WorldWideScience

Sample records for green fluorescent protein-expressing

  1. Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system.

    Science.gov (United States)

    Limaye, Arati; Koya, Vijay; Samsam, Mohtashem; Daniell, Henry

    2006-05-01

    Oral delivery of biopharmaceutical proteins expressed in plant cells should reduce their cost of production, purification, processing, cold storage, transportation, and delivery. However, poor intestinal absorption of intact proteins is a major challenge. To overcome this limitation, we investigate here the concept of receptor-mediated oral delivery of chloroplast-expressed foreign proteins. Therefore, the transmucosal carrier cholera toxin B-subunit and green fluorescent protein (CTB-GFP), separated by a furin cleavage site, was expressed via the tobacco chloroplast genome. Polymerase chain reaction (PCR) and Southern blot analyses confirmed site-specific transgene integration and homoplasmy. Immunoblot analysis and ELISA confirmed expression of monomeric and pentameric forms of CTB-GFP, up to 21.3% of total soluble proteins. An in vitro furin cleavage assay confirmed integrity of the engineered furin cleavage site, and a GM1 binding assay confirmed the functionality of CTB-GFP pentamers. Following oral administration of CTB-GFP expressing leaf material to mice, GFP was observed in the mice intestinal mucosa, liver, and spleen in fluorescence and immunohistochemical studies, while CTB remained in the intestinal cell. This report of receptor-mediated oral delivery of a foreign protein into the circulatory system opens the door for low-cost production and delivery of human therapeutic proteins.

  2. Transgenic nude mouse with green fluorescent protein expression-based human glioblastoma multiforme animal model with EGFR expression and invasiveness.

    Science.gov (United States)

    Tan, Guo-Wei; Lan, Fo-Lin; Gao, Jian-Guo; Jiang, Cai-Mou; Zhang, Yi; Huang, Xiao-Hong; Ma, Yue-Hong; Shao, He-Dui; He, Xue-Yang; Chen, Jin-Long; Long, Jian-Wu; Xiao, Hui-Sheng; Guo, Zhi-Tong; Diao, Yi

    2012-08-01

    Previously, we developed an orthotopic xenograft model of human glioblastoma multiforme (GBM) with high EGFR expression and invasiveness in Balb/c nu/nu nude mice. Now we also developed the same orthotopic xenograft model in transgenic nude mice with green fluorescent protein (GFP) expression. The present orthotopic xenografts labeled by phycoerythrin fluorescing red showed high EGFR expression profile, and invasive behavior under a bright green-red dual-color fluorescence background. A striking advantage in the present human GBM model is that the change of tumor growth can be observed visually instead of sacrificing animals in our further antitumor therapy studies.

  3. Monitoring of phytopathogenic Ralstonia solanacearum cells using green fluorescent protein-expressing plasmid derived from bacteriophage phiRSS1.

    Science.gov (United States)

    Kawasaki, Takeru; Satsuma, Hideki; Fujie, Makoto; Usami, Shoji; Yamada, Takashi

    2007-12-01

    A green fluorescent protein (GFP)-expressing plasmid was constructed from a filamentous bacteriophage phiRSS1 that infects the phytopathogen Ralstonia solanacearum. This plasmid designated as pRSS12 (4.7 kbp in size) consists of an approximately 2248 bp region of the phiRSS1 RF DNA, including ORF1-ORF3 and the intergenic region (IG), and a Km cassette in addition to the GFP gene. It was easily introduced by electroporation and stably maintained even without selective pressure in strains of R. solanacearum of different races and biovars. Strong green fluorescence emitted from pRSS12-transformed bacterial cells was easily monitored in tomato tissues (stem, petiole, and root) after infection as well as from soil samples. These results suggest that pRSS12 can serve as an easy-to-use GFP-tagging tool for any given strain of R. solanacearum in cytological as well as field studies.

  4. Green fluorescent protein expression from recombinant lettuce infectious yellows virus-defective RNAs originating from RNA 2.

    Science.gov (United States)

    Yeh, H H; Tian, T; Medina, V; Falk, B W

    2001-10-10

    Lettuce infectious yellows virus (LIYV) RNA 2 defective RNAs (D RNAs) were compared in protoplasts for their ability to replicate and to express the green fluorescent protein (GFP) from recombinant D RNA constructs. Initially four LIYV D RNAs of different genetic composition were compared, but only two (LIYV D RNA M5 and M18) replicated to high levels. Both of these contained at least two complete ORFs, one being the 3'-terminal ORF encoding P26. Northern hybridization analysis using probes corresponding to 3' regions of LIYV RNA 2 detected the P26 subgenomic RNA from protoplasts infected with LIYV RNAs 1 and 2 or protoplasts inoculated only with RNA 1 plus either the LIYV D RNA M5 or M18, suggesting that these LIYV D RNAs served as templates to generate the P26 subgenomic RNA. The GFP coding region was inserted as an in-frame insertion into the P26 coding region of the LIYV M5 and M18 D RNAs, yielding M5gfp and M18gfp. When transcripts of M5gfp and M18gfp were used to inoculate protoplasts, bright fluorescence was seen only when they were co-inoculated with LIYV RNA 1. The percentage of fluorescent protoplasts ranged from experiment to experiment, but was as high as 5.8%. Time course analyses showed that fluorescence was not detected before 48 h pi, and this correlated with the timing of LIYV RNA 2 and RNA 2 D RNA accumulation, but not with that of LIYV RNA 1. Copyright 2001 Academic Press.

  5. A Novel Prokaryotic Green Fluorescent Protein Expression System for Testing Gene Editing Tools Activity Like Zinc Finger Nuclease.

    Science.gov (United States)

    Sabzehei, Faezeh; Kouhpayeh, Shirin; Dastjerdeh, Mansoureh Shahbazi; Khanahmad, Hossein; Salehi, Rasoul; Naderi, Shamsi; Taghizadeh, Razieh; Rabiei, Parisa; Hejazi, Zahra; Shariati, Laleh

    2017-01-01

    Gene editing technology has created a revolution in the field of genome editing. The three of the most famous tools in gene editing technology are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated systems. As their predictable nature, it is necessary to assess their efficiency. There are some methods for this purpose, but most of them are time labor and complicated. Here, we introduce a new prokaryotic reporter system, which makes it possible to evaluate the efficiency of gene editing tools faster, cheaper, and simpler than previous methods. At first, the target sites of a custom ZFN, which is designed against a segment of ampicillin resistance gene, were cloned on both sides of green fluorescent protein (GFP) gene to construct pPRO-GFP. Then pPRO-GFP was transformed into Escherichia coli TOP10F' that contains pZFN (contains expression cassette of a ZFN against ampicillin resistant gene), or p15A-KanaR as a negative control. The transformed bacteria were cultured on three separate media that contained ampicillin, kanamycin, and ampicillin + kanamycin; then the resulted colonies were assessed by flow cytometry. The results of flow cytometry showed a significant difference between the case (bacteria contain pZFN) and control (bacteria contain p15A, KanaR) in MFI (Mean Fluorescence Intensity) ( P < 0.0001). According to ZFN efficiency, it can bind and cut the target sites, the bilateral cutting can affect the intensity of GFP fluorescence. Our flow cytometry results showed that this ZFN could reduce the intensity of GFP color and colony count of bacteria in media containing amp + kana versus control sample.

  6. Trade-Offs Associated with Photoprotective Green Fluorescent Protein Expression as Potential Drivers of Balancing Selection for Color Polymorphism in Reef Corals

    Directory of Open Access Journals (Sweden)

    Cathryn Quick

    2018-02-01

    Full Text Available Photodamage of symbiotic algae exposed to thermal stress is involved in mass coral bleaching, a major cause of reef decline. Photoprotection is therefore a vital part of coral stress physiology. Corals produce a variety of green fluorescent protein (GFP-like proteins, some of which screen the symbiotic algae from excess sun light. Different tissue concentrations of these GFP-like proteins distinguish color morphs that are characteristic for many coral species. The question arises whether these pigmentation differences may diversify the niches that can be occupied by corals along the steep light gradient that structures coral reef communities. We assessed the implications of GFP-like protein expression in two color morphs of the symbiotic coral Hydnophora grandis, both associated with the same Symbiodinium sp. (subclade C40. The color morphs of this species (high fluorescent, HF; and low fluorescent, LF, characterized by markedly different contents of a cyan fluorescent protein, were exposed to different quantities of blue light (470 nm that matched the major absorption band of the host pigment (473 nm. High intensities of blue light caused less photodamage to the symbiotic algae of the HF morph and resulted in higher growth rates of these corals compared to representatives of the LF morph. In contrast, under low intensities of blue light, the HF morph showed lower growth rates than the LF morph, indicating that trade-offs are associated with high levels of fluorescent protein expression under this condition. Both morphs showed highest growth rates at medium light intensities with no obvious influence of the tissue pigmentation. Reef coral color polymorphism caused by photoprotective GFP-like proteins may therefore be a product of balancing selection in which high pigment contents may be beneficial at the upper and detrimental at the lower end of the depth distribution range of symbiotic corals. Conversely, color morphs with GFP-like proteins

  7. Effect of aqueous media on the copper-ion-mediated phototoxicity of CuO nanoparticles toward green fluorescent protein-expressing Escherichia coli.

    Science.gov (United States)

    Shang, Enxiang; Li, Yang; Niu, Junfeng; Guo, Huiyuan; Zhou, Yijing; Liu, Han; Zhang, Xinqi

    2015-12-01

    Quantitative comparison of different aqueous media on the phototoxicity of copper oxide nanoparticles (CuO NPs) is crucial for understanding their ecological effects. In this study, the phototoxicity of CuO NPs toward the green fluorescent protein-expressing Escherichia coli (GFP-E. coli) under UV irradiation (365 nm) was investigated in Luria-Bertani medium (LB), NaCl solution, deionized water (DI) and phosphate-buffered saline (PBS). The phototoxicity of CuO NPs toward GFP-E. coli decreased in the order of DI>NaCl>PBS>LB because of different released concentrations of Cu(2+). The 3h released Cu(2+) concentrations by 10mg/L CuO NPs in DI water, NaCl solution, LB medium, and PBS were 1946.3 ± 75.6, 1242.5 ± 47.6, 1023.4 ± 41.2, and 1162.1 ± 41.9 μg/L, respectively. Transmission electron microscope and laser scanning confocal microscope images of E. coli exposed to CuO NPs demonstrated that the released Cu(2+) resulted in fragmentation of bacterial cell walls, leakage of intracellular components, and finally death of bacteria in four media after UV light irradiation. In each medium, the bacterial mortality rate logarithmically increased with the releasing concentrations of Cu(2+) by CuO NPs (R(2)>0.90) exposed to 3h UV light. This study highlights the importance of taking into consideration of water chemistry when the phototoxicity of CuO NPs is assessed in nanotoxicity research. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Genetic Transformation of an Obligate Anaerobe, P. gingivalis for FMN-Green Fluorescent Protein Expression in Studying Host-Microbe Interaction

    OpenAIRE

    Choi, Chul Hee; DeGuzman, Jefferson V.; Lamont, Richard J.; Yilmaz, Özlem

    2011-01-01

    The recent introduction of "oxygen-independent" flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) is of major interest to both eukaryotic and prokaryotic microbial biologists. Accordingly, we demonstrate for the first time that an obligate anaerobe, the successful opportunistic pathogen of the oral cavity, Porphyromonas gingivalis, can be genetically engineered for expression of the non-toxic green FbFP. The resulting transformants are functional for studying dynamic bacterial pr...

  9. Genetic transformation of an obligate anaerobe, P. gingivalis for FMN-green fluorescent protein expression in studying host-microbe interaction.

    Directory of Open Access Journals (Sweden)

    Chul Hee Choi

    Full Text Available The recent introduction of "oxygen-independent" flavin mononucleotide (FMN-based fluorescent proteins (FbFPs is of major interest to both eukaryotic and prokaryotic microbial biologists. Accordingly, we demonstrate for the first time that an obligate anaerobe, the successful opportunistic pathogen of the oral cavity, Porphyromonas gingivalis, can be genetically engineered for expression of the non-toxic green FbFP. The resulting transformants are functional for studying dynamic bacterial processes in living host cells. The visualization of the transformed P. gingivalis (PgFbFP revealed strong fluorescence that reached a maximum emission at 495 nm as determined by fluorescence microscopy and spectrofluorometry. Human primary gingival epithelial cells (GECs were infected with PgFbFP and the bacterial invasion of host cells was analyzed by a quantitative fluorescence microscopy and antibiotic protection assays. The results showed similar levels of intracellular bacteria for both wild type and PgFbFP strains. In conjunction with organelle specific fluorescent dyes, utilization of the transformed strain provided direct and accurate determination of the live/metabolically active P. gingivalis' trafficking in the GECs over time. Furthermore, the GECs were co-infected with PgFbFP and the ATP-dependent Clp serine protease-deficient mutant (ClpP- to study the differential fates of the two strains within the same host cells. Quantitative co-localization analyses displayed the intracellular PgFbFP significantly associated with the endoplasmic reticulum network, whereas the majority of ClpP- organisms trafficked into the lysosomes. Hence, we have developed a novel and reliable method to characterize live host cell-microbe interactions and demonstrated the adaptability of FMN-green fluorescent protein for studying persistent host infections induced by obligate anaerobic organisms.

  10. Genetic transformation of an obligate anaerobe, P. gingivalis for FMN-green fluorescent protein expression in studying host-microbe interaction.

    Science.gov (United States)

    Choi, Chul Hee; DeGuzman, Jefferson V; Lamont, Richard J; Yilmaz, Özlem

    2011-04-15

    The recent introduction of "oxygen-independent" flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) is of major interest to both eukaryotic and prokaryotic microbial biologists. Accordingly, we demonstrate for the first time that an obligate anaerobe, the successful opportunistic pathogen of the oral cavity, Porphyromonas gingivalis, can be genetically engineered for expression of the non-toxic green FbFP. The resulting transformants are functional for studying dynamic bacterial processes in living host cells. The visualization of the transformed P. gingivalis (PgFbFP) revealed strong fluorescence that reached a maximum emission at 495 nm as determined by fluorescence microscopy and spectrofluorometry. Human primary gingival epithelial cells (GECs) were infected with PgFbFP and the bacterial invasion of host cells was analyzed by a quantitative fluorescence microscopy and antibiotic protection assays. The results showed similar levels of intracellular bacteria for both wild type and PgFbFP strains. In conjunction with organelle specific fluorescent dyes, utilization of the transformed strain provided direct and accurate determination of the live/metabolically active P. gingivalis' trafficking in the GECs over time. Furthermore, the GECs were co-infected with PgFbFP and the ATP-dependent Clp serine protease-deficient mutant (ClpP-) to study the differential fates of the two strains within the same host cells. Quantitative co-localization analyses displayed the intracellular PgFbFP significantly associated with the endoplasmic reticulum network, whereas the majority of ClpP- organisms trafficked into the lysosomes. Hence, we have developed a novel and reliable method to characterize live host cell-microbe interactions and demonstrated the adaptability of FMN-green fluorescent protein for studying persistent host infections induced by obligate anaerobic organisms.

  11. Immunohistochemistry of connexin 43 throughout anterior pituitary gland in a transgenic rat with green fluorescent protein-expressing folliculo-stellate cells.

    Science.gov (United States)

    Horiguchi, Kotaro; Fujiwara, Ken; Kouki, Tom; Kikuchi, Motoshi; Yashiro, Takashi

    2008-12-01

    Folliculo-stellate (FS) cells in the anterior pituitary gland have been speculated to possess multifunctional properties. Because gap junctions (GJ) have been identified between FS cells, FS cells may be interconnected electrophysiologically by GJ and serve as signal transmission networks to modulate hormone release in the anterior pituitary gland. But whether GJ are localized among FS cells from the pars tuberalis through the pars distalis is unclear. The S100b-GFP transgenic rat has recently been generated, which expresses green fluorescent protein (GFP) specifically in FS cells in the anterior pituitary. This model is expected to be a powerful tool for studies of FS cells. The purpose of the present paper was therefore to examine the localization of GJ on connexin 43 immunohistochemistry throughout the anterior pituitary gland of S100b-GFP rats under confocal laser microscopy. The localization patterns of FS cells was also observed in primary culture of anterior pituitary cells and the question of whether GJ between FS cells are reconstructed in vitro was investigated. In vivo studies showed that GJ were present specifically between FS cells from the pars tuberalis to the pars distalis in the anterior pituitary gland. The appearance of FS cells was distinguished into two types, with localization of GJ differing between types. In vitro, it was observed for the first time that FS cells in primary culture could be categorized into two types. In vivo localization of GJ between FS cells was reconstructed in vitro. These morphological observations are consistent with the hypothesis that FS cells form an electrophysiological network throughout the anterior pituitary for signal transmission.

  12. Engineering and Characterization of a Superfolder Green Fluorescent Protein

    International Nuclear Information System (INIS)

    Pedelacq, J.; Cabantous, S.; Tran, T.; Terwilliger, T.; Waldo, G.

    2006-01-01

    Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP

  13. Red and Green Fluorescence from Oral Biofilms.

    Science.gov (United States)

    Volgenant, Catherine M C; Hoogenkamp, Michel A; Krom, Bastiaan P; Janus, Marleen M; Ten Cate, Jacob M; de Soet, Johannes J; Crielaard, Wim; van der Veen, Monique H

    2016-01-01

    Red and green autofluorescence have been observed from dental plaque after excitation by blue light. It has been suggested that this red fluorescence is related to caries and the cariogenic potential of dental plaque. Recently, it was suggested that red fluorescence may be related to gingivitis. Little is known about green fluorescence from biofilms. Therefore, we assessed the dynamics of red and green fluorescence in real-time during biofilm formation. In addition, the fluorescence patterns of biofilm formed from saliva of eight different donors are described under simulated gingivitis and caries conditions. Biofilm formation was analysed for 12 hours under flow conditions in a microfluidic BioFlux flow system with high performance microscopy using a camera to allow live cell imaging. For fluorescence images dedicated excitation and emission filters were used. Both green and red fluorescence were linearly related with the total biomass of the biofilms. All biofilms displayed to some extent green and red fluorescence, with higher red and green fluorescence intensities from biofilms grown in the presence of serum (gingivitis simulation) as compared to the sucrose grown biofilms (cariogenic simulation). Remarkably, cocci with long chain lengths, presumably streptococci, were observed in the biofilms. Green and red fluorescence were not found homogeneously distributed within the biofilms: highly fluorescent spots (both green and red) were visible throughout the biomass. An increase in red fluorescence from the in vitro biofilms appeared to be related to the clinical inflammatory response of the respective saliva donors, which was previously assessed during an in vivo period of performing no-oral hygiene. The BioFlux model proved to be a reliable model to assess biofilm fluorescence. With this model, a prediction can be made whether a patient will be prone to the development of gingivitis or caries.

  14. Red and Green Fluorescence from Oral Biofilms.

    Directory of Open Access Journals (Sweden)

    Catherine M C Volgenant

    Full Text Available Red and green autofluorescence have been observed from dental plaque after excitation by blue light. It has been suggested that this red fluorescence is related to caries and the cariogenic potential of dental plaque. Recently, it was suggested that red fluorescence may be related to gingivitis. Little is known about green fluorescence from biofilms. Therefore, we assessed the dynamics of red and green fluorescence in real-time during biofilm formation. In addition, the fluorescence patterns of biofilm formed from saliva of eight different donors are described under simulated gingivitis and caries conditions. Biofilm formation was analysed for 12 hours under flow conditions in a microfluidic BioFlux flow system with high performance microscopy using a camera to allow live cell imaging. For fluorescence images dedicated excitation and emission filters were used. Both green and red fluorescence were linearly related with the total biomass of the biofilms. All biofilms displayed to some extent green and red fluorescence, with higher red and green fluorescence intensities from biofilms grown in the presence of serum (gingivitis simulation as compared to the sucrose grown biofilms (cariogenic simulation. Remarkably, cocci with long chain lengths, presumably streptococci, were observed in the biofilms. Green and red fluorescence were not found homogeneously distributed within the biofilms: highly fluorescent spots (both green and red were visible throughout the biomass. An increase in red fluorescence from the in vitro biofilms appeared to be related to the clinical inflammatory response of the respective saliva donors, which was previously assessed during an in vivo period of performing no-oral hygiene. The BioFlux model proved to be a reliable model to assess biofilm fluorescence. With this model, a prediction can be made whether a patient will be prone to the development of gingivitis or caries.

  15. Red and green fluorescence from oral biofilms

    NARCIS (Netherlands)

    Volgenant, C.M.C.; Hoogenkamp, M.A.; Krom, B.P.; Janus, M.M.; ten Cate, J.M.; de Soet, J.J.; Crielaard, W.; van der Veen, M.H.

    2016-01-01

    Red and green autofluorescence have been observed from dental plaque after excitation by blue light. It has been suggested that this red fluorescence is related to caries and the cariogenic potential of dental plaque. Recently, it was suggested that red fluorescence may be related to gingivitis.

  16. Mechanistic background and clinical applications of indocyanine green fluorescence imaging of hepatocellular carcinoma.

    Science.gov (United States)

    Ishizawa, Takeaki; Masuda, Koichi; Urano, Yasuteru; Kawaguchi, Yoshikuni; Satou, Shouichi; Kaneko, Junichi; Hasegawa, Kiyoshi; Shibahara, Junji; Fukayama, Masashi; Tsuji, Shingo; Midorikawa, Yutaka; Aburatani, Hiroyuki; Kokudo, Norihiro

    2014-02-01

    Although clinical applications of intraoperative fluorescence imaging of liver cancer using indocyanine green (ICG) have begun, the mechanistic background of ICG accumulation in the cancerous tissues remains unclear. In 170 patients with hepatocellular carcinoma cells (HCC), the liver surfaces and resected specimens were intraoperatively examined by using a near-infrared fluorescence imaging system after preoperative administration of ICG (0.5 mg/kg i.v.). Microscopic examinations, gene expression profile analysis, and immunohistochemical staining were performed for HCCs, which showed ICG fluorescence in the cancerous tissues (cancerous-type fluorescence), and HCCs showed fluorescence only in the surrounding non-cancerous liver parenchyma (rim-type fluorescence). ICG fluorescence imaging enabled identification of 273 of 276 (99%) HCCs in the resected specimens. HCCs showed that cancerous-type fluorescence was associated with higher cancer cell differentiation as compared with rim-type HCCs (P Fluorescence microscopy identified the presence of ICG in the canalicular side of the cancer cell cytoplasm, and pseudoglands of the HCCs showed a cancerous-type fluorescence pattern. The ratio of the gene and protein expression levels in the cancerous to non-cancerous tissues for Na(+)/taurocholate cotransporting polypeptide (NTCP) and organic anion-transporting polypeptide 8 (OATP8), which are associated with portal uptake of ICG by hepatocytes that tended to be higher in the HCCs that showed cancerous-type fluorescence than in those that showed rim-type fluorescence. Preserved portal uptake of ICG in differentiated HCC cells by NTCP and OATP8 with concomitant biliary excretion disorders causes accumulation of ICG in the cancerous tissues after preoperative intravenous administration. This enables highly sensitive identification of HCC by intraoperative ICG fluorescence imaging.

  17. Differential tissue expression of enhanced green fluorescent protein in ‘Green mice’

    OpenAIRE

    Ma, De-Fu; Tezuka, Hideo; Kondo, Tetsuo; Sudo, Katsuko; Niu, Dong-Feng; Nakazawa, Tadao; Kawasaki, Tomonori; Yamane, Tetsu; Nakamura, Nobuki; Katoh, Ryohei

    2010-01-01

    In order to clarify tissue expression of enhanced green fluorescent protein (EGFP) in ‘green mice’ from a transgenic line having an EGFP cDNA under the control of a chicken beta-actin promoter and cytomegalovirus enhancer, we studied the expression of EGFP in various organs and tissues from these ‘green mice’ by immunohistochemistry with anti- EGFP antibody in conjunction with direct observation for EGFP fluorescence using confocal laser scanning microscopy. On i...

  18. Evaluation of gene amplification and protein expression of HER-2/neu in esophageal squamous cell carcinoma using Fluorescence in situ Hybridization (FISH) and immunohistochemistry

    International Nuclear Information System (INIS)

    Sato-Kuwabara, Yukie; Neves, José I; Fregnani, José HTG; Sallum, Rubens A; Soares, Fernando A

    2009-01-01

    Esophageal squamous cell carcinoma (ESCC) is the sixth most frequent neoplasia in Brazil. It is usually associated with a poor prognosis because it is often at an advanced stage when diagnosed and there is a high frequency of lymph node metastases. It is important to know what prognostic factors can facilitate diagnosis, optimize therapeutic decisions, and improve the survival of these patients. A member of the epidermal growth factor receptor (EGFR) family, c-erbB-2, has received much attention because of its therapeutic implications; however, few studies involving fluorescence in situ hybridization (FISH) analysis of HER-2/neu gene amplification and protein expression in ESCC have been conducted. The aim of this study was to verify the presence of HER-2/neu gene amplification using FISH, and to correlate the results with immunohistochemical expression and clinical-pathological findings. One hundred and ninety-nine ESCC cases were evaluated using the Tissue Microarray (TMA) technique. A polyclonal antibody against c-erbB-2 was used for immunohistochemistry. Analyses were based on the membrane staining pattern. The results were classified according to the Herceptest criteria (DAKO): negative (0/1+), potential positive (2+) and positive (3+). The FISH reactions were performed according to the FISH HER2 PharmDx (DAKO) protocol. In each case, 100 tumor nuclei were evaluated. Cases showing a gene/CEN17 fluorescence ratio ≥ 2 were considered positive for gene amplification. The c-erbB-2 expression was negative in 117/185 cases (63.2%) and positive in 68 (36.8%), of which 56 (30.3%) were 2+ and 12 (6.5%) were 3+. No significant associations were found among protein expression, clinicopathological data and overall survival. Among the 47 cases analyzed, 38 (80.9%) showed no gene amplification while 9 (19.1%) showed amplification, as demonstrated by FISH. Cases that were negative (0/1+) and potential positive (2+) for c-erbB-2 expression by immunohistochemistry showed no

  19. Development of ultrasound-assisted fluorescence imaging of indocyanine green.

    Science.gov (United States)

    Morikawa, Hiroyasu; Toyota, Shin; Wada, Kenji; Uchida-Kobayashi, Sawako; Kawada, Norifumi; Horinaka, Hiromichi

    2017-01-01

    Indocyanine green (ICG) accumulation in hepatocellular carcinoma means tumors can be located by fluorescence. However, because of light scattering, it is difficult to detect ICG fluorescence from outside the body. We propose a new fluorescence imaging method that detects changes in the intensity of ICG fluorescence by ultrasound-induced temperature changes. ICG fluorescence intensity decreases as the temperature rises. Therefore, it should theoretically be possible to detect tissue distribution of ICG using ultrasound to heat tissue, moving the point of ultrasound transmission, and monitoring changes in fluorescence intensity. A new probe was adapted for clinical application. It consisted of excitation light from a laser, fluorescence sensing through a light pipe, and heating by ultrasound. We applied the probe to bovine liver to image the accumulation of ICG. ICG emits fluorescence (820 nm) upon light irradiation (783 nm). With a rise in temperature, the fluorescence intensity of ICG decreased by 0.85 %/°C. The distribution of fluorescent ICG was detected using an ultrasonic warming method in a new integrated probe. Modulating fluorescence by changing the temperature using ultrasound can determine where ICG accumulates at a depth, highlighting its potential as a means to locate hepatocellular carcinoma.

  20. Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy

    NARCIS (Netherlands)

    van Manen, Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; van den Berg, Timo K; Roos, Dirk; Otto, Cees

    2008-01-01

    We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91(phox), which are both subunits of the phagocyte NADPH

  1. Molecular quantification of genes encoding for green-fluorescent proteins

    DEFF Research Database (Denmark)

    Felske, A; Vandieken, V; Pauling, B V

    2003-01-01

    A quantitative PCR approach is presented to analyze the amount of recombinant green fluorescent protein (gfp) genes in environmental DNA samples. The quantification assay is a combination of specific PCR amplification and temperature gradient gel electrophoresis (TGGE). Gene quantification...... PCR strategy is a highly specific and sensitive way to monitor recombinant DNA in environments like the efflux of a biotechnological plant....

  2. A Review of Indocyanine Green Fluorescent Imaging in Surgery

    Directory of Open Access Journals (Sweden)

    Jarmo T. Alander

    2012-01-01

    Full Text Available The purpose of this paper is to give an overview of the recent surgical intraoperational applications of indocyanine green fluorescence imaging methods, the basics of the technology, and instrumentation used. Well over 200 papers describing this technique in clinical setting are reviewed. In addition to the surgical applications, other recent medical applications of ICG are briefly examined.

  3. Expression of green fluorescent protein (GFPuv) in Escherichia coli ...

    African Journals Online (AJOL)

    Administrator

    The recombinant green fluorescent protein (GFPuv) was expressed by transformed cells of Escherichia coli DH5-α grown in LB/amp broth at 37oC, for 8 h and 24 h. To evaluate the effectiveness of different parameters to improve the expression of GFPuv by E. coli, four variable culturing conditions were set up for assays by ...

  4. Molecular Iodine Fluorescence Using a Green Helium-Neon Laser

    Science.gov (United States)

    Williamson, J. Charles

    2011-01-01

    Excitation of molecular iodine vapor with a green (543.4 nm) helium-neon laser produces a fluorescence spectrum that is well suited for the upper-level undergraduate physical chemistry laboratory. Application of standard evaluation techniques to the spectrum yields ground electronic-state molecular parameters in good agreement with literature…

  5. An optical method for reducing green fluorescence from urine during fluorescence-guided cystoscopy

    DEFF Research Database (Denmark)

    Lindvold, Lars René; Hermann, Gregers G

    2016-01-01

    Photodynamic diagnosis (PDD) of bladder tumour tissue significantly improves endoscopic diagnosis and treatment of bladder cancer in rigid cystoscopes in the operating theatre and thus reduces tumour recurrence. PDD comprises the use of blue light, which unfortunately excites green fluorescence...... this light source also is useful for exciting autofluorescence in healthy bladder mucosa. This autofluorescence then provides a contrast to the sensitized fluorescence (PDD) of tumours in the bladder....

  6. An optical method for reducing green fluorescence from urine during fluorescence-guided cystoscopy

    Science.gov (United States)

    Lindvold, Lars R.; Hermann, Gregers G.

    2016-12-01

    Photodynamic diagnosis (PDD) of bladder tumour tissue significantly improves endoscopic diagnosis and treatment of bladder cancer in rigid cystoscopes in the operating theatre and thus reduces tumour recurrence. PDD comprises the use of blue light, which unfortunately excites green fluorescence from urine. As this green fluorescence confounds the desired red fluorescence of the PDD, methods for avoiding this situation particularly in cystoscopy using flexible cystoscopes are desirable. In this paper we demonstrate how a tailor made high power LED light source at 525 nm can be used for fluorescence assisted tumour detection using both a flexible and rigid cystoscope used in the outpatient department (OPD) and operating room (OR) respectively. It is demonstrated both in vitro and in vivo how this light source can significantly reduce the green fluorescence problem with urine. At the same time this light source also is useful for exciting autofluorescence in healthy bladder mucosa. This autofluorescence then provides a contrast to the sensitized fluorescence (PDD) of tumours in the bladder.

  7. Differential tissue expression of enhanced green fluorescent protein in 'green mice'.

    Science.gov (United States)

    Ma, De-Fu; Tezuka, Hideo; Kondo, Tetsuo; Sudo, Katsuko; Niu, Dong-Feng; Nakazawa, Tadao; Kawasaki, Tomonori; Yamane, Tetsu; Nakamura, Nobuki; Katoh, Ryohei

    2010-06-01

    In order to clarify tissue expression of enhanced green fluorescent protein (EGFP) in 'green mice' from a transgenic line having an EGFP cDNA under the control of a chicken beta-actin promoter and cytomegalovirus enhancer, we studied the expression of EGFP in various organs and tissues from these 'green mice' by immunohistochemistry with anti- EGFP antibody in conjunction with direct observation for EGFP fluorescence using confocal laser scanning microscopy. On immunohistochemical examination and on direct observation by confocal laser scanning microscopy, the level of EGFP expression varied among organs and tissues. EGFP expression was diffusely and strongly observed in the skin, pituitary, thyroid gland, parathyroid gland, heart, gall bladder, pancreas, adrenals and urinary bladder. There was only sporadic and weak expression of EGFP in the epithelium of the trachea, bronchus of the lung, stratified squamous epithelium and gastric glands of the stomach, hepatic bile ducts of the liver, glomeruli and renal tubules of the kidney and endo-metrial glands of the uterus. Furthermore, EGFP was only demonstrated within the goblet and paneth cells in the colon and small intestine, the tall columnar cells in the ductus epididymis, and the leydig cells in the testis. In conclusion, our results show that EGFP is differentially expressed in organs and tissues of 'green mice', which indicates that 'green mice' may prove useful for research involving transplantation and tissue clonality.

  8. Using Fluorescence Intensity of Enhanced Green Fluorescent Protein to Quantify Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Erin Wilson

    2018-05-01

    Full Text Available A variety of direct and indirect methods have been used to quantify planktonic and biofilm bacterial cells. Direct counting methods to determine the total number of cells include plate counts, microscopic cell counts, Coulter cell counting, flow cytometry, and fluorescence microscopy. However, indirect methods are often used to supplement direct cell counting, as they are often more convenient, less time-consuming, and require less material, while providing a number that can be related to the direct cell count. Herein, an indirect method is presented that uses fluorescence emission intensity as a proxy marker for studying bacterial accumulation. A clinical strain of Pseudomonas aeruginosa was genetically modified to express a green fluorescent protein (PA14/EGFP. The fluorescence intensity of EGFP in live cells was used as an indirect measure of live cell density, and was compared with the traditional cell counting methods of optical density (OD600 and plate counting (colony-forming units (CFUs. While both OD600 and CFUs are well-established methods, the use of fluorescence spectroscopy to quantify bacteria is less common. This study demonstrates that EGFP intensity is a convenient reporter for bacterial quantification. In addition, we demonstrate the potential for fluorescence spectroscopy to be used to measure the quantity of PA14/EGFP biofilms, which have important human health implications due to their antimicrobial resistance. Therefore, fluorescence spectroscopy could serve as an alternative or complementary quick assay to quantify bacteria in planktonic cultures and biofilms.

  9. Changes of the laser-induced blue, green and red fluorescence signatures during greening of etiolated leaves of wheat

    International Nuclear Information System (INIS)

    Stober, F.; Lichtenthaler, H.K.

    1992-01-01

    The UV-laser-induced blue, green and red fluorescence-emission spectra were used to characterize the pigment status of etiolated leaves of wheat (Triticum aestivum L.) during a 48 h greening period under white light conditions. Upon UV-light excitation (337 nm) leaves not only show a fluorescence emission in the red spectral region between 650 and 800nm (chlorophyll fluorescence with maxima near 690nm and 735 nm), but also in the blue and green regions between 400 to 570 nm with maxima or shoulders near 450 nm (blue) and 530 nm (green). During greening of etiolated leaves the chlorophyll-fluorescence ratio F690/F735 strongly correlated with the total chlorophyll content and the ratio of the chlorophylls to the carotenoids (a+b/x+c). The ratio of the blue to the green fluorescence F450/F530 was also correlated with the total chlorophyll content and the ratio of chlorophylls to total carotenoids (a+b/x+c). Consequently, there also existed a correlation between the chlorophyll-fluorescence ratio F690/F735 and the ratio of the blue to green fluorescence F450/F530. In contrast, the ratios of the blue to red fluorescences F450/F690 and F450/F735 did not show clear relations to the pigment content of the investigated plants. The particular shape of the UV-laser-induced-fluorescence emission spectra of wheat leaves as well as the dependencies of the fluorescence ratios on the pigment content are due to a partial and differential reabsorption of the emitted fluorescences by the photosynthetic pigments

  10. Detection of ALK gene rearrangement in non-small cell lung cancer: a comparison of fluorescence in situ hybridization and chromogenic in situ hybridization with correlation of ALK protein expression.

    Science.gov (United States)

    Kim, Hyojin; Yoo, Seol-Bong; Choe, Ji-Young; Paik, Jin Ho; Xu, Xianhua; Nitta, Hiroaki; Zhang, Wenjun; Grogan, Thomas M; Lee, Choon-Taek; Jheon, Sanghoon; Chung, Jin-Haeng

    2011-08-01

    Accurate determination of ALK rearrangement is important in lung cancer patients, especially in determining their eligibility for crizotinib therapy. Fluorescence in situ hybridization (FISH) has been regarded as the gold standard method for detecting ALK rearrangement. However, FISH requires a fluorescence microscope, and the signals are labile and rapidly fade over time. This study evaluates the concordance between ALK gene rearrangement in non-small cell lung cancer assessed by ALK FISH and a newly developed ALK chromogenic in situ hybridization (CISH) and correlates the results with ALK protein expression assessed by immunohistochemistry. A total of 465 formalin-fixed, paraffin-embedded non-small cell lung cancer samples were analyzed by ALK FISH (PathVysion, Vysis, Abbott) and ALK CISH. For comparison, all specimens were stained by immunohistochemistry (clone 5A4, Novocastra) and interobserver reproducibility was assessed. We found that agreement between the pathologists on the CISH-determined ALK status was achieved in 449 patients (96.6%), and ALK rearrangement was identified in 18 patients (4.0%) in CISH method. Among these cases, 443 cases (95.3%) had results matching the corresponding FISH results: 17 rearranged, 425 wild types, and 1 discordant case. There was high concordance in the assessment of ALK gene rearrangement between FISH and CISH techniques (κ = 0.92) and between observers (κ = 0.97). In addition, there was high concordance in the ALK gene status and ALK protein expression between CISH and IHC tests (κ = 0.82). CISH is a highly reproducible and practical method to detect ALK gene rearrangement and correlated well with ALK protein expression. Here, we present a diagnostic algorithm (Chung's SNUBH ALK protocol) to detect lung cancer with ALK rearrangements using IHC, FISH and CISH. Because CISH allows a concurrent analysis of histological features of the tumors and gene rearrangement, it appears to be a useful method in determining ALK gene

  11. Green synthesis, structure and fluorescence spectra of new azacyanine dyes

    Science.gov (United States)

    Enchev, Venelin; Gadjev, Nikolai; Angelov, Ivan; Minkovska, Stela; Kurutos, Atanas; Markova, Nadezhda; Deligeorgiev, Todor

    2017-11-01

    A series of symmetric and unsymmetric monomethine azacyanine dyes (monomethine azacyanine and merocyanine sulfobetaines) were synthesized with moderate to high yields via a novel method using microwave irradiation. The compounds are derived from a condensation reaction between 2-thiomethylbenzotiazolium salts and 2-imino-3-methylbenzothiazolines proceeded under microwave irradiation. The synthetic approach involves the use of green solvent and absence of basic reagent. TD-DFT calculations were performed to simulate absorption and fluorescent spectra of synthesized dyes. Absorption maxima, λmax, of the studied dyes were found in the range 364-394 nm. Molar absorbtivities were evaluated in between 40300 and 59200 mol-1 dm3 cm-1. Fluorescence maxima, λfl, were registered around 418-448 nm upon excitation at 350 nm. A slight displacements of theoretically estimated absorption maxima according to experimental ones is observed. The differences are most probably due to the fact that the DFT calculations were carried out without taking into account the solvent effect. In addition, the merocyanine sulfobetaines also fluorescence in blue optical range (420-480 nm) at excitation in red range (630-650 nm).

  12. Correlation of Hypoxia and Pro-senescence Protein Expression in Green Sea Turtle (Chelonia mydas Lung Epithelial and Dermal Fibroblast Cell Culture

    Directory of Open Access Journals (Sweden)

    Anggraini Barlian

    2018-03-01

    Full Text Available Recent studies have shown hypoxia-induced gene expression correlated with cellular senescence. HIF-1α (hypoxia-inducible factor 1-alpha, p53, and pRB were induced under hypoxia and correlated with cellular senescence. The localization and expression of HIF-1α, p53, and pRB in Chelonia mydas lung epithelial and dermal fibroblast cell cultures were analyzed under normoxic and hypoxic conditions (at 4 and 24 hours. Human dermal fibroblast was used for comparison purposes. Protein localization was analyzed with immunocytochemistry, while protein expression was analyzed with the Western blot and enhanced chemiluminescence (ECL method. HIF-1α, p53, and pRB were localized in the nuclei of the C. mydas cell cultures treated with hypoxia. The C. mydas lung epithelial cell cultures had a higher increase of HIF-1α expression than the human dermal fibroblast cell culture. The hypoxic conditions did not affect p53 expression significantly in C. mydas lung epithelial and dermal fibroblast cell cultures. Meanwhile, pRB expression changed significantly under hypoxia in the C. mydas dermal fibroblast cells. Expression of p53 and pRB in the human cell cultures was higher than in the C. mydas cell cultures. This research suggests that C. mydas and human cell cultures have different pro-senescence protein expression responses under hypoxic conditions.

  13. The structure of mAG, a monomeric mutant of the green fluorescent protein Azami-Green, reveals the structural basis of its stable green emission

    International Nuclear Information System (INIS)

    Ebisawa, Tatsuki; Yamamura, Akihiro; Kameda, Yasuhiro; Hayakawa, Kou; Nagata, Koji; Tanokura, Masaru

    2010-01-01

    The crystal structure of a monomeric mutant of Azami-Green (mAG) from G. fascicularis was determined at 2.2 Å resolution. Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first known monomeric green-emitting fluorescent protein that is not a variant of Aequorea victoria green fluorescent protein (avGFP). These two green fluorescent proteins are only 27% identical in their amino-acid sequences. mAG is more similar in its amino-acid sequence to four fluorescent proteins: Dendra2 (a green-to-red irreversibly photoconverting fluorescent protein), Dronpa (a bright-and-dark reversibly photoswitchable fluorescent protein), KikG (a tetrameric green-emitting fluorescent protein) and Kaede (another green-to-red irreversibly photoconverting fluorescent protein). To reveal the structural basis of stable green emission by mAG, the 2.2 Å crystal structure of mAG has been determined and compared with the crystal structures of avGFP, Dronpa, Dendra2, Kaede and KikG. The structural comparison revealed that the chromophore formed by Gln62-Tyr63-Gly64 (QYG) and the fixing of the conformation of the imidazole ring of His193 by hydrogen bonds and van der Waals contacts involving His193, Arg66 and Thr69 are likely to be required for the stable green emission of mAG. The crystal structure of mAG will contribute to the design and development of new monomeric fluorescent proteins with faster maturation, brighter fluorescence, improved photostability, new colours and other preferable properties as alternatives to avGFP and its variants

  14. Green Fluorescent Protein (GFP) as a reporter gene for the plant pathogenic oomycete Phytophthora ramorum

    Science.gov (United States)

    Marko Riedel; Gautier Calmin; Lassaad Belbahri; Francois Lefort; Monika Gotz; Stefan Wagner; Sabine. Werres

    2009-01-01

    Transgenic Phytophthora ramorum strains that produce green fluorescent protein (GFP) constitutively were obtained after stable DNA integration using a polyethylene glycol and CaCl2-based transformation protocol. Green fluorescent protein production was studied in developing colonies and in different propagules of the pathogen...

  15. Refractive Index Sensing of Green Fluorescent Proteins in Living Cells Using Fluorescence Lifetime Imaging Microscopy

    Science.gov (United States)

    van Manen, Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; van den Berg, Timo K.; Roos, Dirk; Otto, Cees

    2008-01-01

    We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91phox, which are both subunits of the phagocyte NADPH oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91phox are targeted to the cytosol and to membranes, respectively. Frequency-domain FLIM experiments on these PLB-985 cells resulted in average fluorescence lifetimes of 2.70 ns for cytosolic GFP-Rac2 and 2.31 ns for membrane-bound GFP-gp91phox. By comparing these lifetimes with a calibration curve obtained by measuring GFP lifetimes in PBS/glycerol mixtures of known refractive index, we found that the local refractive indices of cytosolic GFP-Rac2 and membrane-targeted GFP-gp91phox are ∼1.38 and ∼1.46, respectively, which is in good correspondence with reported values for the cytosol and plasma membrane measured by other techniques. The ability to measure the local refractive index of proteins in living cells by FLIM may be important in revealing intracellular spatial heterogeneities within organelles such as the plasma and phagosomal membrane. PMID:18223002

  16. Green Fluorescent Organic Light Emitting Device with High Luminance

    Directory of Open Access Journals (Sweden)

    Ning YANG

    2014-06-01

    Full Text Available In this work, we fabricated the small molecule green fluorescent bottom-emission organic light emitting device (OLED with the configuration of glass substrate/indium tin oxide (ITO/Copper Phthalocyanine (CuPc 25 nm/ N,N’-di(naphthalen-1-yl-N,N’-diphenyl-benzidine (NPB 45 nm/ tris(8-hydroxyquinoline aluminium (Alq3 60 nm/ Lithium fluoride (LiF 1 nm/Aluminum (Al 100 nm where CuPc and NPB are the hole injection layer and the hole transport layer, respectively. CuPc is introduced in this device to improve carrier injection and efficiency. The experimental results indicated that the turn-on voltage is 2.8 V with a maximum luminance of 23510 cd/m2 at 12 V. The maximum current efficiency and power efficiency are 4.8 cd/A at 100 cd/m2 and 4.2 lm/W at 3 V, respectively. The peak of electroluminance (EL spectrum locates at 530 nm which is typical emission peak of green light. In contrast, the maximum current efficiency and power efficiency of the device without CuPc are only 4.0 cd/A at 100 mA/cm2 and 4.2 lm/W at 3.6 V, respectively.

  17. Interferences of Silica Nanoparticles in Green Fluorescent Protein Folding Processes.

    Science.gov (United States)

    Klein, Géraldine; Devineau, Stéphanie; Aude, Jean Christophe; Boulard, Yves; Pasquier, Hélène; Labarre, Jean; Pin, Serge; Renault, Jean Philippe

    2016-01-12

    We investigated the relationship between unfolded proteins, silica nanoparticles and chaperonin to determine whether unfolded proteins could stick to silica surfaces and how this process could impair heat shock protein activity. The HSP60 catalyzed green fluorescent protein (GFP) folding was used as a model system. The adsorption isotherms and adsorption kinetics of denatured GFP were measured, showing that denaturation increases GFP affinity for silica surfaces. This affinity is maintained even if the surfaces are covered by a protein corona and allows silica NPs to interfere directly with GFP folding by trapping it in its unstructured state. We determined also the adsorption isotherms of HSP60 and its chaperonin activity once adsorbed, showing that SiO2 NP can interfere also indirectly with protein folding through chaperonin trapping and inhibition. This inhibition is specifically efficient when NPs are covered first with a layer of unfolded proteins. These results highlight for the first time the antichaperonin activity of silica NPs and ask new questions about the toxicity of such misfolded proteins/nanoparticles assembly toward cells.

  18. Production of yellow-green fluorescent pigment by Pseudomonas fluorescens

    Directory of Open Access Journals (Sweden)

    Gildo Almeida da Silva

    2006-05-01

    Full Text Available A medium was prepared from brewery waste yeast with and without mineral salts to study growth and yellow-green fluorescent pigment production (YGFP by Pseudomonas fluorescens. The King's medium used for detection of siderophore production were expressively weaker inductors of YGFP formation when compared to FYE medium. Although FYE and CYE could be used for growth of P. fluorescens, only FYE was an attractive medium for detection of YGFP strain producers.Diversos microrganismos secretam substâncias com alta afinidade por ferro. Estas moléculas, sideróforos, transportam ferro para o interior das células. Como a produção destas moléculas depende da composição do meio, foi avaliada a influência do extrato de levedura (FYE, proveniente de resíduo de cervejaria, com e sem adição de sais minerais, sobre o crescimento de Pseudomonas fluorescens e sobre a formação de pigmento fluorescente verde-amarelado (YGFP. Observou-se que (i FYE com sacarose (G7 e o extrato de levedura comercial (CYE possuem um pico bem definido próximo a 260 nm; (ii FYE, mas não CYE, promove alta formação de YGFP. Os meios de King's, usados para detectar a formação de sideróforo, se comportaram como indutores expressivamente mais fracos de YGFP que o meio FYE. Embora FYE e CYE possam ser usados para o crescimento de P. fluorescens, apenas FYE pode ser usado como meio para a detecção de linhagens formadoras de YGFP.

  19. Green Fluorescent Protein (GFP-Based Overexpression Screening and Characterization of AgrC, a Receptor Protein of Quorum Sensing in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Shengdi Fan

    2013-09-01

    Full Text Available Staphylococcus aureus AgrC is an important component of the agr quorum-sensing system. AgrC is a membrane-embedded histidine kinase that is thought to act as a sensor for the recognition of environmental signals and the transduction of signals into the cytoplasm. However, the difficulty of expressing and purifying functional membrane proteins has drastically hindered in-depth understanding of the molecular structures and physiological functions of these proteins. Here, we describe the high-yield expression and purification of AgrC, and analyze its kinase activity. A C-terminal green fluorescent protein (GFP fusion to AgrC served as a reporter for monitoring protein expression levels in real time. Protein expression levels were analyzed by the microscopic assessment of the whole-cell fluorescence. The expressed AgrC-GFP protein with a C-terminal His-tagged was purified using immobilized metal affinity chromatography (IMAC and size exclusion chromatography (SEC at yields of ≥10 mg/L, following optimization. We also assessed the effects of different detergents on membrane solubilization and AgrC kinase activity, and polyoxyethylene-(23-lauryl-ether (Brij-35 was identified as the most suitable detergent. Furthermore, the secondary structural stability of purified AgrC was analyzed using circular dichroism (CD spectroscopy. This study may serve as a general guide for improving the yields of other membrane protein preparations and selecting the appropriate detergent to stabilize membrane proteins for biophysical and biochemical analyses.

  20. Microspectroscopic analysis of green fluorescent proteins infiltrated into mesoporous silica nanochannels

    NARCIS (Netherlands)

    Ma, Yujie; Rajendran, Prayanka; Blum, Christian; Cesa, Yanina; Gartmann, Nando; Brühwiler, Dominik; Subramaniam, Vinod

    2011-01-01

    The infiltration of enhanced green fluorescent protein (EGFP) into nanochannels of different diameters in mesoporous silica particles was studied in detail by fluorescence microspectroscopy at room temperature. Silica particles from the MCM-41, ASNCs and SBA-15 families possessing nanometer-sized

  1. Optimization of mNeonGreen for Homo sapiens increases its fluorescent intensity in mammalian cells.

    Science.gov (United States)

    Tanida-Miyake, Emiko; Koike, Masato; Uchiyama, Yasuo; Tanida, Isei

    2018-01-01

    Green fluorescent protein (GFP) is tremendously useful for investigating many cellular and intracellular events. The monomeric GFP mNeonGreen is about 3- to 5-times brighter than GFP and monomeric enhanced GFP and shows high photostability. The maturation half-time of mNeonGreen is about 3-fold faster than that of monomeric enhanced GFP. However, the cDNA sequence encoding mNeonGreen contains some codons that are rarely used in Homo sapiens. For better expression of mNeonGreen in human cells, we synthesized a human-optimized cDNA encoding mNeonGreen and generated an expression plasmid for humanized mNeonGreen under the control of the cytomegalovirus promoter. The resultant plasmid was introduced into HEK293 cells. The fluorescent intensity of humanized mNeonGreen was about 1.4-fold higher than that of the original mNeonGreen. The humanized mNeonGreen with a mitochondria-targeting signal showed mitochondrial distribution of mNeonGreen. We further generated an expression vector of humanized mNeonGreen with 3xFLAG tags at its carboxyl terminus as these tags are useful for immunological analyses. The 3xFLAG-tagged mNeonGreen was recognized well with an anti-FLAG-M2 antibody. These plasmids for the expression of humanized mNeonGreen and mNeonGreen-3xFLAG are useful tools for biological studies in mammalian cells using mNeonGreen.

  2. Green Fluorescence of Cytaeis Hydroids Living in Association with Nassarius Gastropods in the Red Sea

    KAUST Repository

    Prudkovsky, Andrey A.; Ivanenko, Viatcheslav N.; Nikitin, Mikhail A.; Lukyanov, Konstantin A.; Belousova, Anna; Reimer, James D.; Berumen, Michael L.

    2016-01-01

    Green Fluorescent Proteins (GFPs) have been reported from a wide diversity of medusae, but only a few observations of green fluorescence have been reported for hydroid colonies. In this study, we report on fluorescence displayed by hydroid polyps of the genus Cytaeis Eschscholtz, 1829 (Hydrozoa: Anthoathecata: Filifera) found at night time in the southern Red Sea (Saudi Arabia) living on shells of the gastropod Nassarius margaritifer (Dunker, 1847) (Neogastropoda: Buccinoidea: Nassariidae). We examined the fluorescence of these polyps and compare with previously reported data. Intensive green fluorescence with a spectral peak at 518 nm was detected in the hypostome of the Cytaeis polyps, unlike in previous reports that reported fluorescence either in the basal parts of polyps or in other locations on hydroid colonies. These results suggest that fluorescence may be widespread not only in medusae, but also in polyps, and also suggests that the patterns of fluorescence localization can vary in closely related species. The fluorescence of polyps may be potentially useful for field identification of cryptic species and study of geographical distributions of such hydroids and their hosts.

  3. Green Fluorescence of Cytaeis Hydroids Living in Association with Nassarius Gastropods in the Red Sea

    KAUST Repository

    Prudkovsky, Andrey A.

    2016-02-03

    Green Fluorescent Proteins (GFPs) have been reported from a wide diversity of medusae, but only a few observations of green fluorescence have been reported for hydroid colonies. In this study, we report on fluorescence displayed by hydroid polyps of the genus Cytaeis Eschscholtz, 1829 (Hydrozoa: Anthoathecata: Filifera) found at night time in the southern Red Sea (Saudi Arabia) living on shells of the gastropod Nassarius margaritifer (Dunker, 1847) (Neogastropoda: Buccinoidea: Nassariidae). We examined the fluorescence of these polyps and compare with previously reported data. Intensive green fluorescence with a spectral peak at 518 nm was detected in the hypostome of the Cytaeis polyps, unlike in previous reports that reported fluorescence either in the basal parts of polyps or in other locations on hydroid colonies. These results suggest that fluorescence may be widespread not only in medusae, but also in polyps, and also suggests that the patterns of fluorescence localization can vary in closely related species. The fluorescence of polyps may be potentially useful for field identification of cryptic species and study of geographical distributions of such hydroids and their hosts.

  4. Transformation of Sclerotinia Sclerotiorum with the Green Fluorescent Protein Gene and Fluorescence of Hyphae in Four Inoculated Hosts

    Science.gov (United States)

    Sclerotinia sclerotiorum is an important pathogen of a wide variety of crops. To obtain a genetic marker to observe and study the interaction of the pathogen with its hosts, isolates ND30 and ND21 were transformed using pCT74 and gGFP constructs both containing genes for the green fluorescent protei...

  5. A study of the interaction between malachite green and lysozyme by steady-state fluorescence.

    Science.gov (United States)

    Ding, Fei; Liu, Wei; Liu, Feng; Li, Zhi-Yuan; Sun, Ying

    2009-09-01

    The interaction of a N-methylated diaminotriphenylmethane dye, malachite green, with lysozyme was investigated by fluorescence spectroscopic techniques under physiological conditions. The binding parameters have been evaluated by fluorescence quenching methods. The results revealed that malachite green caused the fluorescence quenching of lysozyme through a static quenching procedure. The thermodynamic parameters like DeltaH and DeltaS were calculated to be -15.33 kJ mol(-1) and 19.47 J mol(-1) K(-1) according to van't Hoff equation, respectively, which proves main interaction between malachite green and lysozyme is hydrophobic forces and hydrogen bond contact. The distance r between donor (lysozyme) and acceptor (malachite green) was obtained to be 3.82 nm according to Frster's theory. The results of synchronous fluorescence, UV/vis and three-dimensional fluorescence spectra showed that binding of malachite green with lysozyme can induce conformational changes in lysozyme. In addition, the effects of common ions on the constants of lysozyme-malachite green complex were also discussed.

  6. ERG protein expression over time

    DEFF Research Database (Denmark)

    Berg, Kasper Drimer; Brasso, Klaus; Thomsen, Frederik Birkebæk

    2015-01-01

    AIMS: We evaluated the consistency in ERG protein expression from diagnostic specimens through rebiopsies to radical prostatectomies in patients with clinically localised prostate cancer to investigate the validity of ERG status in biopsies. METHODS: ERG expression was assessed by immunohistochem......AIMS: We evaluated the consistency in ERG protein expression from diagnostic specimens through rebiopsies to radical prostatectomies in patients with clinically localised prostate cancer to investigate the validity of ERG status in biopsies. METHODS: ERG expression was assessed...

  7. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria

    DEFF Research Database (Denmark)

    Andersen, Jens Bo; Sternberg, Claus; Poulsen, Lars K.

    1998-01-01

    Use of the green fluorescent protein (Gfp) from the jellyfish Aequorea victoria ia is a powerful method for nondestructive in situ monitoring, since expression of green fluorescence does not require any substrate addition. To expand the use of Gfp as a reporter protein, new variants have been...... constructed by the addition of short peptide sequences to the C-terminal end of intact Gfp. This rendered the Gfp susceptible to the action of indigenous housekeeping proteases, resulting in protein variants with half-lives ranging from 40 min to a few hours when synthesized in Escherichia coli...

  8. Ratiometric Matryoshka biosensors from a nested cassette of green- and orange-emitting fluorescent proteins.

    Science.gov (United States)

    Ast, Cindy; Foret, Jessica; Oltrogge, Luke M; De Michele, Roberto; Kleist, Thomas J; Ho, Cheng-Hsun; Frommer, Wolf B

    2017-09-05

    Sensitivity, dynamic and detection range as well as exclusion of expression and instrumental artifacts are critical for the quantitation of data obtained with fluorescent protein (FP)-based biosensors in vivo. Current biosensors designs are, in general, unable to simultaneously meet all these criteria. Here, we describe a generalizable platform to create dual-FP biosensors with large dynamic ranges by employing a single FP-cassette, named GO-(Green-Orange) Matryoshka. The cassette nests a stable reference FP (large Stokes shift LSSmOrange) within a reporter FP (circularly permuted green FP). GO- Matryoshka yields green and orange fluorescence upon blue excitation. As proof of concept, we converted existing, single-emission biosensors into a series of ratiometric calcium sensors (MatryoshCaMP6s) and ammonium transport activity sensors (AmTryoshka1;3). We additionally identified the internal acid-base equilibrium as a key determinant of the GCaMP dynamic range. Matryoshka technology promises flexibility in the design of a wide spectrum of ratiometric biosensors and expanded in vivo applications.Single fluorescent protein biosensors are susceptible to expression and instrumental artifacts. Here Ast et al. describe a dual fluorescent protein design whereby a reference fluorescent protein is nested within a reporter fluorescent protein to control for such artifacts while preserving sensitivity and dynamic range.

  9. Photoacoustic tomography of human hepatic malignancies using intraoperative indocyanine green fluorescence imaging.

    Science.gov (United States)

    Miyata, Akinori; Ishizawa, Takeaki; Kamiya, Mako; Shimizu, Atsushi; Kaneko, Junichi; Ijichi, Hideaki; Shibahara, Junji; Fukayama, Masashi; Midorikawa, Yutaka; Urano, Yasuteru; Kokudo, Norihiro

    2014-01-01

    Recently, fluorescence imaging following the preoperative intravenous injection of indocyanine green has been used in clinical settings to identify hepatic malignancies during surgery. The aim of this study was to evaluate the ability of photoacoustic tomography using indocyanine green as a contrast agent to produce representative fluorescence images of hepatic tumors by visualizing the spatial distribution of indocyanine green on ultrasonographic images. Indocyanine green (0.5 mg/kg, intravenous) was preoperatively administered to 9 patients undergoing hepatectomy. Intraoperatively, photoacoustic tomography was performed on the surface of the resected hepatic specimens (n = 10) under excitation with an 800 nm pulse laser. In 4 hepatocellular carcinoma nodules, photoacoustic imaging identified indocyanine green accumulation in the cancerous tissue. In contrast, in one hepatocellular carcinoma nodule and five adenocarcinoma foci (one intrahepatic cholangiocarcinoma and 4 colorectal liver metastases), photoacoustic imaging delineated indocyanine green accumulation not in the cancerous tissue but rather in the peri-cancerous hepatic parenchyma. Although photoacoustic tomography enabled to visualize spatial distribution of ICG on ultrasonographic images, which was consistent with fluorescence images on cut surfaces of the resected specimens, photoacoustic signals of ICG-containing tissues decreased approximately by 40% even at 4 mm depth from liver surfaces. Photoacoustic tomography using indocyanine green also failed to identify any hepatocellular carcinoma nodules from the body surface of model mice with non-alcoholic steatohepatitis. In conclusion, photoacoustic tomography has a potential to enhance cancer detectability and differential diagnosis by ultrasonographic examinations and intraoperative fluorescence imaging through visualization of stasis of bile-excreting imaging agents in and/or around hepatic tumors. However, further technical advances are needed

  10. Photoacoustic tomography of human hepatic malignancies using intraoperative indocyanine green fluorescence imaging.

    Directory of Open Access Journals (Sweden)

    Akinori Miyata

    Full Text Available Recently, fluorescence imaging following the preoperative intravenous injection of indocyanine green has been used in clinical settings to identify hepatic malignancies during surgery. The aim of this study was to evaluate the ability of photoacoustic tomography using indocyanine green as a contrast agent to produce representative fluorescence images of hepatic tumors by visualizing the spatial distribution of indocyanine green on ultrasonographic images. Indocyanine green (0.5 mg/kg, intravenous was preoperatively administered to 9 patients undergoing hepatectomy. Intraoperatively, photoacoustic tomography was performed on the surface of the resected hepatic specimens (n = 10 under excitation with an 800 nm pulse laser. In 4 hepatocellular carcinoma nodules, photoacoustic imaging identified indocyanine green accumulation in the cancerous tissue. In contrast, in one hepatocellular carcinoma nodule and five adenocarcinoma foci (one intrahepatic cholangiocarcinoma and 4 colorectal liver metastases, photoacoustic imaging delineated indocyanine green accumulation not in the cancerous tissue but rather in the peri-cancerous hepatic parenchyma. Although photoacoustic tomography enabled to visualize spatial distribution of ICG on ultrasonographic images, which was consistent with fluorescence images on cut surfaces of the resected specimens, photoacoustic signals of ICG-containing tissues decreased approximately by 40% even at 4 mm depth from liver surfaces. Photoacoustic tomography using indocyanine green also failed to identify any hepatocellular carcinoma nodules from the body surface of model mice with non-alcoholic steatohepatitis. In conclusion, photoacoustic tomography has a potential to enhance cancer detectability and differential diagnosis by ultrasonographic examinations and intraoperative fluorescence imaging through visualization of stasis of bile-excreting imaging agents in and/or around hepatic tumors. However, further technical

  11. Chromophore-protein coupling beyond nonpolarizable models: understanding absorption in green fluorescent protein

    NARCIS (Netherlands)

    Daday, C.; Curutchet, C.; Sinicropi, A.; Mennucci, B.; Filippi, Claudia

    2015-01-01

    The nature of the coupling of the photoexcited chromophore with the environment in a prototypical system like green fluorescent protein (GFP) is to date not understood, and its description still defies state-of-the-art multiscale approaches. To identify which theoretical framework of the

  12. Absorption tuning of the green fluorescent protein chromophore: synthesis and studies of model compounds

    DEFF Research Database (Denmark)

    Brøndsted Nielsen, Mogens; Andersen, Lars Henrik; Rinza, Tomás Rocha

    2011-01-01

    The green fluorescent protein (GFP) chromophore is a heterocyclic compound containing a p-hydroxybenzylidine attached to an imidazol-5(4H)-one ring. This review covers the synthesis of a variety of model systems for elucidating the intrinsic optical properties of the chromophore in the gas phase ...

  13. Ultrafast excited and ground-state dynamics of the green fluorescent protein chromophore in solution

    NARCIS (Netherlands)

    Vengris, M.; van Stokkum, I.H.M.; He, X.; Bell, A.F.; Tonge, P.J.; van Grondelle, R.; Larsen, D.S.

    2004-01-01

    Ultrafast dispersed pump-dump-probe spectroscopy was applied to HBDI (4′-hydroxybenzylidene-2,3-dimethylimidazolinone), a model green fluorescent protein (GFP) chromophore in solution with different protonation states. The measured three-dimensional data was analyzed using a global analysis method

  14. A Practical Teaching Course in Directed Protein Evolution Using the Green Fluorescent Protein as a Model

    Science.gov (United States)

    Ruller, Roberto; Silva-Rocha, Rafael; Silva, Artur; Schneider, Maria Paula Cruz; Ward, Richard John

    2011-01-01

    Protein engineering is a powerful tool, which correlates protein structure with specific functions, both in applied biotechnology and in basic research. Here, we present a practical teaching course for engineering the green fluorescent protein (GFP) from "Aequorea victoria" by a random mutagenesis strategy using error-prone polymerase…

  15. Effect of pH on the Heat-Induced Denaturation and Renaturation of Green Fluorescent Protein: A Laboratory Experiment

    Science.gov (United States)

    Flores, Rosa V.; Sola, Hilda M.; Torres, Juan C.; Torres, Rafael E.; Guzman, Ernick E.

    2013-01-01

    A fluorescence spectroscopy experiment is described where students integrated biochemistry and instrumental analysis, while characterizing the green fluorescent protein excitation and emission spectra in terms of its phenolic and phenolate chromophores. Students studied the combined effect of pH and temperature on the protein's fluorescence,…

  16. Labeling RNAs in Live Cells Using Malachite Green Aptamer Scaffolds as Fluorescent Probes.

    Science.gov (United States)

    Yerramilli, V Siddartha; Kim, Kyung Hyuk

    2018-03-16

    RNAs mediate many different processes that are central to cellular function. The ability to quantify or image RNAs in live cells is very useful in elucidating such functions of RNA. RNA aptamer-fluorogen systems have been increasingly used in labeling RNAs in live cells. Here, we use the malachite green aptamer (MGA), an RNA aptamer that can specifically bind to malachite green (MG) dye and induces it to emit far-red fluorescence signals. Previous studies on MGA showed a potential for the use of MGA for genetically tagging other RNA molecules in live cells. However, these studies also exhibited low fluorescence signals and high background noise. Here we constructed and tested RNA scaffolds containing multiple tandem repeats of MGA as a strategy to increase the brightness of the MGA aptamer-fluorogen system as well as to make the system fluoresce when tagging various RNA molecules, in live cells. We demonstrate that our MGA scaffolds can induce fluorescence signals by up to ∼20-fold compared to the basal level as a genetic tag for other RNA molecules. We also show that our scaffolds function reliably as genetically encoded fluorescent tags for mRNAs of fluorescent proteins and other RNA aptamers.

  17. Protein Expression Analyses at the Single Cell Level

    Directory of Open Access Journals (Sweden)

    Masae Ohno

    2014-09-01

    Full Text Available The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level.

  18. The retardation by gamma irradiation of greening in potatoes exposed to fluorescent lighting

    International Nuclear Information System (INIS)

    Beyers, M.

    1981-01-01

    The optimum gamma irradiation treatments for the inhibition of greening of unwashed Up-to-Date potatoes exposed to continuous fluorescent lighting were 0,15 and 0,20 kGy. The 0,15 and 0,20 kGy treated potatoes took 8,7 and 10,3 d longer respectively than the controls for 50% of the potatoes to turn green. The results were verified by chlorophyll determinations. The solanine content of the γ-irradiated potatoes did not differ significantly from that of the controls during the period of exposure. Gamma irradiated tubers which were removed from continuous fluorescent lighting after 7 d to 'household' conditions of daylight and fluorescent light alternated with darkness maintained the quality of day 7 for at least another 16 d. Factors such as washing, packaging, display temperature, post-irradiation pre-illumination storage and cultivar differences did not detract from the effectiveness of γ-irradiation in retarding the greening of potatoes. A comparison of γ-irradiation with dipping inedible oil showed the latter treatment to be more effective than irradiation in inhibiting greening but the treatment caused serious rotting. No difference in the taste or colour of irradiated and nonirradiated potatoes cooked in various ways could be detected [af

  19. Near-infrared-fluorescence imaging of lymph nodes by using liposomally formulated indocyanine green derivatives.

    Science.gov (United States)

    Toyota, Taro; Fujito, Hiromichi; Suganami, Akiko; Ouchi, Tomoki; Ooishi, Aki; Aoki, Akira; Onoue, Kazutaka; Muraki, Yutaka; Madono, Tomoyuki; Fujinami, Masanori; Tamura, Yutaka; Hayashi, Hideki

    2014-01-15

    Liposomally formulated indocyanine green (LP-ICG) has drawn much attention as a highly sensitive near-infrared (NIR)-fluorescence probe for tumors or lymph nodes in vivo. We synthesized ICG derivatives tagged with alkyl chains (ICG-Cn), and we examined NIR-fluorescence imaging for lymph nodes in the lower extremities of mice by using liposomally formulated ICG-Cn (LP-ICG-Cn) as well as conventional liposomally formulated ICG (LP-ICG) and ICG. Analysis with a noninvasive preclinical NIR-fluorescence imaging system revealed that LP-ICG-Cn accumulates in only the popliteal lymph node 1h after injection into the footpad, whereas LP-ICG and ICG accumulate in the popliteal lymph node and other organs like the liver. This result indicates that LP-ICG-Cn is a useful NIR-fluorescence probe for noninvasive in vivo bioimaging, especially for the sentinel lymph node. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Application of indocyanine green-fluorescence imaging to full-thickness cholecystectomy.

    Science.gov (United States)

    Morita, Kiyomi; Ishizawa, Takeaki; Tani, Keigo; Harada, Nobuhiro; Shimizu, Atsushi; Yamamoto, Satoshi; Takemura, Nobuyuki; Kaneko, Junichi; Aoki, Taku; Sakamoto, Yoshihiro; Sugawara, Yasuhiko; Hasegawa, Kiyoshi; Kokudo, Norihiro

    2014-05-01

    Fluorescence imaging using indocyanine green (ICG) has recently been applied to laparoscopic surgery to identify cancerous tissues, lymph nodes, and vascular anatomy. Here we report the application of ICG-fluorescence imaging to visualize the boundary between the liver and subserosal tissues of the gallbladder during laparoscopic full-thickness cholecystectomy. A patient with a potentially malignant gallbladder lesion was administered 2.5-mg intravenous ICG just before laparoscopic full-thickness cholecystectomy. Intraoperative fluorescence imaging enabled the real-time delineation of both extrahepatic bile duct anatomy and hepatic parenchyma throughout the procedure, which resulted in complete removal of subserosal tissues between liver and gallbladder. Safe and feasible ICG-fluorescence imaging can be widely applied to laparoscopic hepatobiliary surgery by utilizing a biliary excretion property of ICG. © 2014 Japan Society for Endoscopic Surgery, Asia Endosurgery Task Force and Wiley Publishing Asia Pty Ltd.

  1. Essential alterations of heparan sulfate during the differentiation of embryonic stem cells to Sox1-enhanced green fluorescent protein-expressing neural progenitor cells.

    NARCIS (Netherlands)

    Johnson, C.E.; Crawford, B.E.; Stavridis, M.; Dam, G.B. ten; Wat, A.L.; Rushton, G.; Ward, C.M.; Wilson, V.; Kuppevelt, A.H.M.S.M. van; Esko, J.D.; Smith, A.; Gallagher, J.T.; Merry, C.L.

    2007-01-01

    Embryonic stem (ES) cells can be cultured in conditions that either maintain pluripotency or allow differentiation to the three embryonic germ layers. Heparan sulfate (HS), a highly polymorphic glycosaminoglycan, is a critical cell surface coreceptor in embryogenesis, and in this paper we describe

  2. Quantification of two forms of green sulfur bacteria in their natural habitat using bacteriochlorophyll fluorescence spectra

    Science.gov (United States)

    Kharcheva, Anastasia V.; Zhiltsova, Anna A.; Lunina, Olga N.; Savvichev, Alexander S.; Patsaeva, Svetlana V.

    2016-04-01

    Detection of phototropic organisms in their natural habitat using optical instruments operating under water is urgently needed for many tasks of ecological monitoring. While fluorescence methods are widely applied nowadays to detect and characterize phytoplankton communities, the techniques for detection and recognition of anoxygenic phototrophs are considered challenging. Differentiation of the forms of anoxygenic green sulfur bacteria in natural water using spectral techniques remains problematic. Green sulfur bacteria could be found in two forms, green-colored (containing BChl d in pigment compound) and brown-colored (containing BChl e), have the special ecological niche in such reservoirs. Separate determination of these microorganisms by spectral methods is complicated because of similarity of spectral characteristics of their pigments. We describe the novel technique of quantification of two forms of green sulfur bacteria directly in water using bacteriochlorophyll fluorescence without pigment extraction. This technique is noninvasive and could be applied in remote mode in the water bodies with restricted water circulation to determine simultaneously concentrations of two forms of green sulfur bacteria in their natural habitat.

  3. A green fluorescent protein with photoswitchable emission from the deep sea.

    Directory of Open Access Journals (Sweden)

    Alexander Vogt

    Full Text Available A colorful variety of fluorescent proteins (FPs from marine invertebrates are utilized as genetically encoded markers for live cell imaging. The increased demand for advanced imaging techniques drives a continuous search for FPs with new and improved properties. Many useful FPs have been isolated from species adapted to sun-flooded habitats such as tropical coral reefs. It has yet remained unknown if species expressing green fluorescent protein (GFP-like proteins also exist in the darkness of the deep sea. Using a submarine-based and -operated fluorescence detection system in the Gulf of Mexico, we discovered ceriantharians emitting bright green fluorescence in depths between 500 and 600 m and identified a GFP, named cerFP505, with bright fluorescence emission peaking at 505 nm. Spectroscopic studies showed that approximately 15% of the protein bulk feature reversible ON/OFF photoswitching that can be induced by alternating irradiation with blue und near-UV light. Despite being derived from an animal adapted to essentially complete darkness and low temperatures, cerFP505 maturation in living mammalian cells at 37 degrees C, its brightness and photostability are comparable to those of EGFP and cmFP512 from shallow water species. Therefore, our findings disclose the deep sea as a potential source of GFP-like molecular marker proteins.

  4. Fish with red fluorescent eyes forage more efficiently under dim, blue-green light conditions.

    Science.gov (United States)

    Harant, Ulrike Katharina; Michiels, Nicolaas Karel

    2017-04-20

    Natural red fluorescence is particularly conspicuous in the eyes of some small, benthic, predatory fishes. Fluorescence also increases in relative efficiency with increasing depth, which has generated speculation about its possible function as a "light organ" to detect cryptic organisms under bluish light. Here we investigate whether foraging success is improved under ambient conditions that make red fluorescence stand out more, using the triplefin Tripterygion delaisi as a model system. We repeatedly presented 10 copepods to individual fish (n = 40) kept under a narrow blue-green spectrum and compared their performance with that under a broad spectrum with the same overall brightness. The experiment was repeated for two levels of brightness, a shaded one representing 0.4% of the light present at the surface and a heavily shaded one with about 0.01% of the surface brightness. Fish were 7% more successful at catching copepods under the narrow, fluorescence-friendly spectrum than under the broad spectrum. However, this effect was significant under the heavily shaded light treatment only. This outcome corroborates previous predictions that fluorescence may be an adaptation to blue-green, heavily shaded environments, which coincides with the opportunistic biology of this species that lives in the transition zone between exposed and heavily shaded microhabitats.

  5. Visualization of subcapsular hepatic malignancy by indocyanine-green fluorescence imaging during laparoscopic hepatectomy.

    Science.gov (United States)

    Kudo, Hiroki; Ishizawa, Takeaki; Tani, Keigo; Harada, Nobuhiro; Ichida, Akihiko; Shimizu, Atsushi; Kaneko, Junichi; Aoki, Taku; Sakamoto, Yoshihiro; Sugawara, Yasuhiko; Hasegawa, Kiyoshi; Kokudo, Norihiro

    2014-08-01

    Although laparoscopic hepatectomy has increasingly been used to treat cancers in the liver, the accuracy of intraoperative diagnosis may be inferior to that of open surgery because the ability to visualize and palpate the liver surface during laparoscopy is relatively limited. Fluorescence imaging has the potential to provide a simple compensatory diagnostic tool for identification of cancers in the liver during laparoscopic hepatectomy. In 17 patients who were to undergo laparoscopic hepatectomy, 0.5 mg/kg body weight of indocyanine green (ICG) was administered intravenously within the 2 weeks prior to surgery. Intraoperatively, a laparoscopic fluorescence imaging system obtained fluorescence images of its surfaces during mobilization of the liver. In all, 16 hepatocellular carcinomas (HCCs) and 16 liver metastases (LMs) were resected. Of these, laparoscopic ICG fluorescence imaging identified 12 HCCs (75%) and 11 LMs (69%) on the liver surfaces distributed over Couinaud's segments 1-8, including the 17 tumors that had not been identified by visual inspections of normal color images. The 23 tumors that were identified by fluorescence imaging were located closer to the liver surfaces than another nine tumors that were not identified by fluorescence imaging (median [range] depth 1 [0-5] vs. 11 [8-30] mm; p fluorescence imaging enables real-time identification of subcapsular liver cancers, thus facilitating estimation of the required extent of hepatic mobilization and determination of the location of an appropriate hepatic transection line.

  6. Application of green fluorescent protein for monitoring phenol-degrading strains

    Directory of Open Access Journals (Sweden)

    Ana Milena Valderrama F.

    2001-07-01

    Full Text Available Several methods have been developed for detecting microorganisms in environmental samples. Some systems for incorporating reporter genes, such as lux or the green fluorescent protein (GFP gene, have been developed recently This study describes gfp gene marking of a phenol degrading strain, its evaluation and monitoring in a bioreactor containing refinery sour water. Tagged strains were obtained having the same physiological and metabolic characteristics as the parent strain. Fluorescent expression was kept stable with no selection for more than 50 consecutive generations and tagged strains were recovered from the bioreactor after forty-five days of phenol-degradation treatment.

  7. A green method for the preparation of fluorescent hybrid structures of gold and corrole

    Energy Technology Data Exchange (ETDEWEB)

    Pereira, Ângela S., E-mail: aspereira@ua.pt; Barata, Joana F. B. [University of Aveiro, CICECO – Chemistry Department, Aveiro Institute of Materials (Portugal); Vaz Serra, Vanda I. R. C. [University of Aveiro, QOPNA Chemistry Department (Portugal); Pereira, Sérgio; Trindade, Tito [University of Aveiro, CICECO – Chemistry Department, Aveiro Institute of Materials (Portugal)

    2015-10-15

    Gold/soap nanostructures were prepared by a green methodology using saponified household sunflower oil, as reducing and organic dispersing agent of auric acid. The incorporation of hydrophobic molecules on the novel water-soluble gold nanoparticles was followed by fluorescence lifetime imaging microscopy, using as model hydrophobic compound 5,10,15-tris-(pentafluorophenyl)corrolatogallium(III)(pyridine) (GaPFC), a highly fluorescent corrole. The results showed the hydrophobic GaPFC located between the organic bilayer surrounding several Au nanoparticles, which in turn were coated with fatty acids salts anchored by the double bond at the gold’s surface.

  8. Photoabsorption of green and red fluorescent protein chromophore anions in vacuo.

    Science.gov (United States)

    Wan, Songbo; Liu, Shasha; Zhao, Guangjiu; Chen, Maodu; Han, Keli; Sun, Mengtao

    2007-09-01

    Photoabsorption properties of green and red fluorescent protein chromophore anions in vacuo were investigated theoretically, based on the experimental results in gas phase [Phys. Rev. Lett. 2001, 87, 228102; Phys. Rev. Lett. 2003, 90, 118103]. Their calculated transition energies in absorption with TD-DFT and ZINDO methods are directly compared to the experimental reports in gas phase, and the calculations with ZINDO method can correctly reproduce the absorption spectra. The orientation and strength of their transition dipole moments were revealed with transition density. We also showed the orientation and result of their intramolecular charge transfer with transition difference density. The calculated results show that with the increase of the extended conjugated system, the orientation of transition dipole moments and the orientation of charge transfer can be reversed. They are the linear responds with the external electric fields. These theoretical results reveal the insight understanding of the photoinduced dynamics of green and red fluorescent protein chromophore anions and cations in vacuo.

  9. Use of green fluorescent protein to monitor Lactobacillus plantarum in the gastrointestinal tract of goats.

    Science.gov (United States)

    Han, Xufeng; Wang, Lei; Li, Wei; Li, Bibo; Yang, Yuxin; Yan, Hailong; Qu, Lei; Chen, Yulin

    2015-01-01

    The experiment aimed to specifically monitor the passage of lactobacilli in vivo after oral administration. The green fluorescent protein (GFP) gene was cloned downstream from the constitutive p32 promoter from L. lactis subsp. cremoris Wg2. The recombinant expression vector, pLEM415-gfp-p32, was electroporated into Lactobacillus plantarum (L. plantarum) isolated from goat. Green fluorescent protein (GFP) was successfully expressed in L. plantarum. After 2 h post-administration, transformed Lactobacillus could be detectable in all luminal contents. In the rumen, bacteria concentration initially decreased, reached the minimum at 42 h post-oral administration and then increased. However, this concentration decreased constantly in the duodenum. This result indicated that L. plantarum could colonize in the rumen but not in the duodenum.

  10. Hyperspectral fluorescence imaging coupled with multivariate image analysis techniques for contaminant screening of leafy greens

    Science.gov (United States)

    Everard, Colm D.; Kim, Moon S.; Lee, Hoyoung

    2014-05-01

    The production of contaminant free fresh fruit and vegetables is needed to reduce foodborne illnesses and related costs. Leafy greens grown in the field can be susceptible to fecal matter contamination from uncontrolled livestock and wild animals entering the field. Pathogenic bacteria can be transferred via fecal matter and several outbreaks of E.coli O157:H7 have been associated with the consumption of leafy greens. This study examines the use of hyperspectral fluorescence imaging coupled with multivariate image analysis to detect fecal contamination on Spinach leaves (Spinacia oleracea). Hyperspectral fluorescence images from 464 to 800 nm were captured; ultraviolet excitation was supplied by two LED-based line light sources at 370 nm. Key wavelengths and algorithms useful for a contaminant screening optical imaging device were identified and developed, respectively. A non-invasive screening device has the potential to reduce the harmful consequences of foodborne illnesses.

  11. Early history, discovery, and expression of Aequorea green fluorescent protein, with a note on an unfinished experiment.

    Science.gov (United States)

    Tsuji, Frederick I

    2010-08-01

    The bioluminescent hydromedusan jellyfish, Aequorea victoria, emits a greenish light (lambda(max) = 508 nm) when stimulated electrically or mechanically. The light comes from photocytes located along the margin of its umbrella. The greenish light depends on two intracellular proteins working in consort: aequorin (21.4 kDa) and a green fluorescent protein (27 kDa). An excited state green fluorescent protein molecule results, which, on returning to the ground state, emits a greenish light. Similarly, a green light emission may be induced in the green fluorescent protein by exposing it to ultraviolet or blue light. Because the green light can be readily detected under a fluorescence microscope, the green fluorescent protein, tagged to a protein of interest, has been used widely as a marker to locate proteins in cells and to monitoring gene expression. This article reviews the work that took place leading to the discovery, cloning, and expression of the green fluorescent protein, with a note on an unfinished experiment. (c) 2010 Wiley-Liss, Inc.

  12. Gastric Tube Reconstruction with Superdrainage Using Indocyanine Green Fluorescence During Esophagectomy

    OpenAIRE

    KITAGAWA, HIROYUKI; NAMIKAWA, TSUTOMU; IWABU, JUN; HANAZAKI, KAZUHIRO

    2017-01-01

    We report a case of gastric tube reconstruction with superdrainage using indocyanine green fluorescence during esophagectomy for esophageal cancer. A 53-year-old man with a history of early esophageal cancer treated with endoscopic mucosal dissection experienced esophageal cancer recurrence. There was no evidence of lymph node involvement or distant metastasis on computed tomography; therefore, we performed thoracoscopic esophagectomy. After thoracoscopic esophagectomy, we created a gastric t...

  13. Construction of green fluorescent protein-tagged recombinant iridovirus to assess viral replication.

    Science.gov (United States)

    Huang, Youhua; Huang, Xiaohong; Cai, Jia; Ye, Fuzhou; Guan, Liya; Liu, Hong; Qin, Qiwei

    2011-09-01

    Green fluorescent protein-tagged recombinant virus has been successfully applied to observing the infective dynamics and evaluating viral replication. Here, we identified soft-shelled turtle iridovirus (STIV) ORF55 as an envelope protein (VP55), and developed a recombinant STIV expressing an enhanced green fluorescent protein (EGFP) fused to VP55 (EGFP-STIV). Recombinant EGFP-STIV shared similar single-step growth curves and ultrastructural morphology with wild type STIV (wt-STIV). The green fluorescence distribution during EGFP-STIV infection was consistent with the intracellular distribution of VP55 which was mostly co-localized with virus assembly sites. Furthermore, EGFP-STIV could be used to evaluate viral replication conveniently under drug treatment, and the result showed that STIV replication was significantly inhibited after the addition of antioxidant pyrrolidine dithiocarbamate (PDTC). Thus, the EGFP-tagged recombinant iridovirus will not only be useful for further investigations on the viral replicative dynamics, but also provide an alternative simple strategy to screen for antiviral substances. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Gastric Tube Reconstruction with Superdrainage Using Indocyanine Green Fluorescence During Esophagectomy.

    Science.gov (United States)

    Kitagawa, Hiroyuki; Namikawa, Tsutomu; Iwabu, Jun; Hanazaki, Kazuhiro

    2017-01-01

    We report a case of gastric tube reconstruction with superdrainage using indocyanine green fluorescence during esophagectomy for esophageal cancer. A 53-year-old man with a history of early esophageal cancer treated with endoscopic mucosal dissection experienced esophageal cancer recurrence. There was no evidence of lymph node involvement or distant metastasis on computed tomography; therefore, we performed thoracoscopic esophagectomy. After thoracoscopic esophagectomy, we created a gastric tube. When pulling up the gastric tube through the post-mediastinum route, a root of the right gastroepiploic vein was injured. We subsequently performed superdrainage to avoid congestion of the gastric tube with omental vein and pre-tracheal vein anastomosis at the neck, and confirmed venous flow using the indocyanine green fluorescence method. No postoperative anastomotic leakage was observed, and the patient was discharged 22 days after surgery. Thus, we recommend the indocyanine green fluorescence method in cases involving superdrainage during esophagectomy. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  15. Green Synthesis of Fluorescent Carbon Dots for Selective Detection of Tartrazine in Food Samples.

    Science.gov (United States)

    Xu, Hua; Yang, Xiupei; Li, Gu; Zhao, Chuan; Liao, Xiangjun

    2015-08-05

    A simple, economical, and green method for the preparation of water-soluble, high-fluorescent carbon quantum dots (C-dots) has been developed via hydrothermal process using aloe as a carbon source. The synthesized C-dots were characterized by atomic force microscope (AFM), transmission electron microscopy (TEM), fluorescence spectrophotometer, UV-vis absorption spectra as well as Fourier transform infrared spectroscopy (FTIR). The results reveal that the as-prepared C-dots were spherical shape with an average diameter of 5 nm and emit bright yellow photoluminescence (PL) with a quantum yield of approximately 10.37%. The surface of the C-dots was rich in hydroxyl groups and presented various merits including high fluorescent quantum yield, excellent photostability, low toxicity and satisfactory solubility. Additionally, we found that one of the widely used synthetic food colorants, tartrazine, could result in a strong fluorescence quenching of the C-dots through a static quenching process. The decrease of fluorescence intensity made it possible to determine tartrazine in the linear range extending from 0.25 to 32.50 μM, This observation was further successfully applied for the determination of tartrazine in food samples collected from local markets, suggesting its great potential toward food routine analysis. Results from our study may shed light on the production of fluorescent and biocompatible nanocarbons due to our simple and environmental benign strategy to synthesize C-dots in which aloe was used as a carbon source.

  16. Lipid nanoparticle vectorization of indocyanine green improves fluorescence imaging for tumor diagnosis and lymph node resection.

    Science.gov (United States)

    Navarro, Fabrice P; Berger, Michel; Guillermet, Stéphanie; Josserand, Véronique; Guyon, Laurent; Neumann, Emmanuelle; Vinet, Françoise; Texier, Isabelle

    2012-10-01

    Fluorescence imaging is opening a new era in image-guided surgery and other medical applications. The only FDA approved contrast agent in the near infrared is IndoCyanine Green (ICG), which despites its low toxicity, displays poor chemical and optical properties for long-term and sensitive imaging applications in human. Lipid nanoparticles are investigated for improving ICG optical properties and in vivo fluorescence imaging sensitivity. 30 nm diameter lipid nanoparticles (LNP) are loaded with ICG. Their characterization and use for tumor and lymph node imaging are described. Nano-formulation benefits dye optical properties (6 times improved brightness) and chemical stability (>6 months at 4 degrees C in aqueous buffer). More importantly, LNP vectorization allows never reported sensitive and prolonged (>1 day) labeling of tumors and lymph nodes. Composed of human-use approved ingredients, this novel ICG nanometric formulation is foreseen to expand rapidly the field of clinical fluorescence imaging applications.

  17. IR-FEL-induced green fluorescence protein (GFP) gene transfer into plant cell

    CERN Document Server

    Awazu, K; Tamiya, E

    2002-01-01

    A Free Electron Laser (FEL) holds potential for various biotechnological applications due to its characteristics such as flexible wavelength tunability, short pulse and high peak power. We could successfully introduce the Green Fluorescent Protein (GFP) gene into tobacco BY2 cells by IR-FEL laser irradiation. The irradiated area of the solution containing BY2 cells and plasmid was about 0.1 mm sup 2. FEL irradiation at a wavelength of 5.75 and 6.1 mu m, targeting absorption by the ester bond of the lipid and the amide I bond of the protein, respectively, was shown to cause the introduction of the fluorescent dye into the cell. On the other hand, transient expression of the GFP fluorescence was only observed after irradiation at 5.75 mu m. The maximum transfer efficiency was about 0.5%.

  18. Recombination-stable multimeric green fluorescent protein for characterization of weak promoter outputs in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Rugbjerg, Peter; Knuf, Christoph; Förster, Jochen

    2015-01-01

    a less leaky Cu2+-inducible promoter based on CUP1. The basal expression level of the new promoter was approx. 61% below the wild-type CUP1 promoter, thus expanding the absolute range of Cu2+-based gene control. The stability of 3vGFP towards direct-repeat recombination was assayed in S. cerevisiae......Green fluorescent proteins (GFPs) are widely used for visualization of proteins to track localization and expression dynamics. However, phenotypically important processes can operate at too low expression levels for routine detection, i.e. be overshadowed by autofluorescence noise. While GFP...... functions well in translational fusions, the use of tandem GFPs to amplify fluorescence signals is currently avoided in Saccharomyces cerevisiae and many other microorganisms due to the risk of loop-out by direct-repeat recombination. We increased GFP fluorescence by translationally fusing three different...

  19. Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein

    DEFF Research Database (Denmark)

    Østergaard, H.; Henriksen, A.; Hansen, Flemming G.

    2001-01-01

    To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease...... in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivoby non- invasive fluorimetric measurements. The 1.5 Angstrom crystal structure of the oxidized protein revealed a disulfide bond- induced distortion of the beta -barrel, as well...... the physiological range for redox-active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway....

  20. Intradermal indocyanine green for in vivo fluorescence laser scanning microscopy of human skin: a pilot study.

    Directory of Open Access Journals (Sweden)

    Constanze Jonak

    Full Text Available BACKGROUND: In clinical diagnostics, as well as in routine dermatology, the increased need for non-invasive diagnosis is currently satisfied by reflectance laser scanning microscopy. However, this technique has some limitations as it relies solely on differences in the reflection properties of epidermal and dermal structures. To date, the superior method of fluorescence laser scanning microscopy is not generally applied in dermatology and predominantly restricted to fluorescein as fluorescent tracer, which has a number of limitations. Therefore, we searched for an alternative fluorophore matching a novel skin imaging device to advance this promising diagnostic approach. METHODOLOGY/PRINCIPAL FINDINGS: Using a Vivascope®-1500 Multilaser microscope, we found that the fluorophore Indocyanine-Green (ICG is well suited as a fluorescent marker for skin imaging in vivo after intradermal injection. ICG is one of few fluorescent dyes approved for use in humans. Its fluorescence properties are compatible with the application of a near-infrared laser, which penetrates deeper into the tissue than the standard 488 nm laser for fluorescein. ICG-fluorescence turned out to be much more stable than fluorescein in vivo, persisting for more than 48 hours without significant photobleaching whereas fluorescein fades within 2 hours. The well-defined intercellular staining pattern of ICG allows automated cell-recognition algorithms, which we accomplished with the free software CellProfiler, providing the possibility of quantitative high-content imaging. Furthermore, we demonstrate the superiority of ICG-based fluorescence microscopy for selected skin pathologies, including dermal nevi, irritant contact dermatitis and necrotic skin. CONCLUSIONS/SIGNIFICANCE: Our results introduce a novel in vivo skin imaging technique using ICG, which delivers a stable intercellular fluorescence signal ideal for morphological assessment down to sub-cellular detail. The application of

  1. Crystallization and preliminary X-ray analysis of a monomeric mutant of Azami-Green (mAG), an Aequorea victoria green fluorescent protein-like green-emitting fluorescent protein from the stony coral Galaxea fascicularis

    International Nuclear Information System (INIS)

    Ebisawa, Tatsuki; Yamamura, Akihiro; Kameda, Yasuhiro; Hayakawa, Kou; Nagata, Koji; Tanokura, Masaru

    2009-01-01

    A monomeric mutant of Azami-Green from G. fascicularis was expressed, purified and crystallized using the sitting-drop vapour-diffusion method. The crystal belonged to space group P1 and diffracted X-rays to 2.20 Å resolution. Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first monomeric green-emitting fluorescent protein that is not a derivative of Aequorea victoria green fluorescent protein (avGFP). mAG and avGFP are 27% identical in amino-acid sequence. Diffraction-quality crystals of recombinant mAG were obtained by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The mAG crystal diffracted X-rays to 2.20 Å resolution on beamline AR-NW12A at the Photon Factory (Tsukuba, Japan). The crystal belonged to space group P1, with unit-cell parameters a = 41.78, b = 51.72, c = 52.89 Å, α = 90.96, β = 103.41, γ = 101.79°. The Matthews coefficient (V M = 2.10 Å 3 Da −1 ) indicated that the crystal contained two mAG molecules per asymmetric unit

  2. Development of a green fluorescent protein metastatic-cancer chick-embryo drug-screen model.

    Science.gov (United States)

    Bobek, Vladimir; Plachy, Jiri; Pinterova, Daniela; Kolostova, Katarina; Boubelik, Michael; Jiang, Ping; Yang, Meng; Hoffman, Robert M

    2004-01-01

    The chick-embryo model has been an important tool to study tumor growth, metastasis, and angiogenesis. However, an imageable model with a genetic fluorescent tag in the growing and spreading cancer cells that is stable over time has not been developed. We report here the development of such an imageable fluorescent chick-embryo metastatic cancer model with the use of green fluorescent protein (GFP). Lewis lung carcinoma cells, stably expressing GFP, were injected on the 12th day of incubation in the chick embryo. GFP-Lewis lung carcinoma metastases were visualized by fluorescence, after seven days additional incubation, in the brain, heart, and sternum of the developing chick embryo, with the most frequent site being the brain. The combination of streptokinase and gemcitabine was evaluated in this GFP metastatic model. Twelve-day-old chick embryos were injected intravenously with GFP-Lewis lung cancer cells, along with these two agents either alone or in combination. The streptokinase-gemcitabine combination inhibited metastases at all sites. The effective dose of gemcitabine was found to be 10 mg/kg and streptokinase 2000 IU per embryo. The data in this report suggest that this new stably fluorescent imageable metastatic-cancer chick-embryo model will enable rapid screening of new antimetastatic agents.

  3. Intraoperative Detection of Superficial Liver Tumors by Fluorescence Imaging Using Indocyanine Green and 5-aminolevulinic Acid.

    Science.gov (United States)

    Kaibori, Masaki; Matsui, Kosuke; Ishizaki, Morihiko; Iida, Hiroya; Okumura, Tadayoshi; Sakaguchi, Tatsuma; Inoue, Kentaro; Ikeura, Tsukasa; Asano, Hiroaki; Kon, Masanori

    2016-04-01

    Indocyanine green (ICG) and the porphyrin precursor 5-aminolevulinic acid (5-ALA) have been approved as fluorescence imaging agents in the clinical setting. This study evaluated the usefulness of fluorescence imaging with both ICG and 5-ALA for intraoperative identification of latent small liver tumors. There were 48 patients who had main tumors within 5 mm of the liver surface. 5-ALA hydrochloride was orally administered to patients 3 h before surgery. ICG had been intravenously injected within 14 days prior to surgery. Intraoperatively, after visual inspection, manual palpation and ultrasonography fluorescence images of the liver surface were obtained with ICG and 5-ALA prior to resection. With ICG, the sensitivity, specificity and accuracy for detecting the preoperatively identified main tumors were 96%, 50% and 94%, respectively. Twelve latent small tumors were newly detected on the liver surface using ICG, five of which proved to be carcinomas. With 5-ALA, the sensitivity, specificity and accuracy for detecting the main tumors were 57%, 100% and 58%, respectively. Five latent small tumors were newly detected using 5-ALA; all were carcinomas. Overall, five new tumors were detected by both ICG and 5-ALA fluorescence imaging; two were hepatocellular carcinomas (HCCs) and three were metastases of colorectal cancer. The sensitivity and specificity of ICG fluorescence imaging for main tumor detection were relatively high and low, respectively, but the opposite was true of 5-ALA imaging. Fluorescence imaging using 5-ALA may provide greater specificity in the detection of surface-invisible malignant liver tumors than using ICG fluorescence imaging alone. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  4. Split green fluorescent protein as a modular binding partner for protein crystallization

    International Nuclear Information System (INIS)

    Nguyen, Hau B.; Hung, Li-Wei; Yeates, Todd O.; Terwilliger, Thomas C.; Waldo, Geoffrey S.

    2013-01-01

    A strategy using a new split green fluorescent protein (GFP) as a modular binding partner to form stable protein complexes with a target protein is presented. The modular split GFP may open the way to rapidly creating crystallization variants. A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP β-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10–11) hairpin in complex with GFP(1–9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10–11) hairpin with a variety of GFP(1–9) mutants engineered for favorable crystallization

  5. Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering.

    Science.gov (United States)

    Close, Devin W; Paul, Craig Don; Langan, Patricia S; Wilce, Matthew C J; Traore, Daouda A K; Halfmann, Randal; Rocha, Reginaldo C; Waldo, Geoffery S; Payne, Riley J; Rucker, Joseph B; Prescott, Mark; Bradbury, Andrew R M

    2015-07-01

    In this article, we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization. © 2014 Wiley Periodicals, Inc.

  6. Broadband photon pair generation in green fluorescent proteins through spontaneous four-wave mixing

    Science.gov (United States)

    Shi, Siyuan; Thomas, Abu; Corzo, Neil V.; Kumar, Prem; Huang, Yuping; Lee, Kim Fook

    2016-01-01

    Recent studies in quantum biology suggest that quantum mechanics help us to explore quantum processes in biological system. Here, we demonstrate generation of photon pairs through spontaneous four-wave mixing process in naturally occurring fluorescent proteins. We develop a general empirical method for analyzing the relative strength of nonlinear optical interaction processes in five different organic fluorophores. Our results indicate that the generation of photon pairs in green fluorescent proteins is subject to less background noises than in other fluorophores, leading to a coincidence-to-accidental ratio ~145. As such proteins can be genetically engineered and fused to many biological cells, our experiment enables a new platform for quantum information processing in a biological environment such as biomimetic quantum networks and quantum sensors. PMID:27076032

  7. In Vivo Photoacoustic and Fluorescence Cystography Using Clinically Relevant Dual Modal Indocyanine Green

    Directory of Open Access Journals (Sweden)

    Sungjo Park

    2014-10-01

    Full Text Available Conventional X-ray-based cystography uses radio-opaque materials, but this method uses harmful ionizing radiation and is not sensitive. In this study, we demonstrate nonionizing and noninvasive photoacoustic (PA and fluorescence (FL cystography using clinically relevant indocyanine green (ICG in vivo. After transurethral injection of ICG into rats through a catheter, their bladders were photoacoustically and fluorescently visualized. A deeply positioned bladder below the skin surface (i.e., ~1.5–5 mm was clearly visible in the PA and FL image using a laser pulse energy of less than 2 mJ/cm2 (1/15 of the safety limit. Then, the in vivo imaging results were validated through in situ studies. Our results suggest that dual modal cystography can provide a nonionizing and noninvasive imaging tool for bladder mapping.

  8. Utility of Indocyanine Green Fluorescence Imaging for Intraoperative Localization in Reoperative Parathyroid Surgery.

    Science.gov (United States)

    Sound, Sara; Okoh, Alexis; Yigitbas, Hakan; Yazici, Pinar; Berber, Eren

    2015-10-27

    Due to the variations in anatomic location, the identification of parathyroid glands may be challenging. Although there have been advances in preoperative imaging modalities, there is still a need for an accurate intraoperative guidance. Indocyanine green (ICG) is a new agent that has been used for intraoperative fluorescence imaging in a number of general surgical procedures. Its utility for parathyroid localization in humans has not been reported in the literature. We report 3 patients who underwent reoperative neck surgery for primary hyperparathyroidism. Using a video-assisted technique with intraoperative ICG fluorescence imaging, the parathyroid glands were recognized and removed successfully in all cases. Surrounding soft tissue structures remained nonfluorescent, and could be distinguished from the parathyroid glands. This report suggests a potential utility of ICG imaging in intraoperative localization of parathyroid glands in reoperative neck surgery. Future work is necessary to assess its benefit for first-time parathyroid surgery. © The Author(s) 2015.

  9. Application of split-green fluorescent protein for topology mapping membrane proteins in Escherichia coli

    DEFF Research Database (Denmark)

    Toddo, Stephen; Soderstrom, Bill; Palombo, Isolde

    2012-01-01

    A topology map of a membrane protein defines the location of transmembrane helices and the orientation of soluble domains relative to the membrane. In the absence of a high-resolution structure, a topology map is an essential guide for studying structurefunction relationships. Although these maps....../periplasmic location of the N-terminus of a protein. Here, we show that the bimolecular split-green fluorescent protein complementation system can overcome this limitation and can be used to determine the location of both the N- and C-termini of inner membrane proteins in Escherichia coli....

  10. Multi-state lasing in self-assembled ring-shaped green fluorescent protein microcavities

    Energy Technology Data Exchange (ETDEWEB)

    Dietrich, Christof P., E-mail: cpd3@st-andrews.ac.uk; Höfling, Sven; Gather, Malte C., E-mail: mcg6@st-andrews.ac.uk [SUPA, School of Physics and Astronomy, University of St Andrews, St Andrews KY16 9SS (United Kingdom)

    2014-12-08

    We demonstrate highly efficient lasing from multiple photonic states in microcavities filled with self-assembled rings of recombinant enhanced green fluorescent protein (eGFP) in its solid state form. The lasing regime is achieved at very low excitation energies of 13 nJ and occurs from cavity modes dispersed in both energy and momentum. We attribute the momentum distribution to very efficient scattering of incident light at the surface of the eGFP rings. The distribution of lasing states in energy is induced by the large spectral width of the gain spectrum of recombinant eGFP (FWHM ≅ 25 nm)

  11. Direct and Indirect Electron Emission from the Green Fluorescent Protein Chromophore

    Science.gov (United States)

    Toker, Y.; Rahbek, D. B.; Klærke, B.; Bochenkova, A. V.; Andersen, L. H.

    2012-09-01

    Photoelectron spectra of the deprotonated green fluorescent protein chromophore have been measured in the gas phase at several wavelengths within and beyond the S0-S1 photoabsorption band of the molecule. The vertical detachment energy (VDE) was determined to be 2.68±0.1eV. The data show that the first electronically excited state is bound in the Franck-Condon region, and that electron emission proceeds through an indirect (resonant) electron-emission channel within the corresponding absorption band.

  12. Preparation and Observation of Fresh-frozen Sections of the Green Fluorescent Protein Transgenic Mouse Head

    International Nuclear Information System (INIS)

    Tada, Masahito; Shinohara, Yoshinori; Kato, Ichiro; Hiraga, Koichi; Aizawa, Tomoyasu; Demura, Makoto; Mori, Yoshihiro; Shinoda, Hiroyuki; Mizuguchi, Mineyuki; Kawano, Keiichi

    2006-01-01

    Hard tissue decalcification can cause variation in the constituent protein characteristics. This paper describes a method of preparating of frozen mouse head sections so as to clearly observe the nature of the constituent proteins. Frozen sections of various green fluorescent protein (GFP) transgenic mouse heads were prepared using the film method developed by Kawamoto and Shimizu. This method made specimen dissection without decalcification possible, wherein GFP was clearly observed in an undamaged state. Conversely, using the same method with decalcification made GFP observation in the transgenic mouse head difficult. This new method is suitable for observing GFP marked cells, enabling us to follow the transplanted GFP marked cells within frozen head sections

  13. Indocyanine green fluorescence angiography for intraoperative assessment of gastrointestinal anastomotic perfusion

    DEFF Research Database (Denmark)

    Degett, Thea Helene; Andersen, Helene Schou; Gögenur, Ismail

    2016-01-01

    PURPOSE: Anastomotic leakage following gastrointestinal surgery remains a frequent and serious complication associated with a high morbidity and mortality. Indocyanine green fluorescence angiography (ICG-FA) is a newly developed technique to measure perfusion intraoperatively. The aim of this paper...... included in the review if they assessed anastomotic perfusion intraoperatively with ICG-FA in order to predict anastomotic leakage in humans. RESULTS: Of 790 screened papers 14 studies were included in this review. Ten studies (n = 916) involved patients with colorectal anastomoses and four studies (n...

  14. Recent advances in near-infrared fluorescence-guided imaging surgery using indocyanine green.

    Science.gov (United States)

    Namikawa, Tsutomu; Sato, Takayuki; Hanazaki, Kazuhiro

    2015-12-01

    Near-infrared (NIR) fluorescence imaging has better tissue penetration, allowing for the effective rejection of excitation light and detection deep inside organs. Indocyanine green (ICG) generates NIR fluorescence after illumination by an NIR ray, enabling real-time intraoperative visualization of superficial lymphatic channels and vessels transcutaneously. The HyperEye Medical System (HEMS) can simultaneously detect NIR rays under room light to provide color imaging, which enables visualization under bright light. Thus, NIR fluorescence imaging using ICG can provide for excellent diagnostic accuracy in detecting sentinel lymph nodes in cancer and microvascular circulation in various ischemic diseases, to assist us with intraoperative decision making. Including HEMS in this system could further improve the sentinel lymph node mapping and intraoperative identification of blood supply in reconstructive organs and ischemic diseases, making it more attractive than conventional imaging. Moreover, the development of new laparoscopic imaging systems equipped with NIR will allow fluorescence-guided surgery in a minimally invasive setting. Future directions, including the conjugation of NIR fluorophores to target specific cancer markers might be realistic technology with diagnostic and therapeutic benefits.

  15. [Place of indocyanine green coupled with fluorescence imaging in research of breast cancer sentinel node].

    Science.gov (United States)

    Vermersch, Charlotte; Raia Barjat, Tiphaine; Perrot, Marianne; Lima, Suzanne; Chauleur, Céline

    2016-04-01

    The sentinel node has a fundamental role in the management of early breast cancer. Currently, the double detection of blue and radioisotope is recommended. But in common practice, many centers use a single method. However, with a single detection, the risk of false negatives and the identification failure rate increase to a significant extent and the number of sentinel lymph node detected and removed is not enough. Furthermore, the tracers used until now show inconveniences. The purpose of this work is to present a new method of detection, using the green of indocyanine coupled with fluorescence imaging, and to compare it with the already existing methods. The method combined by fluorescence and isotopic is reliable, sure, of fast learning and could constitute a good strategy of detection. The major interest is to obtain a satisfactory number of sentinel nodes. The profit could be even more important for overweight patients. The fluorescence used alone is at the moment not possible. Wide ranging studies are necessary. The FLUOTECH, randomized study of 100 patients, comparing the isotopic method of double isotope technique and fluorescence, is underway to confirm these data. Copyright © 2016 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

  16. Using green fluorescent malaria parasites to screen for permissive vector mosquitoes

    Directory of Open Access Journals (Sweden)

    Martin Beatrice

    2006-03-01

    Full Text Available Abstract Background The Plasmodium species that infect rodents, particularly Plasmodium berghei and Plasmodium yoelii, are useful to investigate host-parasite interactions. The mosquito species that act as vectors of human plasmodia in South East Asia, Africa and South America show different susceptibilities to infection by rodent Plasmodium species. P. berghei and P. yoelii infect both Anopheles gambiae and Anopheles stephensi, which are found mainly in Africa and Asia, respectively. However, it was reported that P. yoelii can infect the South American mosquito, Anopheles albimanus, while P. berghei cannot. Methods P. berghei lines that express the green fluorescent protein were used to screen for mosquitoes that are susceptible to infection by P. berghei. Live mosquitoes were examined and screened for the presence of a fluorescent signal in the abdomen. Infected mosquitoes were then examined by time-lapse microscopy to reveal the dynamic behaviour of sporozoites in haemolymph and extracted salivary glands. Results A single fluorescent oocyst can be detected in live mosquitoes and P. berghei can infect A. albimanus. As in other mosquitoes, P. berghei sporozoites can float through the haemolymph and invade A. albimanus salivary glands and they are infectious in mice after subcutaneous injection. Conclusion Fluorescent Plasmodium parasites can be used to rapidly screen susceptible mosquitoes. These results open the way to develop a laboratory model in countries where importation of A. gambiae and A. stephensi is not allowed.

  17. Development and validation of a custom made indocyanine green fluorescence lymphatic vessel imager

    Science.gov (United States)

    Pallotta, Olivia J.; van Zanten, Malou; McEwen, Mark; Burrow, Lynne; Beesley, Jack; Piller, Neil

    2015-06-01

    Lymphoedema is a chronic progressive condition often producing significant morbidity. An in-depth understanding of an individual's lymphatic architecture is valuable both in the understanding of underlying pathology and for targeting and tailoring treatment. Severe lower limb injuries resulting in extensive loss of soft tissue require transposition of a flap consisting of muscle and/or soft tissue to close the defect. These patients are at risk of lymphoedema and little is known about lymphatic regeneration within the flap. Indocyanine green (ICG), a water-soluble dye, has proven useful for the imaging of lymphatic vessels. When injected into superficial tissues it binds to plasma proteins in lymph. By exposing the dye to specific wavelengths of light, ICG fluoresces with near-infrared light. Skin is relatively transparent to ICG fluorescence, enabling the visualization and characterization of superficial lymphatic vessels. An ICG fluorescence lymphatic vessel imager was manufactured to excite ICG and visualize real-time fluorescence as it travels through the lymphatic vessels. Animal studies showed successful ICG excitation and detection using this imager. Clinically, the imager has assisted researchers to visualize otherwise hidden superficial lymphatic pathways in patients postflap surgery. Preliminary results suggest superficial lymphatic vessels do not redevelop in muscle flaps.

  18. Green synthesis of highly fluorescent carbon quantum dots from sugarcane bagasse pulp

    Energy Technology Data Exchange (ETDEWEB)

    Thambiraj, S. [Nano-Bio Materials and Sensors Laboratory, PSG Institute of Advanced Studies, Coimbatore, 641 004, Tamil Nadu (India); Ravi Shankaran, D., E-mail: dravishankaran@hotmail.com [Nano-Bio Materials and Sensors Laboratory, PSG Institute of Advanced Studies, Coimbatore, 641 004, Tamil Nadu (India); National Centre for Nanoscience and Nanotechnology, University of Madras, Guindy Campus, Chennai, 600 025, Tamil Nadu (India)

    2016-12-30

    Graphical abstract: Schematic representation of CQDs from sugarcane bagasse carbon. - Highlights: • CQDs were synthesised from sugarcane bagasse waste with top down approaches. • Synthesis method is green, simple and efficient process. • CQDs possess high quantum yield, good stability and highly fluorescent in nature. • The morphological and topographical study of CQDs was done by HR-TEM and AFM and was observed that the average size is 4.1 ± 0.17 nm and surface thickness is 5 nm. - Abstract: Carbon quantum dots (CQDs) have great potential due to its advantageous characteristics of highly fluorescent nature and good stability. In this study, we aimed to develop a simple and efficient method for the green synthesis of fluorescent CQDs from sugarcane bagasse, a renewable and sustainable resource. The process involves the top down approach of chemical oxidation followed by exfoliation of sugarcane carbon. The synthesized CQDs was characterized by UV–vis absorption spectroscopy, Spectrofluorophotometry, Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), Raman spectroscopy, X-ray photon spectroscopy (XPS), Atomic force microscopy (AFM) and High-resolution transmission electron microscopy (HR-TEM). The synthesized CQDs possess stable fluorescent properties, good bio-compatibility and high quantum yield. The CQDs are highly crystalline with longitudinal dimensions of 4.1 ± 0.17 nm with an average roughness of around 5 nm. The XRD and TEM analysis indicates that the synthesized CQDs possess face centred cubic crystal structure. The results suggest that the proposed CQDs could be utilized for bio-sensor, bio-imaging and drug delivery applications.

  19. Thermal stability of chemically denatured green fluorescent protein (GFP) A preliminary study

    Energy Technology Data Exchange (ETDEWEB)

    Nagy, Attila; Malnasi-Csizmadia, Andras; Somogyi, Bela; Lorinczy, Denes

    2004-02-09

    Green fluorescent protein (GFP) is a light emitter in the bioluminescence reaction of the jellyfish Aequorea victoria. The protein consist of 238 amino acids and produces green fluorescent light ({lambda}{sub max}=508 nm), when irradiated with near ultraviolet light. The fluorescence is due to the presence of chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser{sup 65}-Tyr{sup 66}-Gly{sup 67}-, which buried into {beta}-barrel. GFP is extremely compact and heat stable molecule. In this work, we present data for the effect of chemical denaturing agent on the thermal stability of GFP. When denaturing agent is applied, global thermal stability and the melting point of the molecule is decreases, that can be monitored with differential scanning calorimetry. The results indicate, that in 1-6 M range of GuHCl the melting temperature is decreasing continuously from 83 to 38 deg. C. Interesting finding, that the calculated calorimetric enthalpy decreases with GuHCl concentration up to 3 M (5.6-0.2 kJ mol{sup -1}), but at 4 M it jumps to 8.4 and at greater concentration it is falling down to 1.1 kJ mol{sup -1}. First phenomena, i.e. the decrease of melting point with increasing GuHCl concentration can be easily explained by the effect of the extended chemical denaturation, when less and less amount of heat required to diminish the remaining hydrogen bonds in {beta}-barrel. The surprising increase of calorimetric enthalpy at 4 M concentration of GuHCl could be the consequence of a dimerization or a formation of stable complex between GFP and denaturing agent as well as a precipitation at an extreme GuHCl concentration. We are planning further experiments to elucidate fluorescent consequence of these processes.

  20. New fluorescence spectroscopic method for the simultaneous determination of alkaloids in aqueous extract of green coffee beans.

    Science.gov (United States)

    Yisak, Hagos; Redi-Abshiro, Mesfin; Chandravanshi, Bhagwan Singh

    2018-05-11

    There is no fluorescence spectroscopic method for the determination of trigonelline and theobromine in green coffee beans. Therefore, the objective of this study was to develop a new fluorescence spectroscopic method to determine the alkaloids simultaneously in the aqueous extract of green coffee beans. The calibration curves were linear in the range 2-6, 1-6, 1-5 mg/L for caffeine, theobromine and trigonelline, respectively, with R 2  ≥ 0.9987. The limit of detection and limit of quantification were 2, 6 and 7 µg/L and 40, 20 and 20 µg/L for caffeine, theobromine and trigonelline, respectively. Caffeine and trigonelline exhibited well separated fluorescence excitation spectra and therefore the two alkaloids were selectively quantified in the aqueous extract of green coffee. While theobromine showed overlapping fluorescence excitation spectra with caffeine and hence theobromine could not be determined in the aqueous extract of green coffee beans. The amount of caffeine and trigonelline in the three samples of green coffee beans were found to be 0.95-1.10 and 1.00-1.10% (w/w), respectively. The relative standard deviations (RSD ≤ 4%) of the method for the three compounds of interest were of very good. The accuracy of the developed analytical method was evaluated by spiking standard caffeine and trigonelline to green coffee beans and the average recoveries were 99 ± 2% for both the alkaloids. A fast, sensitive and reliable fluorescence method for the simultaneous determination of caffeine and trigonelline in the aqueous extract of green coffee beans was developed and validated. The developed method reflected an effective performance to the direct determination of the two alkaloids in the aqueous extract of green coffee beans.

  1. Transcript and protein expression profile of PF11_0394, a Plasmodium falciparum protein expressed in salivary gland sporozoites

    Directory of Open Access Journals (Sweden)

    Schlarman Maggie S

    2012-03-01

    Full Text Available Abstract Background Plasmodium falciparum malaria is a significant problem around the world today, thus there is still a need for new control methods to be developed. Because the sporozoite displays dual infectivity for both the mosquito salivary glands and vertebrate host tissue, it is a good target for vaccine development. Methods The P. falciparum gene, PF11_0394, was chosen as a candidate for study due to its potential role in the invasion of host tissues. This gene, which was selected using a data mining approach from PlasmoDB, is expressed both at the transcriptional and protein levels in sporozoites and likely encodes a putative surface protein. Using reverse transcription-polymerase chain reaction (RT-PCR and green fluorescent protein (GFP-trafficking studies, a transcript and protein expression profile of PF11_0394 was determined. Results The PF11_0394 protein has orthologs in other Plasmodium species and Apicomplexans, but none outside of the group Apicomplexa. PF11_0394 transcript was found to be present during both the sporozoite and erythrocytic stages of the parasite life cycle, but no transcript was detected during axenic exoerythrocytic stages. Despite the presence of transcript throughout several life cycle stages, the PF11_0394 protein was only detected in salivary gland sporozoites. Conclusions PF11_0394 appears to be a protein uniquely detected in salivary gland sporozoites. Even though a specific function of PF11_0394 has not been determined in P. falciparum biology, it could be another candidate for a new vaccine.

  2. Application of silver nanoparticles in the detection of SYBR Green I by surface enhanced Raman and surface-enhanced fluorescence

    Science.gov (United States)

    Guo, Wei; Wu, Jian; Wang, Chunyan; Zhang, Tian; Chen, Tao

    2018-05-01

    Silver nanomaterials have remarkable application in biomedical detection due to their unique surface plasmon resonance (SPR) characteristics. It can be used for surface-enhanced Raman scattering (SERS) and surface-enhanced fluorescence (SEF). Current research elaborates a technique for improvement of SYBR Green I detection obtained from surface-enhanced Raman scattering (SERS) and surface-enhanced fluorescence (SEF) by silver nanoparticles with the average size about 70 nm. Primarily, SYBR Green I is an important fluorescent dye used in polymerase chain reaction (PCR). It is found that both Raman and fluorescence can be used for detection of this dye. Furthermore, the enhanced efficiency of the Raman and fluorescence by SERS and SEF is observed in this study, the enhancement factor for Raman signals is 3.2 × 103, and the fluorescence intensity bincreased two times by SEF. The quantitative detection of SYBR Green I by SERS and SEF can be achieved. The present work can be used to improve the detection of SYBR Green I by SERS and SEF. It would also be employed for high-sensitive detection of other materials in the future.

  3. Expression of pH-sensitive green fluorescent protein in Arabidopsis thaliana

    Science.gov (United States)

    Moseyko, N.; Feldman, L. J.

    2001-01-01

    This is the first report on using green fluorescent protein (GFP) as a pH reporter in plants. Proton fluxes and pH regulation play important roles in plant cellular activity and therefore, it would be extremely helpful to have a plant gene reporter system for rapid, non-invasive visualization of intracellular pH changes. In order to develop such a system, we constructed three vectors for transient and stable transformation of plant cells with a pH-sensitive derivative of green fluorescent protein. Using these vectors, transgenic Arabidopsis thaliana and tobacco plants were produced. Here the application of pH-sensitive GFP technology in plants is described and, for the first time, the visualization of pH gradients between different developmental compartments in intact whole-root tissues of A. thaliana is reported. The utility of pH-sensitive GFP in revealing rapid, environmentally induced changes in cytoplasmic pH in roots is also demonstrated.

  4. Modulating fluorescence quantum yield of highly concentrated fluorescein using differently shaped green synthesized gold nanoparticles

    International Nuclear Information System (INIS)

    John, Jisha; Thomas, Lincy; Kurian, Achamma; George, Sajan D.

    2016-01-01

    The interaction of dye molecules with differently shaped nanoparticles is of great interest owing to the potential applications in areas of bioimaging, sensing and photodynamic therapy (biology) as well as solar cells (photonics) applications. For such applications, noble metallic nanoparticles are commonly employed to either enhance or quench the luminescence of a nearby fluorophore. However, in most of the studies, the dye concentration is limited to avoid self-quenching. This paper reports the influence of differently shaped gold nanoparticles (spherical, bean and star), prepared via green synthesis, on the emission behavior as well as on the fluorescence quantum yield of fluorescein dye at concentrations for which self-quenching occurs. The emission behavior is probed via laser based steady state fluorescence whereas quantum yield is measured using a dual beam laser based thermal lens technique. The experimentally observed fluorescence quenching with a concomitant increase in thermal lens signal in the vicinity of nanoparticles are explained in terms of nonradiative energy transfer between the donor and the acceptor. Further, the influence of pH of the prepared gold nanofluid on the absorption, emission as well as quantum yield are also accounted. These studies elucidate that even at high concentrations of dye, the gold nanoparticle and its shape clearly influences the optical properties of nearby dye molecules and thus can be exploited for future applications. - Highlights: • Green synthesis of differently shaped gold nanoparticles. • Tailoring emission properties of fluorescein with respect to nanoparticle concentration and shape. • Tailoring the quantum yield of highly concentrated fluorescein with nanoparticles.

  5. Green Fluorescent Protein as a Model for Protein Crystal Growth Studies

    Science.gov (United States)

    Agena, Sabine; Smith, Lori; Karr, Laurel; Pusey, Marc

    1998-01-01

    Green fluorescent protein (GFP) from jellyfish Aequorea Victoria has become a popular marker for e.g. mutagenesis work. Its fluorescent property, which originates from a chromophore located in the center of the molecule, makes it widely applicable as a research too]. GFP clones have been produced with a variety of spectral properties, such as blue and yellow emitting species. The protein is a single chain of molecular weight 27 kDa and its structure has been determined at 1.9 Angstrom resolution. The combination of GFP's fluorescent property, the knowledge of its several crystallization conditions, and its increasing use in biophysical and biochemical studies, all led us to consider it as a model material for macromolecular crystal growth studies. Initial preparations of GFP were from E.coli with yields of approximately 5 mg/L of culture media. Current yields are now in the 50 - 120 mg/L range, and we hope to further increase this by expression of the GFP gene in the Pichia system. The results of these efforts and of preliminary crystal growth studies will be presented.

  6. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting

    OpenAIRE

    Bajar, Bryce T.; Wang, Emily S.; Lam, Amy J.; Kim, Bongjae B.; Jacobs, Conor L.; Howe, Elizabeth S.; Davidson, Michael W.; Lin, Michael Z.; Chu, Jun

    2016-01-01

    Many genetically encoded biosensors use F?rster resonance energy transfer (FRET) to dynamically report biomolecular activities. While pairs of cyan and yellow fluorescent proteins (FPs) are most commonly used as FRET partner fluorophores, respectively, green and red FPs offer distinct advantages for FRET, such as greater spectral separation, less phototoxicity, and lower autofluorescence. We previously developed the green-red FRET pair Clover and mRuby2, which improves responsiveness in intra...

  7. Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research.

    Directory of Open Access Journals (Sweden)

    Takuya Kobayashi

    Full Text Available BACKGROUND: Genetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming. METHODOLOGY/PRINCIPAL FINDINGS: Here we report a novel multifunctional green fluorescent protein (mfGFP tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8xHis, streptavidin-binding peptide (SBP, and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP with 8xHis and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM. These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry. CONCLUSIONS AND SIGNIFICANCE: The multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies.

  8. In Vitro Osteogenic Potential of Green Fluorescent Protein Labelled Human Embryonic Stem Cell-Derived Osteoprogenitors

    Directory of Open Access Journals (Sweden)

    Intekhab Islam

    2016-01-01

    Full Text Available Cellular therapy using stem cells in bone regeneration has gained increasing interest. Various studies suggest the clinical utility of osteoprogenitors-like mesenchymal stem cells in bone regeneration. However, limited availability of mesenchymal stem cells and conflicting evidence on their therapeutic efficacy limit their clinical application. Human embryonic stem cells (hESCs are potentially an unlimited source of healthy and functional osteoprogenitors (OPs that could be utilized for bone regenerative applications. However, limited ability to track hESC-derived progenies in vivo greatly hinders translational studies. Hence, in this study, we aimed to establish hESC-derived OPs (hESC-OPs expressing green fluorescent protein (GFP and to investigate their osteogenic differentiation potential in vitro. We fluorescently labelled H9-hESCs using a plasmid vector encoding GFP. The GFP-expressing hESCs were differentiated into hESC-OPs. The hESC-OPsGFP+ stably expressed high levels of GFP, CD73, CD90, and CD105. They possessed osteogenic differentiation potential in vitro as demonstrated by increased expression of COL1A1, RUNX2, OSTERIX, and OPG transcripts and mineralized nodules positive for Alizarin Red and immunocytochemical expression of osteocalcin, alkaline phosphatase, and collagen-I. In conclusion, we have demonstrated that fluorescently labelled hESC-OPs can maintain their GFP expression for the long term and their potential for osteogenic differentiation in vitro. In future, these fluorescently labelled hESC-OPs could be used for noninvasive assessment of bone regeneration, safety, and therapeutic efficacy.

  9. Predictable tuning of protein expression in bacteria

    DEFF Research Database (Denmark)

    Bonde, Mads; Pedersen, Margit; Klausen, Michael Schantz

    2016-01-01

    We comprehensively assessed the contribution of the Shine-Dalgarno sequence to protein expression and used the data to develop EMOPEC (Empirical Model and Oligos for Protein Expression Changes; http://emopec.biosustain.dtu.dk). EMOPEC is a free tool that makes it possible to modulate the expressi...

  10. Ultrastable green fluorescence carbon dots with a high quantum yield for bioimaging and use as theranostic carriers

    DEFF Research Database (Denmark)

    Yang, Chuanxu; Thomsen, Rasmus Peter; Ogaki, Ryosuke

    2015-01-01

    to widely used semiconductor quantum dots. However, it remains a great challenge to prepare highly stable, water-soluble green luminescent Cdots with a high quantum yield. Herein we report a new synthesis route for green luminescent Cdots imbuing these desirable properties and demonstrate their potential...... in biomedical applications. Oligoethylenimine (OEI)–β-cyclodextrin (βCD) Cdots were synthesised using a simple and fast heating method in phosphoric acid. The synthesised Cdots showed strong green fluorescence under UV excitation with a 30% quantum yield and exhibited superior stability over a wide pH range. We...

  11. Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii

    Science.gov (United States)

    Wu, Jinxia; Hu, Zhangli; Wang, Chaogang; Li, Shuangfei; Lei, Anping

    2008-08-01

    To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein (GFP) was expressed in C. reinhardtii under the control of promoters: RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae: RBCS2 mediated strain Tran-I and HSP70A-RBCS2 mediated strain Tran-II. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-II was at least double of that in Tran-I. In addition, a threefold increase of GFP in Tran-II was induced by heat shock at 40°C. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.

  12. Transient expression of green fluorescent protein in parasitic dodder as a tool for studying of cytoskeleton

    Directory of Open Access Journals (Sweden)

    Kaštier Peter

    2017-06-01

    Full Text Available Dodder (Cuscuta species cause severe agricultural damage in many countries throughout the world. To establish strategies for control of its growth and spreading it is important to study its life cycle and survival strategies. For these efforts genetic modification would represent a powerful tool. Here we report on Agrobacteriummediated transformation of dodder using green fluorescent protein (GFP fused to actin-binding protein as a vital marker. Since the shoot of germinating C. europaea contains a functional apical meristem and grows quickly comparing to the root-like structure, the shoot apex was used here as explant. The transgene expression was only transient, nevertheless it enabled to detect allocation of actin filaments and studying the cytoskeleton organization in dodder shoot apex. Transient expression of GFP appears to be a suitable method for studying Cuscuta development through cytoskeleton organisation that is presently largely unexplored.

  13. Legionella clemsonensis sp. nov.: a green fluorescing Legionella strain from a patient with pneumonia.

    Science.gov (United States)

    Palmer, Allison; Painter, Joseph; Hassler, Hayley; Richards, Vincent P; Bruce, Terri; Morrison, Shatavia; Brown, Ellen; Kozak-Muiznieks, Natalia A; Lucas, Claressa; McNealy, Tamara L

    2016-10-01

    A novel Legionella species was identified based on sequencing, cellular fatty acid analysis, biochemical reactions, and biofilm characterization. Strain D5610 was originally isolated from the bronchial wash of a patient in Ohio, USA. The bacteria were gram-negative, rod-shaped, and exhibited green fluorescence under long wave UV light. Phylogenetic analysis and fatty acid composition revealed a distinct separation within the genus. The strain grows between 26-45°C and forms biofilms equivalent to L. pneumophila Philadelphia 1. These characteristics suggest that this isolate is a novel Legionella species, for which the name Legionella clemsonensis sp nov. is proposed. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

  14. Identification of a functional nuclear export signal in the green fluorescent protein asFP499

    International Nuclear Information System (INIS)

    Mustafa, Huseyin; Strasser, Bernd; Rauth, Sabine; Irving, Robert A.; Wark, Kim L.

    2006-01-01

    The green fluorescent protein (GFP) asFP499 from Anemonia sulcata is a distant homologue of the GFP from Aequorea victoria. We cloned the asFP499 gene into a mammalian expression vector and showed that this protein was expressed in the human lymphoblast cell line Ramos RA1 and in the embryonic kidney 293T cell line (HEK 293T). In HEK 293T cells, asFP499 was localized mainly in the cytoplasm, suggesting that the protein was excluded from the nucleus. We identified 194 LRMEKLNI 201 as a candidate nuclear export signal in asFP499 and mutated the isoleucine at position 201 to an alanine. Unlike the wildtype form, the mutant protein was distributed throughout the cytoplasm and nucleus. This is First report of a GFP that contains a functional NES

  15. Differential diagnosis of feline leukemia virus subgroups using pseudotype viruses expressing green fluorescent protein.

    Science.gov (United States)

    Nakamura, Megumi; Sato, Eiji; Miura, Tomoyuki; Baba, Kenji; Shimoda, Tetsuya; Miyazawa, Takayuki

    2010-06-01

    Feline leukemia virus (FeLV) is classified into three receptor interference subgroups, A, B and C. In this study, to differentiate FeLV subgroups, we developed a simple assay system using pseudotype viruses expressing green fluorescent protein (GFP). We prepared gfp pseudotype viruses, named gfp(FeLV-A), gfp(FeLV-B) and gfp(FeLV-C) harboring envelopes of FeLV-A, B and C, respectively. The gfp pseudotype viruses completely interfered with the same subgroups of FeLV reference strains on FEA cells (a feline embryonic fibroblast cell line). We also confirmed that the pseudotype viruses could differentiate FeLV subgroups in field isolates. The assay will be useful for differential diagnosis of FeLV subgroups in veterinary diagnostic laboratories in the future.

  16. Evidence of green fluorescent protein and growth hormone expression in red abalone (Haliotis rufescens larvae

    Directory of Open Access Journals (Sweden)

    Mancilla-Sánchez Edgar

    2017-02-01

    Full Text Available The red abalone Haliotis rufescens is a highly appreciated mollusk in the national and international markets. Due to its natural over-exploitation and low growth rate, several genetic improvements were made, however special efforts are needed to increase its production. This study presents transgenic abalone’s larvae expressing green fluorescent protein (GFP fused to Cobia (Rachycentron canadum Growth Hormone (GH using sperm media transgenesis technique (SMT, pAcGFP1-N vector under the control of cytomegalovirus (CMV promoter. Sperms were exposed to three voltages (0.5, 0.75 and 1.0 Kv using a micropulser electroporator (Bio-Rad®. The highest GFP-GH expression average (40% was obtained in abalone larvae at 0.75 v. GFP and GH transgenes were positively detected by PCR, western blot and confocal microscope, respectively.

  17. Induction of the arginine vasopressin-enhanced green fluorescent protein fusion transgene in the rat locus coeruleus

    Czech Academy of Sciences Publication Activity Database

    Todoroki, M.; Ueta, Y.; Fujihara, H.; Otsubo, H.; Shibata, M.; Hashimoto, H.; Kabayashi, M.; Sakamoto, H.; Kawata, M.; Dayanithi, Govindan; Murphy, D.; Hiro, H.; Takahashi, E.; Nagata, S.

    2010-01-01

    Roč. 13, č. 4 (2010), s. 281-292 ISSN 1025-3890 Institutional research plan: CEZ:AV0Z50390703 Keywords : colchicine * green fluorescent protein * hypothalamus Subject RIV: FH - Neurology Impact factor: 2.553, year: 2010

  18. Amphibious fluorescent carbon dots: one-step green synthesis and application for light-emitting polymer nanocomposites.

    Science.gov (United States)

    Zhou, Li; He, Benzhao; Huang, Jiachang

    2013-09-21

    A facile and green approach for the synthesis of amphibious fluorescent carbon dots (CDs) from natural polysaccharide is reported. Light-emitting polymer nanocomposites with excellent optical performance can be easily prepared by incorporation of the amphibious CDs into the polymer matrix.

  19. Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for Live Cell Imaging

    NARCIS (Netherlands)

    Overkamp, Wout; Beilharz, Katrin; Weme, Ruud Detert Oude; Solopova, Ana; Karsens, Harma; Kovacs, Akos T.; Kok, Jan; Kuipers, Oscar P.; Veening, Jan-Willem

    2013-01-01

    Green fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localization in vivo, and many different variants are currently available to study bacterial cell biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental

  20. Effect of Solvation on Electron Detachment and Excitation Energies of a Green Fluorescent Protein Chromophore Variant.

    Science.gov (United States)

    Bose, Samik; Chakrabarty, Suman; Ghosh, Debashree

    2016-05-19

    Hybrid quantum mechanics/molecular mechanics (QM/MM) is applied to the fluorinated green fluorescent protein (GFP) chromophore (DFHBDI) in its deprotonated form to understand the solvatochromic shifts in its vertical detachment energy (VDE) and vertical excitation energy (VEE). This variant of the GFP chromophore becomes fluorescent in an RNA environment and has a wide range of applications in biomedical and biochemical fields. From microsolvation studies, we benchmark (with respect to full QM) the accuracy of our QM/MM calculations with effective fragment potential (EFP) as the MM method of choice. We show that while the solvatochromic shift in the VEE is minimal (0.1 eV blue shift) and its polarization component is only 0.03 eV, the effect of the solvent on the VDE is quite large (3.85 eV). We also show by accurate calculations on the solvatochromic shift of the VDE that polarization accounts for ∼0.23 eV and therefore cannot be neglected. The effect of the counterions on the VDE of the deprotonated chromophore in solvation is studied in detail, and a charge-smearing scheme is suggested for charged chromophores.

  1. Functional incorporation of green fluorescent protein into hepatitis B virus envelope particles

    International Nuclear Information System (INIS)

    Lambert, Carsten; Thome, Nicole; Kluck, Christoph J.; Prange, Reinhild

    2004-01-01

    The envelope of hepatitis B virus (HBV), containing the L, M, and S proteins, is essential for virus entry and maturation. For direct visualization of HBV, we determined whether envelope assembly could accommodate the green fluorescent protein (GFP). While the C-terminal addition of GFP to S trans-dominant negatively inhibited empty envelope particle secretion, the N-terminal GFP fusion to S (GFP.S) was co-integrated into the envelope, giving rise to fluorescent particles. Microscopy and topogenesis analyses demonstrated that the proper intracellular distribution and folding of GFP.S, required for particle export were rescued by interprotein interactions with wild-type S. Thereby, a dual location of GFP, inside and outside the envelope, was observed. GFP.S was also efficiently packaged into the viral envelope, and these GFP-tagged virions retained the capacity for attachment to HBV receptor-positive cells in vitro. Together, GFP-tagged virions should be suitable to monitor HBV uptake and egress in live hepatocytes

  2. Controllable synthesis of green and blue fluorescent carbon nanodots for pH and Cu(2+) sensing in living cells.

    Science.gov (United States)

    Shi, Lihong; Li, Yanyan; Li, Xiaofeng; Zhao, Bo; Wen, Xiangping; Zhang, Guomei; Dong, Chuan; Shuang, Shaomin

    2016-03-15

    We report a controllable strategy for fabrication of green and blue fluorescent carbon nanodots (CDs), and demonstrate their applications for pH and Cu(2+) sensing in living cells. Green and blue fluorescent CDs have been synthesized by hydrothermal method and pyrolysis of leeks, respectively, providing an easy way for the production of CDs without the request of tedious synthetic methodology or the use of toxic/expensive solvents and starting materials. Green fluorescent CDs (G-CDs) exhibit high tolerance to pH values and external cations. Blue fluorescent CDs (B-CDs) can be applied to pH and Cu(2+) sensing. The linear range of Cu(2+) detection is 0.01-10.00 μM and the detection limit is 0.05 μM. For pH detection, there is a good linearity in the pH range of 3.5-10.0. The linear and rapid response of B-CDs to Cu(2+) and pH is valuable for Cu(2+) and pH sensing in living cells. Confocal fluorescent imaging of human cervical carcinoma cells indicates that B-CDs could visualize Cu(2+) and pH fluctuations in living cells with negligible autofluorescence. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Experimental Evolution of a Green Fluorescent Protein Composed of 19 Unique Amino Acids without Tryptophan

    Science.gov (United States)

    Kawahara-Kobayashi, Akio; Hitotsuyanagi, Mitsuhiro; Amikura, Kazuaki; Kiga, Daisuke

    2014-04-01

    At some stage of evolution, genes of organisms may have encoded proteins that were synthesized using fewer than 20 unique amino acids. Similar to evolution of the natural 19-amino-acid proteins GroEL/ES, proteins composed of 19 unique amino acids would have been able to evolve by accumulating beneficial mutations within the 19-amino-acid repertoire encoded in an ancestral genetic code. Because Trp is thought to be the last amino acid included in the canonical 20-amino-acid repertoire, this late stage of protein evolution could be mimicked by experimental evolution of 19-amino-acid proteins without tryptophan (Trp). To further understand the evolution of proteins, we tried to mimic the evolution of a 19-amino-acid protein involving the accumulation of beneficial mutations using directed evolution by random mutagenesis on the whole targeted gene sequence. We created active 19-amino-acid green fluorescent proteins (GFPs) without Trp from a poorly fluorescent 19-amino-acid mutant, S1-W57F, by using directed evolution with two rounds of mutagenesis and selection. The N105I and S205T mutations showed beneficial effects on the S1-W57F mutant. When these two mutations were combined on S1-W57F, we observed an additive effect on the fluorescence intensity. In contrast, these mutations showed no clear improvement individually or in combination on GFPS1, which is the parental GFP mutant composed of 20 amino acids. Our results provide an additional example for the experimental evolution of 19-amino-acid proteins without Trp, and would help understand the mechanisms underlying the evolution of 19-amino-acid proteins. (236 words)

  4. Review of fluorescence guided surgery systems: identification of key performance capabilities beyond indocyanine green imaging

    Science.gov (United States)

    DSouza, Alisha V.; Lin, Huiyun; Henderson, Eric R.; Samkoe, Kimberley S.; Pogue, Brian W.

    2016-08-01

    There is growing interest in using fluorescence imaging instruments to guide surgery, and the leading options for open-field imaging are reviewed here. While the clinical fluorescence-guided surgery (FGS) field has been focused predominantly on indocyanine green (ICG) imaging, there is accelerated development of more specific molecular tracers. These agents should help advance new indications for which FGS presents a paradigm shift in how molecular information is provided for resection decisions. There has been a steady growth in commercially marketed FGS systems, each with their own differentiated performance characteristics and specifications. A set of desirable criteria is presented to guide the evaluation of instruments, including: (i) real-time overlay of white-light and fluorescence images, (ii) operation within ambient room lighting, (iii) nanomolar-level sensitivity, (iv) quantitative capabilities, (v) simultaneous multiple fluorophore imaging, and (vi) ergonomic utility for open surgery. In this review, United States Food and Drug Administration 510(k) cleared commercial systems and some leading premarket FGS research systems were evaluated to illustrate the continual increase in this performance feature base. Generally, the systems designed for ICG-only imaging have sufficient sensitivity to ICG, but a fraction of the other desired features listed above, with both lower sensitivity and dynamic range. In comparison, the emerging research systems targeted for use with molecular agents have unique capabilities that will be essential for successful clinical imaging studies with low-concentration agents or where superior rejection of ambient light is needed. There is no perfect imaging system, but the feature differences among them are important differentiators in their utility, as outlined in the data and tables here.

  5. Review of fluorescence guided surgery systems: identification of key performance capabilities beyond indocyanine green imaging

    Science.gov (United States)

    DSouza, Alisha V.; Lin, Huiyun; Henderson, Eric R.; Samkoe, Kimberley S.; Pogue, Brian W.

    2016-01-01

    Abstract. There is growing interest in using fluorescence imaging instruments to guide surgery, and the leading options for open-field imaging are reviewed here. While the clinical fluorescence-guided surgery (FGS) field has been focused predominantly on indocyanine green (ICG) imaging, there is accelerated development of more specific molecular tracers. These agents should help advance new indications for which FGS presents a paradigm shift in how molecular information is provided for resection decisions. There has been a steady growth in commercially marketed FGS systems, each with their own differentiated performance characteristics and specifications. A set of desirable criteria is presented to guide the evaluation of instruments, including: (i) real-time overlay of white-light and fluorescence images, (ii) operation within ambient room lighting, (iii) nanomolar-level sensitivity, (iv) quantitative capabilities, (v) simultaneous multiple fluorophore imaging, and (vi) ergonomic utility for open surgery. In this review, United States Food and Drug Administration 510(k) cleared commercial systems and some leading premarket FGS research systems were evaluated to illustrate the continual increase in this performance feature base. Generally, the systems designed for ICG-only imaging have sufficient sensitivity to ICG, but a fraction of the other desired features listed above, with both lower sensitivity and dynamic range. In comparison, the emerging research systems targeted for use with molecular agents have unique capabilities that will be essential for successful clinical imaging studies with low-concentration agents or where superior rejection of ambient light is needed. There is no perfect imaging system, but the feature differences among them are important differentiators in their utility, as outlined in the data and tables here. PMID:27533438

  6. Optical absorption and fluorescence spectroscopy studies of Artepillin C, the major component of green propolis

    Science.gov (United States)

    Camuri, Isamara Julia; Costa, Adriano Batista; Ito, Amando Siuiti; Pazin, Wallance Moreira

    2018-06-01

    The bioactivity of propolis against several pathogens is well established, leading to the extensive consumption of that bee product to prevent diseases. Brazilian green propolis, collected by the species Apis mellifera, is one of the most consumed in the world. The chemical composition of green propolis is complex and it has been shown that it displays antioxidant, antimicrobial, anti-inflammatory and antitumor activities, especially due to the high content of Artepillin C. The molecule is a derivative of cinnamic acid with two prenylated groups, responsible for the improvement of the affinity of the compound for lipophilic environment. A carboxylic group (COOH) is also present in the molecule, making it a pH-sensitive compound and the pH-dependent structure of Artepillin C, may modulate its biological activity related to interactions with the cellular membrane of organisms and tissues. Molecular properties of Artepillin C on aqueous solution were examined by optical absorption, steady state and time-resolved fluorescence spectroscopies. Acid-base titration based on the spectral position of the near UV absorption band, resulted in the pKa value of 4.65 for the carboxylic group in Artepillin C. In acidic pH, below the pKa value, an absorption band raised around 350 nm at Artepillin C concentration above 50 μM, due to aggregation of the molecule. In neutral pH, with excitation at 310 nm, Artepillin C presents dual emission at 400 and 450 nm. In pH close to the pKa, the optical spectra show contribution from both protonated and deprotonated species. A three-exponential function was necessary to fit the intensity decays at the different pHs, dominated by a very short lifetime component, around 0.060 ns. The fast decay resulted in emission before fluorescence depolarization, and in values of fluorescence anisotropy higher than could be expected for monomeric forms of the compound. The results give fundamental knowledge about the protonation-deprotonation state of the

  7. Investigation on the infection mechanism of the fungus Clonostachys rosea against nematodes using the green fluorescent protein.

    Science.gov (United States)

    Zhang, Lin; Yang, Jinkui; Niu, Qiuhong; Zhao, Xuna; Ye, Fengping; Liang, Lianming; Zhang, Ke-Qin

    2008-04-01

    The fungus Clonostachys rosea (syn. Gliocladium roseum) is a potential biocontrol agent. It can suppress the sporulation of the plant pathogenic fungus Botrytis cinerea and kill pathogenic nematodes, but the process of nematode pathogenesis is poorly understood. To help understand the underlying mechanism, we constructed recombinant strains containing a plasmid with both the enhanced green fluorescent protein gene egfp and the hygromycin resistance gene hph. Expression of the green fluorescent protein (GFP) was monitored using fluorescence microscopy. Our observations reveal that the pathogenesis started from the adherence of conidia to nematode cuticle for germination, followed by the penetration of germ tubes into the nematode body and subsequent death and degradation of the nematodes. These are the first findings on the infection process of the fungal pathogen marked with GFP, and the developed method can become an important tool for studying the molecular mechanisms of nematode infection by C. rosea.

  8. Temporal protein expression pattern in intracellular signalling ...

    Indian Academy of Sciences (India)

    Supplementary figure 1. Protein expression dynamics observed in Experiment, Synchronous and. Asynchronous simulation. .... molecular basis for T cell suppression by IL-10: CD28-asso- ciated IL-10 receptor inhibits CD28 tyrosine ...

  9. Comparison between the indocyanine green fluorescence and blue dye methods for sentinel lymph node biopsy using novel fluorescence image-guided resection equipment in different types of hospitals.

    Science.gov (United States)

    He, Kunshan; Chi, Chongwei; Kou, Deqiang; Huang, Wenhe; Wu, Jundong; Wang, Yabing; He, Lifang; Ye, Jinzuo; Mao, Yamin; Zhang, Guo-Jun; Wang, Jiandong; Tian, Jie

    2016-12-01

    Sentinel lymph node biopsy (SLNB) has become a standard of care to detect axillary lymph metastasis in early-stage breast cancer patients with clinically negative axillary lymph nodes. Current SLNB detection modalities comprising a blue dye, a radioactive tracer, or a combination of both have advantages as well as disadvantages. Thus, near-infrared fluorescence imaging using indocyanine green (ICG) has recently been regarded as a novel method that has generated interest for SLNB around the world. However, the lack of appropriate fluorescence imaging systems has hindered further research and wide application of this method. Therefore, we developed novel fluorescence image-guided resection equipment (FIRE) to detect sentinel lymph nodes (SLNs). Moreover, to compare the ICG fluorescence imaging method with the blue dye method and to explore the universal feasibility of the former, a different type of hospital study was conducted. Ninety-nine eligible patients participated in the study at 3 different types of hospitals. After subcutaneous ICG allergy testing, all the patients were subcutaneously injected with methylene blue and ICG into the subareolar area. Consequently, 276 SLNs (range 1-7) were identified in 98 subjects (detection rate: 99%) by using the ICG fluorescence imaging method. In contrast, the blue dye method only identified 202 SLNs (range 1-7) in 91 subjects (detection rate: 91.92%). Besides, the results of the fluorescence imaging method were similar in the 3 hospitals. Our findings indicate the universal feasibility of the ICG fluorescence imaging method for SLNB using the fluorescence image-guided resection equipment in early breast cancer detection. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Green Fluorescent Protein Purification as a Didactic Tool During Practical Classes For Undergraduates Students of UFAM

    Directory of Open Access Journals (Sweden)

    J.A.Q.A Faria

    2017-07-01

    Full Text Available INTRODUCTION: The Green Fluorescent Protein (GFP, originated from the jellyfish Aequorea victoria has broadly applicability for cellular and molecular biology research. Its spectral characteristics make it practical  to be detect by UV-A (black light lamp during the purification procedure. Moreover, this approach implementation during a practical class allows the exploring of fluorescence features. OBJETIVES: the purpose of this investigation was to teach the concepts and principles of protein purification during a practical class using recombinant GFP protein. MATERIAL E METHODS: Transformed E. coli JM110 expressing GFP were resuspended in buffer solution (Tris-HCl 20 mM pH 8.0, 150 mM NaCl, 5 mM EDTA, 20% (NH42SO4 following the sonication step. The lysate was submitted to the purification through hydrophobic interaction chromatography column (HIC. After analysis of chromatogram, some collected fractions were quantified by Bradford assay and evaluated by SDS-PAGE. Besides that, the GFP presences were measured at an excitation wavelength of 488 nm on a spectrofluorimeter. RESULTS AND DISCUSSION: Before the experiments, the students were encouraged to explore the biochemistry characteristics of GFP, assessing protein data banks and published articles. These guided questions conducted to discussion of the purification strategy choosen. The GFP purification enabled the visual observation of chromatography principles necessary for the theory assimilation. During the chromatography running, we used a UV-A lamp which allowed a greatly exploration of concepts beyond this technique such as the sample injection, the GFP column retention, and the elution step. The chromatogram obtaneid were analysed and correlated to the collected fractions. Our next step was the efficiency analysis generated by the GFP measurement, total protein quantification and the analytical method SDS-PAGE. CONCLUSION: Collectively, we observed in this class the clear development

  11. Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding

    Energy Technology Data Exchange (ETDEWEB)

    Kitamura, Akira; Nakayama, Yusaku; Kinjo, Masataka, E-mail: kinjo@sci.hokudai.ac.jp

    2015-07-31

    Classical nuclear localization signal (NLS) sequences have been used for artificial localization of green fluorescent protein (GFP) in the nucleus as a positioning marker or for measurement of the nuclear-cytoplasmic shuttling rate in living cells. However, the detailed mechanism of nuclear retention of GFP-NLS remains unclear. Here, we show that a candidate mechanism for the strong nuclear retention of GFP-NLS is via the RNA-binding ability of the NLS sequence. GFP tagged with a classical NLS derived from Simian virus 40 (GFP-NLS{sup SV40}) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLS{sup SV40} in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLS{sup SV40} formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLS{sup SV40} likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLS{sup SV40} can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells. - Highlights: • Nuclear localization signal-tagged GFP (GFP-NLS) showed clear nuclear localization. • The GFP-NLS dynamically localized not only in the nucleoplasm, but also to the nucleolus. • The nuclear localization of GFP-NLS results from transient oligomerization mediated via RNA binding. • Our NLS-tagging procedure is ideal for use in artificial sequestration of proteins in the nucleus.

  12. Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding

    International Nuclear Information System (INIS)

    Kitamura, Akira; Nakayama, Yusaku; Kinjo, Masataka

    2015-01-01

    Classical nuclear localization signal (NLS) sequences have been used for artificial localization of green fluorescent protein (GFP) in the nucleus as a positioning marker or for measurement of the nuclear-cytoplasmic shuttling rate in living cells. However, the detailed mechanism of nuclear retention of GFP-NLS remains unclear. Here, we show that a candidate mechanism for the strong nuclear retention of GFP-NLS is via the RNA-binding ability of the NLS sequence. GFP tagged with a classical NLS derived from Simian virus 40 (GFP-NLS SV40 ) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLS SV40 in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLS SV40 formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLS SV40 likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLS SV40 can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells. - Highlights: • Nuclear localization signal-tagged GFP (GFP-NLS) showed clear nuclear localization. • The GFP-NLS dynamically localized not only in the nucleoplasm, but also to the nucleolus. • The nuclear localization of GFP-NLS results from transient oligomerization mediated via RNA binding. • Our NLS-tagging procedure is ideal for use in artificial sequestration of proteins in the nucleus

  13. Efficient and Scalable Synthesis of 4-Carboxy-Pennsylvania Green Methyl Ester: A Hydrophobic Building Block for Fluorescent Molecular Probes.

    Science.gov (United States)

    Woydziak, Zachary R; Fu, Liqiang; Peterson, Blake R

    2014-01-01

    Fluorinated fluorophores are valuable tools for studies of biological systems. However, amine-reactive single-isomer derivatives of these compounds are often very expensive. To provide an inexpensive alternative, we report a practical synthesis of 4-carboxy-Pennsylvania Green methyl ester. Derivatives of this hydrophobic fluorinated fluorophore, a hybrid of the dyes Oregon Green and Tokyo Green, are often cell permeable, enabling labeling of intracellular targets and components. Moreover, the low pKa of Pennsylvania Green (4.8) confers bright fluorescence in acidic cellular compartments such as endosomes, enhancing its utility for chemical biology investigations. To improve access to the key intermediate 2,7-difluoro-3,6-dihydroxyxanthen-9-one, we subjected bis-(2,4,5-trifluorophenyl)methanone to iterative nucleophilic aromatic substitution by hydroxide on scales of > 40 g. This intermediate was used to prepare over 15 grams of pure 4-carboxy-Pennsylvania Green methyl ester in 28% overall yield without requiring chromatography. This compound can be converted into the amine reactive N -hydroxysuccinimidyl ester in essentially quantitative yield for the synthesis of a wide variety of fluorescent molecular probes.

  14. Benzylglucosinolate Derived Isothiocyanate from Tropaeolum majus Reduces Gluconeogenic Gene and Protein Expression in Human Cells.

    Directory of Open Access Journals (Sweden)

    Valentina Guzmán-Pérez

    Full Text Available Nasturtium (Tropaeolum majus L. contains high concentrations of benzylglcosinolate. We found that a hydrolysis product of benzyl glucosinolate-the benzyl isothiocyanate (BITC-modulates the intracellular localization of the transcription factor Forkhead box O 1 (FOXO1. FoxO transcription factors can antagonize insulin effects and trigger a variety of cellular processes involved in tumor suppression, longevity, development and metabolism. The current study evaluated the ability of BITC-extracted as intact glucosinolate from nasturtium and hydrolyzed with myrosinase-to modulate i the insulin-signaling pathway, ii the intracellular localization of FOXO1 and, iii the expression of proteins involved in gluconeogenesis, antioxidant response and detoxification. Stably transfected human osteosarcoma cells (U-2 OS with constitutive expression of FOXO1 protein labeled with GFP (green fluorescent protein were used to evaluate the effect of BITC on FOXO1. Human hepatoma HepG2 cell cultures were selected to evaluate the effect on gluconeogenic, antioxidant and detoxification genes and protein expression. BITC reduced the phosphorylation of protein kinase B (AKT/PKB and FOXO1; promoted FOXO1 translocation from cytoplasm into the nucleus antagonizing the insulin effect; was able to down-regulate the gene and protein expression of gluconeogenic enzymes; and induced the gene expression of antioxidant and detoxification enzymes. Knockdown analyses with specific siRNAs showed that the expression of gluconeogenic genes was dependent on nuclear factor (erythroid derived-like2 (NRF2 and independent of FOXO1, AKT and NAD-dependent deacetylase sirtuin-1 (SIRT1. The current study provides evidence that BITC might have a role in type 2 diabetes T2D by reducing hepatic glucose production and increasing antioxidant resistance.

  15. Enhanced green fluorescent protein is a nearly ideal long-term expression tracer for hematopoietic stem cells, whereas DsRed-express fluorescent protein is not.

    Science.gov (United States)

    Tao, Wen; Evans, Barbara-Graham; Yao, Jing; Cooper, Scott; Cornetta, Kenneth; Ballas, Christopher B; Hangoc, Giao; Broxmeyer, Hal E

    2007-03-01

    Validated gene transfer and expression tracers are essential for elucidating functions of mammalian genes. Here, we have determined the suitability and unintended side effects of enhanced green fluorescent protein (EGFP) and DsRed-Express fluorescent protein as expression tracers in long-term hematopoietic stem cells (HSCs). Retrovirally transduced mouse bone marrow cells expressing either EGFP or DsRed-Express in single or mixed dual-color cell populations were clearly discerned by flow cytometry and fluorescence microscopy. The results from in vivo competitive repopulation assays demonstrated that EGFP-expressing HSCs were maintained nearly throughout the lifespan of the transplanted mice and retained long-term multilineage repopulating potential. All mice assessed at 15 months post-transplantation were EGFP positive, and, on average, 24% total peripheral white blood cells expressed EGFP. Most EGFP-expressing recipient mice lived at least 22 months. In contrast, Discosoma sp. red fluorescent protein (DsRed)-expressing donor cells dramatically declined in transplant-recipient mice over time, particularly in the competitive setting, in which mixed EGFP- and DsRed-expressing cells were cotransplanted. Moreover, under in vitro culture condition favoring preservation of HSCs, purified EGFP-expressing cells grew robustly, whereas DsRed-expressing cells did not. Therefore, EGFP has no detectable deteriorative effects on HSCs, and is nearly an ideal long-term expression tracer for hematopoietic cells; however, DsRed-Express fluorescent protein is not suitable for these cells.

  16. Green fluorescent protein as indicator of nonviral transient transfection efficiency in endometrial and testicular biopsies.

    Science.gov (United States)

    Zizzi, Antonio; Minardi, Daniele; Ciavattini, Andrea; Giantomassi, Federica; Montironi, Rodolfo; Muzzonigro, Giovanni; Di Primio, Roberto; Lucarini, Guendalina

    2010-03-01

    In the last years, physical and chemical methods of plasmid delivery have revolutionized the efficiency of nonviral gene transfer, and the success of gene therapy is largely dependent upon the development of gene-delivery methods. The nonviral techniques that lead to a direct transfer of DNA into tissue fragments, like electroporation (EP) and lipofection delivery systems are still insufficiently investigated. Our aim was to test the efficiency of EP and lipofection protocols in endometrial and testicular tissue fragments, using a naked plasmid DNA encoding green fluorescent protein (GFP). Because the transfection efficiency depends upon several factors, we tried to optimize the transfection conditions by testing different lipofectamine 2000 and plasmid ratios, electrical parameters, and culture after transfection. Our results show that these two nonviral methods of gene delivery are feasible and efficient in gene transfection of endometrial and testicular tissue biopsies. We found that the most performing ratio of plasmid:lipofectamine was 10:50 for transient lipofection, whereas two pulses for 10 s at 960 microF of capacitance, 200 V of voltage were the most favorable electrical parameters for EP efficiency in the presence of 5 microL of phMGFP plasmid. After lipofection and EP, the highest GFP intensity was observed respectively after 48 and 72 h of tissue fragment culturing. In conclusion, nonviral methods are attractive for an improvement of the gene therapy and our protocol could provide useful indications for in vivo gene therapy applications.

  17. New Wistar Kyoto and spontaneously hypertensive rat transgenic models with ubiquitous expression of green fluorescent protein

    Directory of Open Access Journals (Sweden)

    Ana Isabel Garcia Diaz

    2016-04-01

    Full Text Available The Wistar Kyoto (WKY rat and the spontaneously hypertensive (SHR rat inbred strains are well-established models for human crescentic glomerulonephritis (CRGN and metabolic syndrome, respectively. Novel transgenic (Tg strains add research opportunities and increase scientific value to well-established rat models. We have created two novel Tg strains using Sleeping Beauty transposon germline transgenesis, ubiquitously expressing green fluorescent protein (GFP under the rat elongation factor 1 alpha (EF1a promoter on the WKY and SHR genetic backgrounds. The Sleeping Beauty system functioned with high transgenesis efficiency; 75% of new rats born after embryo microinjections were transgene positive. By ligation-mediated PCR, we located the genome integration sites, confirming no exonic disruption and defining a single or low copy number of the transgenes in the new WKY-GFP and SHR-GFP Tg lines. We report GFP-bright expression in embryos, tissues and organs in both lines and show preliminary in vitro and in vivo imaging data that demonstrate the utility of the new GFP-expressing lines for adoptive transfer, transplantation and fate mapping studies of CRGN, metabolic syndrome and other traits for which these strains have been extensively studied over the past four decades.

  18. Illuminating the origins of spectral properties of green fluorescent proteins via proteochemometric and molecular modeling.

    Science.gov (United States)

    Nantasenamat, Chanin; Simeon, Saw; Owasirikul, Wiwat; Songtawee, Napat; Lapins, Maris; Prachayasittikul, Virapong; Wikberg, Jarl E S

    2014-10-15

    Green fluorescent protein (GFP) has immense utility in biomedical imaging owing to its autofluorescent nature. In efforts to broaden the spectral diversity of GFP, there have been several reports of engineered mutants via rational design and random mutagenesis. Understanding the origins of spectral properties of GFP could be achieved by means of investigating its structure-activity relationship. The first quantitative structure-property relationship study for modeling the spectral properties, particularly the excitation and emission maximas, of GFP was previously proposed by us some years ago in which quantum chemical descriptors were used for model development. However, such simplified model does not consider possible effects that neighboring amino acids have on the conjugated π-system of GFP chromophore. This study describes the development of a unified proteochemometric model in which the GFP chromophore and amino acids in its vicinity are both considered in the same model. The predictive performance of the model was verified by internal and external validation as well as Y-scrambling. Our strategy provides a general solution for elucidating the contribution that specific ligand and protein descriptors have on the investigated spectral property, which may be useful in engineering novel GFP variants with desired characteristics. Copyright © 2014 Wiley Periodicals, Inc.

  19. Study of the antimalarial properties of hydroxyethylamine derivatives using green fluorescent protein transformed Plasmodium berghei

    Directory of Open Access Journals (Sweden)

    Mariana Conceição Souza

    2015-06-01

    Full Text Available A rapid decrease in parasitaemia remains the major goal for new antimalarial drugs and thus, in vivo models must provide precise results concerning parasitaemia modulation. Hydroxyethylamine comprise an important group of alkanolamine compounds that exhibit pharmacological properties as proteases inhibitors that has already been proposed as a new class of antimalarial drugs. Herein, it was tested the antimalarial property of new nine different hydroxyethylamine derivatives using the green fluorescent protein (GFP-expressing Plasmodium berghei strain. By comparing flow cytometry and microscopic analysis to evaluate parasitaemia recrudescence, it was observed that flow cytometry was a more sensitive methodology. The nine hydroxyethylamine derivatives were obtained by inserting one of the following radical in the para position: H, 4Cl, 4-Br, 4-F, 4-CH3, 4-OCH3, 4-NO2, 4-NH2 and 3-Br. The antimalarial test showed that the compound that received the methyl group (4-CH3 inhibited 70% of parasite growth. Our results suggest that GFP-transfected P. berghei is a useful tool to study the recrudescence of novel antimalarial drugs through parasitaemia examination by flow cytometry. Furthermore, it was demonstrated that the insertion of a methyl group at the para position of the sulfonamide ring appears to be critical for the antimalarial activity of this class of compounds.

  20. Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source

    Science.gov (United States)

    Kawarasaki, Tatsuo; Uchiyama, Kazuhiko; Hirao, Atsushi; Azuma, Sadahiro; Otake, Masayoshi; Shibata, Masatoshi; Tsuchiya, Seiko; Enosawa, Shin; Takeuchi, Koichi; Konno, Kenjiro; Hakamata, Yoji; Yoshino, Hiroyuki; Wakai, Takuya; Ookawara, Shigeo; Tanaka, Hozumi; Kobayashi, Eiji; Murakami, Takashi

    2009-09-01

    Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation.

  1. Parts Characterization for Tunable Protein Expression

    DEFF Research Database (Denmark)

    Klausen, Michael Schantz; Sommer, Morten Otto Alexander

    2018-01-01

    Flow-seq combines flexible genome engineering methods with flow cytometry-based cell sorting and deep DNA sequencing to enable comprehensive interrogation of genotype to phenotype relationships. One application is to study the effect of specific regulatory elements on protein expression. Construc......Flow-seq combines flexible genome engineering methods with flow cytometry-based cell sorting and deep DNA sequencing to enable comprehensive interrogation of genotype to phenotype relationships. One application is to study the effect of specific regulatory elements on protein expression...

  2. TRPM4 protein expression in prostate cancer

    DEFF Research Database (Denmark)

    Berg, Kasper Drimer; Soldini, Davide; Jung, Maria

    2016-01-01

    BACKGROUND: Transient receptor potential cation channel, subfamily M, member 4 (TRPM4) messenger RNA (mRNA) has been shown to be upregulated in prostate cancer (PCa) and might be a new promising tissue biomarker. We evaluated TRPM4 protein expression and correlated the expression level.......79-2.62; p = 0.01-0.03 for the two observers) when compared to patients with a lower staining intensity. CONCLUSIONS: TRPM4 protein expression is widely expressed in benign and cancerous prostate tissue, with highest staining intensities found in PCa. Overexpression of TRPM4 in PCa (combination of high...

  3. New cell line development for antibody-producing Chinese hamster ovary cells using split green fluorescent protein

    Directory of Open Access Journals (Sweden)

    Kim Yeon-Gu

    2012-05-01

    Full Text Available Abstract Background The establishment of high producer is an important issue in Chinese hamster ovary (CHO cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene. Fluorescence-activated cell sorting (FACS-based cell line development is an efficient strategy for the selection of CHO cells in high therapeutic protein production. Results An internal ribosome entry site (IRES was introduced for using two green fluorescence protein (GFP fragments as a reporter to both antibody chains, the heavy chain and the light chain. The cells co-transfected with two GFP fragments showed the emission of green fluorescence by the reconstitution of split GFP. The FACS-sorted pool with GFP expression had a higher specific antibody productivity (qAb than that of the unsorted pool. The qAb was highly correlated with the fluorescence intensity with a high correlation coefficient, evidenced from the analysis of median GFP and qAb in individual selected clones. Conclusions This study proved that the fragment complementation for split GFP could be an efficient indication for antibody production on the basis of high correlation of qAb with reconstitution of GFP. Taken together, we developed an efficient FACS-based screening method for high antibody-producing CHO cells with the benefits of the split GFP system.

  4. [Sentinel node detection using optonuclear probe (gamma and fluorescence) after green indocyanine and radio-isotope injections].

    Science.gov (United States)

    Poumellec, M-A; Dejode, M; Figl, A; Darcourt, J; Haudebourg, J; Sabah, Y; Voury, A; Martaens, A; Barranger, E

    2016-04-01

    Assess the biopsy's feasibility of the sentinel lymph node biopsy (SLNB) using optonuclear probe after of indocyanine green (ICG) and radio-isotope (RI) injections. Twenty-one patients with a localized breast cancer and unsuspicious axillary nodes underwent a SLNB after both injections of ICG and radio-isotope. One or more SLN were identified on the 21 patients (identification rate of 100%). The median number SLN was 2 (1-3). Twenty SLN were both radio-actives and fluorescents (54.1%), 11 fluorescent only (29.7%) and 6 were only radio-actives (16.2%). Seven patients had a metastatic SLN (8 SLN overall). Among them, only one had a micrometastasic SLN, 5 others had a macrometastatic SLN and one patient had two macrometastatic SLNs. Among the 8 metastatic SLN, 5 were both fluorescent and radioactive, 2 were only fluorescent and 1 was only radioactive. Detection SLN using optonuclear probe after indocyanine green and radio-isotope injections is effective and could be, after validation by randomized trial, a reliable alternative to the blue dye injection for teams who consider that combined detection as the reference. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  5. A dark green fluorescent protein as an acceptor for measurement of Förster resonance energy transfer.

    Science.gov (United States)

    Murakoshi, Hideji; Shibata, Akihiro C E; Nakahata, Yoshihisa; Nabekura, Junichi

    2015-10-15

    Measurement of Förster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) is a powerful method for visualization of intracellular signaling activities such as protein-protein interactions and conformational changes of proteins. Here, we developed a dark green fluorescent protein (ShadowG) that can serve as an acceptor for FLIM-FRET. ShadowG is spectrally similar to monomeric enhanced green fluorescent protein (mEGFP) and has a 120-fold smaller quantum yield. When FRET from mEGFP to ShadowG was measured using an mEGFP-ShadowG tandem construct with 2-photon FLIM-FRET, we observed a strong FRET signal with low cell-to-cell variability. Furthermore, ShadowG was applied to a single-molecule FRET sensor to monitor a conformational change of CaMKII and of the light oxygen voltage (LOV) domain in HeLa cells. These sensors showed reduced cell-to-cell variability of both the basal fluorescence lifetime and response signal. In contrast to mCherry- or dark-YFP-based sensors, our sensor allowed for precise measurement of individual cell responses. When ShadowG was applied to a separate-type Ras FRET sensor, it showed a greater response signal than did the mCherry-based sensor. Furthermore, Ras activation and translocation of its effector ERK2 into the nucleus could be observed simultaneously. Thus, ShadowG is a promising FLIM-FRET acceptor.

  6. Green synthesis of fluorescence carbon nanoparticles from yum and application in sensitive and selective detection of ATP.

    Science.gov (United States)

    Zhan, Zixuan; Cai, Jiao; Wang, Qi; Su, Yingying; Zhang, Lichun; Lv, Yi

    2016-05-01

    Fluorescent carbon nanoparticles (CPs), a fascinating class of recently discovered nanocarbons, have been widely known as some of the most promising sensing probes in biological or chemical analysis. In this study, we demonstrate a green synthetic methodology for generating water-soluble CPs with a quantum yield of approximately 24% via a simple heating process using yum mucilage as a carbon source. The prepared carbon nanoparticles with an ~10 nm size possessed excellent fluorescence properties, and the fluorescence of the CPs was strongly quenched by Fe(3+), and recovered by adenosine triphosphate (ATP), thus, an 'off' and 'on' system can be easily established. This 'CPs-Fe(3+)-ATP' strategy was sensitive and selective at detecting ATP with the linear range of 0.5 µmol L(-1) to 50 µmol L(-1) and with a detection limit of 0.48 µmol L(-1). Copyright © 2015 John Wiley & Sons, Ltd.

  7. Selective Permeation and Organic Extraction of Recombinant Green Fluorescent Protein (gfpuv from Escherichia coli

    Directory of Open Access Journals (Sweden)

    Ishii Marina

    2002-04-01

    Full Text Available Abstract Background Transformed cells of Escherichia coli DH5-α with pGFPuv, induced by IPTG (isopropyl-β-d-thiogalactopyranoside, express the green fluorescent protein (gfpuv during growth phases. E. coli subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-phase partitioning extraction (TPP method were compared to the direct application of TPP to the same culture of E. coli on releasing gfpuv from the over-expressing cells. Material and Methods Cultures (37°C/100 rpm/ 24 h; μ = 0.99 h-1 - 1.10 h-1 of transformed (pGFP Escherichia coli DH5-α, expressing the green fluorescent protein (gfpuv, absorbance at 394 nm and emission at 509 nm were sonicated in successive intervals of sonication (25 vibrations/pulse to determine the maximum amount of gfpuv released from the cells. For selective permeation, the transformed previously frozen (-75°C cells were subjected to three freeze/thaw (-20°C/ 0.83°C/min cycles interlaid by sonication (3 pulses/ 6 seconds/ 25 vibrations. The intracellular permeate with gfpuv in extraction buffer (TE solution (25 mM Tris-HCl, pH 8.0, 1 mM β-mercaptoethanol β-ME, 0.1 mM PMSF was subjected to the three-phase partitioning (TPP method with t-butanol and 1.6 M ammonium sulfate. Sonication efficiency was verified on the application to the cells previously treated by the TPP method. The intra-cell releases were mixed and eluted through methyl HIC column with a buffer solution (10 mM Tris-HCl, 10 mM EDTA, pH 8.0. Results The sonication maximum released amount obtained from the cells was 327.67 μg gfpuv/mL (20.73 μg gfpuv/mg total proteins – BSA, after 9 min of treatment. Through the selective permeation by three repeated freezing/thawing/sonication cycles applied to the cells, a close content of 241.19 μg gfpuv/mL (29.74 μg gfpuv/mg BSA was obtained. The specific mass range of gfpuv released from the same cultures, by the three-phase partitioning (TPP

  8. Looking at the Green Fluorescent Protein (GFP) chromophore from a different perspective: A computational insight

    Science.gov (United States)

    Paul, Bijan Kumar; Guchhait, Nikhil

    2013-02-01

    In the present contribution Density Functional Theory (DFT) has been applied to explore molecular dipole moment, frontier molecular orbital (FMO) features, chemical hardness, and the molecular electrostatic potential surface (MEPS) characteristics for optimized molecular geometry of the Green Fluorescent Protein (GFP) chromophore p-hydroxybenzylideneimidazolinone (HBDI) both in its protonated (neutral) and deprotonated (anion) forms. The distribution of atomic charges over the entire molecular framework as obtained from Natural Bond Orbital (NBO) analysis is found to faithfully replicate the predictions from the MEP map in respect of reactivity map of HBDI (neutral and anion) and possible sites for hydrogen bonding interactions etc. The three dimensional MEP map encompassing the entire molecule yields a reliable reactivity map of HBDI molecule also displaying the most probable regions for non-covalent interactions. The differential distribution of the electrostatic potential over the neutral and anionic species of HBDI is authentically reflected on MEP map and NBO charge distribution analysis. Thermodynamic properties such as heat capacity, thermal energy, enthalpy, entropy have been calculated and the correlation of the various thermodynamic functions with temperature has been established for neutral molecule. More importantly, however, the computational approach has been employed to unveil the nonlinear optical (NLO) properties of protonated (neutral) and deprotonated (anion) HBDI. Also in an endeavor to achieve a fuller understanding on this aspect the effect of basis set on the NLO properties of the title molecule has been investigated. Our computations delineate the discernible differences in NLO properties between the neutral and anionic species of HBDI whereby indicating the possibility of development of photoswitchable NLO device.

  9. Accurate thermometry based on the red and green fluorescence intensity ratio in NaYF4: Yb, Er nanocrystals for bioapplication.

    Science.gov (United States)

    Liu, Lixin; Qin, Feng; Lv, Tianquan; Zhang, Zhiguo; Cao, Wenwu

    2016-10-15

    A biological temperature measurement method based on the fluorescence intensity ratio (FIR) was developed to reduce uncertainty. The upconversion luminescence of NaYF4:Yb, Er nanocrystals was studied as a function of temperature around the physiologically relevant range of 300-330 K. We found that the green-green FIR Fe and red-green FIR (I660/I540) varied linearly as temperature increased. The thermometric uncertainties using the two FIRs were discussed and were determined to be almost constant at 0.6 and 0.09 K for green-green and red-green, respectively. The lower thermometric uncertainty comes from the intense signal-to-noise ratio of the measured FIRs owing to their comparable fluorescence intensities.

  10. Tunable recombinant protein expression with E. coli in a mixed-feed environment.

    Science.gov (United States)

    Sagmeister, Patrick; Schimek, Clemens; Meitz, Andrea; Herwig, Christoph; Spadiut, Oliver

    2014-04-01

    Controlling the recombinant protein production rate in Escherichia coli is of utmost importance to ensure product quality and quantity. Up to now, only the genetic construct, introduced into E. coli, and the specific growth rate of the culture were used to influence and stir the productivity. However, bioprocess technological means to control or even tune the productivity of E. coli are scarce. Here, we present a novel method for the process-technological control over the recombinant protein expression rate in E. coli. A mixed-feed fed-batch bioprocess based on the araBAD promoter expression system using both D-glucose and L-arabinose as assimilable C-sources was designed. Using the model product green fluorescent protein, we show that the specific product formation rate can be efficiently tuned even on the cellular level only via the uptake rate of L-arabinose. This novel approach introduces an additional degree of freedom for the design of recombinant bioprocesses with E. coli. We anticipate that the presented method will result in significant quality and robustness improvement as well as cost and process time reduction for recombinant bacterial bioprocesses in the future.

  11. A novel duct-lobular segmentectomy for breast tumors with nipple discharge using near-infrared indocyanine green fluorescence imaging

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Ohno

    2013-10-01

    Full Text Available A 44-year-old woman was referred to our hospital with pathological nipple discharge from her left breast. Ultrasonography revealed a solid tumor beneath her left areola that measured 17 mm in diameter with a dilated mammary duct. Contrast-enhanced magnetic resonance imaging showed an early-enhanced cystic tumor and a dilated mammary duct. We performed a duct-lobular segmentectomy using near-infrared indocyanine green (ICG-fluorescence imaging. Under general anesthesia, a silicone tube was inserted into an orifice of a fluid-discharging mammary duct, and 1 mL dye-fluorescence liquid containing ICG and indigo carmine was injected into the mammary duct. A periareolar incision was made, and the fluorescence image of the demarcated mammary duct segment was obtained. The mammary duct segment was dissected, along with the demarcation line. The cystic lesion and dilated mammary duct were fully resected, and the pathological diagnosis was intraductal papilloma of the breast. We report that near-infrared ICG fluorescence could be applied for imaging of the mammary duct segment, and the fluorescence image allowed for easier duct-lobular segmentectomy for nipple discharge.

  12. Development of CRTEIL and CETRIZ, Cre-loxP-Based Systems, Which Allow Change of Expression of Red to Green or Green to Red Fluorescence upon Transfection with a Cre-Expression Vector

    Directory of Open Access Journals (Sweden)

    Masato Ohtsuka

    2009-01-01

    Full Text Available We developed Cre-loxP-based systems, termed CRTEIL and CETRIZ, which allow gene switching in a noninvasive manner. Single transfection with pCRTEIL resulted in predominant expression of red fluorescence. Cotransfection with pCRTEIL and Cre-expression plasmid (pCAG/NCre caused switching from red to green fluorescence. Similarly, cotransfection with pCETRIZ and pCAG/NCre resulted in change of green to red fluorescence. These noninvasive systems will be useful in cell lineage analysis, since descendants of cells exhibiting newly activated gene expression can be continuously monitored in noninvasive fashion.

  13. Crystal structure of the fluorescent protein from Dendronephthya sp. in both green and photoconverted red forms

    Energy Technology Data Exchange (ETDEWEB)

    Pletneva, Nadya V.; Pletnev, Sergei; Pakhomov, Alexey A.; Chertkova, Rita V.; Martynov, Vladimir I.; Muslinkina, Liya; Dauter, Zbigniew; Pletnev, Vladimir Z.

    2016-07-13

    The fluorescent protein fromDendronephthyasp. (DendFP) is a member of the Kaede-like group of photoconvertible fluorescent proteins with a His62-Tyr63-Gly64 chromophore-forming sequence. Upon irradiation with UV and blue light, the fluorescence of DendFP irreversibly changes from green (506 nm) to red (578 nm). The photoconversion is accompanied by cleavage of the peptide backbone at the Cα—N bond of His62 and the formation of a terminal carboxamide group at the preceding Leu61. The resulting double Cα=Cβbond in His62 extends the conjugation of the chromophore π system to include imidazole, providing the red fluorescence. Here, the three-dimensional structures of native green and photoconverted red forms of DendFP determined at 1.81 and 2.14 Å resolution, respectively, are reported. This is the first structure of photoconverted red DendFP to be reported to date. The structure-based mutagenesis of DendFP revealed an important role of positions 142 and 193: replacement of the original Ser142 and His193 caused a moderate red shift in the fluorescence and a considerable increase in the photoconversion rate. It was demonstrated that hydrogen bonding of the chromophore to the Gln116 and Ser105 cluster is crucial for variation of the photoconversion rate. The single replacement Gln116Asn disrupts the hydrogen bonding of Gln116 to the chromophore, resulting in a 30-fold decrease in the photoconversion rate, which was partially restored by a further Ser105Asn replacement.

  14. Construction of a plasmid coding for green fluorescent protein tagged cathepsin L and data on expression in colorectal carcinoma cells

    Directory of Open Access Journals (Sweden)

    Tripti Tamhane

    2015-12-01

    Full Text Available The endo-lysosomal cysteine cathepsin L has recently been shown to have moonlighting activities in that its unexpected nuclear localization in colorectal carcinoma cells is involved in cell cycle progression (Tamhane et al., 2015 [1]. Here, we show data on the construction and sequence of a plasmid coding for human cathepsin L tagged with an enhanced green fluorescent protein (phCL-EGFP in which the fluorescent protein is covalently attached to the C-terminus of the protease. The plasmid was used for transfection of HCT116 colorectal carcinoma cells, while data from non-transfected and pEGFP-N1-transfected cells is also shown. Immunoblotting data of lysates from non-transfected controls and HCT116 cells transfected with pEGFP-N1 and phCL-EGFP, showed stable expression of cathepsin L-enhanced green fluorescent protein chimeras, while endogenous cathepsin L protein amounts exceed those of hCL-EGFP chimeras. An effect of phCL-EGFP expression on proliferation and metabolic states of HCT116 cells at 24 h post-transfection was observed.

  15. A Rapid Label-Free Fluorescent Aptasensor PicoGreen-Based Strategy for Aflatoxin B₁ Detection in Traditional Chinese Medicines.

    Science.gov (United States)

    Zhang, Cheng; Dou, Xiaowen; Zhang, Lei; Sun, Meifeng; Zhao, Ming; OuYang, Zhen; Kong, Dandan; Antonio, F Logrieco; Yang, Meihua

    2018-02-28

    Aflatoxin B₁ (AFB₁) is a very hazardous carcinogen, readily contaminating foodstuffs and traditional Chinese medicines (TCMs) that has inspired increasing health concerns due to dietary exposure. Colloidal nanocrystals have been proposed as optical labels for aptasensor assembly, but these typically require tedious multistep conjugation and suffer from unsatisfactory robustness when used for complex matrices. In the present study, we report a rapid and sensitive method for screening for trace AFB₁ levels in TCMs using a label-free fluorescent aptasensor PicoGreen dye-based strategy. Using PicoGreen to selectively measure complementary double-stranded DNA, fluorescence enhancement due to dsDNA is 'turned off' in the presence of AFB₁ due binding of aptamer target over complementary sequence. Self-assembly of a label-free fluorescent aptasensor based on AFB₁ aptamer and PicoGreen dye was performed. Due to competition between the complementary sequence and AFB₁ target, this rapid method was capable of highly sensitive and selective screening for AFB₁ in five types of TCMs. This proposed approach had a limit of detection as low as 0.1 μg·L -1 and good linearity with a range of 0.1-10 μg·L -1 (0.1-10 ppb). Among the 20 samples tested, 6 batches were found to be contaminated with AFB₁ using this method, which was confirmed using sophisticated liquid chromatography-electrospray ionization-tandem mass spectrometry/mass spectrometry analysis. The results of this study indicate the developed method has the potential to be a simple, quick, and sensitive tool for detecting AFB₁ in TCMs.

  16. Peptide aptamer-assisted immobilization of green fluorescent protein for creating biomolecule-complexed carbon nanotube device

    Science.gov (United States)

    Nii, Daisuke; Nozawa, Yosuke; Miyachi, Mariko; Yamanoi, Yoshinori; Nishihara, Hiroshi; Tomo, Tatsuya; Shimada, Yuichiro

    2017-10-01

    Carbon nanotubes are a novel material for next-generation applications. In this study, we generated carbon nanotube and green fluorescent protein (GFP) conjugates using affinity binding peptides. The carbon nanotube-binding motif was introduced into the N-terminus of the GFP through molecular biology methods. Multiple GFPs were successfully aligned on a single-walled carbon nanotube via the molecular recognition function of the peptide aptamer, which was confirmed through transmission electron microscopy and optical analysis. Fluorescence spectral analysis results also suggested that the carbon nanotube-GFP complex was autonomously formed with orientation and without causing protein denaturation during immobilization. This simple process has a widespread potential for fabricating carbon nanotube-biomolecule hybrid devices.

  17. Design of ortho-Substituted Donor-Acceptor Molecules as Highly Efficient Green Thermally Activated Delayed Fluorescent Emitters

    Science.gov (United States)

    Cha, Jae-Ryung; Gong, Myoung-Seon; Lee, Tak Jae; Ha, Tae Hoon; Lee, Chil Won

    2018-04-01

    The ortho-substituted donor-acceptor molecules 2-(4,6-diphenyl-1, 3, 5-triazin-2-yl)- N,Ndiphenylaniline (DPA- o-Trz) and 2-(4,6-diphenyl-1, 3, 5-triazine-2-yl)- N,N-di- p-tolylaniline (MPA- o-Trz) were designed, synthesized, and found to exhibit green fluorescence characteristics. Notably, the singlet-triplet energy gap was less than 0.1 eV, indicating that reverse intersystem crossing gave rise to thermally activated delayed fluorescence (TADF). The organic light-emitting device performance of MPA- o-Trz showed a high external quantum efficiency of 16.3% and good color stability from 0.1 cd/m2 to 5000 cd/m2.

  18. Study on transferring improved green fluorescent protein gene into wheat via low energy Ar+ implantation

    International Nuclear Information System (INIS)

    Wu Lifang; Li Hong; Song Daojun

    2000-01-01

    An improved GFP gene (mGFP4) was introduced into mature embryo cells of wheat cultivars Wan 9210 and Wanmai 32 via low energy ion beam-mediated delivery technique. Resistant calli were selected on medium containing paromomycin (100-140 mg/L). Five green plants were regenerated from resistant calli of Wan 9210 derived from 387 implated mature embryos. 32 green plants were obtained from 776 irradiated mature embryos in Wanmai 32. No green plant was regenerated from calli of 200 non-transformed embryos. PCR assays of 37 green plants showed that they all obtained the expected size of amplified DNA fragment (600 bp). Southern blot of 4 well-developed green plants confirmed stable integration of GFP gene into wheat genome. The average transformation frequencies of Wan 9210 and Wanmai 32 were 1.3% and 4.1%, respectively, according to the results of PCR assays

  19. Expression and characterization of insulin growth factor-I-enhanced green fluorescent protein fused protein as a tracer for immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Shi Ruina [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Huang Yong [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Wang Dan [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Zhao Meiping [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Li Yuanzong [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China)]. E-mail: yzli@pku.edu.cn

    2006-09-25

    The insulin-like growth factor-I (IGF-I) is an important polypeptide hormone under investigation for body metabolism study and for doping detection. Here, we describe for the first time the expression of a recombinant fusion protein of IGF-I and the enhanced green fluorescent protein (EGFP). The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and homogeneous structure. The fused protein (EGFP-IGF-I) was expressed as a soluble protein in cytoplasm of Escherichia coli and its fluorescence and immunoreaction properties were thoroughly characterized. Finally, we demonstrated the utility of the EGFP-IGF-I fusion protein for the fluorescence immunoassay of IGF-1. The linear range of the assay is 1.6 x 10{sup -8} to 2.0 x 10{sup -6} M with a detection limit of 1.6 x 10{sup -8} M. To our knowledge, this is the first time that EGFP has been used as a quantitative label in a fusion protein to develop a quantitative assay for IGF-I. Furthermore, the use of genetically engineered fusion proteins, which combine peptide hormones with fluorescent protein, can lead to a new labeling approach to a number of bioanalytical applications.

  20. Expression and characterization of insulin growth factor-I-enhanced green fluorescent protein fused protein as a tracer for immunoassay

    International Nuclear Information System (INIS)

    Shi Ruina; Huang Yong; Wang Dan; Zhao Meiping; Li Yuanzong

    2006-01-01

    The insulin-like growth factor-I (IGF-I) is an important polypeptide hormone under investigation for body metabolism study and for doping detection. Here, we describe for the first time the expression of a recombinant fusion protein of IGF-I and the enhanced green fluorescent protein (EGFP). The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and homogeneous structure. The fused protein (EGFP-IGF-I) was expressed as a soluble protein in cytoplasm of Escherichia coli and its fluorescence and immunoreaction properties were thoroughly characterized. Finally, we demonstrated the utility of the EGFP-IGF-I fusion protein for the fluorescence immunoassay of IGF-1. The linear range of the assay is 1.6 x 10 -8 to 2.0 x 10 -6 M with a detection limit of 1.6 x 10 -8 M. To our knowledge, this is the first time that EGFP has been used as a quantitative label in a fusion protein to develop a quantitative assay for IGF-I. Furthermore, the use of genetically engineered fusion proteins, which combine peptide hormones with fluorescent protein, can lead to a new labeling approach to a number of bioanalytical applications

  1. Multiwavelength time-resolved detection of fluorescence during the inflow of indocyanine green into the adult's brain

    Science.gov (United States)

    Gerega, Anna; Milej, Daniel; Weigl, Wojciech; Botwicz, Marcin; Zolek, Norbert; Kacprzak, Michal; Wierzejski, Wojciech; Toczylowska, Beata; Mayzner-Zawadzka, Ewa; Maniewski, Roman; Liebert, Adam

    2012-08-01

    Optical technique based on diffuse reflectance measurement combined with indocyanine green (ICG) bolus tracking is extensively tested as a method for clinical assessment of brain perfusion in adults at the bedside. Methodology of multiwavelength and time-resolved detection of fluorescence light excited in the ICG is presented and advantages of measurements at multiple wavelengths are discussed. Measurements were carried out: 1. on a physical homogeneous phantom to study the concentration dependence of the fluorescence signal, 2. on the phantom to simulate the dynamic inflow of ICG at different depths, and 3. in vivo on surface of the human head. Pattern of inflow and washout of ICG in the head of healthy volunteers after intravenous injection of the dye was observed for the first time with time-resolved instrumentation at multiple emission wavelengths. The multiwavelength detection of fluorescence signal confirms that at longer emission wavelengths, probability of reabsorption of the fluorescence light by the dye itself is reduced. Considering different light penetration depths at different wavelengths, and the pronounced reabsorption at longer wavelengths, the time-resolved multiwavelength technique may be useful in signal decomposition, leading to evaluation of extra- and intracerebral components of the measured signals.

  2. Variable selection based on clustering analysis for improvement of polyphenols prediction in green tea using synchronous fluorescence spectra

    Science.gov (United States)

    Shan, Jiajia; Wang, Xue; Zhou, Hao; Han, Shuqing; Riza, Dimas Firmanda Al; Kondo, Naoshi

    2018-04-01

    Synchronous fluorescence spectra, combined with multivariate analysis were used to predict flavonoids content in green tea rapidly and nondestructively. This paper presented a new and efficient spectral intervals selection method called clustering based partial least square (CL-PLS), which selected informative wavelengths by combining clustering concept and partial least square (PLS) methods to improve models’ performance by synchronous fluorescence spectra. The fluorescence spectra of tea samples were obtained and k-means and kohonen-self organizing map clustering algorithms were carried out to cluster full spectra into several clusters, and sub-PLS regression model was developed on each cluster. Finally, CL-PLS models consisting of gradually selected clusters were built. Correlation coefficient (R) was used to evaluate the effect on prediction performance of PLS models. In addition, variable influence on projection partial least square (VIP-PLS), selectivity ratio partial least square (SR-PLS), interval partial least square (iPLS) models and full spectra PLS model were investigated and the results were compared. The results showed that CL-PLS presented the best result for flavonoids prediction using synchronous fluorescence spectra.

  3. Different visible colors and green fluorescence were obtained from the mutated purple chromoprotein isolated from sea anemone.

    Science.gov (United States)

    Chiang, Cheng-Yi; Chen, Yi-Lin; Tsai, Huai-Jen

    2014-08-01

    Green fluorescent protein (GFP)-like proteins have been studied with the aim of developing fluorescent proteins. Since the property of color variation is understudied, we isolated a novel GFP-like chromoprotein from the carpet anemone Stichodactyla haddoni, termed shCP. Its maximum absorption wavelength peak (λ(max)) is located at 574 nm, resulting in a purple color. The shCP protein consists of 227 amino acids (aa), sharing 96 % identity with the GFP-like chromoprotein of Heteractis crispa. We mutated aa residues to examine any alteration in color. When E63, the first aa of the chromophore, was replaced by serine (E63S), the λ(max) of the mutated protein shCP-E63S was shifted to 560 nm and exhibited a pink color. When Q39, T194, and I196, which reside in the surrounding 5 Å of the chromophore's microenvironment, were mutated, we found that (1) the λ(max) of the mutated protein shCP-Q39S was shifted to 518 nm and exhibited a red color, (2) shCP-T194I exhibited a purple-blue color, and (3) an additional mutation at I196H of the mutated protein shCP-E63L exhibited green fluorescence. In contrast, when the aa located neither at the chromophore nor within its microenvironment were mutated, the resultant proteins shCP-L122H, -E138G, -S137D, -T95I, -D129N, -T194V, -E138Q, -G75E, -I183V, and -I70V never altered their purple color, suggesting that mutations at the shCP chromophore and the surrounding 5 Å microenvironment mostly control changes in color expression or cause fluorescence to develop. Additionally, we found that the cDNAs of shCP and its mutated varieties are faithfully and stably expressed both in Escherichia coli and zebrafish embryos.

  4. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting.

    Science.gov (United States)

    Bajar, Bryce T; Wang, Emily S; Lam, Amy J; Kim, Bongjae B; Jacobs, Conor L; Howe, Elizabeth S; Davidson, Michael W; Lin, Michael Z; Chu, Jun

    2016-02-16

    Many genetically encoded biosensors use Förster resonance energy transfer (FRET) to dynamically report biomolecular activities. While pairs of cyan and yellow fluorescent proteins (FPs) are most commonly used as FRET partner fluorophores, respectively, green and red FPs offer distinct advantages for FRET, such as greater spectral separation, less phototoxicity, and lower autofluorescence. We previously developed the green-red FRET pair Clover and mRuby2, which improves responsiveness in intramolecular FRET reporters with different designs. Here we report the engineering of brighter and more photostable variants, mClover3 and mRuby3. mClover3 improves photostability by 60% and mRuby3 by 200% over the previous generation of fluorophores. Notably, mRuby3 is also 35% brighter than mRuby2, making it both the brightest and most photostable monomeric red FP yet characterized. Furthermore, we developed a standardized methodology for assessing FP performance in mammalian cells as stand-alone markers and as FRET partners. We found that mClover3 or mRuby3 expression in mammalian cells provides the highest fluorescence signals of all jellyfish GFP or coral RFP derivatives, respectively. Finally, using mClover3 and mRuby3, we engineered an improved version of the CaMKIIα reporter Camuiα with a larger response amplitude.

  5. [Artificial Cysteine Bridges on the Surface of Green Fluorescent Protein Affect Hydration of Its Transition and Intermediate States].

    Science.gov (United States)

    Melnik, T N; Nagibina, G S; Surin, A K; Glukhova, K A; Melnik, B S

    2018-01-01

    Studying the effect of cysteine bridges on different energy levels of multistage folding proteins will enable a better understanding of the process of folding and functioning of globular proteins. In particular, it will create prospects for directed change in the stability and rate of protein folding. In this work, using the method of differential scanning microcalorimetry, we have studied the effect of three cysteine bridges introduced in different structural elements of the green fluorescent protein on the denaturation enthalpies, activation energies, and heat-capacity increments when this protein passes from native to intermediate and transition states. The studies have allowed us to confirm that, with this protein denaturation, the process hardly damages the structure initially, but then changes occur in the protein structure in the region of 4-6 beta sheets. The cysteine bridge introduced in this region decreases the hydration of the second transition state and increases the hydration of the second intermediate state during the thermal denaturation of the green fluorescent protein.

  6. Permethylated-β-Cyclodextrin Capped CdTe Quantum Dot and its Sensitive Fluorescence Analysis of Malachite Green.

    Science.gov (United States)

    Cao, Yujuan; Wei, Jiongling; Wu, Wei; Wang, Song; Hu, Xiaogang; Yu, Ying

    2015-09-01

    In the present work, the CdTe quantum dots were covalently conjugated with permethylated-β-cyclodextrin (OMe-β-CD) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride as cross-linking reagent. The obtained functional quantum dots (OMe-β-CD/QDs) showed highly luminescent, water solubility and photostability as well as good inclusion ability to malachite green. A sensitive fluorescence method was developed for the analysis of malachite green in different samples. The good linearity was 2.0 × 10(-7)-1.0 × 10(-5) mol/L and the limit of detect was 1.7 × 10(-8) mol/L. The recoveries for three environmental water samples were 92.0-108.2 % with relative standard deviation (RSD) of 0.24-1.87 %, while the recovery for the fish sample was 94.3 % with RSD of 1.04 %. The results showed that the present method was sensitive and convenient to determine malachite green in complex samples. Graphical Abstract The analytical mechanism of OMe-β-CD/QDs and its linear response to MG.

  7. Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH fusion to gfp (green fluorescent protein

    Directory of Open Access Journals (Sweden)

    Bat-Erdene Jugder

    2016-07-01

    Full Text Available Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2. Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni–Fe] uptake hydrogenase (SH produced by Cupriavidus necator H16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP reporter system to characterise PSH promoter activity using several gene cloning approaches. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSH promoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinant C. necator H16 cells. Here we report the first successful fluorescent reporter system to study PSH promoter activity in C. necator H16. The fusion construct allowed for the design of a simple screening assay to evaluate PSH activity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression.

  8. Deployment of a Fully-Automated Green Fluorescent Protein Imaging System in a High Arctic Autonomous Greenhouse

    Directory of Open Access Journals (Sweden)

    Alain Berinstain

    2013-03-01

    Full Text Available Higher plants are an integral part of strategies for sustained human presence in space. Space-based greenhouses have the potential to provide closed-loop recycling of oxygen, water and food. Plant monitoring systems with the capacity to remotely observe the condition of crops in real-time within these systems would permit operators to take immediate action to ensure optimum system yield and reliability. One such plant health monitoring technique involves the use of reporter genes driving fluorescent proteins as biological sensors of plant stress. In 2006 an initial prototype green fluorescent protein imager system was deployed at the Arthur Clarke Mars Greenhouse located in the Canadian High Arctic. This prototype demonstrated the advantageous of this biosensor technology and underscored the challenges in collecting and managing telemetric data from exigent environments. We present here the design and deployment of a second prototype imaging system deployed within and connected to the infrastructure of the Arthur Clarke Mars Greenhouse. This is the first imager to run autonomously for one year in the un-crewed greenhouse with command and control conducted through the greenhouse satellite control system. Images were saved locally in high resolution and sent telemetrically in low resolution. Imager hardware is described, including the custom designed LED growth light and fluorescent excitation light boards, filters, data acquisition and control system, and basic sensing and environmental control. Several critical lessons learned related to the hardware of small plant growth payloads are also elaborated.

  9. Deployment of a Fully-Automated Green Fluorescent Protein Imaging System in a High Arctic Autonomous Greenhouse

    Science.gov (United States)

    Abboud, Talal; Bamsey, Matthew; Paul, Anna-Lisa; Graham, Thomas; Braham, Stephen; Noumeir, Rita; Berinstain, Alain; Ferl, Robert

    2013-01-01

    Higher plants are an integral part of strategies for sustained human presence in space. Space-based greenhouses have the potential to provide closed-loop recycling of oxygen, water and food. Plant monitoring systems with the capacity to remotely observe the condition of crops in real-time within these systems would permit operators to take immediate action to ensure optimum system yield and reliability. One such plant health monitoring technique involves the use of reporter genes driving fluorescent proteins as biological sensors of plant stress. In 2006 an initial prototype green fluorescent protein imager system was deployed at the Arthur Clarke Mars Greenhouse located in the Canadian High Arctic. This prototype demonstrated the advantageous of this biosensor technology and underscored the challenges in collecting and managing telemetric data from exigent environments. We present here the design and deployment of a second prototype imaging system deployed within and connected to the infrastructure of the Arthur Clarke Mars Greenhouse. This is the first imager to run autonomously for one year in the un-crewed greenhouse with command and control conducted through the greenhouse satellite control system. Images were saved locally in high resolution and sent telemetrically in low resolution. Imager hardware is described, including the custom designed LED growth light and fluorescent excitation light boards, filters, data acquisition and control system, and basic sensing and environmental control. Several critical lessons learned related to the hardware of small plant growth payloads are also elaborated. PMID:23486220

  10. Efficient fluorescent red, green, and blue organic light-emitting devices with a blue host of spirobifluorene derivative

    Energy Technology Data Exchange (ETDEWEB)

    Lee, R.-H. [Department of Chemical and Material Engineering, National Yunlin University of Science and Technology, Yunlin 640, Taiwan (China)], E-mail: lerongho@yuntech.edu.tw; Huang, Y.-W.; Wang, Y.-Y. [Department of Chemical and Material Engineering, National Yunlin University of Science and Technology, Yunlin 640, Taiwan (China); Chang, H.-Y. [EChem Hightech CO., LTD, Hsin-Chu Industrial Park, Hu-Kou, Hsin-Chu, Taiwan (China)

    2008-06-02

    Efficient fluorescent blue, green, and red (RGB) organic light-emitting devices (OLEDs) were fabricated using a blue host material of pyrimidine-containing spirobifluorene derivative 2,7-bis[2-(4-tert-butylphenyl)pyrimidine-5-yl]-9,9'-spirobifluorene (TBPSF) doped with blue dye perylene, green dye 10-(2-benzothiazolyl)-1,1,7,7-tetramethyl-2,3,6,7-tetrahydro-1H,5H, 11H-benzo[l] pyrano[6,7,8-ij] quinolizin-11-one (C545T), and red dye 4-(dicyanomethylene)-2-t-butyl-6-(1,1,7,7-tetramethyljulolidyl-9-enyl) -4H-pyran (DCJTB), respectively. The brightness and current efficiency of the perylene doped blue device reached 10117 cd/m{sup 2} and 2.97 cd/A. Green emission of the C545T doped device reached 8500 cd/m{sup 2} and 13.0 cd/A. Red emission of the DCJTB doped device can be as high as 9000 cd/m{sup 2} and 2.0 cd/A, respectively. High color purity of the blue (Commission Internationale de L'Eclairage (CIE{sub x,y}) coordinates (CIE, x = 0.27, y = 0.24)), green (CIE, x = 0.19, y = 0.63) and red (CIE, x = 0.62, y = 0.37) emissions were achieved for RGB dyes doped TBPSF OLEDs. High brightness, large current efficiency, and good color purity of TBPSF-based RGB OLEDs were obtained by the configuration optimization device, such as inserting the hole and electron-injection materials, and suitable dopant content and light emitting layer thickness.

  11. [A hydroponic cultivation system for rapid high-yield transient protein expression in Nicotiana plants under laboratory conditions].

    Science.gov (United States)

    Mo, Qianzhen; Mai, Rongjia; Yang, Zhixiao; Chen, Minfang; Yang, Tiezhao; Lai, Huafang; Yang, Peiliang; Chen, Qiang; Zhou, Xiaohong

    2012-06-01

    To develop a hydroponic Nicotiana cultivation system for rapid and high-yield transient expression of recombinant proteins under laboratory conditions. To establish the hydroponic cultivation system, several parameters were examined to define the optimal conditions for the expression of recombinant proteins in plants. We used the green fluorescent protein (GFP) and the geminiviral plant transient expression vector as the model protein/expression vector. We examined the impact of Nicotiana species, the density and time of Agrobacterium infiltration, and the post-infiltration growth period on the accumulation of GFP. The expression levels of GFP in Nicotiana leaves were then examined by Western blotting and ELISA. Our data indicated that a hydroponic Nicotiana cultivation system with a light intensity of 9000 LX/layer, a light cycle of 16 h day/8 h night, a temperature regime of 28 degrees celsius; day/21 degrees celsius; night, and a relative humidity of 80% could support the optimal plant growth and protein expression. After agroinfiltration with pBYGFPDsRed.R/LBA4404, high levels of GFP expression were observed in both N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants cultured with this hydroponic cultivation system. An optimal GFP expression was achieved in both Nicotiana species leaves 4 days after infiltration by Agrobacterium with an OD(600) of 0.8. At a given time point, the average biomass of N. tobaccum (cv. Yuyan No.5) was significantly higher than that of N. benthamiana. The leaves from 6-week-old N. benthamiana plants and 5-week-old N. tobaccum (cv. Yuyan No.5) plants could be the optimal material for agroinfiltration. We have established a hydroponic cultivation system that allows robust growth of N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants and the optimal GFP expression in the artificial climate box.

  12. Determination of Mercury in Milk by Cold Vapor Atomic Fluorescence: A Green Analytical Chemistry Laboratory Experiment

    Science.gov (United States)

    Armenta, Sergio; de la Guardia, Miguel

    2011-01-01

    Green analytical chemistry principles were introduced to undergraduate students in a laboratory experiment focused on determining the mercury concentration in cow and goat milk. In addition to traditional goals, such as accuracy, precision, sensitivity, and limits of detection in method selection and development, attention was paid to the…

  13. Dual-channel (green and red) fluorescence microendoscope with subcellular resolution

    Science.gov (United States)

    de Paula D'Almeida, Camila; Fortunato, Thereza Cury; Teixeira Rosa, Ramon Gabriel; Romano, Renan Arnon; Moriyama, Lilian Tan; Pratavieira, Sebastião.

    2018-02-01

    Usually, tissue images at cellular level need biopsies to be done. Considering this, diagnostic devices, such as microendoscopes, have been developed with the purpose of do not be invasive. This study goal is the development of a dual-channel microendoscope, using two fluorescent labels: proflavine and protoporphyrin IX (PpIX), both approved by Food and Drug Administration. This system, with the potential to perform a microscopic diagnosis and to monitor a photodynamic therapy (PDT) session, uses a halogen lamp and an image fiber bundle to perform subcellular image. Proflavine fluorescence indicates the nuclei of the cell, which is the reference for PpIX localization on image tissue. Preliminary results indicate the efficacy of this optical technique to detect abnormal tissues and to improve the PDT dosimetry. This was the first time, up to our knowledge, that PpIX fluorescence was microscopically observed in vivo, in real time, combined to other fluorescent marker (Proflavine), which allowed to simultaneously observe the spatial localization of the PpIX in the mucosal tissue. We believe this system is very promising tool to monitor PDT in mucosa as it happens. Further experiments have to be performed in order to validate the system for PDT monitoring.

  14. Vibrational energy flow through the green fluorescent protein-water interface: communication maps and thermal boundary conductance.

    Science.gov (United States)

    Xu, Yao; Leitner, David M

    2014-07-17

    We calculate communication maps for green fluorescent protein (GFP) to elucidate energy transfer pathways between the chromophore and other parts of the protein in the ground and excited state. The approach locates energy transport channels from the chromophore to remote regions of the protein via residues and water molecules that hydrogen bond to the chromophore. We calculate the thermal boundary conductance between GFP and water over a wide range of temperature and find that the interface between the protein and the cluster of water molecules in the β-barrel poses negligible resistance to thermal flow, consistent with facile vibrational energy transfer from the chromophore to the β-barrel waters observed in the communication maps.

  15. Intraoperative indocyanine green fluorescent angiography-assisted modified superior gluteal artery perforator flap for reconstruction of sacral pressure sores.

    Science.gov (United States)

    Chang, Chun-Kai; Wu, Chien-Ju; Chen, Chun-Yu; Wang, Chi-Yu; Chu, Tzi-Shiang; Hsu, Kuo-Feng; Chiu, Han-Ting; Liu, Hung-Hui; Chou, Chang-Yi; Wang, Chih-Hsin; Lin, Chin-Ta; Dai, Niann-Tzyy; Tzeng, Yuan-Sheng

    2017-12-01

    Pressure sores are often observed in patients who are bedridden. They can be a severe problem not only for patients and their caregivers but also for plastic surgeons. Here, we describe a new method of superior gluteal artery perforator flap harvesting and anchoring with the assistance of intraoperative indocyanine green fluorescent angiography. In this report, we describe the procedure and outcomes for 19 patients with grades III and IV sacral pressure sores who underwent the operation between September 2015 and November 2016. All flaps survived, and two experienced wound-edge partial dehiscence. With the assistance of this imaging device, we were able to acquire a reliable superior gluteal artery perforator flap and perform modified operations with it that are safe, easy to learn and associated with fewer complications than are traditional. © 2017 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

  16. La proteína verde fluorescente ilumina la biociencia The Green Fluorescent Protein that glows in Bioscience

    Directory of Open Access Journals (Sweden)

    María Inés Pérez Millán

    2009-06-01

    Full Text Available La proteína verde fluorescente (o GFP, por sus siglas en inglés, Green Fluorescent Protein es una proteína producida por la medusa Aequorea victoria que emite bioluminiscencia en la zona verde del espectro visible. El gen que codifica esta proteína ha sido clonado y se utiliza habitualmente en biología molecular como marcador. Los descubrimientos relacionados a la GFP merecieron el Premio Nobel de Química 2008, en conjunto a los tres investigadores, Dres Shimomura, Chalfie y Tsien que participaron escalonadamente en dilucidar la estructura y función de la proteína. El Dr. Shimomura descubrió y estudió las propiedades de GFP, el Dr. Chalfie usando técnicas de biología molecular logró introducir el gen que codificaba para la GFP en el ADN del gusano transparente C. elegans, e inició la era de GFP como marcador de procesos en células y organismos. Finalmente el Dr. Tsien modificó la estructura de la proteína para producir moléculas que emiten luz a distintas longitudes de onda, extendiendo la paleta de colores de las proteínas. Las proteínas fluorescentes, entre las cuales se encuentra la GFP, son muy versátiles y se utilizan en diversos campos como la microbiología, ingeniería genética, fisiología, e ingeniería ambiental. Permiten ver procesos previamente invisibles, como el desarrollo de neuronas, cómo se diseminan las células cancerosas, o la contaminación de agua con arsénico, por mencionar algunos usos. Con la obtención de proteínas de muchos colores complejas redes biológicas pueden ser marcadas diferencialmente, lo que permite visualizar la biología celular en acción.Green fluorescent protein (GFP is a protein produced by the jellyfish Aequorea victoria, that emits bioluminescence in the green zone of the visible spectrum. The GFP gene has been cloned and is used in molecular biology as a marker. The three researchers that participated independently in elucidating the structure and function of this and its

  17. Green fluorescent protein (GFP) leakage from microbial biosensors provides useful information for the evaluation of the scale-down effect

    DEFF Research Database (Denmark)

    Delvigne, Frank; Brognaux, Alison; Francis, Frédéric

    2011-01-01

    Mixing deficiencies can be potentially detected by the use of a dedicated whole cell microbial biosensor. In this work, a csiE promoter induced under carbon-limited conditions was involved in the elaboration of such biosensor. The cisE biosensor exhibited interesting response after up and down......-shift of the dilution rate in chemostat mode. Glucose limitation was accompanied by green fluorescent protein (GFP) leakage to the extracellular medium. In order to test the responsiveness of microbial biosensors to substrate fluctuations in large-scale, a scale-down reactor (SDR) experiment was performed. The glucose...... fluctuations were characterized at the single cell level and tend to decrease the induction of GFP. Simulations run on the basis of a stochastic hydrodynamic model have shown the variability and the frequencies at which biosensors are exposed to glucose gradient in the SDR. GFP leakage was observed to a great...

  18. Assessing boreal forest photosynthetic dynamics through space-borne measurements of greenness, chlorophyll fluorescence and model GPP

    Science.gov (United States)

    Walther, Sophia; Guanter, Luis; Voigt, Maximilian; Köhler, Philipp; Jung, Martin; Joiner, Joanna

    2015-04-01

    sophia.walther@gfz-potsdam.de The seasonality of photosynthesis of boreal forests is an essential driver of the terrestrial carbon, water and energy cycles. However, current carbon cycle model results only poorly represent interannual variability and predict very different magnitudes and timings of carbon fluxes between the atmosphere and the land surface (e.g. Jung et al. 2011, Richardson et al. 2012). Reflectance-based satellite measurements, which give an indication of the amount of green biomass on the Earth's surface, have so far been used as input to global carbon cycle simulations, but they have limitations as they are not directly linked to instantaneous photosynthesis. As an alternative, space-borne retrievals of sun-induced chlorophyll fluorescence (SIF) boast the potential to provide a direct indication of the seasonality of boreal forest photosynthetic activity and thus to improve carbon model performances. SIF is a small electromagnetic signal that is re-emitted from the photosystems in the chloroplasts, which results in a direct relationship to photosynthetic efficiency. In this contribution we examine the seasonality of the boreal forests with three different vegetation parameters, namely greenness, SIF and model simulations of gross primary production (gross carbon flux into the plants by photosynthesis, GPP). We use the enhanced vegetation index (EVI) to represent green biomass. EVI is calculated from NBAR MODIS reflectance measurements (0.05deg, 16 days temporal resolution) for the time from January 2007-May 2013. SIF data originate from GOME-2 measurements on board the MetOp-A satellite in a spatial resolution of 0.5deg for the time from 2007-2011 (Joiner et al. (2013), Köhler et al. (2014)). As a third data source, data-driven GPP model results are used for the time from 2006-2012 with 0.5deg spatial resolution. The method to quantify phenology developed by Gonsamo et al. (2013) is applied to infer the main phenological phases (greenup/onset of

  19. Development and characterization of enhanced green fluorescent protein and luciferase expressing cell line for non-destructive evaluation of tissue engineering constructs.

    NARCIS (Netherlands)

    Blum, J.S.; Temenoff, J.S.; Park, H.; Jansen, J.A.; Mikos, A.G.; Barry, M.A.

    2004-01-01

    This study investigates the utility of genetically modified cells developed for the qualitative and quantitative non-destructive evaluation of cells on biomaterials. The Fisher rat fibroblastic cell line has been genetically modified to stably express the reporter genes enhanced green fluorescence

  20. A flow cytometry-optimized assay using an SOS-green fluorescent protein (SOS-GFP) whole-cell biosensor for the detection of genotoxins in complex environments

    DEFF Research Database (Denmark)

    Norman, Anders; Hansen, Lars H.; Sørensen, Søren Johannes

    2006-01-01

    /mL, and proved far more sensitive than a previously published assay using the same biosensor strain. By applying the SOS-green fluorescent protein (GFP) whole-cell biosensor directly to soil microcosms we were also able to evaluate both the applicability and sensitivity of a biosensor based on SOS...

  1. Green fluorescent protein-mtalin causes defects in actin organization and cell expansion in Arabidopsis and inhibits actin depolymerizing factor's actin depolymerizing activity in vitro

    NARCIS (Netherlands)

    Ketelaar, T.; Anthony, R.G.; Hussey, P.J.

    2004-01-01

    Expression of green fluorescent protein (GFP) linked to an actin binding domain is a commonly used method for live cell imaging of the actin cytoskeleton. One of these chimeric proteins is GFP-mTalin (GFP fused to the actin binding domain of mouse talin). Although it has been demonstrated that

  2. Introduction of Red-Green-Blue Fluorescent Dyes into a Metal-Organic Framework for Tunable White Light Emission.

    Science.gov (United States)

    Wen, Yuehong; Sheng, Tianlu; Zhu, Xiaoquan; Zhuo, Chao; Su, Shaodong; Li, Haoran; Hu, Shengmin; Zhu, Qi-Long; Wu, Xintao

    2017-10-01

    The unique features of the metal-organic frameworks (MOFs), including ultrahigh porosities and surface areas, tunable pores, endow the MOFs with special utilizations as host matrices. In this work, various neutral and ionic guest dye molecules, such as fluorescent brighteners, coumarin derivatives, 4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran (DCM), and 4-(p-dimethylaminostyryl)-1-methylpyridinium (DSM), are encapsulated in a neutral MOF, yielding novel blue-, green-, and red-phosphors, respectively. Furthermore, this study introduces the red-, green-, and blue-emitting dyes into a MOF together for the first time, producing white-light materials with nearly ideal Commission International ed'Eclairage (CIE) coordinates, high color-rendering index values (up to 92%) and quantum yields (up to 26%), and moderate correlated color temperature values. The white light is tunable by changing the content or type of the three dye guests, or the excitation wavelength. Significantly, the introduction of blue-emitting guests in the methodology makes the available MOF host more extensive, and the final white-light output more tunable and high-quality. Such strategy can be widely adopted to design and prepare white-light-emitting materials. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. A set of enhanced green fluorescent protein concatemers for quantitative determination of nuclear localization signal strength.

    Science.gov (United States)

    Böhm, Jennifer; Thavaraja, Ramya; Giehler, Susanne; Nalaskowski, Marcus M

    2017-09-15

    Regulated transport of proteins between nucleus and cytoplasm is an important process in the eukaryotic cell. In most cases, active nucleo-cytoplasmic protein transport is mediated by nuclear localization signal (NLS) and/or nuclear export signal (NES) motifs. In this study, we developed a set of vectors expressing enhanced GFP (EGFP) concatemers ranging from 2 to 12 subunits (2xEGFP to 12xEGFP) for analysis of NLS strength. As shown by in gel GFP fluorescence analysis and αGFP Western blotting, EGFP concatemers are expressed as fluorescent full-length proteins in eukaryotic cells. As expected, nuclear localization of concatemeric EGFPs decreases with increasing molecular weight. By oligonucleotide ligation this set of EGFP concatemers can be easily fused to NLS motifs. After determination of intracellular localization of EGFP concatemers alone and fused to different NLS motifs we calculated the size of a hypothetic EGFP concatemer showing a defined distribution of EGFP fluorescence between nucleus and cytoplasm (n/c ratio = 2). Clear differences of the size of the hypothetic EGFP concatemer depending on the fused NLS motif were observed. Therefore, we propose to use the size of this hypothetic concatemer as quantitative indicator for comparing strength of different NLS motifs. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Distribution and Spectroscopy of Green Fluorescent Protein and Acyl-CoA: Cholesterol Acytransferase in Sf21 Insect Cells

    Science.gov (United States)

    Richmond, R. C.; Mahtani, H.; Lu, X.; Chang, T. Y.; Malak, H.; Rose, M. Franklin (Technical Monitor)

    2001-01-01

    Acyl-CoA: cholesterol acyltransferase (ACAT) is thought to significantly participate in the pathway of cholesterol esterification that underlies the pathology of artherosclerosis. This enzyme is a membrane protein known to be preferentially bound within the endoplasmic reticulum of mammalian cells, from which location it esterifies cholesterol derived from low density lipoprotein. Cultures of insect cells were separately infected with baculovirus containing the gene for green fluroescent protein (GFP) and with baculovirus containing tandem genes for GFP and ACAT. These infected cultures expressed GFP and the fusion protein GCAT, respectively, with maximum expression occurring on the fourth day after infection. Extraction of GFP- and of GCAT-expressing cells with urea and detergent resulted in recovery of fluorescent protein in aqueous solution. Fluorescence spectra at neutral pH were identical for both GFP and GCAT extracts in aqueous solution, indicating unperturbed tertiary structure for the GFP moiety within GCAT. In a cholesterol esterification assay, GCAT demonstrated ACAT activity, but with less efficiency compared to native ACAT. It was hypothesized that the membrane protein ACAT would lead to differences in localization of GCAT compared to GFP within the respective expressing insect cells. The GFP marker directly and also within the fusion protein GCAT was accordingly used as the intracellular probe that was fluorescently analyzed by the new biophotonics technique of hyperspectral imaging. In that technique, fluorescence imaging was obtained from two dimensional arrays of cells, and regions of interest from within those images were then retrospectively analyzed for the emission spectra that comprises the image. Results of hyperspectral imaging of insect cells on day 4 postinfection showed that GCAT was preferentially localized to the cytoplasm of these cells compared to GFP. Furthermore, the emission spectra obtained for the localized GCAT displayed a peak

  5. Near-infrared fluorescence cholangiography with indocyanine green for biliary atresia. Real-time imaging during the Kasai procedure: a pilot study.

    Science.gov (United States)

    Hirayama, Yutaka; Iinuma, Yasushi; Yokoyama, Naoyuki; Otani, Tetsuya; Masui, Daisuke; Komatsuzaki, Naoko; Higashidate, Naruki; Tsuruhisa, Shiori; Iida, Hisataka; Nakaya, Kengo; Naito, Shinichi; Nitta, Koju; Yagi, Minoru

    2015-12-01

    Hepatoportoenterostomy (HPE) with the Kasai procedure is the treatment of choice for biliary atresia (BA) as the initial surgery. However, the appropriate level of dissection level of the fibrous cone (FC) of the porta hepatis (PH) is frequently unclear, and the procedure sometimes results in unsuccessful outcomes. Recently, indocyanine green near-infrared fluorescence imaging (ICG-FCG) has been developed as a form of real-time cholangiography. We applied this technique in five patients with BA to visualize the biliary flow at the PH intraoperatively. ICG was injected intravenously the day before surgery as the liver function test, and the liver was observed with a near-infrared camera system during the operation while the patient's feces was also observed. In all patients, the whole liver fluoresced diffusely with ICG-containing stagnant bile, whereas no extrahepatic structures fluoresced. The findings of the ICG fluorescence pattern of the PH after dissection of the FC were classified into three types: spotty fluorescence, one patient; diffuse weak fluorescence, three patients; and diffuse strong fluorescence, one patient. In all five patients, the feces evacuated after HPE showed distinct fluorescent spots, although that obtained before surgery showed no fluorescence. One patient with diffuse strong fluorescence who did not achieve JF underwent living related liver transplantation six months after the initial HPE procedure. Four patients, including three cases involving diffuse weak fluorescence and one case involving spotty fluorescence showed weak fluorescence compared to that of the surrounding liver surface. We were able to detect the presence of bile excretion at the time of HPE intraoperatively and successfully evaluated the extent of bile excretion using this new technique. Furthermore, the ICG-FCG findings may provide information leading to a new classification and potentially function as an indicator predicting the clinical outcomes after HPE.

  6. Green fluorescent protein changes the conductance of connexin 43 (Cx43) hemichannels reconstituted in planar lipid bilayers.

    Science.gov (United States)

    Carnarius, Christian; Kreir, Mohamed; Krick, Marcel; Methfessel, Christoph; Moehrle, Volker; Valerius, Oliver; Brüggemann, Andrea; Steinem, Claudia; Fertig, Niels

    2012-01-20

    In mammalian tissues, connexin 43 (Cx43) is the most prominent member of the connexin family. In a single lipid bilayer, six connexin subunits assemble into a hemichannel (connexon). Direct communication of apposing cells is realized by two adjacent hemichannels, which can form gap junction channels. Here, we established an expression system in Pichia pastoris to recombinantly produce and purify Cx43 as well as Cx43 fused to green fluorescent protein (GFP). Proteins were isolated from crude cell membrane fractions via affinity chromatography. Cx43 and Cx43-GFP hemichannels were reconstituted in giant unilamellar vesicles as proven by fluorescence microscopy, and their electrophysiological behavior was analyzed on the single channel level by planar patch clamping. Cx43 and Cx43-GFP both showed an ohmic behavior and a voltage-dependent open probability. Cx43 hemichannels exhibited one major mean conductance of 224 ± 26 picosiemens (pS). In addition, a subconductance state at 124 ± 5 pS was identified. In contrast, the analysis of Cx43-GFP single channels revealed 10 distinct conductance states in the range of 15 to 250 pS, with a larger open probability at 0 mV as compared with Cx43, which suggests that intermolecular interactions between the GFP molecules alter the electrophysiology of the protein.

  7. Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayers*

    Science.gov (United States)

    Carnarius, Christian; Kreir, Mohamed; Krick, Marcel; Methfessel, Christoph; Moehrle, Volker; Valerius, Oliver; Brüggemann, Andrea; Steinem, Claudia; Fertig, Niels

    2012-01-01

    In mammalian tissues, connexin 43 (Cx43) is the most prominent member of the connexin family. In a single lipid bilayer, six connexin subunits assemble into a hemichannel (connexon). Direct communication of apposing cells is realized by two adjacent hemichannels, which can form gap junction channels. Here, we established an expression system in Pichia pastoris to recombinantly produce and purify Cx43 as well as Cx43 fused to green fluorescent protein (GFP). Proteins were isolated from crude cell membrane fractions via affinity chromatography. Cx43 and Cx43-GFP hemichannels were reconstituted in giant unilamellar vesicles as proven by fluorescence microscopy, and their electrophysiological behavior was analyzed on the single channel level by planar patch clamping. Cx43 and Cx43-GFP both showed an ohmic behavior and a voltage-dependent open probability. Cx43 hemichannels exhibited one major mean conductance of 224 ± 26 picosiemens (pS). In addition, a subconductance state at 124 ± 5 pS was identified. In contrast, the analysis of Cx43-GFP single channels revealed 10 distinct conductance states in the range of 15 to 250 pS, with a larger open probability at 0 mV as compared with Cx43, which suggests that intermolecular interactions between the GFP molecules alter the electrophysiology of the protein. PMID:22139870

  8. Effect of lentiviruses carrying enhanced green fluorescent protein injected into the scala media through a cochleostomy in rats.

    Science.gov (United States)

    Wei, Yan; Fu, Yong; Liu, Shaosheng; Xia, Guihua; Pan, Song

    2013-01-01

    The purposes of the current study were to assess the feasibility of post-auricular microinjection of lentiviruses carrying enhanced green fluorescent protein (EGFP) into the scala media through cochleostomies in rats, determine the expression of viral gene in the cochlea, and record the post-operative changes in the number and auditory function of cochlear hair cells (HCs). Healthy rats were randomly divided into two groups. The left ears of the animals in group I were injected with lentivirus carrying EGFP (n=10) via scala media lateral wall cochleostomies, and the left ears of the animals in group II were similarly injected with artificial endolymph (n=10). Prior to and 30 days post-injection, auditory function was assessed with click-auditory brainstem response (ABR) testing, EGFP expression was determined with cochlear frozen sections under fluorescence microscopy, and survival of HCs was estimated based on whole mount preparations. Thirty days after surgery, click-ABR testing revealed that there were significant differences in the auditory function, EGFP expression, and survival of HCs in the left ears before and after surgery in the same rats from each group. In group I, EGFP was noted in the strial marginal cells of the scala media, the organ of Corti, spiral nerves, and spiral ganglion cells. Lentiviruses were successfully introduced into the scala media through cochleostomies in rats, and the EGFP reporter gene was efficiently expressed in the organ of Corti, spiral nerves, and spiral ganglion cells. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Screening by coral green fluorescent protein (GFP)-like chromoproteins supports a role in photoprotection of zooxanthellae

    Science.gov (United States)

    Smith, E. G.; D'Angelo, C.; Salih, A.; Wiedenmann, J.

    2013-06-01

    Green fluorescent protein (GFP)-like pigments are responsible for the vivid colouration of many reef-building corals and have been proposed to act as photoprotectants. Their role remains controversial because the functional mechanism has not been elucidated. We provide direct evidence to support a photoprotective role of the non-fluorescent chromoproteins (CPs) that form a biochemically and photophysically distinct group of GFP-like proteins. Based on observations of Acropora nobilis from the Great Barrier Reef, we explored the photoprotective role of CPs by analysing five coral species under controlled conditions. In vitro and in hospite analyses of chlorophyll excitation demonstrate that screening by CPs leads to a reduction in chlorophyll excitation corresponding to the spectral properties of the specific CPs present in the coral tissues. Between 562 and 586 nm, the CPs maximal absorption range, there was an up to 50 % reduction of chlorophyll excitation. The screening was consistent for established and regenerating tissue and amongst symbiont clades A, C and D. Moreover, among two differently pigmented morphs of Acropora valida grown under identical light conditions and hosting subclade type C3 symbionts, high CP expression correlated with reduced photodamage under acute light stress.

  10. Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein

    Energy Technology Data Exchange (ETDEWEB)

    Albariño, César G., E-mail: calbarino@cdc.gov; Wiggleton Guerrero, Lisa; Lo, Michael K.; Nichol, Stuart T.; Towner, Jonathan S.

    2015-10-15

    Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014–2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral compounds and measuring neutralizing antibodies. - Highlights: • Recombinant Ebola virus (EBOV) derived from Makona variant was rescued. • New protocol for viral rescue allows 100% efficiency. • Modified EBOV expresses a green fluorescent protein from a VP40-fused protein. • Modified EBOV was tested as tool to screen antiviral compounds and measure neutralizing antibodies.

  11. Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces

    Science.gov (United States)

    Szent-Gyorgyi, Chris; Stanfield, Robyn L.; Andreko, Susan; Dempsey, Alison; Ahmed, Mushtaq; Capek, Sara; Waggoner, Alan; Wilson, Ian A.; Bruchez, Marcel P.

    2013-01-01

    We report that a symmetric small molecule ligand mediates the assembly of antibody light chain variable domains (VLs) into a correspondent symmetric ternary complex with novel interfaces. The L5* Fluorogen Activating Protein (FAP) is a VL domain that binds malachite green dye (MG) to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two VL domains. Unliganded L5* VL domains crystallize as a similar antiparallel VL/VL homodimer. The complementarity determining regions (CDRs) are spatially oriented to form novel VL/VL and VL/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable VL/MG/VL complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two VL domains into a single protein tightens MG binding over 1,000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high affinity protein tags and capture reagents. PMID:23978698

  12. Facile green synthesis of fluorescent N-doped carbon dots from Actinidia deliciosa and their catalytic activity and cytotoxicity applications

    Science.gov (United States)

    Arul, Velusamy; Sethuraman, Mathur Gopalakrishnan

    2018-04-01

    Green synthesis of fluorescent nitrogen doped carbon dots (N-CDs) using Actinidia deliciosa (A. deliciosa) fruit extract as a carbon precursor and aqueous ammonia as a nitrogen dopant is reported here. The synthesized N-CDs were characterized by high resolution transmission electron microscopy (HR-TEM), energy dispersive spectroscopy (EDS), selected area electron diffraction (SAED), UV-Visible spectroscopy (UV-Vis), fluorescence spectroscopy, Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), Raman spectroscopy and X-ray photoelectron spectroscopy (XPS). The average size of the N-CDs was approximately 3.59 nm and the calculated inter layer distance was found to be 0.21 nm. Raman spectroscopy and SAED pattern revealed the graphitic nature of the synthesized N-CDs. The N-CDs were found to emit intense blue color at 405 nm under the excitation of 315 nm. The doping of nitrogen over the surface of the N-CDs was confirmed by EDS, FT-IR and XPS studies. The synthesized N-CDs were found to exhibit excellent catalytic activity in the reduction of Rhodamine-B using sodium borohydrate. The MTT assay was used to evaluate the cytotoxicity and biocompatibility of N-CDs towards L-929 and MCF-7 cells. From the results obtained, it was found that the N-CDs exhibit low cytotoxicity and superior biocompatibility on both L-929 and MCF-7 cells.

  13. Effects of nanoencapsulation and PEGylation on biodistribution of indocyanine green in healthy mice: quantitative fluorescence imaging and analysis of organs

    Directory of Open Access Journals (Sweden)

    Bahmani B

    2013-04-01

    Full Text Available Baharak Bahmani,1 Christian Y Lytle,2 Ameae M Walker,2 Sharad Gupta,1 Valentine I Vullev,1 Bahman Anvari1 1Department of Bioengineering, 2Division of Biomedical Sciences, University of California, Riverside, CA, USA Abstract: Near-infrared nanoconstructs present a potentially effective platform for site-specific and deep tissue optical imaging and phototherapy. We have engineered a polymeric nanocapsule composed of polyallylamine hydrochloride (PAH chains cross-linked with sodium phosphate and doped with indocyanine green (ICG toward such endeavors. The ICG-doped nanocapsules were coated covalently with polyethylene glycol (5000 daltons through reductive amination. We administrated the constructs by tail vein injection to healthy mice. To characterize the biodistribution of the constructs, we performed in vivo quantitative fluorescence imaging and subsequently analyzed the various extracted organs. Our results suggest that encapsulation of ICG in these PEGylated constructs is an effective approach to prolong the circulation time of ICG and delay its hepatic accumulation. Increased bioavailability of ICG, due to encapsulation, offers the potential of extending the clinical applications of ICG, which are currently limited due to rapid elimination of ICG from the vasculature. Our results also indicate that PAH and ICG-doped nanocapsules (ICG-NCs are not cytotoxic at the levels used in this study. Keywords: cancer, fluorescent imaging, nanoprobes, near infrared, pharmacokinetics, phototherapy, vascular imaging

  14. Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein

    International Nuclear Information System (INIS)

    Albariño, César G.; Wiggleton Guerrero, Lisa; Lo, Michael K.; Nichol, Stuart T.; Towner, Jonathan S.

    2015-01-01

    Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014–2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral compounds and measuring neutralizing antibodies. - Highlights: • Recombinant Ebola virus (EBOV) derived from Makona variant was rescued. • New protocol for viral rescue allows 100% efficiency. • Modified EBOV expresses a green fluorescent protein from a VP40-fused protein. • Modified EBOV was tested as tool to screen antiviral compounds and measure neutralizing antibodies

  15. Fiscal 1999 achievement report on research and development project on intellectual infrastructure creation and utilization technologies. Development of efficient protein expression system (Development of efficient protein expression system utilizing protein folding mechanism of hyperthermophilic bacteria); 1999 nendo kokoritsu tanpakushitsu hatsugen system no kaihatsu seika hokokusho. Chokonetsukin no tanpakushitsu oritatami kiko wo riyoshita kokoritsu tanpakushitsu hatsugen system no kaihatsu

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2000-03-01

    Efforts were exerted to achieve efficient expression of proteins of hyperthermophilic bacteria, hyperthermophilic archaeabacteria in particular, using a heterogene expression system in which Escherichia coli was the host. In an effort to search for genes related to protein folding and to elucidate the mechanism of folding, chaperonin and prefoldin subunit genes, out of various factors participating in protein folding in hyperthermophilic archaeabacteria, were cloned, and expressed in Escherichia coli. As a system for analyzing protein folding reaction, an experimental system was established on a substrate comprising isopropyl malate dehydrogenase, citrate synthase, glucose dehydrogenase, and a green fluorescent protein. Studies were further conducted to elucidate the mechanism of expression of enzyme genes in Escherichia coli for the establishment of a mass production method for useful enzymes. Also carried out was the research and development of an element technology evaluation system involving protein expression. (NEDO)

  16. green

    Directory of Open Access Journals (Sweden)

    Elena Grigoryeva

    2011-02-01

    Full Text Available The “green” topic follows the “youngsters”, which is quite natural for the Russian language.Traditionally these words put together sound slightly derogatory. However, “green” also means fresh, new and healthy.For Russia, and for Siberia in particular, “green” architecture does sound new and fresh. Forced by the anxious reality, we are addressing this topic intentionally. The ecological crisis, growing energy prices, water, air and food deficits… Alexander Rappaport, our regular author, writes: “ It has been tolerable until a certain time, but under transition to the global civilization, as the nature is destroyed, and swellings of megapolises expand incredibly fast, the size and the significance of all these problems may grow a hundredfold”.However, for this very severe Siberian reality the newness of “green” architecture may turn out to be well-forgotten old. A traditional Siberian house used to be built on principles of saving and environmental friendliness– one could not survive in Siberia otherwise.Probably, in our turbulent times, it is high time to fasten “green belts”. But we should keep from enthusiastic sticking of popular green labels or repainting of signboards into green color. We should avoid being drowned in paper formalities under “green” slogans. And we should prevent the Earth from turning into the planet “Kin-dza-dza”.

  17. One-pot green synthesis of carbon dots by using Saccharum officinarum juice for fluorescent imaging of bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae) cells

    Energy Technology Data Exchange (ETDEWEB)

    Mehta, Vaibhavkumar N. [Applied Chemistry Department, S. V. National Institute of Technology, Surat, 395 007 (India); Jha, Sanjay [Gujarat Agricultural Biotechnology Institute, Navsari Agricultural University, Surat, 395007 (India); Kailasa, Suresh Kumar, E-mail: sureshkumarchem@gmail.com [Applied Chemistry Department, S. V. National Institute of Technology, Surat, 395 007 (India)

    2014-05-01

    We are reporting highly economical plant-based hydrothermal method for one-pot green synthesis of water-dispersible fluorescent carbon dots (CDs) by using Saccharum officinarum juice as precursor. The synthesized CDs were characterized by UV-visible, fluorescence, Fourier transform infrared (FT-IR), dynamic light scattering (DLS), high-resolution transmission electron microscopic (HR-TEM), and laser scanning confocal microscopic techniques. The CDs are well dispersed in water with an average size of ∼ 3 nm and showed bright blue fluorescence under UV-light (λ{sub ex} = 365 nm). These CDs acted as excellent fluorescent probes in cellular imaging of bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae). - Highlights: • One-pot green synthesis was used for fluorescent CDs. • FT-IR, DLS, and TEM were used for the characterization of CDs. • The CDs are well dispersed in water with an average size of ∼ 3 nm. • The CDs acted as fluorescent probes for imaging of bacteria and yeast cells.

  18. Pilot Assessment of the Repeatability of Indocyanine Green Fluorescence Imaging and Correlation with Traditional Foot Perfusion Assessments.

    Science.gov (United States)

    Venermo, M; Settembre, N; Albäck, A; Vikatmaa, P; Aho, P-S; Lepäntalo, M; Inoue, Y; Terasaki, H

    2016-10-01

    Ankle brachial index (ABI), toe pressures (TP), and transcutaneous oxygen pressure (TcPO 2 ) are traditionally used in the assessment of critical limb ischemia (CLI). Indocyanine green (ICG) fluorescence imaging can be used to evaluate local circulation in the foot and to evaluate the severity of ischemia. This prospective study analyzed the suitability of a fluorescence imaging system (photodynamic eye [PDE]) in CLI. Forty-one patients with CLI were included. Of the patients, 66% had diabetes and there was an ischemic tissue lesion in 70% of the limbs. ABI, toe pressures, TcPO 2 and ICG-fluorescence imaging (ICG-FI) were measured in each leg. To study the repeatability of the ICG-FI, each patient underwent the study twice. After the procedure, foot circulation was measured using a time-intensity curve, where T1/2 (the time needed to achieve half of the maximum fluorescence intensity) and PDE10 (increase of the intensity during the first 10 s) were determined. A time-intensity curve was plotted using the same areas as for the TcPO 2 probes (n=123). The mean ABI was 0.43, TP 21 mmHg, TcPO 2 23 mmHg, T1/2 38 s, and PDE10 19 AU. Time-intensity curves were repeatable. In a Bland-Altman scatter plot, the 95% limits of agreement of PDE10 was 9.9 AU and the corresponding value of T1/2 was 14 s. Correlation between ABI and TP was significant (R=.73, p<.001), and it was weaker in diabetic patients (R=.47, p=.048) compared with non-diabetic patients (R=.89, p=.002). Correlations between ABI and TcPO 2 and TP and TcPO 2 were weak (R=.37, p=.05 and R=.43, p=.037, respectively). Correlation between TcPO 2 and PDE10 was strong in diabetic patients (R=.70, p=.003). According to this pilot study, ICG-FI with PDE can be used in the assessment of blood supply in the ischemic foot. Copyright © 2016 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

  19. Flash fluorescence with indocyanine green videoangiography to identify the recipient artery for bypass with distal middle cerebral artery aneurysms: operative technique.

    Science.gov (United States)

    Rodríguez-Hernández, Ana; Lawton, Michael T

    2012-06-01

    Distal middle cerebral artery (MCA) aneurysms frequently have nonsaccular morphology that necessitates trapping and bypass. Bypasses can be difficult because efferent arteries lie deep in the opercular cleft and may not be easily identifiable. We introduce the "flash fluorescence" technique, which uses videoangiography with indocyanine green (ICG) dye to identify an appropriate recipient artery on the cortical surface for the bypass, enabling a more superficial and easier anastomosis. Flash fluorescence requires 3 steps: (1) temporary clip occlusion of the involved afferent artery; (2) videoangiography demonstrating fluorescence in uninvolved arteries on the cortical surface; and (3) removal of the temporary clip with flash fluorescence in the involved efferent arteries on the cortical surface, thereby identifying a recipient. Alternatively, temporary clips can occlude uninvolved arteries, and videoangiography will demonstrate initial fluorescence in efferent arteries during temporary occlusion and flash fluorescence in uninvolved arteries during reperfusion. From a consecutive series of 604 MCA aneurysms treated microsurgically, 22 (3.6%) were distal aneurysms and 11 required a bypass. The flash fluorescence technique was used in 3 patients to select the recipient artery for 2 superficial temporal artery-to-MCA bypasses and 1 MCA-MCA bypass. The correct recipient was selected in all cases. The flash fluorescence technique provides quick, reliable localization of an appropriate recipient artery for bypass when revascularization is needed for a distal MCA aneurysm. This technique eliminates the need for extensive dissection of the efferent artery and enables a superficial recipient site that makes the anastomosis safer, faster, and less demanding.

  20. Model and methods to assess hepatic function from indocyanine green fluorescence dynamical measurements of liver tissue.

    Science.gov (United States)

    Audebert, Chloe; Vignon-Clementel, Irene E

    2018-03-30

    The indocyanine green (ICG) clearance, presented as plasma disappearance rate is, presently, a reliable method to estimate the hepatic "function". However, this technique is not instantaneously available and thus cannot been used intra-operatively (during liver surgery). Near-infrared spectroscopy enables to assess hepatic ICG concentration over time in the liver tissue. This article proposes to extract more information from the liver intensity dynamics by interpreting it through a dedicated pharmacokinetics model. In order to account for the different exchanges between the liver tissues, the proposed model includes three compartments for the liver model (sinusoids, hepatocytes and bile canaliculi). The model output dependency to parameters is studied with sensitivity analysis and solving an inverse problem on synthetic data. The estimation of model parameters is then performed with in-vivo measurements in rabbits (El-Desoky et al. 1999). Parameters for different liver states are estimated, and their link with liver function is investigated. A non-linear (Michaelis-Menten type) excretion rate from the hepatocytes to the bile canaliculi was necessary to reproduce the measurements for different liver conditions. In case of bile duct ligation, the model suggests that this rate is reduced, and that the ICG is stored in the hepatocytes. Moreover, the level of ICG remains high in the blood following the ligation of the bile duct. The percentage of retention of indocyanine green in blood, which is a common test for hepatic function estimation, is also investigated with the model. The impact of bile duct ligation and reduced liver inflow on the percentage of ICG retention in blood is studied. The estimation of the pharmacokinetics model parameters may lead to an evaluation of different liver functions. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. Enhanced visualization of the bile duct via parallel white light and indocyanine green fluorescence laparoscopic imaging

    Science.gov (United States)

    Demos, Stavros G.; Urayama, Shiro

    2014-03-01

    Despite best efforts, bile duct injury during laparoscopic cholecystectomy is a major potential complication. Precise detection method of extrahepatic bile duct during laparoscopic procedures would minimize the risk of injury. Towards this goal, we have developed a compact imaging instrumentation designed to enable simultaneous acquisition of conventional white color and NIR fluorescence endoscopic/laparoscopic imaging using ICG as contrast agent. The capabilities of this system, which offers optimized sensitivity and functionality, are demonstrated for the detection of the bile duct in an animal model. This design could also provide a low-cost real-time surgical navigation capability to enhance the efficacy of a variety of other image-guided minimally invasive procedures.

  2. Economical and green synthesis of bagasse-derived fluorescent carbon dots for biomedical applications

    International Nuclear Information System (INIS)

    Du, Fengyi; Zhang, Miaomiao; Li, Xiaofeng; Jiang, Xinyi; Li, Zhang; Hua, Ye; Shao, Genbao; Jin, Jie; Shao, Qixiang; Gong, Aihua; Li, Jianan; Zhou, Ming

    2014-01-01

    Carbon quantum dots (CDs) are promising nanomaterials in biomedical, photocatalytical and photoelectronic applications. However, determining how to explore an ideal precursor for a renewable carbon resource is still an interesting challenge. Here, for the first time, we report that renewable wastes of bagasse as a new precursor were prepared for fluorescent CDs by a hydrothermal carbonization (HTC) process. The characterization results show that such bagasse-derived CDs are monodispersed, contain quasi spherical particles with a diameter of about 1.8 nm and exhibit favorable photoluminescence properties, super-high photostability and good dispersibility in water. Most importantly, bagasse-derived CDs have good biocompatibility and can be easily and quickly internalized by living cancer cells; they can also be used for multicolour biolabeling and bioimaging in cancer cells. It is suggested that bagasse-derived CDs might have potential applications in biomedical and photoelectronic fields. (paper)

  3. Photoconversion and fluorescence properties of a red/green-type cyanobacteriochrome AM1_C0023g2 that binds not only phycocyanobilin but also biliverdin.

    Directory of Open Access Journals (Sweden)

    Keiji eFushimi

    2016-04-01

    Full Text Available Cyanobacteriochromes (CBCRs are distantly related to the red/far-red responsive phytochromes. Red/green-type CBCRs are widely distributed among various cyanobacteria. The red/green-type CBCRs covalently bind phycocyanobilin (PCB and show red/green reversible photoconversion. Recent studies revealed that some red/green-type CBCRs from chlorophyll d-bearing cyanobacterium Acaryochloris marina covalently bind not only PCB but also biliverdin (BV. The BV-binding CBCRs show far-red/orange reversible photoconversion. Here, we identified another CBCR (AM1_C0023g2 from A. marina that also covalently binds not only PCB but also BV with high binding efficiencies, although BV chromophore is unstable in the presence of urea. Replacement of Ser334 with Gly resulted in significant improvement in the yield of the BV-binding holoprotein, thereby ensuring that the mutant protein is a fine platform for future development of optogenetic switches. We also succeeded in detecting near-infrared fluorescence from mammalian cells harboring PCB-binding AM1_C0023g2 whose fluorescence quantum yield is 3.0%. Here the PCB-binding holoprotein is shown as a platform for future development of fluorescent probes.

  4. Study of cell-differentiation and assembly of photosynthetic proteins during greening of etiolated Zea mays leaves using confocal fluorescence microspectroscopy at liquid-nitrogen temperature.

    Science.gov (United States)

    Shibata, Yutaka; Katoh, Wataru; Tahara, Yukari

    2013-04-01

    Fluorescence microspectroscopy observations were used to study the processes of cell differentiation and assemblies of photosynthesis proteins in Zea mays leaves under the greening process. The observations were done at 78K by setting the sample in a cryostat to avoid any undesired progress of the greening process during the measurements. The lateral and axial spatial resolutions of the system were 0.64μm and 4.4μm, respectively. The study revealed the spatial distributions of protochlorophyllide (PChld) in both the 632-nm-emitting and 655-nm-emitting forms within etiolated Zea mays leaves. The sizes of the fluorescence spots attributed to the former were larger than those of the latter, validating the assignment of the former and latter to the prothylakoid and prolamellar bodies, respectively. In vivo microspectroscopy observations of mature Zea mays leaves confirmed the different photosystem II (PS I)/photosystem I (PS II) ratio between the bundle sheath (BS) and mesophyll (MS) cells, which is specific for C4-plants. The BS cells in Zea mays leaves 1h after the initiation of the greening process tended to show fluorescence spectra at shorter wavelength side (at around 679nm) than the MS cells (at around 682nm). The 679-nm-emitting chlorophyll-a form observed mainly in the BS cells was attributed to putative precursor complexes to PS I. The BS cells under 3-h greening showed higher relative intensities of the PS I fluorescence band at around 735nm, suggesting the reduced PS II amount in the BS cells in this greening stage. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Synthesis and formation mechanistic investigation of nitrogen-doped carbon dots with high quantum yields and yellowish-green fluorescence

    Science.gov (United States)

    Hou, Juan; Wang, Wei; Zhou, Tianyu; Wang, Bo; Li, Huiyu; Ding, Lan

    2016-05-01

    Heteroatom doped carbon dots (CDs) have received increasing attention due to their unique properties and related applications. However, previously reported CDs generally show strong emission only in the blue-light region, thus restricting their further applications. And the fundamental investigation on the preparation process is always neglected. Herein, we have developed a simple and solvent-free synthetic strategy to fabricate nitrogen-doped CDs (N-CDs) from citric acid and dicyandiamide. The as-prepared N-CDs exhibited a uniform size distribution, strong yellowish-green fluorescence emission and a high quantum yield of 73.2%. The products obtained at different formation stages were detailedly characterized by transmission electron microscopy, X-ray diffraction spectrometer, X-ray photoelectron spectroscopy and UV absorbance spectroscopy. A possible formation mechanism has thus been proposed including dehydration, polymerization and carbonization. Furthermore, the N-CDs could serve as a facile and label-free probe for the detection of iron and fluorine ions with detection limits of 50 nmol L-1 and 75 nmol L-1, respectively.Heteroatom doped carbon dots (CDs) have received increasing attention due to their unique properties and related applications. However, previously reported CDs generally show strong emission only in the blue-light region, thus restricting their further applications. And the fundamental investigation on the preparation process is always neglected. Herein, we have developed a simple and solvent-free synthetic strategy to fabricate nitrogen-doped CDs (N-CDs) from citric acid and dicyandiamide. The as-prepared N-CDs exhibited a uniform size distribution, strong yellowish-green fluorescence emission and a high quantum yield of 73.2%. The products obtained at different formation stages were detailedly characterized by transmission electron microscopy, X-ray diffraction spectrometer, X-ray photoelectron spectroscopy and UV absorbance spectroscopy. A

  6. Recombinant canine distemper virus strain Snyder Hill expressing green or red fluorescent proteins causes meningoencephalitis in the ferret.

    Science.gov (United States)

    Ludlow, M; Nguyen, D T; Silin, D; Lyubomska, O; de Vries, R D; von Messling, V; McQuaid, S; De Swart, R L; Duprex, W P

    2012-07-01

    The propensity of canine distemper virus (CDV) to spread to the central nervous system is one of the primary features of distemper. Therefore, we developed a reverse genetics system based on the neurovirulent Snyder Hill (SH) strain of CDV (CDV(SH)) and show that this virus rapidly circumvents the blood-brain and blood-cerebrospinal fluid (CSF) barriers to spread into the subarachnoid space to induce dramatic viral meningoencephalitis. The use of recombinant CDV(SH) (rCDV(SH)) expressing enhanced green fluorescent protein (EGFP) or red fluorescent protein (dTomato) facilitated the sensitive pathological assessment of routes of virus spread in vivo. Infection of ferrets with these viruses led to the full spectrum of clinical signs typically associated with distemper in dogs during a rapid, fatal disease course of approximately 2 weeks. Comparison with the ferret-adapted CDV(5804P) and the prototypic wild-type CDV(R252) showed that hematogenous infection of the choroid plexus is not a significant route of virus spread into the CSF. Instead, viral spread into the subarachnoid space in rCDV(SH)-infected animals was triggered by infection of vascular endothelial cells and the hematogenous spread of virus-infected leukocytes from meningeal blood vessels into the subarachnoid space. This resulted in widespread infection of cells of the pia and arachnoid mater of the leptomeninges over large areas of the cerebral hemispheres. The ability to sensitively assess the in vivo spread of a neurovirulent strain of CDV provides a novel model system to study the mechanisms of virus spread into the CSF and the pathogenesis of acute viral meningitis.

  7. Near-infrared fluorescence imaging and photodynamic therapy with indocyanine green lactosome has antineoplastic effects for hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Takumi Tsuda

    Full Text Available Anticancer agents and operating procedures have been developed for hepatocellular carcinoma (HCC patients, but their prognosis remains poor. It is necessary to develop novel diagnostic and therapeutic strategies for HCC to improve its prognosis. Lactosome is a core-shell-type polymeric micelle, and enclosing labeling or anticancer agents into this micelle enables drug delivery. In this study, we investigated the diagnostic and therapeutic efficacies of indocyanine green (ICG-loaded lactosome for near-infrared fluorescence (NIF imaging and photodynamic therapy (PDT for HCC.The human HCC cell line HuH-7 was treated with ICG or ICG-lactosome, followed by PDT, and the cell viabilities were measured (in vitro PDT efficiency. For NIF imaging, HuH-7 cells were subcutaneously transplanted into BALB/c nude mice, followed by intravenous administration of ICG or ICG-lactosome. The transplanted animals were treated with PDT, and the antineoplastic effects were analyzed (in vivo PDT efficiency.PDT had toxic effects on HuH-7 cells treated with ICG-lactosome, but not ICG alone. NIF imaging revealed that the fluorescence of tumor areas in ICG-lactosome-treated animals was higher than that of contralateral regions at 24 h after injection and thereafter. PDT exerted immediate and continuous phototoxic effects in the transplanted mice treated with ICG-lactosome.Our results demonstrate that ICG-lactosome accumulated in xenograft tumors, and that PDT had antineoplastic effects on these malignant implants. NIF imaging and PDT with ICG-lactosome could be useful diagnostic and/or therapeutic strategies for HCC.

  8. Crystal Structure of Green Fluorescent Protein Clover and Design of Clover-Based Redox Sensors.

    Science.gov (United States)

    Campbell, Benjamin C; Petsko, Gregory A; Liu, Ce Feng

    2018-02-06

    We have determined the crystal structure of Clover, one of the brightest fluorescent proteins, and found that its T203H/S65G mutations relative to wild-type GFP lock the critical E222 side chain in a fixed configuration that mimics the major conformer of that in EGFP. The resulting equilibrium shift to the predominantly deprotonated chromophore increases the extinction coefficient (EC), opposes photoactivation, and is responsible for the bathochromic shift. Clover's brightness can further be attributed to a π-π stacking interaction between H203 and the chromophore. Consistent with these observations, the Clover G65S mutant reversed the equilibrium shift, dramatically decreased the EC, and made Clover photoactivatable under conditions that activated photoactivatable GFP. Using the Clover structure, we rationally engineered a non-photoactivatable redox sensor, roClover1, and determined its structure as well as that of its parental template, roClover0.1. These high-resolution structures provide deeper insights into structure-function relationships in GFPs and may aid the development of excitation-improved ratiometric biosensors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Heterogeneity mapping of protein expression in tumors using quantitative immunofluorescence.

    Science.gov (United States)

    Faratian, Dana; Christiansen, Jason; Gustavson, Mark; Jones, Christine; Scott, Christopher; Um, InHwa; Harrison, David J

    2011-10-25

    -quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ. We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system(6). Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index(7). To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

  10. Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen dye.

    Science.gov (United States)

    Moreno, Luis A; Cox, Kendra L

    2010-11-05

    Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit. The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 μL sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode. Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows

  11. Indocyanine green fluorescence angiography during laparoscopic low anterior resection: results of a case-matched study.

    Science.gov (United States)

    Boni, Luigi; Fingerhut, Abe; Marzorati, Alessandro; Rausei, Stefano; Dionigi, Gianlorenzo; Cassinotti, Elisa

    2017-04-01

    Colorectal anastomoses after anterior resection for cancer carry a high risk of leakage. Different factors might influence the correct healing of anastomosis, but adequate perfusion of the bowel is highlighted as one of the most important elements. Fluorescence angiography (FA) is a new technique that allows the surgeon to perform real-time intraoperative angiography to evaluate the perfusion of the anastomosis and hence, potentially, reduce leak rate. The aim of this study was to evaluate the impact of FA of the bowel on postoperative complications and anastomotic leakage after laparoscopic anterior resection with total mesorectal excision (TME). FA was performed in all patients undergoing laparoscopic anterior resection with TME for cancer followed by colorectal or coloanal anastomosis. Results were compared to a historical controls group of 38 patients previously operated by the same surgeon for the same indication but without the use of FA. From October 2014 to November 2015, 42 patients underwent laparoscopic anterior resection with TME and FA of the bowel. The surgeon subjectively decided to change the planned anastomotic level of the descending colon due to hypoperfused distal segment in two out of 42 patients in the FA group (4.7 %). Anastomotic leakage, confirmed by postoperative CT scan and water-soluble contrast enema, was found in two cases of a historical controls group and none in the FA group. No adverse events (side effects or allergic reaction) related to FA were recorded. All the other postoperative complications were comparable between the two groups. In our experience, ICG FA was safe and effective in low rectal cancer resection, possibly leading to a reduction in the anastomotic leakage rate after TME.

  12. Use of invisible near infrared light fluorescence with indocyanine green and methylene blue in urology. Part 2.

    Science.gov (United States)

    Polom, Wojciech; Markuszewski, Marcin; Rho, Young Soo; Matuszewski, Marcin

    2014-01-01

    In the second part of this paper, concerning the use of invisible near infrared light (NIR) fluorescence with indocyanine green (ICG) and methylene blue (MB) in urology, other possible uses of this new technique will be presented. In kidney transplantation, this concerns allograft perfusion and real time NIR-guided angiography; moreover, perfusion angiography of tissue flaps, NIRF visualization of ureters, NIR-guided visualization of urinary calcifications, NIRF in male infertility and semen quality assessment. In this part, we have also analysed cancer targeting and imaging fluorophores as well as cost benefits associated with the use of these new techniques. PubMed and Medline databases were searched for ICG and MB use in urological settings, along with data published in abstracts of urological conferences. Although NIR-guided ICG and MB are still in their initial phases, there have been significant developments in a few more major domains of urology, including 1) kidney transplantation: kidney allograft perfusion and vessel reconstruction; 2) angiography perfusion of tissue flaps; 3) visualization of ureters; 4) visualization of urinary calcifications; and 5) NIRF in male infertility and semen quality assessment. Near infrared technology in urology is at its early stages. More studies are needed to assess the true potential and limitations of the technology. Initial studies show that this pioneering tool may influence various aspects of urology.

  13. Assessment of heavy metal bioavailability in contaminated sediments and soils using green fluorescent protein-based bacterial biosensors

    International Nuclear Information System (INIS)

    Liao, V.H.-C.; Chien, M.-T.; Tseng, Y.-Y.; Ou, K.-L.

    2006-01-01

    A green fluorescent protein (GFP)-based bacterial biosensor Escherichia coli DH5α (pVLCD1) was developed based on the expression of gfp under the control of the cad promoter and the cadC gene of Staphylococcus aureus plasmid pI258. DH5α (pVLCD1) mainly responded to Cd(II), Pb(II), and Sb(III), the lowest detectable concentrations being 0.1 nmol L -1 , 10 nmol L -1 , and 0.1 nmol L -1 , respectively, with 2 h exposure. The biosensor was field-tested to measure the relative bioavailability of the heavy metals in contaminated sediments and soil samples. The results showed that the majority of heavy metals remained adsorbed to soil particles: Cd(II)/Pb(II) was only partially available to the biosensor in soil-water extracts. Our results demonstrate that the GFP-based bacterial biosensor is useful and applicable in determining the bioavailability of heavy metals with high sensitivity in contaminated sediment and soil samples and suggests a potential for its inexpensive application in environmentally relevant sample tests. - Nonpathogenic GFP-based bacterial biosensor is applicable in determining the bioavailability of heavy metals in environmental samples

  14. Quality evaluation of the edible blue-green alga Nostoc flagelliforme using a chlorophyll fluorescence parameter and several biochemical markers.

    Science.gov (United States)

    Gao, Xiang; Yang, Yiwen; Ai, Yufeng; Luo, Hongyi; Qiu, Baosheng

    2014-01-15

    Nostoc flagelliforme is an edible blue-green alga with herbal and dietary values. Due to the diminishing supply of natural N. flagelliforme and the large investment on the development of its cultivation technology, it is anticipated that artificially cultured N. flagelliforme will soon sustain the market supply. Once this change occurs, the storage-associated quality problem will become the focus of attention for future trade. In this paper, we used a chlorophyll fluorescence parameter, maximum quantum efficiency of Photosystem II (Fv/Fm), and several biomarkers to evaluate the quality of several N. flagelliforme samples. It was found that longer storage times resulted in darker coloured solutions (released pigments) and decreased amounts of chlorophyll a (Chl a) and water-soluble sugars (WSS). Additionally, a higher Fv/Fm value suggests better physiological recovery and quality. In actual application, determination of Fv/Fm would be the first step for evaluating the quality of N. flagelliforme, and the biochemical indexes would serve as good secondary markers. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. A quasi-lentiviral green fluorescent protein reporter exhibits nuclear export features of late human immunodeficiency virus type 1 transcripts

    International Nuclear Information System (INIS)

    Graf, Marcus; Ludwig, Christine; Kehlenbeck, Sylvia; Jungert, Kerstin; Wagner, Ralf

    2006-01-01

    We have previously shown that Rev-dependent expression of HIV-1 Gag from CMV immediate early promoter critically depends on the AU-rich codon bias of the gag gene. Here, we demonstrate that adaptation of the green fluorescent protein (GFP) reporter gene to HIV codon bias is sufficient to turn this hivGFP RNA into a quasi-lentiviral message following the rules of late lentiviral gene expression. Accordingly, GFP expression was significantly decreased in transfected cells strictly correlating with reduced RNA levels. In the presence of the HIV 5' major splice donor, the hivGFP RNAs were stabilized in the nucleus and efficiently exported to the cytoplasm following fusion of the 3' Rev-responsive element (RRE) and coexpression of HIV-1 Rev. This Rev-dependent translocation was specifically inhibited by leptomycin B suggesting export via the CRM1-dependent pathway used by late lentiviral transcripts. In conclusion, this quasi-lentiviral reporter system may provide a new platform for developing sensitive Rev screening assays

  16. Comparison of indocyanine green fluorescence and parathyroid autofluorescence imaging in the identification of parathyroid glands during thyroidectomy.

    Science.gov (United States)

    Kahramangil, Bora; Berber, Eren

    2017-12-01

    Indocyanine green fluorescence (ICGF) and parathyroid autofluorescence (AF) are two new techniques that aid in the identification of parathyroid glands (PG) intraoperatively during thyroidectomy. There is no study comparing the efficacy of these techniques. This was an IRB-approved clinical study comparing the utility of ICGF and AF for identification of PGs during thyroidectomy. Data were collected prospectively. Both techniques were compared to naked eye (NE) for PG detection. Standard statistical methods were used for data analysis. Twenty-two patients in each group underwent a total of 39 total thyroidectomies and 5 thyroid lobectomies. AF and ICGF had similar detection rates for PGs [98% (61 of 62) and 95% (60 of 63) of PGs, respectively; P=0.31]. The location of PGs was suggested before detection with NE more frequently by AF than ICGF [52% (32 of 62) vs. 6% (4 of 63) of PGs; P0.99]. To the best of our knowledge, this is the first comparative study between parathyroid AF and ICGF in detection of PGs during thyroidectomy. Our data suggest both techniques have similarly high detection rates and that the main difference lies in the timing of detection. AF more frequently detects PGs before recognition with NE compared to ICGF.

  17. Assessing pharmacokinetics of indocyanine green-loaded nanoparticle in tumor with a dynamic diffuse fluorescence tomography system

    Science.gov (United States)

    Zhang, Yanqi; Yin, Guoyan; Zhao, Huijuan; Ma, Wenjuan; Gao, Feng; Zhang, Limin

    2018-02-01

    Real-time and continuous monitoring of drug release in vivo is an important task in pharmaceutical development. Here, we devoted to explore a real-time continuous study of the pharmacokinetics of free indocyanine green (ICG) and ICG loaded in the shell-sheddable nanoparticles in tumor based on a dynamic diffuse fluorescence tomography (DFT) system: A highly-sensitive dynamic DFT system of CT-scanning mode generates informative and instantaneous sampling datasets; An analysis procedure extracts the pharmacokinetic parameters from the reconstructed time curves of the mean ICG concentration in tumor, using the Gauss-Newton scheme based on two-compartment model. Compared with the pharmacokinetic parameters of free ICG in tumor, the ICG loaded in the shell-sheddable nanoparticles shows efficient accumulation in tumor. The results demonstrate our proposed dynamic-DFT can provide an integrated and continuous view of the drug delivery of the injected agents in different formulations, which is helpful for the development of diagnosis and therapy for tumors.

  18. Fusions between green fluorescent protein and beta-glucuronidase as sensitive and vital bifunctional reporters in plants.

    Science.gov (United States)

    Quaedvlieg, N E; Schlaman, H R; Admiraal, P C; Wijting, S E; Stougaard, J; Spaink, H P

    1998-11-01

    By fusing the genes encoding green fluorescent protein (GFP) and beta-glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.

  19. investigating acid production by Streptococcus mutans with a surface-displayed pH-sensitive green fluorescent protein.

    Directory of Open Access Journals (Sweden)

    Lihong Guo

    Full Text Available Acidogenicity and aciduricity are the main virulence factors of the cavity-causing bacterium Streptococcus mutans. Monitoring at the individual cell level the temporal and spatial distribution of acid produced by this important oral pathogen is central for our understanding of these key virulence factors especially when S. mutans resides in multi-species microbial communities. In this study, we explored the application of pH-sensitive green fluorescent proteins (pHluorins to investigate these important features. Ecliptic pHluorin was functionally displayed on the cell surface of S. mutans as a fusion protein with SpaP. The resulting strain (O87 was used to monitor temporal and spatial pH changes in the microenvironment of S. mutans cells under both planktonic and biofilm conditions. Using strain O87, we revealed a rapid pH drop in the microenviroment of S. mutans microcolonies prior to the decrease in the macro-environment pH following sucrose fermentation. Meanwhile, a non-uniform pH distribution was observed within S. mutans biofilms, reflecting differences in microbial metabolic activity. Furthermore, strain O87 was successfully used to monitor the S. mutans acid production profiles within dual- and multispecies oral biofilms. Based on these findings, the ecliptic pHluorin allows us to investigate in vivo and in situ acid production and distribution by the cariogenic species S. mutans.

  20. The role of bone marrow-derived cells in bone fracture repair in a green fluorescent protein chimeric mouse model

    International Nuclear Information System (INIS)

    Taguchi, Kazuhiro; Ogawa, Rei; Migita, Makoto; Hanawa, Hideki; Ito, Hiromoto; Orimo, Hideo

    2005-01-01

    We investigated the role of bone marrow cells in bone fracture repair using green fluorescent protein (GFP) chimeric model mice. First, the chimeric model mice were created: bone marrow cells from GFP-transgenic C57BL/6 mice were injected into the tail veins of recipient wild-type C57BL/6 mice that had been irradiated with a lethal dose of 10 Gy from a cesium source. Next, bone fracture models were created from these mice: closed transverse fractures of the left femur were produced using a specially designed device. One, three, and five weeks later, fracture lesions were extirpated for histological and immunohistochemical analyses. In the specimens collected 3 and 5 weeks after operation, we confirmed calluses showing intramembranous ossification peripheral to the fracture site. The calluses consisted of GFP- and osteocalcin-positive cells at the same site, although the femur consisted of only osteocalcin-positive cells. We suggest that bone marrow cells migrated outside of the bone marrow and differentiated into osteoblasts to make up the calluses

  1. Stable transformation of sunflower (Helianthus annuus L.) using a non-meristematic regeneration protocol and green fluorescent protein as a vital marker.

    Science.gov (United States)

    Müller, A; Iser, M; Hess, D

    2001-10-01

    Stable transformation of sunflower was achieved using a non-meristematic hypocotyl explant regeneration protocol of public inbred HA300B. Uniformly transformed shoots were obtained after co-cultivation with Agrobacterium tumefaciens carrying a gfp (green fluorescent protein) gene containing an intron that blocks expression of gfp in Agrobacterium. Easily detectable, bright green fluorescence of transformed tissues was used to establish an optimal regeneration and transformation procedure. By Southern blot analysis, integration of the gfp and nptll genes was confirmed. Stable transformation efficiency was 0.1%. From 68 T1 plants analyzed, 17 showed transmission of transgene DNA and 15 of them contained the intact gfp gene. Expression of gfp was detected in 10 T1 plants carrying the intact gfp gene using a fluorimetric assay or western blot analysis. Expression of the nptll gene was confirmed in 13 T1 plants. The transformation system enables the rapid transfer of agronomically important genes.

  2. Turn-off fluorescence sensor for the detection of ferric ion in water using green synthesized N-doped carbon dots and its bio-imaging.

    Science.gov (United States)

    Edison, Thomas Nesakumar Jebakumar Immanuel; Atchudan, Raji; Shim, Jae-Jin; Kalimuthu, Senthilkumar; Ahn, Byeong-Cheol; Lee, Yong Rok

    2016-05-01

    This paper reports turn-off fluorescence sensor for Fe(3+) ion in water using fluorescent N-doped carbon dots as a probe. A simple and efficient hydrothermal carbonization of Prunus avium fruit extract for the synthesis of fluorescent nitrogen-doped carbon dots (N-CDs) is described. This green approach proceeds quickly and provides good quality N-CDs. The mean size of synthesized N-CDs was approximately 7nm calculated from the high-resolution transmission electron microscopic images. X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy revealed the presence of -OH, -NH2, -COOH, and -CO functional groups over the surface of CDs. The N-CDs showed excellent fluorescent properties, and emitted blue fluorescence at 411nm upon excitation at 310nm. The calculated quantum yield of the synthesized N-CDs is 13% against quinine sulfate as a reference fluorophore. The synthesized N-CDs were used as a fluorescent probe towards the selective and sensitive detection of biologically important Fe(3+) ions in water by fluorescence spectroscopy and for bio-imaging of MDA-MB-231 cells. The limit of detection (LOD) and the Stern-Volmer quenching constant for the synthesized N-CDs were 0.96μM and 2.0958×10(3)M of Fe(3+) ions. The green synthesized N-CDs are efficiently used as a promising candidate for the detection of Fe(3+) ions and bio-imaging. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. A vector carrying the GFP gene (Green fluorescent protein as a yeast marker for fermentation processes Um vetor com o gene da GFP (Green fluorescent protein para a marcação de leveduras em processos fermentativos

    Directory of Open Access Journals (Sweden)

    Luiz Humberto Gomes

    2000-12-01

    Full Text Available Contaminant yeasts spoil pure culture fermentations and cause great losses in quality and product yields. They can be detected by a variety of methods although none being so efficient for early detection of contaminant yeast cells that appear at low frequency. Pure cultures bearing genetic markers can ease the direct identification of cells and colonies among contaminants. Fast and easy detection are desired and morphological markers would even help the direct visualization of marked pure cultures among contaminants. The GFP gene for green fluorescent protein of Aquorea victoria, proved to be a very efficient marker to visualize transformed cells in mixed populations and tissues. To test this marker in the study of contaminated yeast fermentations, the GFP gene was used to construct a vector under the control of the ADH2 promoter (pYGFP3. Since ADH2 is repressed by glucose the expression of the protein would not interfere in the course of fermentation. The transformed yeasts with the vector pYGFP3 showed high stability and high bioluminescence to permit identification of marked cells among a mixed population of cells. The vector opens the possibility to conduct further studies aiming to develop an efficient method for early detection of spoilage yeasts in industrial fermentative processes.Leveduras contaminantes podem causar grandes perdas em processos fermentativos quando infectam culturas puras e degradam a qualidade do produto final. Estas leveduras podem ser detectadas por diversos métodos mas nenhum deles oferece resultados com a exatidão e precisão necessárias, quando os contaminantes estão em baixa freqüência. Culturas puras contendo um gene marcador podem ser utilizadas para a direta identificação de células e colônias contaminantes. Detecção rápida e fácil é desejada e marcadores morfológicos podem auxiliar na visualização da cultura marcada. O gene da GFP (green fluorescent protein extraído da Aequorea victoria

  4. Development and characterization of a green fluorescent protein-based rat cell bioassay system for detection of AH receptor ligands

    Energy Technology Data Exchange (ETDEWEB)

    Zhao Bin; Denison, M. [California Univ., Davis, CA (United States). Dept. of Environmental Toxicology

    2004-09-15

    Proper epidemiological, risk assessment and exposure analysis of TCDD and related HAHs requires accurate measurements of these chemicals both in the species of interest and in various exposure matrices (i.e. biological, environmental, food and feed). While high-resolution instrumental analysis techniques are established for these chemicals, these procedures are very costly, time-consuming and are impractical for large scale sampling studies. Accordingly, numerous bioanalytical methods have been developed for the detection of these chemicals in extracts from a variety of matrices, the majority of which take the advantage of the ability of these chemicals to activate one or more aspects of the AhR-dependent mechanism of action. One of the most sensitive bioassay systems developed to date is the so-called CALUX (Chemically Activated Luciferase Expression) assay, which is based on novel recombinant cell lines that contain a stably transfected dioxin (AhR)-responsive firefly luciferase gene. Treatment of these cells with TCDD and related HAHs and polycyclic aromatic hydrocarbons (PAHs), as well as other AhR ligands, results in induction of reporter gene expression in a time-, dose-, AhR-, and chemical-specific manner. The level of reporter gene expression correlates with the total concentration of the TCDD-like AhR inducers (agonists) present in the sample. Although the firefly luciferase reporter gene contributes to the high degree of sensitivity of the assay, it also has limitations with respect to our need for a rapid and inexpensive bioassay for high-throughput screening analysis. Accordingly, we previously developed a stably transfected murine cell line containing an AhRresponsive enhanced green fluorescent protein (EGFP) reporter gene. This cell line provided us with a high-throughput cell bioassay system for identification and characterization of AhR agonists and antagonists. Here we have extended these studies and describe the development, optimization, and

  5. Visualization of plant viral suppressor silencing activity in intact leaf lamina by quantitative fluorescent imaging

    Directory of Open Access Journals (Sweden)

    Francis Kevin P

    2011-08-01

    Full Text Available Abstract Background Transient expression of proteins in plants has become a favoured method over the production of stably transformed plants because, in addition to enabling high protein yields, it is both fast and easy to apply. An enhancement of transient protein expression can be achieved by plant virus-encoded RNA silencing suppressor proteins. Since viral suppressor proteins differ in their efficiency to enhance transient protein expression in plants, we developed a whole-leaf green fluorescent protein (GFP-based imaging assay to quantitatively assess suppressor protein activity. Results In a transient GFP-expression assay using wild-type and GFP-transgenic N. benthamiana, addition of the plant viral suppressors Beet mild yellowing virus (BMYV-IPP P0 or Plum pox virus (PPV HC-Pro was shown to increase fluorescent protein expression 3-4-fold, 7 days post inoculation (dpi when compared to control plants. In contrast, in agroinfiltrated patches without suppressor activity, near complete silencing of the GFP transgene was observed in the transgenic N. benthamiana at 21 dpi. Both co-infiltrated suppressors significantly enhanced GFP expression over time, with HC-Pro co-infiltrations leading to higher short term GFP fluorescence (at 7 dpi and P0 giving higher long term GFP fluorescence (at 21 dpi. Additionally, in contrast to HC-Pro co-infiltrations, an area of complete GFP silencing was observed at the edge of P0 co-infiltrated areas. Conclusions Fluorescence imaging of whole intact leaves proved to be an easy and effective method for spatially and quantitatively observing viral suppressor efficiency in plants. This suppressor assay demonstrates that plant viral suppressors greatly enhanced transient GFP expression, with P0 showing a more prolonged suppressor activity over time than HC-Pro. Both suppressors could prove to be ideal candidates for enhancing target protein expression in plants.

  6. Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions.

    Science.gov (United States)

    Tsachaki, Maria; Birk, Julia; Egert, Aurélie; Odermatt, Alex

    2015-07-01

    Membrane proteins of the endoplasmic reticulum (ER) are involved in a wide array of essential cellular functions. Identification of the topology of membrane proteins can provide significant insight into their mechanisms of action and biological roles. This is particularly important for membrane enzymes, since their topology determines the subcellular site where a biochemical reaction takes place and the dependence on luminal or cytosolic co-factor pools and substrates. The methods currently available for the determination of topology of proteins are rather laborious and require post-lysis or post-fixation manipulation of cells. In this work, we have developed a simple method for defining intracellular localization and topology of ER membrane proteins in living cells, based on the fusion of the respective protein with redox-sensitive green-fluorescent protein (roGFP). We validated the method and demonstrated that roGFP fusion proteins constitute a reliable tool for the study of ER membrane protein topology, using as control microsomal 11β-hydroxysteroid dehydrogenase (11β-HSD) proteins whose topology has been resolved, and comparing with an independent approach. We then implemented this method to determine the membrane topology of six microsomal members of the 17β-hydroxysteroid dehydrogenase (17β-HSD) family. The results revealed a luminal orientation of the catalytic site for three enzymes, i.e. 17β-HSD6, 7 and 12. Knowledge of the intracellular location of the catalytic site of these enzymes will enable future studies on their biological functions and on the role of the luminal co-factor pool. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Ectopic bone formation cannot occur by hydroxyapatite/β-tricalcium phosphate bioceramics in green fluorescent protein chimeric mice

    Science.gov (United States)

    Cheng, Lijia; Duan, Xin; Xiang, Zhou; Shi, Yujun; Lu, Xiaofeng; Ye, Feng; Bu, Hong

    2012-12-01

    Many studies have shown that calcium phosphate ceramics (CP) have osteoconductive and osteoinductive properties; however, the exact mechanism of bone induction has not yet been reported. This study was performed to investigate if destroying immunological function will influence osteogenesis, to explain the mechanism which is unclear. In this study, twenty C57BL/6 mice were divided into two groups (n = 10), in group 1, a hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) ceramic was implanted into both the left and right leg muscles of each mouse; in group 2, ten mice experienced lethal irradiation, then were injected bone marrow (BM) cells from green fluorescent protein (GFP) transgenic mice by tail veil, after bone marrow transplantation (BMT), heart, liver, spleen, lung, kidney, and muscle were harvested for biological analysis, after the GFP chimera model was established successfully, the same HA/β-TCP ceramic was implanted into both leg muscles of each mouse immediately after irradiation. 45 and 90 days after implantation, the ceramics of the two groups were harvested to perform with hematoxylin and eosin (HE) and immunohistochemistry (IHC) staining; the results showed that there was no bone formation in group 2, while new bone tissues were detected in group 1. Our findings suggest that the BM cell from GFP transgenic mice is a good biomarker and it could set a good platform for chimera model; it also shows that BM cell is one of cell resources of bone induction, and destruction of immune function will impede osteoinduction by CP. Overall, our results may shed light on clear mechanism study of bone induction in the future.

  8. Semi-Automated Hydrophobic Interaction Chromatography Column Scouting Used in the Two-Step Purification of Recombinant Green Fluorescent Protein

    Science.gov (United States)

    Murphy, Patrick J. M.

    2014-01-01

    Background Hydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported. Methods and Results Bacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation. Conclusions Conducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in

  9. Ectopic bone formation cannot occur by hydroxyapatite/{beta}-tricalcium phosphate bioceramics in green fluorescent protein chimeric mice

    Energy Technology Data Exchange (ETDEWEB)

    Cheng Lijia [Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu (China); Duan Xin [Department of Orthopaedics, Chengdu Second People' s Hospital, Chengdu (China); Department of Orthopaedics, West China Hospital, Sichuan University, Chengdu (China); Xiang Zhou [Department of Orthopaedics, West China Hospital, Sichuan University, Chengdu (China); Shi Yujun; Lu Xiaofeng; Ye Feng [Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu (China); Bu Hong, E-mail: hongbu@scu.edu.cn [Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu (China); Department of Pathology, West China Hospital, Sichuan University, Chengdu (China)

    2012-12-01

    Highlights: Black-Right-Pointing-Pointer Firstly, chimeric mouse model could be established successfully by bone marrow transplantation after irradiation. Black-Right-Pointing-Pointer Secondly, bone induction can occur in wild-type mice 90 days after implantation, but not occur in chimeric mice. Black-Right-Pointing-Pointer Thirdly, destruction of immune function will block osteoinduction by calcium phosphate ceramics. - Abstract: Many studies have shown that calcium phosphate ceramics (CP) have osteoconductive and osteoinductive properties; however, the exact mechanism of bone induction has not yet been reported. This study was performed to investigate if destroying immunological function will influence osteogenesis, to explain the mechanism which is unclear. In this study, twenty C57BL/6 mice were divided into two groups (n = 10), in group 1, a hydroxyapatite/{beta}-tricalcium phosphate (HA/{beta}-TCP) ceramic was implanted into both the left and right leg muscles of each mouse; in group 2, ten mice experienced lethal irradiation, then were injected bone marrow (BM) cells from green fluorescent protein (GFP) transgenic mice by tail veil, after bone marrow transplantation (BMT), heart, liver, spleen, lung, kidney, and muscle were harvested for biological analysis, after the GFP chimera model was established successfully, the same HA/{beta}-TCP ceramic was implanted into both leg muscles of each mouse immediately after irradiation. 45 and 90 days after implantation, the ceramics of the two groups were harvested to perform with hematoxylin and eosin (HE) and immunohistochemistry (IHC) staining; the results showed that there was no bone formation in group 2, while new bone tissues were detected in group 1. Our findings suggest that the BM cell from GFP transgenic mice is a good biomarker and it could set a good platform for chimera model; it also shows that BM cell is one of cell resources of bone induction, and destruction of immune function will impede

  10. Characterization of the influence of 1-butyl-3-methylimidazolium chloride on the structure and thermal stability of green fluorescent protein

    International Nuclear Information System (INIS)

    Heller, William T.; O'Neill, Hugh Michael; Zhang, Qiu; Baker, Gary A.

    2010-01-01

    Ionic liquids (ILs) are finding a vast array of applications as novel solvents for a wide variety of processes that include enzymatic chemistry, particularly as more biocompatible ILs are designed and discovered. While it is assumed that a native or near-native structure is required for enzymatic activity, there is some evidence that ILs alter protein structure and oligomerization states in a manner than can negatively impact function. The IL 1-butyl-3-methylimidazolium chloride, (bmim)Cl, is a well-studied, water-miscible member of the popular 1-alkyl-3-methylimidazolium IL family. To improve our understanding of the impact of water-miscible ILs on proteins, we have characterized the structure and oligomerization state of green fluorescent protein (GFP) in aqueous solutions containing 25 and 50 vol % (bmim)Cl using a combination of optical spectroscopy and small-angle neutron scattering (SANS). Measurements were also performed as a function of temperature to provide insight into the effect of the IL on the thermal stability of GFP. While GFP exists as a dimer in water, the presence of 25 vol % (bmim)Cl causes GFP to transition to a monomeric state. The SANS data indicate that GFP is a great deal less compact in 50 vol % (bmim)Cl than in neat water, indicative of unfolding from the native structure. The oligomerization state of the protein in IL-containing aqueous solution changes from a dimer to a monomer in response to the IL, but does not change as a function of temperature in the IL-containing solution. The SANS and spectroscopic results also demonstrate that the addition of (bmim)Cl to the solution decreases the thermal stability of GFP, allowing the protein to unfold at lower temperatures than in aqueous solution.

  11. Survival of salmonella transformed to express green fluorescent protein on Italian parsley as affected by processing and storage.

    Science.gov (United States)

    Duffy, E A; Cisneros-Zevallos, L; Castillo, A; Pillai, S D; Ricke, S C; Acuff, G R

    2005-04-01

    To study the effect of processing and storage parameters on the survival of Salmonella on fresh Italian parsley, parsley bunches were dipped for 3 or 15 min in suspensions that were preequilibrated to 5, 25, or 35 degrees C and inoculated with Salmonella transformed to express enhanced green fluorescent protein. Loosely attached and/or associated, strongly attached and/or associated, and internalized and/or entrapped Salmonella cells were enumerated over 0, 1, and 7 days of storage at 25 degrees C and over 0, 1, 7, 14, and 30 days of storage at 4 degrees C using surface-plating procedures. Leaf sections obtained from samples after 0, 1, and 7 days of storage were examined using confocal scanning laser microscopy. Temperature of the dip suspension had little effect on the attachment and survival of Salmonella cells on parsley. Regardless of the temperature or duration of dip, Salmonella was internalized. Immersion for longer times resulted in higher numbers of attached and internalized cells. Microscopic observations supported these results and revealed Salmonella cells near the stomata and within cracks in the cuticle. Storage temperature had the greatest impact on the survival of Salmonella cells on parsley. When stored at 25 degrees C, parsley had a shelf life of 7 days, and Salmonella populations significantly increased over the 7 days of storage. For parsley stored at 4 degrees C, numbers of Salmonella cells decreased over days 0, 1, and 7. After 7 days of storage, there were no viable internalized Salmonella cells detected. Storage temperature represents an important control point for the safety of fresh parsley.

  12. Use of Green Fluorescent Protein-Transgenic Strains to Study Pathogenic and Nonpathogenic Lifestyles in Colletotrichum acutatum.

    Science.gov (United States)

    Horowitz, Sigal; Freeman, Stanley; Sharon, Amir

    2002-07-01

    ABSTRACT Colletotrichum acutatum, which causes anthracnose disease on strawberry, can also persist on several other plant species without causing disease symptoms. The genetic and molecular bases that determine pathogenic and nonpathogenic lifestyles in C. acutatum are unclear. We developed a transformation system for C. acutatum by electroporation of germinating conidia, and transgenic isolates that express the green fluorescent protein (GFP) were produced. Details of the pathogenic and nonpathogenic lifestyles of C. acutatum were determined by using GFP-transgenic isolates. Major differences between colonization-mediating processes of strawberry and of other plants were observed. On the main host, strawberry, the germinating conidia formed branched, thick hyphae, and large numbers of appressoria were produced that were essential for plant penetration. In strawberry, the fungus developed rapidly, filling the mesophyll with dense mycelium that invaded the cells and caused necrosis of the tissue. In nonpathogenic interactions on pepper, eggplant, and tomato, the conidia germinated, producing thin, straight germ tubes. Appressoria were produced but failed to germinate and penetrate leaf tissue, resulting in epiphytic growth without invasion of the plant. Penetration of the plant occurred only several days after inoculation and was restricted to the intercellular spaces of the first cell layers of infected tissue without causing any visible damage. Much of the new fungal biomass continued to develop on the surface of inoculated organs in the nonpathogenic interaction. The differences in fungal development on strawberry compared with the other plant species suggest that signal molecules, which may be present only in strawberry, trigger appressorial germination and penetration of the primary host.

  13. Green fluorescent protein labeling of Listeria, Salmonella, and Escherichia coli O157:H7 for safety-related studies.

    Directory of Open Access Journals (Sweden)

    Li Ma

    Full Text Available Many food safety-related studies require tracking of introduced foodborne pathogens to monitor their fate in complex environments. The green fluorescent protein (GFP gene (gfp provides an easily detectable phenotype so has been used to label many microorganisms for ecological studies. The objectives of this study were to label major foodborne pathogens and related bacteria, including Listeria monocytogenes, Listeria innocua, Salmonella, and Escherichia coli O157:H7 strains, with GFP and characterize the labeled strains for stability of the GFP plasmid and the plasmid's effect on bacterial growth. GFP plasmids were introduced into these strains by a CaCl(2 procedure, conjugation or electroporation. Stability of the label was determined through sequential propagation of labeled strains in the absence of selective pressure, and rates of plasmid-loss were calculated. Stability of the GFP plasmid varied among the labeled species and strains, with the most stable GFP label observed in E. coli O157:H7. When grown in nonselective media for two consecutive subcultures (ca. 20 generations, the rates of plasmid loss among labeled E. coli O157:H7, Salmonella and Listeria strains ranged from 0%-30%, 15.8%-99.9% and 8.1%-93.4%, respectively. Complete loss (>99.99% of the plasmid occurred in some labeled strains after five consecutive subcultures in the absence of selective pressure, whereas it remained stable in others. The GFP plasmid had an insignificant effect on growth of most labeled strains. E. coli O157:H7, Salmonella and Listeria strains can be effectively labeled with the GFP plasmid which can be stable in some isolates for many generations without adversely affecting growth rates.

  14. Magnetic solid-phase extraction for determination of the total malachite green, gentian violet and leucomalachite green, leucogentian violet in aquaculture water by high-performance liquid chromatography with fluorescence detection.

    Science.gov (United States)

    Zhao, Jiao; Wei, Daqiao; Yang, Yaling

    2016-06-01

    In this study, magnetic multi-walled carbon nanotube nanoparticles were synthesized and used as the adsorbent for the sums of malachite green, gentian violet and leucomalachite green, leucogentian violet in aquaculture water samples followed by high performance liquid chromatography with fluorescence detection. This method was based on in situ reduction of chromic malachite green, gentian violet to colorless leucomalachite green, leucogentian violet with potassium borohydride, respectively. The obtained adsorbent combines the advantages of carbon nanotubes and Fe3 O4 nanoparticles in one material for separation and preconcentration of the reductive dyes in aqueous media. The structure and properties of the prepared nanoparticles were characterized by transmission and scanning electron microscopy, X-ray diffraction, and Fourier-transform infrared spectroscopy. The main parameters affecting the adsorption recoveries were investigated and optimized, including reducing agent concentration, type and amount of sorbent, sample pH, and eluting conditions. Under the optimum conditions, the limits of detection in this method were 0.22 and 0.09 ng/mL for malachite green and gentian violet, respectively. Product recoveries ranged from 87.0 to 92.8% with relative standard deviations from 4.6 to 5.9%. The results indicate that the sorbent is a suitable material for the removal and concentration of triphenylmethane dyes from polluted environmental samples. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Blue-green fluorescence and visible-infrared reflectance of corn (Zea mays L.) grain for in situ field detection of nitrogen supply

    International Nuclear Information System (INIS)

    McMurtrey, J.E. III; Chappelle, E.W.; Kim, M.S.; Corp, L.A.; Daughtry, C.S.T.

    1996-01-01

    The sensing of spectral attributes of corn (Zea mays L.) grain from site specific areas of the field during the harvest process may be useful in managing agronomic inputs and production practices on those areas of the field in subsequent growing seasons. Eight levels of nitrogen (N) fertilization were applied to field grown corn at Beltsville, Maryland. These N treatments produced a range of chlorophyll levels, biomass and physiological condition in the live plant canopies. After harvest, spectra were obtained in the laboratory on whole grain samples. Fluorescence emissions were acquired from 400 to 600 nm and percent reflectance were measured in the visible (VIS) near infrared (NIR) and mid-infrared (MIR) regions from 400 nm to 2400 nm. A ultraviolet (UV) excitation band centered at 385 nm was the most effective in producing fluorescence emission differences in the blue-green region of the fluorescence spectrum with maxima centered from 430-470nm in the blue and with an intense shoulder centered at around 530-560 nm in the green region. Reflectance showed the most spectral differences in the NIR and MIR (970-2330 nm) regions

  16. Automated Analysis of Protein Expression and Gene Amplification within the Same Cells of Paraffin-Embedded Tumour Tissue

    Directory of Open Access Journals (Sweden)

    Timo Gaiser

    2010-01-01

    Full Text Available Background: The simultaneous detection of protein expression and gene copy number changes in patient samples, like paraffin-embedded tissue sections, is challenging since the procedures of immunohistochemistry (IHC and Fluorescence in situ Hybridization (FISH negatively influence each other which often results in suboptimal staining. Therefore, we developed a novel automated algorithm based on relocation which allows subsequent detection of protein content and gene copy number changes within the same cell.

  17. The E1 mechanism in photo-induced beta-elimination reactions for green-to-red conversion of fluorescent proteins.

    Science.gov (United States)

    Tsutsui, Hidekazu; Shimizu, Hideaki; Mizuno, Hideaki; Nukina, Nobuyuki; Furuta, Toshiaki; Miyawaki, Atsushi

    2009-11-25

    KikGR is a fluorescent protein engineered to display green-to-red photoconvertibility that is induced by irradiation with ultraviolet or violet light. Similar to Kaede and EosFP, two naturally occurring photoconvertible proteins, KikGR contains a His(62)-Tyr(63)-Gly(64) tripeptide sequence, which forms a green chromophore that can be photoconverted to a red one via formal beta-elimination and subsequent extension of a pi-conjugated system. Using a crystallizable variant of KikGR, we determined the structures of both the green and red state at 1.55 A resolution. The double bond between His(62)-C(alpha) and His(62)-C(beta) in the red chromophore is in a cis configuration, indicating that rotation along the His(62) C(alpha)-C(beta) bond occurs following cleavage of the His(62) N(alpha)-C(alpha) bond. This structural rearrangement provides evidence that the beta-elimination reaction governing the green-to-red photoconversion of KikGR follows an E1 (elimination, unimolecular) mechanism.

  18. Fluorescent minerals - A potential source of UV protection and visible light for the growth of green algae and cyanobacteria in extreme cosmic environments

    Science.gov (United States)

    Omairi, Tareq; Wainwright, Milton

    2015-07-01

    We propose that green algae (Chlorella variabilis and Dunaliella tertiolecta) and cyanobacteria (Synechococcus elongatus and Nostoc commune) can grow inside fluorescent rock minerals which convert damaging UV light to visible light, thereby allowing these organisms to survive and thrive in UV-rich environments without (or with limited) visible light, which would otherwise be inimical to them. The four microorganisms were incubated inside fluorescent rocks composed of fluorite, calcite and pyrite. The resultant growth was then measured following exposure to UV radiation, with the use of optical density and measurement of chlorophyll concentration. Results show that the microorganisms were shielded from harmful UV in these semi-transparent rocks, while at the same time benefiting from the fact that the minerals converted UV to visible light; this have been shown by a statistically significant increase in their growth, which although lower than when the cells were incubated in sunlight, was significantly higher than in controls incubated in the dark.

  19. Imaging of pharmacokinetic rates of indocyanine green in mouse liver with a hybrid fluorescence molecular tomography/x-ray computed tomography system.

    Science.gov (United States)

    Zhang, Guanglei; Liu, Fei; Zhang, Bin; He, Yun; Luo, Jianwen; Bai, Jing

    2013-04-01

    Pharmacokinetic rates have the potential to provide quantitative physiological and pathological information for biological studies and drug development. Fluorescence molecular tomography (FMT) is an attractive imaging tool for three-dimensionally resolving fluorophore distribution in small animals. In this letter, pharmacokinetic rates of indocyanine green (ICG) in mouse liver are imaged with a hybrid FMT and x-ray computed tomography (XCT) system. A recently developed FMT method using structural priors from an XCT system is adopted to improve the quality of FMT reconstruction. In the in vivo experiments, images of uptake and excretion rates of ICG in mouse liver are obtained, which can be used to quantitatively evaluate liver function. The accuracy of the results is validated by a fiber-based fluorescence measurement system.

  20. Comparison of High Performance Liquid Chromatography with Fluorescence Detector and with Tandem Mass Spectrometry methods for detection and quantification of Ochratoxin A in green and roasted coffee beans

    Directory of Open Access Journals (Sweden)

    Raquel Duarte da Costa Cunha Bandeira

    2013-12-01

    Full Text Available Two analytical methods for the determination and confirmation of ochratoxin A (OTA in green and roasted coffee samples were compared. Sample extraction and clean-up were based on liquid-liquid phase extraction and immunoaffinity column. The detection of OTA was carried out with the high performance liquid chromatography (HPLC combined either with fluorescence detection (FLD, or positive electrospray ionization (ESI+ coupled with tandem mass spectrometry (MS-MS. The results obtained with the LC-ESI-MS/MS were specific and more sensitive, with the advantages in terms of unambiguous analyte identification, when compared with the HPLC-FLD.

  1. Superselective intra-arterial hepatic injection of indocyanine green (ICG) for fluorescence image-guided segmental positive staining: experimental proof of the concept.

    Science.gov (United States)

    Diana, Michele; Liu, Yu-Yin; Pop, Raoul; Kong, Seong-Ho; Legnèr, Andras; Beaujeux, Remy; Pessaux, Patrick; Soler, Luc; Mutter, Didier; Dallemagne, Bernard; Marescaux, Jacques

    2017-03-01

    Intraoperative liver segmentation can be obtained by means of percutaneous intra-portal injection of a fluorophore and illumination with a near-infrared light source. However, the percutaneous approach is challenging in the minimally invasive setting. We aimed to evaluate the feasibility of fluorescence liver segmentation by superselective intra-hepatic arterial injection of indocyanine green (ICG). Eight pigs (mean weight: 26.01 ± 5.21 kg) were involved. Procedures were performed in a hybrid experimental operative suite equipped with the Artis Zeego ® , multiaxis robotic angiography system. A pneumoperitoneum was established and four laparoscopic ports were introduced. The celiac trunk was catheterized, and a microcatheter was advanced into different segmental hepatic artery branches. A near-infrared laparoscope (D-Light P, Karl Storz) was used to detect the fluorescent signal. To assess the correspondence between arterial-based fluorescence demarcation and liver volume, metallic markers were placed along the fluorescent border, followed by a 3D CT-scanning, after injecting intra-arterial radiological contrast (n = 3). To assess the correspondence between arterial and portal supplies, percutaneous intra-portal angiography and intra-arterial angiography were performed simultaneously (n = 1). Bright fluorescence signal enhancing the demarcation of target segments was obtained from 0.1 mg/mL, in matter of seconds. Correspondence between the volume of hepatic segments and arterial territories was confirmed by CT angiography. Higher background fluorescence noise was found after positive staining by intra-portal ICG injection, due to parenchymal accumulation and porto-systemic shunting. Intra-hepatic arterial ICG injection, rapidly highlights hepatic target segment borders, with a better signal-to-background ratio as compared to portal vein injection, in the experimental setting.

  2. Cucurbitacin delta 23-reductase from the fruit of Cucurbita maxima var. Green Hubbard. Physicochemical and fluorescence properties and enzyme-ligand interactions.

    Science.gov (United States)

    Dirr, H W; Schabort, J C; Weitz, C

    1986-02-01

    Cucurbitacin delta 23-reductase from Cucurbita maxima var. Green Hubbard fruit displays an apparent Mr of 32,000, a Stokes radius of 263 nm and a diffusion coefficient of 8.93 X 10(-7) cm2 X s-1. The enzyme appears to possess a homogeneous dimeric quaternary structure with a subunit Mr of 15,000. Two tryptophan and fourteen tyrosine residues per dimer were found. Emission spectral properties of the enzyme and fluorescence quenching by iodide indicate the tryptophan residues to be buried within the protein molecule. In the pH range 5-7, where no conformational changes were detected, protonation of a sterically related ionizable group with a pK of approx. 6.0 markedly influenced the fluorescence of the tryptophan residues. Protein fluorescence quenching was employed to determine the dissociation constants for binding of NADPH (Kd 17 microM), NADP+ (Kd 30 microM) and elaterinide (Kd 227 microM). Fluorescence energy transfer between the tryptophan residues and enzyme-bound NADPH was observed.

  3. The effect of excess expression of GFP in a novel heart-specific green fluorescence zebrafish regulated by nppa enhancer at early embryonic development.

    Science.gov (United States)

    Huang, Wen; Deng, Yun; Dong, Wei; Yuan, Wuzhou; Wan, Yongqi; Mo, Xiaoyan; Li, Yongqing; Wang, Zequn; Wang, Yuequn; Ocorr, Karen; Zhang, Bo; Lin, Shuo; Wu, Xiushan

    2011-02-01

    In order to study the impalpable effect of GFP in homozygous heart-specific GFP-positive zebrafish during the early stage, the researchers analyzed the heart function of morphology and physiology at the first 3 days after fertilization. This zebrafish line was produced by a large-scale Tol2 transposon mediated enhancer trap screen that generated a transgenic zebrafish with a heart-specific expression of green fluorescent protein (GFP)-tagged under control of the nppa enhancer. In situ hybridization experiments showed that the nppa:GFP line faithfully recapitulated both the spatial and temporal expressions of the endogenous nppa. Green fluorescence was intensively and specifically expressed in the myocardial cells located both in the heart chambers and in the atrioventricular canal. The embryonic heart of nppa:GFP line developed normally compared with those in the wild type. There was no difference between the nappa:GFP and wild type lines with respect to heart rate, overall size, ejection volume, and fractional shortening. Thus the excess expression of GFP in this transgenic line seemed to exert no detrimental effects on zebrafish hearts during the early stages.

  4. Establishment of a hepatocellular carcinoma cell line expressing dual reporter genes: sodium iodide symporter (NIS) and enhanced green fluorescence protein (EGFP)

    International Nuclear Information System (INIS)

    Kwak, Won Jung; Koo, Bon Chul; Kwon, Mo Sun

    2007-01-01

    Dual reporter gene imaging has several advantages for more sophisticated molecular imaging studies such as gene therapy monitoring. Herein, we have constructed hepatoma cell line expressing dual reporter genes of sodium iodide symporter (NIS) and enhanced green fluorescence protein (EGFP), and the functionalities of the genes were evaluated in vivo by nuclear and optical imaging. A pRetro-PN vector was constructed after separating NIS gene from pcDNA-NIS. RSV-EGFP-WPRE fragment separated from pLNRGW was cloned into pRetro-PN vector. The final vector expressing dual reporter genes was named pRetro-PNRGW. A human hepatoma (HepG2) cells were transfected by the retrovirus containing NIS and EGFP gene (HepG2-NE). Expression of NIS gene was confirmed by RT-PCR, radioiodine uptake and efflux studies. Expression of EGFP was confirmed by RT-PCR and fluorescence microscope. The HepG2 and HepG2-NE cells were implanted in shoulder and hindlimb of nude mice, then fluorescence image, gamma camera image and I-124 microPET image were undertaken. The HepG2-NE cell was successfully constructed. RT-PCR showed NIS and EGFP mRNA expression. About 50% of cells showed fluorescence. The iodine uptake of NIS-expressed cells was about 9 times higher than control. In efflux study, T 1/2 of HepG2-NE cells was 9 min. HepG2-NE xenograft showed high signal-to-background fluorescent spots and higher iodine-uptake compared to those of HepG2 xenograft. A hepatoma cell line expressing NIS and EGFP dual reporter genes was successfully constructed and could be used as a potential either by therapeutic gene or imaging reporter gene

  5. An efficient tag derived from the common epitope of tospoviral NSs proteins for monitoring recombinant proteins expressed in both bacterial and plant systems.

    Science.gov (United States)

    Cheng, Hao-Wen; Chen, Kuan-Chun; Raja, Joseph A J; Li, Jian-Xian; Yeh, Shyi-Dong

    2013-04-15

    NSscon (23 aa), a common epitope in the gene silencing suppressor NSs proteins of the members of the Watermelon silver mottle virus (WSMoV) serogroup, was previously identified. In this investigation, we expressed different green fluorescent protein (GFP)-fused deletions of NSscon in bacteria and reacted with NSscon monoclonal antibody (MAb). Our results indicated that the core 9 amino acids, "(109)KFTMHNQIF(117)", denoted as "nss", retain the reactivity of NSscon. In bacterial pET system, four different recombinant proteins labeled with nss, either at N- or C-extremes, were readily detectable without position effects, with sensitivity superior to that for the polyhistidine-tag. When the nss-tagged Zucchini yellow mosaic virus (ZYMV) helper component-protease (HC-Pro) and WSMoV nucleocapsid protein were transiently expressed by agroinfiltration in tobacco, they were readily detectable and the tag's possible efficacy for gene silencing suppression was not noticed. Co-immunoprecipitation of nss-tagged and non-tagged proteins expressed from bacteria confirmed the interaction of potyviral HC-Pro and coat protein. Thus, we conclude that this novel nss sequence is highly valuable for tagging recombinant proteins in both bacterial and plant expression systems. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Construction of a ColD cda Promoter-Based SOS-Green Fluorescent Protein Whole-Cell Biosensor with Higher Sensitivity toward Genotoxic Compounds than Constructs Based on recA, umuDC, or sulA Promoters

    DEFF Research Database (Denmark)

    Norman, Anders; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

    2005-01-01

    Four different green fluorescent protein (GFP)-based whole-cell biosensors were created based on the DNA damage inducible SOS response of Escherichia coli in order to evaluate the sensitivity of individual SOS promoters toward genotoxic substances. Treatment with the known carcinogen N-methyl-N'-......Four different green fluorescent protein (GFP)-based whole-cell biosensors were created based on the DNA damage inducible SOS response of Escherichia coli in order to evaluate the sensitivity of individual SOS promoters toward genotoxic substances. Treatment with the known carcinogen N......-cell biosensor which is not only able to detect minute levels of genotoxins but, due to its use of the green fluorescent protein, also a reporter system which should be applicable in high-throughput screening assays as well as a wide variety of in situ detection studies....

  7. Bcl-2 protein expression is associated with p27 and p53 protein expressions and MIB-1 counts in breast cancer

    International Nuclear Information System (INIS)

    Tsutsui, Shinichi; Yasuda, Kazuhiro; Suzuki, Kosuke; Takeuchi, Hideya; Nishizaki, Takashi; Higashi, Hidefumi; Era, Shoichi

    2006-01-01

    Recent experimental studies have shown that Bcl-2, which has been established as a key player in the control of apoptosis, plays a role in regulating the cell cycle and proliferation. The aim of this study was to investigate the relationship between Bcl-2 and p27 protein expression, p53 protein expression and the proliferation activity as defined by the MIB-1 counts. The prognostic implication of Bcl-2 protein expression in relation to p27 and p53 protein expressions and MIB-1 counts for breast cancer was also evaluated. The immunohistochemical expression of Bcl-2 protein was evaluated in a series of 249 invasive ductal carcinomas of the breast, in which p27 and p53 protein expressions and MIB-1 counts had been determined previously. The Bcl-2 protein expression was found to be decreased in 105 (42%) cases. A decreased Bcl-2 protein expression was significantly correlated with a nuclear grade of III, a negative estrogen receptor, a decreased p27 protein expression, a positive p53 protein expression, positive MIB-1 counts and a positive HER2 protein expression. The incidence of a nuclear grade of III and positive MIB-1 counts increased as the number of abnormal findings of Bcl-2, p27 and p53 protein expressions increased. A univariate analysis indicated a decreased Bcl-2 protein expression to be significantly (p = 0.0089) associated with a worse disease free survival (DFS), while a multivariate analysis indicated the lymph node status and MIB-1 counts to be independently significant prognostic factors for the DFS. The Bcl-2 protein expression has a close correlation with p27 and p53 protein expressions and the proliferation activity determined by MIB-1 counts in invasive ductal carcinoma of the breast. The prognostic value of Bcl-2 as well as p27 and p53 protein expressions was dependent on the proliferation activity in breast cancer

  8. Bcl-2 protein expression is associated with p27 and p53 protein expressions and MIB-1 counts in breast cancer

    Directory of Open Access Journals (Sweden)

    Nishizaki Takashi

    2006-07-01

    Full Text Available Abstract Background Recent experimental studies have shown that Bcl-2, which has been established as a key player in the control of apoptosis, plays a role in regulating the cell cycle and proliferation. The aim of this study was to investigate the relationship between Bcl-2 and p27 protein expression, p53 protein expression and the proliferation activity as defined by the MIB-1 counts. The prognostic implication of Bcl-2 protein expression in relation to p27 and p53 protein expressions and MIB-1 counts for breast cancer was also evaluated. Methods The immunohistochemical expression of Bcl-2 protein was evaluated in a series of 249 invasive ductal carcinomas of the breast, in which p27 and p53 protein expressions and MIB-1 counts had been determined previously. Results The Bcl-2 protein expression was found to be decreased in 105 (42% cases. A decreased Bcl-2 protein expression was significantly correlated with a nuclear grade of III, a negative estrogen receptor, a decreased p27 protein expression, a positive p53 protein expression, positive MIB-1 counts and a positive HER2 protein expression. The incidence of a nuclear grade of III and positive MIB-1 counts increased as the number of abnormal findings of Bcl-2, p27 and p53 protein expressions increased. A univariate analysis indicated a decreased Bcl-2 protein expression to be significantly (p = 0.0089 associated with a worse disease free survival (DFS, while a multivariate analysis indicated the lymph node status and MIB-1 counts to be independently significant prognostic factors for the DFS. Conclusion The Bcl-2 protein expression has a close correlation with p27 and p53 protein expressions and the proliferation activity determined by MIB-1 counts in invasive ductal carcinoma of the breast. The prognostic value of Bcl-2 as well as p27 and p53 protein expressions was dependent on the proliferation activity in breast cancer.

  9. Tuning protein expression using synonymous codon libraries targeted to the 5' mRNA coding region

    DEFF Research Database (Denmark)

    Goltermann, Lise; Borch Jensen, Martin; Bentin, Thomas

    2011-01-01

    intermediate expression levels of green fluorescent protein in Escherichia coli. At least in one case, no apparent effect on protein stability was observed, pointing to RNA level effects as the principal reason for the observed expression differences. Targeting a synonymous codon library to the 5' coding...

  10. Real-time navigation system for sentinel lymph node biopsy in breast cancer patients using projection mapping with indocyanine green fluorescence.

    Science.gov (United States)

    Takada, Masahiro; Takeuchi, Megumi; Suzuki, Eiji; Sato, Fumiaki; Matsumoto, Yoshiaki; Torii, Masae; Kawaguchi-Sakita, Nobuko; Nishino, Hiroto; Seo, Satoru; Hatano, Etsuro; Toi, Masakazu

    2018-05-09

    Inability to visualize indocyanine green fluorescence images in the surgical field limits the application of current near-infrared fluorescence imaging (NIR) systems for real-time navigation during sentinel lymph node (SLN) biopsy in breast cancer patients. The aim of this study was to evaluate the usefulness of the Medical Imaging Projection System (MIPS), which uses active projection mapping, for SLN biopsy. A total of 56 patients (59 procedures) underwent SLN biopsy using the MIPS between March 2016 and November 2017. After SLN biopsy using the MIPS, residual SLNs were removed using a conventional NIR camera and/or radioisotope method. The primary endpoint of this study was identification rate of SLNs using the MIPS. In all procedures, at least one SLN was detected by the MIPS, giving an SLN identification rate of 100% [95% confidence interval (CI) 94-100%]. SLN biopsy was successfully performed without operating lights in all procedures. In total, 3 positive SLNs were excised using MIPS, but were not included in the additional SLNs excised by other methods. The median number of SLNs excised using the MIPS was 3 (range 1-7). Of procedures performed after preoperative systemic therapy, the median number of SLNs excised using the MIPS was 3 (range 2-6). The MIPS is effective in detecting SLNs in patients with breast cancer, providing continuous and accurate projection of fluorescence signals in the surgical field, without need for operating lights, and could be useful in real-time navigation surgery for SLN biopsy.

  11. Nanoscale orientation and lateral organization of chimeric metal-binding green fluorescent protein on lipid membrane determined by epifluorescence and atomic force microscopy

    International Nuclear Information System (INIS)

    Prachayasittikul, Virapong; Isarankura Na Ayudhya, Chartchalerm; Tantimongcolwat, Tanawut; Galla, Hans-Joachim

    2005-01-01

    Epifluorescence microscopy as well as atomic force microscopy was successfully applied to explore the orientation and lateral organization of a group of chimeric green fluorescent proteins (GFPs) on lipid membrane. Incorporation of the chimeric GFP carrying Cd-binding region (His6CdBP4GFP) to the fluid phase of DPPC monolayer resulted in a strong fluorescence intensity at the air-water interface. Meanwhile, non-specific adsorption of the GFP having hexahistidine (His6GFP) led to the perturbation of the protein structure in which very low fluorescence was observed. Specific binding of both of the chimeric GFPs to immobilized zinc ions underneath the metal-chelating lipid membrane was revealed. This specific binding could be reversibly controlled by addition of metal ions or metal chelator. Binding of the chimeric GFPs to the metal-chelating lipid membrane was proven to be the end-on orientation while the side-on adsorption was contrarily noted in the absence of metal ions. Increase of lateral mobility owing to the fluidization effect on the chelating lipid membrane subsequently facilitated crystal formation. All these findings have opened up a potential approach for a specific orientation of immobilization of protein at the membrane interface. This could have accounted for a better opportunity of sensor development

  12. Scale-up from microtiter plate to laboratory fermenter: evaluation by online monitoring techniques of growth and protein expression in Escherichia coli and Hansenula polymorpha fermentations

    Directory of Open Access Journals (Sweden)

    Engelbrecht Christoph

    2009-12-01

    Full Text Available Abstract Background In the past decade, an enormous number of new bioprocesses have evolved in the biotechnology industry. These bioprocesses have to be developed fast and at a maximum productivity. Up to now, only few microbioreactors were developed to fulfill these demands and to facilitate sample processing. One predominant reaction platform is the shaken microtiter plate (MTP, which provides high-throughput at minimal expenses in time, money and work effort. By taking advantage of this simple and efficient microbioreactor array, a new online monitoring technique for biomass and fluorescence, called BioLector, has been recently developed. The combination of high-throughput and high information content makes the BioLector a very powerful tool in bioprocess development. Nevertheless, the scalabilty of results from the micro-scale to laboratory or even larger scales is very important for short development times. Therefore, engineering parameters regarding the reactor design and its operation conditions play an important role even on a micro-scale. In order to evaluate the scale-up from a microtiter plate scale (200 μL to a stirred tank fermenter scale (1.4 L, two standard microbial expression systems, Escherichia coli and Hansenula polymorpha, were fermented in parallel at both scales and compared with regard to the biomass and protein formation. Results Volumetric mass transfer coefficients (kLa ranging from 100 to 350 1/h were obtained in 96-well microtiter plates. Even with a suboptimal mass transfer condition in the microtiter plate compared to the stirred tank fermenter (kLa = 370-600 1/h, identical growth and protein expression kinetics were attained in bacteria and yeast fermentations. The bioprocess kinetics were evaluated by optical online measurements of biomass and protein concentrations exhibiting the same fermentation times and maximum signal deviations below 10% between the scales. In the experiments, the widely applied green

  13. Structural Basis of X-ray-Induced Transient Photo-bleaching in a Photoactivatable Green Fluorescent Protein

    Energy Technology Data Exchange (ETDEWEB)

    Adam, V. [European Synchrotron Radiation Facility, 6 rue Jules Horowitz, BP 220, 38043 Grenoble Cedex (France); Carpentier, Ph.; Lelimousin, M.; Darnault, C.; Bourgeois, D. [IBS, Institut de Biologie Structurale Jean-Pierre Ebel, CEA, CNRS, UniVersite Joseph Fourier, 41 rue Jules Horowitz, 38027 Grenoble (France); Violot, S. [Laboratoire de Physiologie Cellulaire Vegetale, Institut de Recherches en Technologie et Sciences pour le ViVant, CEA, CNRS, INRA, UniVersite Joseph Fourier, 17 rue des Martyrs, F-38054 Grenoble (France); Nienhaus, U. [Institute of Applied Physics and Center for Functional nano-structures (CFN), Karlsruhe Institute of Technology, 76128 Karlsruhe (Germany); Nienhaus, U. [Department of Physics, UniVersity of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (US)

    2009-07-01

    We have observed the photoactivatable fluorescent protein IrisFP in a transient dark state with near-atomic resolution. This dark state is assigned to a radical species that either relaxes to the ground state or evolves into a permanently bleached chromophore. We took advantage of X-rays to populate the radical, which presumably forms under illumination with visible light by an electron-transfer reaction in the triplet state. The combined X-ray diffraction and in crystallo UV-vis absorption, fluorescence, and Raman data reveal that radical formation in IrisFP involves pronounced but reversible distortion of the chromophore, suggesting a transient loss of {pi} conjugation. These results reveal that the methylene bridge of the chromophore is the Achilles' heel of fluorescent proteins and help unravel the mechanisms of blinking and photo-bleaching in FPs, which are of importance in the rational design of photo-stable variants. and is also partly reversible. (authors)

  14. One-pot evaporation–condensation strategy for green synthesis of carbon nitride quantum dots: An efficient fluorescent probe for ion detection and bioimaging

    International Nuclear Information System (INIS)

    Yin, Ying; Zhang, Yumin; Gao, Tangling; Yao, Tai; Han, Jiecai; Han, Zhengbin; Zhang, Zhihua; Wu, Qiong; Song, Bo

    2017-01-01

    Herein, highly blue graphitic carbon nitride quantum dots (g-CNQDs) were synthesized by one-step microwave-assisted evaporation–condensation strategy using bulk g-C_3N_4 as the precursor within 5 min. In contrast with conventional chemical routes, the as-synthesized g-CNQDs exhibited a high crystalline quality, excellent fluorescence characteristics, and a narrow size distribution with an average diameter of 3.5 ± 0.5 nm. More importantly, by using a household microwave oven, this method has the advantages of wide accessibility, environmental friendliness, a high yield of ∼40%, and can be facilely synthesized in a large scale (scaled up to a gram scale). Notably, owing to the absence of any organic reagents, the blueas-prepared g-CNQDs show the excitation wavelength-independent photoluminescence (PL) behavior. Moreover, benefiting from the stable PL emission, good water solubility, and extraordinary biocompatibility with a high quantum yield of ∼17%, the fluorescent g-CNQDs can serve as a potential sensitive and selective probe for Fe"3"+ detection with a super low detection limit of 2 nM and an effective labeling agent for live-cell imaging. This work provides a unique opportunity to obtain g-CNQDs in large scale via a facile route, which may pave the way for the further design of g-CNQDs with other applications. - Highlights: • Green synthesis of g-CNQDs via one-step evaporation-condensation method. • The g-CNQDs have shown high crystalline quality and intrinsic fluorescence features. • The fluorescent g-CNQDs can serve as a sensitive and selective probe to detect Fe"3"+ ions with a low detection limit of 2 nM. • g-CNQDs can serve as an effective labeling agent for live-cell imaging with extraordinary biocompatibility.

  15. Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei.

    Science.gov (United States)

    Su, Shengchang; Bangar, Hansraj; Saldanha, Roland; Pemberton, Adin; Aronow, Bruce; Dean, Gary E; Lamkin, Thomas J; Hassett, Daniel J

    2014-10-01

    Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253-, P2cysZK-, and P1-driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP- and RFP-tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild-type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high-throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  16. One-pot evaporation–condensation strategy for green synthesis of carbon nitride quantum dots: An efficient fluorescent probe for ion detection and bioimaging

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Ying; Zhang, Yumin [Center for Composite Materials, Harbin Institute of Technology, Harbin 150001 (China); Gao, Tangling [Institute of Petrochemistry, Heilongjiang Academy of Sciences, Harbin 150040 (China); Yao, Tai [Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology, Harbin 150001 (China); Han, Jiecai [Center for Composite Materials, Harbin Institute of Technology, Harbin 150001 (China); Han, Zhengbin, E-mail: hanzhengbin@hit.edu.cn [School of Life Science and Technology, Harbin Institute of Technology, Harbin 150001 (China); Zhang, Zhihua [Liaoning Key Materials Laboratory for Railway, School of Materials Science and Engineering, Dalian Jiaotong University, Dalian 116028 (China); Wu, Qiong [School of Life Science and Technology, Harbin Institute of Technology, Harbin 150001 (China); Song, Bo, E-mail: songbo@hit.edu.cn [Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology, Harbin 150001 (China); Department of Physics, Harbin Institute of Technology, Harbin 150001 (China)

    2017-06-15

    Herein, highly blue graphitic carbon nitride quantum dots (g-CNQDs) were synthesized by one-step microwave-assisted evaporation–condensation strategy using bulk g-C{sub 3}N{sub 4} as the precursor within 5 min. In contrast with conventional chemical routes, the as-synthesized g-CNQDs exhibited a high crystalline quality, excellent fluorescence characteristics, and a narrow size distribution with an average diameter of 3.5 ± 0.5 nm. More importantly, by using a household microwave oven, this method has the advantages of wide accessibility, environmental friendliness, a high yield of ∼40%, and can be facilely synthesized in a large scale (scaled up to a gram scale). Notably, owing to the absence of any organic reagents, the blueas-prepared g-CNQDs show the excitation wavelength-independent photoluminescence (PL) behavior. Moreover, benefiting from the stable PL emission, good water solubility, and extraordinary biocompatibility with a high quantum yield of ∼17%, the fluorescent g-CNQDs can serve as a potential sensitive and selective probe for Fe{sup 3+} detection with a super low detection limit of 2 nM and an effective labeling agent for live-cell imaging. This work provides a unique opportunity to obtain g-CNQDs in large scale via a facile route, which may pave the way for the further design of g-CNQDs with other applications. - Highlights: • Green synthesis of g-CNQDs via one-step evaporation-condensation method. • The g-CNQDs have shown high crystalline quality and intrinsic fluorescence features. • The fluorescent g-CNQDs can serve as a sensitive and selective probe to detect Fe{sup 3+} ions with a low detection limit of 2 nM. • g-CNQDs can serve as an effective labeling agent for live-cell imaging with extraordinary biocompatibility.

  17. Development of a keratinocyte-based screening model for antipsoriatic drugs using green fluorescent protein under the control of an endogenous promoter.

    Science.gov (United States)

    Pol, Arno; van Ruissen, Fred; Schalkwijk, Joost

    2002-08-01

    Inflamed epidermis (psoriasis, wound healing, ultraviolet-irradiated skin) harbors keratinocytes that are hyperproliferative and display an abnormal differentiation program. A distinct feature of this so-called regenerative maturation pathway is the expression of proteins such as the cytokeratins CK6, CK16, and CK17 and the antiinflammatory protein SKALP/elafin. These proteins are absent in normal skin but highly induced in lesional psoriatic skin. Expression of these genes can be used as a surrogate marker for psoriasis in drug-screening procedures of large compound libraries. The aim of this study was to develop a keratinocyte cell line that contained a reporter gene under the control of a psoriasis-associated endogenous promoter and demonstrate its use in an assay suitable for screening. We generated a stably transfected keratinocyte cell line that expresses enhanced green fluorescent protein (EGFP), under the control of a 0.8-kb fragment derived from the promoter of the SKALP/elafin gene, which confers high levels of tissue-specific expression at the mRNA level. Induction of the SKALP promoter by tumor necrosis factor-alpha resulted in increased expression levels of the secreted SKALP-EGFP fusion protein as assessed by direct readout of fluorescence and fluorescence polarization in 96-well cell culture plates. The fold stimulation of the reporter gene was comparable to that of the endogenous SKALP gene as assessed by enzyme-linked immunosorbent assay. Although the dynamic range of the screening system is limited, the small standard deviation yields a Z factor of 0.49. This indicates that the assay is suitable as a high-throughput screen, and provides proof of the concept that a secreted EGFP fusion protein under the control of a physiologically relevant endogenous promoter can be used as a fluorescence-based high-throughput screen for differentiation-modifying or antiinflammatory compounds that act via the keratinocyte.

  18. Kiss-1/GPR54 protein expression in breast cancer.

    Science.gov (United States)

    Papaoiconomou, Eleni; Lymperi, Maria; Petraki, Constantina; Philippou, Anastassios; Msaouel, Pavlos; Michalopoulou, Fani; Kafiri, Georgia; Vassilakos, George; Zografos, Georgios; Koutsilieris, Michael

    2014-03-01

    Numerous studies have shown that the Kiss-1 gene countervails the metastatic aptitude of several cancer cell lines and solid-tumor neoplasias. However, there still remains ambiguity regarding its role in breast cancer and literature has arisen asserting that Kiss-1 expression may be linked to an aggressive phenotype and malignant progression. Herein, we investigated the protein expression of Kiss-1 and its receptor GPR54 in breast cancer tissues compared to non-cancerous mammary tissues. Paraffin-fixed cancer tissues from 43 women with resected breast adenocarcinomas and 11 specimens derived from women suffering from fibrocystic disease, serving as controls, were immunostained with Kiss-1 and GPR54 antibodies. Kiss-1 and GPR54 protein expression levels were significantly higher in breast cancer compared to fibrocystic tissues (pbreast cancer and fibrocystic disease specimens. Kiss-1/GPR54 expression was found to be significantly higher in breast cancer compared to non-malignant mammary tissues.

  19. Monitoring prion protein expression in complex biological samples by SERS for diagnostic applications

    International Nuclear Information System (INIS)

    Manno, D; Filippo, E; Fiore, R; Serra, A; Urso, E; Rizzello, A; Maffia, M

    2010-01-01

    Surface-enhanced Raman spectroscopy (SERS) allows a new insight into the analysis of cell physiology. In this work, the difficulty of producing suitable substrates that, besides permitting the amplification of the Raman signal, do not interact with the biological material causing alteration, has been overcome by a combined method of hydrothermal green synthesis and thermal annealing. The SERS analysis of the cell membrane has been performed with special attention to the cellular prion protein PrP C . In addition, SERS has also been used to reveal the prion protein-Cu(II) interaction in four different cell models (B104, SH-SY5Y, GN11, HeLa), expressing PrP C at different levels. A significant implication of the current work consists of the intriguing possibility of revealing and quantifying prion protein expression in complex biological samples by a cheap SERS-based method, replacing the expensive and time-consuming immuno-assay systems commonly employed.

  20. Bcl-2 Protein Expression in Egyptian Acute Myeloid Leukemia

    International Nuclear Information System (INIS)

    El-Shakankiry, N.; El-Sayed, Gh.M.M.; El-Maghraby, Sh.; Moneer, M.M.

    2009-01-01

    Objective: The primary cause of treatment failure in acute myeloid leukemia (AML) is the emergence of both resistant disease and early relapse. The bcl-2 gene encodes a 26-kDa protein that promotes cell survival by blocking programmed cell death (apoptosis). In the present study, bcl-2 protein expression was evaluated in newly diagnosed AML patients and correlated with the induction of remission and overall survival (OS), in an attempt to define patients who might benefit from modified therapeutic strategies. Patients and methods: Pretreatment cellular bcl-2 protein expression was measured in bone marrow samples obtained from 68 patients of newly diagnosed acute myeloid leukemia and 10 healthy controls by western blotting. Results: The mean bcl-2 protein expression was significantly higher in patients (0.68610.592) compared to controls (0.313±0.016) (p=0.002). The overall survival for patients with mean bcl-2 expression of less, and more than or equal to 0.315, was 67% and 56%, respectively, with no significant difference between the two groups 0»=0.86). Conclusion: Even though we did not observe a significant difference in overall survival between patients with high and low levels of bcl-2, modulation of this protein might still be considered as an option for enhancing the effectiveness of conventional chemotherapy.

  1. Green and Selective Fluorescent Sensor for Detection of Sn (IV) and Mo (VI) Based on Boron and Nitrogen-Co-Doped Carbon Dots.

    Science.gov (United States)

    Tabaraki, Reza; Abdi, Omran; Yousefipour, Sedigheh

    2017-03-01

    A green and simple microwave-assisted method was used to synthesis water-soluble boron and nitrogen-co-doped carbon dots (B-N-CDs). These B-N-CDs were successfully used for the fluorescent determination of Sn 4+ and Mo 6+ ions. This probe had a fast response time at pH = 4 with high sensitivity and selectivity. Linear correlation between F 0 /F and the concentration was seen in the range of 0.2-18 μM and 0.2-25 μM for Sn 4+ and Mo 6+ , respectively. Under optimum condition, the limit of detection (LOD) for Sn 4+ and Mo 6+ were 150 nM and 132 nM, respectively. The performance of the sensor was evaluated by different real samples such as tap, river and mineral water, canned fish sample and tomato samples.

  2. Expression, purification, crystallization and preliminary X-ray analysis of eCGP123, an extremely stable monomeric green fluorescent protein with reversible photoswitching properties

    International Nuclear Information System (INIS)

    Don Paul, Craig; Traore, Daouda A. K.; Byres, Emma; Rossjohn, Jamie; Devenish, Rodney J.; Kiss, Csaba; Bradbury, Andrew; Wilce, Matthew C. J.; Prescott, Mark

    2011-01-01

    eCGP123, an extremely stable GFP with photoswitching properties, has been expressed, purified and crystallized. A diffraction data set has been collected at 2.10 Å resolution. Enhanced consensus green protein variant 123 (eCGP123) is an extremely thermostable green fluorescent protein (GFP) that exhibits useful negative reversible photoswitching properties. eCGP123 was derived by the application of both a consensus engineering approach and a recursive evolutionary process. Diffraction-quality crystals of recombinant eCGP123 were obtained by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The eCGP123 crystal diffracted X-rays to 2.10 Å resolution. The data were indexed in space group P1, with unit-cell parameters a = 74.63, b = 75.38, c = 84.51 Å, α = 90.96, β = 89.92, γ = 104.03°. The Matthews coefficient (V M = 2.26 Å 3 Da −1 ) and a solvent content of 46% indicated that the asymmetric unit contained eight eCGP123 molecules

  3. Biolistic co-transformation of Metarhizium anisopliae var. acridum strain CG423 with green fluorescent protein and resistance to glufosinate ammonium.

    Science.gov (United States)

    Inglis, P W; Aragão, F J; Frazão, H; Magalhães, B P; Valadares-Inglis, M C

    2000-10-15

    Metarhizium anisopliae var. acridum (syn. M. flavoviride) is recognized as a highly specific and virulent mycopathogen of locusts and grasshoppers and is currently being developed as a biological control agent for this group of insects in Brazil. Intact conidia of M. anisopliae var. acridum strain CG423 were transformed using microparticle bombardment. Plasmids used were: (1) pBARKS1 carrying the bar gene of Streptomyces hygroscopicus fused to the Aspergillus nidulans trpC promoter, encoding resistance to glufosinate ammonium (or phosphinothricin) and modified by addition of the telomeric repeat (TTAGGG)(18) of Fusarium oxysporum and 2.pEGFP/gpd/tel carrying a red-shifted variant gene for Aequorea victoria green fluorescent protein (EGFP) which we have fused to the A. nidulans gpd promoter and trpC terminator. Highly fluorescent co-transformants were selected on solid minimal medium containing 100 microg ml(-1) glufosinate ammonium using an inverted microscope with 450-490 nm excitation/510 nm emission filter set. Southern blot analysis of co-transformants revealed varying multiple chromosomal integrations of both bar and egfp genes at both telomeric and non-telomeric loci. Transformants retained pathogenicity in bioassays against Rhammatocerus schistocercoides and showed unaltered lack of pathogenicity against larvae of the non-target insect Anticarsia gemmatalis. One co-transformant from four tested, however, showed a significant, but non-dose-dependent, elevation in virulence against Tenebrio molitor.

  4. Visualization of the African swine fever virus infection in living cells by incorporation into the virus particle of green fluorescent protein-p54 membrane protein chimera

    International Nuclear Information System (INIS)

    Hernaez, Bruno; Escribano, Jose M.; Alonso, Covadonga

    2006-01-01

    Many stages of African swine fever virus infection have not yet been studied in detail. To track the behavior of African swine fever virus (ASFV) in the infected cells in real time, we produced an infectious recombinant ASFV (B54GFP-2) that expresses and incorporates into the virus particle a chimera of the p54 envelope protein fused to the enhanced green fluorescent protein (EGFP). The incorporation of the fusion protein into the virus particle was confirmed immunologically and it was determined that p54-EGFP was fully functional by confirmation that the recombinant virus made normal-sized plaques and presented similar growth curves to the wild-type virus. The tagged virus was visualized as individual fluorescent particles during the first stages of infection and allowed to visualize the infection progression in living cells through the viral life cycle by confocal microscopy. In this work, diverse potential applications of B54GFP-2 to study different aspects of ASFV infection are shown. By using this recombinant virus it was possible to determine the trajectory and speed of intracellular virus movement. Additionally, we have been able to visualize for first time the ASFV factory formation dynamics and the cytophatic effect of the virus in live infected cells. Finally, we have analyzed virus progression along the infection cycle and infected cell death as time-lapse animations

  5. Fluorescent minerals--A potential source of UV protection and visible light for the growth of green algae and cyanobacteria in extreme cosmic environments.

    Science.gov (United States)

    Omairi, Tareq; Wainwright, Milton

    2015-07-01

    We propose that green algae (Chlorella variabilis and Dunaliella tertiolecta) and cyanobacteria (Synechococcus elongatus and Nostoc commune) can grow inside fluorescent rock minerals which convert damaging UV light to visible light, thereby allowing these organisms to survive and thrive in UV-rich environments without (or with limited) visible light, which would otherwise be inimical to them. The four microorganisms were incubated inside fluorescent rocks composed of fluorite, calcite and pyrite. The resultant growth was then measured following exposure to UV radiation, with the use of optical density and measurement of chlorophyll concentration. Results show that the microorganisms were shielded from harmful UV in these semi-transparent rocks, while at the same time benefiting from the fact that the minerals converted UV to visible light; this have been shown by a statistically significant increase in their growth, which although lower than when the cells were incubated in sunlight, was significantly higher than in controls incubated in the dark. Copyright © 2015 The Committee on Space Research (COSPAR). Published by Elsevier Ltd. All rights reserved.

  6. Detection of peritoneal dissemination with near-infrared fluorescence laparoscopic imaging using a liposomal formulation of a synthesized indocyanine green liposomal derivative.

    Science.gov (United States)

    Hoshino, Isamu; Maruyama, Tetsuro; Fujito, Hiromichi; Tamura, Yutaka; Suganami, Akiko; Hayashi, Hideki; Toyota, Taro; Akutsu, Yasunori; Murakami, Kentaro; Isozaki, Yuka; Akanuma, Naoki; Takeshita, Nobuyoshi; Toyozumi, Takeshi; Komatsu, Aki; Matsubara, Hisahiro

    2015-03-01

    Although conventional staging laparoscopy (SL) has improved the diagnostic accuracy of peritoneal dissemination, novel technology is needed to increase the sensitivity of SL. We herein describe a new imaging method employing near-infrared (NIR) fluorescence imaging using a liposomal synthesized indocyanine green (ICG) liposomal derivative, LP-ICG-C18. LP-ICG-C18 is a NIR-photoactivating probe in which an ICG fluorophore is covalently conjugated with a phospholipid moiety. Nude mice were intraperitoneally injected with gastric cancer cells. Twelve days later, the mice were given intravenous injections of LP-ICG-C18 at a dose of 0.15 mg/kg. A NIR imaging system was used to identify the disseminated tumors. The disseminated nodules in mice were detected without any difficulties. Disseminated tumor nodules were collected from mice with or without injections of liposomal formulation and were transferred into the swine peritoneal cavity. The nodules in the swine peritoneal cavity were clearly and promptly defined by the NIR imaging system. NIR-fluorescing liposomal probes can effectively target peritoneal disseminated tumors and can be easily detected by a NIR imaging system. These results warrant future clinical trials of our imaging system and may contribute to a more precise diagnosis and therapeutic approach for gastric cancer patients. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  7. Trypanosoma cruzi Coexpressing Ornithine Decarboxylase and Green Fluorescence Proteins as a Tool to Study the Role of Polyamines in Chagas Disease Pathology

    Directory of Open Access Journals (Sweden)

    Jeremías José Barclay

    2011-01-01

    Full Text Available Polyamines are essential for Trypanosoma cruzi, the causative agent of Chagas disease. As T. cruzi behaves as a natural auxotrophic organism, it relies on host polyamines biosynthesis. In this paper we obtained a double-transfected T. cruzi parasite that expresses the green fluorescent protein (GFP and a heterologous ornithine decarboxylase (ODC, used itself as a novel selectable marker. These autotrophic and fluorescent parasites were characterized; the ODC presented an apparent Km for ornithine of 0.51 ± 0.16 mM and an estimated Vmax value of 476.2 nmoles/h/mg of protein. These expressing ODC parasites showed higher metacyclogenesis capacity than the auxotrophic counterpart, supporting the idea that polyamines are engaged in this process. This double-transfected T. cruzi parasite results in a powerful tool—easy to follow by its fluorescence—to study the role of polyamines in Chagas disease pathology and in related processes such as parasite survival, invasion, proliferation, metacyclogenesis, and tissue spreading.

  8. Identification of a progenitor cell population destined to form fracture fibrocartilage callus in Dickkopf-related protein 3-green fluorescent protein reporter mice.

    Science.gov (United States)

    Mori, Yu; Adams, Douglas; Hagiwara, Yusuke; Yoshida, Ryu; Kamimura, Masayuki; Itoi, Eiji; Rowe, David W

    2016-11-01

    Fracture healing is a complex biological process involving the proliferation of mesenchymal progenitor cells, and chondrogenic, osteogenic, and angiogenic differentiation. The mechanisms underlying the proliferation and differentiation of mesenchymal progenitor cells remain unclear. Here, we demonstrate Dickkopf-related protein 3 (Dkk3) expression in periosteal cells using Dkk3-green fluorescent protein reporter mice. We found that proliferation of mesenchymal progenitor cells began in the periosteum, involving Dkk3-positive cell proliferation near the fracture site. In addition, Dkk3 was expressed in fibrocartilage cells together with smooth muscle α-actin and Col3.6 in the early phase of fracture healing as a cell marker of fibrocartilage cells. Dkk3 was not expressed in mature chondrogenic cells or osteogenic cells. Transient expression of Dkk3 disappeared in the late phase of fracture healing, except in the superficial periosteal area of fracture callus. The Dkk3 expression pattern differed in newly formed type IV collagen positive blood vessels and the related avascular tissue. This is the first report that shows Dkk3 expression in the periosteum at a resting state and in fibrocartilage cells during the fracture healing process, which was associated with smooth muscle α-actin and Col3.6 expression in mesenchymal progenitor cells. These fluorescent mesenchymal lineage cells may be useful for future studies to better understand fracture healing.

  9. Green-fluorescent protein+ Astrocytes Attach to beta-Amyloid Plaques in an Alzheimer Mouse Model and GFPare Sensitive for Clasmatodendrosis

    Directory of Open Access Journals (Sweden)

    Christian eHumpel

    2016-04-01

    Full Text Available Alzheimer’s disease (AD is pathologically characterized by beta-amyloid (Aβ plaques and Tau pathology. It is well-established that Aβ plaques are surrounded by reactive astrocytes, highly expressing glial fibrillary acidic protein (GFAP. In order to study the cellular interaction of reactive astrocytes with Aβ plaques, we crossbred mice overexpressing amyloid precursor protein (APP with the Swedish-Dutch-Iowa mutations (APP-SweDI with mice expressing green fluorescent protein (GFP under the GFAP-promotor. Three-dimensional confocal microscopy revealed a tight association and intense sprouting of astrocytic fine branched processes towards Aβ plaques in 12 month old mice. In order to study phagocytosis, 110 µm thick brain slices from 12 month old crossbred mice were cultured overnight, however, we found that the GFP fluorescence faded away, distal processes degenerated and a complete loss of astrocytic morphology was seen (clasmatodendrosis. In summary, our data show that GFP+ reactive astrocytes make intense contact with Aβ plaques but these cells are highly vulnerable for degeneration.

  10. Development of a Novel Green Fluorescent Protein-Based Binding Assay to Study the Association of Plakins with Intermediate Filament Proteins.

    Science.gov (United States)

    Favre, Bertrand; Begré, Nadja; Bouameur, Jamal-Eddine; Borradori, Luca

    2016-01-01

    Protein-protein interactions are fundamental for most biological processes, such as the formation of cellular structures and enzymatic complexes or in signaling pathways. The identification and characterization of protein-protein interactions are therefore essential for understanding the mechanisms and regulation of biological systems. The organization and dynamics of the cytoskeleton, as well as its anchorage to specific sites in the plasma membrane and organelles, are regulated by the plakins. These structurally related proteins anchor different cytoskeletal networks to each other and/or to other cellular structures. The association of several plakins with intermediate filaments (IFs) is critical for maintenance of the cytoarchitecture. Pathogenic mutations in the genes encoding different plakins can lead to dramatic manifestations, occurring principally in the skin, striated muscle, and/or nervous system, due to cytoskeletal disorganization resulting in abnormal cell fragility. Nevertheless, it is still unclear how plakins bind to IFs, although some general rules are slowly emerging. We here describe in detail a recently developed protein-protein fluorescence binding assay, based on the production of recombinant proteins tagged with green fluorescent protein (GFP) and their use as fluid-phase fluorescent ligands on immobilized IF proteins. Using this method, we have been able to assess the ability of C-terminal regions of GFP-tagged plakin proteins to bind to distinct IF proteins and IF domains. This simple and sensitive technique, which is expected to facilitate further studies in this area, can also be potentially employed for any kind of protein-protein interaction studies. © 2016 Elsevier Inc. All rights reserved.

  11. Detection of malachite green in fish based on magnetic fluorescent probe of CdTe QDs/nano-Fe3O4@MIPs

    Science.gov (United States)

    Wu, Le; Lin, Zheng-Zhong; Zeng, Jun; Zhong, Hui-Ping; Chen, Xiao-Mei; Huang, Zhi-Yong

    2018-05-01

    A magnetic fluorescent probe of CdTe QDs/nano-Fe3O4@MIPs was prepared using CdTe QDs and Fe3O4 nanoparticles as co-nucleus and molecularly imprinted polymers (MIPs) as specific recognition sites based on a reverse microemulsion method. With the specific enrichment and magnetic separation properties, the probe of CdTe QDs/nano-Fe3O4@MIPs was used to detect malachite green (MG) in fish samples. The TEM analysis showed that the particles of CdTe QDs/nano-Fe3O4@MIPs were spherical with average diameter around 53 nm, and a core-shell structure was well-shaped with several Fe3O4 nanoparticles and CdTe QDs embedded in each of the microsphere. Quick separation of the probes from solutions could be realized with a magnet, indicating the excellent magnetic property of CdTe QDs/nano-Fe3O4@MIPs. The probe exhibited high specific adsorption towards MG and excellent fluorescence emission at λem 598 nm. The fluorescence of CdTe QDs/nano-Fe3O4@MIPs could be linearly quenched by MG at the concentrations from 0.025 to 1.5 μmol L-1. The detection limit was 0.014 μmol L-1. The average recovery of spiked MG in fish samples was 105.2%. The result demonstrated that the as-prepared CdTe QDs/nano-Fe3O4@MIPs could be used as a probe to the detection of trace MG in fish samples.

  12. Quantification of green fluorescent protein by in vivo imaging, PCR, and flow cytometry: comparison of transgenic strains and relevance for fetal cell microchimerism.

    Science.gov (United States)

    Fujiki, Yutaka; Tao, Kai; Bianchi, Diana W; Giel-Moloney, Maryann; Leiter, Andrew B; Johnson, Kirby L

    2008-02-01

    Animal models are increasingly being used for the assessment of fetal cell microchimerism in maternal tissue. We wished to determine the optimal transgenic mouse strain and analytic technique to facilitate the detection of rare transgenic microchimeric fetal cells amongst a large number of maternal wild-type cells. We evaluated two strains of mice transgenic for the enhanced green fluorescent protein (EGFP): a commercially available, commonly used strain (C57BL/6-Tg(ACTB-EGFP)10sb/J) (CAG) and a newly created strain (ROSA26-EGFP) using three different techniques: in vivo and ex vivo fluorescent imaging (for whole body and dissected organs, respectively), PCR amplification of gfp, and flow cytometry (FCM). By fluorescent imaging, organs from CAG mice were 10-fold brighter than organs from ROSA26-EGFP mice (P characteristics that make it useful under specific experimental circumstances. The CAG mouse model is preferable when experiments require brighter cells, whereas ROSA26-EGFP is more appropriate when uniform or ubiquitous expression is more important than brightness. Investigators must carefully select the transgenic strain most suited to the experimental design to obtain the most consistent and reproducible data. In vivo imaging allows for phenotypic evaluation of whole animals and intact organs; however, we did not evaluate its utility for the detection of rare, fetal microchimeric cells in the maternal organs. Finally, while PCR amplification of a paternally inherited transgene does allow for the quantitative determination of rare microchimeric cells, FCM allows for both quantitative and qualitative evaluations of fetal cells at very high sensitivity in a plethora of maternal organs. (c) 2008 International Society for Analytical Cytology

  13. Establishment of A Malignant Pleural Effusion Mouse Model with Lewis Lung 
Carcinoma Cell Lines Expressing Enhanced Green Fluorescent Protein

    Directory of Open Access Journals (Sweden)

    Xingqun MA

    2012-06-01

    Full Text Available Background and objective Malignant pleural effusion (MPE is a poor prognosis factor in patients with advanced lung cancer. The aim of this study is to establish a mouse model of MPE using Lewis lung carcinoma (LLC cell lines expressing enhanced green fluorescent protein (EGFP. Methods The mouse model was created by injecting LLC-EGFP cells directly into the pleural cavity of mice that were sacrificed periodically. The dynamic growth and metastasis of tumor cells were screened using in vivo fluorescence imaging. The remaining mice were subjected to transverse computed tomography (CT imaging periodically to analyze the formation rate of pleural effusion. The survival rate and tumor metastasis were also observed. Pleural fluid was gently aspirated using a 1 mL syringe and its volume was measured. When two or more mice bore pleural effusion at the same time, we calculated the average volume. The correlation of pleural effusion with the integrated optical density (IOD were analyzed. Results Four days after the inoculation of LLC-EGFP cells, green fluorescence was observed by opening the chest wall. The tumor formation rate was 100%, and the IOD gradually increased after inoculation. The metastasis sites were mediastinal, and the hilar lymph nodes were contralateral pleural as well as pericardial. The metastasis rates were 87%, 73% and 20%, respectively. The CT scan revealed that the formation rates of pleural effusion on days 7, 14 and 21 were 13%, 46% and 53%, respectively. The average volume of pleural effusion increased obviously on day 10 and peaked on day 16 with a value of 0.5 mL. The mean survival time of nude mice was 28.8 days. The volume of pleural effusion and IOD were significantly correlated (r=0.91, P<0.000,1. Conclusion A mouse model of lung cancer malignant pleural effusion was successfully established by injecting LLC lines expressing EGFP into the pleural cavity under a microscope. The model can enable dynamic observations of the

  14. Differential Protein Expression in Congenital and Acquired Cholesteatomas.

    Directory of Open Access Journals (Sweden)

    Seung-Ho Shin

    Full Text Available Congenital cholesteatomas are epithelial lesions that present as an epithelial pearl behind an intact eardrum. Congenital and acquired cholesteatomas progress quite differently from each other and progress patterns can provide clues about the unique origin and pathogenesis of the abnormality. However, the exact pathogenic mechanisms by which cholesteatomas develop remain unknown. In this study, key proteins that directly affect cholesteatoma pathogenesis are investigated with proteomics and immunohistochemistry. Congenital cholesteatoma matrices and retroauricular skin were harvested during surgery in 4 patients diagnosed with a congenital cholesteatoma. Tissue was also harvested from the retraction pocket in an additional 2 patients during middle ear surgery. We performed 2-dimensional (2D electrophoresis to detect and analyze spots that are expressed only in congenital cholesteatoma and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS to separate proteins by molecular weight. Protein expression was confirmed by immunohistochemical staining. The image analysis of 2D electrophoresis showed that 4 congenital cholesteatoma samples had very similar protein expression patterns and that 127 spots were exclusively expressed in congenital cholesteatomas. Of these 127 spots, 10 major spots revealed the presence of titin, forkhead transcription activator homolog (FKH 5-3, plectin 1, keratin 10, and leucine zipper protein 5 by MALDI-TOF/MS analysis. Immunohistochemical staining showed that FKH 5-3 and titin were expressed in congenital cholesteatoma matrices, but not in acquired cholesteatomas. Our study shows that protein expression patterns are completely different in congenital cholesteatomas, acquired cholesteatomas, and skin. Moreover, non-epithelial proteins, including FKH 5-3 and titin, were unexpectedly expressed in congenital cholesteatoma tissue. Our data indicates that congenital cholesteatoma origins

  15. Effects of immunosuppressive treatment on protein expression in rat kidney

    Directory of Open Access Journals (Sweden)

    Kędzierska K

    2014-09-01

    Full Text Available Karolina Kędzierska,1 Katarzyna Sporniak-Tutak,2 Krzysztof Sindrewicz,2 Joanna Bober,3 Leszek Domański,1 Mirosław Parafiniuk,4 Elżbieta Urasińska,5 Andrzej Ciechanowicz,6 Maciej Domański,1 Tomasz Smektała,2 Marek Masiuk,5 Wiesław Skrzypczak,6 Małgorzata Ożgo,6 Joanna Kabat-Koperska,1 Kazimierz Ciechanowski1 1Department of Nephrology, Transplantology, and Internal Medicine, 2Department of Dental Surgery, 3Department of Medical Chemistry, 4Department of Forensic Medicine, 5Department of Pathomorphology, Pomeranian Medical University, 6Department of Physiology, Cytobiology, and Proteomics, West Pomeranian University of Technology, Szczecin, Poland Abstract: The structural proteins of renal tubular epithelial cells may become a target for the toxic metabolites of immunosuppressants. These metabolites can modify the properties of the proteins, thereby affecting cell function, which is a possible explanation for the mechanism of immunosuppressive agents' toxicity. In our study, we evaluated the effect of two immunosuppressive strategies on protein expression in the kidneys of Wistar rats. Fragments of the rat kidneys were homogenized after cooling in liquid nitrogen and then dissolved in lysis buffer. The protein concentration in the samples was determined using a protein assay kit, and the proteins were separated by two-dimensional electrophoresis. The obtained gels were then stained with Coomassie Brilliant Blue, and their images were analyzed to evaluate differences in protein expression. Identification of selected proteins was then performed using mass spectrometry. We found that the immunosuppressive drugs used in popular regimens induce a series of changes in protein expression in target organs. The expression of proteins involved in drug, glucose, amino acid, and lipid metabolism was pronounced. However, to a lesser extent, we also observed changes in nuclear, structural, and transport proteins' synthesis. Very slight differences

  16. Gateway-compatible vectors for high-throughput protein expression in pro- and eukaryotic cell-free systems.

    Science.gov (United States)

    Gagoski, Dejan; Mureev, Sergey; Giles, Nichole; Johnston, Wayne; Dahmer-Heath, Mareike; Škalamera, Dubravka; Gonda, Thomas J; Alexandrov, Kirill

    2015-02-10

    Although numerous techniques for protein expression and production are available the pace of genome sequencing outstrips our ability to analyze the encoded proteins. To address this bottleneck, we have established a system for parallelized cloning, DNA production and cell-free expression of large numbers of proteins. This system is based on a suite of pCellFree Gateway destination vectors that utilize a Species Independent Translation Initiation Sequence (SITS) that mediates recombinant protein expression in any in vitro translation system. These vectors introduce C or N terminal EGFP and mCherry fluorescent and affinity tags, enabling direct analysis and purification of the expressed proteins. To maximize throughput and minimize the cost of protein production we combined Gateway cloning with Rolling Circle DNA Amplification. We demonstrate that as little as 0.1 ng of plasmid DNA is sufficient for template amplification and production of recombinant human protein in Leishmania tarentolae and Escherichia coli cell-free expression systems. Our experiments indicate that this approach can be applied to large gene libraries as it can be reliably performed in multi-well plates. The resulting protein expression pipeline provides a valuable new tool for applications of the post genomic era. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Green analytical determination of emerging pollutants in environmental waters using excitation-emission photoinduced fluorescence data and multivariate calibration.

    Science.gov (United States)

    Hurtado-Sánchez, María Del Carmen; Lozano, Valeria A; Rodríguez-Cáceres, María Isabel; Durán-Merás, Isabel; Escandar, Graciela M

    2015-03-01

    An eco-friendly strategy for the simultaneous quantification of three emerging pharmaceutical contaminants is presented. The proposed analytical method, which involves photochemically induced fluorescence matrix data combined with second-order chemometric analysis, was used for the determination of carbamazepine, ofloxacin and piroxicam in water samples of different complexity without the need of chromatographic separation. Excitation-emission photoinduced fluorescence matrices were obtained after UV irradiation, and processed with second-order algorithms. Only one of the tested algorithms was able to overcome the strong spectral overlapping among the studied pollutants and allowed their successful quantitation in very interferent media. The method sensitivity in superficial and underground water samples was enhanced by a simple solid-phase extraction with C18 membranes, which was successful for the extraction/preconcentration of the pollutants at trace levels. Detection limits in preconcentrated (1:125) real water samples ranged from 0.04 to 0.3 ng mL(-1). Relative prediction errors around 10% were achieved. The proposed strategy is significantly simpler and greener than liquid chromatography-mass spectrometry methods, without compromising the analytical quality of the results. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. BAX protein expression and clinical outcome in epithelial ovarian cancer.

    Science.gov (United States)

    Tai, Y T; Lee, S; Niloff, E; Weisman, C; Strobel, T; Cannistra, S A

    1998-08-01

    Expression of the pro-apoptotic protein BAX sensitizes ovarian cancer cell lines to paclitaxel in vitro by enhancing the pathway of programmed cell death. The present study was performed to determine the relationship between BAX expression and clinical outcome in 45 patients with newly diagnosed ovarian cancer. BAX protein expression was analyzed by immunohistochemistry, and its relationship with clinical outcome was determined. Assessment of BAX mRNA transcript levels and mutational analysis of the BAX coding region were also performed. BAX protein was expressed at high levels (defined as > or = 50% of tumor cells positive) in tumor tissue from 60% of newly diagnosed patients. All patients whose tumors expressed high levels of BAX achieved a complete response (CR) to first-line chemotherapy that contained paclitaxel plus a platinum analogue, compared with 57% of patients in the low-BAX group (P = .036). After a median follow-up of 1.9 years, the median disease-free survival (DFS) of patients in the high-BAX group has not been reached, compared with a median DFS of 1.1 years for low-BAX expressors (P = .0061). BAX retained independent prognostic significance in multivariate analysis when corrected for stage and histology. BAX mRNA transcripts were easily detected in samples with low BAX protein expression, and no BAX mutations were identified. The correlation between high BAX levels and improved clinical outcome suggests that an intact apoptotic pathway is an important determinant of chemoresponsiveness in ovarian cancer patients who receive paclitaxel.

  19. Genome engineering for improved recombinant protein expression in Escherichia coli.

    Science.gov (United States)

    Mahalik, Shubhashree; Sharma, Ashish K; Mukherjee, Krishna J

    2014-12-19

    A metabolic engineering perspective which views recombinant protein expression as a multistep pathway allows us to move beyond vector design and identify the downstream rate limiting steps in expression. In E.coli these are typically at the translational level and the supply of precursors in the form of energy, amino acids and nucleotides. Further recombinant protein production triggers a global cellular stress response which feedback inhibits both growth and product formation. Countering this requires a system level analysis followed by a rational host cell engineering to sustain expression for longer time periods. Another strategy to increase protein yields could be to divert the metabolic flux away from biomass formation and towards recombinant protein production. This would require a growth stoppage mechanism which does not affect the metabolic activity of the cell or the transcriptional or translational efficiencies. Finally cells have to be designed for efficient export to prevent buildup of proteins inside the cytoplasm and also simplify downstream processing. The rational and the high throughput strategies that can be used for the construction of such improved host cell platforms for recombinant protein expression is the focus of this review.

  20. Evaluation of the pH- and Thermal Stability of the Recombinant Green Fluorescent Protein (GFP) in the Presence of Sodium Chloride

    Science.gov (United States)

    Ishii, Marina; Kunimura, Juliana Sayuri; Jeng, Hélio Tallon; Vessoni Penna, Thereza Christina; Cholewa, Olivia

    The thermal stability of recombinant green fluorescent protein (GFP) in sodium chloride (NaCl) solutions at different concentrations, pH, and temperatures was evaluated by assaying the loss of fluorescence intensity as a measure of denaturation. GFP, extracted from Escherichia coli cells by the three-phase partitioning method and purified through a butyl hydrophobic interaction chromatography (HIC) column, was diluted in water for injection (WFI) (pH 6.0-7.0) and in 10 mM buffer solutions (acetate, pH 5.0; phosphate, pH 7.0; and Tris-EDTA, pH 8.0) with 0.9-30% NaCl or without and incubated at 80-95°C. The extent of protein denaturation was expressed as a percentage of the calculated decimal reduction time (D-value). In acetate buffer (pH 4.84 ±0.12), the mean D-values for 90% reduction in GFP fluorescence ranged from 2.3 to 3.6 min, independent of NaCl concentration and temperature. GFP thermal stability diluted in WFI (pH 5.94±0.60) was half that observed in phosphate buffer (pH 6.08±0.60); but in both systems, D-values decreased linearly with increasing NaCl concentration, with D-values (at 80°C) ranging from 3.44, min (WFI) to 6.1 min (phosphate buffer), both with 30% NaCl. However, D-values in Tris-EDTA (pH 7.65±0.17) were directly dependent on the NaCl concentration and 5-10 times higher than D-values for GFP in WFI at 80°C. GFP pH-and thermal stability can be easily monitored by the convenient measure of fluorescence intensity and potentially be used as an indicator to monitor that processing times and temperatures were attained.

  1. Translational up-regulation and high-level protein expression from plasmid vectors by mTOR activation via different pathways in PC3 and 293T cells.

    Directory of Open Access Journals (Sweden)

    Prashanthi Karyala

    Full Text Available BACKGROUND: Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines. METHODOLOGY/PRINCIPAL FINDINGS: In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B, β-galactosidase (β-gal and green fluorescent protein (GFP from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational up-regulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3. CONCLUSIONS/SIGNIFICANCE: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to

  2. General theory for integrated analysis of growth, gene, and protein expression in biofilms.

    Science.gov (United States)

    Zhang, Tianyu; Pabst, Breana; Klapper, Isaac; Stewart, Philip S

    2013-01-01

    A theory for analysis and prediction of spatial and temporal patterns of gene and protein expression within microbial biofilms is derived. The theory integrates phenomena of solute reaction and diffusion, microbial growth, mRNA or protein synthesis, biomass advection, and gene transcript or protein turnover. Case studies illustrate the capacity of the theory to simulate heterogeneous spatial patterns and predict microbial activities in biofilms that are qualitatively different from those of planktonic cells. Specific scenarios analyzed include an inducible GFP or fluorescent protein reporter, a denitrification gene repressed by oxygen, an acid stress response gene, and a quorum sensing circuit. It is shown that the patterns of activity revealed by inducible stable fluorescent proteins or reporter unstable proteins overestimate the region of activity. This is due to advective spreading and finite protein turnover rates. In the cases of a gene induced by either limitation for a metabolic substrate or accumulation of a metabolic product, maximal expression is predicted in an internal stratum of the biofilm. A quorum sensing system that includes an oxygen-responsive negative regulator exhibits behavior that is distinct from any stage of a batch planktonic culture. Though here the analyses have been limited to simultaneous interactions of up to two substrates and two genes, the framework applies to arbitrarily large networks of genes and metabolites. Extension of reaction-diffusion modeling in biofilms to the analysis of individual genes and gene networks is an important advance that dovetails with the growing toolkit of molecular and genetic experimental techniques.

  3. Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein.

    Science.gov (United States)

    Haarmeyer, Carolyn N; Smith, Matthew D; Chundawat, Shishir P S; Sammond, Deanne; Whitehead, Timothy A

    2017-04-01

    Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue toward energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure-function relationships driving this adsorption. In this work, we have systematically investigated the role of protein surface potential on lignin adsorption using a model monomeric fluorescent protein. We have designed and experimentally characterized 16 model protein variants spanning the physiological range of net charge (-24 to +16 total charges) and total charge density (0.28-0.40 charges per sequence length) typical for natural proteins. Protein designs were expressed, purified, and subjected to in silico and in vitro biophysical measurements to evaluate the relationship between protein surface potential and lignin adsorption properties. The designs were comparable to model fluorescent protein in terms of thermostability and heterologous expression yield, although the majority of the designs unexpectedly formed homodimers. Protein adsorption to lignin was studied at two different temperatures using Quartz Crystal Microbalance with Dissipation Monitoring and a subtractive mass balance assay. We found a weak correlation between protein net charge and protein-binding capacity to lignin. No other single characteristic, including apparent melting temperature and 2nd virial coefficient, showed correlation with lignin binding. Analysis of an unrelated cellulase dataset with mutations localized to a family I carbohydrate-binding module showed a similar correlation between net charge and lignin binding capacity. Overall, our study provides strategies to identify highly active, low

  4. Preservação da proteína verde fluorescente no tecido ósseo descalcificado Preservation of the green fluorescent protein on decalcified bone tissue

    Directory of Open Access Journals (Sweden)

    Jankerle Neves Boeloni

    2010-10-01

    Full Text Available A proteína verde fluorescente (GFP foi originalmente descoberta no cnidário Aequorea victoria. Células-tronco GFP positivas podem ser rastreadas in vivo quando usadas na terapia de doenças. No entanto, no osso, a fluorescência gerada pela GFP pode ser perdida durante o processo de descalcificação, dificultando o rastreamento das células-tronco usadas no tratamento de doenças ou defeitos ósseos. O objetivo deste estudo foi comparar diferentes técnicas de preservação da GFP no tecido ósseo descalcificado. Foram utilizados fêmures de ratas GFP Lewis distribuídos em quatro grupos: 1 descalcificado em ácido fórmico e incluído em parafina; 2 descalcificado em ácido fórmico e submetido à criomicrotomia; 3 descalcificado em EDTA e incluído em parafina; e 4 descalcificado em EDTA com criomicrotomia. Secções de tecido ósseo de todos os grupos foram analisadas para identificação da fluorescência natural e posteriormente submetidas à imunofluorescência, sendo utilizados anti-GFP e Alexa Flúor 555. As imagens foram obtidas por microscopia confocal. Osteócitos, osteoblastos e células da medula óssea de ratos GFP somente tiveram sua fluorescência natural preservada no tecido ósseo descalcificado em EDTA e submetido à microtomia por congelação. Nos demais grupos, houve perda da fluorescência natural, e as células GFP somente puderam ser identificadas com o uso da reação de imunofluorescência com anti-GFP. Conclui-se que a descalcificação em EDTA e a criomicrotomia são as melhores técnicas para preservar a fluorescência natural das células GFP no tecido ósseo e que a visualização de células GFP em tecido ósseo descalcificado em ácido fórmico e incluído em parafina somente pode ser realizada com o uso da técnica de imunofluorescência.Green fluorescent protein (GFP was originally derived from the cnidarians Aequorea victoria. GFP-positive stem cells can be tracked in vivo when used in the therapy of

  5. Use of green fluorescent protein fusions to analyse the N- and C-terminal signal peptides of GPI-anchored cell wall proteins in Candida albicans.

    Science.gov (United States)

    Mao, Yuxin; Zhang, Zimei; Wong, Brian

    2003-12-01

    Glycophosphatidylinositol (GPI)-anchored proteins account for 26-35% of the Candida albicans cell wall. To understand the signals that regulate these proteins' cell surface localization, green fluorescent protein (GFP) was fused to the N- and C-termini of the C. albicans cell wall proteins (CWPs) Hwp1p, Als3p and Rbt5p. C. albicans expressing all three fusion proteins were fluorescent at the cell surface. GFP was released from membrane fractions by PI-PLC and from cell walls by beta-glucanase, which implied that GFP was GPI-anchored to the plasma membrane and then covalently attached to cell wall glucans. Twenty and 25 amino acids, respectively, from the N- and C-termini of Hwp1p were sufficient to target GFP to the cell surface. C-terminal substitutions that are permitted by the omega rules (G613D, G613N, G613S, G613A, G615S) did not interfere with GFP localization, whereas some non-permitted substitutions (G613E, G613Q, G613R, G613T and G615Q) caused GFP to accumulate in intracellular ER-like structures and others (G615C, G613N/G615C and G613D/G615C) did not. These results imply that (i) GFP fusions can be used to analyse the N- and C-terminal signal peptides of GPI-anchored CWPs, (ii) the omega amino acid in Hwp1p is G613, and (iii) C can function at the omega+2 position in C. albicans GPI-anchored proteins.

  6. Ultrasonic selectivity on depressing photosynthesis of cyanobacteria and green algae probed by chlorophyll-a fluorescence transient.

    Science.gov (United States)

    Duan, Zhipeng; Tan, Xiao; Li, Niegui

    2017-10-01

    Ultrasound can inhibit cyanobacterial growth through rupturing cells, but this pathway frequently has the risk to release intercellular toxin (e.g., microcystin). Depressing photosynthesis without cell disruption may provide a new strategy to control cyanobacterial blooms using ultrasound, especially Microcystis blooms. In this work, Microcystis aeruginosa (toxic cyanobacteria) and Chlorella pyrenoidosa (typical green algae) were chosen as model microalgae to verify this hypothesis. Results showed that ultrasound has the ability to inhibit cyanobacterial photosynthesis significantly and selectively. Specifically, sonication damaged Q A , a tightly bound one-electron acceptor, and blocked electron flow at Q B , a two-electron acceptor, in the photosystem II (PSII) of M. aeruginosa when it was exposed for 60 s (35 kHz, 0.043 W/cm 3 ). Moreover, 44.8% of the reaction centers (RCs) in the PSII of M. aeruginosa were transferred into inactive ones (RC si s), and the cell concentration decreased by 32.5% after sonication for 300 s. By contrast, only 7.9% of RC si occurred in C. pyrenoidosa, and cell concentration and chlorophyll-a content reduced by 18.7% and 9.3%, respectively. Differences in both species (i.e., cell structures) might be responsible for the varying levels to sonication. This research suggests that cyanobacteria, especially Microcystis, could be controlled by ultrasound via damaging their PSIIs.

  7. Mechanical stimulation increases proliferation, differentiation and protein expression in culture

    DEFF Research Database (Denmark)

    Grossi, Alberto; Yadav, Kavita; Lawson, Moira Ann

    2007-01-01

    Myogenesis is a complex sequence of events, including the irreversible transition from the proliferation-competent myoblast stage into fused, multinucleated myotubes. Myogenic differentiation is regulated by positive and negative signals from surrounding tissues. Stimulation due to stretch- or load...... to elucidate also the signaling pathway by which this mechanical stimulation can causes an increase in protein expression. When mechanically stimulated via laminin receptors on cell surface, C(2)C(12) cells showed an increase in cell proliferation and differentiation. Populations undergoing mechanical...... stimulation through laminin receptors show an increase in expression of Myo-D, myogenin and an increase in ERK1/2 phosphorylation. Cells stimulated via fibronectin receptors show no significant increases in fusion competence. We conclude that load induced signalling through integrin containing laminin...

  8. Time-resolved spectral studies of blue-green fluorescence of artichoke (Cynara cardunculus L. Var. Scolymus) leaves: identification of chlorogenic acid as one of the major fluorophores and age-mediated changes.

    Science.gov (United States)

    Morales, Fermín; Cartelat, Aurélie; Alvarez-Fernández, Ana; Moya, Ismael; Cerovic, Zoran G

    2005-12-14

    Synchrotron radiation and the time-correlated single-photon counting technique were used to investigate the spectral and time-resolved characteristics of blue-green fluorescence (BGF) of artichoke leaves. Leaves emitted BGF under ultraviolet (UV) excitation; the abaxial side was much more fluorescent than the adaxial side, and in both cases, the youngest leaves were much more fluorescent than the oldest ones. The BGF of artichoke leaves was dominated by the presence of hydroxycinnamic acids. A decrease in the percentage of BGF attributable to the very short kinetic component (from 42 to 20%), in the shape of the BGF excitation spectra, and chlorogenic acid concentrations indicate that there is a loss of hydroxycinnamic acid with leaf age. Studies on excitation, emission, and synchronized fluorescence spectra of leaves and trichomes and chlorogenic acid contents indicate that chlorogenic acid is one of the main blue-green fluorophores in artichoke leaves. Results of the present study indicate that 20-42% (i.e., the very short kinetic component) of the overall BGF is emitted by chlorogenic acid. Time-resolved BGF measurements could be a means to extract information on chlorogenic acid fluorescence from the overall leaf BGF.

  9. SGLT2 Protein Expression Is Increased in Human Diabetic Nephropathy

    Science.gov (United States)

    Wang, Xiaoxin X.; Levi, Jonathan; Luo, Yuhuan; Myakala, Komuraiah; Herman-Edelstein, Michal; Qiu, Liru; Wang, Dong; Peng, Yingqiong; Grenz, Almut; Lucia, Scott; Dobrinskikh, Evgenia; D'Agati, Vivette D.; Koepsell, Hermann; Kopp, Jeffrey B.; Rosenberg, Avi Z.; Levi, Moshe

    2017-01-01

    There is very limited human renal sodium gradient-dependent glucose transporter protein (SGLT2) mRNA and protein expression data reported in the literature. The first aim of this study was to determine SGLT2 mRNA and protein levels in human and animal models of diabetic nephropathy. We have found that the expression of SGLT2 mRNA and protein is increased in renal biopsies from human subjects with diabetic nephropathy. This is in contrast to db-db mice that had no changes in renal SGLT2 protein expression. Furthermore, the effect of SGLT2 inhibition on renal lipid content and inflammation is not known. The second aim of this study was to determine the potential mechanisms of beneficial effects of SGLT2 inhibition in the progression of diabetic renal disease. We treated db/db mice with a selective SGLT2 inhibitor JNJ 39933673. We found that SGLT2 inhibition caused marked decreases in systolic blood pressure, kidney weight/body weight ratio, urinary albumin, and urinary thiobarbituric acid-reacting substances. SGLT2 inhibition prevented renal lipid accumulation via inhibition of carbohydrate-responsive element-binding protein-β, pyruvate kinase L, SCD-1, and DGAT1, key transcriptional factors and enzymes that mediate fatty acid and triglyceride synthesis. SGLT2 inhibition also prevented inflammation via inhibition of CD68 macrophage accumulation and expression of p65, TLR4, MCP-1, and osteopontin. These effects were associated with reduced mesangial expansion, accumulation of the extracellular matrix proteins fibronectin and type IV collagen, and loss of podocyte markers WT1 and synaptopodin, as determined by immunofluorescence microscopy. In summary, our study showed that SGLT2 inhibition modulates renal lipid metabolism and inflammation and prevents the development of nephropathy in db/db mice. PMID:28196866

  10. Quantification of endogenous and exogenous protein expressions of Na,K-ATPase with super-resolution PALM/STORM imaging.

    Science.gov (United States)

    Bernhem, Kristoffer; Blom, Hans; Brismar, Hjalmar

    2018-01-01

    Transient transfection of fluorescent fusion proteins is a key enabling technology in fluorescent microscopy to spatio-temporally map cellular protein distributions. Transient transfection of proteins may however bypass normal regulation of expression, leading to overexpression artefacts like misallocations and excess amounts. In this study we investigate the use of STORM and PALM microscopy to quantitatively monitor endogenous and exogenous protein expression. Through incorporation of an N-terminal hemagglutinin epitope to a mMaple3 fused Na,K-ATPase (α1 isoform), we analyze the spatial and quantitative changes of plasma membrane Na,K-ATPase localization during competitive transient expression. Quantification of plasma membrane protein density revealed a time dependent increase of Na,K-ATPase, but no increase in size of protein clusters. Results show that after 41h transfection, the total plasma membrane density of Na,K-ATPase increased by 63% while the endogenous contribution was reduced by 16%.

  11. Compact whole-body fluorescent imaging of nude mice bearing EGFP expressing tumor

    Science.gov (United States)

    Chen, Yanping; Xiong, Tao; Chu, Jun; Yu, Li; Zeng, Shaoqun; Luo, Qingming

    2005-01-01

    Issue of tumor has been a hotspot of current medicine. It is important for tumor research to detect tumors bearing in animal models easily, fast, repetitively and noninvasivly. Many researchers have paid their increasing interests on the detecting. Some contrast agents, such as green fluorescent protein (GFP) and Discosoma red fluorescent protein (Dsred) were applied to enhance image quality. Three main kinds of imaging scheme were adopted to visualize fluorescent protein expressing tumors in vivo. These schemes based on fluorescence stereo microscope, cooled charge-coupled-device (CCD) or camera as imaging set, and laser or mercury lamp as excitation light source. Fluorescence stereo microscope, laser and cooled CCD are expensive to many institutes. The authors set up an inexpensive compact whole-body fluorescent imaging tool, which consisted of a Kodak digital camera (model DC290), fluorescence filters(B and G2;HB Optical, Shenyang, Liaoning, P.R. China) and a mercury 50-W lamp power supply (U-LH50HG;Olympus Optical, Japan) as excitation light source. The EGFP was excited directly by mercury lamp with D455/70 nm band-pass filter and fluorescence was recorded by digital camera with 520nm long-pass filter. By this easy operation tool, the authors imaged, in real time, fluorescent tumors growing in live mice. The imaging system is external and noninvasive. For half a year our experiments suggested the imaging scheme was feasible. Whole-body fluorescence optical imaging for fluorescent expressing tumors in nude mouse is an ideal tool for antitumor, antimetastatic, and antiangiogenesis drug screening.

  12. Evaluation of an mRNA lipofection procedure for human dendritic cells and induction of cytotoxic T lymphocytes against enhanced green fluorescence protein.

    Science.gov (United States)

    Okano, Kozue; Fukui, Mikiko; Suehiro, Yutaka; Hamanaka, Yuichiro; Imai, Kohzoh; Hinoda, Yuji

    2003-01-01

    We utilized an mRNA lipofection procedure in human dendritic cells (DCs) and attempted to induce cytotoxic T lymphocytes (CTLs) against enhanced green fluorescence protein (EGFP). EGFP mRNA was transfected into phytohemagglutinin (PHA)-stimulated lymphocytes or adherent peripheral blood mononuclear cell-derived DCs using a liposomal reagent. Lipofection efficiency was measured by flow cytometry. In PHA-stimulated lymphocytes, increasing concentrations of liposome or mRNA increased EGFP expression levels by up to 64.4%, but caused a decrease in cell viability. A similar trend was also observed in DCs. For 70% DC viability, the concentration of liposomes was 24 microl/ml, and the mRNA concentration was 6 microg/ml. Under these conditions, ELISPOT and (51)Cr release assays were performed on CD8+ T cells stimulated twice with EGFP mRNA-transfected DCs. The number of interferon-gamma-producing cells was increased when the CD8+ T cells were cocultured for 24 h with PHA-stimulated lymphocytes transfected with EGFP mRNA. The level of specific lysis of EGFP mRNA-transfected DCs also increased to approximately 80%, with an effector to target ratio of 40:1. These data suggest that EGFP is immunogenic for human T cells, confirming that our lipofection procedure may be of use for inducing specific CTLs. Copyright 2003 S. Karger AG, Basel

  13. Interaction analysis of chimeric metal-binding green fluorescent protein and artificial solid-supported lipid membrane by quartz crystal microbalance and atomic force microscopy

    International Nuclear Information System (INIS)

    Prachayasittikul, Virapong; Na Ayudhya, Chartchalerm Isarankura; Hilterhaus, Lutz; Hinz, Andreas; Tantimongcolwat, Tanawut; Galla, Hans-Joachim

    2005-01-01

    Non-specific adsorption and specific interaction between a chimeric green fluorescent protein (GFP) carrying metal-binding region and the immobilized zinc ions on artificial solid-supported lipid membranes was investigated using the quartz crystal microbalance technique and the atomic force microscopy (AFM). Supported lipid bilayer, composed of octanethiol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-[N- (5-amino-1-carboxypentyl iminodiacetic acid)succinyl] (NTA-DOGS)-Zn 2+ , was formed on the gold electrode of quartz resonator (5 MHz). Binding of the chimeric GFP to zinc ions resulted in a rapid decrease of resonance frequency. Reversibility of the process was demonstrated via the removal of metal ions by EDTA. Nanoscale structural orientation of the chimeric GFP on the membrane was imaged by AFM. Association constant of the specific binding to metal ions was 2- to 3-fold higher than that of the non-specific adsorption, which was caused by the fluidization effect of the metal-chelating lipid molecules as well as the steric hindrance effect. This infers a possibility for a further development of biofunctionalized membrane. However, maximization is needed in order to attain closer advancement to a membrane-based sensor device

  14. Gene expression of a green fluorescent protein homolog as a host-specific biomarker of heat stress within a reef-building coral.

    Science.gov (United States)

    Smith-Keune, C; Dove, S

    2008-01-01

    Recent incidences of mass coral bleaching indicate that major reef building corals are increasingly suffering thermal stress associated with climate-related temperature increases. The development of pulse amplitude modulated (PAM) fluorometry has enabled rapid detection of the onset of thermal stress within coral algal symbionts, but sensitive biomarkers of thermal stress specific to the host coral have been slower to emerge. Differential display reverse transcription polymerase chain reaction (DDRT-PCR) was used to produce fingerprints of gene expression for the reef-building coral Acropora millepora exposed to 33 degrees C. Changes in the expression of 23 out of 399 putative genes occurred within 144 h. Down-regulation of one host-specific gene (AmA1a) occurred within just 6 h. Full-length sequencing revealed the product of this gene to be an all-protein chromatophore (green fluorescent protein [GFP]-homolog). RT-PCR revealed consistent down-regulation of this GFP-homolog for three replicate colonies within 6 h at both 32 degrees C and 33 degrees C but not at lower temperatures. Down-regulation of this host gene preceded significant decreases in the photosynthetic activity of photosystem II (dark-adapted F (v)/F (m)) of algal symbionts as measured by PAM fluorometry. Gene expression of host-specific genes such as GFP-homologs may therefore prove to be highly sensitive indicators for the onset of thermal stress within host coral cells.

  15. Use of Blue-Green Fluorescence and Thermal Imaging in the Early Detection of Sunflower Infection by the Root Parasitic Weed Orobanche cumana Wallr.

    Science.gov (United States)

    Ortiz-Bustos, Carmen M; Pérez-Bueno, María L; Barón, Matilde; Molinero-Ruiz, Leire

    2017-01-01

    Although the impact of Orobanche cumana Wallr. on sunflower ( Helianthus annuus L.) becomes evident with emergence of broomrape shoots aboveground, infection occurs early after sowing, the host physiology being altered during underground parasite stages. Genetic resistance is the most effective control method and one of the main goals of sunflower breeding programmes. Blue-green fluorescence (BGF) and thermal imaging allow non-destructive monitoring of plant diseases, since they are sensitive to physiological disorders in plants. We analyzed the BGF emission by leaves of healthy sunflower plantlets, and we implemented BGF and thermal imaging in the detection of the infection by O. cumana during underground parasite development. Increases in BGF emission were observed in leaf pairs of healthy sunflowers during their development. Lower BGF was consistently detected in parasitized plants throughout leaf expansion and low pigment concentration was detected at final time, supporting the interpretation of a decrease in secondary metabolites upon infection. Parasite-induced stomatal closure and transpiration reduction were suggested by warmer leaves of inoculated sunflowers throughout the experiment. BGF imaging and thermography could be implemented for fast screening of sunflower breeding material. Both techniques are valuable approaches to assess the processes by which O. cumana alters physiology (secondary metabolism and photosynthesis) of sunflower.

  16. Generation of knock-in mice that express nuclear enhanced green fluorescent protein and tamoxifen-inducible Cre recombinase in the notochord from Foxa2 and T loci.

    Science.gov (United States)

    Imuta, Yu; Kiyonari, Hiroshi; Jang, Chuan-Wei; Behringer, Richard R; Sasaki, Hiroshi

    2013-03-01

    The node and the notochord are important embryonic signaling centers that control embryonic pattern formation. Notochord progenitor cells present in the node and later in the posterior end of the notochord move anteriorly to generate the notochord. To understand the dynamics of cell movement during notochord development and the molecular mechanisms controlling this event, analyses of cell movements using time-lapse imaging and conditional manipulation of gene activities are required. To achieve this goal, we generated two knock-in mouse lines that simultaneously express nuclear enhanced green fluorescent protein (EGFP) and tamoxifen-inducible Cre, CreER(T2) , from two notochord gene loci, Foxa2 and T (Brachury). In Foxa2(nEGFP-CreERT2/+) and T(nEGFP-CreERT2/+) embryos, nuclei of the Foxa2 or T-expressing cells, which include the node, notochord, and endoderm (Foxa2) or wide range of posterior mesoderm (T), were labeled with EGFP at intensities that can be used for live imaging. Cre activity was also induced in cells expressing Foxa2 and T 1 day after tamoxifen administration. These mice are expected to be useful tools for analyzing the mechanisms of notochord development. Copyright © 2013 Wiley Periodicals, Inc.

  17. Use of Blue-Green Fluorescence and Thermal Imaging in the Early Detection of Sunflower Infection by the Root Parasitic Weed Orobanche cumana Wallr.

    Directory of Open Access Journals (Sweden)

    Carmen M. Ortiz-Bustos

    2017-05-01

    Full Text Available Although the impact of Orobanche cumana Wallr. on sunflower (Helianthus annuus L. becomes evident with emergence of broomrape shoots aboveground, infection occurs early after sowing, the host physiology being altered during underground parasite stages. Genetic resistance is the most effective control method and one of the main goals of sunflower breeding programmes. Blue-green fluorescence (BGF and thermal imaging allow non-destructive monitoring of plant diseases, since they are sensitive to physiological disorders in plants. We analyzed the BGF emission by leaves of healthy sunflower plantlets, and we implemented BGF and thermal imaging in the detection of the infection by O. cumana during underground parasite development. Increases in BGF emission were observed in leaf pairs of healthy sunflowers during their development. Lower BGF was consistently detected in parasitized plants throughout leaf expansion and low pigment concentration was detected at final time, supporting the interpretation of a decrease in secondary metabolites upon infection. Parasite-induced stomatal closure and transpiration reduction were suggested by warmer leaves of inoculated sunflowers throughout the experiment. BGF imaging and thermography could be implemented for fast screening of sunflower breeding material. Both techniques are valuable approaches to assess the processes by which O. cumana alters physiology (secondary metabolism and photosynthesis of sunflower.

  18. Leading coordinate analysis of reaction pathways in proton chain transfer: Application to a two-proton transfer model for the green fluorescent protein

    International Nuclear Information System (INIS)

    Wang Sufan; Smith, Sean C.

    2006-01-01

    The 'leading coordinate' approach to computing an approximate reaction pathway, with subsequent determination of the true minimum energy profile, is applied to a two-proton chain transfer model based on the chromophore and its surrounding moieties within the green fluorescent protein (GFP). Using an ab initio quantum chemical method, a number of different relaxed energy profiles are found for several plausible guesses at leading coordinates. The results obtained for different trial leading coordinates are rationalized through the calculation of a two-dimensional relaxed potential energy surface (PES) for the system. Analysis of the 2-D relaxed PES reveals that two of the trial pathways are entirely spurious, while two others contain useful information and can be used to furnish starting points for successful saddle-point searches. Implications for selection of trial leading coordinates in this class of proton chain transfer reactions are discussed, and a simple diagnostic function is proposed for revealing whether or not a relaxed pathway based on a trial leading coordinate is likely to furnish useful information

  19. Comparing Avocado, Swamp Bay, and Camphortree as Hosts of Raffaelea lauricola Using a Green Fluorescent Protein (GFP)-Labeled Strain of the Pathogen.

    Science.gov (United States)

    Campbell, A S; Ploetz, R C; Rollins, J A

    2017-01-01

    Raffaelea lauricola, a fungal symbiont of the ambrosia beetle Xyleborus glabratus, causes laurel wilt in members of the Lauraceae plant family. North American species in the family, such as avocado (Persea americana) and swamp bay (P. palustris), are particularly susceptible to laurel wilt, whereas the Asian camphortree (Cinnamomum camphora) is relatively tolerant. To determine whether susceptibility is related to pathogen colonization, a green fluorescent protein-labeled strain of R. lauricola was generated and used to inoculate avocado, swamp bay, and camphortree. Trees were harvested 3, 10, and 30 days after inoculation (DAI), and disease severity was rated on a 1-to-10 scale. By 30 DAI, avocado and swamp bay developed significantly more severe disease than camphortree (mean severities of 6.8 and 5.5 versus 1.6, P < 0.003). The extent of xylem colonization was recorded as the percentage of lumena that were colonized by the pathogen. More xylem was colonized in avocado than camphortree (0.9% versus 0.1%, P < 0.03) but colonization in swamp bay (0.4%) did not differ significantly from either host. Although there were significant correlations between xylem colonization and laurel wilt severity in avocado (r = 0.74), swamp bay (r = 0.82), and camphortree (r = 0.87), even severely affected trees of all species were scarcely colonized by the pathogen.

  20. A Real-Time Near-Infrared Fluorescence Imaging Method for the Detection of Oral Cancers in Mice Using an Indocyanine Green-Labeled Podoplanin Antibody.

    Science.gov (United States)

    Ito, Akihiro; Ohta, Mitsuhiko; Kato, Yukinari; Inada, Shunko; Kato, Toshio; Nakata, Susumu; Yatabe, Yasushi; Goto, Mitsuo; Kaneda, Norio; Kurita, Kenichi; Nakanishi, Hayao; Yoshida, Kenji

    2018-01-01

    Podoplanin is distinctively overexpressed in oral squamous cell carcinoma than oral benign neoplasms and plays a crucial role in the pathogenesis and metastasis of oral squamous cell carcinoma but its diagnostic application is quite limited. Here, we report a new near-infrared fluorescence imaging method using an indocyanine green (ICG)-labeled anti-podoplanin antibody and a desktop/a handheld ICG detection device for the visualization of oral squamous cell carcinoma-xenografted tumors in nude mice. Both near-infrared imaging methods using a desktop (in vivo imaging system: IVIS) and a handheld device (photodynamic eye: PDE) successfully detected oral squamous cell carcinoma tumors in nude mice in a podoplanin expression-dependent manner with comparable sensitivity. Of these 2 devices, only near-infrared imaging methods using a handheld device visualized oral squamous cell carcinoma xenografts in mice in real time. Furthermore, near-infrared imaging methods using the handheld device (PDE) could detect smaller podoplanin-positive oral squamous cell carcinoma tumors than a non-near-infrared, autofluorescence-based imaging method. Based on these results, a near-infrared imaging method using an ICG-labeled anti-podoplanin antibody and a handheld detection device (PDE) allows the sensitive, semiquantitative, and real-time imaging of oral squamous cell carcinoma tumors and therefore represents a useful tool for the detection and subsequent monitoring of malignant oral neoplasms in both preclinical and some clinical settings.

  1. Synthesis and green up-conversion fluorescence of colloidal La0.78Yb0.20Er0.02F3/SiO2 core/shell nanocrystals

    International Nuclear Information System (INIS)

    Wang Yan; Qin Weiping; Zhang Jisen; Cao Chunyan; Zhang Jishuang; Jin Ye; Zhu Peifen; Wei Guodong; Wang Guofeng; Wang Lili

    2007-01-01

    Water-soluble PVP-stabilized hexagonal-phase La 0.78 Yb 0.20 Er 0.02 F 3 nanocrystals (NCs) were synthesized by hydrothermal method. The NCs were coated with a very thin silica shell, and amino groups were introduced to the surface of silica shells by copolymerization of 3-aminopropyl(triethoxy)silane. The core/shell NCs can be dispersed in ethanol and water to form stable colloidal solution. The transmission electron microscopy (TEM), selected area electron diffraction (SAED), powder X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FT-IR) were used to characterize the core/shell materials. In addition, the green up-conversion fluorescence mechanism of La 0.78 Yb 0.20 Er 0.02 F 3 /SiO 2 NCs was studied with a 980-nm diode laser as excitation source. The water solubility, small core/shell particles size, and well colloidal stability mean the green up-conversion fluorescence NCs have potential applications in bioassay. - Graphical abstract: Colloidal La 0.78 Yb 0.20 Er 0.02 F 3 /SiO 2 Core/Shell nanocrystals (NCs) were synthesized and the free amino groups were introduced to the surface of silica shells by copolymerization 3-aminopropyl(triethoxy)silane. The NCs can be dispersed in ethanol and water to form stable colloidal solution. In addition, the NCs exhibit green up-conversion fluorescence under 980-nm excitation

  2. Monitoring prion protein expression in complex biological samples by SERS for diagnostic applications

    Energy Technology Data Exchange (ETDEWEB)

    Manno, D; Filippo, E; Fiore, R; Serra, A [Dipartimento di Scienza dei Materiali, Universita del Salento, Lecce (Italy); Urso, E; Rizzello, A; Maffia, M [Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita del Salento, Lecce (Italy)

    2010-04-23

    Surface-enhanced Raman spectroscopy (SERS) allows a new insight into the analysis of cell physiology. In this work, the difficulty of producing suitable substrates that, besides permitting the amplification of the Raman signal, do not interact with the biological material causing alteration, has been overcome by a combined method of hydrothermal green synthesis and thermal annealing. The SERS analysis of the cell membrane has been performed with special attention to the cellular prion protein PrP{sup C}. In addition, SERS has also been used to reveal the prion protein-Cu(II) interaction in four different cell models (B104, SH-SY5Y, GN11, HeLa), expressing PrP{sup C} at different levels. A significant implication of the current work consists of the intriguing possibility of revealing and quantifying prion protein expression in complex biological samples by a cheap SERS-based method, replacing the expensive and time-consuming immuno-assay systems commonly employed.

  3. Heterogeneity of proteins expressed by Brazilian Sporothrix schenckii isolates.

    Science.gov (United States)

    Fernandes, Geisa Ferreira; Do Amaral, Cristiane Candida; Sasaki, Alexandre; Godoy, Patrício Martinez; De Camargo, Zoilo Pires

    2009-12-01

    The profiles of proteins present in the exoantigens of Brazilian Sporothrix schenckii isolates were studied and compared by electrophoresis (SDS-PAGE). Thirteen isolates from five different regions of Brazil (1,000 to 2,000 km apart) and ten from a more limited region (200 to 400 km apart within the state of São Paulo) were cultured in Sabouraud, M199 and minimum (MM) media. Qualitative and quantitative differences in the expression of proteins, which varied according to the medium and the isolate, were observed. Fractions with the same MW but varying in intensity were detected, as well as fractions present in 1 isolate but absent in others. Dendrograms were constructed and isolates grouped based on the fractions obtained, irrespective of the intensity. The results showed that Brazilian S. schenckii isolates express different protein profiles, a feature also present in isolates from a more restricted region. The exoantigens were found to have a maximum of 15 protein fractions, ranging in MW from 19-220 KDaltons depending on the medium used for the cultures. These data show the great heterogeneity of Brazilian S. schenckii protein expression.

  4. HER 2/neu protein expression in colorectal cancer

    International Nuclear Information System (INIS)

    Schuell, B; Gruenberger, T; Scheithauer, W; Zielinski, Ch; Wrba, F

    2006-01-01

    Conflicting data exist about the prevalence of HER-2/neu overexpression in colorectal cancer ranging from 0 to 83 %. In our study we tried to clarify the extent of expression and its relationship to clinicopathological parameters. This study involved 77 specimens of malignant colorectal cancer lesions of surgically resected patients. HER-2/neu immunohistochemistry was performed using the Hercep-Test Kit. Out of 77 specimens, 56 were Her-2/neu negative (70%), 20 (26%) showed a barely immunostaining (1+), only 1 (1%) was moderately (2+) and 2 (3%) were strongly positive (3+). Her-2/neu staining (moderately and strongly positive) was only detected in primary tumours of patients with confirmed metastases. No relationship was found between membranous HER-2 expression and patients' gender or differentiation. The median survival time of patients with positive HER-2/neu immunostaining was 21 versus 39 months in patients without HER-2/neu expression (p = 0.088). The c-erbB protein expression was observed in colorectal cancer but rarely in the therapeutic range (2+ and 3+). There was no significant association with tumour grade, gender, localization of the primary tumour or survival. These data indicate that c-erbB-2 is unlikely to play a major role in the therapeutic management of colorectal cancer

  5. Synthesis and properties of the para-trimethylammonium analogues of green fluorescence protein (GFP) chromophore: The mimic of protonated GFP chromophore.

    Science.gov (United States)

    Fanjiang, Ming-Wei; Li, Ming-Ju; Sung, Robert; Sung, Kuangsen

    2018-04-01

    At low pH, protons from the external, bulk solution can protonate the phenoxide group of the p-HBDI chromophore in wild-type green fluorescent protein (wtGFP) and its mutants, and likely continue to tentatively protonate the phenol hydroxyl group of the same chromophores. Because the protonated GFP chromophore is a transient, we prepare the stable p-trimethylammonium analogues (2a and 2b) of the GFP chromophore to mimic it and explore their properties. What we found is that the p-trimethylammonium analogues of the GFP chromophore have the highly electrophilic amidine carbon, blue-shifted electronic absorption, smaller molar absorptivity, smaller fluorescent quantum yield, and faster E-Z thermoisomerization rate. The amidine carbon of the p-trimethylammonium analogue (2b) of the GFP chromophore is the only site that is attacked by very weak nucleophile of water, resulting in ring-opening of the imidazolinone moiety. The half-life of its decay rate in D 2 O is around 33 days. Actually, acid-catalyzed hydrolysis of p-HBDI also results in ring-opening of the imidazolinone moiety. The ratio of the acid-catalyzed hydrolysis rate constants [k obs (p-HBDI)/k obs (1b)] between p-HBDI and 1b (p-dimethylammonium analogue of the GFP chromophore) is dramatically increased from 0.30 at pH = 2 to 0.63 at pH = 0. This is the evidence that more and more phenol hydroxyl groups of p-HBDI are tentatively protonated in a low-pH aqueous solution and that accelerates hydrolysis of p-HBDI in the way similar to the quaternary ammonium derivatives 2a and 2b in water. With this view point, 2a and 2b still can partially mimic the cationic p-HBDI with the protonated phenol hydroxyl group. Implication of the experiment is that the amidine carbon of the chromophore in wtGFP and its mutants at very low pH should be highly electrophilic. Whether ring-opening of the imidazolinone moiety of the GFP chromophore would occur or not depends on if water molecules can reach the amidine carbon of

  6. Stimulation of Protein Expression Through the Harmonic Resonance of Frequency-Specific Music.

    Science.gov (United States)

    Orhan, Ibrahim Y; Gulbahar, Burak A

    2016-12-01

    The use of specific frequencies for specific individual amino acids may increase the potential energy of protein molecules in the medium [1]. The resonance would also increase the movement of particles in the cytosol, increasing the collisions necessary for the conduction of protein expression. The clash of two waves that share frequencies will exhibit an increase in energy through an increase in amplitude [2]. The increase in energy would in turn increase the number of collisions forming the tRNA-amino acid, increasing the amino acid acquiry for ribosomes to improve intracellular efficiency in gene expression. To test the hypothesis, Red Fluorescent Protein (RFP) in transformated BL-21 strains of E. coli and p53 protein of MCF-7 were examined after exposure to sounds of specific frequencies. Through the exposure of the experimental systems to a sequence of sounds that match the frequencies of specific amino acids, the levels of RFP exhibition respective to the control groups in the bacterial medium increased two-fold in terms of RFU. The experiments that targeted the p53 protein with the 'music' showed a decrease in the cell prevalence in the MCF-7 type breast cancer cells by 28%, by decreasing the speed of tumour formation. Exposure to 'music' that was designed through assigning a musical note for every single one of the twenty unique amino acids, produced both an analytical and a visible shift in protein synthesis, making it as potential tool for reducing procedural time uptake.

  7. Differential protein expression in alligator leukocytes in response to bacterial lipopolysaccharide injection.

    Science.gov (United States)

    Merchant, Mark; Kinney, Clint; Sanders, Paige

    2009-12-01

    Blood was collected from three juvenile alligators (Alligator mississippiensis) before, and again 24h after, injection with bacterial lipopolysaccharide (LPS). The leukocytes were collected from both samples, and the proteins were extracted. Each group of proteins was labeled with a different fluorescent dye and the differences in protein expression were analyzed by two dimensional differential in-gel expressions (2D-DIGE). The proteins which appeared to be increased or decreased by treatment with LPS were selected and analyzed by MALDI-TOF to determine mass and LC-MS/MS to acquire the partial protein sequences. The peptide sequences were compared to the NCBI protein sequence database to determine homology with other sequences from other species. Several proteins of interest appeared to be increased upon LPS stimulation. Proteins with homology to human transgelin-2, fish glucose-6-phosphate dehydrogenase, amphibian α-enolase, alligator lactate dehydrogenase, fish ubiquitin-activating enzyme, and fungal β-tubulin were also increased after LPS injection. Proteins with homology to fish vimentin 4, murine heterogeneous nuclear ribonucleoprotein A3, and avian calreticulin were found to be decreased in response to LPS. In addition, five proteins, four of which were up-regulated (827, 560, 512, and 650%) and one that exhibited repressed expression (307%), did not show homology to any protein in the database, and thus may represent newly discovered proteins. We are using this biochemical approach to isolate and characterize alligator proteins with potential relevant immune function.

  8. Transmission of Mannheimia haemolytica from domestic sheep (Ovis aries) to bighorn sheep (Ovis canadensis): unequivocal demonstration with green fluorescent protein-tagged organisms.

    Science.gov (United States)

    Lawrence, Paulraj K; Shanthalingam, Sudarvili; Dassanayake, Rohana P; Subramaniam, Renuka; Herndon, Caroline N; Knowles, Donald P; Rurangirwa, Fred R; Foreyt, William J; Wayman, Gary; Marciel, Ann Marie; Highlander, Sarah K; Srikumaran, Subramaniam

    2010-07-01

    Previous studies demonstrated that bighorn sheep (Ovis canadensis) died of pneumonia when commingled with domestic sheep (Ovis aries) but did not conclusively prove that the responsible pathogens were transmitted from domestic to bighorn sheep. The objective of this study was to determine, unambiguously, whether Mannheimia haemolytica can be transmitted from domestic to bighorn sheep when they commingle. Four isolates of M. haemolytica were obtained from the pharynx of two of four domestic sheep and tagged with a plasmid carrying the genes for green fluorescent protein (GFP) and ampicillin resistance (AP(R)). Four domestic sheep, colonized with the tagged bacteria, were kept about 10 m apart from four bighorn sheep for 1 mo with no clinical signs of pneumonia observed in the bighorn sheep during that period. The domestic and bighorn sheep were then allowed to have fence-line contact for 2 mo. During that period, three bighorn sheep acquired the tagged bacteria from the domestic sheep. At the end of the 2 mo of fence-line contact, the animals were allowed to commingle. All four bighorn sheep died 2 days to 9 days following commingling. The lungs from all four bighorn sheep showed gross and histopathologic lesions characteristic of M. haemolytica pneumonia. Tagged M. haemolytica were isolated from all four bighorn sheep, as confirmed by growth in ampicillin-containing culture medium, PCR-amplification of genes encoding GFP and Ap(R), and immunofluorescent staining of GFP. These results unequivocally demonstrate transmission of M. haemolytica from domestic to bighorn sheep, resulting in pneumonia and death of bighorn sheep.

  9. Non-Target Effects of Green Fluorescent Protein (GFP-Derived Double-Stranded RNA (dsRNA-GFP Used in Honey Bee RNA Interference (RNAi Assays

    Directory of Open Access Journals (Sweden)

    Francis M. F. Nunes

    2013-01-01

    Full Text Available RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP-derived double-stranded RNA (dsRNA-GFP is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control.

  10. Non-Target Effects of Green Fluorescent Protein (GFP)-Derived Double-Stranded RNA (dsRNA-GFP) Used in Honey Bee RNA Interference (RNAi) Assays.

    Science.gov (United States)

    Nunes, Francis M F; Aleixo, Aline C; Barchuk, Angel R; Bomtorin, Ana D; Grozinger, Christina M; Simões, Zilá L P

    2013-01-04

    RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP)-derived double-stranded RNA (dsRNA-GFP) is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding) on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control.

  11. Early postnatal virus inoculation into the scala media achieved extensive expression of exogenous green fluorescent protein in the inner ear and preserved auditory brainstem response thresholds.

    Science.gov (United States)

    Wang, Yunfeng; Sun, Yu; Chang, Qing; Ahmad, Shoeb; Zhou, Binfei; Kim, Yeunjung; Li, Huawei; Lin, Xi

    2013-01-01

    Gene transfer into the inner ear is a promising approach for treating sensorineural hearing loss. The special electrochemical environment of the scala media raises a formidable challenge for effective gene delivery at the same time as keeping normal cochlear function intact. The present study aimed to define a suitable strategy for preserving hearing after viral inoculation directly into the scala media performed at various postnatal developmental stages. We assessed transgene expression of green fluorescent protein (GFP) mediated by various types of adeno-associated virus (AAV) and lentivirus (LV) in the mouse cochlea. Auditory brainstem responses were measured 30 days after inoculation to assess effects on hearing. Patterns of GFP expression confirmed extensive exogenous gene expression in various types of cells lining the endolymphatic space. The use of different viral vectors and promoters resulted in specific cellular GFP expression patterns. AAV2/1 with cytomegalovirus promoter apparently gave the best results for GFP expression in the supporting cells. Histological examination showed normal cochlear morphology and no hair cell loss after either AAV or LV injections. We found that hearing thresholds were not significantly changed when the injections were performed in mice younger than postnatal day 5, regardless of the type of virus tested. Viral inoculation and expression in the inner ear for the restoration of hearing must not damage cochlear function. Using normal hearing mice as a model, we have achieved this necessary step, which is required for the treatment of many types of congenital deafness that require early intervention. Copyright © 2013 John Wiley & Sons, Ltd.

  12. Protein expression analysis of inflammation-related colon carcinogenesis

    Directory of Open Access Journals (Sweden)

    Yasui Yumiko

    2009-01-01

    Full Text Available Background: Chronic inflammation is a risk factor for colorectal cancer (CRC development. The aim of this study was to determine the differences in protein expression between CRC and the surrounding nontumorous colonic tissues in the mice that received azoxymethane (AOM and dextran sodium sulfate (DSS using a proteomic analysis. Materials and Methods: Male ICR mice were given a single intraperitoneal injection of AOM (10 mg/kg body weight, followed by 2% (w/v DSS in their drinking water for seven days, starting one week after the AOM injection. Colonic adenocarcinoma developed after 20 weeks and a proteomics analysis based on two-dimensional gel electrophoresis and ultraflex TOF/TOF mass spectrometry was conducted in the cancerous and nontumorous tissue specimens. Results: The proteomic analysis revealed 21 differentially expressed proteins in the cancerous tissues in comparison to the nontumorous tissues. There were five markedly increased proteins (beta-tropomyosin, tropomyosin 1 alpha isoform b, S100 calcium binding protein A9, and an unknown protein and 16 markedly decreased proteins (Car1 proteins, selenium-binding protein 1, HMG-CoA synthase, thioredoxin 1, 1 Cys peroxiredoxin protein 2, Fcgbp protein, Cytochrome c oxidase, subunit Va, ETHE1 protein, and 7 unknown proteins. Conclusions: There were 21 differentially expressed proteins in the cancerous tissues of the mice that received AOM and DSS. Their functions include metabolism, the antioxidant system, oxidative stress, mucin production, and inflammation. These findings may provide new insights into the mechanisms of inflammation-related colon carcinogenesis and the establishment of novel therapies and preventative strategies to treat carcinogenesis in the inflamed colon.

  13. The proteome response to amyloid protein expression in vivo.

    Directory of Open Access Journals (Sweden)

    Ricardo A Gomes

    Full Text Available Protein misfolding disorders such as Alzheimer, Parkinson and transthyretin amyloidosis are characterized by the formation of protein amyloid deposits. Although the nature and location of the aggregated proteins varies between different diseases, they all share similar molecular pathways of protein unfolding, aggregation and amyloid deposition. Most effects of these proteins are likely to occur at the proteome level, a virtually unexplored reality. To investigate the effects of an amyloid protein expression on the cellular proteome, we created a yeast expression system using human transthyretin (TTR as a model amyloidogenic protein. We used Saccharomyces cerevisiae, a living test tube, to express native TTR (non-amyloidogenic and the amyloidogenic TTR variant L55P, the later forming aggregates when expressed in yeast. Differential proteome changes were quantitatively analyzed by 2D-differential in gel electrophoresis (2D-DIGE. We show that the expression of the amyloidogenic TTR-L55P causes a metabolic shift towards energy production, increased superoxide dismutase expression as well as of several molecular chaperones involved in protein refolding. Among these chaperones, members of the HSP70 family and the peptidyl-prolyl-cis-trans isomerase (PPIase were identified. The latter is highly relevant considering that it was previously found to be a TTR interacting partner in the plasma of ATTR patients but not in healthy or asymptomatic subjects. The small ubiquitin-like modifier (SUMO expression is also increased. Our findings suggest that refolding and degradation pathways are activated, causing an increased demand of energetic resources, thus the metabolic shift. Additionally, oxidative stress appears to be a consequence of the amyloidogenic process, posing an enhanced threat to cell survival.

  14. Protein expression of saccharomyces cerevisiae in response to uranium exposure

    International Nuclear Information System (INIS)

    Sakamoto, Fuminori; Nankawa, Takuya; Kozai, Naofumi; Ohnuki, Toshihiko; Fujii, Tsutomu; Iefuji, Haruyuki; Francis, A.J.

    2007-01-01

    Protein expression of Saccharomyces cerevisiae grown in the medium containing 238 U (VI) and 233 U (VI) was examined by two-dimensional gel electrophoresis. Saccharomyces cerevisiae of BY4743 was grown in yeast nitrogen base medium containing glucose and glycerol 2-phosphate and 238 U of 0, 2.0, and 5.0 x 10 -4 M or 233 U of 2.5 x 10 -6 M (radioactivity was higher by 350 times than 2.0 x 10 -4 M 238 U) and 5.0 x 10 -6 M for 112 h at 30 degC. The growth of Saccharomyces cerevisiae was monitored by measuring OD 600 at 112 h after the inoculation. Uranium concentrations in the media also were measured by radiometry using a liquid scintillation counter. The growths of the yeast grown in the above media were in the following order: control>2.5 x 10 -6 M 233 U>2.0 x 10 -4 M 238 U>5.0 x 10 -6 M 233 U>5.0 x 10 -4 M 238 U. This result indicated that not only radiological but also chemical effect of U reduced the growth of the yeast. The concentrations of U in the medium containing 238 U or 233 U decreased, suggesting U accumulation by the yeast cells. The 2-D gel electrophoresis analysis showed the appearance of several spots after exposure to 238 U or to 233 U but not in the control containing no uranium. These results show that the yeast cells exposed to U express several specific proteins. (author)

  15. Intrafocal heterogeneity of ERG protein expression and gene fusion pattern in prostate cancer.

    Science.gov (United States)

    Suh, Ja Hee; Park, Jeong Hwan; Lee, Cheol; Moon, Kyung Chul

    2017-10-01

    Prostate cancer is considered to be highly heterogeneous, with various morphologic features and biologic behaviors. The TMPRSS2-ERG gene fusion is the most frequently observed genetic aberration in prostate cancer. The aim of this study was to elucidate the intrafocal heterogeneity of ERG gene fusion status. ERG immunohistochemistry (IHC) was performed in samples from 168 prostate cancer patients who had undergone radical prostatectomy, and 40 cases showing ERG-positive IHC staining were selected for tissue microarray (TMA) construction. Two to six representative cores were selected from each tumor focus. In the cases with heterogeneous ERG IHC staining intensity, the areas showing different intensities were separately selected. Using the TMA blocks, IHC and fluorescence in situ hybridization (FISH) were conducted to evaluate the heterogeneity of ERG protein expression and ERG fusion gene patterns, respectively, in a single tumor focus. Heterogeneity of ERG IHC staining was defined as the simultaneous presence of negative and positive cores in the same tumor focus. Heterogeneity of ERG FISH was defined by the presence of cores with positive and negative FISH signals or cores with break-apart and interstitial deletion FISH signals in the same tumor focus. A total of 202 TMA cores were isolated from 40 ERG-positive cases. Of the 202 total cores, 19 were negative for ERG IHC staining, and 46 showed 1+, 52 showed 2+, and 85 showed 3+ ERG staining intensity. Eleven cores were negative for ERG FISH signal, 119 cores showed ERG break-apart FISH signals, and the remaining 72 cores revealed interstitial deletion. Intrafocal heterogeneity of ERG IHC staining was found in 20% (8/40) of cases, and intrafocal heterogeneity of ERG gene fusion pattern was found in 32.5% (13/40) of cases. In summary, this study showed significantly frequent intrafocal heterogeneity of ERG protein expression, gene fusion status and fusion pattern. This heterogeneity can be caused by the development

  16. Construction of genetically engineered bacteria that degrades organophosphorus pesticide residues and can be easily detected by the fluorescence.

    Science.gov (United States)

    Li, Qin; Wang, Pan; Chen, Rui; Li, Wei; Wu, Yi-Jun

    2014-01-01

    Organophosphorus compounds (OPs) are widely used in agriculture and industry and there is increased concern about their toxicological effects in the environment. Bioremediation can offer an efficient and cost-effective option for the removal of OPs. Herein, we describe the construction of a genetically engineered microorganism (GEM) that can degrade OPs and be directly detected and monitored in the environment using an enhanced green fluorescent protein (EGFP) fusion strategy. The coding regions of EGFP, a reporter protein that can fluoresce by itself, and organophosphorus hydrolase (OPH), which has a broad substrate specificity and is able to hydrolyse a number of organophosphorus pesticides, were cloned into the expression vector pET-28b. The fusion protein of EGFP-OPH was expressed in E. coli BL21 (DE3) and the protein expression reached the highest level at 11 h after isopropyl beta-D-thiogalactopyranoside induction. The fluorescence of the GEM was detected by fluorescence spectrophotometry and microscopy, and its ability to degrade OPs was determined by OPH activity assay. Those GEM that express the fusion protein (EGFP and OPH) exhibited strong fluorescence intensity and also potent hydrolase activity, which could be used to degrade organophosphorus pesticide residues in the environment and can also be directly monitored by fluorescence.

  17. Nitrile Probes of Electric Field Agree with Independently Measured Fields in Green Fluorescent Protein Even in the Presence of Hydrogen Bonding.

    Science.gov (United States)

    Slocum, Joshua D; Webb, Lauren J

    2016-05-25

    There is growing interest in using the nitrile vibrational oscillation as a site-specific probe of local environment to study dynamics, folding, and electrostatics in biological molecules such as proteins. Nitrile probes have been used extensively as reporters of electric field using vibrational Stark effect spectroscopy. However, the analysis of frequencies in terms of electric fields is potentially complicated by the large ground state dipole moment of the nitrile, which may irrevocably perturb the protein under investigation, and the ability of nitriles to accept hydrogen bonds, which causes frequency shifts that are not described by the Stark effect. The consequence of this is that vibrational spectroscopy of nitriles in biomolecules could be predominately sensitive to their local hydration status, not electrostatic environment, and have the potential to be particularly destabilizing to the protein. Here, we introduce green fluorescent protein (GFP) as a model system for addressing these concerns using biosynthetically incorporated p-cyanophenylalanine (pCNF) residues in the interior of GFP and measuring absorption energies of both the intrinsic GFP fluorophore and pCNF residues in response to a series of amino acid mutations. We show that observed changes in emission energy of GFP due to the mutations strongly correlate with changes in electric field experienced by both the nitrile probes and the intrinsic fluorophore. Additionally, we show that changes in electric field measured from the intrinsic fluorophore due to amino acid mutations are unperturbed by the addition of pCNF residues inserted nearby. Finally, we show that changes in electric field experienced by the vibrational probes trend monotonically with changes in field experienced by the native fluorophore even though the nitrile probe is engaged in moderate hydrogen bonding to nearby water molecules, indicated by the temperature dependence of the nitrile's absorption energy. Together these results

  18. Green fluorescent protein (GFP color reporter gene visualizes parvovirus B19 non-structural segment 1 (NS1 transfected endothelial modification.

    Directory of Open Access Journals (Sweden)

    Thomas Wurster

    Full Text Available BACKGROUND: Human Parvovirus B19 (PVB19 has been associated with myocarditis putative due to endothelial infection. Whether PVB19 infects endothelial cells and causes a modification of endothelial function and inflammation and, thus, disturbance of microcirculation has not been elucidated and could not be visualized so far. METHODS AND FINDINGS: To examine the PVB19-induced endothelial modification, we used green fluorescent protein (GFP color reporter gene in the non-structural segment 1 (NS1 of PVB19. NS1-GFP-PVB19 or GFP plasmid as control were transfected in an endothelial-like cell line (ECV304. The endothelial surface expression of intercellular-adhesion molecule-1 (CD54/ICAM-1 and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147 were evaluated by flow cytometry after NS-1-GFP or control-GFP transfection. To evaluate platelet adhesion on NS-1 transfected ECs, we performed a dynamic adhesion assay (flow chamber. NS-1 transfection causes endothelial activation and enhanced expression of ICAM-1 (CD54: mean ± standard deviation: NS1-GFP vs. control-GFP: 85.3 ± 11.2 vs. 61.6 ± 8.1; P<0.05 and induces endothelial expression of EMMPRIN/CD147 (CD147: mean ± SEM: NS1-GFP vs. control-GFP: 114 ± 15.3 vs. 80 ± 0.91; P<0.05 compared to control-GFP transfected cells. Dynamic adhesion assays showed that adhesion of platelets is significantly enhanced on NS1 transfected ECs when compared to control-GFP (P<0.05. The transfection of ECs was verified simultaneously through flow cytometry, immunofluorescence microscopy and polymerase chain reaction (PCR analysis. CONCLUSIONS: GFP color reporter gene shows transfection of ECs and may help to visualize NS1-PVB19 induced endothelial activation and platelet adhesion as well as an enhanced monocyte adhesion directly, providing in vitro evidence of possible microcirculatory dysfunction in PVB19-induced myocarditis and, thus, myocardial tissue damage.

  19. Orthogonal Electric Field Measurements near the Green Fluorescent Protein Fluorophore through Stark Effect Spectroscopy and pKa Shifts Provide a Unique Benchmark for Electrostatics Models.

    Science.gov (United States)

    Slocum, Joshua D; First, Jeremy T; Webb, Lauren J

    2017-07-20

    Measurement of the magnitude, direction, and functional importance of electric fields in biomolecules has been a long-standing experimental challenge. pK a shifts of titratable residues have been the most widely implemented measurements of the local electrostatic environment around the labile proton, and experimental data sets of pK a shifts in a variety of systems have been used to test and refine computational prediction capabilities of protein electrostatic fields. A more direct and increasingly popular technique to measure electric fields in proteins is Stark effect spectroscopy, where the change in absorption energy of a chromophore relative to a reference state is related to the change in electric field felt by the chromophore. While there are merits to both of these methods and they are both reporters of local electrostatic environment, they are fundamentally different measurements, and to our knowledge there has been no direct comparison of these two approaches in a single protein. We have recently demonstrated that green fluorescent protein (GFP) is an ideal model system for measuring changes in electric fields in a protein interior caused by amino acid mutations using both electronic and vibrational Stark effect chromophores. Here we report the changes in pK a of the GFP fluorophore in response to the same mutations and show that they are in excellent agreement with Stark effect measurements. This agreement in the results of orthogonal experiments reinforces our confidence in the experimental results of both Stark effect and pK a measurements and provides an excellent target data set to benchmark diverse protein electrostatics calculations. We used this experimental data set to test the pK a prediction ability of the adaptive Poisson-Boltzmann solver (APBS) and found that a simple continuum dielectric model of the GFP interior is insufficient to accurately capture the measured pK a and Stark effect shifts. We discuss some of the limitations of this

  20. Scaffold preferences of mesenchymal stromal cells and adipose-derived stem cells from green fluorescent protein transgenic mice influence the tissue engineering of bone.

    Science.gov (United States)

    Wittenburg, Gretel; Flade, Viktoria; Garbe, Annette I; Lauer, Günter; Labudde, Dirk

    2014-05-01

    We have analysed the growth and differentiation of mesenchymal stromal cells (MSC) from bone marrow, and of adipose derived stem cells (ASC) from murine abdominal fat tissue, of green fluorescent protein (GFP) transgenic animals grown directly on two types of hydroxyapatite ceramic bone substitutes. BONITmatrix® and NanoBone® have specific mechanical and physiochemical properties such as porosity and an inner surface that influence cellular growth. Both MSC and ASC were separately seeded on 200mg of each biomaterial and cultured for 3 weeks under osteogenic differentiation conditions. The degree of mineralisation was assessed by alizarin red dye and the specific alkaline phosphatase activity of the differentiated cells. The morphology of the cells was examined by scanning electron microscopy and confocal microscopy. The osteoblastic phenotype of the cells was confirmed by analysing the expression of bone-specific genes (Runx2, osteocalcin, osteopontin, and osteonectin) by semiquantitative reverse transcriptase polymerase chain reaction (PCR). Comparison of BONITmatrix® and NanoBone® showed cell type-specific preferences in terms of osteogenic differentiation. MSC-derived osteoblast-like cells spread optimally on the surface of NanoBone® but not BONITmatrix® granules. In contrast BONITmatrix® granules conditioned the growth of osteoblast-like cells derived from ASC. The osteoblastic phenotype of the cultured cells on all matrices was confirmed by specific gene expression. Our results show that the in vitro growth and osteogenic differentiation of murine MSC or ASC of GFP transgenic mice are distinctly influenced by the ceramic substratum. While NanoBone® granules support the proliferation and differentiation of murine MSC isolated from bone marrow, the growth of murine ASC is supported by BONITmatrix® granules. NanoBone® is therefore recommended for use as scaffold in tissue engineering that requires MSC, whereas ASC can be combined with BONITmatrix® for

  1. Construction of a subgenomic CV-B3 replicon expressing emerald green fluorescent protein to assess viral replication of a cardiotropic enterovirus strain in cultured human cells.

    Science.gov (United States)

    Wehbe, Michel; Huguenin, Antoine; Leveque, Nicolas; Semler, Bert L; Hamze, Monzer; Andreoletti, Laurent; Bouin, Alexis

    2016-04-01

    Coxsackieviruses B (CV-B) (Picornaviridae) are a common infectious cause of acute myocarditis in children and young adults, a disease, which is a precursor to 10-20% of chronic myocarditis and dilated cardiomyopathy (DCM) cases. The mechanisms involved in the disease progression from acute to chronic myocarditis phase and toward the DCM clinical stage are not fully understood but are influenced by both viral and host factors. Subgenomic replicons of CV-B can be used to assess viral replication mechanisms in human cardiac cells and evaluate the effects of potential antiviral drugs on viral replication activities. Our objectives were to generate a reporter replicon from a cardiotropic prototype CV-B3/28 strain and to characterize its replication properties into human cardiac primary cells. To obtain this replicon, a cDNA plasmid containing the full CV-B3/28 genome flanked by a hammerhead ribozyme sequence and an MluI restriction site was generated and used as a platform for the insertion of sequences encoding emerald green fluorescent protein (EmGFP) in place of those encoding VP3. In vitro transcribed RNA from this plasmid was transfected into HeLa cells and human primary cardiac cells and was able to produce EmGFP and VP1-containing polypeptides. Moreover, non-structural protein biological activity was assessed by the specific cleavage of eIF4G1 by viral 2A(pro). Viral RNA replication was indirectly demonstrated by inhibition assays, fluoxetine was added to cell culture and prevented the EmGFP synthesis. Our results indicated that the EmGFP CV-B3 replicon was able to replicate and translate as well as the CV-B3/28 prototype strain. Our EmGFP CV-B3 replicon will be a valuable tool to readily investigate CV-B3 replication activities in human target cell models. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Identification of the pigment responsible for the blue fluorescence band in the laser induced fluorescence (LIF) spectra of green plants, and the potential use of this band in remotely estimating rates of photosynthesis

    International Nuclear Information System (INIS)

    Chappelle, E.W.; McMurtrey, J.E. III; Kim, M.S.

    1991-01-01

    The laser-induced fluorescence (LIF) of vegetation is being investigated in this laboratory for use as a technique for the remote detection of the effects of environmental stress upon vegetation, as well as for plant identification. The fluorescence band with a maximum at 440 nm, in conjunction with the chlorophyll bands with maxima at 685 and 740 nm, has been found to be a critical band in the development of algorithms for detecting stress, and identifying plant types. The identification of the plant constituent responsible for this band is vital to understanding the mechanism underlying its fluorescence changes in response to environmental and physiological changes. The identification was achieved as follows: The laser induced fluorescence (LIF) spectra of pure plant pigments were determined. Fluorescence bands with maxima at 420 nm, 440 nm, 490 nm, and 525 nm were observed for vitamin K 1 , reduced nicotinamide adenine dinucleotide (NADPH), beta-carotene, and riboflavin, respectively. The LIF spectra of water extracts and acetone extracts of clover leaves were also measured. It was found that the blue fluorescence band was associated with the water extract. NADPH which is a water-soluble compound, and the water extract of clover had no fluorescence after oxidation by potassium ferricyanide, while the fluorescence of water insoluble vitamin K 1 was unchanged by the oxidizing agent. It was also found that the absorption maximum of NADPH was the same as the absorption maximum of the aqueous extract of clover. The above findings indicated that the compound responsible for the blue fluorescence at 440 nm is in the reduced state and is water-soluble. It was concluded that NADPH was responsible for the blue fluorescence at 440 nm. The strong linear relationship between the fluorescence at 440 nm and the rate of photosynthesis suggests the possible use of LIF measurements in the remote estimation of photosynthetic rates. (author)

  3. In vitro protein expression: an emerging alternative to cell-based approaches.

    Science.gov (United States)

    He, Mingyue

    2011-04-30

    Protein expression remains a bottleneck in the production of proteins. Owing to several advantages, cell-free translation is emerging as an alternative to cell-based methods for the generation of proteins. Recent advances have led to many novel applications of cell-free systems in biotechnology, proteomics and fundamental biological research. This special issue of New Biotechnology describes recent advances in cell-free protein expression systems and their applications. Copyright © 2010 Elsevier B.V. All rights reserved.

  4. Use of sperm plasmid DNA lipofection combined with REMI (restriction enzyme-mediated insertion) for production of transgenic chickens expressing eGFP (enhanced green fluorescent protein) or human follicle-stimulating hormone.

    Science.gov (United States)

    Harel-Markowitz, Eliane; Gurevich, Michael; Shore, Laurence S; Katz, Adi; Stram, Yehuda; Shemesh, Mordechai

    2009-05-01

    Linearized p-eGFP (plasmid-enhanced green fluorescent protein) or p-hFSH (plasmid human FSH) sequences with the corresponding restriction enzyme were lipofected into sperm genomic DNA. Sperm transfected with p-eGFP were used for artificial insemination in hens, and in 17 out of 19 of the resultant chicks, the exogenous DNA was detected in their lymphocytes as determined by PCR and expressed in tissues as determined by (a) PCR, (b) specific emission of green fluorescence by the eGFP, and (c) Southern blot analysis. A complete homology was found between the Aequorea Victoria eGFP DNA and a 313-bp PCR product of extracted DNA from chick blood cells. Following insemination with sperm lipofected with p-hFSH, transgenic offspring were obtained for two generations as determined by detection of the transgene for human FSH (PCR) and expression of the gene (RT-PCR and quantitative real-time PCR) and the presence of the protein in blood (radioimmunoassay). Data demonstrate that lipofection of plasmid DNA with restriction enzyme is a highly efficient method for the production of transfected sperm to produce transgenic offspring by direct artificial insemination.

  5. Comparison of secretory signal peptides for heterologous protein expression in microalgae: Expanding the secretion portfolio for Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    João Vitor Dutra Molino

    Full Text Available Efficient protein secretion is a desirable trait for any recombinant protein expression system, together with simple, low-cost, and defined media, such as the typical media used for photosynthetic cultures of microalgae. However, low titers of secreted heterologous proteins are usually obtained, even with the most extensively studied microalga Chlamydomonas reinhardtii, preventing their industrial application. In this study, we aimed to expand and evaluate secretory signal peptides (SP for heterologous protein secretion in C. reinhardtii by comparing previously described SP with untested sequences. We compared the SPs from arylsulfatase 1 and carbonic anhydrase 1, with those of untried SPs from binding protein 1, an ice-binding protein, and six sequences identified in silico. We identified over 2000 unique SPs using the SignalP 4.0 software. mCherry fluorescence was used to compare the protein secretion of up to 96 colonies for each construct, non-secretion construct, and parental wild-type cc1690 cells. Supernatant fluorescence varied according to the SP used, with a 10-fold difference observed between the highest and lowest secretors. Moreover, two SPs identified in silico secreted the highest amount of mCherry. Our results demonstrate that the SP should be carefully selected and that efficient sequences can be coded in the C. reinhardtii genome. The SPs described here expand the portfolio available for research on heterologous protein secretion and for biomanufacturing applications.

  6. Metaphysical green

    DEFF Research Database (Denmark)

    Earon, Ofri

    2011-01-01

    to adapt to urban environment. It explores the potential of Sensation of Green in the city. The paper questions whether the Sensation of Green could introduce a new spectrum of greens, beside the real green. It develops the term of metaphysical green – does green have to be green or can it be only...

  7. Effect of fluorescent pseudomonades and Trichoderma sp. and their combination with two chemicals on Penicillium digitatum caused agent of citrus green mold.

    Science.gov (United States)

    Zamani, M; Tehrani, A Sharifi; Ahmadzadeh, M; Abadi, A Alizadeh Ali

    2006-01-01

    Citrus green mold (Penicillium digitatum) causes economic losses. Chemical fungicides such as imazalil provide the primary means for controlling green mold decay of citrus fruits. Continuous use of fungicides has faced two major obstacles- increasing public concern regarding contamination of perishables with fungicidal residues, and proliferation of resistance in the pathogen populations. The aim of this research was to determine if the attacks of green mold on orange could be reduced by usage of biocontrol agent alone or in combination with low dosage of imazalil or sodium bicarbonate. Pseudomonas fluorescens isolate PN, P. fluorescens isolate PS and Trichoderma virens isolate TE were evaluated as potential biological agents for control of green mold of oranges caused by P. digitatum. Increasing concentration of SB decreased spore germination of P. digitatum. In laboratory tests, a cell suspension (10(8) cells per ml.) of bacterial strains reduced the incidence of green mold. On fruits surface biocontrol activity of antagonistic isolates was significantly increased when combined with low dosage of imazalil (500ppm) or sodium carbonate (5%). Effect of Trichoderma virens on controlling P. digitatum was better than others with or without these chemicals.

  8. Electron-phonon coupling in solubilized LHC II complexes of green plants investigated by line-narrowing and temperature-dependent fluorescence spectroscopy

    CERN Document Server

    Pieper, J K; Renger, G; Schödel, R; Voigt, J

    2001-01-01

    Line-narrowed and temperature-dependent fluorescence spectra are reported for the solubilized trimeric light-harvesting complex of Photosystem II (LHC II). Special attention has been paid to eliminate effects owing to reabsorption and to ensure that the line-narrowed fluorescence spectra are virtually unaffected by hole burning or scattering artifacts. Analysis of line-narrowed fluorescence spectra at 4.2 K indicates that the lowest Q//y-state of LHC II is characterized by weak electron-phonon coupling with a Huang-Rhys factor of similar to 0.9 and a broad and strongly asymmetric one- phonon profile with a peak frequency omega//m of 15 cm**-**1 and a width of Gamma = 105 cm**-**1. The 4.2 K fluorescence data are further consistent with the assignment of the lowest Q//y-state at similar to 680.0 nm and an inhomogeneous width of similar to 80 cm**- **1 gathered from a recent hole-burning study (Pieper et al. J. Phys. Chem. A 1999, 103, 2412). The temperature dependence of the fluorescence spectra of LHC II is s...

  9. Live-cell topology assessment of URG7, MRP6102 and SP-C using glycosylatable green fluorescent protein in mammalian cells

    International Nuclear Information System (INIS)

    Lee, Hunsang; Lara, Patricia; Ostuni, Angela; Presto, Jenny; Johansson, Janne; Nilsson, IngMarie; Kim, Hyun

    2014-01-01

    Highlights: • Glycosylatable GFP (gGFP) is developed for the use in mammalian cells. • gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. • Differential fluorescence/glycosylation pattern probes membrane protein topology. • Membrane topology of URG7, MRP6 102 , and SP-C was determined by gGFP tagging in vivo. - Abstract: Experimental tools to determine membrane topology of a protein are rather limited in higher eukaryotic organisms. Here, we report the use of glycosylatable GFP (gGFP) as a sensitive and versatile membrane topology reporter in mammalian cells. gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. Thus, positive fluorescence signal assigns location of gGFP to the cytosol whereas no fluorescence signal and a glycosylated status of gGFP map the location of gGFP to the ER lumen. By using mammalian gGFP, the membrane topology of disease-associated membrane proteins, URG7, MRP6 102 , SP-C(Val) and SP-C(Leu) was confirmed. URG7 is partially targeted to the ER, and inserted in C in form. MRP6 102 and SP-C(Leu/Val) are inserted into the membrane in C out form. A minor population of untargeted SP-C is removed by proteasome dependent quality control system

  10. Live-cell topology assessment of URG7, MRP6{sub 102} and SP-C using glycosylatable green fluorescent protein in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hunsang [School of Biological Sciences, Seoul National University, Seoul 151-747 (Korea, Republic of); Lara, Patricia [Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm (Sweden); Ostuni, Angela [Department of Sciences, University of Basilicata, Viale dell’Ateneo Lucano 10, 85100 Potenza (Italy); Presto, Jenny [Karolinska Institutet, Dept of Neurobiology, Care Sciences and Society, Novum 5th Floor, 141 86 Stockholm (Sweden); Johansson, Janne [Karolinska Institutet, Dept of Neurobiology, Care Sciences and Society, Novum 5th Floor, 141 86 Stockholm (Sweden); Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, 751 23 Uppsala (Sweden); Institute of Mathematics and Natural Sciences, Tallinn University, Narva mnt 25, 101 20 Tallinn (Estonia); Nilsson, IngMarie [Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm (Sweden); Kim, Hyun, E-mail: joy@snu.ac.kr [School of Biological Sciences, Seoul National University, Seoul 151-747 (Korea, Republic of)

    2014-08-08

    Highlights: • Glycosylatable GFP (gGFP) is developed for the use in mammalian cells. • gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. • Differential fluorescence/glycosylation pattern probes membrane protein topology. • Membrane topology of URG7, MRP6{sub 102}, and SP-C was determined by gGFP tagging in vivo. - Abstract: Experimental tools to determine membrane topology of a protein are rather limited in higher eukaryotic organisms. Here, we report the use of glycosylatable GFP (gGFP) as a sensitive and versatile membrane topology reporter in mammalian cells. gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. Thus, positive fluorescence signal assigns location of gGFP to the cytosol whereas no fluorescence signal and a glycosylated status of gGFP map the location of gGFP to the ER lumen. By using mammalian gGFP, the membrane topology of disease-associated membrane proteins, URG7, MRP6{sub 102}, SP-C(Val) and SP-C(Leu) was confirmed. URG7 is partially targeted to the ER, and inserted in C{sub in} form. MRP6{sub 102} and SP-C(Leu/Val) are inserted into the membrane in C{sub out} form. A minor population of untargeted SP-C is removed by proteasome dependent quality control system.

  11. Downregulation of ATM Gene and Protein Expression in Canine Mammary Tumors.

    Science.gov (United States)

    Raposo-Ferreira, T M M; Bueno, R C; Terra, E M; Avante, M L; Tinucci-Costa, M; Carvalho, M; Cassali, G D; Linde, S D; Rogatto, S R; Laufer-Amorim, R

    2016-11-01

    The ataxia telangiectasia mutated (ATM) gene encodes a protein associated with DNA damage repair and maintenance of genomic integrity. In women, ATM transcript and protein downregulation have been reported in sporadic breast carcinomas, and the absence of ATM protein expression has been associated with poor prognosis. The aim of this study was to evaluate ATM gene and protein expression in canine mammary tumors and their association with clinical outcome. ATM gene and protein expression was evaluated by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively, in normal mammary gland samples (n = 10), benign mammary tumors (n = 11), nonmetastatic mammary carcinomas (n = 19), and metastatic mammary carcinomas (n = 11). Lower ATM transcript levels were detected in benign mammary tumors and carcinomas compared with normal mammary glands (P = .011). Similarly, lower ATM protein expression was observed in benign tumors (P = .0003), nonmetastatic mammary carcinomas (P ATM gene or protein levels were detected among benign tumors and nonmetastatic and metastatic mammary carcinomas (P > .05). The levels of ATM gene or protein expression were not significantly associated with clinical and pathological features or with survival. Similar to human breast cancer, the data in this study suggest that ATM gene and protein downregulation is involved in canine mammary gland tumorigenesis. © The Author(s) 2016.

  12. Green(ing) infrastructure

    CSIR Research Space (South Africa)

    Van Wyk, Llewellyn V

    2014-03-01

    Full Text Available the generation of electricity from renewable sources such as wind, water and solar. Grey infrastructure – In the context of storm water management, grey infrastructure can be thought of as the hard, engineered systems to capture and convey runoff..., pumps, and treatment plants.  Green infrastructure reduces energy demand by reducing the need to collect and transport storm water to a suitable discharge location. In addition, green infrastructure such as green roofs, street trees and increased...

  13. Generation of fluorescence quenchers from the triplet states of chlorophylls in the major light-harvesting complex II from green plants

    NARCIS (Netherlands)

    Barzda, V.; Vengris, M.; Valkunas, L.; van Amerongen, H.; van Grondelle, R.

    2000-01-01

    Laser flash-induced changes of the fluorescence yield were studied in aggregates of light-harvesting complex II (LHCII) on a time scale ranging from microseconds to seconds. Carotenoid (Car) and chlorophyll (Chl) triplet states, decaying with lifetimes of several microseconds and hundreds of

  14. Methods for efficient high-throughput screening of protein expression in recombinant Pichia pastoris strains.

    Science.gov (United States)

    Camattari, Andrea; Weinhandl, Katrin; Gudiminchi, Rama K

    2014-01-01

    The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a whole-cell assay for a fungal cytochrome P450; and (3) a nonenzymatic assay for detection and quantification of tagged protein secreted into the supernatant.

  15. Synthesis and Properties of the p-Sulfonamide Analogue of the Green Fluorescent Protein (GFP) Chromophore: The Mimic of GFP Chromophore with Very Strong N-H Photoacid Strength.

    Science.gov (United States)

    Chen, Yi-Hui; Sung, Robert; Sung, Kuangsen

    2018-04-06

    The para-sulfonamide analogue ( p-TsABDI) of a green fluorescent protein (GFP) chromophore was synthesized to mimic the GFP chromophore. Its S 1 excited-state p K a * value in dimethylsulfoxide (DMSO) is -1.5, which is strong enough to partially protonate dipolar aprotic solvents and causes excited-state proton transfer (ESPT), so it can partially mimic the GFP chromophore to further study the ESPT-related photophysics and the blinking phenomenon of GFP. In comparison with 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) (p K a = 7.4, p K a * = 1.3 in water), p-TsABDI (p K a = 6.7, p K a * = -1.5 in DMSO) is a better photoacid for pH-jump studies.

  16. Combination of the Endogenous lhcsr1 Promoter and Codon Usage Optimization Boosts Protein Expression in the Moss Physcomitrella patens

    Directory of Open Access Journals (Sweden)

    Manuel Hiss

    2017-10-01

    Full Text Available The moss Physcomitrella patens is used both as an evo-devo model and biotechnological production system for metabolites and pharmaceuticals. Strong in vivo expression of genes of interest is important for production of recombinant proteins, e.g., selectable markers, fluorescent proteins, or enzymes. In this regard, the choice of the promoter sequence as well as codon usage optimization are two important inside factors to consider in order to obtain optimum protein accumulation level. To reliably quantify fluorescence, we transfected protoplasts with promoter:GFP fusion constructs and measured fluorescence intensity of living protoplasts in a plate reader system. We used the red fluorescent protein mCherry under 2x 35S promoter control as second reporter to normalize for different transfection efficiencies. We derived a novel endogenous promoter and compared deletion variants with exogenous promoters. We used different codon-adapted green fluorescent protein (GFP genes to evaluate the influence of promoter choice and codon optimization on protein accumulation in P. patens, and show that the promoter of the gene of P. patens chlorophyll a/b binding protein lhcsr1 drives expression of GFP in protoplasts significantly (more than twofold better than the commonly used 2x 35S promoter or the rice actin1 promoter. We identified a shortened 677 bp version of the lhcsr1 promoter that retains full activity in protoplasts. The codon optimized GFP yields significantly (more than twofold stronger fluorescence signals and thus demonstrates that adjusting codon usage in P. patens can increase expression strength. In combination, new promotor and codon optimized GFP conveyed sixfold increased fluorescence signal.

  17. An Evaluation of Quantitative PCR Assays (TaqMan® and SYBR Green for the Detection of Babesia bigemina and Babesia bovis, and a Novel Fluorescent-ITS1-PCR Capillary Electrophoresis Method for Genotyping B. bovis Isolates

    Directory of Open Access Journals (Sweden)

    Bing Zhang

    2016-09-01

    Full Text Available Babesia spp. are tick-transmitted haemoparasites causing tick fever in cattle. In Australia, economic losses to the cattle industry from tick fever are estimated at AUD$26 Million per annum. If animals recover from these infections, they become immune carriers. Here we describe a novel multiplex TaqMan qPCR targeting cytochrome b genes for the identification of Babesia spp. The assay shows high sensitivity, specificity and reproducibility, and allows quantification of parasite DNA from Babesia bovis and B. bigemina compared to standard PCR assays. A previously published cytochrome b SYBR Green qPCR was also tested in this study, showing slightly higher sensitivity than the Taqman qPCRs but requires melting curve analysis post-PCR to confirm specificity. The SYBR Green assays were further evaluated using both diagnostic submissions and vaccinated cattle (at 7, 9, 11 and 14 days post-inoculation showed that B. bigemina can be detected more frequently than B. bovis. Due to fewer circulating parasites, B. bovis detection in carrier animals requires higher DNA input. Preliminary data for a novel fluorescent PCR genotyping based on the Internal Transcribed Spacer 1 region to detect vaccine and field alleles of B. bovis are described. This assay is capable of detecting vaccine and novel field isolate alleles in a single sample.

  18. Evaluation the vigour of urban green lawn grown under long-term shade conditions by the use of chlorophyll fluorescence technique

    Directory of Open Access Journals (Sweden)

    Dąbrowski Piotr

    2015-09-01

    Full Text Available Unfavorable light conditions in urban areas are one of the most important cause of inappropriate grass communities condition. The possibility to detect the plant stress caused by shade is an important element in shaping the environment. The answer to following questions: what is the ability to detect the stress caused by shade in chosen lawn varieties of Perennial ryegrass by using the chlorophyll a fluorescence (O-J-I-P test and which of tested varieties has the best properties to create grasslands in reduced light conditions is the aim of this work. Two-factor experimental micro-plot was conducted with three varieties and three different shadowing variants. Chlorophyll a fluorescence measurements were provided and were compared to leaf density. Our results explored significant difference between selected varieties in the terms of their photosynthetic apparatus adaption to light conditions. During May, all tested varieties were characterized by the rise of all fluorescence curve points under lower light intensity. The largest changes under shade conditions were noticed for the variety ‘Taya’. During next months a declining trend of photosynthetic efficiency for this variety was observed. On the basis of our results, we assume that each variety has unique threshold and needs of light intensity.

  19. In Vitro and In Vivo Characterization of a Dual-Function Green Fluorescent Protein–HSV1-Thymidine Kinase Reporter Gene Driven by the Human Elongation Factor 1α Promoter

    Directory of Open Access Journals (Sweden)

    Gary D. Luker

    2002-04-01

    Full Text Available Toward the goal of monitoring activity of native mammalian promoters with molecular imaging techniques, we stably transfected DU145 prostate carcinoma cells with a fusion construct of enhanced green fluorescent protein (EGFP and wild-type herpes simplex virus-1 thymidine kinase (HSV1-TK as a reporter gene driven by the promoter for human elongation factor 1α (EF-1α-EGFP-TK. Using this model system, expression of EGFP was quantified by flow cytometry and fluorescence microscopy, while the HSV1-TK component of the reporter was quantified with 8-[3H]ganciclovir (8-[3H]GCV. As analyzed by flow cytometry, passage of EGFP-TK-DU145 transfected cells (ETK in vitro resulted in populations of cells with high and low expression of EGFP over time. High and low ETK cells retained 23-fold and 5-fold more GCV, respectively, than control. While differences in uptake and retention of GCV corresponded to relative expression of the reporter gene in each subpopulation of cells as determined by both flow cytometry (EGFP and quantitative RT-PCR, the correlation was not linear. Furthermore, in high ETK cells, net retention of various radiolabeled nucleoside analogues varied; the rank order was 8-[3H]GCV < 9-(4-fluoro-3-hydroxymethylbutylguanine ([18F]FHBG ≈ 8-[3H]penciclovir (8-[3H]PCV < 2′-fluoro-2′-deoxy-5-iodouracil-beta-d-arabinofuranoside (2-[14C]FIAU. Xenograft tumors of ETK cells in vivo accumulated 2.5-fold more 8-[3H]GCV per gram of tissue and showed greater fluorescence from EGFP than control DU145 cells, demonstrating that the reporter gene functioned in vivo. These data extend previous reports by showing that a human promoter can be detected in vitro and in vivo with a dual-function reporter exploiting optical and radiotracer techniques.

  20. Urothelial dysfunction and sensory protein expressions in patients with urological or systemic diseases and hypersensitive bladder

    Directory of Open Access Journals (Sweden)

    Hueih-Ling Ong

    2017-09-01

    Conclusion: Patients with OAB or HSB showed increased urothelial inflammation and lower barrier protein expression. Increased M3/β3-AR or M2/β3-AR in the urothelium was associated with OAB, whereas decreased M3/β3-AR or M2/β3-AR was associated with poor voiding efficiency and large PVR in LUTD.

  1. Glycoprofiling of N-linked glycans of erythropoietin therapeutic protein expressed in Yarrowia lipolytica

    CSIR Research Space (South Africa)

    Kahari, D

    2008-11-01

    Full Text Available profiling techniques. The gene encoding Lip2 was cloned as a C-terminally His-tagged protein, expressed in Yarrowia lipolytica (Madzak, C et al;2004) and the glycan composition of the purified protein was analysed by HPLC and MALDITOF. The HPLC techniques...

  2. C-fos protein expression in central nervous system. Effects of acute whole-body irradiation

    International Nuclear Information System (INIS)

    Martin, C.; Chollat, S.; Mahfoudi, H.; Lambert, F.; Baille Le Crom, V.; Fatome, M.

    1995-01-01

    Study of c-Fos protein expression in the rat striatum after gamma or (neutron-gamma) irradiation was carried on. c-Fos protein is expressed one hour after gamma exposure at the dose of 15 Gy but specificity of the response must be verified. (author)

  3. Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris.

    Science.gov (United States)

    Vogl, Thomas; Gebbie, Leigh; Palfreyman, Robin W; Speight, Robert

    2018-03-15

    Pichia pastoris (syn. Komagataella phaffii ) is one of the most common eukaryotic expression systems for heterologous protein production. Expression cassettes are typically integrated in the genome to obtain stable expression strains. In contrast to Saccharomyces cerevisiae , where short overhangs are sufficient to target highly specific integration, long overhangs are more efficient in P. pastoris and ectopic integration of foreign DNA can occur. Here, we aimed to elucidate the influence of ectopic integration by high-throughput screening of >700 transformants and whole-genome sequencing of 27 transformants. Different vector designs and linearization approaches were used to mimic the most common integration events targeted in P. pastoris Fluorescence of an enhanced green fluorescent protein (eGFP) reporter protein was highly uniform among transformants when the expression cassettes were correctly integrated in the targeted locus. Surprisingly, most nonspecifically integrated transformants showed highly uniform expression that was comparable to specific integration, suggesting that nonspecific integration does not necessarily influence expression. However, a few clones (integrated cassettes showed a greater variation spanning a 25-fold range, surpassing specifically integrated reference strains up to 6-fold. High-expression strains showed a correlation between increased gene copy numbers and high reporter protein fluorescence levels. Our results suggest that for comparing expression levels between strains, the integration locus can be neglected as long as a sufficient numbers of transformed strains are compared. For expression optimization of highly expressible proteins, increasing copy number appears to be the dominant positive influence rather than the integration locus, genomic rearrangements, deletions, or single-nucleotide polymorphisms (SNPs). IMPORTANCE Yeasts are commonly used as biotechnological production hosts for proteins and metabolites. In the yeast

  4. Evolutionary Divergence of Gene and Protein Expression in the Brains of Humans and Chimpanzees.

    Science.gov (United States)

    Bauernfeind, Amy L; Soderblom, Erik J; Turner, Meredith E; Moseley, M Arthur; Ely, John J; Hof, Patrick R; Sherwood, Chet C; Wray, Gregory A; Babbitt, Courtney C

    2015-07-10

    Although transcriptomic profiling has become the standard approach for exploring molecular differences in the primate brain, very little is known about how the expression levels of gene transcripts relate to downstream protein abundance. Moreover, it is unknown whether the relationship changes depending on the brain region or species under investigation. We performed high-throughput transcriptomic (RNA-Seq) and proteomic (liquid chromatography coupled with tandem mass spectrometry) analyses on two regions of the human and chimpanzee brain: The anterior cingulate cortex and caudate nucleus. In both brain regions, we found a lower correlation between mRNA and protein expression levels in humans and chimpanzees than has been reported for other tissues and cell types, suggesting that the brain may engage extensive tissue-specific regulation affecting protein abundance. In both species, only a few categories of biological function exhibited strong correlations between mRNA and protein expression levels. These categories included oxidative metabolism and protein synthesis and modification, indicating that the expression levels of mRNA transcripts supporting these biological functions are more predictive of protein expression compared with other functional categories. More generally, however, the two measures of molecular expression provided strikingly divergent perspectives into differential expression between human and chimpanzee brains: mRNA comparisons revealed significant differences in neuronal communication, ion transport, and regulatory processes, whereas protein comparisons indicated differences in perception and cognition, metabolic processes, and organization of the cytoskeleton. Our results highlight the importance of examining protein expression in evolutionary analyses and call for a more thorough understanding of tissue-specific protein expression levels. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular

  5. Methylation status and protein expression of RASSF1A in breast cancer patients.

    Science.gov (United States)

    Hagrass, Hoda A; Pasha, Heba F; Shaheen, Mohamed A; Abdel Bary, Eman H; Kassem, Rasha

    2014-01-01

    Recently genetics and epigenetics alterations have been found to be characteristic of malignancy and hence can be used as targets for detection of neoplasia. RAS association domain family protein 1A (RASSF1A) gene hypermethylation has been a subject of interest in recent researches on cancer breast patients. The aim of the present study was to evaluate whether RASSF1A methylation status and RASSF1A protein expression are associated with the major clinico-pathological parameters. One hundred and twenty breast cancer Egyptian patients and 100-control subjects diagnosed with benign lesions of the breast were enrolled in this study. We evaluated RASSF1A methylation status in tissue and serum samples using Methyl specific PCR together with RASSF1A protein expression in tissues by immunohistochemistry. Results were studied in relation to known prognostic clinicopathological features in breast cancer. Frequency of RASSF1A methylation in tissues and serum were 70 and 63.3 % respectively and RASSF1A protein expression showed frequency of 46.7 %. There was an association between RASSF1A methylation in tissues, serum and loss of protein expression in tissues with invasive carcinoma, advanced stage breast cancer, L.N. metastasis, ER/PR and HER2 negativity. RASSF1A methylation in serum showed high degree of concordance with methylation in tissues (Kappa = 0.851, P < 0.001). RASSF1A hypermethylation in tissues and serum and its protein expression may be a valid, reliable and sensitive tool for detection and follow up of breast cancer patients.

  6. Improvement of the green fluorescent protein reporter system in Leishmania spp. for the in vitro and in vivo screening of antileishmanial drugs.

    Science.gov (United States)

    Pulido, Sergio A; Muñoz, Diana L; Restrepo, Adriana M; Mesa, Carol V; Alzate, Juan F; Vélez, Iván D; Robledo, Sara M

    2012-04-01

    Development of new therapeutic approaches for leishmaniasis treatment requires new high throughput screening methodologies for the antileishmanial activity of the new compounds both in vitro and in vivo. Reporter genes as the GFP have become one of the most promissory and widely used tools for drug screening in several models, since it offers live imaging, high sensibility, specificity and flexibility; additionally, the use of GFP as a reporter gene in screening assays eliminates all the drawbacks presented in conventional assays and also those technical problems found using other reporter genes. The utility of the GFP as a reporter gene in drug screening assays with Leishmania parasites depends on the homogeneity and stability of the GFP transfected strains. Stable expression of the GFP in the Old World Leishmania species has been demonstrated using integration vectors; however, no reports exist yet about the success of this methodology in the New World species. Here we report the generation of New World Leishmania strains expressing the GFP protein from an integration vector, which replaces one copy of the 18S RNA in the chromosome with the GFP coding sequence by homologous recombination. We also prove that the expression of the integrated GFP is stable and homogeneous in the transfected parasites after months in culture without selective pressure or during its use in hamster infection assays. The fluorescent strains are useful for in vitro, ex vivo and in vivo drug screening assays since no considerable variations in virulence or infectivity where seen attributable to the genetic manipulation during both in vitro and in vivo infection experiments. The platform described here for drug testing assays based on the use of stable fluorescent Leishmania strains coupled to flow cytometry and fluorescent microscopy is more sensitive, more specific and faster than conventional assays used normally for the evaluation of compounds with potential antileishmanial activity

  7. Feasibility of dual reporter gene in rat myoblast cell line using human sodium iodide symporter (hNIS) and enhanced green fluorescent protein (EGFP) gene

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Yong Jin; Lee, You La; Ahn, Sohn Joo; Choi, Chang Ik; Lee, Sang Woo; Ahn, Byeong Cheol; Lee, Jae Tae [School of Medicine, Kyungpook National University, Daegu (Korea, Republic of)

    2007-07-01

    To develop a non-invasive combined imaging method of gamma camera and optical imaging to assess rat myoblast cell line, H9c2, we constructed retrovirus containing hNIS and EGFP gene, and transfected to rat myoblast cell and monitored hNIS and EGFP expression. Rat myoblast cell line, H9C2, was transfected with hNIS and EGFP gene using retrovirus (H9C2-NG). The expression of hNIS and EGFP gene was determined by RT-PCR and fluorescence microscopy, respectively. The uptake and efflux of I-125 were measured in the transfected and wild type cell lines. Each cell line was injected to 4 flank sites (H9c2: 1X107 or 2X107, H9C2-NG: 1X107 or 2X107) in nude mouse. Scintigraphic image was performed at 3h, 1 day after H9C2 and H9C2-NG cell inoculation. We performed gamma camera and animal PET imaging to evaluate NIS expression. Also, GFP image obtained using optical imaging system. The expression of hNIS and EGFP gene was confirmed by RT-PCR. In iodide uptake, H9C2-NG cells accumulated 274.52.2 pmol/ mg protein at 30 min. But wild type cell line did not uptake iodide. In fluorescent microscopy, H9C2-NG cells were highly fluorescent than that of H9C2 cells. In iodide efflux study, 50% of radioactivity flowed out during the first 10min. Scintigraphy showed increased uptake of Tc-99m in H9c2-NG than in H9C2 for 1 day. Also, H9C2-NG cells showed high signal-to-background fluorescent spots in animal body. In this study, NIS and EGFP reporter gene were successfully transfected by a retrovirus in myoblast cell line, and the transfected cell can be easily visualized in vivo. These results suggest that NIS and EGFP gene has an excellent feasibility as a reporter gene, and it can be used to monitor cell trafficking for monitoring.

  8. Demonstrating Fluorescence with Neon Paper and Plastic

    Science.gov (United States)

    Birriel, Jennifer J.; Roe, Clarissa

    2015-01-01

    Several papers in this journal have dealt with the fluorescence in orange neon plastic, olive oil, and soda. In each case, the fluorescent emission was excited by either green or violet-blue laser light. In this paper, we examine the fluorescent emission spectra of so-called neon colored papers and plastic clipboards available in department and…

  9. Correlation between quantitative HER-2 protein expression and risk for brain metastases in HER-2+ advanced breast cancer patients receiving trastuzumab-containing therapy.

    Science.gov (United States)

    Duchnowska, Renata; Biernat, Wojciech; Szostakiewicz, Barbara; Sperinde, Jeff; Piette, Fanny; Haddad, Mojgan; Paquet, Agnes; Lie, Yolanda; Czartoryska-Arłukowicz, Bogumiła; Wysocki, Piotr; Jankowski, Tomasz; Radecka, Barbara; Foszczynska-Kłoda, Małgorzata; Litwiniuk, Maria; Debska, Sylwia; Weidler, Jodi; Huang, Weidong; Buyse, Marc; Bates, Michael; Jassem, Jacek

    2012-01-01

    Patients with human epidermal growth factor receptor (HER)-2+ breast cancer are at particularly high risk for brain metastases; however, the biological basis is not fully understood. Using a novel HER-2 assay, we investigated the correlation between quantitative HER-2 expression in primary breast cancers and the time to brain metastasis (TTBM) in HER-2+ advanced breast cancer patients treated with trastuzumab. The study group included 142 consecutive patients who were administered trastuzumab-based therapy for HER-2+ metastatic breast cancer. HER-2/neu gene copy number was quantified as the HER-2/centromeric probe for chromosome 17 (CEP17) ratio by central laboratory fluorescence in situ hybridization (FISH). HER-2 protein was quantified as total HER-2 protein expression (H2T) by the HERmark® assay (Monogram Biosciences, Inc., South San Francisco, CA) in formalin-fixed, paraffin-embedded tumor samples. HER-2 variables were correlated with clinical features and TTBM was measured from the initiation of trastuzumab-containing therapy. A higher H2T level (continuous variable) was correlated with shorter TTBM, whereas HER-2 amplification by FISH and a continuous HER-2/CEP17 ratio were not predictive (p = .013, .28, and .25, respectively). In the subset of patients that was centrally determined by FISH to be HER-2+, an above-the-median H2T level was significantly associated with a shorter TTBM (hazard ratio, [HR], 2.4; p = .005), whereas this was not true for the median HER-2/CEP17 ratio by FISH (p = .4). Correlation between a continuous H2T level and TTBM was confirmed on multivariate analysis (HR, 3.3; p = .024). These data reveal a strong relationship between the quantitative HER-2 protein expression level and the risk for brain relapse in HER-2+ advanced breast cancer patients. Consequently, quantitative assessment of HER-2 protein expression may inform and facilitate refinements in therapeutic treatment strategies for selected subpopulations of patients in this

  10. Towards green loyalty: the influences of green perceived risk, green image, green trust and green satisfaction

    Science.gov (United States)

    Chrisjatmiko, K.

    2018-01-01

    The paper aims to present a comprehensive framework for the influences of green perceived risk, green image, green trust and green satisfaction to green loyalty. The paper also seeks to account explicitly for the differences in green perceived risk, green image, green trust, green satisfaction and green loyalty found among green products customers. Data were obtained from 155 green products customers. Structural equation modeling was used in order to test the proposed hypotheses. The findings show that green image, green trust and green satisfaction has positive effects to green loyalty. But green perceived risk has negative effects to green image, green trust and green satisfaction. However, green perceived risk, green image, green trust and green satisfaction also seems to be a good device to gain green products customers from competitors. The contributions of the paper are, firstly, a more complete framework of the influences of green perceived risk, green image, green trust and green satisfaction to green loyalty analyses simultaneously. Secondly, the study allows a direct comparison of the difference in green perceived risk, green image, green trust, green satisfaction and green loyalty between green products customers.

  11. Thermally stable green Ba(3)Y(PO(4))3:Ce(3+),Tb(3+) and red Ca(3)Y(AlO)(3)(BO(3))4:Eu(3+) phosphors for white-light fluorescent lamps.

    Science.gov (United States)

    Huang, Chien-Hao; Kuo, Te-Wen; Chen, Teng-Ming

    2011-01-03

    A class of thermal stable of green-emitting phosphors Ba(3)Y(PO(4))(3):Ce(3+),Tb(3+) (BYP:Ce(3+),Tb(3+)) and red-emitting phosphors Ca(3)Y(AlO)(3)(BO(3))(4):Eu(3+) (CYAB:Eu(3+)) for white-light fluorescent lamps were synthesized by high temperature solid-state reaction. We observed a decay of only 3% at 150 °C for BYP:0.25Ce3+,0.25Tb3+ (3% for LaPO4:Ce(3+),Tb(3+)), and a decay of 4% for CYAB:0.5Eu(3+) (7% for Y(2)O(3):Eu(3+), 24% for Y(2)O(2)S:Eu(3+)). The emission intensity of composition-optimized Ba(3)(Y(0.5)Ce(0.25)Tb(0.25))(PO(4))(3) is 70% of that of commercial LaPO(4):Ce(3+),Tb(3+) phosphors, and the CIE chromaticity coordinates are found to be (0.323, 0.534). The emission intensity of Ca(3)(Y(0.5)Eu(0.5))(AlO)(3)(BO(3))(4) is 70% and 83% of those of Y(2)O(3):Eu(3+) and Y(2)O(2)S:Eu(3+) phosphors, respectively, and the CIE chromaticity coordinates are redder (0.652, 0.342) than those of Y(2)O(3):Eu(3+) (0.645, 0.347) and Y(2)O(2)S:Eu(3+) (0.647, 0.343). A white-light fluorescent lamp is fabricated using composition-optimized Ba(3)(Y(0.5)Ce(0.25)Tb(0.25))(PO(4))(3) and Ca(3)(Y(0.5)Eu(0.5))(AlO)(3)(BO(3))(4) phosphors and matching blue-emitting phosphors. The results indicate that the quality of the brightness and color reproduction is suitable for application in shortwave UV fluorescent lamps. The white-light fluorescent lamp displays CIE chromaticity coordinates of x = 0.33, y = 0.35, a warm white light with a correlated color temperature of 5646 K, and a color-rendering index of Ra = 70.

  12. Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.

    Science.gov (United States)

    Peng, Tao; Hang, Howard C

    2016-11-02

    Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

  13. Yellow fever virus envelope protein expressed in insect cells is capable of syncytium formation in lepidopteran cells and could be used for immunodetection of YFV in human sera

    Directory of Open Access Journals (Sweden)

    Nagata Tatsuya

    2011-05-01

    Full Text Available Abstract Background Yellow fever is an haemorrhagic disease caused by a virus that belongs to the genus Flavivirus (Flaviviridae family and is transmitted by mosquitoes. Among the viral proteins, the envelope protein (E is the most studied one, due to its high antigenic potencial. Baculovirus are one of the most popular and efficient eukaryotic expression system. In this study a recombinant baculovirus (vSynYFE containing the envelope gene (env of the 17D vaccine strain of yellow fever virus was constructed and the recombinant protein antigenicity was tested. Results Insect cells infected with vSynYFE showed syncytium formation, which is a cytopathic effect characteristic of flavivirus infection and expressed a polypeptide of around 54 kDa, which corresponds to the expected size of the recombinant E protein. Furthermore, the recombinant E protein expression was also confirmed by fluorescence microscopy of vSynYFE-infected insect cells. Total vSynYFE-infected insect extracts used as antigens detected the presence of antibodies for yellow fever virus in human sera derived from yellow fever-infected patients in an immunoassay and did not cross react with sera from dengue virus-infected patients. Conclusions The E protein expressed by the recombinant baculovirus in insect cells is antigenically similar to the wild protein and it may be useful for different medical applications, from improved diagnosis of the disease to source of antigens for the development of a subunit vaccine.

  14. Low birthweight is associated with specific changes in muscle insulin-signalling protein expression

    DEFF Research Database (Denmark)

    Ozanne, SE; Jensen, CB; Tingey, KJ

    2005-01-01

    muscle in a human cohort and a rat model. METHODS: We recruited 20 young men with low birthweight (mean birthweight 2702+/-202 g) and 20 age-matched control subjects (mean birthweight 3801+/-99 g). Biopsies were obtained from the vastus lateralis muscle and protein expression of selected insulin......-signalling proteins was determined. Rats used for this study were male offspring born to dams fed a standard (20%) protein diet or a low (8%) protein diet during pregnancy and lactation. Protein expression was determined in soleus muscle from adult offspring. RESULTS: Low-birthweight subjects showed reduced muscle...... expression of protein kinase C (PKC)zeta, p85alpha, p110beta and GLUT4. PKCzeta, GLUT4 and p85 were also reduced in the muscle of rats fed a low-protein diet. Other proteins studied were unchanged in low-birthweight humans and in rats fed a low-protein diet when compared with control groups. CONCLUSIONS...

  15. YKL-40 protein expression is not a prognostic marker in patients with primary breast cancer

    DEFF Research Database (Denmark)

    Roslind, Anne; Knoop, Ann; Jensen, Maj-Britt

    2007-01-01

    in tumor tissue was assessed by immunohistochemistry in a cohort of 630 high-risk breast cancer patients with a median estimated potential follow-up time of 10 and 13 years for disease-free (DFS) and overall survival (OS), respectively. YKL-40 protein expression was found in malignant tumor cells......YKL-40 is a new biomarker in serum with a prognostic value in several localized and metastatic malignancies. The current knowledge regarding the biological functions of YKL-40 in cancer links YKL-40 to increased aggressiveness of the tumor. Utilizing tissue microarrays, YKL-40 protein expression...... and in inflammatory cells. High expression was associated with positive estrogen and progesterone receptor status and high tumor differentiation. Contrary to studies on serum YKL-40 as a prognostic biomarker, a high YKL-40 expression in tumor cells was not significantly associated with DSF and OS in univariate...

  16. Nucleic acid programmable protein array a just-in-time multiplexed protein expression and purification platform.

    Science.gov (United States)

    Qiu, Ji; LaBaer, Joshua

    2011-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Do cysteine residues regulate transient receptor potential canonical type 6 (TRPC6) channel protein expression?

    DEFF Research Database (Denmark)

    Thilo, Florian; Liu, Ying; Krueger, Katharina

    2012-01-01

    The regulation of calcium influx through transient receptor potential canonical type 6 channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine or acetylcysteine affect TRPC6 expression in human monocytes. We observed that patie......The regulation of calcium influx through transient receptor potential canonical type 6 channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine or acetylcysteine affect TRPC6 expression in human monocytes. We observed...... that patients with chronic renal failure had significantly elevated homocysteine levels and TRPC6 mRNA expression levels in monocytes compared to control subjects. We further observed that administration of homocysteine or acetylcysteine significantly increased TRPC6 channel protein expression compared...... to control conditions. We therefore hypothesize that cysteine residues increase TRPC6 channel protein expression in humans....

  18. Increase of bcl-2 Protein Expression in Aggressive Basal Cell Carcinoma of Head and Neck

    OpenAIRE

    Cláudia CAZAL; Mariana Roesch ELY; Ana Paula Veras SOBRAL; Wilton Wilney Nascimento PADILHA

    2006-01-01

    Objective: The aim of this study was to verify the bcl-2 protein expression in 22 cutaneous basal cell carcinomas (BCC) of the head and neck, and to compare it with its aggressive behavior. Method: Tumors were histologically classified in non-aggressive (BCC 1) and aggressive (BCC 2) and then submitted to the immunohistochemistry technique with the streptavidin-biotin peroxidase method using the anti-bcl-2 antibody. Results: After proceeding to morphological analysis, sixteen tumors (72.7%) w...

  19. WWOX protein expression varies among ovarian carcinoma histotypes and correlates with less favorable outcome

    International Nuclear Information System (INIS)

    Nunez, María I; Mills, Gordon B; Aldaz, C Marcelo; Rosen, Daniel G; Ludes-Meyers, John H; Abba, Martín C; Kil, Hyunsuk; Page, Robert; Klein-Szanto, Andres JP; Godwin, Andrew K; Liu, Jinsong

    2005-01-01

    The putative tumor suppressor WWOX gene spans the common chromosomal fragile site 16D (FRA16D) at chromosome area 16q23.3-24.1. This region is a frequent target for loss of heterozygosity and chromosomal rearrangement in ovarian, breast, hepatocellular, prostate carcinomas and other neoplasias. The goal of these studies was to evaluate WWOX protein expression levels in ovarian carcinomas to determine if they correlated with clinico-pathological parameters, thus providing additional support for WWOX functioning as a tumor suppressor. We performed WWOX protein expression analyses by means of immunobloting and immunohistochemistry on normal ovaries and specific human ovarian carcinoma Tissue Microarrays (n = 444). Univariate analysis of clinical-pathological parameters based on WWOX staining was determined by χ 2 test with Yates' correction. The basic significance level was fixed at p < 0.05. Immunoblotting analysis from normal ovarian samples demonstrated consistently strong WWOX expression while 37% ovarian carcinomas showed reduced or undetectable WWOX protein expression levels. The immunohistochemistry of normal human ovarian tissue sections confirmed strong WWOX expression in ovarian surface epithelial cells and in epithelial inclusion cysts within the cortex. Out of 444 ovarian carcinoma samples analyzed 30% of tumors showed lack of or barely detectable WWOX expression. The remaining ovarian carcinomas (70%) stained moderately to strongly positive for this protein. The two histotypes showing significant loss of WWOX expression were of the Mucinous (70%) and Clear Cell (42%) types. Reduced WWOX expression demonstrated a significant association with clinical Stage IV (FIGO) (p = 0.007), negative Progesterone Receptor (PR) status (p = 0.008) and shorter overall survival (p = 0.03). These data indicate that WWOX protein expression is highly variable among ovarian carcinoma histotypes. It was also observed that subsets of ovarian tumors demonstrated loss of

  20. High SRPX2 protein expression predicts unfavorable clinical outcome in patients with prostate cancer

    Science.gov (United States)

    Zhang, Meng; Li, Xiaoli; Fan, Zhirui; Zhao, Jing; Liu, Shuzheng; Zhang, Mingzhi; Li, Huixiang; Goscinski, Mariusz Adam; Fan, Huijie; Suo, Zhenhe

    2018-01-01

    Background Sushi repeat-containing protein X-linked 2 (SRPX2) is overexpressed in a variety of different tumor tissues and correlated with poor prognosis in patients. Little research focuses on the role of SRPX2 expression in prostate cancer (PCa), and the clinicopathological significance of the protein expression in this tumor is relatively unknown. However, our previous transcriptome data from those cancer stem-like cells indicated the role of SRPX2 in PCa. Materials and methods In this study, RT-PCR and Western blotting were firstly used to examine the SRPX2 expression in three PCa cell lines including LNCaP, DU145, and PC3, and then SRPX2 protein expression was immunohistochemically investigated and statistically analyzed in a series of 106 paraffin-embedded PCa tissue specimens. Results Significantly lower levels of SRPX2 expression were verified in the LNCaP cells, compared with the expression in the aggressive DU145 and PC3 cells, in both mRNA and protein levels. Immunohistochemically, there were variable SRPX2 protein expressions in the clinical samples. Moreover, high levels of SRPX2 expression in the PCa tissues were significantly associated with Gleason score (P=0.008), lymph node metastasis (P=0.009), and distant metastasis (P=0.021). Furthermore, higher levels of SRPX2 expression in the PCa tissues were significantly associated with shorter overall survival (OS) (P<0.001). Conclusion Our results demonstrate that SRPX2 is highly expressed in aggressive PCa cells in vitro, and its protein expression in PCa is significantly associated with malignant clinical features and shorter OS, strongly indicating its prognostic value in prostate cancers. PMID:29881288

  1. Changes in protein expression in p53 deleted spontaneous thymic lymphomas

    DEFF Research Database (Denmark)

    Honoré, Bent; Vorum, Henrik; Pedersen, Anders Elm

    2004-01-01

    with the protein expression in p53+/+ and p53-/- thymocytes. Only a minority (13 proteins) of the quantitatively changed proteins were common for the two thymic lymphoma cell lines, suggesting that the p53 deficiency mainly results in genetic dysfunctions which are individual for a given tumor. Two of the detected...... structure containing motifs of the glyoxalase-bleomycin resistance protein family (MDR) as deduced from the cDNA....

  2. Methods for Using Small Non-Coding RNAs to Improve Recombinant Protein Expression in Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Sarah Inwood

    2018-01-01

    Full Text Available The ability to produce recombinant proteins by utilizing different “cell factories” revolutionized the biotherapeutic and pharmaceutical industry. Chinese hamster ovary (CHO cells are the dominant industrial producer, especially for antibodies. Human embryonic kidney cells (HEK, while not being as widely used as CHO cells, are used where CHO cells are unable to meet the needs for expression, such as growth factors. Therefore, improving recombinant protein expression from mammalian cells is a priority, and continuing effort is being devoted to this topic. Non-coding RNAs are RNA segments that are not translated into a protein and often have a regulatory role. Since their discovery, major progress has been made towards understanding their functions. Non-coding RNA has been investigated extensively in relation to disease, especially cancer, and recently they have also been used as a method for engineering cells to improve their protein expression capability. In this review, we provide information about methods used to identify non-coding RNAs with the potential of improving recombinant protein expression in mammalian cell lines.

  3. Exercise alters myostatin protein expression in sedentary and exercised streptozotocin-diabetic rats.

    Science.gov (United States)

    Bassi, Daniela; Bueno, Patricia de Godoy; Nonaka, Keico Okino; Selistre-Araujo, Heloisa Sobreiro; Leal, Angela Merice de Oliveira

    2015-04-01

    The aim of this study was to analyze the effect of exercise on the pattern of muscle myostatin (MSTN) protein expression in two important metabolic disorders, i.e., obesity and diabetes mellitus. MSTN, is a negative regulator of skeletal muscle mass. We evaluated the effect of exercise on MSTN protein expression in diabetes mellitus and high fat diet-induced obesity. MSTN protein expression in gastrocnemius muscle was analyzed by Western Blot. P sedentary or exercised obese animals. Diabetes reduced gastrocnemius muscle weight in sedentary animals. However, gastrocnemius muscle weight increased in diabetic exercised animals. Both the precursor and processed forms of muscle MSTN protein were significantly higher in sedentary diabetic rats than in control rats. The precursor form was significantly lower in diabetic exercised animals than in diabetic sedentary animals. However, the processed form did not change. These results demonstrate that exercise can modulate the muscle expression of MSTN protein in diabetic rats and suggest that MSTN may be involved in energy homeostasis.

  4. Actual Proliferating Index and p53 protein expression as prognostic marker in odontogenic cysts.

    Science.gov (United States)

    Gadbail, A R; Chaudhary, M; Patil, S; Gawande, M

    2009-10-01

    The purpose of this study was to evaluate the biological aggressiveness of odontogenic keratocyst/keratocystic odontogenic tumour (KCOT), radicular cyst (RC) and dentigerous cyst (DC) by observing the actual proliferative activity of epithelium, and p53 protein expression. The actual proliferative activity was measured by Ki-67 Labelling Index and argyrophilic nucleolar organizing regions (AgNOR) count per nucleus. The p53 protein expression was also evaluated. Ki-67 positive cells were observed higher in suprabasal cell layers of KCOT with uniform distribution, a few of them were predominantly observed in basal cell layer in RC and DC. The AgNOR count was significantly higher in suprabasal cell layers of KCOT. The actual proliferative activity was noted to be higher in suprabasal cell layers of KCOT. The p53 immunolabelling was dense and scattered in basal and suprabasal cell layers in KCOT. The weakly stained p53 positive cells were observed diffusely distributed in KCOT, whereas they were mainly seen in basal cell layer of RC and DC. The quantitative and qualitative differences of the proliferative activity and the p53 protein expression in sporadic KCOT may be associated with intrinsic growth potential that could play a role in its development and explain locally aggressive biological behaviour. AgNOR count and p53 protein detection in odontogenic lesions can be of great consequence to predict the biological behaviour and prognosis.

  5. Protein expression in the nucleus accumbens of rats exposed to developmental vitamin D deficiency.

    Directory of Open Access Journals (Sweden)

    John McGrath

    Full Text Available INTRODUCTION: Developmental vitamin D (DVD deficiency is a candidate risk factor for schizophrenia. Animal models have confirmed that DVD deficiency is associated with a range of altered genomic, proteomic, structural and behavioural outcomes in the rat. Because the nucleus accumbens has been implicated in neuropsychiatric disorders, in the current study we examined protein expression in this region in adult rats exposed to DVD deficiency METHODS: Female Sprague Dawley rats were maintained on a vitamin D deficient diet for 6 weeks, mated and allowed to give birth, after which a diet containing vitamin D was reintroduced. Male adult offspring (n = 8 were compared to control male (n = 8. 2-D gel electrophoresis-based proteomics and mass spectroscopy were used to investigate differential protein expression. RESULTS: There were 35 spots, mapped to 33 unique proteins, which were significantly different between the two groups. Of these, 22 were down-regulated and 13 up-regulated. The fold changes were uniformly small, with the largest FC being -1.67. Within the significantly different spots, three calcium binding proteins (calbindin1, calbindin2 and hippocalcin were altered. Other proteins associated with DVD deficiency related to mitochondrial function, and the dynamin-like proteins. CONCLUSIONS: Developmental vitamin D deficiency was associated with subtle changes in protein expression in the nucleus accumbens. Disruptions in pathways related to calcium-binding proteins and mitochondrial function may underlie some of the behavioural features associated with animal models of developmental vitamin D deficiency.

  6. A Breast Tissue Protein Expression Profile Contributing to Early Parity-Induced Protection Against Breast Cancer

    Directory of Open Access Journals (Sweden)

    Christina Marie Gutierrez

    2015-11-01

    Full Text Available Background/Aims: Early parity reduces breast cancer risk, whereas, late parity and nulliparity increase breast cancer risk. Despite substantial efforts to understand the protective effects of early parity, the precise molecular circuitry responsible for these changes is not yet fully defined. Methods: Here, we have conducted the first study assessing protein expression profiles in normal breast tissue of healthy early parous, late parous, and nulliparous women. Breast tissue biopsies were obtained from 132 healthy parous and nulliparous volunteers. These samples were subjected to global protein expression profiling and immunohistochemistry. GeneSpring and MetaCore bioinformatics analysis software were used to identify protein expression profiles associated with early parity (low risk versus late/nulliparity (high risk. Results: Early parity reduces expression of key proteins involved in mitogenic signaling pathways in breast tissue through down regulation of EGFR1/3, ESR1, AKT1, ATF, Fos, and SRC. Early parity is also characterized by greater genomic stability and reduced tissue inflammation based on differential expression of aurora kinases, p53, RAD52, BRCA1, MAPKAPK-2, ATF-1, ICAM1, and NF-kappaB compared to late and nulli parity. Conclusions: Early parity reduces basal cell proliferation in breast tissue, which translates to enhanced genomic stability, reduced cellular stress/inflammation, and thus reduced breast cancer risk.

  7. The predictive nature of transcript expression levels on protein expression in adult human brain.

    Science.gov (United States)

    Bauernfeind, Amy L; Babbitt, Courtney C

    2017-04-24

    Next generation sequencing methods are the gold standard for evaluating expression of the transcriptome. When determining the biological implications of such studies, the assumption is often made that transcript expression levels correspond to protein levels in a meaningful way. However, the strength of the overall correlation between transcript and protein expression is inconsistent, particularly in brain samples. Following high-throughput transcriptomic (RNA-Seq) and proteomic (liquid chromatography coupled with tandem mass spectrometry) analyses of adult human brain samples, we compared the correlation in the expression of transcripts and proteins that support various biological processes, molecular functions, and that are located in different areas of the cell. Although most categories of transcripts have extremely weak predictive value for the expression of their associated proteins (R 2 values of < 10%), transcripts coding for protein kinases and membrane-associated proteins, including those that are part of receptors or ion transporters, are among those that are most predictive of downstream protein expression levels. The predictive value of transcript expression for corresponding proteins is variable in human brain samples, reflecting the complex regulation of protein expression. However, we found that transcriptomic analyses are appropriate for assessing the expression levels of certain classes of proteins, including those that modify proteins, such as kinases and phosphatases, regulate metabolic and synaptic activity, or are associated with a cellular membrane. These findings can be used to guide the interpretation of gene expression results from primate brain samples.

  8. Differential Protein Expressions in Virus-Infected and Uninfected Trichomonas vaginalis.

    Science.gov (United States)

    He, Ding; Pengtao, Gong; Ju, Yang; Jianhua, Li; He, Li; Guocai, Zhang; Xichen, Zhang

    2017-04-01

    Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis , we detected 2 strains of T. vaginalis ; the virus-infected (V + ) and uninfected (V - ) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V + compared with V - isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V + isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V + and V - isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.

  9. A comparative study of cell cycle mediator protein expression patterns in anaplastic and papillary thyroid carcinoma.

    Science.gov (United States)

    Evans, Juanita J; Crist, Henry S; Durvesh, Saima; Bruggeman, Richard D; Goldenberg, David

    2012-07-01

    Anaplastic thyroid carcinoma (ATC) is an extremely aggressive and rapidly fatal neoplasm. The aim of this study was to identify a limited cell cycle associated protein expression pattern unique to ATC and to correlate that pattern with clinical outcome. This represents one of the largest tissue micro-array projects comparing the cell cycle protein expression data of ATC to other well-differentiated tumors in the literature. Tissue microarrays were created from 21 patients with ATC and an age and gender matched cohort of patients with papillary thyroid carcinoma (PTC). Expression of epidermal growth factor receptor, cyclin D1, cyclin E, p53, p21, p16, aurora kinase A, opioid growth factor (OGF), OGF-receptor, thyroglobulin and Ki-67 was evaluated in a semi-quantitative fashion. Differences in protein expression between the cohorts were evaluated using chi-square tests with Bonferroni adjustments. Survival time and presence of metastasis at presentation were collected. The ATC cohort showed a statistically significant decrease (p cycle with aberrant expression of multiple protein markers suggesting increased proliferative activity and loss of control of cell cycle progression to G₁ phase. These findings support the assertion that ATC may represent the furthest end of a continuum of thyroid carcinoma dedifferentiation.

  10. Novel approach for transient protein expression in primary cultures of human dental pulp-derived cells.

    Science.gov (United States)

    Suguro, Hisashi; Mikami, Yoshikazu; Koshi, Rieko; Ogiso, Bunnai; Watanabe, Eri; Watanabe, Nobukazu; Honda, Masaki J; Asano, Masatake; Komiyama, Kazuo

    2011-08-01

    Transfection is a powerful method for investigating variable biological functions of desired genes. However, the efficiency of transfection into primary cultures of dental pulp-derived cells (DPDC) is low. Therefore, using a recombinant vaccinia virus (vTF7-3), which contains T7 RNA polymerase, we have established a transient protein expression system in DPDCs. In this study, we used the human polymeric immunoglobulin receptor (pIgR) cDNA as a model gene. pIgR expression by the vTF7-3 expression system was confirmed by flow cytometry analysis and Western blotting. Furthermore, exogenous pIgR protein localized at the cell surface in DPDCs and formed a secretory component (SC). This suggests that exogenous pIgR protein expressed by the vTF7-3 expression system acts like endogenous pIgR protein. These results indicate the applicability of the method for cells outgrown from dental pulp tissue. In addition, as protein expression could be detected shortly after transfection (approximately 5h), this experimental system has been used intensely for experiments examining very early steps in protein exocytosis. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Activation of p44/42 in Human Natural Killer Cells Decreases Cell-surface Protein Expression: Relationship to Tributyltin-induced alterations of protein expression

    Science.gov (United States)

    Dudimah, Fred D.; Abraha, Abraham; Wang, Xiaofei; Whalen, Margaret M.

    2010-01-01

    Tributyltin (TBT) activates the mitogen activated protein kinase (MAPK), p44/42 in human natural killer (NK) cells. TBT also reduces NK cytotoxic function and decreases the expression of several NK-cell proteins. To understand the role that p44/42 activation plays in TBT-induced loss of NK cell function, we have investigated how selective activation of p44/42 by phorbol 12-myristate 13-acetate (PMA) affects NK cells. Previously we showed that PMA caused losses of lytic function similar to those seen with TBT exposures. Here we examined activation of p44/42 in the regulation of NK-cell protein expression and how this regulation may explain the protein expression changes seen with TBT exposures. NK cells exposed to PMA were examined for levels of cell-surface proteins, granzyme mRNA, and perforin mRNA expression. The expression of CD11a, CD16, CD18, and CD56 were reduced, perforin mRNA levels were unchanged and granzyme mRNA levels were increased. To verify that activation of p44/42 was responsible for the alterations seen in CD11a, CD16, CD18, and CD56 with PMA, NK cells were treated with the p44/42 pathway inhibitor (PD98059) prior to PMA exposures. In the presence of PD98059, PMA caused no decreases in the expression of the cell-surface proteins. Results of these studies indicate that the activation of p44/42 may lead to the loss of NK cell cytotoxic function by decreasing the expression of CD11a, CD16, CD18, and CD56. Further, activation of p44/42 appears to be at least in part responsible for the TBT-induced decreases in expression of CD16, CD18, and CD56. PMID:20883105

  12. Highly fluorescent benzofuran derivatives of the GFP chromophore

    DEFF Research Database (Denmark)

    Christensen, Mikkel Andreas; Jennum, Karsten Stein; Abrahamsen, Peter Bæch

    2012-01-01

    Intramolecular cyclization reactions of Green Fluorescent Protein chromophores (GFPc) containing an arylethynyl ortho-substituent at the phenol ring provide new aryl-substituted benzofuran derivatives of the GFPc. Some of these heteroaromatic compounds exhibit significantly enhanced fluorescence...

  13. Evolutionary tuning of protein expression levels of a positively autoregulated two-component system.

    Directory of Open Access Journals (Sweden)

    Rong Gao

    2013-10-01

    Full Text Available Cellular adaptation relies on the development of proper regulatory schemes for accurate control of gene expression levels in response to environmental cues. Over- or under-expression can lead to diminished cell fitness due to increased costs or insufficient benefits. Positive autoregulation is a common regulatory scheme that controls protein expression levels and gives rise to essential features in diverse signaling systems, yet its roles in cell fitness are less understood. It remains largely unknown how much protein expression is 'appropriate' for optimal cell fitness under specific extracellular conditions and how the dynamic environment shapes the regulatory scheme to reach appropriate expression levels. Here, we investigate the correlation of cell fitness and output response with protein expression levels of the E. coli PhoB/PhoR two-component system (TCS. In response to phosphate (Pi-depletion, the PhoB/PhoR system activates genes involved in phosphorus assimilation as well as genes encoding themselves, similarly to many other positively autoregulated TCSs. We developed a bacteria competition assay in continuous cultures and discovered that different Pi conditions have conflicting requirements of protein expression levels for optimal cell fitness. Pi-replete conditions favored cells with low levels of PhoB/PhoR while Pi-deplete conditions selected for cells with high levels of PhoB/PhoR. These two levels matched PhoB/PhoR concentrations achieved via positive autoregulation in wild-type cells under Pi-replete and -deplete conditions, respectively. The fitness optimum correlates with the wild-type expression level, above which the phosphorylation output saturates, thus further increase in expression presumably provides no additional benefits. Laboratory evolution experiments further indicate that cells with non-ideal protein levels can evolve toward the optimal levels with diverse mutational strategies. Our results suggest that the natural

  14. Spatiotemporal relationships between growth and microtubule orientation as revealed in living root cells of Arabidopsis thaliana transformed with green-fluorescent-protein gene construct GFP-MBD

    Science.gov (United States)

    Granger, C. L.; Cyr, R. J.

    2001-01-01

    Arabidopsis thaliana plants were transformed with GFP-MBD (J. Marc et al., Plant Cell 10: 1927-1939, 1998) under the control of a constitutive (35S) or copper-inducible promoter. GFP-specific fluorescence distributions, levels, and persistence were determined and found to vary with age, tissue type, transgenic line, and individual plant. With the exception of an increased frequency of abnormal roots of 35S GFP-MBD plants grown on kanamycin-containing media, expression of GFP-MBD does not appear to affect plant phenotype. The number of leaves, branches, bolts, and siliques as well as overall height, leaf size, and seed set are similar between wild-type and transgenic plants as is the rate of root growth. Thus, we conclude that the transgenic plants can serve as a living model system in which the dynamic behavior of microtubules can be visualized. Confocal microscopy was used to simultaneously monitor growth and microtubule behavior within individual cells as they passed through the elongation zone of the Arabidopsis root. Generally, microtubules reoriented from transverse to oblique or longitudinal orientations as growth declined. Microtubule reorientation initiated at the ends of the cell did not necessarily occur simultaneously in adjacent neighboring cells and did not involve complete disintegration and repolymerization of microtubule arrays. Although growth rates correlated with microtubule reorientation, the two processes were not tightly coupled in terms of their temporal relationships, suggesting that other factor(s) may be involved in regulating both events. Additionally, microtubule orientation was more defined in cells whose growth was accelerating and less stringent in cells whose growth was decelerating, indicating that microtubule-orienting factor(s) may be sensitive to growth acceleration, rather than growth per se.

  15. Knock-in of Enhanced Green Fluorescent Protein or/and Human Fibroblast Growth Factor 2 Gene into β-Casein Gene Locus in the Porcine Fibroblasts to Produce Therapeutic Protein.

    Science.gov (United States)

    Lee, Sang Mi; Kim, Ji Woo; Jeong, Young-Hee; Kim, Se Eun; Kim, Yeong Ji; Moon, Seung Ju; Lee, Ji-Hye; Kim, Keun-Jung; Kim, Min-Kyu; Kang, Man-Jong

    2014-11-01

    Transgenic animals have become important tools for the production of therapeutic proteins in the domestic animal. Production efficiencies of transgenic animals by conventional methods as microinjection and retrovirus vector methods are low, and the foreign gene expression levels are also low because of their random integration in the host genome. In this study, we investigated the homologous recombination on the porcine β-casein gene locus using a knock-in vector for the β-casein gene locus. We developed the knock-in vector on the porcine β-casein gene locus and isolated knock-in fibroblast for nuclear transfer. The knock-in vector consisted of the neomycin resistance gene (neo) as a positive selectable marker gene, diphtheria toxin-A gene as negative selection marker, and 5' arm and 3' arm from the porcine β-casein gene. The secretion of enhanced green fluorescent protein (EGFP) was more easily detected in the cell culture media than it was by western blot analysis of cell extract of the HC11 mouse mammary epithelial cells transfected with EGFP knock-in vector. These results indicated that a knock-in system using β-casein gene induced high expression of transgene by the gene regulatory sequence of endogenous β-casein gene. These fibroblasts may be used to produce transgenic pigs for the production of therapeutic proteins via the mammary glands.

  16. Cryopreservation of green fluorescent protein (GFP)-labeled primordial germ cells with GFP fused to the 3' untranslated region of the nanos gene by vitrification of Japanese eel (Anguilla japonica) somite stage embryos.

    Science.gov (United States)

    Kawakami, Y; Ishihara, M; Saito, T; Fujimoto, T; Adachi, S; Arai, K; Yamaha, E

    2012-12-01

    Primordial germ cells (PGC) are the only cell type in developing embryos with the potential to transmit genetic information to the next generation. In this study, PGC of Japanese eel (Anguilla japonica) were visualized by injection of mRNA synthesized from a construct carrying the green fluorescent protein (GFP) gene fused to the 3' untranslated region of the Japanese eel nanos gene. We investigated the feasibility of cryopreserving Japanese eel PGC by vitrification of dechorionated whole somite stage embryos. The GFP-labeled PGC were rapidly cooled using liquid nitrogen after exposure to a pretreatment solution containing 1.5 M cryoprotectant (methanol, dimethyl sulfoxide, and glycerol for 10 min and ethylene glycol for 10, 20, and 30 min) and a vitrification solution containing 3 M cryoprotectant and 0.5 M sucrose for 1, 5, and 10 min. Ethylene glycerol is an effective cryoprotectant for embryonic cells and shows no evidence of ice formation after thawing. Vitrified and thawed PGC were transplanted into blastula stage embryos from zebrafish (Danio rerio). The GFP-labeled PGC migrated toward the host gonadal ridge, suggesting maintenance of their normal migration motility. These techniques may assist in achieving inter- and intraspecies germ-line chimers using donor Japanese eel PGC.

  17. Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions

    Directory of Open Access Journals (Sweden)

    Puisieux Alain

    2003-10-01

    Full Text Available Abstract Background Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive. Results We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC in peripheral blood mononuclear cells; the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy. Conclusion Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

  18. Sentinel Node Mapping Using Indocyanine Green and Near-infrared Fluorescence Imaging Technology for Uterine Malignancies: Preliminary Experience With the Da Vinci Xi System.

    Science.gov (United States)

    Siesto, Gabriele; Romano, Fabrizio; Fiamengo, Barbara; Vitobello, Domenico

    2016-01-01

    Sentinel lymph node (SLN) mapping has emerged as the new frontier for the surgical staging of apparently early-stage cervical and endometrial cancer. Different colorimetric and radioactive tracers, alone and in combination, have been proposed with encouraging results. Fluorometric mapping using indocyanine green (ICG) appears to be a suitable and attractive alternative to provide reliable staging [1-4]. In this video, we present the technique of SLN mapping in 2 cases (1 endometrial and 1 cervical cancer, respectively) using ICG and the near-infrared technology provided by the newest Da Vinci Xi robotic system (Intuitive Surgical Inc., Sunnyvale, CA). Together we report the results of our preliminary experience on the first 20 cases performed. The new robotic Da Vinci Xi system was available at our institution since May 2015. Upon institutional review board/ethical committee approval, all consecutive patients with early-stage endometrial and cervical cancer who were judged suitable for robotic surgery have been enrolled for SLN mapping with ICG. We adopted the Memorial Sloan Kettering Cancer Center SLN algorithm; the tracer was delivered into the cervix in all cases. Four milliliters (1.25 mg/mL) of ICG was injected divided into the 3- and 9-o'clock positions of the cervix alone, with 1 mL deep into the stroma and 1 mL submucosally at the skin incision. Sentinel lymph nodes were examined with a protocol including both ultrastaging with immunohistochemistry [3] and 1-step nucleic acid amplification assay [5,6] under a parallel protocol of study. During the study period, 20 cases were managed; 14 and 6 patients had endometrial and cervical cancer, respectively. SLN was detected in all cases (20/20, 100%). Bilateral SLNs were detected in 17 of 20 (85.0%) cases. Based on preoperative and intraoperative findings, 13 (65.0%) patients received systematic pelvic lymphadenectomy after SLN mapping. Three (15.0%) patients had microscopic nodal metastases on SLN. No

  19. A human monocytic NF-κB fluorescent reporter cell line for detection of microbial contaminants in biological samples.

    Directory of Open Access Journals (Sweden)

    Claire Battin

    Full Text Available Sensing of pathogens by innate immune cells is essential for the initiation of appropriate immune responses. Toll-like receptors (TLRs, which are highly sensitive for various structurally and evolutionary conserved molecules derived from microbes have a prominent role in this process. TLR engagement results in the activation of the transcription factor NF-κB, which induces the expression of cytokines and other inflammatory mediators. The exquisite sensitivity of TLR signalling can be exploited for the detection of bacteria and microbial contaminants in tissue cultures and in protein preparations. Here we describe a cellular reporter system for the detection of TLR ligands in biological samples. The well-characterized human monocytic THP-1 cell line was chosen as host for an NF-ᴋB-inducible enhanced green fluorescent protein reporter gene. We studied the sensitivity of the resultant reporter cells for a variety of microbial components and observed a strong reactivity towards TLR1/2 and TLR2/6 ligands. Mycoplasma lipoproteins are potent TLR2/6 agonists and we demonstrate that our reporter cells can be used as reliable and robust detection system for mycoplasma contaminations in cell cultures. In addition, a TLR4-sensitive subline of our reporters was engineered, and probed with recombinant proteins expressed in different host systems. Bacterially expressed but not mammalian expressed proteins induced strong reporter activity. We also tested proteins expressed in an E. coli strain engineered to lack TLR4 agonists. Such preparations also induced reporter activation in THP-1 cells highlighting the importance of testing recombinant protein preparations for microbial contaminations beyond endotoxins. Our results demonstrate the usefulness of monocytic reporter cells for high-throughput screening for microbial contaminations in diverse biological samples, including tissue culture supernatants and recombinant protein preparations. Fluorescent reporter

  20. 4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression

    Directory of Open Access Journals (Sweden)

    Katherine D. Shives

    2016-10-01

    Full Text Available West Nile virus (WNV is a (+ sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7GpppNm 5′ cap with 2′-O-methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1 for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K and eukaryotic translation initiation factor 4E-binding protein (4EBP pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E interaction and eukaryotic initiation factor 4F (eIF4F complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6 and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome.

  1. PTEN gene and phosphorylation of Akt protein expression in the LPS-induced lung fibroblast

    Directory of Open Access Journals (Sweden)

    Mao-lin HUANG

    2014-09-01

    Full Text Available Objective: To investigate PTEN gene expression and the Akt phosphorylation of protein expression in the LPS-induced lung fibroblast, to initially reveal the relation between PTEN gene and the Akt phosphorylated proteins to LPS-induced lung fibroblast proliferation mechanism. Methods: BrdU experiments was performed to evaluate the LPS-induced lung fibroblast proliferation,  RT-PCR and Western Blot analysis were used to analyze the PTEN gene expression and Western blot was performed to analyze Akt phosphorylated protein expression. Results: PTEN mRNA level of the experimental group were significantly lower than the control group (P<0.05 with LPS simulation for 24h and 72h , and there were no significant difference between the experimental group and control group the experimental group and control group (P>0.05 . PTEN protein expression levels of the experimental group were significantly lower than the control group (P<0.05 , at 72h, and PTEN mRNA levels had no significant differences between these of the experimental and control group at 6h,12h and 24h(p>0.05. Phosphorylation Akt protein level (relative to total Akt protein was significantly higer than the control group (P<0.05 at 24h and 72h, and phosphorylation Akt protein levels had no significant differences between these of the experimental and control group at 6h and 12h (P>0.05 .Conclusion: PTEN gene and phosphorylation Akt protein involve in LPS-induced lung fibroblast proliferation signal transduction pathway.

  2. Effects of Diabetes on Salivary Gland Protein Expression of Tetrahydrobiopterin and Nitric Oxide Synthesis and Function.

    Science.gov (United States)

    Stewart, Cassandra R; Obi, Nneka; Epane, Elodie C; Akbari, Alexander A; Halpern, Leslie; Southerland, Janet H; Gangula, Pandu R

    2016-06-01

    Xerostomia is defined as dry mouth resulting from a change in the amount or composition of saliva and is often a major oral health complication associated with diabetes mellitus (DM). Studies have shown that xerostomia is more common in females at the onset of DM. Evidence suggests that nitric oxide (NO) plays a critical role in healthy salivary gland function. However, the specific mechanisms by which NO regulates salivary gland function at the onset of DM have yet to be determined. This study has two aims: 1) to determine whether protein expression or dimerization of NO synthase enzymes (neuronal [nNOS] and endothelial [eNOS]) are altered in the onset of diabetic xerostomia; and 2) to determine whether the changes in nNOS/eNOS protein expression or dimerization are correlated with changes in NO cofactor tetrahydrobiopterin (BH4) biosynthetic enzymes (guanosine triphosphate cyclohydrolase-1 and dihydrofolate reductase). Functional and Western blot studies were performed in streptozotocin-induced and control Sprague-Dawley female rats with DM (type 1 [t1DM]) using standardized protocols. Confirmation of xerostomia was determined by increased water intake and decreased salivary flow rate. The results showed that in female rats with DM, salivary hypofunction is correlated with decreased submandibular and parotid gland sizes. The results also show a decrease in NOS and BH4 biosynthetic enzyme in submandibular glands. These results indicate that a decrease in submandibular NO-BH4 protein expression may provide insight pertaining to mechanisms for the development of hyposalivation in DM-induced xerostomia. Furthermore, understanding the role of the NO-BH4 pathway may give insight into possible treatment options for the patient with DM experiencing xerostomia.

  3. 4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression.

    Science.gov (United States)

    Shives, Katherine D; Massey, Aaron R; May, Nicholas A; Morrison, Thomas E; Beckham, J David

    2016-10-18

    West Nile virus (WNV) is a (+) sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7 GpppN m 5' cap with 2'- O -methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1) for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4EBP) pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E) interaction and eukaryotic initiation factor 4F (eIF4F) complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6) and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome.

  4. NM23 protein expression in colorectal carcinoma using TMA (tissue microarray: association with metastases and survival

    Directory of Open Access Journals (Sweden)

    Levindo Alves de Oliveira

    2010-12-01

    Full Text Available CONTEXT: NM23, a metastasis suppressor gene, may be associated with prognosis in patients with colorectal carcinoma. OBJECTIVE: To analyze NM23 expression and its association with the presence of lymph node and liver metastases and survival in patients operated on for colorectal carcinoma. METHODS: One hundred thirty patients operated on for colorectal carcinoma were investigated. Tissue microarray blocks containing neoplastic tissue and tumor-adjacent non-neoplastic mucosa were obtained and analyzed by immunohistochemical staining using a monoclonal anti-NM23 antibody. Immunohistochemical expression was assessed using a semiquantitative scoring method, counting the percentage of stained cells. The results were compared regarding morphological and histological characteristics of the colorectal carcinoma, presence of lymph node and liver metastases, tumor staging, and patient survival. Statistical analysis was performed using the Mann-Whitney test, the Kruskal-Wallis test and Fisher's exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. RESULTS: NM23 expression was higher in colorectal carcinoma tissue than in adjacent non-neoplastic mucosa (P<0.0001. NM23 protein expression did not correlate with degree of cell differentiation (P = 0.57, vascular invasion (P = 0.85, lymphatic invasion (P = 0.41, perineural infiltration (P = 0.46, staging (P = 0.19, lymph node metastases (P = 0.08, or liver metastases (P = 0.59. Disease-free survival showed significant association (P = 0.01 with the intensity of NM23 protein immunohistochemical expression in colorectal carcinoma tissue, whereas overall survival showed no association with NM23 protein expression (P = 0.13. CONCLUSIONS: NM23 protein expression was higher in neoplastic colorectal carcinoma tissue than in adjacent non-neoplastic mucosa, showing no correlation with morphological aspects, presence of lymph node or liver metastases, colorectal carcinoma

  5. Heterologous Protein Expression in Pichia pastoris: Latest Research Progress and Applications.

    Science.gov (United States)

    Juturu, Veeresh; Wu, Jin Chuan

    2018-01-04

    Pichia pastoris is a well-known platform strain for heterologous protein expression. Over the past five years, different strategies to improve the efficiency of recombinant protein expression by this yeast strain have been developed; these include a patent-free protein expression kit, construction of the P. pastoris CBS7435Ku70 platform strain with its high efficiency in site-specific recombination of plasmid DNA into the genomic DNA, the design of synthetic promoters and their variants by combining different core promoters with multiple putative transcription factors, the generation of mutant GAP promoter variants with various promoter strengths, codon optimization, engineering the α-factor signal sequence by replacing the native glutamic acid at the Kex2 cleavage site with the other 19 natural amino acids and the addition of mammalian signal sequence to the yeast signal sequence, and the co-expression of single chaperones, multiple chaperones or helper proteins that aid in recombinant protein folding. Publically available high-quality genome data from multiple strains of P. pastoris GS115, DSMZ 70382, and CBS7435 and the continuous development of yeast expression kits have successfully promoted the metabolic engineering of this strain to produce carotenoids, xanthophylls, nootkatone, ricinoleic acid, dammarenediol-II, and hyaluronic acid. The cell-surface display of enzymes has obviously increased enzyme stability, and high-level intracellular expression of acyl-CoA and ethanol O-acyltransferase, lipase and d-amino acid oxidase has opened up applications in whole-cell biocatalysis for producing flavor molecules and biodiesel, as well as the deracemization of racemic amino acids. High-level expression of various food-grade enzymes, cellulases, and hemicellulases for applications in the food, feed and biorefinery industries is in its infancy and needs strengthening. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. A new kymogram-based method reveals unexpected effects of marker protein expression and spatial anisotropy of cytoskeletal dynamics in plant cell cortex

    Czech Academy of Sciences Publication Activity Database

    Cvrčková, F.; Oulehlová, Denisa

    2017-01-01

    Roč. 13, MAR 27 (2017), č. článku 19. ISSN 1746-4811 Institutional support: RVO:61389030 Keywords : green fluorescent protein * cortical microtubule arrays * actin stochastic dynamics * arabidopsis-thaliana * epidermal-cells * quantitative-analysis * binding domain * gfp-tubulin * f-actin * microscopy * Actin * Microtubules * Lifeact * Variable angle fluorescence microscopy * Spinning disc confocal microscopy * Kymogram * Structure stability * Lateral mobility * Anisotropy * FH1 (At3g25500) Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 3.510, year: 2016

  7. Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris

    International Nuclear Information System (INIS)

    Clark, Lindsay; Zahm, Jacob A.; Ali, Rustam; Kukula, Maciej; Bian, Liangqiao; Patrie, Steven M.; Gardner, Kevin H.; Rosen, Michael K.; Rosenbaum, Daniel M.

    2015-01-01

    13 C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific 13 C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient 13 C isoleucine δ1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets

  8. Differential protein expression in mussels Mytilus galloprovincialis exposed to nano and ionic Ag

    International Nuclear Information System (INIS)

    Gomes, Tânia; Pereira, Catarina G.; Cardoso, Cátia; Bebianno, Maria João

    2013-01-01

    Highlights: •Different protein expression profiles between tissues and Ag forms. •Ag NPs and Ag + presented different mechanisms of toxic action. •Ag NPs toxicity is mediated by oxidative stress-induced cell signalling cascades. •New biomarkers for Ag NPs were proposed, i.e. MVP, ras partial and precol-P. -- Abstract: Ag NPs are one of the most commonly used NPs in nanotechnology whose environmental impacts are to date unknown and the information about bioavailability, mechanisms of biological uptake and toxic implications in organisms is scarce. So, the main objective of this study was to investigate differences in protein expression profiles in gills and digestive gland of mussels Mytilus galloprovincialis exposed to Ag NPs and Ag + (10 μg L −1 ) for a period of 15 days. Protein expression profiles of exposed gills and digestive glands were compared to those of control mussels using two–dimensional electrophoresis to discriminate differentially expressed proteins. Different patterns of protein expression were obtained for exposed mussels, dependent not only on the different redox requirements of each tissue but also to the Ag form used. Unique sets of differentially expressed proteins were affected by each silver form in addition to proteins that were affected by both Ag NPs and Ag + . Fifteen of these proteins were subsequently identified by MALDI–TOF–TOF and database search. Ag NPs affected similar cellular pathways as Ag + , with common response mechanisms in cytoskeleton and cell structure (catchin, myosin heavy chain), stress response (heat shock protein 70), oxidative stress (glutathione s-transferase), transcriptional regulation (nuclear receptor subfamily 1G), adhesion and mobility (precollagen-P) and energy metabolism (ATP synthase F0 subunit 6 and NADH dehydrogenase subunit 2). Exposure to Ag NPs altered the expression of two proteins associated with stress response (major vault protein and ras partial) and one protein involved in

  9. Machine learning in computational biology to accelerate high-throughput protein expression

    DEFF Research Database (Denmark)

    Sastry, Anand; Monk, Jonathan M.; Tegel, Hanna

    2017-01-01

    and machine learning identifies protein properties that hinder the HPA high-throughput antibody production pipeline. We predict protein expression and solubility with accuracies of 70% and 80%, respectively, based on a subset of key properties (aromaticity, hydropathy and isoelectric point). We guide...... the selection of protein fragments based on these characteristics to optimize high-throughput experimentation. Availability and implementation: We present the machine learning workflow as a series of IPython notebooks hosted on GitHub (https://github.com/SBRG/Protein_ML). The workflow can be used as a template...

  10. Differential protein expression in mussels Mytilus galloprovincialis exposed to nano and ionic Ag

    Energy Technology Data Exchange (ETDEWEB)

    Gomes, Tânia; Pereira, Catarina G.; Cardoso, Cátia; Bebianno, Maria João, E-mail: mbebian@ualg.pt

    2013-07-15

    Highlights: •Different protein expression profiles between tissues and Ag forms. •Ag NPs and Ag{sup +} presented different mechanisms of toxic action. •Ag NPs toxicity is mediated by oxidative stress-induced cell signalling cascades. •New biomarkers for Ag NPs were proposed, i.e. MVP, ras partial and precol-P. -- Abstract: Ag NPs are one of the most commonly used NPs in nanotechnology whose environmental impacts are to date unknown and the information about bioavailability, mechanisms of biological uptake and toxic implications in organisms is scarce. So, the main objective of this study was to investigate differences in protein expression profiles in gills and digestive gland of mussels Mytilus galloprovincialis exposed to Ag NPs and Ag{sup +} (10 μg L{sup −1}) for a period of 15 days. Protein expression profiles of exposed gills and digestive glands were compared to those of control mussels using two–dimensional electrophoresis to discriminate differentially expressed proteins. Different patterns of protein expression were obtained for exposed mussels, dependent not only on the different redox requirements of each tissue but also to the Ag form used. Unique sets of differentially expressed proteins were affected by each silver form in addition to proteins that were affected by both Ag NPs and Ag{sup +}. Fifteen of these proteins were subsequently identified by MALDI–TOF–TOF and database search. Ag NPs affected similar cellular pathways as Ag{sup +}, with common response mechanisms in cytoskeleton and cell structure (catchin, myosin heavy chain), stress response (heat shock protein 70), oxidative stress (glutathione s-transferase), transcriptional regulation (nuclear receptor subfamily 1G), adhesion and mobility (precollagen-P) and energy metabolism (ATP synthase F0 subunit 6 and NADH dehydrogenase subunit 2). Exposure to Ag NPs altered the expression of two proteins associated with stress response (major vault protein and ras partial) and one

  11. In situ protein expression in tumour spheres: development of an immunostaining protocol for confocal microscopy

    International Nuclear Information System (INIS)

    Weiswald, Louis-Bastien; Guinebretière, Jean-Marc; Richon, Sophie; Bellet, Dominique; Saubaméa, Bruno; Dangles-Marie, Virginie

    2010-01-01

    Multicellular tumour sphere models have been shown to closely mimic phenotype characteristics of in vivo solid tumours, or to allow in vitro propagation of cancer stem cells (CSCs). CSCs are usually characterized by the expression of specific membrane markers using flow cytometry (FC) after enzymatic dissociation. Consequently, the spatial location of positive cells within spheres is not documented. Confocal microscopy is the best technique for the imaging of thick biological specimens after multi-labelling but suffers from poor antibody penetration. Thus, we describe here a new protocol for in situ confocal imaging of protein expression in intact spheroids. Protein expression in whole spheroids (150 μm in diameter) from two human colon cancer cell lines, HT29 and CT320X6, has been investigated with confocal immunostaining, then compared with profiles obtained through paraffin immunohistochemistry (pIHC) and FC. Target antigens, relevant for colon cancer and with different expression patterns, have been studied. We first demonstrate that our procedure overcomes the well-known problem of antibody penetration in compact structures by performing immunostaining of EpCAM, a membrane protein expressed by all cells within our spheroids. EpCAM expression is detected in all cells, even the deepest ones. Likewise, antibody access is confirmed with CK20 and CD44 immunostaining. Confocal imaging shows that 100% of cells express β-catenin, mainly present in the plasma membrane with also cytoplasmic and nuclear staining, in agreement with FC and pIHC data. pIHC and confocal imaging show similar CA 19-9 cytoplasmic and membranar expression profile in a cell subpopulation. CA 19-9 + cell count confirms confocal imaging as a highly sensitive method (75%, 62% and 51%, for FC, confocal imaging and pIHC, respectively). Finally, confocal imaging reveals that the weak expression of CD133, a putative colon CSC marker, is restricted to the luminal cell surface of colorectal cancer acini

  12. Effect of 17-allylamino-17-demethoxygeldanamycin (17-AAG) on Akt protein expression is more effective in head and neck cancer cell lineages that retain PTEN protein expression.

    Science.gov (United States)

    Pontes, Flávia Sirotheau C; Pontes, Hélder A R; de Souza, Lucas L; de Jesus, Adriana S; Joaquim, Andrea M C; Miyahara, Ligia A N; Fonseca, Felipe P; Pinto Junior, Décio S

    2018-03-01

    The aim of this study was to evaluate the expression of Akt, PTEN, Mdm2 and p53 proteins in three different head and neck squamous cell carcinoma (HNSCC) cell lines (HN6, HN19 and HN30), all of them treated with epidermal growth factor (EGF) and 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of Hsp90 protein. Immunofluorescence and western blot were performed in order to analyze the location and quantification, respectively, of proteins under the action 17-AAG and EGF. Treatment with EGF resulted in increased levels of Akt, PTEN and p53 in all cell lineages. The expression of Mdm2 was constant in HN30 and HN6 lineages, while in HN19 showed slightly decreased expression. Under the action 17-AAG, in HN6 and HN19, the expression of PTEN and p53 proteins was suppressed, while Akt and Mdm2 expression was reduced. Finally, in the HN30 cell lineage were absolute absence of expression of Akt, Mdm2 and p53 and decreased expression of PTEN. These data allow us to speculate on the particular utility of 17-AAG for HNSCC treatment through the inhibition of Akt protein expression, especially in the cases that retain the expression of PTEN protein. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Effect of polysaccharides from Angelica sinensis on Bcl-2 and Bax protein expression of irradiated liver cells

    International Nuclear Information System (INIS)

    Sun Yuanlin; Tang Jian; Gu Xiaohong; Li Deyuan

    2009-01-01

    Objective: To investigate the effect of polysaccharides from Angelica sinensis (ASP3) on Bcl-2 and Bax protein expression of irradiated liver cells from mice. Methods: Bcl-2 and Bax protein expression of liver cells in vitro exposed to 2.0 Gy rays were examined by using immunohistochemistry method. Results: The expression of apoptosis-accelerating protein Bax in the irradiation group was enhanced obviously (70.83%), while apoptosis inhibiting protein Bcl-2 tended to decline (55.60%), with the statistically significant difference (P <0.01) compared with that of the control. ASP3 pretreatment could regulate Bcl-2 and Bax protein expression of liver cells, inhibiting Bax protein expression(64.14/58.37%) and increasing Bcl-2 protein expression(59.21%/ 67.45%). The differences between the high dosage (100 mg/L of ASP3) and the irradiation group were statistically significant (P<0.05). Conclusions: ASP3 pretreatment could prohibit the apoptosis of radiation- damaged liver cells due to abnormal expression of Bcl-2 and Bax, and reduce the cell apoptosis by increasing Bcl-2/Bax protein expression so as to enhance the radiation endurance of liver cells. (authors)

  14. On-line solid-phase enrichment coupled to packed reactor flow injection analysis in a green analytical procedure to determine low levels of folic acid using fluorescence detection

    Directory of Open Access Journals (Sweden)

    Emara Samy

    2012-12-01

    Full Text Available Abstract Background Analysis of folic acid (FA is not an easy task because of its presence in lower concentrations, its lower stability under acidic conditions, and its sensitiveness against light and high temperature. The present study is concerned with the development and validation of an automated environmentally friendly pre-column derivatization combined by solid-phase enrichment (SPEn to determine low levels of FA. Results Cerium (IV trihydroxyhydroperoxide (CTH as a packed oxidant reactor has been used for oxidative cleavage of FA into highly fluorescent product, 2-amino-4-hydroxypteridine-6-carboxylic acid. FA was injected into a carrier stream of 0.04 M phosphate buffer, pH 3.4 at a flow-rate of 0.25 mL/min. The sample zone containing the analyte was passed through the CTH reactor thermostated at 40°C, and the fluorescent product was trapped and enriched on a head of small ODS column (10 mm x 4.6 mm i.d., 5 μm particle size. The enriched product was then back-flush eluted by column-switching from the small ODS column to the detector with a greener mobile phase consisting of ethanol and phosphate buffer (0.04M, pH 3.4 in the ratio of 5:95 (v/v. The eluent was monitored fluorimetrically at emission and excitation wavelengths of 463 and 367 nm, respectively. The calibration graph was linear over concentrations of FA in the range of 1.25-50 ng/mL, with a detection limit of 0.49 ng/mL. Conclusion A new simple and sensitive green analytical procedure including on-line pre-column derivatization combined by SPEn has been developed for the routine quality control and dosage form assay of FA at very low concentration level. The method was a powerful analytical technique that had excellent sensitivity, sufficient accuracy and required relatively simple and inexpensive instrumentation.

  15. Green Tourism

    OpenAIRE

    Hasan, Ali

    2014-01-01

    Green tourism is defined as environmentally friendly tourism activities with various focuses and meanings. In a broad term, green tourism is about being an environmentally friendly tourist or providing environmentally friendly tourist services. The green tourism concept would be highly appealing to tourism enterprises and operators owing to increasing governmental pressure to improve environmental performance by adopting effective and tangible environmental management techniques. Green to...

  16. Metaphysical green

    OpenAIRE

    Earon, Ofri

    2011-01-01

    “Sensation of Green is about the mental process like touching, seeing, hearing, or smelling, resulting from the immediate stimulation of landscape forms, plants, trees, wind and water. Sensation of Green triggers a feeling of scale, cheerfulness, calmness and peace. The spatial performance of Sensation of Green is created by a physical interaction between the language of space and the language of nature” The notion of Sensation of Green was developed through a previous study ‘Learning from th...

  17. Parasitization by Scleroderma guani influences protein expression in Tenebrio molitor pupae.

    Science.gov (United States)

    Zhu, Jia-Ying; Wu, Guo-Xing; Ze, Sang-Zi; Stanley, David W; Yang, Bin

    2014-07-01

    Ectoparasitoid wasps deposit their eggs onto the surface and inject venom into their hosts. Venoms are chemically complex and they exert substantial impact on hosts, including permanent or temporary paralysis and developmental arrest. These visible venom effects are due to changes in expression of genes encoding physiologically relevant proteins. While the influence of parasitization on gene expression in several lepidopterans has been reported, the molecular details of parasitoid/beetle relationships remain mostly unknown. This shortcoming led us to pose the hypothesis that envenomation by the ectoparasitic ant-like bethylid wasp Scleroderma guani leads to changes in protein expression in the yellow mealworm beetle Tenebrio molitor. We tested our hypothesis by comparing the proteomes of non-parasitized and parasitized host pupae using iTRAQ-based proteomics. We identified 41 proteins that were differentially expressed (32↑- and 9↓-regulated) in parasitized pupae. We assigned these proteins to functional categories, including immunity, stress and detoxification, energy metabolism, development, cytoskeleton, signaling and others. We recorded parallel changes in mRNA levels and protein abundance in 14 selected proteins following parasitization. Our findings support our hypothesis by documenting changes in protein expression in parasitized hosts. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Oestradiol and progesterone differentially alter cytoskeletal protein expression and flame cell morphology in Taenia crassiceps.

    Science.gov (United States)

    Ambrosio, Javier R; Ostoa-Saloma, Pedro; Palacios-Arreola, M Isabel; Ruíz-Rosado, Azucena; Sánchez-Orellana, Pedro L; Reynoso-Ducoing, Olivia; Nava-Castro, Karen E; Martínez-Velázquez, Nancy; Escobedo, Galileo; Ibarra-Coronado, Elizabeth G; Valverde-Islas, Laura; Morales-Montor, Jorge

    2014-09-01

    We examined the effects of oestradiol (E2) and progesterone (P4) on cytoskeletal protein expression in the helminth Taenia crassiceps - specifically actin, tubulin and myosin. These proteins assemble into flame cells, which constitute the parasite excretory system. Total protein extracts were obtained from E2- and P4-treated T. crassiceps cysticerci and untreated controls, and analysed by one- and two-dimensional protein electrophoresis, flow cytometry, immunofluorescence and videomicroscopy. Exposure of T. crassiceps cysticerci to E2 and P4 induced differential protein expression patterns compared with untreated controls. Changes in actin, tubulin and myosin expression were confirmed by flow cytometry of parasite cells and immunofluorescence. In addition, parasite morphology was altered in response to E2 and P4 versus controls. Flame cells were primarily affected at the level of the ciliary tuft, in association with the changes in actin, tubulin and myosin. We conclude that oestradiol and progesterone act directly on T. crassiceps cysticerci, altering actin, tubulin and myosin expression and thus affecting the assembly and function of flame cells. Our results increase our understanding of several aspects of the molecular crosstalk between host and parasite, which might be useful in designing anthelmintic drugs that exclusively impair parasitic proteins which mediate cell signaling and pathogenic reproduction and establishment. Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  19. AR-v7 protein expression is regulated by protein kinase and phosphatase

    Science.gov (United States)

    Li, Yinan; Xie, Ning; Gleave, Martin E.; Rennie, Paul S.; Dong, Xuesen

    2015-01-01

    Failure of androgen-targeted therapy and progression of castration-resistant prostate cancer (CRPC) are often attributed to sustained expression of the androgen receptor (AR) and its major splice variant, AR-v7. Although the new generation of anti-androgens such as enzalutamide effectively inhibits AR activity, accumulating pre-clinical and clinical evidence indicates that AR-v7 remains constitutively active in driving CRPC progression. However, molecular mechanisms which control AR-v7 protein expression remain unclear. We apply multiple prostate cancer cell models to demonstrate that enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the gene context of cancer cells. The balance between PP-1 and Akt activation governs AR phosphorylation status and activation of the Mdm2 ubiquitin ligase. Mdm2 recognizes phosphorylated serine 213 of AR-v7, and induces AR-v7 ubiquitination and protein degradation. These findings highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked. PMID:26378044

  20. iQuantitator: A tool for protein expression inference using iTRAQ

    Directory of Open Access Journals (Sweden)

    Comte-Walters Susana

    2009-10-01

    Full Text Available Abstract Background Isobaric Tags for Relative and Absolute Quantitation (iTRAQ™ [Applied Biosystems] have seen increased application in differential protein expression analysis. To facilitate the growing need to analyze iTRAQ data, especially for cases involving multiple iTRAQ experiments, we have developed a modeling approach, statistical methods, and tools for estimating the relative changes in protein expression under various treatments and experimental conditions. Results This modeling approach provides a unified analysis of data from multiple iTRAQ experiments and links the observed quantity (reporter ion peak area to the experiment design and the calculated quantity of interest (treatment-dependent protein and peptide fold change through an additive model under log transformation. Others have demonstrated, through a case study, this modeling approach and noted the computational challenges of parameter inference in the unbalanced data set typical of multiple iTRAQ experiments. Here we present the development of an inference approach, based on hierarchical regression with batching of regression coefficients and Markov Chain Monte Carlo (MCMC methods that overcomes some of these challenges. In addition to our discussion of the underlying method, we also present our implementation of the software, simulation results, experimental results, and sample output from the resulting analysis report. Conclusion iQuantitator's process-based modeling approach overcomes limitations in current methods and allows for application in a variety of experimental designs. Additionally, hypertext-linked documents produced by the tool aid in the interpretation and exploration of results.

  1. Biological activity of Bt proteins expressed in different structures of transgenic corn against Spodoptera frugiperda

    Directory of Open Access Journals (Sweden)

    Daniel Bernardi

    2016-06-01

    Full Text Available ABSTRACT: Spodoptera frugiperda (J. E. Smith is the main target pest of Bt corn technologies, such as YieldGard VT PRO(tm (Cry1A.105/Cry2Ab2 and PowerCore(tm (Cry1A.105/Cry2Ab2/Cry1F. In this study, it was evaluated the biological activity of Bt proteins expressed in different plant structures of YieldGard VT PRO(tm and PowerCore(tm corn against S. frugiperda . Complete mortality of S. frugiperda neonates was observed on leaf-disc of both Bt corn technologies. However, the mortality in silks and grains was lower than 50 and 6%, respectively. In addition, more than 49% of the surviving larvae in silks and grains completed the biological cycle. However, all life table parameters were negatively affected in insects that developed in silks and grains of both Bt corn events. In summary, the low biological activity of Bt proteins expressed on silks and grains of YieldGard VT PRO(tm and PowerCore(tm corn can contribute to the resistance evolution in S. frugiperda populations.

  2. Protein Expression Landscape of Mouse Embryos during Pre-implantation Development

    Directory of Open Access Journals (Sweden)

    Yawei Gao

    2017-12-01

    Full Text Available Pre-implantation embryo development is an intricate and precisely regulated process orchestrated by maternally inherited proteins and newly synthesized proteins following zygotic genome activation. Although genomic and transcriptomic studies have enriched our understanding of the genetic programs underlying this process, the protein expression landscape remains unexplored. Using quantitative mass spectrometry, we identified nearly 5,000 proteins from 8,000 mouse embryos of each stage (zygote, 2-cell, 4-cell, 8-cell, morula, and blastocyst. We found that protein expression in zygotes, morulas, and blastocysts is distinct from 2- to 8-cell embryos. Analysis of protein phosphorylation identified critical kinases and signal transduction pathways. We highlight key factors and their important roles in embryo development. Combined analysis of transcriptomic and proteomic data reveals coordinated control of RNA degradation, transcription, and translation and identifies previously undefined exon-junction-derived peptides. Our study provides an invaluable resource for further mechanistic studies and suggests core factors regulating pre-implantation embryo development.

  3. Induction of Ski Protein Expression upon Luteinization in Rat Granulosa Cells

    Directory of Open Access Journals (Sweden)

    Hyun Kim

    2012-05-01

    Full Text Available Ski protein is implicated in proliferation/differentiation in a variety of cells. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells; however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to locate Ski protein in the rat ovary during luteinizationto predict the possible role of Ski. In order to examine the expression pattern of Ski protein along with the progress of luteinization, follicular growth was induced by administration of equine chorionic gonadtropin to immature female rats, and luteinization was induced by human chorionic gonadtropin treatment to mimic luteinizing hormone (LH surge. While no Ski-positive granulosa cells were present in preovulatory follicle, Ski protein expression was induced in response to LH surge, and was maintained after the formation of the corpus luteum (CL. Though Ski protein is absent in granulosa cells of preovulatory follicle, its mRNA (c-Ski was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggests that its expression is regulated post-transcriptionally.

  4. RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

    International Nuclear Information System (INIS)

    López, Claudia S.; Sloan, Rachel; Cylinder, Isabel; Kozak, Susan L.; Kabat, David; Barklis, Eric

    2014-01-01

    The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag protein expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export

  5. Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

    Directory of Open Access Journals (Sweden)

    Walchli John

    2009-04-01

    Full Text Available Abstract Background With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. Results In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38α, viral polymerase (HCV NS5B, and bacterial structural protein (FtsZ were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. Conclusion The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.

  6. Use of nonpathogenic, green fluorescent protein-marked Escherichia coli Biotype I cultures to evaluate the self-cleansing capabilities of a commercial beef grinding system after a contamination event.

    Science.gov (United States)

    Wages, Jennifer A; Williams, Jennifer; Adams, Jacquelyn; George, Bruce; Oxford, Eric; Zelenka, Dan

    2014-11-01

    Inoculated beef trim containing a cocktail of green fluorescent protein-marked Escherichia coli biotype I cultures as surrogates for E. coli O157:H7 was introduced into two large, commercial grinding facilities capable of producing 180,000 kg of ground product in 1 day. Three repetitions were performed over 3 days. Sampling occurred at three different points within the process: postprimary grind, postsecondary grind-blender, and postpackaging. Resulting data show that, as the inoculated meat passes through the system, the presence of the marked surrogate quickly diminishes. The depletion rates are directly related to the amount of product in kilograms (represented by time) that has passed through the system, but these rates vary with each step of the process. The primary grinder appears to rid itself of the contaminant the most quickly; in all repetitions, the contaminant was not detected within 5 min of introduction of the contaminated combo bin into the system, which in all cases, was prior to the introduction of a second combo bin and within 1,800 kg of product. After the blending step and subsequent secondary grinding, the contaminant was detected in product produced from both the parent combo and the combo bin added directly after the parent combo bin; however, for those days on which three combo bins (approximately 2,700 kg) were available for sampling, the contaminant was not detected from product representing the third combo bin. Similarly, at the packaging step, the contaminant was detected in the product produced by both the parent and second combo bins; however, on those days when a third combo bin was available for sampling (repetitions 2 and 3), the contaminant was not detected from product produced from the third combo bin.

  7. Clinical Significance of "Double-hit" and "Double-protein" expression in Primary Gastric B-cell Lymphomas.

    Science.gov (United States)

    He, Miaoxia; Chen, Keting; Li, Suhong; Zhang, Shimin; Zheng, Jianming; Hu, Xiaoxia; Gao, Lei; Chen, Jie; Song, Xianmin; Zhang, Weiping; Wang, Jianmin; Yang, Jianmin

    2016-01-01

    Primary gastric B-cell lymphoma is the second most common malignancy of the stomach. There are many controversial issues about its diagnosis, treatment and clinical management. "Double-hit" and "double-protein" involving gene rearrangement and protein expression of c-Myc and bcl2/bcl6 are the most used terms to describe DLBCL poor prognostic factors in recent years. However, very little is known about the role of these prognostic factors in primary gastric B-cell lymphomas. This study aims to obtain a molecular pathology prognostic model of gastric B-cell lymphoma for clinical stratified management by evaluating how the "double-hit" and "double-protein" in tumor cells as well as microenvironmental reaction of tumor stromal tissue affect clinical outcome in primary gastric B-cell lymphomas. Data and tissues of 188 cases diagnosed with gastric B-cell lymphomas were used in this study. Tumor tissue microarray (TMA) of formalin fixed and paraffin embedded (FFPE) tissues was constructed for fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) analysis with a serial of biomarkers containing MYC, BCL2, BCL6, CD31, SPARC, CD10, MUM1 and Ki-67. Modeled period analysis was used to estimate 3-year and 5-year overall survival (OS) and disease-free survival (DFS) distributions. There was no definite "double-hit" case though the gene rearrangement of c-Myc (5.9%), bcl2 (0.1%) and bcl6 (7.4%) was found in gastric B-cell lymphomas. The gene amplification or copy gains of c-Myc (10.1%), bcl-2 (17.0%) and bcl-6 (0.9%) were present in these lymphomas. There were 12 cases of the lymphomas with the "double-protein" expression of MYC and BCL2/BCL6. All patients with "double-protein" gastric B-cell lymphomas had poor outcome compared with those without. More importantly, "MYC-BCL2-BCL6" negative group of gastric B-cell lymphoma patients had favorable clinical outcome regardless clinical stage, pathological types and therapeutic modalities. And the similar better

  8. Energy transfer from carotenoids to chlorophyll in blue-green, red and green algae and greening bean leaves

    NARCIS (Netherlands)

    Goedheer, J.C.

    1969-01-01

    From fluorescence action spectra, fluorescence spectra and absorption spectra measured at room temperature and at 77 °K of light petroleum (b.p. 40–60°)-treated and normal chloroplasts, it is concluded that: 1. 1. In blue-green and red algae energy transfer from β-carotene to chlorophyll occurs

  9. Ultrafast Proton Shuttling in Psammocora Cyan Fluorescent Protein

    NARCIS (Netherlands)

    Kennis, J.T.M.; van Stokkum, I.H.M.; Peterson, D.S.; Pandit, A.; Wachter, R.M.

    2013-01-01

    Cyan, green, yellow, and red fluorescent proteins (FPs) homologous to green fluorescent protein (GFP) are used extensively as model systems to study fundamental processes in photobiology, such as the capture of light energy by protein-embedded chromophores, color tuning by the protein matrix, energy

  10. Metaphysical green

    DEFF Research Database (Denmark)

    Earon, Ofri

    2011-01-01

    example is a tiny Danish summer house from 1918 . The second example is ‘House before House’ , in Tokyo. The third example is a prefabricated house ‘CHU’ . The analysis evaluates the characteristics of diverse tones of green – from green image to green sensation. The analysis is based on the original...... of Sensation of Green is created by a physical interaction between the language of space and the language of nature” The notion of Sensation of Green was developed through a previous study ‘Learning from the Summer House’ investigating the unique architectural characteristics of the Danish summer houses...... the Sensation of Green? Three existing examples are agents to this discussion. The first example is a Danish summer house. The other two are international urban examples. While the summer house articulates the original meaning of Sensation of Green, the urban examples illustrate its urban context. The first...

  11. Nine New Fluorescent Probes

    Science.gov (United States)

    Lin, Tsung-I.; Jovanovic, Misa V.; Dowben, Robert M.

    1989-06-01

    Absorption and fluorescence spectroscopic studies are reported here for nine new fluorescent probes recently synthesized in our laboratories: four pyrene derivatives with substituents of (i) 1,3-diacetoxy-6,8-dichlorosulfonyl, (ii) 1,3-dihydroxy-6,8-disodiumsulfonate, (iii) 1,3-disodiumsulfonate, and (iv) l-ethoxy-3,6,8-trisodiumsulfonate groups, and five [7-julolidino] coumarin derivatives with substituents of (v) 3-carboxylate-4-methyl, (vi) 3- methylcarboxylate, (vii) 3-acetate-4-methyl, (viii) 3-propionate-4-methyl, and (ix) 3-sulfonate-4-methyl groups. Pyrene compounds i and ii and coumarin compounds v and vi exhibit interesting absorbance and fluorescence properties: their absorption maxima are red shifted compared to the parent compound to the blue-green region, and the band width broadens considerably. All four blue-absorbing dyes fluoresce intensely in the green region, and the two pyrene compounds emit at such long wavelengths without formation of excimers. The fluorescence properties of these compounds are quite environment-sensitive: considerable spectral shifts and fluorescence intensity changes have been observed in the pH range from 3 to 10 and in a wide variety of polar and hydrophobic solvents with vastly different dielectric constants. The high extinction and fluorescence quantum yield of these probes make them ideal fluorescent labeling reagents for proteins, antibodies, nucleic acids, and cellular organelles. The pH and hydrophobicity-dependent fluorescence changes can be utilized as optical pH and/or hydrophobicity indicators for mapping environmental difference in various cellular components in a single cell. Since all nine probes absorb in the UV, but emit at different wavelengths in the visible, these two groups of compounds offer an advantage of utilizing a single monochromatic light source (e.g., a nitrogen laser) to achieve multi-wavelength detection for flow cytometry application. As a first step to explore potential application in

  12. Fluorescence spectroscopy

    DEFF Research Database (Denmark)

    Bagatolli, Luis

    2016-01-01

    Fluorescence spectroscopy is a powerful experimental tool used by scientists from many disciplines. During the last decades there have been important developments on distinct fluorescence methods, particularly those related to the study of biological phenomena. This chapter discusses the foundati......Fluorescence spectroscopy is a powerful experimental tool used by scientists from many disciplines. During the last decades there have been important developments on distinct fluorescence methods, particularly those related to the study of biological phenomena. This chapter discusses...

  13. c-MYC amplification and c-myc protein expression in pancreatic acinar cell carcinomas. New insights into the molecular signature of these rare cancers.

    Science.gov (United States)

    La Rosa, Stefano; Bernasconi, Barbara; Vanoli, Alessandro; Sciarra, Amedeo; Notohara, Kenji; Albarello, Luca; Casnedi, Selenia; Billo, Paola; Zhang, Lizhi; Tibiletti, Maria Grazia; Sessa, Fausto

    2018-05-02

    The molecular alterations of pancreatic acinar cell carcinomas (ACCs) and mixed acinar-neuroendocrine carcinomas (MANECs) are not completely understood, and the possible role of c-MYC amplification in tumor development, progression, and prognosis is not known. We have investigated c-MYC gene amplification in a series of 35 ACCs and 4 MANECs to evaluate its frequency and a possible prognostic role. Gene amplification was investigated using interphasic fluorescence in situ hybridization analysis simultaneously hybridizing c-MYC and the centromere of chromosome 8 probes. Protein expression was immunohistochemically investigated using a specific monoclonal anti-c-myc antibody. Twenty cases had clones with different polysomies of chromosome 8 in absence of c-MYC amplification, and 5 cases had one amplified clone and other clones with chromosome 8 polysomy, while the remaining 14 cases were diploid for chromosome 8 and lacked c-MYC amplification. All MANECs showed c-MYC amplification and/or polysomy which were observed in 54% pure ACCs. Six cases (15.3%) showed nuclear immunoreactivity for c-myc, but only 4/39 cases showed simultaneous c-MYC amplification/polysomy and nuclear protein expression. c-myc immunoreactivity as well as c-MYC amplification and/or chromosome 8 polysomy was not statistically associated with prognosis. Our study demonstrates that a subset of ACCs shows c-MYC alterations including gene amplification and chromosome 8 polysomy. Although they are not associated with a different prognostic signature, the fact that these alterations are present in all MANECs suggests a role in the acinar-neuroendocrine differentiation possibly involved in the pathogenesis of MANECs.

  14. S100A10 protein expression is associated with oxaliplatin sensitivity in human colorectal cancer cells

    Directory of Open Access Journals (Sweden)

    Suzuki Sayo

    2011-12-01

    Full Text Available Abstract Background Individual responses to oxaliplatin (L-OHP-based chemotherapy remain unpredictable. The objective of our study was to find candidate protein markers for tumor sensitivity to L-OHP from intracellular proteins of human colorectal cancer (CRC cell lines. We performed expression difference mapping (EDM analysis of whole cell lysates from 11 human CRC cell lines with different sensitivities to L-OHP by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS, and identified a candidate protein by liquid chromatography/mass spectrometry ion trap time-of-flight (LCMS-IT-TOF. Results Of the qualified mass peaks obtained by EDM analysis, 41 proteins were differentially expressed in 11 human colorectal cancer cell lines. Among these proteins, the peak intensity of 11.1 kDa protein was strongly correlated with the L-OHP sensitivity (50% inhibitory concentrations (P R2 = 0.80. We identified this protein as Protein S100-A10 (S100A10 by MS/MS ion search using LCMS-IT-TOF. We verified its differential expression and the correlation between S100A10 protein expression levels in drug-untreated CRC cells and their L-OHP sensitivities by Western blot analyses. In addition, S100A10 protein expression levels were not correlated with sensitivity to 5-fluorouracil, suggesting that S100A10 is more specific to L-OHP than to 5-fluorouracil in CRC cells. S100A10 was detected in cell culture supernatant, suggesting secretion out of cells. Conclusions By proteomic approaches including SELDI technology, we have demonstrated that intracellular S100A10 protein expression levels in drug-untreated CRC cells differ according to cell lines and are significantly correlated with sensitivity of CRC cells to L-OHP exposure. Our findings provide a new clue to searching predictive markers of the response to L-OHP, suggesting that S100A10 is expected to be one of the candidate protein markers.

  15. The protein expression landscape of mitosis and meiosis in diploid budding yeast.

    Science.gov (United States)

    Becker, Emmanuelle; Com, Emmanuelle; Lavigne, Régis; Guilleux, Marie-Hélène; Evrard, Bertrand; Pineau, Charles; Primig, Michael

    2017-03-06

    Saccharomyces cerevisiae is an established model organism for the molecular analysis of fundamental biological processes. The genomes of numerous strains have been sequenced, and the transcriptome and proteome ofmajor phases during the haploid and diploid yeast life cycle have been determined. However, much less is known about dynamic changes of the proteome when cells switch from mitotic growth to meiotic development. We report a quantitative protein profiling analysis of yeast cell division and differentiation based on mass spectrometry. Information about protein levels was integrated with strand-specific tiling array expression data. We identified a total of 2366 proteins in at least one condition, including 175 proteins showing a statistically significant>5-fold change across the sample set, and 136 proteins detectable in sporulating but not respiring cells. We correlate protein expression patterns with biological processes and molecular function by Gene Ontology term enrichment, chemoprofiling, transcription interference and the formation of double stranded RNAs by overlapping sense/antisense transcripts. Our work provides initial quantitative insight into protein expression in diploid respiring and differentiating yeast cells. Critically, it associates developmentally regulated induction of antisense long noncoding RNAs and double stranded RNAs with fluctuating protein concentrations during growth and development. This integrated genomics analysis helps better understand how the transcriptome and the proteome correlate in diploid yeast cells undergoing mitotic growth in the presence of acetate (respiration) versus meiotic differentiation (Meiosis I and II). The study (i) provides quantitative expression data for 2366 proteins and their cognate mRNAs in at least one sample, (ii) shows strongly fluctuating protein levels during growth and differentiation for 175 cases, and (iii) identifies 136 proteins absent in mitotic but present in meiotic yeast cells. We

  16. Differences in protein expression among five species of stream stonefly (Plecoptera) along a latitudinal gradient in Japan.

    Science.gov (United States)

    Gamboa, Maribet; Tsuchiya, Maria Claret; Matsumoto, Suguru; Iwata, Hisato; Watanabe, Kozo

    2017-11-01

    Proteome variation among natural populations along an environmental gradient may provide insights into how the biological functions of species are related to their local adaptation. We investigated protein expression in five stream stonefly species from four geographic regions along a latitudinal gradient in Japan with varying climatic conditions. The extracted proteins were separated by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization of time-of-flight (MALDI TOF/TOF), yielding 446 proteins. Low interspecies variation in the proteome profiles was observed among five species within geographical regions, presumably due to the co-occurring species sharing the environments. However, large spatial variations in protein expression were found among four geographic regions, suggesting strong regulation of protein expression in heterogeneous environments, where the spatial variations were positively correlated with water temperature. We identified 21 unique proteins expressed specifically in a geographical region and six common proteins expressed throughout all regions. In warmer regions, metabolic proteins were upregulated, whereas proteins related to cold stress, the photoperiod, and mating were downregulated. Oxygen-related and energy-production proteins were upregulated in colder regions with higher altitudes. Thus, our proteomic approach is useful for identifying and understanding important biological functions related to local adaptations by populations of stoneflies. © 2017 Wiley Periodicals, Inc.

  17. Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits.

    Science.gov (United States)

    Freed, Emily F; Winkler, James D; Weiss, Sophie J; Garst, Andrew D; Mutalik, Vivek K; Arkin, Adam P; Knight, Rob; Gill, Ryan T

    2015-11-20

    The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions.

  18. p16(INK4a) promoter methylation and protein expression in breast fibroadenoma and carcinoma.

    Science.gov (United States)

    Di Vinci, Angela; Perdelli, Luisa; Banelli, Barbara; Salvi, Sandra; Casciano, Ida; Gelvi, Ilaria; Allemanni, Giorgio; Margallo, Edoardo; Gatteschi, Beatrice; Romani, Massimo

    2005-04-10

    The potential role of p16(INK4a) methylation in breast cancer is controversial whereas there are no data on fibroadenoma. To assess if inactivation of p16(INK4a) by promoter hypermethylation occurs in this hyperproliferative benign breast lesion or, on the contrary, it is strictly related to the carcinogenic process, we have tested the different histological components of 15 cases of fibroadenoma and the intraductal and infiltrating components of 15 cases of carcinoma and their adjacent non-tumoral epithelium. All samples were obtained by laser-assisted microdissection. The relationship between promoter methylation status, immunohistochemical protein expression and ki67 proliferative activity was evaluated for each lesion. Our data demonstrate that hypermethylation of p16(INK4a) promoter is a common event occurring at similar frequency in all the different histological areas of the benign and malignant breast lesions taken into exam. Conversely, protein p16 expression, although heterogeneously distributed within the section, is considerably higher in breast carcinoma as compared to fibroadenoma in both tumoral and non-tumoral epithelia and stroma. The protein localization was almost exclusively nuclear in fibroadenoma and non-tumoral epithelia whereas, in carcinoma, the staining was both nuclear and cytoplasmic or cytoplasmic alone. Furthermore, in a subset of fibroadenoma with higher proliferative activity, p16 protein expression was substantially decreased as compared to those showing lower proliferation. We did not observe this association in carcinomas. Our data demonstrate that the hypermethylation of the p16(INK4a) promoter is not specifically associated with malignancy and that, on the contrary, the overexpression of p16 and its cytoplasmic sequestration is a feature of breast carcinoma. (c) 2004 Wiley-Liss, Inc.

  19. HPV16 E6 regulates annexin 1 (ANXA1) protein expression in cervical carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Calmon, Marilia Freitas [Department of Biology, Institute of Bioscience, Language and Exact Science, São Paulo State University, São Jose do Rio Preto (Brazil); Sichero, Laura [Molecular Biology Laboratory, Centre for Translational Research in Oncology, Instituto do Câncer do Estado de São Paulo (ICESP), São Paulo (Brazil); Boccardo, Enrique [Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo., São Paulo (Brazil); Villa, Luisa Lina [Department of Radiology and Oncology, Faculdade de Medicina, Universidade de São Paulo, São Paulo (Brazil); Rahal, Paula, E-mail: rahalp@yahoo.com.br [Department of Biology, Institute of Bioscience, Language and Exact Science, São Paulo State University, São Jose do Rio Preto (Brazil)

    2016-09-15

    Annexin 1 (ANXA1) is a substrate for E6AP mediated ubiquitylation. It has been hypothesized that HPV 16 E6 protein redirects E6AP away from ANXA1, increasing its stability and possibly contributing to viral pathogenesis. We analyzed ANXA1 expression in HPV-positive and negative cervical carcinoma-derived cells, in cells expressing HPV-16 oncogenes and in cells transduced with shRNA targeting E6AP. We observed that ANXA1 protein expression increased in HPV-16-positive tumor cells, in keratinocytes expressing HPV-16 E6wt (wild-type) or E6/E7 and C33 cells expressing HPV-16 E6wt. ANXA1 protein expression decreased in cells transfected with E6 Dicer-substrate RNAs (DsiRNA) and C33 cells cotransduced with HPV-16 E6wt and E6AP shRNA. Moreover, colony number and proliferation rate decreased in HPV16-positive cells transduced with ANXA1 shRNA. We observed that in cells infected with HPV16, the E6 binds to E6AP to degrade p53 and upregulate ANXA1. We suggest that ANXA1 may play a role in HPV-mediated carcinogenesis. - Highlights: • ANXA1 upregulation requires the presence of E6 and E6AP and is dependent on E6 integrity. • E6 binds to E6AP to degrade p53 and upregulate ANXA1 in cells infected with HPV16. • ANXA1 plays a role in cell proliferation in HPV-positive cervical cells.

  20. HPV16 E6 regulates annexin 1 (ANXA1) protein expression in cervical carcinoma cell lines

    International Nuclear Information System (INIS)

    Calmon, Marilia Freitas; Sichero, Laura; Boccardo, Enrique; Villa, Luisa Lina; Rahal, Paula

    2016-01-01

    Annexin 1 (ANXA1) is a substrate for E6AP mediated ubiquitylation. It has been hypothesized that HPV 16 E6 protein redirects E6AP away from ANXA1, increasing its stability and possibly contributing to viral pathogenesis. We analyzed ANXA1 expression in HPV-positive and negative cervical carcinoma-derived cells, in cells expressing HPV-16 oncogenes and in cells transduced with shRNA targeting E6AP. We observed that ANXA1 protein expression increased in HPV-16-positive tumor cells, in keratinocytes expressing HPV-16 E6wt (wild-type) or E6/E7 and C33 cells expressing HPV-16 E6wt. ANXA1 protein expression decreased in cells transfected with E6 Dicer-substrate RNAs (DsiRNA) and C33 cells cotransduced with HPV-16 E6wt and E6AP shRNA. Moreover, colony number and proliferation rate decreased in HPV16-positive cells transduced with ANXA1 shRNA. We observed that in cells infected with HPV16, the E6 binds to E6AP to degrade p53 and upregulate ANXA1. We suggest that ANXA1 may play a role in HPV-mediated carcinogenesis. - Highlights: • ANXA1 upregulation requires the presence of E6 and E6AP and is dependent on E6 integrity. • E6 binds to E6AP to degrade p53 and upregulate ANXA1 in cells infected with HPV16. • ANXA1 plays a role in cell proliferation in HPV-positive cervical cells.

  1. Bilirubin Decreases Macrophage Cholesterol Efflux and ATP-Binding Cassette Transporter A1 Protein Expression.

    Science.gov (United States)

    Wang, Dongdong; Tosevska, Anela; Heiß, Elke H; Ladurner, Angela; Mölzer, Christine; Wallner, Marlies; Bulmer, Andrew; Wagner, Karl-Heinz; Dirsch, Verena M; Atanasov, Atanas G

    2017-04-28

    Mild but chronically elevated circulating unconjugated bilirubin is associated with reduced total and low-density lipoprotein cholesterol concentration, which is associated with reduced cardiovascular disease risk. We aimed to investigate whether unconjugated bilirubin influences macrophage cholesterol efflux, as a potential mechanism for the altered circulating lipoprotein concentrations observed in hyperbilirubinemic individuals. Cholesterol efflux from THP-1 macrophages was assessed using plasma obtained from normo- and hyperbilirubinemic (Gilbert syndrome) humans (n=60 per group) or (heterozygote/homozygote Gunn) rats (n=20 per group) as an acceptor. Hyperbilirubinemic plasma from patients with Gilbert syndrome and Gunn rats induced significantly reduced cholesterol efflux compared with normobilirubinemic plasma. Unconjugated bilirubin (3-17.1 μmol/L) exogenously added to plasma- or apolipoprotein A1-supplemented media also decreased macrophage cholesterol efflux in a concentration- and time-dependent manner. We also showed reduced protein expression of the ATP-binding cassette transporter A1 (ABCA1), a transmembrane cholesterol transporter involved in apolipoprotein A1-mediated cholesterol efflux, in THP-1 macrophages treated with unconjugated bilirubin and in peripheral blood mononuclear cells obtained from hyperbilirubinemic individuals. Furthermore, we demonstrated that bilirubin accelerates the degradation rate of the ABCA1 protein in THP-1 macrophages. Cholesterol efflux from THP-1 macrophages is decreased in the presence of plasma obtained from humans and rats with mild hyperbilirubinemia. A direct effect of unconjugated bilirubin on cholesterol efflux was demonstrated and is associated with decreased ABCA1 protein expression. These data improve our knowledge concerning bilirubin's impact on cholesterol transport and represent an important advancement in our understanding of bilirubin's role in cardiovascular disease. © 2017 The Authors. Published on

  2. Changes in HSP gene and protein expression in natural scrapie with brain damage

    Science.gov (United States)

    2011-01-01

    Heat shock proteins (Hsp) perform cytoprotective functions such as apoptosis regulation and inflammatory response control. These proteins can also be secreted to the extracellular medium, acting as inflammatory mediators, and their chaperone activity permits correct folding of proteins and avoids the aggregation of anomalous isoforms. Several studies have proposed the implication of Hsp in prion diseases. We analysed the gene expression and protein distribution of different members of the Hsp27, Hsp70, and Hsp90 families in the central nervous system of sheep naturally infected with scrapie. Different expression profiles were observed in the areas analysed. Whereas changes in transcript levels were not observed in the cerebellum or medulla oblongata, a significant decrease in HSP27 and HSP90 was detected in the prefrontal cortex. In contrast, HSP73 was over-expressed in diencephalons of scrapie animals. Western blotting did not reveal significant differences in Hsp90 and Hsp70 protein expression between scrapie and control animals. Expression rates identified by real-time RT-PCR and western blotting were compared with the extent of classical scrapie lesions using stepwise regression. Changes in Hsp gene and protein expression were associated with prion protein deposition, gliosis and spongiosis rather than with apoptosis. Finally, immunohistochemistry revealed intense Hsp70 and Hsp90 immunolabelling in Purkinje cells of scrapie sheep. In contrast, controls displayed little or no staining in these cells. The observed differences in gene expression and protein distribution suggest that the heat shock proteins analysed play a role in the natural form of the disease. PMID:21314976

  3. Changes in HSP gene and protein expression in natural scrapie with brain damage

    Directory of Open Access Journals (Sweden)

    Serrano Carmen

    2011-01-01

    Full Text Available Abstract Heat shock proteins (Hsp perform cytoprotective functions such as apoptosis regulation and inflammatory response control. These proteins can also be secreted to the extracellular medium, acting as inflammatory mediators, and their chaperone activity permits correct folding of proteins and avoids the aggregation of anomalous isoforms. Several studies have proposed the implication of Hsp in prion diseases. We analysed the gene expression and protein distribution of different members of the Hsp27, Hsp70, and Hsp90 families in the central nervous system of sheep naturally infected with scrapie. Different expression profiles were observed in the areas analysed. Whereas changes in transcript levels were not observed in the cerebellum or medulla oblongata, a significant decrease in HSP27 and HSP90 was detected in the prefrontal cortex. In contrast, HSP73 was over-expressed in diencephalons of scrapie animals. Western blotting did not reveal significant differences in Hsp90 and Hsp70 protein expression between scrapie and control animals. Expression rates identified by real-time RT-PCR and western blotting were compared with the extent of classical scrapie lesions using stepwise regression. Changes in Hsp gene and protein expression were associated with prion protein deposition, gliosis and spongiosis rather than with apoptosis. Finally, immunohistochemistry revealed intense Hsp70 and Hsp90 immunolabelling in Purkinje cells of scrapie sheep. In contrast, controls displayed little or no staining in these cells. The observed differences in gene expression and protein distribution suggest that the heat shock proteins analysed play a role in the natural form of the disease.

  4. Oxidant production and SOD1 protein expression in single skeletal myofibers from Down syndrome mice

    Directory of Open Access Journals (Sweden)

    Patrick M. Cowley

    2017-10-01

    Full Text Available Down syndrome (DS is a genetic condition caused by the triplication of chromosome 21. Persons with DS exhibit pronounced muscle weakness, which also occurs in the Ts65Dn mouse model of DS. Oxidative stress is thought to be an underlying factor in the development of DS-related pathologies including muscle dysfunction. High-levels of oxidative stress have been attributed to triplication and elevated expression of superoxide dismutase 1 (SOD1; a gene located on chromosome 21. The elevated expression of SOD1 is postulated to increase production of hydrogen peroxide and cause oxidative injury and cell death. However, it is unknown whether SOD1 protein expression is associated with greater oxidant production in skeletal muscle from Ts65Dn mice. Thus, our objective was to assess levels of SOD1 expression and oxidant production in skeletal myofibers from the flexor digitorum brevis obtained from Ts65Dn and control mice. Measurements of oxidant production were obtained from myofibers loaded with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH2-DA in the basal state and following 15 min of stimulated unloaded contraction. Ts65Dn myofibers exhibited a significant decrease in basal DCF emissions (p 0.05. Myofibers from Ts65Dn mice tended to be smaller and myonuclear domain was lower (p < 0.05. In summary, myofibers from Ts65Dn mice exhibited decreased basal DCF emissions that were coupled with elevated protein expression of SOD1. Stimulated contraction in isolated myofibers did not affect DCF emissions in either group. These findings suggest the skeletal muscle dysfunction in the adult Ts65Dn mouse is not associated with skeletal muscle oxidative stress.

  5. Fluorescent Protein Approaches in Alpha Herpesvirus Research

    Directory of Open Access Journals (Sweden)

    Ian B. Hogue

    2015-11-01

    Full Text Available In the nearly two decades since the popularization of green fluorescent protein (GFP, fluorescent protein-based methodologies have revolutionized molecular and cell biology, allowing us to literally see biological processes as never before. Naturally, this revolution has extended to virology in general, and to the study of alpha herpesviruses in particular. In this review, we provide a compendium of reported fluorescent protein fusions to herpes simplex virus 1 (HSV-1 and pseudorabies virus (PRV structural proteins, discuss the underappreciated challenges of fluorescent protein-based approaches in the context of a replicating virus, and describe general strategies and best practices for creating new fluorescent fusions. We compare fluorescent protein methods to alternative approaches, and review two instructive examples of the caveats associated with fluorescent protein fusions, including describing several improved fluorescent capsid fusions in PRV. Finally, we present our future perspectives on the types of powerful experiments these tools now offer.

  6. Green Synthesis of a Fluorescent Natural Product

    Science.gov (United States)

    Young, Douglas M.; Welker, Jacob J. C.; Doxsee, Kenneth M.

    2011-01-01

    Synthesis of 4-methylumbelliferone via the acid-catalyzed Pechmann condensation introduces students to several types of organic reactions: transesterification, electrophilic aromatic substitution, and alcohol dehydration. Performed with a recyclable, solid catalyst and under solvent-free conditions, the experiment illustrates many of the…

  7. Green Chemistry

    Energy Technology Data Exchange (ETDEWEB)

    Collison, Melanie

    2011-05-15

    Green chemistry is the science of chemistry used in a way that will not use or create hazardous substances. Dr. Rui Resendes is working in this field at GreenCentre Canada, an offshoot of PARTEQ Innovations in Kingston, Ontario. GreenCentre's preliminary findings suggest their licensed product {sup S}witchable Solutions{sup ,} featuring 3 classes of solvents and a surfactant, may be useful in bitumen oil sands extraction.

  8. Green roofs

    CSIR Research Space (South Africa)

    Van Wyk, Llewellyn V

    2010-04-01

    Full Text Available , beetles and spiders); and the number of birds that nest in vegetated roofs (including kestrels, swallows, and wagtails). Objective The primary objective of a green roof is to create a living habitat in an otherwise barren environment, hence the use... the negative environmental impacts including plant and insect specie loss. Thus at a philosophical level green roofs support the notion “replace what you displace”. Key ecological issues that can be addressed through green roofs include: Negative effects...

  9. Green thunderstorms

    Science.gov (United States)

    Gallagher, Frank Woolsey, III

    Many people around the world have observed green light apparently emanating from severe thunderstorms, but until recently there has been no scientific study of the phenomenon. Green thunderstorms have been observed from time to time in association with deep convection or severe weather events. Some skeptics who have not personally observed a green thunderstorm suggest that they are some kind of illusion. The existence of green thunderstorms has been objectively demonstrated by recording spectra of light from thunderstorms using a handheld spectrophotometer. During the spring and summer of 1995 and the spring of 1996 numerous storms were observed and spectra of the light emanating from these storms were recorded. Observations were made both at the ground and aboard research aircraft. Furthermore, time series of spectra were recorded as the observed color of some storms changed from dark blue to a bluish-green. Several hypotheses have been advanced to explain the occurrence of green light in connection with severe storms. Fankhauser gave some observational support to the belief that green light from thunderstorms is possible and believed that the source of the light is from the blue sky penetrating thin regions in the clouds. Fraser believes that light from the setting sun, in combination with the process of scattering by atmospheric molecules, creates the green light associated with severe weather and the thunderstorm acts only as a black backdrop. Unfortunately, no cloud illuminated by the sun is black and the green airlight produced by the Fraser theory is in reality overwhelmed by light reflected by the cloud. Often the unusual coloration has been attributed to hail or to reflection of light from foliage on the ground. The quantitative measurements made during the observation period fail to support these assumptions. We have observed thunderstorms to be green over ground that was not green and we have observed blue thunderstorms over ground that was green

  10. Enhanced fatty acid oxidation and FATP4 protein expression after endurance exercise training in human skeletal muscle

    DEFF Research Database (Denmark)

    Jeppesen, Jacob; Jordy, Andreas B; Sjøberg, Kim A

    2012-01-01

    ; however, it is not known whether this involves up-regulation of FATP1 and FATP4 protein. Therefore, the aim of this project was to investigate FATP1 and FATP4 protein expression in the vastus lateralis muscle from healthy human individuals and to what extent FATP1 and FATP4 protein expression were......FATP1 and FATP4 appear to be important for the cellular uptake and handling of long chain fatty acids (LCFA). These findings were obtained from loss- or gain of function models. However, reports on FATP1 and FATP4 in human skeletal muscle are limited. Aerobic training enhances lipid oxidation...

  11. Fully integrated high-speed intravascular optical coherence tomography/near-infrared fluorescence structural/molecular imaging in vivo using a clinically available near-infrared fluorescence-emitting indocyanine green to detect inflamed lipid-rich atheromata in coronary-sized vessels.

    Science.gov (United States)

    Lee, Sunki; Lee, Min Woo; Cho, Han Saem; Song, Joon Woo; Nam, Hyeong Soo; Oh, Dong Joo; Park, Kyeongsoon; Oh, Wang-Yuhl; Yoo, Hongki; Kim, Jin Won

    2014-08-01

    Lipid-rich inflamed coronary plaques are prone to rupture. The purpose of this study was to assess lipid-rich inflamed plaques in vivo using fully integrated high-speed optical coherence tomography (OCT)/near-infrared fluorescence (NIRF) molecular imaging with a Food and Drug Administration-approved indocyanine green (ICG). An integrated high-speed intravascular OCT/NIRF imaging catheter and a dual-modal OCT/NIRF system were constructed based on a clinical OCT platform. For imaging lipid-rich inflamed plaques, the Food and Drug Administration-approved NIRF-emitting ICG (2.25 mg/kg) or saline was injected intravenously into rabbit models with experimental atheromata induced by balloon injury and 12- to 14-week high-cholesterol diets. Twenty minutes after injection, in vivo OCT/NIRF imaging of the infrarenal aorta and iliac arteries was acquired only under contrast flushing through catheter (pullback speed up to ≤20 mm/s). NIRF signals were strongly detected in the OCT-visualized atheromata of the ICG-injected rabbits. The in vivo NIRF target-to-background ratio was significantly larger in the ICG-injected rabbits than in the saline-injected controls (Pfluorescence reflectance imaging, which correlated well with the in vivo target-to-background ratios (Pfluorescence microscopy, and histopathology also corroborated the in vivo imaging findings. Integrated OCT/NIRF structural/molecular imaging with a Food and Drug Administration -approved ICG accurately identified lipid-rich inflamed atheromata in coronary-sized vessels. This highly translatable dual-modal imaging approach could enhance our capabilities to detect high-risk coronary plaques. © 2014 American Heart Association, Inc.

  12. Behaviorally Green

    DEFF Research Database (Denmark)

    Sunstein, Cass; Reisch, Lucia A.

    2016-01-01

    of suggestion, inertia, and loss aversion. If well-chosen, green defaults are likely to have large effects in reducing the economic and environmental harms associated with various products and activities. Such defaults may or may not be more expensive to consumers. In deciding whether to establish green...

  13. Green Tea

    Science.gov (United States)

    ... and cancer. Green tea is consumed as a beverage. It is also sold in liquid extracts, capsules, and tablets and is sometimes used in topical products (intended to be applied to the skin). How Much Do We Know? Although many studies have been done on green tea and its ...

  14. Green consumerism

    DEFF Research Database (Denmark)

    de Groot, Judith I.M.; Schuitema, Geertje; Garson, Carrie Lee

    and biospheric values influence the importance of such ‘green’ product characteristics on purchasing intentions. In two within-subjects full-factorial experimental studies (N = 100 and N = 107), we found that purchase intentions of products were only steered by green characteristics if prices were low...... and the brand was familiar. Green product characteristics did not influence purchase intentions at all when these proself product characteristics were not fulfilled (i.e., high prices and unfamiliar brands). The importance of proself and green product characteristics on purchasing intentions was also......Our presentation will focus on the influence of product characteristics and values on green consumerism. Although generally a majority of consumers support the idea of purchasing green products, we argue, based on social dilemma theory, that proself product characteristics and egoistic...

  15. Green lights

    DEFF Research Database (Denmark)

    Fisker, Peter Kielberg

    This study investigates the effect of drought on economic activity globally using remote sensing data. In particular, predicted variation in greenness is correlated with changes in the density of artificial light observed at night on a grid of 0.25 degree latitude-longitude pixels. I define drought...... as greenness estimated by lagged variation in monthly rainfall and temperature. This definition of drought performs well in identifying self-reported drought events since 2000 compared with measures of drought that do not take greenness into account, and the subsequent analysis indicates that predicted...... variation in greenness is positively associated with year-on-year changes in luminosity: If a unit of observation experiences a predicted variation in greenness that lies 1 standard deviation below the global mean, on average 1.5 - 2.5 light pixels out of 900 are extinguished that year. Finally, an attempt...

  16. Evaluation of outer dense fiber-1 and -2 protein expression in asthenozoospermic infertile men

    Directory of Open Access Journals (Sweden)

    Silvia W. Lestari

    2015-07-01

    Full Text Available Background: Most of male infertility are caused by defect in sperm motility (asthenozoospermia. The molecular mechanism of low sperm motility in asthenozoospermic patients has not been fully understood. Sperm motility is strongly related to the axoneme structure which is composed of microtubules and supported by outer dense fiber (ODF and fibrous sheath (FS protein. The objective of this study was to characterize the ODF (ODF1 and ODF2 expression in asthenozoospermic infertile male and control normozoospermic fertile male.Methods: Asthenozoospermic samples (n=18 were collected from infertile patients at Andrology Lab, Cipto Mangunkusumo Hospital Jakarta and control were taken from normozoospermic fertile donor (n=18. After motility analyses by computer-assisted sperm analysis (CASA, semen were divided into two parts, for Western blot and for immunocytochemistry analysis. Antibody against ODF1 and ODF2 protein were used in both analyses.Results: Analysis of ODF1 protein expression showed bands with molecular weight of ~30 kDa and ODF2 ~85 kDa. The mean band intensity of ODF1 and ODF2 protein were lower in the asthenozoospermic group (AG compared to normozoospermic group (NG. Moreover, both ODF proteins were less intense and less localized in the AG than NG. Sperm motility was lower in AG, compared to control NG, i.e. average path velocity (VAP = 32.07 ± 7.03 vs 37.58 ± 8.73 µm/s, p = 0.455; straight line velocity (VSL = 24.17 ± 6.90 vs 27.61 ± 4.50 µm/s, p = 0.317 and curvilinear velocity (VCL = 45.68 ± 7.91 vs 55.55 ± 16.40 µm/s, p = 0.099.Conclusion: There is down-regulation of ODF1 and ODF2 protein expression and less-compact localization in AG sperm compared to the NG. These changes might have caused disturbances in the sperm motility as observed in this study.

  17. Calcium affecting protein expression in longan under simulated acid rain stress.

    Science.gov (United States)

    Pan, Tengfei; Li, Yongyu; Ma, Cuilan; Qiu, Dongliang

    2015-08-01

    Longan (Dimocarpus longana Lour. cv. Wulongling) of uniform one-aged seedlings grown in pots were selected to study specific proteins expressed in leaves under simulated acid rain (SiAR) stress and exogenous Ca(2+) regulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that there was a protein band specifically expressed under SiAR of pH 2.5 stress for 15 days with its molecular weight of about 23 kD. A 17 kD protein band specifically expressed after SiAR stress 5 days. Compared with pH 2.5, the pH 3.5 of SiAR made a less influence to protein expression. Two-dimensional electrophoresis (2-DE) results showed that six new specific proteins including C4 (20.2 kD pI 6.0), F (24 kD pI 6.35), B3 (22.3 kD pI 6.35), B4 (23.5 kD pI 6.5), C5 (21.8 kD pI 5.6), and C6 (20.2 kD pI 5.6) specifically expressed. C4 always expressed during SiAR stress. F expressed under the stress of pH 2.5 for 15 days and expressed in all pH SiAR stress for 20 days. The expression of proteins including B3, C5, and C6 was related to pH value and stress intensity of SiAR. The expression of B4 resulted from synergistic effects of SiAR and Ca. The expression of G1 (Mr 19.3 kD, pI 4.5), G2 (Mr 17.8 kD, pI 4.65), G3 (Mr 16.6 kD, pI 4.6), and G4 (Mr 14.7 kD, pI 4.4) enhanced under the treatment of 5 mM ethylene glycol tetraacetic acid (EGTA) and 2 mM chlorpromazine (CPZ). These proteins showed antagonistic effects and might be relative to the Ca-calmodulin (Ca-CaM) system of longan in response to SiAR stress.

  18. Correlation of histopathologic characteristics to protein expression and function in malignant melanoma.

    Directory of Open Access Journals (Sweden)

    Charlotte Welinder

    Full Text Available Metastatic melanoma is still one of the most prevalent skin cancers, which upon progression has neither a prognostic marker nor a specific and lasting treatment. Proteomic analysis is a versatile approach with high throughput data and results that can be used for characterizing tissue samples. However, such analysis is hampered by the complexity of the disease, heterogeneity of patients, tumors, and samples themselves. With the long term aim of quest for better diagnostics biomarkers, as well as predictive and prognostic markers, we focused on relating high resolution proteomics data to careful histopathological evaluation of the tumor samples and patient survival information.Regional lymph node metastases obtained from ten patients with metastatic melanoma (stage III were analyzed by histopathology and proteomics using mass spectrometry. Out of the ten patients, six had clinical follow-up data. The protein deep mining mass spectrometry data was related to the histopathology tumor tissue sections adjacent to the area used for deep-mining. Clinical follow-up data provided information on disease progression which could be linked to protein expression aiming to identify tissue-based specific protein markers for metastatic melanoma and prognostic factors for prediction of progression of stage III disease.In this feasibility study, several proteins were identified that positively correlated to tumor tissue content including IF6, ARF4, MUC18, UBC12, CSPG4, PCNA, PMEL and MAGD2. The study also identified MYC, HNF4A and TGFB1 as top upstream regulators correlating to tumor tissue content. Other proteins were inversely correlated to tumor tissue content, the most significant being; TENX, EHD2, ZA2G, AOC3, FETUA and THRB. A number of proteins were significantly related to clinical outcome, among these, HEXB, PKM and GPNMB stood out, as hallmarks of processes involved in progression from stage III to stage IV disease and poor survival.In this feasibility

  19. Correlation of histopathologic characteristics to protein expression and function in malignant melanoma

    Science.gov (United States)

    Pawłowski, Krzysztof; Szasz, A. Marcell; Yakovleva, Maria; Sugihara, Yutaka; Malm, Johan; Jönsson, Göran; Ingvar, Christian; Lundgren, Lotta; Baldetorp, Bo; Olsson, Håkan; Rezeli, Melinda; Laurell, Thomas; Wieslander, Elisabet; Marko-Varga, György

    2017-01-01

    Background Metastatic melanoma is still one of the most prevalent skin cancers, which upon progression has neither a prognostic marker nor a specific and lasting treatment. Proteomic analysis is a versatile approach with high throughput data and results that can be used for characterizing tissue samples. However, such analysis is hampered by the complexity of the disease, heterogeneity of patients, tumors, and samples themselves. With the long term aim of quest for better diagnostics biomarkers, as well as predictive and prognostic markers, we focused on relating high resolution proteomics data to careful histopathological evaluation of the tumor samples and patient survival information. Patients and methods Regional lymph node metastases obtained from ten patients with metastatic melanoma (stage III) were analyzed by histopathology and proteomics using mass spectrometry. Out of the ten patients, six had clinical follow-up data. The protein deep mining mass spectrometry data was related to the histopathology tumor tissue sections adjacent to the area used for deep-mining. Clinical follow-up data provided information on disease progression which could be linked to protein expression aiming to identify tissue-based specific protein markers for metastatic melanoma and prognostic factors for prediction of progression of stage III disease. Results In this feasibility study, several proteins were identified that positively correlated to tumor tissue content including IF6, ARF4, MUC18, UBC12, CSPG4, PCNA, PMEL and MAGD2. The study also identified MYC, HNF4A and TGFB1 as top upstream regulators correlating to tumor tissue content. Other proteins were inversely correlated to tumor tissue content, the most significant being; TENX, EHD2, ZA2G, AOC3, FETUA and THRB. A number of proteins were significantly related to clinical outcome, among these, HEXB, PKM and GPNMB stood out, as hallmarks of processes involved in progression from stage III to stage IV disease and poor

  20. A novel approach to sequence validating protein expression clones with automated decision making

    Directory of Open Access Journals (Sweden)

    Mohr Stephanie E

    2007-06-01

    Full Text Available Abstract Background Whereas the molecular assembly of protein expression clones is readily automated and routinely accomplished in high throughput, sequence verification of these clones is still largely performed manually, an arduous and time consuming process. The ultimate goal of validation is to determine if a given plasmid clone matches its reference sequence sufficiently to be "acceptable" for use in protein expression experiments. Given the accelerating increase in availability of tens of thousands of unverified clones, there is a strong demand for rapid, efficient and accurate software that automates clone validation. Results We have developed an Automated Clone Evaluation (ACE system – the first comprehensive, multi-platform, web-based plasmid sequence verification software package. ACE automates the clone verification process by defining each clone sequence as a list of multidimensional discrepancy objects, each describing a difference between the clone and its expected sequence including the resulting polypeptide consequences. To evaluate clones automatically, this list can be compared against user acceptance criteria that specify the allowable number of discrepancies of each type. This strategy allows users to re-evaluate the same set of clones against different acceptance criteria as needed for use in other experiments. ACE manages the entire sequence validation process including contig management, identifying and annotating discrepancies, determining if discrepancies correspond to polymorphisms and clone finishing. Designed to manage thousands of clones simultaneously, ACE maintains a relational database to store information about clones at various completion stages, project processing parameters and acceptance criteria. In a direct comparison, the automated analysis by ACE took less time and was more accurate than a manual analysis of a 93 gene clone set. Conclusion ACE was designed to facilitate high throughput clone sequence

  1. Study of p53 protein expression levels from irradiated peripheral blood lymphocytes for biodosimetry

    International Nuclear Information System (INIS)

    Cavalcanti, M.B.; Fernandes, T.S.; Melo, J.A.; Neves, M.A.B.; Machado, C.G.F

    2005-01-01

    Biodosimetry can be defined as the investigation of radioinduced biological effects in order to correlate them with the absorbed dose. Scoring of unstable chromosomal aberrations and micronuclei, from in vitro irradiated peripheral blood lymphocytes, is commonly used for biodosimetry based on cytogenetic analysis. However, this method of analysis is time-consuming, which may represent a pitfall when fast investigation of a possible exposure to ionizing radiation (IR) is needed. The interaction of IR with the living cell can cause injuries in the DNA molecules. However, normal cells possess mechanisms of repair that are capable to correct those damages. During the repair process of the DNA various proteins are expressed. Among these proteins, p53 plays an important role. This protein is a transcription factor that helps in the maintenance of the genomic integrity. p53 protein is found into the cytoplasm in reduced concentrations and has a short average life. However, expression of p53 protein can be induced by DNA harmful radioinduced, which increases the concentration and the average life of this protein, making possible its detection. Thus, the correlation between the increasing of p53 expression and the irradiation may constitute a fast and reliable method of individual monitoring in cases of accidental or suspected exposures to IR. In this context, the objective of this research was to evaluate the p53 protein expression levels from lymphocytes of the human peripheral blood after in vitro irradiation. For this, samples of peripheral blood from healthy individuals were irradiated with known doses. Lymphocytes were separated on ficoll gradient by centrifugation and re-suspended at 1x 10 6 /mL in RPMI medium enriched with fetal calf serum. Hence, lymphocytes were incubated in 5% CO 2 at 37 deg C prior to the methodology of flow cytometry, using intranuclear antigens for the quantification of p53. In this report, the methodology performed and the results obtained

  2. Study of p53 protein expression levels from irradiated peripheral blood lymphocytes for biodosimetry

    Energy Technology Data Exchange (ETDEWEB)

    Cavalcanti, M.B.; Fernandes, T.S. [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Dept. de Energia Nuclear; Amaral, A. [Universite Paris XII (UPXII) (France); Melo, J.A. [Centro de Radioterapia de Pernambuco (CERAPE), PE (Brazil); Neves, M.A.B.; Machado, C.G.F, E-mail: maribrayner@yahoo.com.br [Fundacao de Hematologia e Hemoterapia de Pernambuco, PE (Brazil)

    2005-07-01

    Biodosimetry can be defined as the investigation of radioinduced biological effects in order to correlate them with the absorbed dose. Scoring of unstable chromosomal aberrations and micronuclei, from in vitro irradiated peripheral blood lymphocytes, is commonly used for biodosimetry based on cytogenetic analysis. However, this method of analysis is time-consuming, which may represent a pitfall when fast investigation of a possible exposure to ionizing radiation (IR) is needed. The interaction of IR with the living cell can cause injuries in the DNA molecules. However, normal cells possess mechanisms of repair that are capable to correct those damages. During the repair process of the DNA various proteins are expressed. Among these proteins, p53 plays an important role. This protein is a transcription factor that helps in the maintenance of the genomic integrity. p53 protein is found into the cytoplasm in reduced concentrations and has a short average life. However, expression of p53 protein can be induced by DNA harmful radioinduced, which increases the concentration and the average life of this protein, making possible its detection. Thus, the correlation between the increasing of p53 expression and the irradiation may constitute a fast and reliable method of individual monitoring in cases of accidental or suspected exposures to IR. In this context, the objective of this research was to evaluate the p53 protein expression levels from lymphocytes of the human peripheral blood after in vitro irradiation. For this, samples of peripheral blood from healthy individuals were irradiated with known doses. Lymphocytes were separated on ficoll gradient by centrifugation and re-suspended at 1x 10{sub 6}/mL in RPMI medium enriched with fetal calf serum. Hence, lymphocytes were incubated in 5% CO{sub 2} at 37 deg C prior to the methodology of flow cytometry, using intranuclear antigens for the quantification of p53. In this report, the methodology performed and the results

  3. Fluorescent sensors based on bacterial fusion proteins

    International Nuclear Information System (INIS)

    Mateu, Batirtze Prats; Pum, Dietmar; Sleytr, Uwe B; Toca-Herrera, José L; Kainz, Birgit

    2014-01-01

    Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins. (paper)

  4. Overexpression of pucC improves the heterologous protein expression level in a Rhodobacter sphaeroides expression system.

    Science.gov (United States)

    Cheng, L; Chen, G; Ding, G; Zhao, Z; Dong, T; Hu, Z

    2015-04-27

    The Rhodobacter sphaeroides system has been used to express membrane proteins. However, its low yield has substantially limited its application. In order to promote the protein expression capability of this system, the pucC gene, which plays a crucial role in assembling the R. sphaeroides light-harvesting 2 complex (LH2), was overexpressed. To build a pucC overexpression strain, a pucC overexpression vector was constructed and transformed into R. sphaeroides CQU68. The overexpression efficiency was evaluated by quantitative real-time polymerase chain reaction. A well-used reporter β-glucuronidase (GUS) was fusion-expressed with LH2 to evaluate the heterologous protein expression level. As a result, the cell culture and protein in the pucC overexpression strain showed much higher typical spectral absorption peaks at 800 and 850 nm compared with the non-overexpression strain, suggesting a higher expression level of LH2-GUS fusion protein in the pucC overexpression strain. This result was further confirmed by Western blot, which also showed a much higher level of heterologous protein expression in the pucC overexpression strain. We further compared GUS activity in pucC overexpression and non-overexpression strains, the results of which showed that GUS activity in the pucC overexpression strain was approximately ten-fold that in the non-overexpression strain. These results demonstrate that overexpressed pucC can promote heterologous protein expression levels in R. sphaeroides.

  5. Comparison of Gene and Protein Expressions in Rats Residing in Standard Cages with Those Having Access to an Exercise Wheel

    Directory of Open Access Journals (Sweden)

    Helaine M. Alessio

    2014-01-01

    Full Text Available Lifelong physical inactivity is associated with morbidity in adulthood, possibly influenced by changes in gene and protein expressions occurring earlier in life. mRNA (Affymetrix gene array and proteomic (2D-DIGE MALDI-TOF/MS analyses were determined in cardiac tissue of young (3 months and old (16 months Sprague-Dawley rats housed with no access to physical activity (SED versus an exercise wheel (EX. Unfavorable phenotypes for body weight, dyslipidemia, and tumorogenesis appeared more often in adult SED versus EX. No differentially expressed genes (DEGs occurred between groups at 3 or 16 months. Within groups, SED and EX shared 215 age-associated DEGs. In SED, ten unique DEGs occurred with age; three had cell adhesion functions (fn1, lgals3, ncam2. In EX, five unique DEGs occurred with age; two involved hypothalamic, pituitary, and gonadal hormone axis (nrob2, xpnpep2. Protein expression involved in binding, sugar metabolic processes, and vascular regulation declined with age in SED (KNT1, ALBU, GPX1, PYGB, LDHB, G3P, PYGM, PGM1, ENOB. Protein expression increased with age in EX for ATP metabolic processes (MYH6, MYH7, ATP5J, ATPA and vascular function (KNT1, ALBU, GPX1. Differences in select gene and protein expressions within sedentary and active animals occurred with age and contributed to distinct health-related phenotypes in adulthood.

  6. Analyses of intricate kinetics of the serum proteome during and after colon surgery by protein expression time series

    NARCIS (Netherlands)

    Roelofsen, Johan; Alvarez Llamas, Gloria; Dijkstra, Martijn; Breitling, Rainer; Havenga, Klaas; Bijzet, Johannes; Zandbergen, Wouter; de Vries, Marcel; Ploeg, Rutger J.; Vonk, Roel J.

    Analyses of intricate kinetics of the serum proteome during and after colon surgery by protein expression time series.Roelofsen H, Alvarez-Llamas G, Dijkstra M, Breitling R, Havenga K, Bijzet J, Zandbergen W, de Vries MP, Ploeg RJ, Vonk RJ. Centre for Medical Biomics, University Medical Centre

  7. An estrogen-responsive plasma protein expression signature in Atlantic cod (Gadus morhua) revealed by SELDI-TOF MS

    DEFF Research Database (Denmark)

    Nielsen, Mari Mæland; Meyer, Sonnich; Larsen, Bodil Katrine

    2011-01-01

    Compound-specific protein expression signatures( PESs) can be revealed by proteomic techniques. The SELDI-TOF MS approach is advantageous due to its simplicity and high-throughput capacity,however, there are concerns regarding the reproducibility of this method. The aim of this study was to define...

  8. Chemokine (C-X-C) ligand 1 (CXCL1) protein expression is increased in aggressive bladder cancers

    International Nuclear Information System (INIS)

    Miyake, Makito; Lawton, Adrienne; Goodison, Steve; Urquidi, Virginia; Gomes-Giacoia, Evan; Zhang, Ge; Ross, Shanti; Kim, Jeongsoon; Rosser, Charles J

    2013-01-01

    Chemokines, including chemokine (C-X-C motif) ligand 1 (CXCL1), may regulate tumor epithelial-stromal interactions that facilitate tumor growth and invasion. Studies have linked CXCL1 expression to gastric, colon and skin cancers, but limited studies to date have described CXCL1 protein expression in human bladder cancer (BCa). CXCL1 protein expression was examined in 152 bladder tissue specimens (142 BCa) by immunohistochemical staining. The expression of CXCL1 was scored by assigning a combined score based on the proportion of cells staining and intensity of staining. CXCL1 expression patterns were correlated with clinicopathological features and follow-up data. CXCL1 protein expression was present in cancerous tissues, but was entirely absent in benign tissue. CXCL1 combined immunostaining score was significantly higher in high-grade tumors relative to low-grade tumors (p = 0.012). Similarly, CXCL1 combined immunostaining score was higher in high stage tumors (T2-T4) than in low stage tumors (Ta-T1) (p < 0.0001). An increase in the combined immunostaining score of CXCL1 was also associated with reduced disease-specific survival. To date, this is the largest study describing increased CXCL1 protein expression in more aggressive phenotypes in human BCa. Further studies are warranted to define the role CXCL1 plays in bladder carcinogenesis and progression

  9. Mechanistic target of rapamycin (MTOR) protein expression in the tumor and its microenvironment correlates with more aggressive pathology at cystectomy

    NARCIS (Netherlands)

    Winters, B.R. (Brian R.); Vakar-Lopez, F. (Funda); Brown, L. (Lisha); Montgomery, B. (Bruce); Seiler, R. (Roland); P.C. Black (Peter C.); J.L. Boormans (Joost); Dall′Era, M. (Marc); Davincioni, E. (Elai); Douglas, J. (James); Gibb, E.A. (Ewan A.); B.W. van Rhijn (Bas); M.S. van der Heijden (Michiel); Hsieh, A.C. (Andrew C.); Wright, J.L. (Jonathan L.); Lam, H.-M. (Hung-Ming)

    2018-01-01

    textabstractBackground: The mechanistic target of rapamycin (mTOR) has been implicated in driving tumor biology in multiple malignancies, including urothelial carcinoma (UC). We investigate how mTOR and phosphorylated mTOR (pmTOR) protein expression correlate with chemoresponsiveness in the tumor

  10. Concordant association validates MGMT methylation and protein expression as favorable prognostic factors in glioma patients on alkylating chemotherapy (Temozolomide).

    Science.gov (United States)

    Pandith, Arshad A; Qasim, Iqbal; Zahoor, Wani; Shah, Parveen; Bhat, Abdul R; Sanadhya, Dheera; Shah, Zafar A; Naikoo, Niyaz A

    2018-04-30

    O 6 -methylguanine-DNA methyltransferase (MGMT) promoter methylation and its subsequent loss of protein expression has been identified to have a variable impact on clinical outcome of glioma patients indicated for chemotherapy with alkylating agents (Temozolomide). This study investigated methylation status of MGMT gene along with in situ protein expression in malignant glioma patients of different histological types to evaluate the associated clinical outcome vis-a-vis use of alkylating drugs and radiotherapy. Sixty three cases of glioma were evaluated for MGMT promoter methylation by methylation-specific PCR (MS-PCR) and protein expression by immunostaining (IHC). Methylation status of MGMT and loss of protein expression showed a very high concordant association with better survival and progression free survival (PFS) (p < 0.0001). Multivariate Cox regression analysis showed both MGMT methylation and loss of protein as significant independent prognostic factors in glioma patients with respect to lower Hazard Ratio (HR) for better OS and PFS) [p < 0.05]. Interestingly concordant MGMT methylation and lack of protein showed better response in TMZ therapy treated patient subgroups with HR of 2.02 and 0.76 (p < 0.05). We found the merits of prognostication of MGMT parameters, methylation as well as loss of its protein as predictive factors for favorable outcome in terms of better survival for TMZ therapy.

  11. Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

    Science.gov (United States)

    Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

    2015-11-01

    Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1. © 2014 Wiley Periodicals, Inc.

  12. The use of fed batch approaches to maximise yields in bacterial fermentation and protein expression

    International Nuclear Information System (INIS)

    McLean, A.

    2001-01-01

    A fermentation facility for the scale up of bacterial and yeast fermentations has been set up at the University of Queensland under the auspices of the ARC Special Research Centre for Functional and Applied Genomics. A major application is the production of recombinant proteins for determination of tertiary structures by X-ray crystallography or nuclear magnetic resonance. For this purpose, large amounts of protein arc needed and the yield from a single fermentation run is crucial to success within constrained laboratory budgets. To achieve maximal yields we are optimising fed batch approaches in bacterial fermentation. Fed batch offers many advantages over batch cultures. Coupled with the ability to monitor online the internal conditions of the fermentation including pH and dissolved oxygen and stirrer cascading functions it is possible to ensure that the nutritional environment of the microorganism is optimised for its growth and or for optimal protein expression. The poster will describe some of our experience in setting up fed batch fermentations and successful applications of fed batches to increasing protein yield. It will also outline services that are available to academic groups outside the University of Queensland For structure determination and functional studies, the production of radiolabelled proteins can also be an advantage. We will describe initial experiments aimed at coupling the principles of fed batch fermentation to the introduction of carbon or nitrogen isotopes into the recombinant protein

  13. Benzo[a]pyrene treatment leads to changes in nuclear protein expression and alternative splicing

    Energy Technology Data Exchange (ETDEWEB)

    Yan Chunlan; Wu Wei [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Li Haiyan [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Huzhou Maternity and Child Care Hospital, Huzhou, Zhejiang 313000 (China); Zhang Guanglin [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Duerksen-Hughes, Penelope J. [Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA 92354 (United States); Zhu Xinqiang, E-mail: zhuxq@zju.edu.cn [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Yang Jun, E-mail: gastate@zju.edu.cn [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Zhejiang-California International Nanosystems Institute, Hangzhou, Zhejiang 310029 (China)

    2010-04-01

    Benzo[a]pyrene (BaP) is a potent pro-carcinogen generated from the combustion of fossil fuel and cigarette smoke. Previously, using a proteomic approach, we have shown that BaP can induce changes in the expression of many cellular proteins, including transcription regulators. In the present study, using a similar approach, we examined the nuclear protein response to BaP in HeLa cells and found that BaP treatment caused expression changes in many nuclear proteins. Twenty-four of these proteins were successfully identified, several of which are involved in the alternative splicing of mRNA, DNA replication, recombination, and repair. The changed expression levels were further confirmed by immunoblot analysis using specific antibodies for two proteins, Lamin A and mitotic checkpoint protein Bub3. The nuclear localization of these two proteins was also confirmed by confocal microscopy. To determine whether alternative splicing was activated following BaP treatment, we examined Fas and CD44, two genes previously shown to be targets of alternative splicing in respond to DNA damage. While no significant activation of alternative splicing was observed for Fas, CD44 splicing variants were found after BaP treatment. Together, these data show that DNA damage induces dramatic changes in nuclear protein expression, and that alternative splicing might be involved in the cellular response to DNA damage.

  14. Lipopolysaccharide regulated protein expression is only partly impaired in monocytes from patients with type I diabetes

    Directory of Open Access Journals (Sweden)

    Abke Sabine

    2006-03-01

    Full Text Available Abstract Background Monocytes play an important role in innate immunity and atherosclerosis. A disturbed secretion of cytokines in lipopolysaccharide (LPS activated monocytes from type 1 diabetes (T1D patients has been described and may contribute to the impaired inflammatory response in these individuals. In the present study the influence of LPS on five different proteins with a function in immunity and atherosclerosis was analyzed in monocytes from controls and T1D patients. Methods Monocytes were isolated from controls and T1D patients and the LPS-stimulated increase of IL-6, CXCL8, monocyte chemotactic protein 1 (CCL2, MCP-1 and superoxide dismutase (SOD 2, as well as the LPS-mediated decrease of apolipoprotein E (Apo E in primary human monocytes from controls and T1D patients was determined. Results CCL2 and IL-6 secretion in response to LPS was found significantly reduced in monocytes from T1D patients when compared to controls whereas basal CCL2 release was similar in control and T1D cells. In contrast, CXCL8 and apolipoprotein E secretion and SOD 2 expression upon LPS stimulation is similar from T1D and control monocytes. Conclusion These data indicate that LPS-mediated protein expression is only partly disturbed in monocytes from T1D patients. Reduced secretion of IL-6 and CCL2 in activated monocytes of these patients may contribute to an impaired inflammatory response and vascular disease.

  15. Machine learning in computational biology to accelerate high-throughput protein expression.

    Science.gov (United States)

    Sastry, Anand; Monk, Jonathan; Tegel, Hanna; Uhlen, Mathias; Palsson, Bernhard O; Rockberg, Johan; Brunk, Elizabeth

    2017-08-15

    The Human Protein Atlas (HPA) enables the simultaneous characterization of thousands of proteins across various tissues to pinpoint their spatial location in the human body. This has been achieved through transcriptomics and high-throughput immunohistochemistry-based approaches, where over 40 000 unique human protein fragments have been expressed in E. coli. These datasets enable quantitative tracking of entire cellular proteomes and present new avenues for understanding molecular-level properties influencing expression and solubility. Combining computational biology and machine learning identifies protein properties that hinder the HPA high-throughput antibody production pipeline. We predict protein expression and solubility with accuracies of 70% and 80%, respectively, based on a subset of key properties (aromaticity, hydropathy and isoelectric point). We guide the selection of protein fragments based on these characteristics to optimize high-throughput experimentation. We present the machine learning workflow as a series of IPython notebooks hosted on GitHub (https://github.com/SBRG/Protein_ML). The workflow can be used as a template for analysis of further expression and solubility datasets. ebrunk@ucsd.edu or johanr@biotech.kth.se. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  16. Effect of crude oil petroleum hydrocarbons on protein expression of the prawn Macrobrachium borellii.

    Science.gov (United States)

    Pasquevich, M Y; Dreon, M S; Gutierrez Rivera, J N; Vázquez Boucard, C; Heras, H

    2013-05-01

    Hydrocarbon pollution is a major environmental threat to ecosystems in marine and freshwater environments, but its toxicological effect on aquatic organisms remains little studied. A proteomic approach was used to analyze the effect of a freshwater oil spill on the prawn Macrobrachium borellii. To this aim, proteins were extracted from midgut gland (hepatopancreas) of male and female prawns exposed 7 days to a sublethal concentration (0.6 ppm) of water-soluble fraction of crude oil (WSF). Exposure to WSF induced responses at the protein expression level. Two-dimensional gel electrophoresis (2-DE) revealed 10 protein spots that were differentially expressed by WSF exposure. Seven proteins were identified using MS/MS and de novo sequencing. Nm23 oncoprotein, arginine methyltransferase, fatty aldehyde dehydrogenase and glutathione S-transferase were down-regulated, whereas two glyceraldehyde-3-phosphate dehydrogenase isoforms and a lipocalin-like crustacyanin (CTC) were up-regulated after WSF exposure. CTC mRNA levels were further analyzed by quantitative real-time PCR showing an increased expression after WSF exposure. The proteins identified are involved in carbohydrate and amino acid metabolism, detoxification, transport of hydrophobic molecules and cellular homeostasis among others. These results provide evidence for better understanding the toxic mechanisms of hydrocarbons. Moreover, some of these differentially expressed proteins would be employed as potential novel biomarkers. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica

    Directory of Open Access Journals (Sweden)

    Singh Upinder

    2009-02-01

    Full Text Available Abstract Background Entamoeba histolytica is an intestinal protozoan parasite of humans. The genome has been sequenced, but the study of individual gene products has been hampered by the lack of the ability to generate gene knockouts. We chose to test the use of RNA interference to knock down gene expression in Entamoeba histolytica. Results An episomal vector-based system, using the E. histolytica U6 promoter to drive expression of 29-basepair short hairpin RNAs, was developed to target protein-encoding genes in E. histolytica. The short hairpin RNAs successfully knocked down protein levels of all three unrelated genes tested with this system: Igl, the intermediate subunit of the galactose- and N-acetyl-D-galactosamine-inhibitable lectin; the transcription factor URE3-BP; and the membrane binding protein EhC2A. Igl levels were reduced by 72%, URE3-BP by 89%, and EhC2A by 97%. Conclusion Use of the U6 promoter to drive expression of 29-basepair short hairpin RNAs is effective at knocking down protein expression for unrelated genes in Entamoeba histolytica, providing a useful tool for the study of this parasite.

  18. Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica.

    Science.gov (United States)

    Linford, Alicia S; Moreno, Heriberto; Good, Katelyn R; Zhang, Hanbang; Singh, Upinder; Petri, William A

    2009-02-17

    Entamoeba histolytica is an intestinal protozoan parasite of humans. The genome has been sequenced, but the study of individual gene products has been hampered by the lack of the ability to generate gene knockouts. We chose to test the use of RNA interference to knock down gene expression in Entamoeba histolytica. An episomal vector-based system, using the E. histolytica U6 promoter to drive expression of 29-basepair short hairpin RNAs, was developed to target protein-encoding genes in E. histolytica. The short hairpin RNAs successfully knocked down protein levels of all three unrelated genes tested with this system: Igl, the intermediate subunit of the galactose- and N-acetyl-D-galactosamine-inhibitable lectin; the transcription factor URE3-BP; and the membrane binding protein EhC2A. Igl levels were reduced by 72%, URE3-BP by 89%, and EhC2A by 97%. Use of the U6 promoter to drive expression of 29-basepair short hairpin RNAs is effective at knocking down protein expression for unrelated genes in Entamoeba histolytica, providing a useful tool for the study of this parasite.

  19. Synthetic Promoters and Transcription Factors for Heterologous Protein Expression in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Fabian Machens

    2017-10-01

    Full Text Available Orthogonal systems for heterologous protein expression as well as for the engineering of synthetic gene regulatory circuits in hosts like Saccharomyces cerevisiae depend on synthetic transcription factors (synTFs and corresponding cis-regulatory binding sites. We have constructed and characterized a set of synTFs based on either transcription activator-like effectors or CRISPR/Cas9, and corresponding small synthetic promoters (synPs with minimal sequence identity to the host’s endogenous promoters. The resulting collection of functional synTF/synP pairs confers very low background expression under uninduced conditions, while expression output upon induction of the various synTFs covers a wide range and reaches induction factors of up to 400. The broad spectrum of expression strengths that is achieved will be useful for various experimental setups, e.g., the transcriptional balancing of expression levels within heterologous pathways or the construction of artificial regulatory networks. Furthermore, our analyses reveal simple rules that enable the tuning of synTF expression output, thereby allowing easy modification of a given synTF/synP pair. This will make it easier for researchers to construct tailored transcriptional control systems.

  20. Time-dependent changes in protein expression in rainbow trout muscle following hypoxia.

    Science.gov (United States)

    Wulff, Tune; Jokumsen, Alfred; Højrup, Peter; Jessen, Flemming

    2012-04-18

    Adaptation to hypoxia is a complex process, and individual proteins will be up- or down-regulated in order to address the main challenges at any given time. To investigate the dynamics of the adaptation, rainbow trout (Oncorhynchus mykiss) was exposed to 30% of normal oxygen tension for 1, 2, 5 and 24 h respectively, after which muscle samples were taken. The successful investigation of numerous proteins in a single study was achieved by selectively separating the sarcoplasmic proteins using 2-DE. In total 46 protein spots were identified as changing in abundance in response to hypoxia using one-way ANOVA and multivariate data analysis. Proteins of interest were subsequently identified by MS/MS following tryptic digestion. The observed regulation following hypoxia in skeletal muscle was determined to be time specific, as only a limited number of proteins were regulated in response to more than one time point. The cellular response to hypoxia included regulation of proteins involved in maintaining iron homeostasis, energy levels and muscle structure. In conclusion, this proteome-based study presents a comprehensive investigation of the expression profiles of numerous proteins at four different time points. This increases our understanding of timed changes in protein expression in rainbow trout muscle following hypoxia. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. p53 protein expression in corneal squamous cell carcinomas of dogs

    Directory of Open Access Journals (Sweden)

    Lucas Bahdour Cossi

    2015-06-01

    Full Text Available Ocular tumors play an increasing concern in veterinary ophthalmology. Corneal squamous cell carcinoma is unfrequent in dogs, and by this way it has little studies. Studies that investigated the carcinogenesis mechanisms wich could help to the development of ocular squamous cell carcinoma (SCC in dog are rare. The aim of this work was to identify by immunohistochemical techniques, the p53 protein expression in the spontaneous dog corneal SCC. For this work, were used five cases of corneal SCC and one case of actinic keratitis. The sections were obtained from paraffin-wax blocks and submitted to histopathological and immunohistochemical analysis. All the six samples showed immunolabeling to cytokeratin and p53 protein. These results support the conclusions that the immunoreactivity of p53 protein by immunohistochemistry is present in canine corneal SCC suppporting its role in carcinogenesis of this tumor, but not provides prognostic indicators in cases of SCC corneal in dog; and can be a association of exposure to solar radiation with the possible mutation of the TP53 gene.

  2. Long interspersed element-1 protein expression is a hallmark of many human cancers.

    Science.gov (United States)

    Rodić, Nemanja; Sharma, Reema; Sharma, Rajni; Zampella, John; Dai, Lixin; Taylor, Martin S; Hruban, Ralph H; Iacobuzio-Donahue, Christine A; Maitra, Anirban; Torbenson, Michael S; Goggins, Michael; Shih, Ie-Ming; Duffield, Amy S; Montgomery, Elizabeth A; Gabrielson, Edward; Netto, George J; Lotan, Tamara L; De Marzo, Angelo M; Westra, William; Binder, Zev A; Orr, Brent A; Gallia, Gary L; Eberhart, Charles G; Boeke, Jef D; Harris, Chris R; Burns, Kathleen H

    2014-05-01

    Cancers comprise a heterogeneous group of human diseases. Unifying characteristics include unchecked abilities of tumor cells to proliferate and spread anatomically, and the presence of clonal advantageous genetic changes. However, universal and highly specific tumor markers are unknown. Herein, we report widespread long interspersed element-1 (LINE-1) repeat expression in human cancers. We show that nearly half of all human cancers are immunoreactive for a LINE-1-encoded protein. LINE-1 protein expression is a common feature of many