BODIPY Conjugate of Epibrassinolide as a Novel Biologically Active Probe for In Vivo Imaging

Brassinosteroids (BRs) are plant hormones of steroid nature, regulating various developmental and adaptive processes. The perception, transport, and signaling of BRs are actively studied nowadays via a wide range of biochemical and genetic tools. However, most of the knowledge about BRs intracellular localization and turnover relies on the visualization of the receptors or cellular compartments using dyes or fluorescent protein fusions. We have previously synthesized a conjugate of epibrassinolide with green fluorescent dye BODIPY (eBL-BODIPY). Here we present a detailed assessment of the compound bioactivity and its suitability as probe for in vivo visualization of BRs. We show that eBL-BODIPY rapidly penetrates epidermal cells of Arabidopsis thaliana roots and after long exposure causes physiological and transcriptomic responses similar to the natural hormone.

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Intracellular localization and in vivo trafficking of p24A and p23

Journal of Cell Science ◽

10.1242/jcs.112.4.537 ◽

1999 ◽

Vol 112 (4) ◽

pp. 537-548 ◽

Cited By ~ 10

Author(s):

R. Blum ◽

F. Pfeiffer ◽

P. Feick ◽

W. Nastainczyk ◽

B. Kohler ◽

...

Keyword(s):

Fluorescent Protein ◽

Intracellular Localization ◽

Maximum Speed ◽

Heterotrimeric G Proteins ◽

P24 Protein ◽

Intermediate Compartment ◽

Copi Vesicle ◽

Green Fluorescent ◽

Golgi Network

Recently, p24A and p23 (also termed Tmp21), two members of the p24 protein family, have been proposed to function as integral receptors for the COPI-vesicle coat. This study describes the intracellular localization and trafficking of p24A in comparison to p23. For immunolocalization of p24A and p23, strong reduction and denaturation conditions were necessary to allow antibody interaction. Both p24A and p23 cycle continuously between intermediate compartment (IC) elements and the cis-Golgi network. In vivo trafficking of p24A and p23 tagged to green fluorescent protein (GFP) revealed that both proteins travel by large (up to 1 micrometer in length) microtubule-dependent pre-Golgi carriers with a maximum speed of up to 1.6 micrometer s-1 from the IC to the Golgi cisternae. Aluminum fluoride, a general activator of heterotrimeric G-proteins, blocked peripheral pre-Golgi movements of GFP-p24A/p23 and inhibited fluorescence recovery after photobleaching in the perinuclear Golgi area. p24A and p23 are predominantly colocalized. Overexpression of GFP-p24A, to an extent which did not destroy the Golgi complex, induced delocalization of part of the proteins into ER elements. This study therefore gives new insights into the localization and trafficking behavior of the two COPI-binding proteins p24A and p23.

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Phosphorylation and/or Presence of Serine 37 in the Movement Protein of Tomato Mosaic Tobamovirus Is Essential for Intracellular Localization and Stability In Vivo

Journal of Virology ◽

10.1128/jvi.73.8.6831-6840.1999 ◽

1999 ◽

Vol 73 (8) ◽

pp. 6831-6840 ◽

Cited By ~ 54

Author(s):

Shigeki Kawakami ◽

Hal S. Padgett ◽

Daijiro Hosokawa ◽

Yoshimi Okada ◽

Roger N. Beachy ◽

...

Keyword(s):

Fluorescent Protein ◽

Movement Protein ◽

Intracellular Localization ◽

Distinct Pattern ◽

Mutant Virus ◽

Wild Type ◽

Tomato Plants ◽

The Stability ◽

Green Fluorescent

ABSTRACT The P30 movement protein (MP) of tomato mosaic tobamovirus (ToMV) is synthesized in the early stages of infection and is phosphorylated in vivo. Here, we determined that serine 37 and serine 238 in the ToMV MP are sites of phosphorylation. MP mutants in which serine was replaced by alanine at positions 37 and 238 (LQ37A238A) or at position 37 only (LQ37A) were not phosphorylated, and mutant viruses did not infect tobacco or tomato plants. By contrast, mutation of serine 238 to alanine did not affect the infectivity of the virus (LQ238A). To investigate the subcellular localization of mutant MPs, we constructed viruses that expressed each mutant MP fused with the green fluorescent protein (GFP) of Aequorea victoria. Wild-type and mutant LQ238A MP fusion proteins showed distinct temporally regulated patterns of MP-GFP localization in protoplasts and formation of fluorescent ring-shaped infection sites on Nicotiana benthamiana. However mutant virus LQ37A MP-GFP did not show a distinct pattern of localization or formation of fluorescent rings. Pulse-chase experiments revealed that MP produced by mutant virus LQ37A was less stable than wild-type and LQ238A MPs. MP which contained threonine at position 37 was phosphorylated, but the stability of the MP in vivo was very low. These studies suggest that the presence of serine at position 37 or phosphorylation of serine 37 is essential for intracellular localization and stability of the MP, which is necessary for the protein to function.

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Visualizing Herpesvirus Procapsids in Living Cells

Journal of Virology ◽

10.1128/jvi.01437-16 ◽

2016 ◽

Vol 90 (22) ◽

pp. 10182-10192 ◽

Cited By ~ 5

Author(s):

Oana Maier ◽

Patricia J. Sollars ◽

Gary E. Pickard ◽

Gregory A. Smith

Keyword(s):

Fluorescent Protein ◽

Living Cells ◽

Capsid Assembly ◽

Wild Type ◽

Capsid Shell ◽

Wide Range ◽

Green Fluorescent ◽

Simplex Virus

ABSTRACTA complete understanding of herpesvirus morphogenesis requires studies of capsid assembly dynamics in living cells. Although fluorescent tags fused to the VP26 and pUL25 capsid proteins are available, neither of these components is present on the initial capsid assembly, the procapsid. To make procapsids accessible to live-cell imaging, we made a series of recombinant pseudorabies viruses that encoded green fluorescent protein (GFP) fused in frame to the internal capsid scaffold and maturation protease. One recombinant, a GFP-VP24 fusion, maintained wild-type propagation kineticsin vitroand approximated wild-type virulencein vivo. The fusion also proved to be well tolerated in herpes simplex virus. Viruses encoding GFP-VP24, along with a traditional capsid reporter fusion (pUL25/mCherry), demonstrated that GFP-VP24 was a reliable capsid marker and revealed that the protein remained capsid associated following entry into cells and upon nuclear docking. These dual-fluorescent viruses made possible the discrimination of procapsids during infection and monitoring of capsid shell maturation kinetics. The results demonstrate the feasibility of imaging herpesvirus procapsids and their morphogenesis in living cells and indicate that the encapsidation machinery does not substantially help coordinate capsid shell maturation.IMPORTANCEThe familyHerpesviridaeconsists of human and veterinary pathogens that cause a wide range of diseases in their respective hosts. These viruses share structurally related icosahedral capsids that encase the double-stranded DNA (dsDNA) viral genome. The dynamics of capsid assembly and maturation have been inaccessible to examination in living cells. This study has overcome this technical hurdle and provides new insights into this fundamental stage of herpesvirus infection.

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Sensitive Bioassay for Detection of Biologically Active Ricin in Food

Journal of Food Protection ◽

10.4315/0362-028x.jfp-11-448 ◽

2012 ◽

Vol 75 (5) ◽

pp. 951-954 ◽

Cited By ~ 14

Author(s):

REUVEN RASOOLY ◽

XIAOHUA HE

Keyword(s):

Large Scale ◽

Fluorescent Protein ◽

Vero Cells ◽

Biologically Active ◽

Green Fluorescent Protein Gene ◽

Mouse Bioassay ◽

Human Embryonic Kidney Cells ◽

Green Fluorescent ◽

And Control

The potential use of ricin as an agent of biological warfare highlights the need to develop fast and effective methods to detect biologically active ricin. The current “gold standard” for ricin detection is an in vivo mouse bioassay; however, this method is not practical to test on a large number of samples and raises ethical concerns with regard to the use of experimental animals. In this work, we generated adenoviral vectors that express the green fluorescent protein gene and used the relative fluorescence units intensity inhibition by transduced cells for quantitative measurement of biologically active ricin. The detection limit of the assay was 200 pg/ml, which is over 500,000 times greater than the adult human lethal oral dose. The inhibition of fluorescence intensity between ricin treatment and control was higher in 72-h posttransduction Vero cells than 24-h human embryonic kidney cells. Therefore, to detect biologically active ricin in food matrices that might influence the assay, we used 72-h posttransduction Vero cells. This simple assay could be used for large-scale screening to detect biologically active ricin in food without added substrates or use of cell fixation methods.

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Role of bacteriophages in STEC infections: new implications for the design of prophylactic and treatment approaches

F1000Research ◽

10.12688/f1000research.3718.1 ◽

2014 ◽

Vol 3 ◽

pp. 74 ◽

Cited By ~ 7

Author(s):

Jaime H. Amorim ◽

Monica R. Jesus ◽

Wilson B. Luiz ◽

Bruna F.M.M. Porchia ◽

Rita C.C. Ferreira ◽

...

Keyword(s):

Shiga Toxin ◽

Fluorescent Protein ◽

Imaging System ◽

Lytic Replication ◽

Biologically Active ◽

Gene Encoding ◽

E Coli ◽

Green Fluorescent

Shiga toxin (Stx) is considered the main virulence factor in Shiga toxin-producing Escherichia coli (STEC) infections. Previously we reported the expression of biologically active Stx by eukaryotic cells in vitro and in vivo following transfection with plasmids encoding Stx under control of the native bacterial promoter. Since stx genes are present in the genome of lysogenic bacteriophages, here we evaluated the relevance of bacteriophages during STEC infection. We used the non-pathogenic E. coli K12 strain carrying a lysogenic 933W mutant bacteriophage in which the stx operon was replaced by a gene encoding the green fluorescent protein (GFP). Tracking GFP expression using an In Vivo Imaging System (IVIS), we detected fluorescence in liver, kidney, and intestine of mice infected with the recombinant E. coli strain after treatment with ciprofloxacin, which induces the lytic replication and release of bacteriophages. In addition, we showed that chitosan, a linear polysaccharide composed of D-glucosamine residues and with a number of commercial and biomedical uses, had strong anti-bacteriophage effects, as demonstrated in vitro and in vivo. These findings bring promising perspectives for the prevention and treatment of hemolytic uremic syndrome (HUS) cases.

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Phytochemical, Analytical and Medicinal Studies of Holoptelea integrifolia Roxb. Planch - A Review

Current Traditional Medicine ◽

10.2174/2215083805666190521103308 ◽

2019 ◽

Vol 5 (4) ◽

pp. 270-277 ◽

Cited By ~ 2

Author(s):

Vijay Kumar ◽

Simranjeet Singh ◽

Ragini Bhadouria ◽

Ravindra Singh ◽

Om Prakash

Keyword(s):

Liquid Chromatography ◽

Thin Layer ◽

Thin Layer Chromatography ◽

High Performance ◽

Biologically Active ◽

Toxicological Studies ◽

Wide Range ◽

Layer Chromatography

Holoptelea integrifolia Roxb. Planch (HI) has been used to treat various ailments including obesity, osteoarthritis, arthritis, inflammation, anemia, diabetes etc. To review the major phytochemicals and medicinal properties of HI, exhaustive bibliographic research was designed by means of various scientific search engines and databases. Only 12 phytochemicals have been reported including biologically active compounds like betulin, betulinic acid, epifriedlin, octacosanol, Friedlin, Holoptelin-A and Holoptelin-B. Analytical methods including the Thin Layer Chromatography (TLC), High-Performance Thin Layer Chromatography (HPTLC), High-Performance Liquid Chromatography (HPLC) and Liquid Chromatography With Mass Spectral (LC-MS) analysis have been used to analyze the HI. From medicinal potency point of view, these phytochemicals have a wide range of pharmacological activities such as antioxidant, antibacterial, anti-inflammatory, and anti-tumor. In the current review, it has been noticed that the mechanism of action of HI with biomolecules has not been fully explored. Pharmacology and toxicological studies are very few. This seems a huge literature gap to be fulfilled through the detailed in-vivo and in-vitro studies.

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Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

Biochemical Journal ◽

10.1042/bj3390299 ◽

1999 ◽

Vol 339 (2) ◽

pp. 299-307 ◽

Cited By ~ 33

Author(s):

Arthur L. KRUCKEBERG ◽

Ling YE ◽

Jan A. BERDEN ◽

Karel van DAM

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Green Fluorescent Protein ◽

Glucose Transport ◽

Cellular Localization ◽

Fluorescent Protein ◽

Hexose Transporter ◽

High Concentrations ◽

Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

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Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

Microorganisms ◽

10.3390/microorganisms9020379 ◽

2021 ◽

Vol 9 (2) ◽

pp. 379

Author(s):

Breanne M. Head ◽

Christopher I. Graham ◽

Teassa MacMartin ◽

Yoav Keynan ◽

Ann Karen C. Brassinga

Keyword(s):

Growth Kinetics ◽

Legionella Pneumophila ◽

Fluorescent Protein ◽

Disease Incidence ◽

Acanthamoeba Castellanii ◽

Infection Model ◽

Intracellular Infection ◽

Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

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Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

The Plant Cell ◽

10.1093/plcell/koab122 ◽

2021 ◽

Author(s):

Noemi Ruiz-Lopez ◽

Jessica Pérez-Sancho ◽

Alicia Esteban del Valle ◽

Richard P Haslam ◽

Steffen Vanneste ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Abiotic Stress ◽

Fluorescent Protein ◽

Mechanical Damage ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact ◽

Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

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CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

Veterinary Research ◽

10.1186/s13567-021-00964-4 ◽

2021 ◽

Vol 52 (1) ◽

Author(s):

Peng-Fei Fu ◽

Xuan Cheng ◽

Bing-Qian Su ◽

Li-Fang Duan ◽

Cong-Rong Wang ◽

...

Keyword(s):

Pseudorabies Virus ◽

Fluorescent Protein ◽

Antiviral Agents ◽

Firefly Luciferase ◽

Inhibitory Effect ◽

Economic Losses ◽

Green Fluorescent ◽

Swine Herds

AbstractPseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

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