per milliliter of rhBMP-7 was added. For the second passage a cell scraper was used to bring the cells into suspension, and 100 microl osteoblasts (at a density of 3.3 x 10(5)) were transferred onto nine blocks of either Bio-Oss, Tutoplast, or PepGen p-15. Blocks incubated with cells that were not treated with rhBMP-7 served as controls. Cell colonization of the biomaterials was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) after a period of 2, 4, and 6 weeks. Throughout the experiment medium, supernatants were collected and collagen was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Finally, the collagen cross-link residues hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) were quantified by HPLC. Within 4 weeks the cells became confluent on all of the studied biomaterials. All samples synthesized bone specific LP and collagen type I. However, in rhBMP-7-stimulated samples, the amount of HP and LP found was increased by 45% compared to non-stimulated samples. Cell proliferation and collagen synthesis was similar on the different biomaterials, but was consistently reduced in specimen not stimulated with rhBMP-7. In vitro stimulation of osteoblasts on Bio-Oss, Tutoplast, or PepGen p-15 with rhBMP-7 and subsequent transplantation of the constructs might lead to an enhanced osseointegration of the biomaterials in vivo.

  8. Studies of protein oxidation as a product quality attribute on a scale-down model for cell culture process development.

    Science.gov (United States)

    Lee, Nacole D; Kondragunta, Bhargavi; Uplekar, Shaunak; Vallejos, Jose; Moreira, Antonio; Rao, Govind

    2015-01-01

    Of importance to the biological properties of proteins produced in cell culture systems are the complex post-translational modifications that are affected by variations in process conditions. Protein oxidation, oxidative modification to intracellular proteins that involves cleavage of the polypeptide chain, and modifications of the amino acid side chains can be affected by such process variations. Dissolved oxygen is a parameter of increasing interest since studies have shown that despite the necessity of oxygen for respiration, there may also be some detrimental effects of oxygen to the cell. Production and accumulation of reactive oxygen species can cause damage to proteins as a result of oxidation of the cell and cellular components. Variation, or changes to cell culture products, can affect function, clearance rate, immunogenicity, and specific activity, which translates into clinical implications. The effect of increasing dissolved oxygen on protein oxidation in immunoglobulin G3-producing mouse hybridoma cells was studied using 50 mL high-throughput mini-bioreactors that employ non-invasive optical sensor technology for monitoring and closed feedback control of pH and dissolved oxygen. Relative protein carbonyl concentration of proteins produced under varying levels of dissolved oxygen was measured by enzyme-linked immunosorbent assay and used as an indicator of oxidative damage. A trend of increasing protein carbonyl content in response to increasing dissolved oxygen levels under controlled conditions was observed. Protein oxidation, oxidative modification to intracellular proteins that involves cleavage of the polypeptide chain, and modifications of the amino acid side chains can be affected by variations in dissolved oxygen levels in cell culture systems. Studies have shown that despite the necessity of oxygen for respiration, there may be detrimental effects of oxygen to the cell. Production and accumulation of reactive oxygen species can cause damage to

  9. Characterization of an immunomodulatory Der p 2-FIP-fve fusion protein produced in transformed rice suspension cell culture.

    Science.gov (United States)

    Su, Chin-Fen; Kuo, I-Chun; Chen, Peng-Wen; Huang, Chiung-Hui; Seow, See Voon; Chua, Kaw Yan; Yu, Su-May

    2012-02-01

    Der p 2, a major allergen of Dermatophagoides pteronyssinus mites, is one of the most clinically relevant allergens to allergic patients worldwide. FIP-fve protein (Fve) from the golden needle mushroom (Flammulina velutipes) is an immunomodulatory protein with potential Th1-skewed adjuvant properties. Here, we produced and immunologically evaluated a Der p 2-Fve fusion protein as a potential immunotherapeutic for allergic diseases. Using an inducible expression system in cultured rice suspension cells, the recombinant Der p 2-Fve fusion protein (designated as OsDp2Fve) was expressed in rice cells under the control of an α-amylase gene (αAmy8) promoter and secreted under sucrose starvation. OsDp2Fve was partially purified from the cultured medium. The conformation of Der p 2 in OsDp2Fve remains intact as reflected by its unaltered allergenicity, as assessed by human IgE ELISA and histamine release assays, compared to non-fusion Der p 2 protein. Furthermore, the Fve protein expressed in OsDp2Fve retains its in vitro lymphoproliferative activity but loses its hemagglutination and lymphoagglutination effects compared to the native protein. Notably, in vivo evaluation showed that mice administered with OsDp2Fve possessed an enhanced production of Der p 2-specific IgG antibodies without potentiating the production of Der p 2-specific IgE and Th2 effector cytokines in comparison with mice co-administered with native Fve and Der p 2 proteins. These results suggest that the recombinant Der p 2-Fve fusion protein produced in rice suspension cell cultures has a great potential for allergy immunotherapy.

  10. The Protective Effects of Erythropoietin on Rat Glomerular Podocytes in Culture are Modulated by Extracellular Matrix Proteins

    Directory of Open Access Journals (Sweden)

    Jan Krtil

    2014-03-01

    Full Text Available Background/Aims: Podocytes are typically cultured on collagen I; however, collagen I is absent from healthy glomerular basement membranes. Erythropoietin (EPO is thought to protect podocytes in vivo. Here, we studied how various types of extracellular matrix (ECM proteins and EPO affect podocytes in culture. Methods: Primary rat podocytes were replated on collagen I, collagen IV, whole ECM extract, laminin, or bare plastic. Cellular adhesion (8 hours after plating, proliferation (5 days, 10 % serum, and resistance to serum deprivation (3 days, 0.5 % serum were assessed. BrdU incorporation and expression of podocyte-specific markers were employed as measures of cellular proliferation and differentiation, respectively. qPCR was used to verify expression of EPO receptor in cultured podocytes. Results: Cellular adhesion was similar on all ECM proteins and unaffected by EPO. Proliferation was accelerated by laminin and the ECM extract, but the final cell density was similar on all ECM surfaces. Collagen IV supported the serum-deprived cells better than the other ECM proteins. EPO (2-20 ng/ml improved viability of serum-deprived podocytes on collagen I, collagen IV, and ECM, but not on laminin or bare plastic. The cells expressed mRNA for EPO receptor. Conclusion: The physiological ECM proteins are more supportive of primary podocytic cultures compared with collagen I. The protective effects of EPO during serum deprivation are modulated by the cultivation surface.

  11. EFFECTS OF ATRAZINE AND AN ATRAZINE METABOLITE MIXTURE ON DIFFERENTIATED MAMMARY EPITHELIAL CELL MILK PROTEIN PRODUCTION IN CULTURE

    Science.gov (United States)

    Effects of Atrazine and an Atrazine Metabolite Mixture on Differentiated Mammary Epithelial Cell Milk Protein Production in CultureE.P. Hines, R. Barbee, M. Blanton, M.S. Pooler, and S.E. Fenton. US EPA, ORD/NHEERL, RTD, RTP, NC, 27711, USA.Previous studies have ...

  12. Mathematical analysis for internal filtration of convection-enhanced high-flux hemodialyzer.

    Science.gov (United States)

    Lee, Jung Chan; Lee, Kyungsoo; Kim, Hee Chan

    2012-10-01

    Structural modifications using a conventional hemodialyzer improved the internal filtration and clearance of middle molecular weight wastes by enhanced convection effect. In this study, we employed a mathematical model describing the internal filtration rate as well as the hemodynamic and hematologic parameters in highflux dialyzer to interpret the previous reported experimental results. Conventional high-flux hemodialysis and convection-enhanced high-flux hemodialysis were configured in the mathematical forms and integrated into the iterative numerical method to predict the internal filtration phenomena inside the dialyzers during dialysis. The distributions of blood pressure, dialysate pressure, oncotic pressure, blood flow rates, dialysate flow rates, local ultrafiltration, hematocrit, protein concentration and blood viscosity along the axial length of dialyzer were calculated in order to estimate the internal filtration volume. The results show that the filtration volumes by internal filtration is two times higher in a convection-enhanced high-flux hemodialyzer than in a conventional high-flux hemodialzer and explains the experimental result of improved clearance of middle molecular size waste in convection-enhanced high-flux hemodialyzer.

  13. Mycobacterium avium and purified protein derivative-specific cytotoxicity mediated by CD4+ lymphocytes from healthy HIV-seropositive and-seronegative individuals

    DEFF Research Database (Denmark)

    Ravn, P; Pedersen, B K

    1996-01-01

    with no history of previous or active mycobacterial infection. Antigen-specific killing of macrophages presenting mycobacterial antigens (purified protein derivative or M. avium culture filtrate) was conducted. The phenotype of the killer cells was determined by a fluorescence-activated cell sorter after antigen...

  14. Quantitation of soluble aggregates in recombinant monoclonal antibody cell culture by pH-gradient protein A chromatography.

    Science.gov (United States)

    Pan, Hai; Chen, Ken; Pulisic, Matt; Apostol, Izydor; Huang, Gang

    2009-05-15

    Monoclonal antibodies (mAbs) produced from mammalian cell culture may contain significant amounts of dimers and higher order aggregates. Quantitation of soluble aggregates in the cell culture is time-consuming and labor-intensive, usually involving a purification step to remove the impurities that interfere with the subsequent size exclusion chromatography (SEC) analysis. We have developed a novel pH-gradient protein A chromatography for rapid, non-size based separation of the aggregates in mAb cell culture samples. Our results demonstrate that this method has excellent correlation with SEC and can be applied to both human immunoglobulin gamma 1 (IgG1) and IgG2 antibodies. This approach can be useful in the quantitation of soluble aggregates in crude cell culture samples.

  15. Nutritional Composition and Protein Profile of Goat Yogurt PE with Double Culture between Streptococcus thermophilus and Lactobacilus species

    Directory of Open Access Journals (Sweden)

    Ismi Kurnia Budiarti

    2013-08-01

    Full Text Available Aim: The aims of this study are to characterize the nutrient compositions and protein profiles of Etawah breed (PE goat yogurt fermented by double cultures . Material and Methods: To accomplish this, we used goat and bovine milk in five treatment groups: (1 fresh milk bovine, (2 goat,(3 milk fermented by L. acidophilus and S. thermophilus (LA + ST, (4 L. bulgaricus and S. thermophilus (LB+ ST,and (5 a comersial mixture. PE goat milk was fermented using 2.5% starting bacterial concentrations at 45oC with a pH ranging from 4.5 to 6.6. Nutrient compositions were measured by proximate analysis.SDS PAGE was conducted using 15% separating and 3% stacking gels. To measure the density of protein bands, we used QuantityOne software. Results: Our results indicated that LA+ST and LB+ST treatments had higher levels of lipids than the control treatment. Conversely, both strain combinations had lower levels of proteins than the control. Organoleptic testing suggests that many attributes (e.g., colour, taste, smell, texture and viscosity differ significantly from the control. Protein profiles revealed that while the LB + ST and commercial cultures contained proteins with a molecular weight of 36 kDa, the LA + ST cultures did not appear to possess this protein.Based on the molecular weight, we suggest that this protein is in the alpha casein group. Conclusion: The protein composition of fermented goat and bovine milk is similiar, but the absence of band with molecular weight 36 kDa from goat milk, LB+ST and mix comercial. [Cukurova Med J 2013; 38(4.000: 681-686

  16. Process cost and facility considerations in the selection of primary cell culture clarification technology.

    Science.gov (United States)

    Felo, Michael; Christensen, Brandon; Higgins, John

    2013-01-01

    The bioreactor volume delineating the selection of primary clarification technology is not always easily defined. Development of a commercial scale process for the manufacture of therapeutic proteins requires scale-up from a few liters to thousands of liters. While the separation techniques used for protein purification are largely conserved across scales, the separation techniques for primary cell culture clarification vary with scale. Process models were developed to compare monoclonal antibody production costs using two cell culture clarification technologies. One process model was created for cell culture clarification by disc stack centrifugation with depth filtration. A second process model was created for clarification by multi-stage depth filtration. Analyses were performed to examine the influence of bioreactor volume, product titer, depth filter capacity, and facility utilization on overall operating costs. At bioreactor volumes cost savings compared to clarification using centrifugation. For bioreactor volumes >5,000 L, clarification using centrifugation followed by depth filtration offers significant cost savings. For bioreactor volumes of ∼ 2,000 L, clarification costs are similar between depth filtration and centrifugation. At this scale, factors including facility utilization, available capital, ease of process development, implementation timelines, and process performance characterization play an important role in clarification technology selection. In the case study presented, a multi-product facility selected multi-stage depth filtration for cell culture clarification at the 500 and 2,000 L scales of operation. Facility implementation timelines, process development activities, equipment commissioning and validation, scale-up effects, and process robustness are examined. © 2013 American Institute of Chemical Engineers.

  17. O-GlcNAcylation as a novel ammonia-induced posttranslational protein modification in cultured rat astrocytes.

    Science.gov (United States)

    Karababa, Ayşe; Görg, Boris; Schliess, Freimut; Häussinger, Dieter

    2014-12-01

    Hepatic encephalopathy (HE) is a clinical manifestation of a low grade cerebral edema with a mutual interrelationship between osmotic- and oxidative stress. This leads to RNA oxidation and posttranslational protein modifications such as protein tyrosine nitration with pathophysiological relevance. Here, we report on O-GlcNAcylation as another ammonia-induced posttranslational protein modification in cultured rat astrocytes. NH4Cl induced O-GlcNAcylation of distinct proteins (25-250 kDa) in astrocytes in a dose- and time-dependent manner. Exposure of astrocytes to NH4Cl (5 mmol/l) for 48 h and 72 h significantly increased protein O-GlcNAcylation by about 2-fold and 4-fold, respectively. NH4Cl at a concentration of 1 mmol/l was sufficient to double protein O-GlcNAcylation in astrocytes after 72 h as compared to untreated controls. Ammonia-induced protein O-GlcNAcylation was sensitive towards glutamine-synthetase inhibition by methionine sulfoximine (MSO), but was not induced by hypoosmolarity (205 mosmol/l) or CH3NH3Cl (5 mmol/l). Increased protein O-GlcNAcylation in NH4Cl (5 mmol/l, 48 h)-treated astrocytes was fully reversible within 24 h after withdrawal of NH4Cl from culture medium. Amongst the proteins which are O-GlcNAcylated in response to ammonia, GAPDH was identified. It is concluded that ammonia induces reversible protein O-GlcNAcylation in astrocytes that depends on glutamine synthesis but not on astrocyte swelling per se or ammonia-induced pH-changes. In view of the complex involvement of O-GlcNAcylation in cell regulation, such as energy metabolism, apoptosis and circadian rhythmicity and in pathologies, such as neurodegenerative diseases, O-GlcNAcylation might contribute to the pathophysiology of hepatic encephalopathy.

  18. Maize genome sequencing by methylation filtration.

    Science.gov (United States)

    Palmer, Lance E; Rabinowicz, Pablo D; O'Shaughnessy, Andrew L; Balija, Vivekanand S; Nascimento, Lidia U; Dike, Sujit; de la Bastide, Melissa; Martienssen, Robert A; McCombie, W Richard

    2003-12-19

    Gene enrichment strategies offer an alternative to sequencing large and repetitive genomes such as that of maize. We report the generation and analysis of nearly 100,000 undermethylated (or methylation filtration) maize sequences. Comparison with the rice genome reveals that methylation filtration results in a more comprehensive representation of maize genes than those that result from expressed sequence tags or transposon insertion sites sequences. About 7% of the repetitive DNA is unmethylated and thus selected in our libraries, but potentially active transposons and unmethylated organelle genomes can be identified. Reverse transcription polymerase chain reaction can be used to finish the maize transcriptome.

  19. The Perspective of Riverbank Filtration in China

    Science.gov (United States)

    Li, J.; Teng, Y.; Zhai, Y.; Zuo, R.

    2014-12-01

    Sustainable drinking water supply can affect the health of people, and the surrounding ecosystems. According to statistics of the monitoring program of drinking water sources in 309 at or above prefecture level of China in 2013, the major pollutants index were total phosphorus, ammonia and manganese in surface drinking water sources, respectively, iron, ammonia and manganese in groundwater drinking water sources, respectively. More than 150 drinking water emergency environmental accidents happened since 2006, 52 of these accidents led to the disruption of water supply in waterworks, and a population of over ten million were affected. It indicated that there is a potential risk for people's health by the use of river water directly and it is necessary to require alternative techniques such as riverbank filtration for improving the drinking water quality. Riverbank filtration is an inexpensive natural process, not only smoothing out normal pollutant concentration found in surface water but also significantly reducing the risk from such emergency events as chemical spill into the river. Riverbank filtration technique has been used in many countries more than 100 years, including China. In China, in 1950s, the bank infiltration technique was first applied in northeast of China. Extensive bank infiltration application was conducted in 1980s, and more than 300 drinking water sources utilities bank infiltration established mainly near the Songhua River Basin, the Yellow River Basin, Haihe River Basin. However, the comparative lack of application and researches on riverbank filtration have formed critical scientific data gap in China. As the performance of riverbank filtration technique depend on not only the design and setting such as well type, pumping rate, but also the local hydrogeology and environmental properties. We recommend more riverbank filtration project and studies to be conducted to collect related significant environmental geology data in China

  20. Salt disposition alternatives filtration at SRTC

    Energy Technology Data Exchange (ETDEWEB)

    Walker, B. W.; Hobbs, D.

    2000-01-27

    Several of the prospective salt disposition alternative technologies require a monosodium titanate (MST) contact to remove strontium and actinides from inorganic salt solution feedstock. This feedstock also contains sludge solids from waste removal operations and may contain defoamers added in the evaporator systems. Filtration is required to remove the sludge and MST solids before sending the salt solution for further processing. This report describes testing performed using the Parallel Theological Experimental Filter (PREF). The PREF contains two single tube Mott sintered metal crossflow filters. For this test one filter was isolated so that the maximum velocities could be achieved. Previous studies showed slurries of MST and sludge in the presence of sodium tetraphenylborate (NaTPB) were filterable since the NaTPB slurry formed a filter cake which aided in removing the smaller MST and sludge particles. Some of the salt disposition alternative technologies do not use NaTPB raising the question of how effective crossflow filtration is with a feed stream containing only sludge and MST. Variables investigated included axial velocity, transmembrane pressure, defoamer effects, and solids concentration (MST and sludge). Details of the tests are outlined in the technical report WSRC-RP-98-O0691. Key conclusions from this study are: (1) Severe fouling of the Mott sintered metal filter did not occur with any of the solutions filtered. (2) The highest fluxes, in the range of .46 to 1.02 gpm/f{sup 2}, were obtained when salt solution decanted from settled solids was fed to the filter. These fluxes would achieve 92 to 204 gpm filtrate production for the current ITP filters. The filtrate fluxes were close to the flux of 0.42 gpm/f{sup 2} reported for In Tank Precipitation Salt Solution by Morrisey. (3) For the range of solids loading studied, the filter flux ranged from .04 to .17 gpm/f{sup 2} which would result in a filtrate production rate of 9 to 31 gpm for the current HP

  1. Industrial investigations of the liquid steel filtration

    Directory of Open Access Journals (Sweden)

    K. Janiszewski

    2014-07-01

    Full Text Available Hitherto existing investigations concerning the ceramic filter use in the steel making processes have given good results. The obtained results of filtration have proved that this method may be used as an effective and cheap way of steel filtration from non-metallic inclusions. Placing filters in the tundish is the best location considering the limitation of the possibility of secondary pollution of steel. Yet, the results presented in this paper, of an experiment prepared and carried out in the industrial environment, are the only positive results obtained, which are connected with so much quantities of liquid steel processed with use of the multi-hole ceramic filters.

  2. Projective Dimension in Filtrated K-Theory

    DEFF Research Database (Denmark)

    Bentmann, Rasmus Moritz

    2013-01-01

    Under mild assumptions, we characterise modules with projective resolutions of length n∈N in the target category of filtrated K-theory over a finite topological space in terms of two conditions involving certain Tor -groups. We show that the filtrated K-theory of any separable C∗dash-algebra over...... any topological space with at most four points has projective dimension 2 or less. We observe that this implies a universal coefficient theorem for rational equivariant KK-theory over these spaces. As a contrasting example, we find a separable C∗dash-algebra in the bootstrap class over a certain five...

  3. Projective Dimension in Filtrated K-Theory

    DEFF Research Database (Denmark)

    Bentmann, Rasmus Moritz

    2013-01-01

    any topological space with at most four points has projective dimension 2 or less. We observe that this implies a universal coefficient theorem for rational equivariant KK-theory over these spaces. As a contrasting example, we find a separable C∗dash-algebra in the bootstrap class over a certain five......-point space, the filtrated K-theory of which has projective dimension 3. Finally, as an application of our investigations, we exhibit Cuntz-Krieger algebras which have projective dimension 2 in filtrated K-theory over their respective primitive spectrum....

  4. The potential of chitosan in enhancing peptide and protein absorption across the TR146 cell culture model-an in vitro model of the buccal epithelium

    DEFF Research Database (Denmark)

    Portero, Ana; Remuñán-López, Carmen; Nielsen, Hanne Mørck

    2002-01-01

    To investigate the potential of chitosan (CS) to enhance buccal peptide and protein absorption, the TR146 cell culture model, a model of the buccal epithelium, was used.......To investigate the potential of chitosan (CS) to enhance buccal peptide and protein absorption, the TR146 cell culture model, a model of the buccal epithelium, was used....

  5. Mechanisms of pressure filtration of liquid aluminum alloys

    Science.gov (United States)

    Cao, X.

    2006-12-01

    The Prefil Footprinter, a portable pressure filtration instrument, is usually used to detect the quality of liquid aluminum alloys. However, no investigations have ever been done to calculate the cake resistance to date. Based on the identification and classification of flow behavior using the first derivative method for filtrate mass vs filtration time curves, conventional filtration equations are successfully employed to understand the filtration behaviors. From the analyses of the variations of cake resistance with filtration time, the filtration mechanisms are discussed in detail over the different filtration stages. During the steady stage, either incompressible or compressible cake mode is the main mechanism. At the initial and terminal transient stages, however, deep-bed filtration, complete straining, and solidification clogging may appear. Solid inclusions in liquid metal have significant influence on the cake structures and properties. Some important issues related to the heterogeneity of filter media and test methodology are highlighted in this work.

  6. Transient expression and cellular localization of recombinant proteins in cultured insect cells

    Science.gov (United States)

    Heterologous protein expression systems are used for production of recombinant proteins, interpretation of cellular trafficking/localization, and for the determination of biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for ...

  7. Molecular and biochemical characterization of the NS1 protein of non-cultured influenza B virus strains circulating in Singapore

    KAUST Repository

    Jumat, Muhammad Raihan

    2016-08-04

    In this study we compared the NS1 protein of Influenza B/Lee/40 and several non-cultured Influenza B virus clinical strains detected in Singapore. In B/Lee/40 virus-infected cells and in cells expressing the recombinant B/Lee/40 NS1 protein a full-length 35 kDa NS1 protein and a 23 kDa NS1 protein species (p23) were detected. Mutational analysis of the NS1 gene indicated that p23 was generated by a novel cleavage event within the linker domain between an aspartic acid and proline at amino acid residues at positions 92 and 93 respectively (DP92–93), and that p23 contained the first 92 amino acids of the NS1 protein. Sequence analysis of the Singapore strains indicated the presence of either DP92–93 or NP92–93 in the NS1 protein, but protein expression analysis showed that p23 was only detected in NS1 proteins with DP92–93.. An additional adjacent proline residue at position 94 (P94) was present in some strains and correlated with increased p23 levels, suggesting that P94 has a synergistic effect on the cleavage of the NS1 protein. The first 145 amino acids of the NS1 protein are required for inhibition of ISG15-mediated ubiquitination, and our analysis showed that Influenza B viruses circulating in Singapore with DP92–93 expressed truncated NS1 proteins and may differ in their capacity to inhibit ISG15 activity. Thus, DP92–93 in the NS1 protein may confer a disadvantage to Influenza B viruses circulating in the human population and interestingly the low frequency of DP92–93detection in the NS1 protein since 2004 is consistent with this suggestion.

  8. Mixed culture of Kluyveromyces marxianus and Candida krusei for single-cell protein production and organic load removal from whey.

    Science.gov (United States)

    Yadav, J S S; Bezawada, J; Ajila, C M; Yan, S; Tyagi, R D; Surampalli, R Y

    2014-07-01

    The study was conducted to evaluate the potential of mixed culture of Kluyveromyces marxianus and Candida krusei to enhance COD removal efficiency, minimize contamination at extreme conditions (high temperature 40°C and low pH 3.5) during batch and continuous aerobic fermentation and to obtain improved quality single-cell protein (SCP) using whey as substrate. The batch fermentation of mono-culture and mixed culture result showed that the mixed culture resulted in 8.8% higher COD removal efficacy with 19% higher biomass yield and 33% increased productivity. The maximum COD removal 80.2% (including residual protein) was obtained at 24h HRT with biomass productivity of 0.17 g/L/h; however, maximum biomass productivity of 0.38 g/L/h and 34% COD removal were obtained at 6h HRT. The results showed that the mixed culture of acid resistance and thermo-tolerant yeasts was a potential way to produce SCP (animal feed) and simultaneous COD removal under extreme operating conditions.

  9. Effects of bone morphogenetic protein-2 on bone cells in primary culture: immunohistochemical and electronmicroscopical studies

    Energy Technology Data Exchange (ETDEWEB)

    Schmitz, I.; Prochnow, N.; Mueller, K.M. [Berufsgenossenschaftliche Kliniken Bergmannsheil, Bochum (Germany). Inst. fuer Pathologie; Wiemann, M.; Schirrmacher, K.; Bingmann, D. [Essen Univ. (Germany). Inst. fuer Physiologie; Sebald, W. [Wuerzburg Univ. (Germany). Inst. fuer Physiologische Chemie II

    2001-02-01

    Bone morphogenetic protein 2 (BMP-2), among other morphogenetic effects on non osseous tissues, promotes bone formation in vivo. Therefore, BMP-2 may accelerate the integration of osseous implants. Although the effects of BMPs on cell proliferation have been studied extensively in vivo or in cell lines, little is published about effects on bone cells in primary cultures, especially on cell differentiation. As such information is a prerequisite to understand and to control effects of BMPs on cells at the surface of implant materials, the present experiments aimed to describe effects of BMP-2 on primary cultures derived from calvarial fragments of neonatal rats. The cells were stimulated with 50 nM BMP-2 added to the nutrient medium for 3 or 6 days. Light- and electronmicroscopical studies showed that cells in the sprouting zones were larger and more often spindle shaped. Stimulated cells had more nucleoli than control cells and the endoplasmic reticulum was widened. They retained properties of typical bone cells: An immunhistochemical analysis showed that stimulated cells increased the activity of alkaline phosphatase, they secreted collagen type I and to a minor extent collagen type III. In BMP-2 treated cells the pattern of cells stained for actin, desmin and vimentin hardly changed whereas extracellular fibronectin appeared to be less cross-linked in BMP-2 treated cultures. The distribution and labeling strength of osteocalcin, a specific marker protein of bone cells did not change markedly. After exposure to BMP-2 cells tended to detach from the cover slips. Electron microscopy showed a reduced number of cell processes possibly facilitating the detachment and/or mobility. Stimulated cells contained an increased number of lamellar bodies which may reflect an increased synthesis and/or membrane turnover. Staining of non-osseous cells with anti-CD68-or anti-myeloid antibodies revealed that the small percentage of these cells regularly occurring in primary cultures

  10. Estimation of single-kidney glomerular filtration rate without exogenous contrast agent.

    Science.gov (United States)

    He, Xiang; Aghayev, Ayaz; Gumus, Serter; Ty Bae, K

    2014-01-01

    Measurement of single-kidney filtration fraction and glomerular filtration rate (GFR) without exogenous contrast is clinically important to assess renal function and pathophysiology, especially for patients with comprised renal function. The objective of this study is to develop a novel MR-based tool for noninvasive quantification of renal function using conventional MR arterial spin labeling water as endogenous tracer. The regional differentiation of the arterial spin labeling water between the glomerular capsular space and the renal parenchyma was characterized and measured according to their MR relaxation properties (T1ρ or T2 ), and applied to the estimation of filtration fraction and single-kidney GFR. The proposed approach was tested to quantify GFR in healthy volunteers at baseline and after a protein-loading challenge. Biexponential decay of the cortical arterial spin labeling water MR signal was observed. The major component decays the same as parenchyma water; the minor component decays much slower as expected from glomerular ultra-filtrates. The mean single-kidney GFR was estimated to be 49 ± 9 mL/min at baseline and increased by 28% after a protein-loading challenge. We developed an arterial spin labeling-based MR imaging method that allows us to estimate renal filtration fraction and singe-kidney GFR without use of exogenous contrast. Copyright © 2013 Wiley Periodicals, Inc.

  11. Membrane fouling and dialysate flow pattern in an internal filtration-enhancing dialyzer.

    Science.gov (United States)

    Yamamoto, Ken-ichiro; Hiwatari, Michihito; Kohori, Fukashi; Sakai, Kiyotaka; Fukuda, Makoto; Hiyoshi, Tatsuo

    2005-01-01

    For efficient removal of large molecular weight solutes by dialysis, several types of internal filtration-enhancing dialyzers (IFEDs) are commercially available. However, in a pressure-driven membrane separation process (i.e., filtration), membrane fouling caused by adhesion of plasma proteins is a severe problem. The objective of the present study is to investigate the effects of internal filtration on membrane fouling based on the membrane's pure-water permeability, diffusive permeability, and sieving coefficient. Hemodialysis experiments were performed with two different dialyzers, IFEDs and non-IFEDs. Local membrane fouling in each dialyzer was evaluated by measuring the pure-water permeability, the diffusive permeability, and the sieving coefficient of native membranes and membranes treated with bovine blood. The effects of packing ratio on dialysate flow pattern were also evaluated by measuring the time required for an ion tracer to reach electrodes placed in the dialyzers. In the IFED, membrane fouling caused by protein adhesion is increased because of enhanced internal filtration only at the early stage of dialysis, and this fouling tends to occur only near the dialysate outlet port. However, enhanced internal filtration has little effect on measured membrane transfer parameters.

  12. Protein improvement in Gari by the use of pure cultures of microorganisms involved in the natural fermentation process.

    Science.gov (United States)

    Ahaotu, I; Ogueke, C C; Owuamanam, C I; Ahaotu, N N; Nwosu, J N

    2011-10-15

    The ability of microorganisms involved in cassava mash fermentation to produce and improve protein value by these microorganisms during fermentation was studied. Standard microbiological procedures were used to isolate, identify and determine the numbers of the organisms. Alcaligenes faecalis, Lactobacillus plantarum, Bacillus subtilis, Leuconostoc cremoris, Aspergillus niger, A. tamari, Geotrichum candidum and Penicillium expansum were isolated and identified from cassava waste water while standard analytical methods were used to determine the ability of the isolates to produce linamarase and the proximate composition, pH and titrable acidity of the fermenting mash. The linamarase activity of the isolates ranged from 0.0416 to 0.2618 micromol mL(-1) nmol(-1). Bacillus subtilis, A. niger, A. tamari and P. expansum did not express any activity for the enzyme. Protein content of mash fermented with mixed fungal culture had the highest protein value (15.4 mg/g/dry matter) while the raw cassava had the least value (2.37 mg/g/dry matter). The naturally fermented sample had the least value for the fermented samples (3.2 mg/g/dry matter). Carbohydrate and fat contents of naturally fermented sample were higher than values obtained from the other fermented samples. Microbial numbers of the sample fermented with mixed bacterial culture was highest and got to their peak at 48 h (57 x 10(8) cfu g(-1)). pH decreased with increase in fermentation time with the mash fermented by the mixed culture of fungi having the lowest pH of 4.05 at the end of fermentation. Titrable acidity increased with increase in fermentation time with the highest value of 1.32% at 96 h of fermentation produced by the mixed culture of fungi. Thus fermentation with the pure cultures significantly increased the protein content of mash.

  13. Coating extracellular matrix proteins on a (3-aminopropyl)triethoxysilane-treated glass substrate for improved cell culture.

    Science.gov (United States)

    Masuda, Hiro-taka; Ishihara, Seiichiro; Harada, Ichiro; Mizutani, Takeomi; Ishikawa, Masayori; Kawabata, Kazushige; Haga, Hisashi

    2014-01-01

    We demonstrate that a (3-aminopropyl)triethoxysilane-treated glass surface is superior to an untreated glass surface for coating with extracellular matrix (ECM) proteins when used as a cell culture substrate to observe cell physiology and behavior. We found that MDCK cells cultured on untreated glass coated with ECM removed the coated ECM protein and secreted different ECM proteins. In contrast, the cells did not remove the coated ECM protein when seeded on (3-aminopropyl)triethoxysilane-treated (i.e., silanized) glass coated with ECM. Furthermore, the morphology and motility of cells grown on silanized glass differed from those grown on non-treated glass, even when both types of glass were initially coated with laminin. We also found that cells on silanized glass coated with laminin had higher motility than those on silanized glass coated with fibronectin. Based on our results, we suggest that silanized glass is a more suitable cell culture substrate than conventional non-treated glass when coated by ECM for observations of ECM effects on cell physiology.

  14. Comparison of gel filtration and ammonium sulphate precipitation in the purification of diphtheria toxin and toxoid.

    Science.gov (United States)

    Møyner, K; Christiansen, G

    1984-02-01

    Crude diphtheria toxin and toxoid were subjected to purification by gel filtration and stepwise ammonium sulphate precipitation. The various fractions obtained by the purification procedures were studied by immunological methods. A high molecular weight fraction of glycoprotein nature was present in all of the crude preparations studied. The fraction was antigenically non-identical with the real toxin or toxoid and did not have its origin in the culture medium. It showed a long flocculation time when tested against equine diphtheria toxoid antiserum. The fraction could be removed from the crude preparations by gel filtration or by precipitation with 21% (w/v) ammonium sulphate. When comparing toxoids purified by each of these methods, the method of gel filtration resulted in a somewhat higher degree of purity, suggesting that this method would be more suitable than the AS precipitation method for the purification of diphtheria toxoid.

  15. Aerosol filtration - performance of filter media; Filtration des aerosols - performances des medias filtrants

    Energy Technology Data Exchange (ETDEWEB)

    Bemer, D.; Regnier, R. [Institut National de Recherche et de Securite (INRS), Dept. Ingenierie des Procedes, 75 - Paris (France); Calle, S.; Thomas, D.; Simon, X.; Appert-Collin, J.Ch. [Centre National de la Recherche Scientifique (CNRS), Lab. des Sciences du Genie Chimique de Nancy, 54 - Vandoeuvre-les-Nancy (France)

    2006-03-15

    Set up in 2000, the Laboratoire de Filtration des Aerosols in Nancy (LFA), associating research teams from INRS (Institut National de Recherche et de Securite and LSGC (Laboratoire des Sciences du Genie Chimique - CNRS), has been commissioned to provide expertise in the aerosol filtration field to both CRAM (Caisse Regionale de l'Assurance Maladie) prevention specialists and industrialists. Following a theoretical review of fibre medium filtration, this paper summarises the different actions undertaken by the LFA: filter performance during clogging with liquid and solid aerosols, modelling, pneumatic unclogging of baghouse dust collectors, influence of micro-leaks, etc. (authors)

  16. Prevention of febrile nonhemolytic transfusion reaction with leucocyte filtrated concentrates

    Institute of Scientific and Technical Information of China (English)

    ZHAO Shu-ming; XIANG Guo-chun; ZHANG Jia-si; CHENG Xiao-ling; LI Ru-qing

    2002-01-01

    Objective: To assess the clinical efficiency of the transfusion of leucocyte filtrated RBC concentrates to prevent febrile nonhemolytic transfusion reactions (FNHTRs). Methods: One hundred patients with liver cirrhosis, gastric ulcer or cancer were subjected to receive RBC concentrates after leucocyte filtration.Another 50 patients with similar diseases were selected to receive non-filtrated RBC concentrates. The incidence of FNHTRs in all patients was investigated. Results: There was no FNHTR in 100 transfusions with leucocyte filtrated RBC concentrates, while FNHTRs occurred in 8 of 50 patients with non-filtrated RBC concentrates, with the incidence of 160%. Conclusion: FNHTRs to RBC transfusion can be prevented with leucocyte filtration.

  17. Plasma discharge self-cleaning filtration system

    Science.gov (United States)

    Cho, Young I.; Fridman, Alexander; Gutsol, Alexander F.; Yang, Yong

    2014-07-22

    The present invention is directed to a novel method for cleaning a filter surface using a plasma discharge self-cleaning filtration system. The method involves utilizing plasma discharges to induce short electric pulses of nanoseconds duration at high voltages. These electrical pulses generate strong Shockwaves that disintegrate and dislodge particulate matter located on the surface of the filter.

  18. Filtration of engineered nanoparticles using porous membranes

    NARCIS (Netherlands)

    Trzaskus, Krzystof

    2016-01-01

    The research presented in this thesis aims at providing a better understanding of the fundamental aspects responsible for nanoparticle removal and fouling development during filtration of engineered nanoparticles. The emphasis is put on the role of interparticle interactions in the feed solution,

  19. Organic micropollutant removal during river bank filtration

    NARCIS (Netherlands)

    Bertelkamp, C.

    2015-01-01

    This study investigated the factors influencing the main removal mechanisms (adsorption and biodegradation) for organic micropollutant (OMP) removal during river bank filtration (RBF) and the possibility of developing a predictive model of this process for OMP removal during RBF. Chapter 2 analysed

  20. Advances in hot gas filtration technology

    Energy Technology Data Exchange (ETDEWEB)

    Weber, C.

    The past decade has seen the introduction of new filter media specifically designed for 'hot-gas' filtration. These media are available as woven or knitted fabrics and as non-wovens, i.e. needled felts. Needlefelted fabrics have proven so highly successful in the dedusting of hot gases that they are widely used nowadays in this new and necessary technology. Hot-gas filtration offers advantages in, for example, the saving or recycling of energy, the elimination of the cooling process, and the short-circuiting of process steps. This paper gives a survey of the types of textile fibres available for hot-gas filtration from the more recently developed organic fibres to refractory fibres. It describes, compares and contrasts their salient properties and lists the uses to which they may be put. It concentrates on such fibres which are generally referred to as 'high performance materials', since they are expected to provide satisfactory performance under extreme conditions of temperature, chemical environment and mechanical stress. It touches on filtration theory governing the collection mechanism. 9 refs., 7 figs., 3 tabs.

  1. Using Digital Filtration for Hurst Parameter Estimation

    Directory of Open Access Journals (Sweden)

    J. Prochaska

    2009-06-01

    Full Text Available We present a new method to estimate the Hurst parameter. The method exploits the form of the autocorrelation function for second-order self-similar processes and is based on one-pass digital filtration. We compare the performance and properties of the new method with that of the most common methods.

  2. Effect of filtration rate on coal-sand dual-media filter performances for microalgae removal.

    Science.gov (United States)

    Sabiri, Nour-Eddine; Monnier, Elodie; Raimbault, Virginie; Massé, Anthony; Séchet, Véronique; Jaouen, Pascal

    2017-02-01

    This study tested the efficiency of granular filtration using a bilayer sand filter for microalgae removal from culture dilutions ranging from 10,000 to 17,000 cells/mL. The objective is to evaluate the removal capacity of the filter without chemical coagulation. Two filter media, sand and anthracite, with mean grain sizes of 0.395 and 1.2 mm, respectively, were used in constant-flow-rate experiments (down-flow mode) with suspensions containing Heterocapsa triquetra microalga. The conventional rapid filtration which usually operates at a constant rate of approximately 5 m(3)/m(2) h is compared to high-rate filtration. Two filtration velocities (5 and 10 m/h) were investigated with bed depth of 1100 mm. Average microalgal cell removal rates were 90% at 5 m/h and 68% at 10 m/h. Turbidity removal was more than 71% at 5 m/h but just 57% at 10 m/h. Head losses did not increase significantly, and values measured at process end were 32 mbar at 5 m/h and 78 mbar at 10 m/h. Retention probabilities were calculated from experimental data. A theoretical model was used to evaluate the contributions of the different drivers of microalgae removal. Hypotheses are developed on the understanding of change in the mechanisms of retention as a function of filtration velocity.

  3. Differential induction of heme oxygenase and other stress proteins in cultured hippocampal astrocytes and neurons by inorganic lead.

    Science.gov (United States)

    Cabell, Leigh; Ferguson, Charles; Luginbill, Deana; Kern, Marcey; Weingart, Adam; Audesirk, Gerald

    2004-07-01

    We examined the effects of exposure to inorganic lead (Pb2+) on the induction of stress proteins in cultured hippocampal neurons and astrocytes, with particular emphasis on the induction of heme oxygenase-1 (HO-1). In radiolabeled neuronal cultures, Pb2+ exposure had no significant effect on the synthesis of any protein at any concentration (up to 250 microM) or duration of exposure (up to 4 days). In radiolabeled astrocyte cultures, however, Pb2+ exposure (100 nM to 100 microM; 1-4 days) increased synthesis of proteins with approximate molecular weights of 23, 32, 45, 57, 72, and 90 kDa. Immunoblot experiments showed that Pb2+ exposure (100 nM to 10 microM, 1-14 days) induces HO-1 synthesis in astrocytes, but not in neurons; this is probably the 32-kDa protein. The other heme oxygenase isoform, HO-2, is present in both neurons and astrocytes, but is not inducible by Pb2+ at concentrations up to 100 microM. HO-1 can be induced by a variety of stimuli. We found that HO-1 induction in astrocytes is increased by combined exposure to Pb2+ and many other stresses, including heat, nitric oxide, H2O2, and superoxide. One of the stimuli that may induce HO-1 is oxidative stress. Lead exposure causes oxidative stress in many cell types, including astrocytes. Induction of HO-1 by Pb2+ is reduced by the hydroxyl radical scavengers dimethylthiourea (DMTU) and mannitol, but not by inhibitors of calmodulin, calmodulin-dependent protein kinases, protein kinase C, or extracellular signal-regulated kinases (ERK). Therefore, we conclude that oxidative stress is an important mechanism by which Pb2+ induces HO-1 synthesis in astrocytes.

  4. The POZ/BTB protein NAC1 interacts with two different histone deacetylases in neuronal-like cultures.

    Science.gov (United States)

    Korutla, L; Wang, P J; Mackler, S A

    2005-08-01

    NAC1 is a cocaine-regulated POZ/BTB (Pox virus and Zinc finger/Bric-a-brac Tramtrack Broad complex) protein. NAC1 is increased by cocaine selectively in the nucleus accumbens, a CNS region important for drug addiction. NAC1's role in the cell, however, is not known. Each of the two NAC1 isoforms, sNAC1 (short NAC1) and lNAC1 (long NAC1), may serve as corepressors for other POZ/BTB proteins. This study investigated whether sNAC1 and lNAC1 demonstrated protein-protein interactions with other corepressors. Histone deacetylase (HDAC) inhibition reversed sNAC1 and lNAC1 repression of Gal4 luciferase, but only in neuronal-like cultures. Because these inhibitors do not distinguish among histone deacetylases, two histone deacetylases were selected for further study. HDAC 3 and 4 both demonstrated protein-protein interactions with sNAC1 and lNAC1. This was shown using coimmunoprecipitations, glutathione-S-transferase (GST) pulldowns and mammalian two-hybrids. Importantly, either the POZ domain or NAC1 without the POZ domain can bind these two HDACs. Other corepressors, specifically NCoR (nuclear receptor corepressor), SMRT (silencing mediator for retinoid and thyroid hormone receptor) and mSin3a, do not exhibit protein-protein interactions with sNAC1 and lNAC1. None showed protein-protein interactions in GST pulldowns or mammalian two-hybrids. Taken together, the results of these experiments indicate sNAC1 and lNAC1 recruit histone deacetylases for transcriptional repression, further enhancing POZ/BTB protein mediated repression.

  5. Double filtration plasmapheresis in a dog with multiple myeloma and hyperviscosity syndrome

    Directory of Open Access Journals (Sweden)

    I. Lippi

    2015-08-01

    Full Text Available A 12 year old, 38 kg, mix-breed, intact male dog presented with a 20 day history of clinical signs consistent with hyperviscosity syndrome secondary to multiple myeloma. The dog received three double filtration plasmapheresis treatments on day 0, 7 and 22 after presentation. A significant (p<0.05 reduction in serum total protein, alpha-2 and gamma globulins was found following each treatment. These reductions were accompanied by a complete resolution, although temporary, of the clinical signs of hyperviscosity syndrome. The present study reported for the first time the use of double filtration plasmapheresis to reduce clinical signs of hyperviscosity syndrome in a dog with multiple myeloma.

  6. The 24-kDa protein from Fusarium oxysporum f.sp. erythroxyli: occurrence in related fungi and the effect of growth medium on its production.

    Science.gov (United States)

    Bailey, B A; Jennings, J C; Anderson, J D

    1997-01-01

    A 24-kDa protein that elicits ethylene production and necrosis in leaves of dicotyledonous plants was previously purified from culture filtrates of Fusarium oxysporum Schlechtend:Fr. f.sp. erythroxyli. Antisera to the denatured 24-kDa protein detected 2.5 ng of the 24-kDa protein on Western blots at 100000-fold dilutions. The antisera cross-reacted with a 24-kDa protein on Western blots of culture filtrates from three other F. oxysporum formae speciales. Of seven Fusarium species, only F. oxysporum, F. acuminatum Ellis and Kellerm., and F. avenaceum (Fr.:Fr.) Sacc. isolates produced an antigenically related 24-kDa protein. Although there were differences in the profiles of proteins extracted from stems of coca (Erythroxylum coca var. coca L. Lam.) infected with F. oxysporum f.sp. erythroxyli compared with uninfected stems, antisera to the 24-kDa protein did not cross-react with any proteins from the infected coca stems. For the fungal isolates studied, the best medium tested for production of the 24-kDa protein contained 1% sucrose and 1% asparagine. Biological activity of the F. oxysporum culture filtrates on sweet basil leaves was consistently correlated with the presence of the 24-kDa protein. Production of the 24-kDa protein was limited in cultures containing pectin or cellulose as the primary carbon source, or in cultures lacking sucrose or casamino acids. Water-soluble extracts from coca stems inhibited production of the 24-kDa protein, whereas cellulose and pectin did not. Components produced by the plant may limit production of the 24-kDa protein in infected plant tissue and thereby limit the response of the plant to the fungus. These results suggest the 24-kDa protein does not function in the symptomatic phase of the F. oxysporum f.sp. erythroxylicoca disease interaction.

  7. OPTIMIZATION OF THE PROCESS OF DRYING THE FILTRATE DISTILLERY DREGS

    Directory of Open Access Journals (Sweden)

    A. A. Shevtsov

    2013-01-01

    Full Text Available The interactions of various factors affecting the process of drying the filtrate distillery dregs are investigated. Rational conditions for the process of drying the filtrate distillery dregs in a spray dryer are obtained.

  8. Growth rate analysis and protein identification of Kappaphycus alvarezii (Rhodophyta, Gigartinales under pH induced stress culture

    Directory of Open Access Journals (Sweden)

    Mian Zi Tee

    2015-11-01

    Full Text Available Environmental pH is one of the factors contributing to abiotic stress which in turn influences the growth and development of macroalgae. This study was conducted in order to assess the growth and physiological changes in Kappaphycus alvarezii under different pH conditions: pHs 6, ∼8.4 (control and 9. K. alvarezii explants exhibited a difference in the daily growth rate (DGR among the different pH treatments (p ≤ 0.05. The highest DGR was observed in control culture with pH ∼8.4 followed by alkaline (pH 9 and acidic (pH 6 induced stress cultures. Protein expression profile was generated from different pH induced K. alvarezii cultures using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE followed by protein identification and analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS and Mascot software. Ribulose bisphosphate carboxylase (Rubisco large chain was identified to be up-regulated under acidic (pH 6 condition during the second and fourth week of culture. The findings indicated that Rubisco can be employed as a biomarker for pH induced abiotic stress. Further study on the association between the expression levels of Rubisco large chain and their underlying mechanisms under pH stress conditions is recommended.

  9. Internal filtration, filtration fraction, and blood flow resistance in high- and low-flux dialyzers.

    Science.gov (United States)

    Schneditz, Daniel; Zierler, Edda; Vanholder, Raymond; Eloot, Sunny

    2014-01-01

    It was the aim to examine the fluid flow in blood and dialysate compartments of highly permeable hollow fiber dialyzers where internal filtration contributes to solute removal but where excessive filtration bears a risk of cell activation and damage. Flow characteristics of high- (HF) and low-flux (LF) dialyzers were studied in lab-bench experiments using whole bovine blood. Measurements obtained under different operating conditions and under zero net ultrafiltration were compared to theoretical calculations obtained from a mathematical model. Experimental resistances in the blood compartment were within ±2% of those calculated from the model when dialysate was used as a test fluid. With whole blood, the experimental resistances in the blood compartment were only 81.8 ± 2.8% and 83.7 ± 4.3% of those calculated for the LF and HF dialyzer, respectively. Surprisingly, measured blood flow resistance slightly but significantly decreased with increasing flow rate (p filtration fraction, while overall internal filtration increased. The increase in internal filtration when increasing blood flow is associated with a beneficial reduction in internal filtration fraction. Concerns of increased hemoconcentration when increasing blood flow therefore appear to be unwarranted.

  10. Single Cell Protein Production by Saccharomyces cerevisiae Using an Optimized Culture Medium Composition in a Batch Submerged Bioprocess.

    Science.gov (United States)

    Hezarjaribi, Mehrnoosh; Ardestani, Fatemeh; Ghorbani, Hamid Reza

    2016-08-01

    Saccharomyces cerevisiae PTCC5269 growth was evaluated to specify an optimum culture medium to reach the highest protein production. Experiment design was conducted using a fraction of the full factorial methodology, and signal to noise ratio was used for results analysis. Maximum cell of 8.84 log (CFU/mL) was resulted using optimized culture composed of 0.3, 0.15, 1, and 50 g L(-1) of ammonium sulfate, iron sulfate, glycine, and glucose, respectively at 300 rpm and 35 °C. Glycine concentration (39.32 % contribution) and glucose concentration (36.15 % contribution) were determined as the most effective factors on the biomass production, while Saccharomyces cerevisiae growth had showed the least dependence on ammonium sulfate (5.2 % contribution) and iron sulfate (19.28 % contribution). The most interaction was diagnosed between ammonium sulfate and iron sulfate concentrations with interaction severity index of 50.71 %, while the less one recorded for glycine and glucose concentration was equal to 8.12 %. An acceptable consistency of 84.26 % was obtained between optimum theoretical cell numbers determined by software of 8.91 log (CFU/mL), and experimentally measured one at optimal condition confirms the suitability of the applied method. High protein content of 44.6 % using optimum culture suggests that Saccharomyces cerevisiae is a good commercial case for single cell protein production.

  11. Technology Review of Modern Gas Turbine Inlet Filtration Systems

    OpenAIRE

    2012-01-01

    An inlet air filtration system is essential for the successful operation of a gas turbine. The filtration system protects the gas turbine from harmful debris in the ambient air, which can lead to issues such as FOD, erosion, fouling, and corrosion. These issues if not addressed will result in a shorter operational life and reduced performance of the gas turbine. Modern day filtration systems are comprised of multiple filtration stages. Each stage is selected based on the local operating envir...

  12. Developing a scalable model of recombinant protein yield from Pichia pastoris: the influence of culture conditions, biomass and induction regime

    Directory of Open Access Journals (Sweden)

    Wilks Martin DB

    2009-07-01

    Full Text Available Abstract Background The optimisation and scale-up of process conditions leading to high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences. Typical experiments rely on varying selected parameters through repeated rounds of trial-and-error optimisation. To rationalise this, several groups have recently adopted the 'design of experiments' (DoE approach frequently used in industry. Studies have focused on parameters such as medium composition, nutrient feed rates and induction of expression in shake flasks or bioreactors, as well as oxygen transfer rates in micro-well plates. In this study we wanted to generate a predictive model that described small-scale screens and to test its scalability to bioreactors. Results Here we demonstrate how the use of a DoE approach in a multi-well mini-bioreactor permitted the rapid establishment of high yielding production phase conditions that could be transferred to a 7 L bioreactor. Using green fluorescent protein secreted from Pichia pastoris, we derived a predictive model of protein yield as a function of the three most commonly-varied process parameters: temperature, pH and the percentage of dissolved oxygen in the culture medium. Importantly, when yield was normalised to culture volume and density, the model was scalable from mL to L working volumes. By increasing pre-induction biomass accumulation, model-predicted yields were further improved. Yield improvement was most significant, however, on varying the fed-batch induction regime to minimise methanol accumulation so that the productivity of the culture increased throughout the whole induction period. These findings suggest the importance of matching the rate of protein production with the host metabolism. Conclusion We demonstrate how a rational, stepwise approach to recombinant protein production screens can reduce process development time.

  13. Stromal cells from human long-term marrow cultures, but not cultured marrow fibroblasts, phagocytose horse serum constituents: studies with a monoclonal antibody that reacts with a species-specific epitope common to multiple horse serum proteins.

    Science.gov (United States)

    Charbord, P; Tippens, D; Wight, T S; Gown, A M; Singer, J W

    1987-01-01

    This report describes an IgG1 mouse monoclonal antibody derived after immunization of mice with washed stromal cells from human, long-term bone marrow cultures. The antigen recognized by the antibody (BMS-1) is a carbohydrate-containing prosthetic group that is common to and specific for multiple horse serum proteins. These proteins are avidly ingested by stromal cells and concentrated in endocytic vesicles. Cultured smooth muscle cells took up the horse proteins in a similar manner to marrow stromal cells while cultured marrow fibroblasts, endothelial cells, and hepatoma cells did not. These data indicate that marrow stromal cells specifically accumulate horse serum proteins which might partially explain the horse serum requirement for long-term marrow culture maintenance. The data also suggest further similarities between marrow stromal and smooth muscle cells and additional differences between marrow fibroblasts and marrow stromal cells.

  14. 40 CFR 141.171 - Criteria for avoiding filtration.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 22 2010-07-01 2010-07-01 false Criteria for avoiding filtration. 141... PROGRAMS (CONTINUED) NATIONAL PRIMARY DRINKING WATER REGULATIONS Enhanced Filtration and Disinfection-Systems Serving 10,000 or More People § 141.171 Criteria for avoiding filtration. In addition to...

  15. 40 CFR 141.174 - Filtration sampling requirements.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 22 2010-07-01 2010-07-01 false Filtration sampling requirements. 141... PROGRAMS (CONTINUED) NATIONAL PRIMARY DRINKING WATER REGULATIONS Enhanced Filtration and Disinfection-Systems Serving 10,000 or More People § 141.174 Filtration sampling requirements. (a)...

  16. 40 CFR 141.71 - Criteria for avoiding filtration.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 22 2010-07-01 2010-07-01 false Criteria for avoiding filtration. 141... PROGRAMS (CONTINUED) NATIONAL PRIMARY DRINKING WATER REGULATIONS Filtration and Disinfection § 141.71 Criteria for avoiding filtration. A public water system that uses a surface water source must meet all...

  17. Purification of hemoglobin by tangential flow filtration with diafiltration.

    Science.gov (United States)

    Elmer, Jacob; Harris, David R; Sun, Guoyong; Palmer, Andre F

    2009-01-01

    A recent study by Palmer, Sun, and Harris (Biotechnol. Prog., 25:189-199, 2009) demonstrated that tangential flow filtration (TFF) can be used to produce HPLC-grade bovine and human hemoglobin (Hb). In this current study, we assessed the quality of bovine Hb (bHb) purified by introducing a 10 L batch-mode diafiltration step to the previously mentioned TFF Hb purification process. The bHb was purified from bovine red blood cells (RBCs) by filtering clarified RBC lysate through 50 nm (stage I) and 500 kDa (stage II) hollow fiber (HF) membranes. The filtrate was then passed through a 100 kDa (stage III) HF membrane with or without an additional 10 L diafiltration step to potentially remove additional small molecular weight impurities. Protein assays, SDS-PAGE, and LC-MS of the purified bHb (stage III retentate) reveal that addition of a diafiltration step has no effect on bHb purity or yield; however, it does increase the methemoglobin level and oxygen affinity of purified bHb. Therefore, we conclude that no additional benefit is gained from diafiltration at stage III and a three stage TFF process is sufficient to produce HPLC-grade bHb.

  18. Inducing effects of macrophage stimulating protein on the expansion of early hematopoietic progenitor cells in liquid culture

    Institute of Scientific and Technical Information of China (English)

    MA Li-xia; HUANG Yan-hong; CHENG La-mei; LEI Jun; WANG Qi-ru

    2007-01-01

    Background Macrophage stimulating protein (MSP) is produced by human bone marrow endothelial cells. In this study,we sought to observe its effects on inducing the expansion of early hematopoietic progenitor cells which were cultured in a liquid culture system in the presence of the combination of stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), granulocyte macrophage-colony stimulating factor (GM-CSF), erythropoietin (EPO) (Cys) and MSP or of Cys and bone marrow endothelial cell conditioned medium (EC-CM).Methods Human bone marrow CD34+ cells were separated and cultured in a liquid culture system for 6 days.Granulocyte-macrophage colony forming unit (CFU-GM) and colony forming unit-granulocyte, erythrocyte, macrophage,megakaryocyte (CFU-GEMM) were employed to assay the effects of different treatment on the proliferation of hematopoeitic stem/progenitor cells. The nitroblue tetrazolium (NBT) reductive test and hoechest 33258 staining were employed to reflect the differentiation and apoptosis of the cells respectively.Results MSP inhibited the proliferation of CFU-GM and CFU-GEMM in semi-solid culture and the inhibitory effect on CFU-GEMM was stronger than on CFU-GM. MSP inhibited the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators. Bone marrow (BM) CFU-GEMM was 2.3-fold or 1.7-fold increase or significantly decreased in either Cys+EC-CM, Cys+MSP or Cys compared with 0 hour control in liquid culture system after 6 days.Conclusion MSP, a hematopoietic inhibitor, inhibits the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators and makes the early hematopoietic progenitor cells expand in a liquid culture system.

  19. A Proteomic Study of Clavibacter Michiganensis Subsp. Michiganensis Culture Supernatants

    Directory of Open Access Journals (Sweden)

    Eva Hiery

    2015-11-01

    Full Text Available Clavibacter michiganensis, subsp. michiganensis is a Gram-positive plant pathogen infecting tomato (Solanum lycopersicum. Despite a considerable economic importance due to significant losses of infected plants and fruits, knowledge about virulence factors of C. michiganensis subsp. michiganensis and host-pathogen interactions on a molecular level are rather limited. In the study presented here, the proteome of culture supernatants from C. michiganensis subsp. michiganensis NCPPB382 was analyzed. In total, 1872 proteins were identified in M9 and 1766 proteins in xylem mimicking medium. Filtration of supernatants before protein precipitation reduced these to 1276 proteins in M9 and 976 proteins in the xylem mimicking medium culture filtrate. The results obtained indicate that C. michiganensis subsp. michiganensis reacts to a sucrose- and glucose-depleted medium similar to the xylem sap by utilizing amino acids and host cell polymers as well as their degradation products, mainly peptides, amino acids and various C5 and C6 sugars. Interestingly, the bacterium expresses the previously described virulence factors Pat-1 and CelA not exclusively after host cell contact in planta but already in M9 minimal and xylem mimicking medium.

  20. A Proteomic Study of Clavibacter Michiganensis Subsp. Michiganensis Culture Supernatants.

    Science.gov (United States)

    Hiery, Eva; Poetsch, Ansgar; Moosbauer, Tanja; Amin, Bushra; Hofmann, Jörg; Burkovski, Andreas

    2015-11-12

    Clavibacter michiganensis, subsp. michiganensis is a Gram-positive plant pathogen infecting tomato (Solanum lycopersicum). Despite a considerable economic importance due to significant losses of infected plants and fruits, knowledge about virulence factors of C. michiganensis subsp. michiganensis and host-pathogen interactions on a molecular level are rather limited. In the study presented here, the proteome of culture supernatants from C. michiganensis subsp. michiganensis NCPPB382 was analyzed. In total, 1872 proteins were identified in M9 and 1766 proteins in xylem mimicking medium. Filtration of supernatants before protein precipitation reduced these to 1276 proteins in M9 and 976 proteins in the xylem mimicking medium culture filtrate. The results obtained indicate that C. michiganensis subsp. michiganensis reacts to a sucrose- and glucose-depleted medium similar to the xylem sap by utilizing amino acids and host cell polymers as well as their degradation products, mainly peptides, amino acids and various C5 and C6 sugars. Interestingly, the bacterium expresses the previously described virulence factors Pat-1 and CelA not exclusively after host cell contact in planta but already in M9 minimal and xylem mimicking medium.

  1. Characterization and Intraorganellar Distribution of Protein Kinases in Amyloplasts Isolated from Cultured Cells of Sycamore (Acer pseudoplatanus L.).

    Science.gov (United States)

    Viale, A M; Ngernprasirtsiri, J; Akazawa, T

    1991-08-01

    Incubation of amyloplasts isolated from cultured cells of sycamore (Acer pseudoplatanus L.) with [gamma-(32)P]ATP resulted in the rapid phosphorylation (half-time of 40 seconds at 25 degrees Celcius) of organellar polypeptides. The preferred substrate for amyloplast protein kinases was Mg(2+). ATP, and recovery of only [(32)P]serine after partial acid hydrolysis indicated the predominance of protein serine kinases in the organelle. These activities were located in the envelope and stromal fractions of the plastid, which showed different specificities toward exogenous protein substrates and distinct patterns of phosphorylation of endogenous polypeptides. A 66-kilodalton polypeptide, inaccessible to an exogenously added protease, was one of the major phosphorylated products found in intact amyloplasts at low [gamma-(32)P] adenosine triphosphate concentrations. This polypeptide represented the major phosphoprotein observed with the isolated envelope fraction. The patterns of polypeptide phosphorylation found in intact amyloplasts and chloroplasts from cultured cell lines of sycamore were clearly distinguishable. The overall results indicate the presence of protein phosphorylation systems unique to this reserve plastid present in nonphotosynthetic tissues.

  2. Intracellular localization of GBF proteins and blue light-induced import of GBF2 fusion proteins into the nucleus of cultured Arabidopsis and soybean cells.

    Science.gov (United States)

    Terzaghi, W B; Bertekap, R L; Cashmore, A R

    1997-05-01

    The G-box is an important regulatory element found in the promoters of many different genes. Four members of an Arabidopsis gene family encoding basic leucine zipper proteins (GBFs) which bind the G-box have previously been cloned. To study GBFs, a polyclonal antibody was raised against GBF1 expressed in bacteria. This antibody also recognized GBF2 and GBF3. Immunoblot analysis of nuclear and cytoplasmic fractions from Arabidopsis and soybean (SB-M) cell cultures indicated that over 90% of proteins detected with anti-GBF1 were cytoplasmic. Electrophoretic mobility shift assays indicated that over 90% of G-box binding activity was cytoplasmic. DNA affinity chromatography demonstrated that each protein detected with anti-GBF1 specifically bound the G-box. To study individual GBFs, DNA constructs fusing GBF1, GBF2 and GBF4 to GUS were made and assayed by transient expression in SB-M protoplasts. Of GUS:GBF1 proteins, 50-62% were localized in the cytoplasm under all conditions tested, while 97% of GUS:GBF4 was localized in the nucleus. By contrast, whereas about 50% of GUS:GBF2 was found in the cytoplasm of dark-grown cells, over 80% of this protein was found in the nucleus in cells cultured under blue light. Deletion analysis of GBF1 identified a region between amino acids 112 and 164 apparently required for cytoplasmic retention. These results suggest the intriguing possibility that limitation of nuclear access may be an important control on GBF activity. In particular, GBF2 is apparently specifically imported into the nucleus in response to light.

  3. Nanoparticles filtration by leaked fibrous filters

    Energy Technology Data Exchange (ETDEWEB)

    Mouret, Guillaume; Calle-Chazelet, Sandrine; Thomas, Dominique; Appert-Collin, Jean-Christophe [Nancy-Universite/LSGC/CNRS - 1 rue Grandville - BP 20451 - F-54001 Nancy Cedex (France)], E-mail: sandrine.calle@ensic.inpl-nancy.fr; Bemer, Denis [INRS - Avenue de Bourgogne - F-54501 Vandoeuvre les Nancy Cedex (France)

    2009-05-01

    The aim of this work is first to measured nanoparticles penetration through three different fiberglass filters intentionally-pierced with calibrated needles at different filtration velocity. Then a semi-empirical model based on the air flow resistances of the new and perforated filter media and on the mechanism of Brownian diffusion for the collection of ultrafine particles by the media enables to well predict the efficiency observed for all tested operating conditions. Results show that the increase of particles penetration is all the more important that the pinhole is large and that the particle diameter is low. Another result is that the filtration efficiency of the new filter media controlled the penetration. A high efficiency filter with a high resistance to air flow will be more damaged than a low efficiency filter when being perforated.

  4. Integrative filtration research and sustainable nanotechnology

    Institute of Scientific and Technical Information of China (English)

    Jing Wang; Drew Thompson; David Y.H.Pui

    2013-01-01

    With the wide applications of nanomaterials in an array of industries,more concerns are being raised about the occupational health and safety of nanoparticles in the workplace,and implications of nanotechnology on the environment and living systems.Studies on environmental,health and safety (EHS) issues of nanomaterials play a significant role in public acceptance,and eventual sustainability,of nanotechnology.We present research results on three aspects of the EHS studies:characterization and measurement of nanoparticles,nanoparticle emission and exposure at workplaces,and control and abatement of nanoparticle release using filtration technology.Measurement of nanoparticle agglomerates using a newly developed instrument,the Universal Nanoparticle Analyzer,is discussed.Nanoparticle emission and exposure measurement results for carbon nanotubes in the manufacture of nanocomposites and for silicon nanoparticles in their production at a pilot scale facility are presented.Filtration of nanoparticles and nanoparticle agglomerates are also studied.

  5. Air filtration and indoor air quality

    DEFF Research Database (Denmark)

    Bekö, Gabriel

    2006-01-01

    Demands for better indoor air quality are increasing, since we spend most of our time indoors and we are more and more aware of indoor air pollution. Field studies in different parts of the world have documented that high percentage of occupants in many offices and buildings find the indoor air...... decent ventilation and air cleaning/air filtration, high indoor air quality cannot be accomplished. The need for effective air filtration has increased with increasing evidence on the hazardous effects of fine particles. Moreover, the air contains gaseous pollutants, removal of which requires various air...... cleaning techniques. Supply air filter is one of the key components in the ventilation system. Studies have shown that used ventilation filters themselves can be a significant source of indoor air pollution with consequent impact on perceived air quality, sick building syndrome symptoms and performance...

  6. Ultrasonic filtration of industrial chemical solutions

    Science.gov (United States)

    Cosma, T.

    1974-01-01

    The practical results obtained as a result of filtering industrial chemical solutions under continuous flow conditions with the aid of an ultrasonic filter are presented. The main part of the assembly consists of an ultrasonic generator with an output power of about 400 W and the filtration assembly, in which there is a magnetostrictive amplifier constructed for 20.5 kHz. In addition to ensuring a continuous flow of filtered solution, ultrasonic filters can be replaced or cleaned at intervals of time that are 8-10 times greater than in the case of mechanical filters. They yield considerably better results as far as the size of the filtered particles is concerned. The parameters on which filtration quality depends are also presented.

  7. Breakthrough of cyanobacteria in bank filtration.

    Science.gov (United States)

    Pazouki, Pirooz; Prévost, Michèle; McQuaid, Natasha; Barbeau, Benoit; de Boutray, Marie-Laure; Zamyadi, Arash; Dorner, Sarah

    2016-10-01

    The removal of cyanobacteria cells in well water following bank filtration was investigated from a source water consisting of two artificial lakes (A and B). Phycocyanin probes used to monitor cyanobacteria in the source and in filtered well water showed an increase of fluorescence values demonstrating a progressive seasonal growth of cyanobacteria in the source water that were correlated with cyanobacterial biovolumes from taxonomic counts (r = 0.59, p cyanobacteria. Of cyanobacteria that passed through bank filtration, smaller cells were significantly more frequent in well water samples (p cyanobacteria to wells. Winter samples demonstrated that cyanobacteria accumulate within bank filters, leading to continued passage of cells beyond the bloom season. Although no concentrations of total microcystin-LR were above detection limits in filtered well water, there is concern that cyanobacterial cells that reach the wells have the potential to contain intracellular toxins. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Radiation-induced p53 protein response in the A549 cell line is culture growth-phase dependent

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, N.F.; Gurule, D.M.; Carpenter, T.R.

    1995-12-01

    One role of the p53 tumor suppressor protein has been recently revealed. Kastan, M.B. reported that p53 protein accumulates in cells exposed to ionizing radiation. The accumulation of p53 protein is in response to DNA damage, most importantly double-strand breaks, that results from exposure to ionizing radiation. The rise in cellular p53 levels is necessary for an arrest in the G{sub 1} phase of the cell cycle to provide additional time for DNA repair. The p53 response has also been demonstrated to enhance PCNA-dependent repair. p53 is thus an important regulator of the cellular response to DNA-damaging radiation. From this data, it can be concluded that the magnitude of the p53 response is not dependent on the phase of culture growth.

  9. Symptomatic hyponatremia during glomerular filtration rate testing

    OpenAIRE

    2010-01-01

    Hyponatremia affects nearly one in five of all hospitalized patients. Severe hyponatremia is associated with significant morbidity and mortality, and is therefore important to recognize. Prior reports have linked duloxetine with hyponatremia, but it is uncommon. In this case report, we describe a research subject taking duloxetine who developed severe symptomatic hyponatremia during glomerular filtration rate testing despite having undergone such testing uneventfully in the past.

  10. Portable Hybrid Powered Water Filtration Device

    Directory of Open Access Journals (Sweden)

    Maria Lourdes V. Balansay

    2015-08-01

    Full Text Available The existing water filtration device has features that can be developed to be more useful and functional during emergency situations. The project’s development has been aided by following provisions in PEC, NEC, NEMA and Philippine National Standard for Safe Drinking Water provide standards for the construction of the project. These standards protect both the prototype and the user. These also served as guide for the maintenance of every component. The design of the portable hybrid powered water filtration device shows that the project has more advanced features such as portability and the power supply used such as photovoltaic module solar cells and manually operated generator. This also shows its effectiveness and reliability based on the results of discharging test, water quality test and water production test. Based on analysis of the overall financial aspects, the machine can be profitable and the amount of revenue and operating cost will increase as years pass. Using the proper machine/ tools and methods of fabrication helps in easy assembly of the project. The materials and components used are cost effective and efficient. The best time for charging the battery using solar panel is 9:00 am onwards while the hand crank generator is too slow because the generated current is little. The water filtration device is very efficient regarding the operating hours and water production. The machine may have a great effect to society and economy in generation of clean available water at less cost.

  11. Lewis lung carcinoma regulation of mechanical stretch-induced protein synthesis in cultured myotubes.

    Science.gov (United States)

    Gao, Song; Carson, James A

    2016-01-01

    Mechanical stretch can activate muscle and myotube protein synthesis through mammalian target of rapamycin complex 1 (mTORC1) signaling. While it has been established that tumor-derived cachectic factors can induce myotube wasting, the effect of this catabolic environment on myotube mechanical signaling has not been determined. We investigated whether media containing cachectic factors derived from Lewis lung carcinoma (LLC) can regulate the stretch induction of myotube protein synthesis. C2C12 myotubes preincubated in control or LLC-derived media were chronically stretched. Protein synthesis regulation by anabolic and catabolic signaling was then examined. In the control condition, stretch increased mTORC1 activity and protein synthesis. The LLC treatment decreased basal mTORC1 activity and protein synthesis and attenuated the stretch induction of protein synthesis. LLC media increased STAT3 and AMP-activated protein kinase phosphorylation in myotubes, independent of stretch. Both stretch and LLC independently increased ERK1/2, p38, and NF-κB phosphorylation. In LLC-treated myotubes, the inhibition of ERK1/2 and p38 rescued the stretch induction of protein synthesis. Interestingly, either leukemia inhibitory factor or glycoprotein 130 antibody administration caused further inhibition of mTORC1 signaling and protein synthesis in stretched myotubes. AMP-activated protein kinase inhibition increased basal mTORC1 signaling activity and protein synthesis in LLC-treated myotubes, but did not restore the stretch induction of protein synthesis. These results demonstrate that LLC-derived cachectic factors can dissociate stretch-induced signaling from protein synthesis through ERK1/2 and p38 signaling, and that glycoprotein 130 signaling is associated with the basal stretch response in myotubes.

  12. Aerosol Filtration Application Using Fibrous Media An Industrial Perspective

    Institute of Scientific and Technical Information of China (English)

    杨传芳

    2012-01-01

    Filtration of aerosol particles using non-woven fibrous media is a common practice for air cleaning. It has found wide applications in industries and our daily lives. This paper overviews some of these applications and provides an industrial perspective. It starts from discussing aerosol filtration theory, followed by a brief review on the advancement of filtration media. After that, filtration applications in respiratory protection, dust collection, and engine in-take air cleaning are elaborated. These are the areas that the author sees as the typical needed ones in China's fast pace economical development endeavor, where air filtration enables the protection of human health, environment and equipment for sustainability.

  13. Protein-engineered scaffolds for in vitro 3D culture of primary adult intestinal organoids.

    Science.gov (United States)

    DiMarco, Rebecca L; Dewi, Ruby E; Bernal, Gabriela; Kuo, Calvin; Heilshorn, Sarah C

    2015-10-15

    Though in vitro culture of primary intestinal organoids has gained significant momentum in recent years, little has been done to investigate the impact of microenvironmental cues provided by the encapsulating matrix on the growth and development of these fragile cultures. In this work, the impact of various in vitro culture parameters on primary adult murine organoid formation and growth are analyzed with a focus on matrix properties and geometric culture configuration. The air-liquid interface culture configuration was found to result in enhanced organoid formation relative to a traditional submerged configuration. Additionally, through use of a recombinantly engineered extracellular matrix (eECM), the effects of biochemical and biomechanical cues were independently studied. Decreasing mechanical stiffness and increasing cell adhesivity were found to increase organoid yield. Tuning of eECM properties was used to obtain organoid formation efficiency values identical to those observed in naturally harvested collagen I matrices but within a stiffer construct with improved ease of physical manipulation. Increased ability to remodel the surrounding matrix through mechanical or enzymatic means was also shown to enhance organoid formation. As the engineering and tunability of recombinant matrices is essentially limitless, continued property optimization may result in further improved matrix performance and may help to identify additional microenvironmental cues that directly impact organoid formation, development, differentiation, and functional behavior. Continued culture of primary organoids in recombinant matrices could therefore prove to be largely advantageous in the field of intestinal tissue engineering for applications in regenerative medicine and in vitro tissue mimics.

  14. Expression, Solubilization, and Purification of Bacterial Membrane Proteins.

    Science.gov (United States)

    Jeffery, Constance J

    2016-02-02

    Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the case of commensal or pathogenic bacteria, interacting with the host organism. Working with membrane proteins in the lab can be more challenging than working with soluble proteins because of difficulties in their recombinant expression and purification. This protocol describes a standard method to express, solubilize, and purify bacterial integral membrane proteins. The recombinant protein of interest with a 6His affinity tag is expressed in E. coli. After harvesting the cultures and isolating cellular membranes, mild detergents are used to solubilize the membrane proteins. Protein-detergent complexes are then purified using IMAC column chromatography. Support protocols are included to help select a detergent for protein solubilization and for use of gel filtration chromatography for further purification.

  15. Filtration Combustion in Smoldering and SHS

    Science.gov (United States)

    Matkowsky, Bernard J.

    2001-01-01

    Smolder waves and SHS (self-propagating high-temperature synthesis) waves are both examples of filtration combustion waves propagating in porous media. Smoldering combustion is important for the study of fire safety. Smoldering itself can cause damage, its products are toxic and it can also lead to the more dangerous gas phase combustion which corresponds to faster propagation at higher temperatures. In SHS , a porous solid sample, consisting of a finely ground powder mixture of reactants, is ignited at one end. A high temperature thermal wave, having a frontal structure, then propagates through the sample converting reactants to products. The SHS technology appears to enjoy a number of advantages over the conventional technology, in which the sample is placed in a furnace and "baked" until it is "well done". The advantages include shorter synthesis times, greater economy, in that the internal energy of the reactions is employed rather than the costly external energy of the furnace, purer products, simpler equipment and no intrinsic limitation on the size of the sample to be synthesized as exists in the conventional technology. When delivery of reactants through the pores to the reaction site is an important aspect of the combustion process, it is referred to as filtration combustion. The two types of filtration combustion have a similar mathematical formulation, describing the ignition, propagation and extinction of combustion waves in porous media. The goal in each case, however, is different. In smoldering the desired goal is to prevent propagation, whereas in SHS the goal is to ensure propagation of the combustion wave, leading to the synthesis of desired products. In addition, the scales in the two areas of application differ. Smoldering generally occurs at lower temperatures and propagation velocities than in SHS nevertheless, the two applications have much in common so that what is learned fit make application can be used to advantage in the other. In porous

  16. Nanofiber filter media for air filtration

    Science.gov (United States)

    Raghavan, Bharath Kumar

    Nanofibers have higher capture efficiencies in comparison to microfibers in the submicron particle size range of 100-500 nm because of small fiber diameter and increased surface area of the fibers. Pressure drop across the filter increases tremendously with decrease in fiber diameter in the continuum flow regime. Nanofibers with fiber diameter less than 300 nm are in the slip flow regime as a consequence of which steep increase in pressure drop is considerably reduced due to slip effect. The outlet or inlet gases have broad range of particle size distribution varying from few micrometers to nanometers. The economic benefits include capture of a wide range of particle sizes in the gas streams using compact filters composed of nanofibers and microfibers. Electrospinning technique was used to successfully fabricate polymeric and ceramic nanofibers. The nanofibers were long, continuous, and flexible with diameters in the range of 200--300 nm. Nanofibers were added to the filter medium either by mixing microfibers and nanofibers or by directly electrospinning nanofibers as thin layer on the surface of the microfiber filter medium. Experimental results showed that either by mixing Nylon 6 nanofibers with B glass fibers or by electrospinning Nylon 6 nanofibers as a thin layer on the surface of the microfiber medium in the surface area ratio of 1 which is 0.06 g of nanofibers for 2 g of microfibers performed better than microfiber filter media in air filtration tests. This improved performance is consistent with numerical modeling. The particle loading on a microfibrous filter were studied for air filtration tests. The experimental and modeling results showed that both pressure drop and capture efficiency increased with loading time. Nanofiber filter media has potential applications in many filtration applications and one of them being hot gas filtration. Ceramic nanofibers made of alumina and titania nanofibers can withstand in the range of 1000°C. Ceramic nanofibers

  17. Filtration Efficiency of Functionalized Ceramic Foam Filters for Aluminum Melt Filtration

    Science.gov (United States)

    Voigt, Claudia; Jäckel, Eva; Taina, Fabio; Zienert, Tilo; Salomon, Anton; Wolf, Gotthard; Aneziris, Christos G.; Le Brun, Pierre

    2017-02-01

    The influence of filter surface chemistry on the filtration efficiency of cast aluminum alloys was evaluated for four different filter coating compositions (Al2O3—alumina, MgAl2O4—spinel, 3Al2O3·2SiO2—mullite, and TiO2—rutile). The tests were conducted on a laboratory scale with a filtration pilot plant, which facilitates long-term filtration tests (40 to 76 minutes). This test set-up allows the simultaneous use of two LiMCAs (before and after the filter) for the determination of the efficiency of inclusion removal. The four tested filter surface chemistries exhibited good thermal stability and mechanical robustness after 750 kg of molten aluminum had been cast. All four filter types exhibited a mean filtration efficiency of at least 80 pct. However, differences were also observed. The highest filtration efficiencies were obtained with alumina- and spinel-coated filter surfaces (>90 pct), and the complete removal of the largest inclusions (>90 µm) was observed. The efficiency was slightly lower with mullite- and rutile-coated filter surfaces, in particular for large inclusions. These observations are discussed in relation to the properties of the filters, in particular in terms of, for example, the surface roughness.

  18. Filtration Efficiency of Functionalized Ceramic Foam Filters for Aluminum Melt Filtration

    Science.gov (United States)

    Voigt, Claudia; Jäckel, Eva; Taina, Fabio; Zienert, Tilo; Salomon, Anton; Wolf, Gotthard; Aneziris, Christos G.; Le Brun, Pierre

    2016-12-01

    The influence of filter surface chemistry on the filtration efficiency of cast aluminum alloys was evaluated for four different filter coating compositions (Al2O3—alumina, MgAl2O4—spinel, 3Al2O3·2SiO2—mullite, and TiO2—rutile). The tests were conducted on a laboratory scale with a filtration pilot plant, which facilitates long-term filtration tests (40 to 76 minutes). This test set-up allows the simultaneous use of two LiMCAs (before and after the filter) for the determination of the efficiency of inclusion removal. The four tested filter surface chemistries exhibited good thermal stability and mechanical robustness after 750 kg of molten aluminum had been cast. All four filter types exhibited a mean filtration efficiency of at least 80 pct. However, differences were also observed. The highest filtration efficiencies were obtained with alumina- and spinel-coated filter surfaces (>90 pct), and the complete removal of the largest inclusions (>90 µm) was observed. The efficiency was slightly lower with mullite- and rutile-coated filter surfaces, in particular for large inclusions. These observations are discussed in relation to the properties of the filters, in particular in terms of, for example, the surface roughness.

  19. The Structural and Functional Organization of the Podocyte Filtration Slits Is Regulated by Tjp1/ZO-1

    Science.gov (United States)

    Itoh, Masahiko; Nakadate, Kazuhiko; Horibata, Yasuhiro; Matsusaka, Taiji; Xu, Jianliang; Hunziker, Walter; Sugimoto, Hiroyuki

    2014-01-01

    Blood filtration in the kidney glomerulus is essential for physiological homeostasis. The filtration apparatus of the kidney glomerulus is composed of three distinct components: the fenestrated endothelial cells, the glomerular basement membrane, and interdigitating foot processes of podocytes that form the slit diaphragm. Recent studies have demonstrated that podocytes play a crucial role in blood filtration and in the pathogenesis of proteinuria and glomerular sclerosis; however, the molecular mechanisms that organize the podocyte filtration barrier are not fully understood. In this study, we suggest that tight junction protein 1 (Tjp1 or ZO-1), which is encoded by Tjp1 gene, plays an essential role in establishing the podocyte filtration barrier. The podocyte-specific deletion of Tjp1 down-regulated the expression of podocyte membrane proteins, impaired the interdigitation of the foot processes and the formation of the slit diaphragm, resulting in glomerular dysfunction. We found the possibility that podocyte filtration barrier requires the integration of two independent units, the pre-existing epithelial junction components and the newly synthesized podocyte-specific components, at the final stage in glomerular morphogenesis, for which Tjp1 is indispensable. Together with previous findings that Tjp1 expression was decreased in glomerular diseases in human and animal models, our results indicate that the suppression of Tjp1 could directly aggravate glomerular disorders, highlights Tjp1 as a potential therapeutic target. PMID:25184792

  20. The structural and functional organization of the podocyte filtration slits is regulated by Tjp1/ZO-1.

    Directory of Open Access Journals (Sweden)

    Masahiko Itoh

    Full Text Available Blood filtration in the kidney glomerulus is essential for physiological homeostasis. The filtration apparatus of the kidney glomerulus is composed of three distinct components: the fenestrated endothelial cells, the glomerular basement membrane, and interdigitating foot processes of podocytes that form the slit diaphragm. Recent studies have demonstrated that podocytes play a crucial role in blood filtration and in the pathogenesis of proteinuria and glomerular sclerosis; however, the molecular mechanisms that organize the podocyte filtration barrier are not fully understood. In this study, we suggest that tight junction protein 1 (Tjp1 or ZO-1, which is encoded by Tjp1 gene, plays an essential role in establishing the podocyte filtration barrier. The podocyte-specific deletion of Tjp1 down-regulated the expression of podocyte membrane proteins, impaired the interdigitation of the foot processes and the formation of the slit diaphragm, resulting in glomerular dysfunction. We found the possibility that podocyte filtration barrier requires the integration of two independent units, the pre-existing epithelial junction components and the newly synthesized podocyte-specific components, at the final stage in glomerular morphogenesis, for which Tjp1 is indispensable. Together with previous findings that Tjp1 expression was decreased in glomerular diseases in human and animal models, our results indicate that the suppression of Tjp1 could directly aggravate glomerular disorders, highlights Tjp1 as a potential therapeutic target.

  1. The structural and functional organization of the podocyte filtration slits is regulated by Tjp1/ZO-1.

    Science.gov (United States)

    Itoh, Masahiko; Nakadate, Kazuhiko; Horibata, Yasuhiro; Matsusaka, Taiji; Xu, Jianliang; Hunziker, Walter; Sugimoto, Hiroyuki

    2014-01-01

    Blood filtration in the kidney glomerulus is essential for physiological homeostasis. The filtration apparatus of the kidney glomerulus is composed of three distinct components: the fenestrated endothelial cells, the glomerular basement membrane, and interdigitating foot processes of podocytes that form the slit diaphragm. Recent studies have demonstrated that podocytes play a crucial role in blood filtration and in the pathogenesis of proteinuria and glomerular sclerosis; however, the molecular mechanisms that organize the podocyte filtration barrier are not fully understood. In this study, we suggest that tight junction protein 1 (Tjp1 or ZO-1), which is encoded by Tjp1 gene, plays an essential role in establishing the podocyte filtration barrier. The podocyte-specific deletion of Tjp1 down-regulated the expression of podocyte membrane proteins, impaired the interdigitation of the foot processes and the formation of the slit diaphragm, resulting in glomerular dysfunction. We found the possibility that podocyte filtration barrier requires the integration of two independent units, the pre-existing epithelial junction components and the newly synthesized podocyte-specific components, at the final stage in glomerular morphogenesis, for which Tjp1 is indispensable. Together with previous findings that Tjp1 expression was decreased in glomerular diseases in human and animal models, our results indicate that the suppression of Tjp1 could directly aggravate glomerular disorders, highlights Tjp1 as a potential therapeutic target.

  2. Effects of extracellular matrix proteins on macrophage differentiation, growth, and function: comparison of liquid and agar culture systems

    Science.gov (United States)

    Armstrong, J. W.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Both spaceflight and skeletal unloading suppress the haematopoietic differentiation of macrophages (Sonnenfeld et al., Aviat. Space Environ. Med., 61:648-653, 1990; Armstrong et al., J. Appl. Physiol., 75:2734-2739, 1993). The mechanism behind this reduction in haematopoiesis has yet to be elucidated. However, changes in bone marrow extracellular matrix (ECM) may be involved. To further understand the role of ECM products in macrophage differentiation, we have performed experiments evaluating the effects of fibronectin, laminin, collagen type I, and collagen type IV on macrophage development and function. Bone marrow-derived macrophages cultured on four different ECM substrates in liquid culture medium showed less growth than those cultured on plastic. Significant morphological differences were seen on each of the substrates used. Phenotypically and functionally, as measured by class II major histocompatibility molecule (MHCII) expression, MAC-2 expression, and the secretion of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), these macrophages were similar. In contrast, bone marrow-derived macrophages cultured in suspension, using agar, showed no difference in growth when exposed to ECM proteins. However, IL-6 and TNF-alpha secretion was affected by fibronectin, laminin, collagen type I, and collagen type IV in a concentration-dependent manner. We conclude that the ECM products fibronectin, laminin, collagen type I, and collagen type IV have profound effects on macrophage development and function. Additionally, we suggest that an ECM-supplemented agar culture system provides an environment more analogous to in vivo bone marrow than does a traditional liquid culture system.

  3. Elevated NF-κB activation is conserved in human myocytes cultured from obese type 2 diabetic patients and attenuated by AMP-activated protein kinase

    DEFF Research Database (Denmark)

    Green, Charlotte Jane; Pedersen, Maria; Pedersen, Bente K;

    2011-01-01

    To examine whether the inflammatory phenotype found in obese and diabetic individuals is preserved in isolated, cultured myocytes and to assess the effectiveness of pharmacological AMP-activated protein kinase (AMPK) activation upon the attenuation of inflammation in these myocytes....

  4. Effect of epidermal growth factor on the labeling of the various RNA species and of nuclear proteins in primary rat astroglial cell cultures

    Energy Technology Data Exchange (ETDEWEB)

    Avola, R.; Condorelli, D.F.; Turpeenoja, L.; Ingrao, F.; Reale, S.; Ragusa, N.; Giuffrida Stella, A.M.

    1988-05-01

    This study investigated the effects of epidermal growth factor (EGF) on the labeling of various RNA species and of nuclear proteins in primary rat astroglial cell cultures. After 12 hours of EGF treatment in serum-free medium or chemically defined medium, significant increase in RNA labeling, and also in acid-soluble radioactivity and RNA content, was observed. The ratio RNA/DNA was significantly higher in EGF-treated cultures compared with controls. Ribosomal RNAs (28S and 18S), polyadenylated, and nonpolyadenylated RNAs showed a higher specific radioactivity in EGF-treated cultures. Among the nuclear proteins, the labeling of basic proteins was enhanced by EGF treatment, whereas that of total nuclear acidic protein (NHPs) was less modified, except for some NHPs separated by gel electrophoresis with a molecular weight (MW) approximately 95-83 and 44 kd, which were significantly more labeled in EGF-treated cultures.

  5. Variations in gene and protein expression in canine chondrodystrophic nucleus pulposus cells following long-term three-dimensional culture.

    Directory of Open Access Journals (Sweden)

    Munetaka Iwata

    Full Text Available Intervertebral disc (IVD degeneration greatly affects quality of life. The nucleus pulposus (NP of chondrodystrophic dog breeds (CDBs is similar to the human NP, because the cells disappear with age and are replaced by fibrochondrocyte-like cells. However, because IVD develops as early as within the first year of life, we used canines as a model to investigate in vitro the mechanisms underlying IVD degeneration. Specifically, we evaluated the potential of a three-dimensional (3D culture of healthy NP as an in vitro model system to investigate the mechanisms of IVD degeneration. Agarose hydrogels were populated with healthy NP cells from beagles after performing magnetic resonance imaging, and mRNA expression profiles and pericellular extracellular matrix (ECM protein distribution were determined. After 25 days of 3D culture, there was a tendency for redifferentiation into the native NP phenotype, and mRNA levels of Col2A1, COMP, and CK18 were not significantly different from those of freshly isolated cells. Our findings suggest that long-term 3D culture promoted chondrodystrophic NP redifferentiation through reconstruction of the pericellular microenvironment. Further, lipopolysaccharide (LPS induced expression of TNF-α, MMP3, MMP13, VEGF, and PGES mRNA in the 3D cultures, creating a molecular milieu that mimics that of degenerated NP. These results suggest that this in vitro model represents a reliable and cost-effective tool for evaluating new therapies for disc degeneration.

  6. Effects of selective inhibition of protein kinase C, cyclic AMP-dependent protein kinase, and Ca(2+)-calmodulin-dependent protein kinase on neurite development in cultured rat hippocampal neurons.

    Science.gov (United States)

    Cabell, L; Audesirk, G

    1993-06-01

    A variety of experimental evidence suggests that calmodulin and protein kinases, especially protein kinase C, may participate in regulating neurite development in cultured neurons, particularly neurite initiation. However, the results are somewhat contradictory. Further, the roles of calmodulin and protein kinases on many aspects of neurite development, such as branching or elongation of axons vs dendrites, have not been extensively studied. Cultured embryonic rat hippocampal pyramidal neurons develop readily identifiable axons and dendrites. We used this culture system and the new generation of highly specific protein kinase inhibitors to investigate the roles of protein kinases and calmodulin in neurite development. Neurons were cultured for 2 days in the continuous presence of calphostin C (a specific inhibitor of protein kinase C), KT5720 (inhibitor of cyclic AMP-dependent protein kinase), KN62 (inhibitor of Ca(2+)-calmodulin-dependent protein kinase II), or calmidazolium (inhibitor of calmodulin), each at concentrations from approximately 1 to 10 times the concentration reported in the literature to inhibit each kinase by 50%. The effects of phorbol 12-myristate 13-acetate (an activator of protein kinase C) and 4 alpha-phorbol 12,13-didecanoate (an inactive phorbol ester) were also tested. At concentrations that had no effect on neuronal viability, calphostin C reduced neurite initiation and axon branching without significantly affecting the number of dendrites per neuron, dendrite branching, dendrite length, or axon length. Phorbol 12-myristate 13-acetate increased axon branching and the number of dendrites per cell, compared to the inactive 4 alpha-phorbol 12,13-didecanoate. KT5720 inhibited only axon branching. KN62 reduced axon length, the number of dendrites per neuron, and both axon and dendrite branching. At low concentrations, calmidazolium had no effect on any aspect of neurite development, but at high concentrations, calmidazolium inhibited every

  7. The development of electro-membrane filtration for the isolation of bioactive peptides: the effect of membrane selection and operating parameters on the transport rate

    NARCIS (Netherlands)

    Bargeman, Gerrald; Koops, G.H.; Houwing, J.; Breebaart, I.; van der Horst, H.C.; Wessling, Matthias

    2002-01-01

    The ability to produce functional food ingredients from natural sources becomes increasingly attractive to the food industry. Antimicrobial (bioactive) ingredients, like peptides and proteins, can be isolated from hydrolysates with membrane filtration and/or chromatography. Electro-membrane filtrati

  8. Quantitative Determination of Lethal Toxin Proteins in Culture Supernatant of Human Live Anthrax Vaccine Bacillus anthracis A16R.

    Science.gov (United States)

    Zai, Xiaodong; Zhang, Jun; Liu, Ju; Liu, Jie; Li, Liangliang; Yin, Ying; Fu, Ling; Xu, Junjie; Chen, Wei

    2016-02-25

    Bacillus anthracis (B. anthracis) is the etiological agent of anthrax affecting both humans and animals. Anthrax toxin (AT) plays a major role in pathogenesis. It includes lethal toxin (LT) and edema toxin (ET), which are formed by the combination of protective antigen (PA) and lethal factor (LF) or edema factor (EF), respectively. The currently used human anthrax vaccine in China utilizes live-attenuated B. anthracis spores (A16R; pXO1+, pXO2-) that produce anthrax toxin but cannot produce the capsule. Anthrax toxins, especially LT, have key effects on both the immunogenicity and toxicity of human anthrax vaccines. Thus, determining quantities and biological activities of LT proteins expressed by the A16R strain is meaningful. Here, we explored LT expression patterns of the A16R strain in culture conditions using another vaccine strain Sterne as a control. We developed a sandwich ELISA and cytotoxicity-based method for quantitative detection of PA and LF. Expression and degradation of LT proteins were observed in culture supernatants over time. Additionally, LT proteins expressed by the A16R and Sterne strains were found to be monomeric and showed cytotoxic activity, which may be the main reason for side effects of live anthrax vaccines. Our work facilitates the characterization of anthrax vaccines components and establishment of a quality control standard for vaccine production which may ultimately help to ensure the efficacy and safety of the human anthrax vaccine A16R.

  9. Dose-dependent differential effect of hemin on protein synthesis and cell proliferation in Leishmania donovani promastigotes cultured in vitro

    Indian Academy of Sciences (India)

    Jayanta K Pal; Manisha Joshi-Purandare

    2001-06-01

    Leishmania donovani requires an exogenous source of heme for growth and transformation. In in vitro culture of the free-living promastigotes, exogenously added hemin enhances cell proliferation. In this investigation, the question of the function of heme with particular reference to protein synthesis and cell proliferation has been addressed. The results of in vitro cell culture experiments demonstrated that hemin (10 M) alone is suitable for supporting optimum level of protein synthesis, and thereby cell proliferation of promastigotes to an extent that it can replace fetal bovine serum. However, in situ labelling experiments along with Western blots revealed that high concentration of hemin (50 M) reduced the level of protein synthesis in general and of -tubulin in particular with a concomitant induction of hsp90, and induced consequent morphological changes that are observed during in situ transformation of promastigotes in mammalian macrophages. These results therefore suggest that sudden exposure to high concentration of heme in mammalian macrophages may be one of the key factors that trigger promastigote to amastigote transformation in L. donovani. Furthermore, hemin with its dual characteristic could be used as a tool to understand molecular mechanism of cell proliferation and transformation in these parasites.

  10. Alterations in protein expression of Arabidopsis thaliana cell cultures during hyper- , simulated and sounding rocket micro-gravity

    Science.gov (United States)

    Hampp, Ruediger; Barjaktarović, Žarko; Babbick, Maren; Magel, Elisabeth; Nordheim, Alfred; Lamkemeyer, Tobias; Hampp, Ruediger

    Callus cell cultures of Arabidopsis thaliana exposed to hypergravity (8g), 2D clinorotation and random positioning exhibit changes in gene expression (Martzivanou et al., Protoplasma 229:155-162, 2003). In a recent investigation we could show that after 2 hrs of exposure also the protein complement shows treatment-related changes. These are indicative for reactive oxygen species being involved in the perception of / response to changes in the gravitational field. In the present study we have extended these investigations for a period of up to 16 hrs of exposure. We report on changes in abundance of 28 proteins which have been identified by nano HPLC-ESI-MS/MS, and which were altered in amount after 2 hrs of treatment. According to changes between 2 and 16 hrs we could distinguish four groups of proteins which either declined, increased from down-regulated to control levels, showed a transient decline or a transient increase. With regard to function, our data indicate stress relief or adaption to a new gravitational steady state under prolonged exposure. The latter assumption is supported by the appearance of a new set of 19 proteins which is changed in abundance after 8 hrs of hypergravity. A comparative analysis of the different treatments showed some similarities in response between 8g centrifugation and 2D clinorotation, while random positioning showed the least responses. In addition, we report on the impact of reduced gravitation on the phospho proteom. Cell cultures exposed to 12 min of microgravity as obtained on board of sounding rockets do not respond with alterations in total protein but in the degree of phosphorylation as demonstrated after 2D SDS PAGE separation and sequencing. On this basis we give evidence for signaling cascades involved in the transduction of gravitational signals.

  11. Elevated NF-κB activation is conserved in human myocytes cultured from obese type 2 diabetic patients and attenuated by AMP-activated protein kinase

    DEFF Research Database (Denmark)

    Green, Charlotte Jane; Pedersen, Maria; Pedersen, Bente K

    2011-01-01

    To examine whether the inflammatory phenotype found in obese and diabetic individuals is preserved in isolated, cultured myocytes and to assess the effectiveness of pharmacological AMP-activated protein kinase (AMPK) activation upon the attenuation of inflammation in these myocytes.......To examine whether the inflammatory phenotype found in obese and diabetic individuals is preserved in isolated, cultured myocytes and to assess the effectiveness of pharmacological AMP-activated protein kinase (AMPK) activation upon the attenuation of inflammation in these myocytes....

  12. Nitration of soluble proteins in organotypic culture models of Parkinson's disease

    DEFF Research Database (Denmark)

    Larsen, Trine R; Söderling, Ann-Sofi; Caidahl, Kenneth

    2008-01-01

    Protein nitration due to oxidative and nitrative stress has been linked to the pathogenesis of Parkinson's disease (PD), but its relationship to the loss of dopamine (DA) or tyrosine hydroxylase (TH) activity is not clear. Here we quantified protein-bound 3-nitrotyrosine (3-NT) by a novel gas...... toxin 1-methyl-4-phenylpyridinium (MPP(+)) or the lipophilic complex I inhibitor rotenone. Incubation with SIN-1 (24h) or MPP(+) treatments (48h) caused dose-dependent protein nitration reaching a maximum of eightfold increase by 10mM SIN-1 or twofold by 10muM MPP(+), but significant DA depletions...... occurred at much lower concentrations of MPP(+) (1muM). Chronic MPP(+) or rotenone treatments (3 weeks) caused maximum protein nitration by 1muM (twofold) or 10nM (fourfold), respectively. Co-treatment with the nitric oxide synthase inhibitor l-NAME (300muM) prevented protein nitration by MPP(+), but did...

  13. Regulation of glutamate dehydrogenase activity in relation to carbon limitation and protein catabolism in carrot cell suspension cultures.

    Science.gov (United States)

    Robinson, S A; Stewart, G R; Phillips, R

    1992-03-01

    Glutamate dehydrogenase (GDH) specific activity and function have been studied in cell suspension cultures of carrot (Daucus carota L. cv Chantenay) in response to carbon and nitrogen supply in the culture medium. The specific activity of GDH was derepressed in sucrose-starved cells concomitant with protein catabolism, ammonium excretion, and the accumulation of metabolically active amino acids. The addition of sucrose led to a rapid decrease in GDH specific activity, an uptake of ammonium from the medium, and a decrease in amino acid levels. The extent of GDH derepression was correlated positively with cellular glutamate concentration. These findings strengthen the view that the function of GDH is the catabolism of glutamate, which under conditions of carbon stress provides carbon skeletons for tricarboxylic acid cycle activity.

  14. Comparison of protein patterns after two-dimensional gel electrophoresis from leaves of in vitro cultures and seedlings of Rubus chamaemorus L.

    Directory of Open Access Journals (Sweden)

    Barbara Thiem

    2014-01-01

    Full Text Available Proteins from leaves of Rubus chamaemorus propagated in vitro were subjected to miniaturized 2-D electrophoresis. The 2-DE patterns of proteins showed qualitative differences between plants propagated in vitro and control seedlings. More proteins of a high molecular weight were observed in leaves of plants from in vitro culture. A two-dimensional map of proteins from leaves provides detailed data concerning both polymorphism and protein patterns of this species. This makes it possible to start constructing a protein map of R. chamaemorus. The reasons for qualitative differences are discussed.

  15. Allelopathic Effects of Cyanobacterial Filtrates on Baltic Diatom

    Science.gov (United States)

    Śliwińska, Sylwia; Latała, Adam

    2012-01-01

    Allelopathy may be one of the factors affecting the formation of massive and harmful algal blooms in aquatic environments. Recent studies indicate that blooms of cyanobacteria in the Baltic Sea has grown significantly in last decades, so it is important to determine the allelopathic interactions between the dominant species of cyanobacteria and microalgae. In this work we investigated the influence of allelopathic compounds on the growth of Skeletonema marinoi by addition of cell-free filtrate of the Baltic cyanobacterium Nodularia spumigena cultures grown under different temperature (15-25°C). Additionally the effects of filtrates of both an exponential and a stationary growing culture of N. spumigena were tested on diatom. These studies indicate that high temperature affected the donor species by increasing its production of allelochemicals. The highest drop of growth of analyzed diatom were observed after the addition of cell-free filtrate obtained from N. spumigena grown at 25°C and constituted 70% of their control. N. spumigena was only allelopathic in exponential growth phase, whereas the cyanobacteria filtrate from stationary phase have any effect on S. marinoi. These findings suggest that N. spumigena may reveal allelopathic activity and that the production of allelopathic substances is influenced by the temperature and growth phase of cyanobacteria. Allelopatia może być kluczowym czynnikiem wpływającym na tworzenie się masowych zakwitów sinic w wielu wodnych ekosystemach. Badania pokazują, że zakwity sinic w Morzu Bałtyckim w ostatnich dekadach znacznie się nasiliły, dlatego tak ważne jest określenie stopnia oddziaływania allelopatycznego dominujących w tym akwenie gatunków fitoplanktonu. W przeprowadzonych badaniach określono wpływ związków allelopatycznych produkowanych przez bałtycką sinicę Nodularia spumigena hodowaną w różnych temperaturach (15-25°C) na wzrost okrzemki Skeletonema marinoi. Dodatkowo w niniejszej pracy por

  16. The tricarboxylic acid cycle activity in cultured primary astrocytes is strongly accelerated by the protein tyrosine kinase inhibitor tyrphostin 23

    DEFF Research Database (Denmark)

    Hohnholt, Michaela C; Blumrich, Eva-Maria; Waagepetersen, Helle S

    2017-01-01

    production. In addition, T23-treatment strongly increased the molecular carbon labeling of the TCA cycle intermediates citrate, succinate, fumarate and malate, and significantly increased the incorporation of (13)C-labelling into the amino acids glutamate, glutamine and aspartate. These results clearly......Tyrphostin 23 (T23) is a well-known inhibitor of protein tyrosine kinases and has been considered as potential anti-cancer drug. T23 was recently reported to acutely stimulate the glycolytic flux in primary cultured astrocytes. To investigate whether T23 also affects the tricarboxylic acid (TCA...

  17. Blockades of mitogen-activated protein kinase and calcineurin both change fibre-type markers in skeletal muscle culture

    DEFF Research Database (Denmark)

    Higginson, James; Wackerhage, Henning; Woods, Niall

    2002-01-01

    A and mitogen-activated protein kinase kinase (MEK1/2) blockade with U0126 upon myosin heavy chain (MHC) isoform mRNA levels and activities of metabolic enzymes after 1 day, 3 days and 7 days of treatment in primary cultures of spontaneously twitching rat skeletal muscle. U0126 treatment significantly decreased......Activation of either the calcineurin or the extracellular signal-regulated kinase (ERK1/2) pathway increases the percentage of slow fibres in vivo suggesting that both pathways can regulate fibre phenotypes in skeletal muscle. We investigated the effect of calcineurin blockade with cyclosporin...

  18. Proteolytic Activation of the Porcine Epidemic Diarrhea Coronavirus Spike Fusion Protein by Trypsin in Cell Culture.

    NARCIS (Netherlands)

    Wicht, Oliver|info:eu-repo/dai/nl/32291177X; Li, Wentao; Willems, Lione; Meuleman, Tom J; Wubbolts, Richard W|info:eu-repo/dai/nl/181688255; van Kuppeveld, Frank J M|info:eu-repo/dai/nl/156614723; Rottier, Peter J M|info:eu-repo/dai/nl/068451954; Bosch, Berend Jan|info:eu-repo/dai/nl/273306049

    2014-01-01

    Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infec

  19. Proteolytic Activation of the Porcine Epidemic Diarrhea Coronavirus Spike Fusion Protein by Trypsin in Cell Culture.

    NARCIS (Netherlands)

    Wicht, Oliver; Li, Wentao; Willems, Lione; Meuleman, Tom J; Wubbolts, Richard W; van Kuppeveld, Frank J M; Rottier, Peter J M; Bosch, Berend Jan

    2014-01-01

    Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infec

  20. Analysis of soybean tissue culture protein dynamics using difference gel electrophoresis

    Science.gov (United States)

    Excised hypocotyls from developing soybean (Glycine max (L.) merr. cv. Jack) were cultivated on agar-solidified medium until callus formed. The calli were then propagated in liquid medium until stable, relatively uniform, finely-divided suspension cultures were obtained. Cells were typically transfe...

  1. Transfer and transport of aluminum in filtration unit

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Aluminum salt coagulants were used prevalently in various water works. In this article, the effects of filtration on residual aluminum concentration and species distribution were researched by determining the concentration of different Aluminum species before and after single layer filter, double layer filter, and membrane filtration units. In the research, size exclusion chromatography (SEC) was used to separate colloidal and soluble aluminum, ion exchange chromatography (IEC) was used to separate organic and inorganic aluminum, and inductivity coupled plasma-atomic emission spectrometry (ICP-AES) was used to determine the aluminum concentration. The results showed that the rapid filtration process had the ability of removing residual aluminum from coagulant effluent water, and that double layer filtration was more effective in residual aluminum removal than single layer filtration, while Nano filtration was more effective than micro filtration. It was found that when the residual aluminum concentration was below 1mg/L in sediment effluent, the residual aluminum concentration in treated water was above 0.2 mg/L. The direct rapid filtration process mainly removed the suspended aluminum. The removal of soluble and colloidal aluminum was always less than 10% and the natural small particles that adsorbed the amount of soluble or small particles aluminum on their surface were difficult to be removed in this process. Micro filtration and nano filtration were good technologies for removing aluminum; the residual aluminum concentration in the effluent was less than 0.05 mg/L.

  2. Enhanced growth and recombinant protein production of Escherichia coli by a perfluorinated oxygen carrier in miniaturized fed-batch cultures

    Directory of Open Access Journals (Sweden)

    Neubauer Peter

    2011-06-01

    Full Text Available Abstract Background Liquid perfluorochemicals (PFCs are interesting oxygen carriers in medicine and biotechnology with a high solubility for oxygen. They have been repeatedly used for improving oxygen transfer into prokaryotic and eukaryotic cell cultures, however their application is still limited. Here we show the great benefit of air/oxygen saturated perfluorodecalin (PFD for high cell density cultivation of Escherichia coli in microwell plates and their positive effect on the soluble production of a correctly folded heterologously expressed alcohol dehydrogenase. Results In EnBase® cultivations the best effect was seen with PFD saturated with oxygen enriched air (appr. 10 μM oxygen per ml when PFD was added at the time of induction. In contrast the effect of PFD was negligible when it was added already at the time of inoculation. Optimisation of addition time and content of loaded oxygen into the PFD resulted in an increased the cell density by 40% compared to control cultures, and correspondingly also the product yield increased, demonstrated at the example of a recombinant alcohol dehydrogenase. Conclusions PFCs are a valuable additive in miniaturized cell culture formats. For production of recombinant proteins in low cell density shaken cultures the addition of oxygen-enriched PFD makes the process more robust, i.e. a high product yield is not any more limited to a very narrow cell density window during which the induction has to be done. The positive effect of PFD was even more obvious when it was added during high cell density cultures. The effect of the PFD phase depends on the amount of oxygen which is loaded into the PFD and which thus is a matter of optimisation.

  3. Benefits and Limitations of Protein Hydrolysates as Components of Serum-Free Media for Animal Cell Culture Applications

    Science.gov (United States)

    Lobo-Alfonso, Juliet; Price, Paul; Jayme, David

    Increased understanding of influential factors for the cultivation of animal cells, combined with heightened regulatory concern over potential transmission of adventitious contaminants associated with serum and other animal-derived components, has elevated interest in using protein hydrolysates as serum replacements or nutrient supplements. This paper reviews the chemistry and biology of various hydrolysates derived from animal, plant and microbial sources. It provides specific examples of a beneficial selection of plant and yeast hydrolysates as ingredients of serum-free nutrient formulations for bioproduction applications of cultured mammalian and insect cells. Strategies for customizing and optimizing nutrients for specialized applications and general benefits and limitations of protein hydrolysates for biopharmaceutical production are also discussed.

  4. Martingale representation property in progressively enlarged filtrations

    CERN Document Server

    Jeanblanc, M

    2012-01-01

    Consider $\\mathbb{G}$ the progressive enlargement of a filtration $\\mathbb{F}$ with a random time $\\tau$. Assuming that, in $\\mathbb{F}$, the martingale representation property holds, we examine conditions under which the martingale representation property holds also in $\\mathbb{G}$. It is noted that the classical results on this subject are no more sufficient to deal with all examples coming from credit risk modeling. In this paper, we introduce a new methodology which extends the various classical results and applies on recent examples.

  5. Supercoiled DNA folded by nonhistone proteins in cultured mouse carcinoma cells.

    Science.gov (United States)

    Nakane, M; Ide, T; Anzai, K; Ohara, S; Andoh, T

    1978-07-01

    Upon gentle lysis of exponentially growing mouse carcinoma cells FM3A by sodium dodecyl sulfate, DNA was released as a "DNA-protein complex" in a folded conformation. No histones could be detected in the DNA-protein complex. The proteins bound to DNA were found to be composed of several kinds of nonhistone proteins with a molecular weight range of 50,000 to 60,000; they appear to play a key role in stabilizing and maintaining the compact and folded structure of the complex. Removal of the proteins by Pronase or 2-mercaptoethanol produced a more relaxed structure sedimenting about half as fast as the original complex in a neutral sucrose gradient. DNA in the folded complex is supercoiled, as indicated by the characteristic biphasic response of its sedimentation rate to increasing concentration of various intercalating agents, actinomycin D, ethidium bromide and acriflavine, with which the cells were treated before lysis. Pronase- or 2-mercaptoethanol-treated relaxed DNA still possessed the characteristic of closed-circular structure as judged from its response to intercalating agents. Nicking with gamma-ray or 4NQO broke these superhelical turns and relaxed the folded complex to slower sedimenting forms equivalent to the relaxed DNA obtained on treatment with Pronase or 2-mercaptoethanol. Viscometric observations of DNA-protein complex were consistent with the above results. A tentative model for the structure of this DNA-protein complex is proposed in which supercoiled DNA is folded into loops by several kinds of nonhistone proteins. Autoradiographic examination of the complex appeared to support this model.

  6. Investigating unsaturated fat, monensin, or bromoethanesulfonate in continuous cultures retaining ruminal protozoa. I. Fermentation, biohydrogenation, and microbial protein synthesis.

    Science.gov (United States)

    Karnati, S K R; Sylvester, J T; Ribeiro, C V D M; Gilligan, L E; Firkins, J L

    2009-08-01

    Methane is an end product of ruminal fermentation that is energetically wasteful and contributes to global climate change. Bromoethanesulfonate, animal-vegetable fat, and monensin were compared with a control treatment to suppress different functional groups of ruminal prokaryotes in the presence or absence of protozoa to evaluate changes in fermentation, digestibility, and microbial N outflow. Four dual-flow continuous culture fermenter systems were used in 4 periods in a 4 x 4 Latin square design split into 2 subperiods. In subperiod 1, a multistage filter system (50-microm smallest pore size) retained most protozoa. At the start of subperiod 2, conventional filters (300-microm pore size) were substituted to efflux protozoa via filtrate pumps over 3 d; after a further 7 d of adaptation, the fermenters were sampled for 3 d. Treatments were retained during both subperiods. Flow of total N and digestibilities of NDF and OM were 18, 16, and 9% higher, respectively, for the defaunated subperiod but were not different among treatments. Ammonia concentration was 33% higher in the faunated fermenters but was not affected by treatment. Defaunation increased the flow of nonammonia N and bacterial N from the fermenters. Protozoal counts were not different among treatments, but bromoethanesulfonate increased the generation time from 43.2 to 55.6 h. Methanogenesis was unaffected by defaunation but tended to be increased by unsaturated fat. Defaunation did not affect total volatile fatty acid production but decreased the acetate:propionate ratio; monensin increased production of isovalerate and valerate. Biohydrogenation of unsaturated fatty acids was impaired in the defaunated fermenters because effluent flows of oleic, linoleic, and linolenic acids were 60, 77, and 69% higher, and the ratio of vaccenic acid:unsaturated FA ratio was decreased by 34% in the effluent. This ratio was increased in both subperiods with the added fat diet, indicating an accumulation of

  7. Impact of 7,12-dimethylbenz[a]anthracene exposure on connexin gap junction proteins in cultured rat ovaries

    Energy Technology Data Exchange (ETDEWEB)

    Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2014-01-15

    7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles in a concentration-dependent manner. The impact of DMBA on connexin (CX) proteins that mediate communication between follicular cell types along with pro-apoptotic factors p53 and Bax were investigated. Postnatal day (PND) 4 Fisher 344 rat ovaries were cultured for 4 days in vehicle medium (1% DMSO) followed by a single exposure to vehicle control (1% DMSO) or DMBA (12.5 nM or 75 nM) and cultured for 4 or 8 days. RT-PCR was performed to quantify Cx37, Cx43, p53 and Bax mRNA level. Western blotting and immunofluorescence staining were performed to determine CX37 or CX43 level and/or localization. Cx37 mRNA and protein increased (P < 0.05) at 4 days of 12.5 nM DMBA exposure. Relative to vehicle control-treated ovaries, mRNA encoding Cx43 decreased (P < 0.05) but CX43 protein increased (P < 0.05) at 4 days by both DMBA exposures. mRNA expression of pro-apoptotic p53 was decreased (P < 0.05) but no changes in Bax expression were observed after 4 days of DMBA exposures. In contrast, after 8 days, DMBA decreased Cx37 and Cx43 mRNA and protein but increased both p53 and Bax mRNA levels. CX43 protein was located between granulosa cells, while CX37 was located at the oocyte cell surface of all follicle stages. These findings support that DMBA exposure impacts ovarian Cx37 and Cx43 mRNA and protein prior to both observed changes in pro-apoptotic p53 and Bax and follicle loss. It is possible that such interference in follicular cell communication is detrimental to follicle viability, and may play a role in DMBA-induced follicular atresia. - Highlights: • DMBA increases Cx37 and Cx43 expression prior to follicle loss. • During follicle loss both Cx37 and Cx43 expressions are reduced. • CX43 protein is absent in follicle remnants lacking an oocyte.

  8. Acrolein stimulates the synthesis of IL-6 and C-reactive protein (CRP) in thrombosis model mice and cultured cells.

    Science.gov (United States)

    Saiki, Ryotaro; Hayashi, Daisuke; Ikuo, Yukiko; Nishimura, Kazuhiro; Ishii, Itsuko; Kobayashi, Kaoru; Chiba, Kan; Toida, Toshihiko; Kashiwagi, Keiko; Igarashi, Kazuei

    2013-12-01

    Measurements of protein-conjugated acrolein (PC-Acro), IL-6, and C-reactive protein (CRP) in plasma were useful for identifying silent brain infarction with high sensitivity and specificity. The aim of this study was to determine whether acrolein causes increased production of IL-6 and CRP in thrombosis model mice and cultured cells. In mice with photochemically induced thrombosis, acrolein produced at the locus of infarction increased the level of IL-6 and then CRP in plasma. This was confirmed in cell culture systems - acrolein stimulated the production of IL-6 in mouse neuroblastoma Neuro-2a cells, mouse macrophage-like J774.1 cells, and human umbilical vein endothelial cells (HUVEC), and IL-6 in turn stimulated the production of CRP in human hepatocarcinoma cells. The level of IL-6 mRNA was increased by acrolein through an increase in phosphorylation of the transcription factors, c-Jun, and NF-κB p65. Furthermore, CRP stimulated IL-6 production in mouse macrophage-like J774.1 cells and HUVEC. IL-6 functioned as a protective factor against acrolein toxicity in Neuro-2a cells and HUVEC. These results show that acrolein stimulates the synthesis of IL-6 and CRP, which function as protecting factors against acrolein toxicity, and that the combined measurement of PC-Acro, IL-6, and CRP is effective for identification of silent brain infarction. The combined measurements of protein-conjugated acrolein (PC-Acro), IL-6, and C-reactive protein (CRP) in plasma were useful for identifying silent brain infarction. The aim of this study was to determine whether acrolein causes increased production of IL-6 and CRP, and indeed acrolein increased IL-6 synthesis and IL-6 in turn increased CRP synthesis. Furthermore, IL-6 decreased acrolein toxicity in several cell lines.

  9. Observations on the use of membrane filtration and liquid impingement to collect airborne microorganisms in various atmospheric environments

    Science.gov (United States)

    Griffin, Dale W.; Gonzalez, C.; Teigell, N.; Petrosky, T.; Northup, D.E.; Lyles, M.

    2011-01-01

    The influence of sample-collection-time on the recovery of culturable airborne microorganisms using a low-flow-rate membrane-filtration unit and a high-flow-rate liquid impinger were investigated. Differences in recoveries were investigated in four different atmospheric environments, one mid-oceanic at an altitude of ~10.0 m, one on a mountain top at an altitude of ~3,000.0 m, one at ~1.0 m altitude in Tallahassee, Florida, and one at ~1.0 m above ground in a subterranean-cave. Regarding use of membrane filtration, a common trend was observed: the shorter the collection period, the higher the recovery of culturable bacteria and fungi. These data also demonstrated that lower culturable counts were common in the more remote mid-oceanic and mountain-top atmospheric environments with bacteria, fungi, and total numbers averaging (by sample time or method categories) filtration for aeromicrobiology studies if start-up costs are not an issue and temperature permits use; (2) although membrane filtration is more cost friendly and has a 'typically' wider operational range, its limits include loss of cell viability with increased sample time and issues with effectively extracting nucleic acids for community-based analyses; (3) the ability to recover culturable microorganisms is limited in 'extreme' atmospheric environments and thus the use of a 'limited' methodology in these environments must be taken into account; and (4) the atmosphere culls, i.e., everything is not everywhere. ?? 2010 US Government.

  10. Reaction of small heat-shock proteins to different kinds of cellular stress in cultured rat hippocampal neurons.

    Science.gov (United States)

    Bartelt-Kirbach, Britta; Golenhofen, Nikola

    2014-01-01

    Upregulation of small heat-shock proteins (sHsps) in response to cellular stress is one mechanism to increase cell viability.We previously described that cultured rat hippocampal neurons express five of the 11 family members but only upregulate two of them (HspB1 and HspB5) at the protein level after heat stress. Since neurons have to cope with many other pathological conditions, we investigated in this study the expression of all five expressed sHsps on mRNA and protein level after sublethal sodium arsenite and oxidative and hyperosmotic stress. Under all three conditions, HspB1, HspB5, HspB6, and HspB8 but not HspB11 were consistently upregulated but showed differences in the time course of upregulation. The increase of sHsps always occurred earlier on mRNA level compared with protein levels. We conclude from our data that these four upregulated sHsps (HspB1, HspB5, HspB6, HspB8) act together in different proportions in the protection of neurons from various stress conditions.

  11. Changes in the effective gravitational field strength affect the state of phosphorylation of stress-related proteins in callus cultures of Arabidopsis thaliana

    OpenAIRE

    Barjaktarović, Žarko; Schütz, Wolfgang; Madlung, Johannes; Fladerer, Claudia; Nordheim, Alfred; Hampp, Rüdiger

    2009-01-01

    In a recent study it was shown that callus cell cultures of Arabidopsis thaliana respond to changes in gravitational field strengths by changes in protein expression. Using ESI-MS/MS for proteins with differential abundance after separation by 2D-PAGE, 28 spots which changed reproducibly and significantly in amount (P

  12. Expression of peroxisome proliferator-activated receptor and CCAAT/enhancer binding protein transcription factors in cultured human sebocytes.

    Science.gov (United States)

    Chen, WenChieh; Yang, Chao-Chun; Sheu, Hamm-Ming; Seltmann, Holger; Zouboulis, Christos C

    2003-09-01

    Lipid synthesis and accumulation represent a major step in sebocyte differentiation and it may be of importance for sebocytes to express two families of transcription factors, CCAAT/enhancer binding proteins (c/EBPs) and peroxisome proliferator-activated receptors (PPARs), which were found to play a crucial role in the differentiation of adipocytes. Using the immortalized human sebaceous gland cell line SZ95 we examined the expression of the molecules before and after treatment with testosterone, 5alpha-dihydrotestosterone, dexamethasone, 17beta-estradiol and genistein, at 6, 12, 24, and 48 h, respectively. Reverse transcription-PCR analysis showed expression of peroxisome proliferator-activated receptors -alpha, -delta, -gamma1, -gamma2 and CCAAT/enhancer binding proteins-alpha, -beta, -gamma-delta in native SZ95 sebocytes. In western blot studies, high levels of CCAAT/enhancer binding proteins-alpha and -beta, and peroxisome proliferator-activated receptors-gamma were expressed at 6, 24, and 12 h, respectively. Immunostaining of the cultured sebocytes showed the CCAAT/enhancer binding proteins-alpha and -beta mainly localized within nuclei, whereas peroxisome proliferator-activated receptors-gamma in the cytoplasm. Strong staining of sebocytes was immunohistochemically revealed in the basal layer of sebaceous glands in human scalp and sebaceous nevus. Genistein down-regulated the expression of CCAAT/enhancer binding proteins-alpha and -beta, and peroxisome proliferator-activated receptors-gamma on the protein level. Treatment with linoleic acid for 48 h induced further differentiation of sebocytes leading to abundant lipid synthesis.

  13. Impact of Cystinosin Glycosylation on Protein Stability by Differential Dynamic Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).

    Science.gov (United States)

    Nevo, Nathalie; Thomas, Lucie; Chhuon, Cerina; Andrzejewska, Zuzanna; Lipecka, Joanna; Guillonneau, François; Bailleux, Anne; Edelman, Aleksander; Antignac, Corinne; Guerrera, Ida Chiara

    2017-03-01

    Cystinosis is a rare autosomal recessive lysosomal storage disorder characterized by intralysosomal accumulation of cystine. The causative gene for cystinosis is CTNS, which encodes the protein cystinosin, a lysosomal proton-driven cystine transporter. Over 100 mutations have been reported, leading to varying disease severity, often in correlation with residual cystinosin activity as a transporter and with maintenance of its protein-protein interactions. In this study, we focus on the ΔITILELP mutation, the only mutation reported that sometimes leads to severe forms, inconsistent with its residual transported activity. ΔITILELP is a deletion that eliminates a consensus site on N66, one of the protein's seven glycosylation sites. Our hypothesis was that the ΔITILELP mutant is less stable and undergoes faster degradation. Our dynamic stable isotope labeling by amino acids in cell culture (SILAC) study clearly showed that wild-type cystinosin is very stable, whereas ΔITILELP is degraded three times more rapidly. Additional lysosome inhibition experiments confirmed ΔITILELP instability and showed that the degradation was mainly lysosomal. We observed that in the lysosome, ΔITILELP is still capable of interacting with the V-ATPase complex and some members of the mTOR pathway, similar to the wild-type protein. Intriguingly, our interactomic and immunofluorescence studies showed that ΔITILELP is partially retained at the endoplasmic reticulum (ER). We proposed that the ΔITILELP mutation causes protein misfolding, ER retention and inability to be processed in the Golgi apparatus, and we demonstrated that ΔITILELP carries high-mannose glycans on all six of its remaining glycosylation sites. We found that the high turnover of ΔITILELP, because of its immature glycosylation state in combination with low transport activity, might be responsible for the phenotype observed in some patients.

  14. Low-cost multi-stage filtration enhanced by coagulation-flocculation in upflow gravel filtration

    NARCIS (Netherlands)

    Sanchez, L.D.; Marin, L.M.; Visscher, J.T.; Rietveld, L.C.

    2012-01-01

    This paper assesses the operational and design aspects of coagulation and flocculation in upflow gravel filters (CF-UGF) in a multi-stage filtration (MSF) plant. This study shows that CF-UGF units improve the performance of MSF considerably, when the system operates with turbidity above 30 NTU. It s

  15. Dynamic optimization of a dead-end filtration trajectory : Non-ideal cake filtration

    NARCIS (Netherlands)

    Blankert, Bastiaan; Kattenbelt, Carolien; Betlem, Ben H.L.; Roffel, Brian

    2007-01-01

    A control strategy aimed at minimizing energy consumption is formulated for non-ideal dead-end cake filtration with an inside-out hollow fiber ultrafiltration membrane system. The non-ideal behavior was assumed to originate from cake compression, non-linear cake resistance and a variable pump effici

  16. Dynamic optimization of a dead-end filtration trajectory : Blocking filtration laws

    NARCIS (Netherlands)

    Blankert, Bastiaan; Betlem, Ben H.L.; Roffel, Brian

    2006-01-01

    An operating model for dead-end membrane filtration is proposed based on the well-known blocking laws. The resulting model contains three parameters representing, the operating strategy, the fouling mechanism and the fouling potential of the feed. The optimal control strategy is determined by minimi

  17. Variability in protein quality used for embryo culture: embryotoxicity of the stabilizer octanoic acid.

    Science.gov (United States)

    Leonard, Phoebe H; Charlesworth, M Cristine; Benson, Linda; Walker, David L; Fredrickson, Jolene R; Morbeck, Dean E

    2013-08-01

    To screen human serum albumin (HSA) preparations for toxicity and investigate causes of variation. Experimental laboratory study. University-based laboratory. FVB and CF1 mice crossed to create embryos used in experiments. Mouse embryo assay performed with 5% or 15% HSA (100 mg/mL albumin) from three samples from three separate manufacturers (A, B, C). Blastocyst rates calculated at 96 hours of culture (experiments repeated in triplicate). The HSA preparations were desalted to remove stabilizers added during HSA processing, then mass spectrometry was used to determine the relative variation in stabilizer concentrations; the effect of the stabilizer octanoic acid on embryo development was tested. At 5% HSA, all samples had blastocyst rates ≥ 70%; at 15% HSA, the blastocyst rates for samples B and C were culture should be monitored and limited. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  18. Immunogenicity of murein-associated proteins from temperature-stressed Streptococcus suis cultures.

    Science.gov (United States)

    Benga, L; Goethe, R; Grosse Beilage, E; Valentin-Weigand, P

    2004-08-01

    We compared immunogenicity in pigs of whole cell lysate proteins (WCP) with murein-associated proteins (MAP) obtained from a virulent serotype 2 strain of Streptococcus (S.) suis grown at 32 or 42 degrees C. Protein fractions were tested for their ability to induce antibodies in 3-week-old piglets by enzyme-linked immunosorbent assay and Western blot analysis. We found a significant increase in the antibody levels in all sera irrespective of the preparation used for immunization. However, alpha-WCP sera showed higher reactivities than alpha-MAP sera, and piglets immunized with 32 degrees C preparations (alpha-32 sera) showed higher responses than those immunized with 42 degrees C preparations (alpha-42 sera). Western blot analysis revealed that alpha-WCP sera in part reacted with different proteins when compared with alpha-MAP sera. Furthermore, some proteins were only detected by alpha-32 but not by alpha-42 sera. In conclusion, the results demonstrate the immunogenicity of cell wall MAP of S. suis, and highlight the importance of considering growth conditions in the preparation of subunit vaccines.

  19. Effects of phenylalanine and threonine oligopeptides on milk protein synthesis in cultured bovine mammary epithelial cells.

    Science.gov (United States)

    Zhou, M M; Wu, Y M; Liu, H Y; Liu, J X

    2015-04-01

    This study was conducted to investigate the effects of phenylalanine (Phe) and threonine (Thr) oligopeptides on αs1 casein gene expression and milk protein synthesis in bovine mammary epithelial cells. Primary mammary epithelial cells were obtained from Holstein dairy cows and incubated in Dulbecco's modified Eagle's medium-F12 medium (DMEM/F12) containing lactogenic hormones (prolactin and glucocorticoids). Free Phe (117 μg/ml) was substituted partly with peptide-bound Phe (phenylalanylphenylalanine, phenylalanyl threonine, threonyl-phenylalanyl-phenylalanine) in the experimental media. After incubation with experimental medium, cells were collected for gene expression analysis and medium was collected for milk protein or amino acid determination. The results showed that peptide-bound Phe at 10% (11.7 μg/ml) significantly enhanced αs1 casein gene expression and milk protein synthesis as compared with equivalent amount of free Phe. When 10% Phe was replaced by phenylalanylphenylalanine, the disappearance of most essential amino acids increased significantly, and gene expression of peptide transporter 2 and some amino acid transporters was significantly enhanced. These results indicate that the Phe and Thr oligopeptides are important for milk protein synthesis, and peptide-bound amino acids could be utilised more efficiently in milk protein synthesis than the equivalent amount of free amino acids.

  20. METC CFD simulations of hot gas filtration

    Energy Technology Data Exchange (ETDEWEB)

    O`Brien, T.J.

    1995-06-01

    Computational Fluid Dynamic (CFD) simulations of the fluid/particle flow in several hot gas filtration vessels will be presented. These simulations have been useful in designing filtration vessels and in diagnosing problems with filter operation. The simulations were performed using the commercial code FLUENT and the METC-developed code MFIX. Simulations of the initial configuration of the Karhula facility indicated that the dirty gas flow over the filter assemblage was very non-uniform. The force of the dirty gas inlet flow was inducing a large circulation pattern that caused flow around the candles to be in opposite directions on opposite sides of the vessel. By introducing a system of baffles, a more uniform flow pattern was developed. This modification may have contributed to the success of the project. Several simulations of configurations proposed by Industrial Filter and Pump were performed, varying the position of the inlet. A detailed resolution of the geometry of the candles allowed determination of the flow between the individual candles. Recent simulations in support of the METC/CeraMem Cooperative Research and Development Agreement have analyzed the flow in the vessel during the cleaning back-pulse. Visualization of experiments at the CeraMem cold-flow facility provided confidence in the use of CFD. Extensive simulations were then performed to assist in the design of the hot test facility being built by Ahlstrom/Pyropower. These tests are intended to demonstrate the CeraMem technology.

  1. Diatomite releases silica during spirit filtration.

    Science.gov (United States)

    Gómez, J; Gil, M L A; de la Rosa-Fox, N; Alguacil, M

    2014-09-15

    The purpose of this study was to ascertain whether diatomite is an inert filter aid during spirit filtration. Surely, any compound with a negative effect on the spirit composition or the consumer's health could be dissolved. In this study different diatomites were treated with 36% vol. ethanol/water mixtures and the amounts and structures of the extracted compounds were determined. Furthermore, Brandy de Jerez was diatomite- and membrane-filtered at different temperatures and the silicon content was analysed. It was found that up to 0.36% by weight of diatomite dissolved in the aqueous ethanol and amorphous silica, in the form of hollow spherical microparticles, was the most abundant component. Silicon concentrations in Brandy de Jerez increased by up to 163.0% after contact with diatomite and these changes were more marked for calcined diatomite. In contrast, reductions of more than 30% in silicon concentrations were achieved after membrane filtration at low temperatures. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Tailoring Supramolecular Nanofibers for Air Filtration Applications.

    Science.gov (United States)

    Weiss, Daniel; Skrybeck, Dominik; Misslitz, Holger; Nardini, David; Kern, Alexander; Kreger, Klaus; Schmidt, Hans-Werner

    2016-06-15

    The demand of new materials and processes for nanofiber fabrication to enhance the performance of air filters is steadily increasing. Typical approaches to obtain nanofibers are based on top-down processes such as melt blowing, centrifugal spinning, and electrospinning of polymer materials. However, fabrication of polymer nanofibers is limited with respect to either a sufficiently high throughput or the smallest achievable fiber diameter. This study reports comprehensively on a fast and simple bottom-up process to prepare supramolecular nanofibers in situ inside viscose/polyester microfiber nonwovens. Here, selected small molecules of the materials class of 1,3,5-benzenetrisamides are employed. The microfiber-nanofiber composites exhibit a homogeneous nanofiber distribution and morphology throughout the entire nonwoven scaffold. Small changes in molecular structure and processing solvent have a strong influence on the final nanofiber diameter and diameter distribution and, consequently, on the filtration performance. Choosing proper processing conditions, microfiber-nanofiber composites with surprisingly high filtration efficiencies of particulate matter are obtained. In addition, the microfiber-nanofiber composite integrity at elevated temperatures was determined and revealed that the morphology of supramolecular nanofibers is maintained compared to that of the utilized polymer nonwoven.

  3. Renal filtration function in patients with gout

    Directory of Open Access Journals (Sweden)

    N. N. Kushnarenko

    2016-01-01

    Full Text Available Aim. To study circadian blood pressure (BP profile in patients with gout depending on the presence of arterial hypertension (HT and their relationship to the renal filtration function.Material and methods. Patients with gout (n=87 were included into the study. All the patients underwent ambulatory BP monitoring (ABPM with the assessment of circadian BP profile, determination of uric acid serum levels, glomerular filtration rate (GFR was evaluated by CKD-EPI method. Depending on GFR level, all the patients were divided into 2 groups - with renal dysfunction or without one.Results. ABPM revealed circadian BP dysregulation in 55% of gout patients both with HT and without HT. Chronic kidney disease (CKD was revealed in 72.4% of male patients, with the prevalence in patients with HT (76.6 vs 61%; p<0.001. Correlations between uric acid levels and some ABPM indicators and GFR were determined.Conclusion. Obtained data suggest the contribution of hyperuricemia in disorders of systemic and renal hemodynamics, leading to the early development of CKD.

  4. Riverbed Clogging and Sustainability of Riverbank Filtration

    Directory of Open Access Journals (Sweden)

    Thomas Grischek

    2016-12-01

    Full Text Available Clogging refers to a reduction of riverbed hydraulic conductivity. Due to difficulties in determining the thickness of the clogging layer, the leakage coefficient (L is introduced and used to quantify the recoverable portion of bank filtrate. L was determined at several riverbank filtration (RBF sites in field tests and using an analytical solution. Results were compared with data from similar experiments in the early 1970s and 1991–1993. In the 1980s, severe river water pollution in conjunction with high water abstraction led to partly unsaturated conditions beneath the riverbed. A leakage coefficient L of 5 × 10−7 s−1 was determined. After water quality improvement, L increased to 1–1.5 × 10−6 s−1. An alternative, cost and time efficient method is presented to estimate accurate leakage coefficients. The analytical solution is based on groundwater level monitoring data from observation wells next to the river, which can later feed into numerical models. The analytical approach was able to reflect long-term changes as well as seasonal variations. Recommendations for its application are given based on experience.

  5. T cell-mediated cytotoxicity against p53-protein derived peptides in bulk and limiting dilution cultures of healthy donors

    DEFF Research Database (Denmark)

    Röpke, M; Regner, M; Claesson, M H

    1995-01-01

    The p53 tumour suppressor gene product plays an important role in the development of most human cancers. Point mutations in the p53 gene are common in malignant states and results in over-expression of wild type and mutant determinants of the p53 protein. This process might generate MHC-I restric......The p53 tumour suppressor gene product plays an important role in the development of most human cancers. Point mutations in the p53 gene are common in malignant states and results in over-expression of wild type and mutant determinants of the p53 protein. This process might generate MHC......-I restricted epitopes for T cell recognition and p53-derived peptides have been suggested as targets for tumour-specific cytotoxic T lymphocytes (CTL). Our primary aim was to estimate the frequencies of p53-peptide reactive CTL precursors (CTLp) in peripheral blood from healthy young individuals. We selected...... wild type and mutated peptides derived from the p53 sequence with a binding motif for HLA-A2.1 molecules. Peripheral blood mononuclear cells (PBMC) from healthy HLA-A2 donors were stimulated in vitro in bulk cultures as well as in limiting dilution cultures using autologous cells pulsed with p53...

  6. A study of dynamic filtration; Um estudo sobre filtracao dinamica

    Energy Technology Data Exchange (ETDEWEB)

    Girao, Joaquim Helder S. [PETROBRAS, Natal, RN (Brazil). Distrito de Perfuracao da Bacia Potiguar. Div. de Tecnicas de Perfuracao

    1989-12-31

    The problems that cause cost increase such as: formation damage and borehole swelling or caving lead us to study the filtration of the liquid part of formation drilling fluid. With the aim of comparing static and dynamic filtration rates, we developed a modest dynamic filtration equipment, consisting of a modified API filter, connected to reservoir by means of a positive injection pump. We carried out various tests, and the results were set in charts and tables. Through these, it is possible to notice how the static and dynamic filtration curves come apart for a same pressure value. We also evaluated the effects of circulation speed, starch concentration and counter pressure. This paper does not include calculations or mathematical models accounting for filtrate invasion radii, but it demonstrates, for example, that cleaning circulation will cause lower filtration rates at lower flows. (author) 5 refs., 11 figs., 14 tabs.

  7. Physicochemical interaction and its influence on deep bed filtration process.

    Science.gov (United States)

    Guo, Jin-long; Meng, Jun; Li, Gui-ping; Luan, Zhao-kun; Tang, Hong-xiao

    2004-01-01

    The capillary model was used to analyze the hydraulic conditions in the deep bed filtration process. The physicochemical interaction forces between the filter media and suspended particles and their influence on deep bed filtration process were also studied theoretically. Through the comparison of the hydraulic and physicochemical forces, the key influencing factors on the filtration process were proposed and investigated. Pilot study of the microflocculation deep bed filtration was carried out in the No. 9 Potable Water Treatment Plant of Beijing, and the experimental results of hydraulic head loss, particle distribution and entrapment were presented. The theoretical prediction was reasonably consistent with the experimental results under different conditions, which indicated that the regulation and control of micro-flocculation and deep bed filtration could be realized by the evaluation of the physicochemical interactions. Further theoretical and experimental research should be carried out to investigate the interaction mechanism and its application in the deep bed filtration and other cases.

  8. Application of Quality by Design to the characterization of the cell culture process of an Fc-Fusion protein.

    Science.gov (United States)

    Rouiller, Yolande; Solacroup, Thomas; Deparis, Véronique; Barbafieri, Marco; Gleixner, Ralf; Broly, Hervé; Eon-Duval, Alex

    2012-06-01

    The production bioreactor step of an Fc-Fusion protein manufacturing cell culture process was characterized following Quality by Design principles. Using scientific knowledge derived from the literature and process knowledge gathered during development studies and manufacturing to support clinical trials, potential critical and key process parameters with a possible impact on product quality and process performance, respectively, were determined during a risk assessment exercise. The identified process parameters were evaluated using a design of experiment approach. The regression models generated from the data allowed characterizing the impact of the identified process parameters on quality attributes. The main parameters having an impact on product titer were pH and dissolved oxygen, while those having the highest impact on process- and product-related impurities and variants were pH and culture duration. The models derived from characterization studies were used to define the cell culture process design space. The design space limits were set in such a way as to ensure that the drug substance material would consistently have the desired quality.

  9. Simulation and prediction of protein production in fed-batch E. coli cultures: An engineering approach.

    Science.gov (United States)

    Calleja, Daniel; Kavanagh, John; de Mas, Carles; López-Santín, Josep

    2016-04-01

    An overall model describing the dynamic behavior of fed-batch E. coli processes for protein production has been built, calibrated and validated. Using a macroscopic approach, the model consists of three interconnected blocks allowing simulation of biomass, inducer and protein concentration profiles with time. The model incorporates calculation of the extra and intracellular inducer concentration, as well as repressor-inducer dynamics leading to a successful prediction of the product concentration. The parameters of the model were estimated using experimental data of a rhamnulose-1-phosphate aldolase-producer strain, grown under a wide range of experimental conditions. After validation, the model has successfully predicted the behavior of different strains producing two different proteins: fructose-6-phosphate aldolase and ω-transaminase. In summary, the presented approach represents a powerful tool for E. coli production process simulation and control.

  10. In-Water Hull Cleaning & Filtration System

    Science.gov (United States)

    George, Dan

    2015-04-01

    Dan George R & D Mining Technology LinkedIn GRD Franmarine have received the following prestigious awards in 2014 for their research & development of an in-water hull cleaning and filtration system "The Envirocart: Golden Gecko Award for Environmental Excellence; WA Innovator of the Year - Growth Sector; Department of Fisheries - Excellence in Marine Biosecurity Award - Innovation Category; Lloyd's List Asia Awards - Environmental Award; The Australian Innovation Challenge - Environment, Agriculture and Food Category; and Australian Shipping and Maritime Industry Award - Environmental Transport Award. The Envirocart developed and patented by GRD Franmarine is a revolutionary new fully enclosed capture and containment in-water hull cleaning technology. The Envirocart enables soft Silicon based antifouling paints and coatings containing pesticides such as Copper Oxide to be cleaned in situ using a contactless cleaning method. This fully containerised system is now capable of being deployed to remote locations or directly onto a Dive Support Vessel and is rated to offshore specifications. This is the only known method of in-water hull cleaning that complies with the Department of Agriculture Fisheries and Forestry (DAFF) and Department of Fisheries WA (DoF) Guidelines. The primary underwater cleaning tool is a hydraulically powered hull cleaning unit fitted with rotating discs. The discs can be fitted with conventional brushes for glass or epoxy based coatings or a revolutionary new patented blade system which can remove marine biofouling without damaging the antifouling paint (silicone and copper oxide). Additionally there are a patented range of fully enclosed hand cleaning tools for difficult to access niche areas such as anodes and sea chests, providing an innovative total solution that enables in-water cleaning to be conducted in a manner that causes no biological risk to the environment. In full containment mode or when AIS are present, material is pumped

  11. The effect of filter cake viscoelasticity on filtration

    DEFF Research Database (Denmark)

    Christensen, Morten Lykkegaard

    Wastewater is usually treated biologically, and a by-product from this treatment is waste-activated sludge with high water content. Sludge is therefore often filtrated to reduce the volume for handling and disposal, and to minimise the water content before incineration of the sludge. However...... a function of pressure as assumed when using the conventional models. Moreover, it is shown how the existing filtration models can be modified and the filtration process for activated sludge simulated....

  12. Analysis of fouling development under dynamic membrane filtration operation

    OpenAIRE

    Saleem, Mubashir; Alibardi, Luca; Cossu, Raffaello; Lavagnolo, Maria Cristina; Spagni, Alessandro

    2016-01-01

    This research is a contribution towards evaluating the appropriate fouling mechanism responsible for the flux decline under dynamic membrane (DM) filtration and its formation mechanism by using gravity-driven filtration in a specifically designed experimental setup. Series of extended short term filtration experiments were performed at varying operating conditions of mixed liquor suspended solids (MLSS) concentrations, trans-membrane pressures (TMP) and mesh pore sizes. Blocking models were a...

  13. Removal of cytomegalovirus DNA from donor blood by filtration.

    Science.gov (United States)

    Smith, K L; Cobain, T; Dunstan, R A

    1993-04-01

    Blood from five donors, previously shown to be positive for cytomegalovirus (CMV) DNA following polymerase chain reaction (PCR) amplification, was filtered through commercially available leucocyte filters. Analysis of pre- and post-filtration samples by PCR with ethidium bromide staining has shown that filtration was successful in removing CMV DNA from all samples. This is evidence that leucocyte filtration of red cell concentrates may greatly decrease the risk of CMV disease following transfusion to susceptible patients.

  14. Human Cells Cultured under Physiological Oxygen Utilize Two Cap-binding Proteins to recruit Distinct mRNAs for Translation.

    Science.gov (United States)

    Timpano, Sara; Uniacke, James

    2016-05-13

    Translation initiation is a focal point of translational control and requires the binding of eIF4E to the 5' cap of mRNA. Under conditions of extreme oxygen depletion (hypoxia), human cells repress eIF4E and switch to an alternative cap-dependent translation mediated by a homolog of eIF4E, eIF4E2. This homolog forms a complex with the oxygen-regulated hypoxia-inducible factor 2α and can escape translation repression. This complex mediates cap-dependent translation under cell culture conditions of 1% oxygen (to mimic tumor microenvironments), whereas eIF4E mediates cap-dependent translation at 21% oxygen (ambient air). However, emerging evidence suggests that culturing cells in ambient air, or "normoxia," is far from physiological or "normal." In fact, oxygen in human tissues ranges from 1-11% or "physioxia." Here we show that two distinct modes of cap-dependent translation initiation are active during physioxia and act on separate pools of mRNAs. The oxygen-dependent activities of eIF4E and eIF4E2 are elucidated by observing their polysome association and the status of mammalian target of rapamycin complex 1 (eIF4E-dependent) or hypoxia-inducible factor 2α expression (eIF4E2-dependent). We have identified oxygen conditions where eIF4E is the dominant cap-binding protein (21% normoxia or standard cell culture conditions), where eIF4E2 is the dominant cap-binding protein (1% hypoxia or ischemic diseases and cancerous tumors), and where both cap-binding proteins act simultaneously to initiate the translation of distinct mRNAs (1-11% physioxia or during development and stem cell differentiation). These data suggest that the physioxic proteome is generated by initiating translation of mRNAs via two distinct but complementary cap-binding proteins.

  15. Microfluidic filtration system to isolate extracellular vesicles from blood.

    Science.gov (United States)

    Davies, Ryan T; Kim, Junho; Jang, Su Chul; Choi, Eun-Jeong; Gho, Yong Song; Park, Jaesung

    2012-12-21

    Extracellular vesicles are released by various cell types, particularly tumor cells, and may be potential targets for blood-based cancer diagnosis. However, studies performed on blood-borne vesicles to date have been limited by lack of effective, standardized purification strategies. Using in situ prepared nanoporous membranes, we present a simple strategy employing a microfluidic filtration system to isolate vesicles from whole blood samples. This method can be applied to purify nano-sized particles from blood allowing isolation of intact extracellular vesicles, avoiding the need for laborious and potentially damaging centrifugation steps or overly specific antibody-based affinity purification. Porous polymer monoliths were integrated as membranes into poly(methyl methacrylate) microfluidic chips by benchtop UV photopolymerization through a mask, allowing precise positioning of membrane elements while preserving simplicity of device preparation. Pore size could be manipulated by changing the ratio of porogenic solvent to prepolymer solution, and was tuned to a size proper for extraction of vesicles. Using the membrane as a size exclusion filter, we separated vesicles from cells and large debris by injecting whole blood under pressure through the microfluidic device. To enhance isolation purity, DC electrophoresis was employed as an alternative driving force to propel particles across the filter and increase the separation efficiency of vesicles from proteins. From the whole blood of melanoma-grown mice, we isolated extracellular vesicles and performed RT-PCR to verify their contents of RNA. Melan A mRNA derived from melanoma tumor cells were found enriched in filtered samples, confirming the recovery of vesicles via their cargo. This filtration system can be incorporated into other on-chip processes enabling integrated sample preparation for the downstream analysis of blood-based extracellular vesicles.

  16. Study of effect of oxygen/glucose-deprived culture on the brain-pancreas relative protein in PC12 cells and the mechanism

    Institute of Scientific and Technical Information of China (English)

    Yan-huaLIN; LuTIE; Ai-huaLIU; Xue-junLI

    2004-01-01

    AIM: To study the effect of oxygen/glucose-deprived (OGD)culture on the expression of a novel protein, brain-pancreas relative protein (BPRP), and the possible regulating mechanism in vitro. BPRP was a key protein found in our previous study of cerebral ischemia. METHODS: PC12 cells was selected and exposed to the Eagle's solution containing 1 mmol/L Na2S2O4 for

  17. Protein A is released into the Staphylococcus aureus culture supernatant with an unprocessed sorting signal.

    Science.gov (United States)

    O'Halloran, Dara P; Wynne, Kieran; Geoghegan, Joan A

    2015-04-01

    The immunoglobulin binding protein A (SpA) of Staphylococcus aureus is synthesized as a precursor with a C-terminal sorting signal. The sortase A enzyme mediates covalent attachment to peptidoglycan so that SpA is displayed on the surface of the bacterium. Protein A is also found in the extracellular medium, but the processes involved in its release are not fully understood. Here, we show that a portion of SpA is released into the supernatant with an intact sorting signal, indicating that it has not been processed by sortase A. Release of SpA was reduced when the native sorting signal of SpA was replaced with the corresponding region of another sortase-anchored protein (SdrE). Similarly, a reporter protein fused to the sorting signal of SpA was released to a greater extent than the same polypeptide fused to the SdrE sorting signal. Released SpA protected bacteria from killing in human blood, indicating that it contributes to immune evasion.

  18. Two Dimensional Electrophoresis of Proteins from Cultures of Erysiphe graminis f.sp. hordei

    DEFF Research Database (Denmark)

    Torp, J.; Andersen, Brian

    1982-01-01

    Conidial proteins from barley powdery mildew, Erysiphe graminis f. sp. hordei, were separated by 2-dimensional electrophoresis in polyacrylamide slab gels. Isoelectric focusing was used in the first dimension and separation according to molecular weight in a gel containing sodium dodecyl sulphate...

  19. Leucine and isoleucine reduce protein degradation in rainbow trout (Oncorhynchus mykiss) primary myoblast cultures

    Science.gov (United States)

    Myogenic precursor cells were isolated from rainbow trout skeletal muscle and incubated in media containing 10% fetal bovine serum for 7 days, thereby differentiating into myoblasts. Rates of protein degradation were determined in response to minimal essential media (MEM) of various amino acid (AA)...

  20. Synthesis of insulin-like growth factor binding protein 3 in vitro in human articular cartilage cultures.

    Science.gov (United States)

    Eviatar, Tamar; Kauffman, Hannah; Maroudas, Alice

    2003-02-01

    To quantify the rate of synthesis of insulin-like growth factor binding protein 3 (IGFBP-3) and insulin-like growth factor 1 (IGF-1) by in vitro cultures of normal and osteoarthritic (OA) human articular cartilage. Levels of IGF-1 and IGFBP-3 in media from in vitro cultures of human cartilage were determined by radioimmunoassay (RIA). IGFBPs were characterized by immunoblots and ligand blots. Ultrafiltration and RIA analysis of synovial fluid (SF) samples and washings of cartilage samples ex vivo were used to calculate partition coefficients and to estimate the amount of IGF-1 and IGFBP-3 in cartilage in vivo. OA cartilage synthesized 150 ng of IGFBP-3 per gm of cartilage per day, compared with 50 ng synthesized by normal cartilage. The surface zone of normal cartilage produced more IGFBP-3 than did the deep zone. Immunoblots and ligand blots confirmed the presence of IGFBP-3. IGFBP-3 synthesis was stimulated by exogenous IGF-1. No freshly synthesized IGF-1 was detected. The quantities of IGF-1 and IGFBP-3 present ex vivo were 11.3 and 78.7 ng/gm of cartilage in normal cartilage and 21.6 and 225.4 ng/gm in OA cartilage. The results show that while IGFBP-3 is synthesized in explant cultures, IGF-1 is not. The rate of IGFBP-3 synthesis is 3 times higher in OA than in normal cartilage. Both IGFBP-3 and IGF-1 penetrate into cartilage from SF in vivo. We estimate that the quantities of IGFBP-3 produced in culture by human cartilage are small compared with the amount supplied in the form of "small complexes" from the circulation. The high value of the partition coefficient of IGFBP-3 implies binding to the matrix.

  1. Effects of inter-culture, arabinogalactan proteins, and hydrogen peroxide on the plant regeneration of wheat immature embryos

    Institute of Scientific and Technical Information of China (English)

    ZHANG Wei; WANG Xin-min; FAN Rong; YIN Gui-xiang; WANG Ke; DU Li-pu; XIAO Le-le; YE Xing-guo

    2015-01-01

    The regeneration rate of wheat immature embryo varies among genotypes, howbeit many elite agriculture wheat varieties have low regeneration rates. Optimization of tissue culture conditions and attempts of adding signal molecules are effective ways to increase plant regeneration rate. Inter-culture is one of ways that have not been investigated in plant tissue culture. Moreover, the use of arabinogalactan proteins (AGPs) and hydrogen peroxide (H2O2) have been reported to increase regeneration rate in a few plant species other than wheat. The current research pioneeringly uses inter-culture of immature embryos of different wheat genotypes, and also investigates impacts of AGP and H2O2 on the induction of embryogenic cali and plant regeneration. As a result, high-frequency regeneration wheat cultivars Kenong 199 (KN199) and Xinchun 9 (XC9), together with low-frequency regeneration wheat line Chinese Spring (CS), presented striking increase in the induction of embryogenic cali and plant regeneration rate of CS through inter-culture strategy, up to 52.19 and 67.98%, respectively. Adding 50 to 200 mg L–1 AGP or 0.005 to 0.01 ‰ H2O2 to the calus induction medium, enhanced growth of embryogenic cali and plant regeneration rate in quite a few wheat genotypes. At 50 mg L–1 AGP application level in calus induction medium plant regeneration rates of 8.49, 409.06 and 283.16% were achieved for Jimai 22 (JM22), Jingdong 18 (JD18) and Yangmai 18 (YM18), respectively; whereas at 100 mg L–1 AGP level, CS (105.44%), Chuannong 16 (CN16) (80.60%) and Ningchun 4 (NC4) (62.87%) acted the best. Moreover CS (79.05%), JM22 (7.55%), CN16 (101.87%), YM18 (365.56%), Yangmai 20 (YM20) (10.48%), and CB301 (187.40%) were more responsive to 0.005 ‰ of H2O2,and NC4 (35.37%) obtained the highest shoot regeneration rates at 0.01 ‰ of H2O2. Overal, these two methods, inter-culture and AGP (or H2O2) application, can be further applied to wheat transgenic research.

  2. The relationship of C-reactive protein levels and positive culture with quality of life in acute rhinosinusitis

    Directory of Open Access Journals (Sweden)

    Schalek P

    2015-01-01

    Full Text Available Petr Schalek, Zuzana Hornáčková, Aleš Hahn Ear, Nose and Throat department, 3rd Medical Faculty of Charles University, Prague, Czech Republic Background: Acute rhinosinusitis (ARS has been shown to significantly reduce patient quality of life (QoL. While the QoL in patients with chronic rhinosinusitis has been the subject of intensive research over the last decade, studies measuring the impact of ARS on patient QoL have remained relatively scarce. The aim of this study was to determine the relationship between the QoL and parameters suggestive of acute bacterial rhinosinusitis (C-reactive protein [CRP] levels and positive culture and to see if measurement of the QoL could be used as an indicator for antibiotic treatment in ARS.Methods: Eighty patients with ARS were enrolled in the study. A novel QoL instrument for patients with ARS, called Measurement of Acute Rhinosinusitis (MARS questionnaire, was given to patients at the time of diagnosis. We assessed patient QoL, obtained endoscopically guided cultures from the middle meatus, and measured levels of CRP. The relationship between QoL MARS scores (QoL-Mscores and CRP was determined using a correlation coefficient. To compare QoL-Mscores, relative to culture-positive and culture-negative patients, the Student’s t-test was used.Results: No correlation between the QoL, assessed using the MARS questionnaire, and positive middle meatus culture was demonstrated (P=0.332. A weak correlation was found between QoL-Mscores and CRP values, with a correlation coefficient of 0.221 and P=0.0498.Conclusion: No correlation between the QoL in ARS patients and positive culture was found in this study. The clinical significance of the correlation between QoL-Mscores and CRP values in the antibiotic decision making process needs further research. Keywords: endoscopy, quality of life, questionnaires, anti-bacterial agents

  3. Isolation and partial characterisation of the relaxation protein from nuclei of cultured mouse and human cells.

    Science.gov (United States)

    Vosberg, H P; Grossman, L I; Vinograd, J

    1975-06-16

    A protein, called relaxation protein because of its ability to remove superhelical turns in closed-circular DNA, has been isolated and partially characterized from the nuclei of LA9 mouse and HeLa cells. The purification was facilitated by an assay method, with PM2 DNA, which used the fluorescence enhancement of the intercalating dye ethidium bromide upon binding to the closed-circular DNA. The amount of dye bound depends upon the degree of the superhelix density of the DNA. The relaxation products were analysed by the buoyant separation method in CsCl containing ethidium bromide and were shown to be completely relaxed. The purification resulted in a single band in a dodecylsulfate gel electrophoresis with an apparent molecular weight of 37000. The pH optimum is 7.0 and the optimal salt concentration is 0.2 M NaCl. The relaxation protein removes negative as well as positive supercoils, the latter generated by the interaction of ethidium bromide with closed-circular DNA. Relaxation of positive supercoils results, after removal of the dye, in the formation of molecules with superhelix densities exceeding that of native PM2 DNA (0.054). The highest negative superhelix density observed was -0.098 +/- 0.001. The corresponding positive superhelix density has been calculated to be + 0.023. A nicking--swivelling--closing mechanism is postulated, but nicked intermediates have so far not been demonstrated. The relaxation protein is not inhibited by known mammalian endonuclease I inhibitors, except for denatured DNA, and does not possess a conventional polynucleotide ligase activity. The relaxation activity was found to be predominantly in the nuclei, with only small amounts present in the cytoplasm and mitochondria. The biological function of transient swivels induced by the relaxation protein is not known. However, transient swivels are considered necessary or useful in the replication of closed-circular DNA or long linear DNA, respectively. Relaxation protein could replace

  4. Microvesicle and tunneling nanotube mediated intercellular transfer of g-protein coupled receptors in cell cultures

    Energy Technology Data Exchange (ETDEWEB)

    Guescini, M. [Department of Biomolecular Sciences, University of Urbino ' Carlo Bo' , 61029 Urbino (Italy); Leo, G.; Genedani, S. [Department Biomedical Sciences, University of Modena and Reggio Emilia (Italy); Carone, C. [Department Biomedical Sciences, University of Modena and Reggio Emilia (Italy); IRCCS San Camillo Lido, Venezia (Italy); Pederzoli, F. [Department Biomedical Sciences, University of Modena and Reggio Emilia (Italy); Ciruela, F. [Departament Patologia i Terapeutica Experimental, Universitat de Barcelona (Spain); Guidolin, D. [Department of Human Anatomy and Physiology, University of Padua (Italy); Stocchi, V.; Mantuano, M. [Department of Biomolecular Sciences, University of Urbino ' Carlo Bo' , 61029 Urbino (Italy); Borroto-Escuela, D.O.; Fuxe, K. [Department of Neuroscience, Karolinska Institutet, Stockholm (Sweden); Agnati, L.F., E-mail: luigiagnati@tin.it [IRCCS San Camillo Lido, Venezia (Italy)

    2012-03-10

    Recent evidence shows that cells exchange collections of signals via microvesicles (MVs) and tunneling nano-tubes (TNTs). In this paper we have investigated whether in cell cultures GPCRs can be transferred by means of MVs and TNTs from a source cell to target cells. Western blot, transmission electron microscopy and gene expression analyses demonstrate that A{sub 2A} and D{sub 2} receptors are present in released MVs. In order to further demonstrate the involvement of MVs in cell-to-cell communication we created two populations of cells (HEK293T and COS-7) transiently transfected with D{sub 2}R-CFP or A{sub 2A}R-YFP. These two types of cells were co-cultured, and FRET analysis demonstrated simultaneously positive cells to the D{sub 2}R-CFP and A{sub 2A}R-YFP. Fluorescence microscopy analysis also showed that GPCRs can move from one cell to another also by means of TNTs. Finally, recipient cells pre-incubated for 24 h with A{sub 2A}R positive MVs were treated with the adenosine A{sub 2A} receptor agonist CGS-21680. The significant increase in cAMP accumulation clearly demonstrated that A{sub 2A}Rs were functionally competent in target cells. These findings demonstrate that A{sub 2A} receptors capable of recognizing and decoding extracellular signals can be safely transferred via MVs from source to target cells.

  5. In vivo evaluation of whey protein-based biofilms as scaffolds for cutaneous cell cultures and biomedical applications

    Energy Technology Data Exchange (ETDEWEB)

    Rouabhia, Mahmoud [Faculte de medecine dentaire, GREB, Universite Laval, Quebec (Ciheam) G1K 7P4 (Canada); Gilbert, Vanessa [Unite de Biotechnologie, Institut des biomateriaux, Hopital Saint-Francois d' Assise, CHUQ, 10 de l' Espinay, Quebec G1L 3L5 (Canada); Wang Hongxum [Unite de Biotechnologie, Institut des biomateriaux, Hopital Saint-Francois d' Assise, CHUQ, 10 de l' Espinay, Quebec G1L 3L5 (Canada); Subirade, Muriel [Chaire de recherche du Canada sur les proteines, bio-systemes et aliments fonctionnels, Centre de Recherche INAF/STELA, Universite Laval, Quebec (Ciheam) G1K 7P4 (Canada)

    2007-03-01

    This study evaluated the toxicity, biodegradability and immunogenicity of newly developed whey protein-based biofilms for possible use as biomaterials for medical applications. Biofilms were prepared using (A) a whey protein isolate plasticized with either diethylene glycol (DEG) or glycerol (GLY), and (B) {beta}-lactoglobulin ({beta}LGA) plasticized with DEG. The biofilms were implanted subcutaneously into Balb/c mice. Analyses were performed at various time points. At 15, 30 and 60 days post-implantation, no necrotic zones or exudates were present at the recipient sites. The biofilms began to degrade as early as 15 days post-implantation, as evidenced by erosion and crumbling. The macroscopic observations were supported by tissue analyses revealing no tissue necrosis or degradation and confirming that the biodegradation of the biofilms began as early as 15 days post-implantation and was almost complete after 60 days. The biodegradation was accompanied by significant leukocyte infiltration at 15 days which significantly decreased at 60 days. The absence of splenomagaly in the implanted mice confirms that these biofilms were not immunogenic. Whey protein-based biofilms are biocompatible and biodegradable and may be of interest for medical applications such as scaffolds for cutaneous cell cultures and skin recovery in burn patients.

  6. Purification of monoclonal antibodies from clarified cell culture fluid using Protein A capture continuous countercurrent tangential chromatography.

    Science.gov (United States)

    Dutta, Amit K; Tran, Travis; Napadensky, Boris; Teella, Achyuta; Brookhart, Gary; Ropp, Philip A; Zhang, Ada W; Tustian, Andrew D; Zydney, Andrew L; Shinkazh, Oleg

    2015-11-10

    Recent studies using simple model systems have demonstrated that continuous countercurrent tangential chromatography (CCTC) has the potential to overcome many of the limitations of conventional Protein A chromatography using packed columns. The objective of this work was to optimize and implement a CCTC system for monoclonal antibody purification from clarified Chinese Hamster Ovary (CHO) cell culture fluid using a commercial Protein A resin. Several improvements were introduced to the previous CCTC system including the use of retentate pumps to maintain stable resin concentrations in the flowing slurry, the elimination of a slurry holding tank to improve productivity, and the introduction of an "after binder" to the binding step to increase antibody recovery. A kinetic binding model was developed to estimate the required residence times in the multi-stage binding step to optimize yield and productivity. Data were obtained by purifying two commercial antibodies from two different manufactures, one with low titer (∼ 0.67 g/L) and one with high titer (∼ 6.9 g/L), demonstrating the versatility of the CCTC system. Host cell protein removal, antibody yields and purities were similar to those obtained with conventional column chromatography; however, the CCTC system showed much higher productivity. These results clearly demonstrate the capabilities of continuous countercurrent tangential chromatography for the commercial purification of monoclonal antibody products.

  7. In vivo evaluation of whey protein-based biofilms as scaffolds for cutaneous cell cultures and biomedical applications.

    Science.gov (United States)

    Rouabhia, Mahmoud; Gilbert, Vanessa; Wang, Hongxum; Subirade, Muriel

    2007-03-01

    This study evaluated the toxicity, biodegradability and immunogenicity of newly developed whey protein-based biofilms for possible use as biomaterials for medical applications. Biofilms were prepared using (A) a whey protein isolate plasticized with either diethylene glycol (DEG) or glycerol (GLY), and (B) beta-lactoglobulin (betaLGA) plasticized with DEG. The biofilms were implanted subcutaneously into Balb/c mice. Analyses were performed at various time points. At 15, 30 and 60 days post-implantation, no necrotic zones or exudates were present at the recipient sites. The biofilms began to degrade as early as 15 days post-implantation, as evidenced by erosion and crumbling. The macroscopic observations were supported by tissue analyses revealing no tissue necrosis or degradation and confirming that the biodegradation of the biofilms began as early as 15 days post-implantation and was almost complete after 60 days. The biodegradation was accompanied by significant leukocyte infiltration at 15 days which significantly decreased at 60 days. The absence of splenomagaly in the implanted mice confirms that these biofilms were not immunogenic. Whey protein-based biofilms are biocompatible and biodegradable and may be of interest for medical applications such as scaffolds for cutaneous cell cultures and skin recovery in burn patients.

  8. Highly protein-resistant coatings and suspension cell culture thereon from amphiphilic block copolymers prepared by RAFT polymerization.

    Science.gov (United States)

    Haraguchi, Kazutoshi; Kubota, Kazuomi; Takada, Tetsuo; Mahara, Saori

    2014-06-09

    Novel amphiphilic block copolymers composed of hydrophobic (poly(2-methoxyethyl acrylate): M) and hydrophilic (poly(N,N-dimethylacrylamide): D) segments were synthesized by living radical polymerization: a reversible addition-fragmentation chain-transfer polymerization. Two types of amphiphilic block copolymers, triblock (MDM) and 4-arm block ((MD)4) copolymers with specific compositions (D/M = (750-1500)/250), were prepared by a versatile one-pot synthesis. These copolymers show good adhesion to various types of substrates (e.g., polystyrene, polycarbonate, polypropylene, Ti, and glass), and the surface coating showed high protein repellency and a low contact angle for water, regardless of the substrate. The two opposing characteristics of high protein repellency and good substrate adhesion were achieved by the combined effects of the molecular architecture of the block copolymers, the high molecular weight, and the characteristics of each segment, that is, low protein adsorption capability of both segments and low glass transition temperature of the hydrophobic segment. Further, a polystyrene dish coated with the MDM block copolymer could be sterilized by γ-ray irradiation and used as a good substrate for a suspension cell culture that exhibits low cell adhesion and good cell growth.

  9. Protein

    Science.gov (United States)

    ... Food Service Resources Additional Resources About FAQ Contact Protein Protein is found throughout the body—in muscle, ... the heart and respiratory system, and death. All Protein Isn’t Alike Protein is built from building ...

  10. Membraneless water filtration using CO2

    Science.gov (United States)

    Shin, Sangwoo; Shardt, Orest; Warren, Patrick; Stone, Howard

    2016-11-01

    Water purification technologies such as ultrafiltration and reverse osmosis utilize porous membranes to remove suspended particles and solutes. These membranes, however, cause many drawbacks such as a high pumping cost and a need for periodic replacement due to fouling. Here we show an alternative membraneless method for separating suspended particles by exposing the colloidal suspension to CO2. Dissolution of CO2 into the suspension creates solute gradients that drive phoretic motion of particles, or so-called diffusiophoresis. Due to the large diffusion potential built up by the dissociation of carbonic acid, colloidal particles move either away from or towards the gas-liquid interface depending on their surface charge. Our findings suggest a means to separate particles without membranes or filters, thus reducing operating and maintenance costs. Using the directed motion of particles induced by exposure to CO2, we demonstrate a scalable, continuous flow, membraneless particle filtration process that exhibits very low pressure drop and is essentially free from fouling.

  11. Pattern recognition using inverse resonance filtration

    CERN Document Server

    Sofina, Olga; Kvetnyy, Roman

    2010-01-01

    An approach to textures pattern recognition based on inverse resonance filtration (IRF) is considered. A set of principal resonance harmonics of textured image signal fluctuations eigen harmonic decomposition (EHD) is used for the IRF design. It was shown that EHD is invariant to textured image linear shift. The recognition of texture is made by transfer of its signal into unstructured signal which simple statistical parameters can be used for texture pattern recognition. Anomalous variations of this signal point on foreign objects. Two methods of 2D EHD parameters estimation are considered with the account of texture signal breaks presence. The first method is based on the linear symmetry model that is not sensitive to signal phase jumps. The condition of characteristic polynomial symmetry provides the model stationarity and periodicity. Second method is based on the eigenvalues problem of matrices pencil projection into principal vectors space of singular values decomposition (SVD) of 2D correlation matrix....

  12. Gravity filtration of suspensions: permeability effects

    Science.gov (United States)

    Soori, Tejaswi; Wang, Mengyu; Ward, Thomas

    2015-11-01

    This paper examines the filtration rates of mono-modal suspensions as a function of time and a cake layer builds up through theory and experimentation. Darcy's Law, which describes fluid flow through porous media, was applied along with the Kynch theory of sedimentation, which provides the basis for analyzing low concentration (ϕ filter media. A CCD camera was used to capture images of the cake formation and fluid drainage processes, and subsequent image and theoretical analysis found the fluid flow experienced a constant pressure loss due to the permeability of the filter media, whereas the experienced pressure loss due to the cake formation varies as a function of time, ϕ and d. The rate of cake formation was also found to be independent of ϕ but dependent on d which can be attributed to a change in porosity affecting permeability. Studies on similar systems with multi-modal suspensions are in-progress.

  13. In vitro differentiation of bone marrow stromal cells into neurons and glial cells and differential protein expression in a two-compartment bone marrow stromal cell/neuron co-culture system.

    Science.gov (United States)

    Qi, Xu; Shao, Ming; Peng, Haisheng; Bi, Zhenggang; Su, Zhiqiang; Li, Hulun

    2010-07-01

    This study was performed to establish a bone marrow stromal cell (BMSC)/neuron two-compartment co-culture model in which differentiation of BMSCs into neurons could occur without direct contact between the two cell types, and to investigate protein expression changes during differentiation of this entirely BMSC-derived population. Cultured BMSCs isolated from Wistar rats were divided into three groups: BMSC culture, BMSC/neuron co-culture and BMSC/neuron two-compartment co-culture. Cells were examined for neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression. The electrophysiological behavior of the BMSCs was examined using patch clamping. Proteins that had significantly different expression levels in BMSCs cultured alone and co-cultured with neurons were studied using a protein chip-mass spectroscopy technique. Expression of NSE and GFAP were significantly higher in co-culture cells than in two-compartment co-culture cells, and significantly higher in both co-culture groups than in BMSCs cultured alone. Five proteins showed significant changes in expression during differentiation: TIP39_RAT and CALC_RAT underwent increases, and INSL6_RAT, PNOC_RAT and PCSK1_RAT underwent decreases in expression. We conclude that BMSCs can differentiate into neurons during both contact co-culture with neurons and two-compartment co-culture with neurons. The rate at which BMSCs differentiated into neurons was higher in contact co-culture than in non-contact co-culture.

  14. Establishment and application of milk fingerprint by gel filtration chromatography.

    Science.gov (United States)

    Gao, P; Li, J; Li, Z; Hao, J; Zan, L

    2016-12-01

    Raw milk adulteration frequently occurs in undeveloped countries. It not only reduces the nutritional value of milk, but it is also harmful to consumers. In this paper, we focused on investigating an efficient method for the quality control of raw milk protein. A gel filtration chromatography (GFC) fingerprint method combined with chemometrics was developed for fingerprint analysis of raw milk. To optimize the GFC conditions, milk fat was removed by centrifugation, and GFC analysis was performed on a Superdex 75 10/300GL column (Just Scientific, Shanghai, China) with 0.2 M NaH2PO4-Na2HPO4 buffer (pH 7.0) as the mobile phase. The flow rate was 0.5mL/min, and the detection wavelength was set at 280 nm. Ten batches of 120 raw milk samples were analyzed to establish the GFC fingerprint under optimal conditions. Six major peaks common to the chromatogram of each raw milk sample were selected for fingerprint analysis, and the characteristic peaks were used to establish a standard chromatographic fingerprint. Principal component analysis was then applied to classify GFC information of adulterated milk and raw milk, allowing adulterated samples to be effectively screened out from the raw milk in principal component analysis scores plot. The fingerprint method demonstrates promising features in detecting milk protein adulteration.

  15. Isolation of recombinant proteins from culture broth by co-precipitation with an amino acid carrier to form stable dry powders.

    Science.gov (United States)

    Moore, Barry D; Deere, Joseph; Edrada-Ebel, RuAngelie; Ingram, Andrew; van der Walle, Christopher F

    2010-08-01

    Protein-coated microcrystals can be generated by co-precipitation of protein and a water-soluble crystalline carrier by addition to excess water miscible organic solvent. We have investigated this novel process for its utility in the concentration and partial purification of a recombinant protein exported into the culture broth during expression by Pichia pastoris. Co-precipitation with a L-glutamine carrier selectively isolated the protein content of the culture broth, with a minimal number of steps, and simultaneously removed contaminants including a novel yeast metabolite. This pigment co-elutes during aqueous chromatography but its elucidation as a benzoylated glycosamine suggested a simple route of removal by partition during the co-precipitation process. Scale-up of the process was readily achieved through in-line mixing and subsequent reconstitution of the dried protein-coated microcrystals yielded natively folded, bioactive protein. Additional washing of the crystals with saturated L-glutamine facilitated further purification of the recombinant protein immobilized on the L-glutamine carrier. Thus, we present a novel method for the harvesting of recombinant protein from culture broth as a dry powder, which may be of general applicability to bioprocessing. (c) 2010 Wiley Periodicals, Inc.

  16. Purification of Peptide Components including Melittin from Bee Venom using gel filtration chromatography and propionic acid/urea polyacrylamide gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Young Chon Choi

    2006-06-01

    Full Text Available Objectives : This study was conducted to carry out Purification of Melittin and other peptide components from Bee Venom using gel filtration chromatography and propionic acid/urea polyacrylamide gel electrophoresis Methods : Melittin and other peptide components were separated from bee venom by using gel filtration chromatography on Sephadex G-50 column in 0.05M ammonium acetate buffer. Results : Melittin and other peptide components were separated from bee venom by using gel filtration chromatography on Sephadex G-50 column in 0.05M ammonium acetate buffer. The fractions obtained from gel filtration chromatography was analyzed by using SDS-PAGE and propionic acid/urea polyacrylamide gel electrophoresis. The melittin obtained from the gel filtration contained residual amount of phospholipase A2 and a protein with molecular weight of 6,000. The contaminating proteins were removed by the second gel filtration chromatography. Conclusion : Gel filtration chromatography and propionic acid/urea polyacrylamide gel electrophoresis are useful to separate peptide components including melittin from bee venom.

  17. Initial ground experiments of silkworm cultures living on different feedstock for provision of high quality animal protein for human in space

    Science.gov (United States)

    Yang, Yunan; Tang, Liman; Tong, Ling; Liu, Yang; Liu, Hong; Li, Xiaomin

    2010-09-01

    Silkworm could be an alternative to provide edible animal protein in Controlled Ecological Life Support System (CELSS) for long-term manned space missions. Silkworms can consume non-edible plant residue and convert plant nutrients to high quality edible animal protein for astronauts. The preliminary investigation of silkworm culture was carried out in earth environment. The silkworms were fed with artificial silkworm diet and the leaves of stem lettuce ( Lactuca sativa L. var. angustana Irish) separately and the nutritional structure of silkworm was investigated and compared, The culture experiments showed that: (1) Stem lettuce leaves could be used as food of silkworm. The protein content of silkworm fed with lettuce leaves can reach 70% of dry mass. (2) The protein content of silkworm powder produced by the fifth instar silkworm larvae was 70%, which was similar to the protein content of silkworm pupae. The powder of the fifth instar silkworm larvae can be utilized by astronaut. (3) The biotransformation rate of silkworm larvae between the third instar and the fifth instar could reach above 70%. The biotransformation cycle of silkworm was determined as 24 days. (4) Using the stem lettuce leaves to raise silkworm, the coarse fiber content of silkworm excrements reached about 33%. The requirements of space silkworm culture equipment, feeding approaches and feeding conditions were also preliminarily designed and calculated. It is estimated that 2.2 m 3 of culture space could satisfy daily animal protein demand for seven astronauts.

  18. RNA and protein synthesis in cultured human fibroblasts derived from donors of various ages.

    Science.gov (United States)

    Chen, J J; Brot, N; Weissbach, H

    1980-07-01

    RNA synthesis in human fibroblasts from donors of various ages was studied in fibroblasts made permeable to nucleoside triphosphates with the nonionic detergent Nonidet P40. Cells from donors of 11 years and older showed a 30-40% decline in total RNA synthesis. The decrease in RNA synthesis was primarily due to a lowering of RNA polymerase II activity (alpha-amanitin sensitive). Studies on the incorporation of leucine into protein also showed a 30-40% decrease in cells from older donors.

  19. Microcarrier culture of lepidopteran cell lines: implications for growth and recombinant protein production.

    Science.gov (United States)

    Ikonomou, Laertis; Drugmand, Jean-Christophe; Bastin, Georges; Schneider, Yves-Jacques; Agathos, Spiros N

    2002-01-01

    Several microcarrier systems were screened with Sf-9 and High-Five cell lines as to their ability to support cell growth and recombinant (beta-galactosidase) protein production. Growth of both cell lines on compact microcarriers, such as Cytodex-1 and glass beads, was minimal, as cells detached easily from the microcarrier surface and grew as single cells in the medium. Cell growth was also problematic on Cytopore-1 and -2 porous microcarriers. Cells remained attached for several days inside the microcarrier pores, but no cell division and proliferation were observed. On the contrary, insect cells grew well in the interior of Fibra-Cel disks mainly as aggregates at points of fiber intersection, reaching final (plateau) densities of about 4 x 10(6) (Sf-9) and 2.7 x 10(6) (High-Five) cells mL(-1) (8 x 10(6) and 5.5 x 10(6) cells per cm(2) of projected disk area, respectively). Their growth was described well by the logistic equation, which takes into account possible inhibition effects. Beta-Galactosidase (beta-gal) production of Sf-9 cells on Fibra-Cel disks (infected at 3.3 x 10(6) cells mL(-1)) was prolonged (192 h), and specific protein production was similar to that of high-density free cell infection. Cultispher-S microcarriers were found to be a very efficient system for the growth of High-Five cells, whereas no growth of Sf-9 cells took place for the same system. Concentrations of about 9 x 10(6) cells mL(-1) were reached within 120 h, with cell growth in both microcarriers and aggregates, appearance of cellular bridges between microcarriers and aggregates, and eventual formation of macroaggregates incorporating several microcarriers. Specific protein productions after beta-gal baculovirus infection at increasing cell concentrations were almost constant, thus leading to elevated volumetric protein production: final beta-gal titers of 946, 1728, and 1484 U mL(-1) were obtained for infection densities of 3.4, 7.2, and 8.9 x 10(6) cells mL(-1), respectively.

  20. Some aspects of applying nanostructured materials in air filtration, water filtration and electrical engineering

    Science.gov (United States)

    Kimmer, Dusan; Vincent, Ivo; Lovecka, Lenka; Kazda, Tomas; Giurg, Adam; Skorvan, Ondrej

    2017-05-01

    Nanostructures prepared from nanofibres and nanostructured composites prepared from nanofibres and fillers are gradually becoming increasingly demanded materials for applications in various industrial branches connected with catalysis, environment protection (air filtration, waste water treatment, sound absorption), in biological engineering, electronics (battery separators, electrode materials), etc. Selected applications of these materials prepared in the company SPUR a.s. are summed up in the following presentation.

  1. Translation and Assembly of Radiolabeled Mitochondrial DNA-Encoded Protein Subunits from Cultured Cells and Isolated Mitochondria.

    Science.gov (United States)

    Formosa, Luke E; Hofer, Annette; Tischner, Christin; Wenz, Tina; Ryan, Michael T

    2016-01-01

    In higher eukaryotes, the mitochondrial electron transport chain consists of five multi-subunit membrane complexes responsible for the generation of cellular ATP. Of these, four complexes are under dual genetic control as they contain subunits encoded by both the mitochondrial and nuclear genomes, thereby adding another layer of complexity to the puzzle of respiratory complex biogenesis. These subunits must be synthesized and assembled in a coordinated manner in order to ensure correct biogenesis of different respiratory complexes. Here, we describe techniques to (1) specifically radiolabel proteins encoded by mtDNA to monitor the rate of synthesis using pulse labeling methods, and (2) analyze the stability, assembly, and turnover of subunits using pulse-chase methods in cultured cells and isolated mitochondria.

  2. Quantitative analysis of a biopharmaceutical protein in cell culture samples using automated capillary electrophoresis (CE) western blot.

    Science.gov (United States)

    Xu, Dong; Marchionni, Kentaro; Hu, Yunli; Zhang, Wei; Sosic, Zoran

    2017-10-25

    An effective control strategy is critical to ensure the safety, purity and potency of biopharmaceuticals. Appropriate analytical tools are needed to realize such goals by providing information on product quality at an early stage to help understanding and control of the manufacturing process. In this work, a fully automated, multi-capillary instrument is utilized for size-based separation and western blot analysis to provide an early readout on product quality in order to enable a more consistent manufacturing process. This approach aims at measuring two important qualities of a biopharmaceutical protein, titer and isoform distribution, in cell culture harvest samples. The acquired data for isoform distribution can then be used to predict the corresponding values of the final drug substance, and potentially provide information for remedy through timely adjustment of the downstream purification process, should the expected values fall out of the accepted range. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  3. Macroautophagy-generated increase of lysosomal amyloid β-protein mediates oxidant-induced apoptosis of cultured neuroblastoma cells

    DEFF Research Database (Denmark)

    Zheng, Lin; Terman, Alexei; Hallbeck, Martin

    2011-01-01

    and accumulation of Aβ within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aβ monomers and oligomers, (2) increased reactive oxygen species production......, and (3) enhanced apoptosis. Oxidant-induced apoptosis positively correlated with cellular Aβ production, being the highest in cells that were stably transfected with APP Swedish KM670/671NL double mutation. Inhibition of γ-secretase, prior and/or in parallel to hyperoxia, suggested that the increase...... and resulting lysosomal Aβ accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide additional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration....

  4. Proteins dominate in the surface layers formed on materials exposed to extracellular polymeric substances from bacterial cultures.

    Science.gov (United States)

    Yang, Yi; Wikieł, Agata J; Dall'Agnol, Leonardo T; Eloy, Pierre; Genet, Michel J; Moura, José J G; Sand, Wolfgang; Dupont-Gillain, Christine C; Rouxhet, Paul G

    2016-01-01

    The chemical compositions of the surface conditioning layers formed by different types of solutions (from isolated EPS to whole culture media), involving different bacterial strains relevant for biocorrosion were compared, as they may influence the initial step in biofilm formation. Different substrata (polystyrene, glass, steel) were conditioned and analyzed by X-ray photoelectron spectroscopy. Peak decomposition and assignment were validated by correlations between independent spectral data and the ubiquitous presence of organic contaminants on inorganic substrata was taken into account. Proteins or peptides were found to be a major constituent of all conditioning layers and polysaccharides were not present in appreciable concentrations; the proportion of nitrogen which may be due to DNA was lower than 15%. There was no significant difference between the compositions of the adlayers formed from different conditioning solutions, except for the adlayers produced with tightly bound EPS extracted from D. alaskensis.

  5. Life Support Filtration System Trade Study for Deep Space Missions

    Science.gov (United States)

    Agui, Juan H.; Perry, Jay L.

    2017-01-01

    The National Aeronautics and Space Administrations (NASA) technical developments for highly reliable life support systems aim to maximize the viability of long duration deep space missions. Among the life support system functions, airborne particulate matter filtration is a significant driver of launch mass because of the large geometry required to provide adequate filtration performance and because of the number of replacement filters needed to a sustain a mission. A trade analysis incorporating various launch, operational and maintenance parameters was conducted to investigate the trade-offs between the various particulate matter filtration configurations. In addition to typical launch parameters such as mass, volume and power, the amount of crew time dedicated to system maintenance becomes an increasingly crucial factor for long duration missions. The trade analysis evaluated these parameters for conventional particulate matter filtration technologies and a new multi-stage particulate matter filtration system under development by NASAs Glenn Research Center. The multi-stage filtration system features modular components that allow for physical configuration flexibility. Specifically, the filtration system components can be configured in distributed, centralized, and hybrid physical layouts that can result in considerable mass savings compared to conventional particulate matter filtration technologies. The trade analysis results are presented and implications for future transit and surface missions are discussed.

  6. Vibrating membrane filtration as improved technology for microalgae dewatering.

    Science.gov (United States)

    Nurra, Claudia; Clavero, Ester; Salvadó, Joan; Torras, Carles

    2014-04-01

    The effect of shear-enhanced filtration by vibratory process in microalgae dewatering is presented in this paper. The aim of this research was to investigate the technical performance and improvement of vibrating membrane filtration compared with conventional tangential cross-flow filtration in microalgae concentration. An industrial-scale available commercial set-up was used. Several membrane materials as polyethersulfone, polyacrylonitrile, etc., and mean pore sizes (from 7000Da to 0.2μm) were tested and compared in both filtration set-ups. Experiments were carried-out with Nannochloropsis gaditana and Phaeodactylum tricornutum microalgae. It has been demonstrated that, even if the choice of the membrane depends on its cut-off, its material and the type of microalgae filtrated, dynamic filtration is always the best technology over a conventional one. If with conventional filtration permeability values were in the vicinity of 10L/h/m(2)/bar in steady state phase, with dynamic filtration these values increased to 30L/h/m(2)/bar or more.

  7. Analysis of the Medium Resistance for Constant Pressure Filtration

    Institute of Scientific and Technical Information of China (English)

    吴燕翔; 王碧玉

    2004-01-01

    A mathematical model for constant pressure filtration is established. The distribution of hydraulic pressure within the cake and the medium resistance are measured. The medium resistance Rm is calculated from the suppositional filtration time Ore. It is demonstrated that Rm is nearly a constant for a given filter cloth.

  8. Purification of contaminated water by filtration through porous glass

    Science.gov (United States)

    Wydeven, T.; Leban, M. I.

    1972-01-01

    Method for purifying water that is contaminated with mineral salts and soluble organic compounds is described. Method consists of high pressure filtration of contaminated water through stabilized porous glass membranes. Procedure for conducting filtration is described. Types of materials by percentage amounts removed from the water are identified.

  9. Filter aids influence on pressure drop across a filtration system

    Science.gov (United States)

    Hajar, S.; Rashid, M.; Nurnadia, A.; Ammar, M. R.; Hasfalina, C. M.

    2017-06-01

    Filter aids is commonly used to reduce pressure drop across air filtration system as it helps to increase the efficiency of filtration of accumulated filter cake. Filtration velocity is one of the main parameters that affect the performance of filter aids material. In this study, a formulated filter aids consisting of PreKot™ and activated carbon mixture (designated as PrekotAC) was tested on PTFE filter media under various filtration velocities of 5, 6, and 8 m/min at a constant material loading of 0.2 mg/mm2. Results showed that pressure drop is highly influenced by filtration velocity where higher filtration velocity leads to a higher pressure drop across the filter cake. It was found that PrekotAC performed better in terms of reducing the pressure drop across the filter cake even at the highest filtration velocity. The diversity in different particle size distribution of non-uniform particle size in the formulated PrekotAC mixture presents a higher permeability causes a lower pressure drop across the accumulated filter cake. The finding suggests that PrekotAC is a promising filter aids material that helps reducing the pressure drop across fabric filtration system.

  10. Scaling and particulate fouling in membrane filtration systems

    NARCIS (Netherlands)

    Boerlage, S.F.E.

    2001-01-01

    In the last decade, pressure driven membrane filtration processes; reverse osmosis, nano, ultra and micro-filtration have undergone steady growth. Drivers for this growth include desalination to combat water scarcity and the removal of various material from water to comply with increasingly stringen

  11. Scaling and particulate fouling in membrane filtration systems

    NARCIS (Netherlands)

    Boerlage, S.F.E.

    2001-01-01

    In the last decade, pressure driven membrane filtration processes; reverse osmosis, nano, ultra and micro-filtration have undergone steady growth. Drivers for this growth include desalination to combat water scarcity and the removal of various material from water to comply with increasingly

  12. Simulation of impaction filtration by a porous filter

    NARCIS (Netherlands)

    Ghazaryan, L.; Lopez Penha, D.J.; Geurts, B.J.; Stolz, S.; Winkelmann, C.; Vafai, K.

    2010-01-01

    We present a new numerical approach for estimating filtration through porous media from first principles. We numerically simulate particle motion as arises in a carrier gas flow. The filtration we look at occurs due to impaction of particles with obstructing surfaces that are contained in the solid

  13. Model and optimization of electromagnetic filtration of metals

    Directory of Open Access Journals (Sweden)

    S. Golak

    2013-04-01

    Full Text Available Electromagnetic buoyancy force causes the movement of non-conducive particles in a conducting liquid under electromagnetic field. The phenomenon allows filtration of small inclusions from molten metals. This paper presents a mathematical model of the filtration process under alternating electromagnetic field and the methodology for maximizing its efficiency.

  14. In vitro amplification of misfolded prion protein using lysate of cultured cells.

    Directory of Open Access Journals (Sweden)

    Charles E Mays

    Full Text Available Protein misfolding cyclic amplification (PMCA recapitulates the prion protein (PrP conversion process under cell-free conditions. PMCA was initially established with brain material and then with further simplified constituents such as partially purified and recombinant PrP. However, availability of brain material from some species or brain material from animals with certain mutations or polymorphisms within the PrP gene is often limited. Moreover, preparation of native PrP from mammalian cells and tissues, as well as recombinant PrP from bacterial cells, involves time-consuming purification steps. To establish a convenient and versatile PMCA procedure unrestricted to the availability of substrate sources, we attempted to conduct PMCA with the lysate of cells that express cellular PrP (PrP(C. PrP(Sc was efficiently amplified with lysate of rabbit kidney epithelial RK13 cells stably transfected with the mouse or Syrian hamster PrP gene. Furthermore, PMCA was also successful with lysate of other established cell lines of neuronal or non-neuronal origins. Together with the data showing that the abundance of PrP(C in cell lysate was a critical factor to drive efficient PrP(Sc amplification, our results demonstrate that cell lysate in which PrP(C is present abundantly serves as an excellent substrate source for PMCA.

  15. Optimization and characterization of an in vitro bovine mammary cell culture system to study regulation of milk protein synthesis and mammary differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Talhouk, R.S.

    1988-01-01

    A long term bovine mammary cell culture system that maintains normal mammary cell function was established and optimized to study milk protein synthesis and secretion and mammary differentiation. This culture system used bovine mammary acini isolated from developing or lactating mammary gland by enzymatic dissociation, and cryopreserved until thawed and plated for growth in vitro for these studies. Cells in M199 with lactogenic hormones {plus minus} fetal calf serum (FCS) were cultured on plastic, 100ul and 500ul type I collagen, and Matrigel, or embedded within type I collagen. Cell morphology, cell number, and total TCA-precipitable {sup 35}S-labelled proteins were monitored. Milk protein ({alpha}{sub s,1}-casein, lactoferrin (LF), {alpha}-lactalbumin, and {beta}-lactoglobulin) secretion and intracellular levels were determined by an ELISA assay.

  16. Glycine cleavage enzyme complex: molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts.

    Science.gov (United States)

    Zay, Agnes; Choy, Francis Y M; Patrick, Chelsea; Sinclair, Graham

    2011-06-01

    The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to

  17. American Ginseng Stimulates Insulin Production and Prevents Apoptosis through Regulation of Uncoupling Protein-2 in Cultured β Cells

    Directory of Open Access Journals (Sweden)

    John Zeqi Luo

    2006-01-01

    Full Text Available American ginseng root displays the ability to achieve glucose homeostasis both experimentally and clinically but the unknown mechanism used by ginseng to achieve its therapeutic effects on diabetes limits its application. Disruption in the insulin secretion of pancreatic β cells is considered the major cause of diabetes. A mitochondrial protein, uncoupling protein-2 (UCP-2 has been found to play a critical role in insulin synthesis and β cell survival. Our preliminary studies found that the extracts of American ginseng inhibit UCP-2 expression which may contribute to the ability of ginseng protecting β cell death and improving insulin synthesis. Therefore, we hypothesized that ginseng extracts suppress UCP-2 in the mitochondria of pancreatic β cells, promoting insulin synthesis and anti-apoptosis (a programmed cell-death mechanism. To test the hypothesis, the serum-deprived quiescent β cells were cultured with or without interleukin-1β (IL-1β, (200 pg ml−1, a cytokine to induce β cell apoptosis and water extracts of American ginseng (25 μg per 5 μl administered to wells of 0.5 ml culture for 24 h. We evaluated effects of ginseng on UCP-2 expression, insulin production, anti-/pro-apoptotic factors Bcl-2/caspase-9 expression and cellular ATP levels. We found that ginseng suppresses UCP-2, down-regulates caspase-9 while increasing ATP and insulin production/secretion and up-regulates Bcl-2, reducing apoptosis. These findings suggest that stimulation of insulin production and prevention of β cell loss by American ginseng extracts can occur via the inhibition of mitochondrial UCP-2, resulting in increase in the ATP level and the anti-apoptotic factor Bcl-2, while down-regulation of pro-apoptotic factor caspase-9 occurs, lowering the occurrence of apoptosis, which support the hypothesis.

  18. Protein-kinase-Cmu expression correlates with enhanced keratinocyte proliferation in normal and neoplastic mouse epidermis and in cell culture.

    Science.gov (United States)

    Rennecke, J; Rehberger, P A; Fürstenberger, G; Johannes, F J; Stöhr, M; Marks, F; Richter, K H

    1999-01-05

    In order to gain insight into the biological function of a PKC iso-enzyme, the protein kinase Cmu, we analyzed the expression pattern of this protein in mouse epidermis and keratinocytes in culture. Daily analysis of neonatal mouse epidermis immediately after birth showed a time-dependent reduction in the PKCmu content. Expression of the proliferating-cell nuclear antigen (PCNA), indicative of the proliferative state of cells, was reduced synchronously with PKCmu as the hyperplastic state of the neonatal tissue declined. In epidermal mouse keratinocytes, fractionated according to their maturation state, PKCmu expression was restricted to PCNA-positive basal-cell fractions. In primary cultures of those cells, growth arrest and induction of terminal differentiation by Ca2+ resulted in strongly reduced PKCmu expression, concomitantly with the loss of PCNA expression. Treatment of PMK-R1 keratinocytes with 100 nM of the mitogen 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in activation of PKCmu, reflected by translocation from the cytosolic to the particulate fraction and by shifts in electrophoretic mobility. DNA synthesis was significantly inhibited by the PKCmu inhibitor Goedecke 6976, while Goedecke 6983 did not inhibit PKCmu. Carcinomas generated according to the 2-stage carcinogenesis protocol in mouse skin consistently exhibited high levels of PKCmu. These data correlate PKCmu expression with the proliferative state of murine keratinocytes and point to a role of PKCmu in growth stimulation. A correlation between PKCmu expression and enhanced cell proliferation was also observed for NIH3T3 fibroblasts transfected with and overexpressing human PKCmu.

  19. Role of protein kinase C signaling in collagen degradation by rabbit corneal fibroblasts cultured in three-dimensional collagen gels.

    Science.gov (United States)

    Nagano, Takashi; Hao, Ji-Long; Nakamura, Masatsugu; Nishida, Teruo

    2002-08-01

    To understand the mechanism of corneal ulceration by characterizing the intracellular signaling pathways that regulate collagen degradation by corneal fibroblasts cultured in three-dimensional type I collagen gels. Specifically, the potential roles of protein kinase C (PKC) and protein kinase A (PKA) in collagen degradation were investigated. Rabbit corneal fibroblasts were cultured in three-dimensional type I collagen gels for 24 hours in the presence of plasminogen and in the absence or presence of activators or inhibitors of PKC or PKA. Degradation of collagen fibrils was then evaluated by measurement of released hydroxyproline, and the production of matrix metalloproteinases (MMPs) was assessed by gelatin zymography and immunoblot analysis. The PKC activator phorbol 12-myristate 13-acetate (PMA) increased the extent of collagen degradation by corneal fibroblasts in a dose-dependent manner, with the maximal effect apparent at a concentration of 0.1 microM. The inactive analog 4alpha-PMA had no effect on collagen degradation. The PKC inhibitor H-7 reduced the extent of collagen degradation by corneal fibroblasts in the absence or presence of PMA. Phorbol 12-myristate 13-acetate also increased the production of proMMP-1, -3, and -9 by corneal fibroblasts, whereas H-7 inhibited this effect. Neither the PKA activators 8-bromo-cAMP, isobutylmethylxanthine, and forskolin nor the PKA inhibitor HA1004 affected collagen degradation by corneal fibroblasts. These results demonstrate that PKC plays an important role in collagen degradation by corneal fibroblasts in three-dimensional type I collagen gels, whereas PKA does not appear to participate in this process.

  20. APE1, the DNA base excision repair protein, regulates the removal of platinum adducts in sensory neuronal cultures by NER

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyun-Suk [Department of Biochemistry and Molecular Biology, Indianapolis, IN 46202 (United States); Guo, Chunlu; Thompson, Eric L. [Department of Pharmacology and Toxicology, Indianapolis, IN 46202 (United States); Jiang, Yanlin [Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Kelley, Mark R. [Department of Biochemistry and Molecular Biology, Indianapolis, IN 46202 (United States); Department of Pharmacology and Toxicology, Indianapolis, IN 46202 (United States); Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Vasko, Michael R. [Department of Pharmacology and Toxicology, Indianapolis, IN 46202 (United States); Lee, Suk-Hee, E-mail: slee@iu.edu [Department of Biochemistry and Molecular Biology, Indianapolis, IN 46202 (United States)

    2015-09-15

    Peripheral neuropathy is one of the major side effects of treatment with the anticancer drug, cisplatin. One proposed mechanism for this neurotoxicity is the formation of platinum adducts in sensory neurons that could contribute to DNA damage. Although this damage is largely repaired by nuclear excision repair (NER), our previous findings suggest that augmenting the base excision repair pathway (BER) by overexpressing the repair protein APE1 protects sensory neurons from cisplatin-induced neurotoxicity. The question remains whether APE1 contributes to the ability of the NER pathway to repair platinum-damage in neuronal cells. To examine this, we manipulated APE1 expression in sensory neuronal cultures and measured Pt-removal after exposure to cisplatin. When neuronal cultures were treated with increasing concentrations of cisplatin for two or three hours, there was a concentration-dependent increase in Pt-damage that peaked at four hours and returned to near baseline levels after 24 h. In cultures where APE1 expression was reduced by ∼80% using siRNA directed at APE1, there was a significant inhibition of Pt-removal over eight hours which was reversed by overexpressing APE1 using a lentiviral construct for human wtAPE1. Overexpressing a mutant APE1 (C65 APE1), which only has DNA repair activity, but not its other significant redox-signaling function, mimicked the effects of wtAPE1. Overexpressing DNA repair activity mutant APE1 (226 + 177APE1), with only redox activity was ineffective suggesting it is the DNA repair function of APE1 and not its redox-signaling, that restores the Pt-damage removal. Together, these data provide the first evidence that a critical BER enzyme, APE1, helps regulate the NER pathway in the repair of cisplatin damage in sensory neurons.

  1. High-Throughput, Automated Protein A Purification Platform with Multiattribute LC-MS Analysis for Advanced Cell Culture Process Monitoring.

    Science.gov (United States)

    Dong, Jia; Migliore, Nicole; Mehrman, Steven J; Cunningham, John; Lewis, Michael J; Hu, Ping

    2016-09-06

    The levels of many product related variants observed during the production of monoclonal antibodies are dependent on control of the manufacturing process, especially the cell culture process. However, it is difficult to characterize samples pulled from the bioreactor due to the low levels of product during the early stages of the process and the high levels of interfering reagents. Furthermore, analytical results are often not available for several days, which slows the process development cycle and prevents "real time" adjustments to the manufacturing process. To reduce the delay and enhance our ability to achieve quality targets, we have developed a low-volume, high-throughput, and high-content analytical platform for at-line product quality analysis. This workflow includes an automated, 96-well plate protein A purification step to isolate antibody product from the cell culture fermentation broth, followed by rapid, multiattribute LC-MS analysis. We have demonstrated quantitative correlations between particular process parameters with the levels of glycosylated and glycated species in a series of small scale experiments, but the platform could be used to monitor other attributes and applied across the biopharmaceutical industry.

  2. Hydrolyzed fish proteins modulates both inflammatory and antioxidant gene expression as well as protein expression in a co culture model of liver and head kidney cells isolated from Atlantic salmon (Salmo salar).

    Science.gov (United States)

    Holen, Elisabeth; He, Juyun; Araujo, Pedro; Seliussen, Jørgen; Espe, Marit

    2016-07-01

    Hydrolyzed fish proteins (H-pro) contain high concentrations of free amino acids and low molecular peptides that potentially may benefit fish health. The following study aimed to test whether the water-soluble phase of H-pro could attenuate lipopolysaccharide (LPS) provoked inflammation in liver cells and head kidney cells isolated from Atlantic salmon. Cells were grown as mono cultures or co cultures to assess possible crosstalk between immune cells and metabolic cells during treatments. Cells were added media with or without H-pro for 2 days before LPS exposure and harvested 24 h post LPS exposure. Respective cells without H-pro and LPS were used as controls. H-pro alone could affect expression of proteins directly as H-pro increased catalase protein expression in head kidney- and liver cells, regardless of culturing methods and LPS treatment. Leukotriene B4 (LTB4) production was also increased by H-pro in head kidney cells co cultured with liver cells. H-pro increased LPS induced interleukin 1β (IL-1β) transcription in liver cells co cultured with head kidney cells. All cultures of head kidney cells showed a significant increase in IL-1β transcription when treated with H-pro + LPS. H-pro decreased caspase-3 transcription in liver cells cultured co cultured with head kidney cells. Peroxisome proliferator activated receptor α (PPAR α) was upregulated, regardless of treatment, in liver cells co cultured with head kidney cells clearly showing that culturing method alone affected gene transcription. H-pro alone and together with LPS as an inflammation inducer, affect both antioxidant and inflammatory responses.

  3. Biofilm Filtrates of Pseudomonas aeruginosa Strains Isolated from Cystic Fibrosis Patients Inhibit Preformed Aspergillus fumigatus Biofilms via Apoptosis.

    Science.gov (United States)

    Shirazi, Fazal; Ferreira, Jose A G; Stevens, David A; Clemons, Karl V; Kontoyiannis, Dimitrios P

    2016-01-01

    Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) colonize cystic fibrosis (CF) patient airways. Pa culture filtrates inhibit Af biofilms, and Pa non-CF, mucoid (Muc-CF) and nonmucoid CF (NMuc-CF) isolates form an ascending inhibitory hierarchy. We hypothesized this activity is mediated through apoptosis induction. One Af and three Pa (non-CF, Muc-CF, NMuc-CF) reference isolates were studied. Af biofilm was formed in 96 well plates for 16 h ± Pa biofilm filtrates. After 24 h, apoptosis was characterized by viability dye DiBAc, reactive oxygen species (ROS) generation, mitochondrial membrane depolarization, DNA fragmentation and metacaspase activity. Muc-CF and NMuc-CF filtrates inhibited and damaged Af biofilm (pbiofilms (3.7- fold) compared to treatment with filtrates from Muc-CF- (2.5- fold) or non-CF Pa (1.7- fold). Depolarization of mitochondrial potential was greater upon exposure to NMuc-CF (2.4-fold) compared to Muc-CF (1.8-fold) or non-CF (1.25-fold) (pbiofilm, compared to control, mediated by metacaspase activation. In conclusion, filtrates from CF-Pa isolates were more inhibitory against Af biofilms than from non-CF. The apoptotic effect involves mitochondrial membrane damage associated with metacaspase activation.

  4. Physicochemical interaction and its influence on deep bed filtration process

    Institute of Scientific and Technical Information of China (English)

    GUO Jin-long; MENG Jun; LI GUI-ping; LUAN Zhao-kun; TANG Hong-xiao

    2004-01-01

    The capillary model was used to analyze the hydraulic conditions in the deep bed filtration process. Thephysicochemical interaction forces between the filter media and suspended particles and their influence on deep bedfiltration process were also studied theoretically. Through the comparison of the hydraulic and physicochemicalforces, the key influencing factors on the filtration process were proposed and investigated. Pilot study of the micro-flocculation deep bed filtration was carried out in the No. 9 Potable Water Treatment Plant of Beijing, and theexperimental results of hydraulic head loss, particle distribution and entrapment were presented. The theoreticalprediction was reasonably consistent with the experimental results under different conditions, which indicated that theregulation and control of micro-flocculation and deep bed filtration could be realized by the evaluation of thephysicochemical interactions. Further theoretical and experimental research should be carried out to investigate theinteraction mechanism and its application in the deep bed filtration and other cases.

  5. Production of (+)-5-deoxystrigol by Lotus japonicus root culture.

    Science.gov (United States)

    Sugimoto, Yukihiro; Ueyama, Tomoki

    2008-01-01

    Lotus japonicus roots, cultured in a modified B5 medium, produced and secreted germination stimulants that induced Striga hermonthica seed germination. The germination-inducing activity was detected both in the roots and the culture filtrate. Following bioassay-guided purification procedures, an active compound was isolated from hexane extracts of the roots and the culture filtrate. Based on chromatographic behaviour on HPLC, and 1H NMR, UV, MS and CD spectroscopic analyses, the germination stimulant was identified as (+)-5-deoxystrigol.

  6. Studies on Single Cell Culture in vitro in Wheat--The variation of grain protein content and its fractions from regenerated plants

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    On the basis of previous studies dealing with the variation of major agronomic and yield characteristics of regenerated plants derived from single cell culture in vitro of common wheat (Triticum aestivum L.Cultivar NE 7742), the grain protein content and its fractions from regenerated plants with stable agronomic characteristics were studied from 1992 to 1995. The results showed that the variation of grain protein content and its fractions in somaclones from single cell culture in vitro were very significant and the range was very wide (11.53~17.70%). Several types of variation were found in the studies, especially the type with higher protein content than that of cultivar NE 7742 (non-culture parent). Among them, -20.69% of lines the grain protein content was significantly higher than that of NE 7742 and combined with high yielding potential. The tendency of variation of the four protein fractions showed that the variation of albumin was not obvious and maintained the same level as NE7742, the content of gliadin increased in some somaclones and decreased in others. However, the percentages both globulin and glutenin tended to increase. The variation of total amount of structural protein and the ratio between globulin and glutenin tended to increase. The variation of total amount of structural protein and the ratio between globulin and albumm was mainly influenced by globulin under the condition of culture in vitro. The variation of total amount of storage protein and the ratio between gliadin and glutenin was mainly affected by glutenin. The results mentioned above demonstrated that the induction and screening of somaclonal variation could be an effective way in wheat improvement in combining high protein content with high yield.

  7. Harvesting of Dunaliella tertiolecta cells by magnetic filtration

    Science.gov (United States)

    Manousakis, Emmanouil; Manariotis, Ioannis D.

    2015-04-01

    The rising cost and reduced reserves of fossil fuels have enhanced the interest for finding alterative energy sources. Microalgae are considered to be the only sustainable option in biodiesel production for two key points. The energy yield from microalgae is much higher than that of oil producing crops, and the cultivation of algae it is not antagonistic with food supply chain. Because of the small size of microalgae and the dilute nature of algal cultures, the harvesting cost of microalgae is so far a limiting step for the scale up of microalgal biofuel production. It is estimated that the algal harvesting cost is at least 20-30% of the total biomass production cost. Traditional methods, which have been employed for the recovery of microalgal biomass, include centrifugation, gravity separation, filtration, flocculation, and flotation. Alternative approaches, other than conventional methods, capable of processing large cultures volume at a low cost, and reducing effluent toxicity are essential for microalgal biomass production. Magnetic separation is a promising technology and has been applied for algal removal in the mid of 1970s. The aim of this study was to investigate the harvesting of microalgae cells using magnetic microparticles (MPs). Dunaliella tertiolecta was selected as a representative for marine microalgae. The cultivation of microalgae was conducted under continuous artificial light, in 20 L flasks. Iron oxide microparticles were prepared by microwave irradiation of FeSO4 7H2O in an alkaline solution. Samples were taken at different operation intervals to conduct harvesting studies. Batch and flow-through experiments were conducted in order to investigate the effect of the magnetic material on microalgae removal. Algal removal in flow through experiments ranged from 70 to 85% depending on the initial MPs concentration even at very short hydraulic retention times (i.e. 2 min). In batch tests, algal removal was up to 97% at MPs concentration of 490 mg/L.

  8. The method used to culture host cells (Sf9 cells) can affect the qualities of baculovirus budding particles expressing recombinant proteins.

    Science.gov (United States)

    Hattori, Tomomi; Nakanishi, Kohei; Mori, Takaaki; Tomita, Masahiro; Tsumoto, Kanta

    2016-01-01

    Budded virus (BV) particles of baculovirus (Autographa californica nucleopolyhedrovirus, AcNPV) are harvested from the supernatant of liquid culture of Sf9 host cells by ultracentrifugation. Using polyacrylamide gel electrophoresis, Western blot and transmission electron microscopy (TEM) of BV samples fractionated closely by sucrose density gradient centrifugation, we observed that BVs exhibited different qualities depending on whether they had been harvested from the supernatant from a standing (static), shaking (suspension), or standing/shaking (pre-/post-infection) culture of Sf9 cells. The amount of BV protein apparently increased in the order of standing, standing/shaking, and shaking procedure, and the yield of intact particles showed an opposite trend. TEM observation clearly showed that appropriate fractions of the standing and standing/shaking cultures contained more intact BV particles than those from the shaking culture. These results suggest that the qualities of recombinant BV particles may be related to the culture conditions of the host cells.

  9. Data on HO-1 and CD200 protein secretion during T-cells and mesenchymal stromal cells co-cultures

    Directory of Open Access Journals (Sweden)

    Mohammad Fayyad-Kazan

    2017-04-01

    Full Text Available In this Data in Brief, we have provided data describing the secretion profile of two main immunoregulatory proteins, heme oxygenase-1 (HO-1 and CD200, from bone marrow (BM, Wharton׳s Jelly (WJ or adipose tissue (AT mesenchymal stromal cells (MSCs being cultivated either in the absence or presence of activated T-cells. Whilst HO-1 is a stress-responsive enzyme displaying diverse cytoprotective effects, CD200 is a membrane glycoprotein delivering immunoregulatory signals following interaction with its receptor (CD200R. Using Enzyme-linked immunosorbent assay (ELISA techniques, these data are presented to show distinct constitutive secretion of both HO-1 and CD200 depending on MSC types. The data presented also demonstrate that the protein levels of HO-1 and CD200 are differentially modulated during co-culture with activated T-cells. All assays were carried out in triplicates and the mean values are reported. The data presented in this article are complementary to our previously published report entitled “The Immunomodulatory Potential of Mesenchymal Stromal Cells: A Story of a Regulatory Network.” [1].

  10. Induction of trans-resveratrol and extracellular pathogenesis-related proteins in elicited suspension cultured cells of Vitis vinifera cv Monastrell.

    Science.gov (United States)

    Belchí-Navarro, Sarai; Almagro, Lorena; Sabater-Jara, Ana Belén; Fernández-Pérez, Francisco; Bru, Roque; Pedreño, Maria Angeles

    2013-02-15

    Suspension-cultured cells of Vitis vinifera cv Monastrell were used to investigate the effects of methyljasmonate, ethylene and salicylic acid separately or in combination with cyclodextrins on both trans-resveratrol production and the induction of defense responses. The results showed that the addition of methyljasmonate or ethylene to suspension-cultured cells jointly treated with cyclodextrins and salicylic acid provoked a decrease of trans-resveratrol levels suggesting that salicylic acid has a negative and antagonistic effect with methyljasmonate or ethylene on trans-resveratrol production. Likewise, the exogenous application of these compounds induced the accumulation of pathogenesis-related proteins. Analysis of the extracellular proteome showed the presence of amino acid sequences homologous to an specific β-1,3-glucanase, class III peroxidases and a β-1,4-mannanase, which suggests that these signal molecules could play a role in mediating defense-related gene product expression in V. vinifera cv Monastrell. Apart from these inducible proteins, other proteins were found in both the control and elicited cell cultures of V. vinifera. These included class IV chitinase, polygalacturonase inhibitor protein and reticuline oxidase-like protein, suggesting that their expression is constitutive being involved in the modification of the cell wall architecture during cell culture growth and in the prevention of pathogen attack. Copyright © 2012 Elsevier GmbH. All rights reserved.

  11. Viability and proliferation of L929, tumour and hybridoma cells in the culture media containing sericin protein as a supplement or serum substitute.

    Science.gov (United States)

    Cao, Ting-Ting; Zhang, Yu-Qing

    2015-09-01

    Cell cultures often require the addition of animal serum and other supplements. In this study, silk sericin, a bioactive protein, recovered from the waste of silk floss production was hydrolysed into three pepsin-degraded sericin peptides with different ranges of molecular mass. Normal animal cells, tumour cells and hybridoma cells were cultured systematically in FBS culture media containing sericin as a supplement or serum substitute. The culture test and microscopic observation of L929 cells showed that the smaller molecular weight of the degraded sericin is most suitable for cell culture. The cell culture results showed that with the degradation of sericin, for normal mouse fibroblast L929 cells, addition of 0.75 % sericin into FBS culture medium yields cell viability that is superior to FBS culture medium alone. When all serum was replaced by sericin, cell viability in the sericin medium could reach about one half of that in FBS medium. When in a medium containing a mixture of FBS: sericin (6:4, v/v), the cell culture effect is about 80 %. For the cultures of four tumour and one hybridoma cells, regardless of the molecular weight range, these degraded sericin peptides could substitute all serum in FBS media. The cell viability and proliferation of these tumour and hybridoma cells are equivalent or superior to that in FBS medium. In other words, cell viability and proliferation of these tumour and hybridoma cells in sericin media are more preferable to serum media. The mechanism of the sericin protein to promote cell growth and proliferation will be further investigated later.

  12. Expression of GPR177 (Wntless/Evi/Sprinter), a Highly Conserved Wnt-Transport Protein, in Rat Tissues, Zebrafish Embryos, and Cultured Human Cells

    OpenAIRE

    Jin, Jay; Morse, Megan; Frey, Colleen; Petko, Jessica; Levenson, Robert

    2010-01-01

    GPR177 is an evolutionarily conserved transmembrane protein necessary for Wnt protein secretion. Little is currently known, however, regarding expression of GPR177, especially in vertebrate species. We have developed an antiserum against GPR177, and used it to examine expression of GPR177 in human tissue culture cells, adult mouse and rat tissues, as well as developing zebrafish embryos. In rodents, GPR177 is expressed in virtually all tissue types and brain regions examined. In zebrafish, GP...

  13. GABAB1 and GABAB2 receptor subunits co-expressed in cultured human RPE cells regulate intracellular Ca2+ via Gi/o-protein and phospholipase C pathways.

    Science.gov (United States)

    Cheng, Z-Y; Wang, X-P; Schmid, K L; Han, X-G

    2014-11-07

    GABAB receptors associate with Gi/o-proteins that regulate voltage-gated Ca(2+) channels and thus the intracellular Ca(2+) concentration ([Ca(2+)]i), there is also reported cross-regulation of phospholipase C. These associations have been studied extensively in the brain and also shown to occur in non-neural cells (e.g. human airway smooth muscle). More recently GABAB receptors have been observed in chick retinal pigment epithelium (RPE). The aims were to investigate whether the GABAB receptor subunits, GABAB1 and GABAB2, are co-expressed in cultured human RPE cells, and then determine if the GABAB receptor similarly regulates the [Ca(2+)]i of RPE cells and if phospholipase C is involved. Human RPE cells were cultured from five donor eye cups. Evidence for GABAB1 and GABAB2 mRNAs and proteins in the RPE cell cultures was investigated using real time polymerase chain reaction, western blots and immunofluorescence. The effects of the GABAB receptor agonist baclofen, antagonist CGP46381, a Gi/o-protein inhibitor pertussis toxin, and the phospholipase C inhibitor U73122 on [Ca(2+)]i in cultured human RPE were demonstrated using Fluo-3. Both GABAB1 and GABAB2 mRNA and protein were identified in cell cultures of human RPE; antibody staining was co-localized to the cell membrane and cytoplasm. One-hundred micromolars of baclofen caused a transient increase in the [Ca(2+)]i of RPE cells regardless of whether Ca(2+) was added to the buffer. Baclofen-induced increases in the [Ca(2+)]i were attenuated by pre-treatment with CGP46381, pertussis toxin, and U73122. GABAB1 and GABAB2 are co-expressed in cell cultures of human RPE. GABAB receptors in RPE regulate the [Ca(2+)]i via a Gi/o-protein and phospholipase C pathway. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  14. Sorghum genome sequencing by methylation filtration.

    Directory of Open Access Journals (Sweden)

    Joseph A Bedell

    2005-01-01

    Full Text Available Sorghum bicolor is a close relative of maize and is a staple crop in Africa and much of the developing world because of its superior tolerance of arid growth conditions. We have generated sequence from the hypomethylated portion of the sorghum genome by applying methylation filtration (MF technology. The evidence suggests that 96% of the genes have been sequence tagged, with an average coverage of 65% across their length. Remarkably, this level of gene discovery was accomplished after generating a raw coverage of less than 300 megabases of the 735-megabase genome. MF preferentially captures exons and introns, promoters, microRNAs, and simple sequence repeats, and minimizes interspersed repeats, thus providing a robust view of the functional parts of the genome. The sorghum MF sequence set is beneficial to research on sorghum and is also a powerful resource for comparative genomics among the grasses and across the entire plant kingdom. Thousands of hypothetical gene predictions in rice and Arabidopsis are supported by the sorghum dataset, and genomic similarities highlight evolutionarily conserved regions that will lead to a better understanding of rice and Arabidopsis.

  15. Capturing phosphates with iron enhanced sand filtration.

    Science.gov (United States)

    Erickson, Andrew J; Gulliver, John S; Weiss, Peter T

    2012-06-01

    Most treatment practices for urban runoff capture pollutants such as phosphorus by either settling or filtration while dissolved phosphorus, typically as phosphates, is untreated. Dissolved phosphorus, however, represents an average 45% of total phosphorus in stormwater runoff and can be more than 95%. In this study, a new stormwater treatment technology to capture phosphate, called the Minnesota Filter, is introduced. The filter comprises iron filings mixed with sand and is tested for phosphate removal from synthetic stormwater. Results indicate that sand mixed with 5% iron filings captures an average of 88% phosphate for at least 200 m of treated depth, which is significantly greater than a sand filter without iron filings. Neither incorporation of iron filings into a sand filter nor capture of phosphates onto iron filings in column experiments had a significant effect on the hydraulic conductivity of the filter at mixtures of 5% or less iron by weight. Field applications with up to 10.7% iron were operated over 1 year without detrimental effects upon hydraulic conductivity. A model is applied and fit to column studies to predict the field performance of iron-enhanced sand filters. The model predictions are verified through the predicted performance of the filters in removing phosphates in field applications. Practical applications of the technology, both existing and proposed, are presented so stormwater managers can begin implementation.

  16. Crosslinked polytriazole membranes for organophilic filtration

    KAUST Repository

    Chisca, Stefan

    2016-12-30

    We report the preparation of crosslinked membranes for organophilic filtration, by reacting a new polytriazole with free OH groups, using non-toxic poly (ethylene glycol) diglycidyl ether (PEGDE). The OH-functionalized polymer was obtained by converting the oxadiazole to triazole rings with high yield (98%). The maximum degree of crosslinking is achieved after 6 h of reaction. The crosslinked polytriazole membranes are stable in a wide range of organic solvents and show high creep recovery, indicating the robustness of crosslinked membranes. The influence of different casting solutions and different crosslinking time on the membrane morphology and membrane performance was investigated. The membranes performance was studied in dimethylformamide (DMF) and (tetrahydrofuran) THF. We achieved a permeance for THF of 49 L m−2 h−1 bar−1 for membranes with molecular weight cut off (MWCO) of 7 kg mol−1 and a permeance for THF of 17.5 L m−2 h−1 bar−1 for membranes with MWCO of 3 kg mol−1. Our data indicate that by using the new polytriazole is possible to adjust the pore dimensions of the membranes to have a MWCO, which covers ultra- and nanofiltration range.

  17. Estimating Glomerular Filtration Rate in Older People

    Directory of Open Access Journals (Sweden)

    Sabrina Garasto

    2014-01-01

    Full Text Available We aimed at reviewing age-related changes in kidney structure and function, methods for estimating kidney function, and impact of reduced kidney function on geriatric outcomes, as well as the reliability and applicability of equations for estimating glomerular filtration rate (eGFR in older patients. CKD is associated with different comorbidities and adverse outcomes such as disability and premature death in older populations. Creatinine clearance and other methods for estimating kidney function are not easy to apply in older subjects. Thus, an accurate and reliable method for calculating eGFR would be highly desirable for early detection and management of CKD in this vulnerable population. Equations based on serum creatinine, age, race, and gender have been widely used. However, these equations have their own limitations, and no equation seems better than the other ones in older people. New equations specifically developed for use in older populations, especially those based on serum cystatin C, hold promises. However, further studies are needed to definitely accept them as the reference method to estimate kidney function in older patients in the clinical setting.

  18. Pulmonary interstitial compliance and microvascular filtration coefficient.

    Science.gov (United States)

    Goldberg, H S

    1980-08-01

    Static and dynamic properties governing the flu