scholarly journals A functional chimaeric S-layer-enhanced green fluorescent protein to follow the uptake of S-layer-coated liposomes into eukaryotic cells

2004 ◽  
Vol 379 (2) ◽  
pp. 441-448 ◽  
Author(s):  
Nicola ILK ◽  
Seta KÜPCÜ ◽  
Gerald MONCAYO ◽  
Sigrid KLIMT ◽  
Rupert C. ECKER ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Hela Cells ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Laser Scanning Microscopy ◽  
Host Cells ◽  
Confocal Laser ◽  
Green Fluorescent ◽  
Egfp Fluorescence

The chimaeric gene encoding a C-terminally truncated form of the S-layer protein SbpA of Bacillus sphaericus CCM 2177 and the EGFP (enhanced green fluorescent protein) was ligated into plasmid pET28a and cloned and expressed in Escherichia coli. Just 1 h after induction of expression an intense EGFP fluorescence was detected in the cytoplasm of the host cells. Expression at 28 °C instead of 37 °C resulted in clearly increased fluorescence intensity, indicating that the folding process of the EGFP moiety was temperature sensitive. To maintain the EGFP fluorescence, isolation of the fusion protein from the host cells had to be performed in the presence of reducing agents. SDS/PAGE analysis, immunoblotting and N-terminal sequencing of the isolated and purified fusion protein confirmed the presence of both the S-layer protein and the EGFP moiety. The fusion protein had maintained the ability to self-assemble in suspension and to recrystallize on peptidoglycan-containing sacculi or on positively charged liposomes, as well as to fluoresce. Comparison of fluorescence excitation and emission spectra of recombinant EGFP and rSbpA31-1068/EGFP revealed identical maxima at 488 and 507 nm respectively. The uptake of liposomes coated with a fluorescent monomolecular protein lattice of rSbpA31-1068/EGFP into HeLa cells was studied by confocal laser-scanning microscopy. The major part of the liposomes was internalized within 2 h of incubation and entered the HeLa cells by endocytosis.

scholarly journals Functional Expression and Subcellular Localization of the Aflatoxin Pathway Enzyme Ver-1 Fused to Enhanced Green Fluorescent Protein

2008 ◽  
Vol 74 (20) ◽  
pp. 6385-6396 ◽  
Author(s):  
Sung-Yong Hong ◽  
John E. Linz
Keyword(s):  
Green Fluorescent Protein ◽  
Subcellular Localization ◽  
Fusion Proteins ◽  
Laser Scanning ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Substrate Surface ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Green Fluorescent

ABSTRACT Aflatoxin, a mycotoxin synthesized by Aspergillus spp., is among the most potent naturally occurring carcinogens known. Little is known about the subcellular organization of aflatoxin synthesis. Previously, we used transmission electron microscopy and immunogold labeling to demonstrate that the late aflatoxin enzyme OmtA localizes primarily to vacuoles in fungal cells on the substrate surface of colonies. In the present work, we monitored subcellular localization of Ver-1 in real time in living cells. Aspergillus parasiticus strain CS10-N2 was transformed with plasmid constructs that express enhanced green fluorescent protein (EGFP) fused to Ver-1. Analysis of transformants demonstrated that EGFP fused to Ver-1 at either the N or C terminus functionally complemented nonfunctional Ver-1 in recipient cells. Western blot analysis detected predominantly full-length Ver-1 fusion proteins in transformants. Confocal laser scanning microscopy demonstrated that Ver-1 fusion proteins localized in the cytoplasm and in the lumen of up to 80% of the vacuoles in hyphae grown for 48 h on solid media. Control EGFP (no Ver-1) expressed in transformants was observed in only 13% of the vacuoles at this time. These data support a model in which middle and late aflatoxin enzymes are synthesized in the cytoplasm and transported to vacuoles, where they participate in aflatoxin synthesis.

Postharvest Handling Conditions Affect Internalization of Salmonella in Baby Spinach during Washing

2013 ◽  
Vol 76 (7) ◽  
pp. 1145-1151 ◽  
Author(s):  
VICENTE M. GÓMEZ-LÓPEZ ◽  
ALICIA MARÍN ◽  
ANA ALLENDE ◽  
LARRY R. BEUCHAT ◽  
MARÍA I. GIL
Keyword(s):  
Green Fluorescent Protein ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Negative Temperature ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Temperature Differential ◽  
Postharvest Handling ◽  
Green Fluorescent ◽  
Spinach Leaves

Internalization of foodborne pathogens in fruits and vegetables is an increasing safety concern. The aim of this research was to assess the potential for internalization of an enteric pathogen (Salmonella enterica serotype Typhimurium) in a leafy vegetable (baby spinach) during washing as influenced by three postharvest handling conditions: (i) illumination, (ii) negative temperature differential, and (iii) relative humidity (RH). To compare these potential postharvest handling conditions, leaves were exposed to different levels of illumination (0, 1,000, and 2,000 lx), temperature differential (5, 11, 14, 20, and 26uC), and RH (99, 85, and 74%) for a short time before or during washing. Washing of baby spinach was carried out in water containing green fluorescent protein–tagged Salmonella Typhimurium (6.5 log CFU/ml) at 5uC for 2 min, followed by surface disinfection with chlorine (10,000 μg/ml) for 1 min, two rinses in water for 10 s, and spin drying for 15 s. Internalization was assessed by enumerating the pathogen on Salmonella-Shigella agar and by confocal laser scanning microscopy. Illumination of spinach leaves before and during washing and a negative temperature differential during washing did not significantly (P > 0.05) increase the number of internalized bacteria. However, exposure of leaves to low-RH conditions before washing, which reduced the tissue water content, decreased internalization of Salmonella compared with internalization in baby spinach exposed to high RH (P ≤ 0.05). Green fluorescent protein–tagged Salmonella Typhimurium was visualized by confocal laser scanning microscopy at a depth of up to 30 μm beneath the surface of spinach leaves after exposure to a high inoculum level (8 log CFU/ml) for an extended time (2 h). Results show that internalization of Salmonella into baby spinach leaves can occur but can be minimized under specific postharvest handling conditions such as low RH.

Application to immunoassays of the fusion protein between protein ZZ and enhanced green fluorescent protein

2006 ◽  
Vol 309 (1-2) ◽  
pp. 130-138 ◽  
Author(s):  
Qi-Lai Huang ◽  
Cheng Chen ◽  
Yun-Zi Chen ◽  
Chen-Guang Gong ◽  
Lin Cao ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent

Migration of Salmonella Enteritidis Phage Type 30 through Almond Hulls and Shells

2008 ◽  
Vol 71 (2) ◽  
pp. 397-401 ◽  
Author(s):  
MICHELLE D. DANYLUK ◽  
MARIA T. BRANDL ◽  
LINDA J. HARRIS
Keyword(s):  
Green Fluorescent Protein ◽  
Laser Scanning ◽  
Salmonella Enteritidis ◽  
Fluorescent Protein ◽  
Phage Type ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Aqueous Environment ◽  
Serovar Typhimurium ◽  
Green Fluorescent

The ability of Salmonella to migrate from an external aqueous environment through the almond hull and shell, and to colonize the kernel, was evaluated in two ways. First, the outer surface of shell halves from five varieties of almonds that differed in shell hardness were placed in contact with a suspension of Salmonella enterica serovar Enteritidis phage type 30 for 24hat24°C. Salmonella Enteritidis was isolated from the inside of these almond shells in 46 and 100% of the samples, by direct swabbing of the inner surface of the shell and by enrichment from the swab, respectively. These findings suggested that hardness of the shell is not a significant factor in the migration of the pathogen through that tissue. In addition, both motile and nonmotile strains of S. enterica serovar Typhimurium migrated through the almond shells to the same extent under the conditions of this assay, indicating that bacterial migration through the wet shell may be a passive process. Second, whole almonds (intact hull, shell, and kernel) were soaked for 24 to 72 h at 24°C in a suspension of Salmonella Enteritidis phage type 30 labeled with the green fluorescent protein. Green fluorescent protein–labeled Salmonella cells were observed on the outer and inner surfaces of both the almond hull and shell, and on the kernel, by confocal laser scanning microscopy. Our data provide direct evidence that wet conditions allow for Salmonella migration through the hull and shell and onto the almond kernel, thus providing a means by which almond kernels may become contaminated in the field.

scholarly journals Systemic Colonization of Potato Plants by a Soilborne, Green Fluorescent Protein-Tagged Strain of Dickeya sp. Biovar 3

2010 ◽  
Vol 100 (2) ◽  
pp. 134-142 ◽  
Author(s):  
Robert Czajkowski ◽  
Waldo J. de Boer ◽  
Henk Velvis ◽  
Jan M. van der Wolf
Keyword(s):  
Green Fluorescent Protein ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Agar Medium ◽  
Lateral Roots ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Potato Plants ◽  
Soil Inoculation ◽  
Green Fluorescent

Colonization of potato plants by soilborne, green fluorescent protein (GFP)-tagged Dickeya sp. IPO2254 was investigated by selective plating, epifluorescence stereo microscopy (ESM), and confocal laser scanning microscopy (CLSM). Replicated experiments were carried out in a greenhouse using plants with an intact root system and plants from which ca. 30% of the lateral roots was removed. One day after soil inoculation, adherence of the pathogen on the roots and the internal colonization of the plants were detected using ESM and CLSM of plant parts embedded in an agar medium. Fifteen days post-soil inoculation, Dickeya sp. was found on average inside 42% of the roots, 13% of the stems, and 13% of the stolons in plants with undamaged roots. At the same time-point, in plants with damaged roots, Dickeya sp. was found inside 50% of the roots, 25% of the stems, and 25% of the stolons. Thirty days postinoculation, some plants showed true blackleg symptoms. In roots, Dickeya sp. was detected in parenchyma cells of the cortex, both inter- and intracellularly. In stems, bacteria were found in xylem vessels and in protoxylem cells. Microscopical observations were confirmed by dilution spread-plating the plant extracts onto agar medium directly after harvest. The implications of infection from soilborne inoculum are discussed.

scholarly journals Fluorescently tagged canine adenovirus via modification with protein IX–enhanced green fluorescent protein

2005 ◽  
Vol 86 (12) ◽  
pp. 3201-3208 ◽  
Author(s):  
Long P. Le ◽  
Jing Li ◽  
Vladimir V. Ternovoi ◽  
Gene P. Siegal ◽  
David T. Curiel
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Neutralizing Antibodies ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Adenovirus Type ◽  
Canine Model ◽  
Heterologous Proteins ◽  
Green Fluorescent ◽  
Protein Ix

Canine adenovirus type 2 (CAV2) has become an attractive vector for gene therapy because of its non-pathogenicity and the lack of pre-existing neutralizing antibodies against this virus in the human population. Additionally, this vector has been proposed as a conditionally replicative adenovirus agent under the control of an osteocalcin promoter for evaluation in a syngeneic, immunocompetent canine model with spontaneous osteosarcoma. In this study, a CAV2 vector labelled with the fluorescent capsid fusion protein IX–enhanced green fluorescent protein (pIX–EGFP) was developed. Expression of the fluorescent fusion-protein label in infected cells with proper nuclear localization, and incorporation into virions, could be detected. The labelled virions could be visualized by fluorescence microscopy; this was applicable to the tracking of CAV2 infection, as well as localizing the distribution of the vector in tissues. Expression of pIX–EGFP could be exploited to detect the replication and spread of CAV2. These results indicate that pIX can serve as a platform for incorporation of heterologous proteins in the context of a canine adenovirus xenotype. It is believed that capsid-labelled CAV2 has utility for vector-development studies and for monitoring CAV2-based oncolytic adenovirus replication.

CXCR2 Inverse Agonism Detected by Arrestin Redistribution

2009 ◽  
Vol 14 (9) ◽  
pp. 1076-1091 ◽  
Author(s):  
Simone Kredel ◽  
Michael Wolff ◽  
Jörg Wiedenmann ◽  
Barbara Moepps ◽  
G. Ulrich Nienhaus ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Large Degree ◽  
Distribution Patterns ◽  
Coated Pits ◽  
Inverse Agonism ◽  
Green Fluorescent

To study CXCR2 modulated arrestin redistribution, the authors employed arrestin as a fusion protein containing either the Aequorea victoria—derived enhanced green fluorescent protein (EGFP) or a recently developed mutant of eqFP611, a red fluorescent protein derived from Entacmaea quadricolor. This mutant, referred to as RFP611, had earlier been found to assume a dimeric quarternary structure. It was therefore employed in this work as a “tandem” (td) construct for pseudo monomeric fusion protein labeling. Both arrestin fusion proteins, containing either td RFP611 (Arr td RFP611) or enhanced green fluorescent protein (EGFP; Arr EGFP), were found to colocalize with internalized fluorescently labeled Gro α a few minutes after Gro α addition. Intriguingly, however, Arr td RFP611 and Arr EGFP displayed distinct cellular distribution patterns in the absence of any CXCR2 activating ligand. Under these conditions, Arr td RFP611 showed a largely homoge neous cytosolic distribution, whereas Arr EGFP segregated, to a large degree, into granular spots. These observations indi cate a higher sensitivity of Arr EGFP to the constitutive activity of CXCR2 and, accordingly, an increased arrestin redistribution to coated pits and endocytic vesicles. In support of this interpretation, the authors found the known CXCR2 antagonist Sch527123 to act as an inverse agonist with respect to Arr EGFP redistribution. The inverse agonistic properties of Sch527123 were confirmed in vitro in a guanine nucleotide binding assay, revealing an IC50 value similar to that observed for Arr EGFP redistribution. Thus, the redistribution assay, when based on Arr EGFP, enables the profiling of antagonistic test compounds with respect to inverse agonism. When based on Arr td RFP611, the assay may be employed to study CXCR2 agonism or neutral antagonism. ( Journal of Biomolecular Screening 2009:1076 1091)

scholarly journals Green Fluorescent Protein-Marked Pseudomonas fluorescens: Localization, Viability, and Activity in the Natural Barley Rhizosphere

1999 ◽  
Vol 65 (10) ◽  
pp. 4646-4651 ◽  
Author(s):  
Bo Normander ◽  
Niels B. Hendriksen ◽  
Ole Nybroe
Keyword(s):  
Green Fluorescent Protein ◽  
Pseudomonas Fluorescens ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Fluorescence ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Protein Fluorescence ◽  
Dividing Cells ◽  
Green Fluorescent

ABSTRACT The gfp-tagged Pseudomonas fluorescensbiocontrol strain DR54-BN14 was introduced into the barley rhizosphere. Confocal laser scanning microscopy revealed that the rhizoplane populations of DR54-BN14 on 3- to 14-day-old roots were able to form microcolonies closely associated with the indigenous bacteria and that a majority of DR54-BN14 cells appeared small and almost coccoid. Information on the viability of the inoculant was provided by a microcolony assay, while measurements of cell volume, the intensity of green fluorescent protein fluorescence, and the ratio of dividing cells to total cells were used as indicators of cellular activity. At a soil moisture close to the water-holding capacity of the soil, the activity parameters suggested that the majority of DR54-BN14 cells were starving in the rhizosphere. Nevertheless, approximately 80% of the population was either culturable or viable but nonculturable during the 3-week incubation period. No impact of root decay on viability was observed, and differences in viability or activity among DR54-BN14 cells located in different regions of the root were not apparent. In dry soil, however, the nonviable state of DR54-BN14 was predominant, suggesting that desiccation is an important abiotic regulator of cell viability.

scholarly journals Spitzenkörper Localization and Intracellular Traffic of Green Fluorescent Protein-Labeled CHS-3 and CHS-6 Chitin Synthases in Living Hyphae of Neurospora crassa

2007 ◽  
Vol 6 (10) ◽  
pp. 1853-1864 ◽  
Author(s):  
Meritxell Riquelme ◽  
Salomon Bartnicki-García ◽  
Juan Manuel González-Prieto ◽  
Eddy Sánchez-León ◽  
Jorge A. Verdín-Ramos ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Neurospora Crassa ◽  
Laser Scanning ◽  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Golgi Body ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Chitin Synthases ◽  
Green Fluorescent

ABSTRACT The subcellular location and traffic of two selected chitin synthases (CHS) from Neurospora crassa, CHS-3 and CHS-6, labeled with green fluorescent protein (GFP), were studied by high-resolution confocal laser scanning microscopy. While we found some differences in the overall distribution patterns and appearances of CHS-3-GFP and CHS-6-GFP, most features were similar and were observed consistently. At the hyphal apex, fluorescence congregated into a conspicuous single body corresponding to the location of the Spitzenkörper (Spk). In distal regions (beyond 40 μm from the apex), CHS-GFP revealed a network of large endomembranous compartments that was predominantly comprised of irregular tubular shapes, while some compartments were distinctly spherical. In the distal subapex (20 to 40 μm from the apex), fluorescence was observed in globular bodies that appeared to disintegrate into vesicles as they advanced forward until reaching the proximal subapex (5 to 20 μm from the apex). CHS-GFP was also conspicuously found delineating developing septa. Analysis of fluorescence recovery after photobleaching suggested that the fluorescence of the Spk originated from the advancing population of microvesicles (chitosomes) in the subapex. The inability of brefeldin A to interfere with the traffic of CHS-containing microvesicles and the lack of colocalization of CHS-GFP with the endoplasmic reticulum (ER)-Golgi body fluorescent dyes lend support to the idea that CHS proteins are delivered to the cell surface via an alternative route distinct from the classical ER-Golgi body secretory pathway.

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