Bax-inhibiting peptides derived from Ku70 and cell-penetrating pentapeptides

2007 ◽  
Vol 35 (4) ◽  
pp. 797-801 ◽  
Author(s):  
J.A. Gomez ◽  
V. Gama ◽  
T. Yoshida ◽  
W. Sun ◽  
P. Hayes ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Protein A ◽  
Cellular Damage ◽  
Toxic Drug ◽  
Cell Penetrating ◽  
Low Toxicity ◽  
Repair Factor ◽  
Green Fluorescent

We found that Ku70, a known DNA repair factor, has a novel function to bind and inhibit Bax (Bcl-2-associated X protein), a key mediator of apoptosis. Pentapeptides derived from the Bax-binding domain of Ku70 were cell-permeable and protected cells from Bax-mediated apoptosis. These pentapeptides were called BIPs (Bax-inhibiting peptides). BIPs may become a useful therapeutic tool to reduce cellular damage. We also generated BIP mutant pentapeptides that do not inhibit Bax, but retain their cell-penetrating activity. Since both BIPs and BIP mutants are cell-permeable, these peptides were designated CPP5s (cell-penetrating pentapeptides). Among the CPP5s discovered, VPTLK (BIP) and KLPVM (BIP mutant) were confirmed to possess protein transduction activity by examination of the delivery of GFP (green fluorescent protein) into cells by these peptides. The mechanism of cell penetration by CPP5s is not known. CPP5s enter the cell at 0 and 4°C. In preliminary studies, various inhibitors of endocytosis and pinocytosis did not show any significant suppression of CPP5 cell entry. CPP5s have very low toxicity in vitro and in vivo and so may be useful tools in order to develop non-toxic drug-delivery technologies.

Development and validation of fourLeishmaniaspecies constitutively expressing GFP protein. A model for drug discovery and disease pathogenesis studies

Parasitology ◽  
2013 ◽  
Vol 141 (4) ◽  
pp. 501-510 ◽  
Author(s):  
ASHA PARBHU PATEL ◽  
ANDREW DEACON ◽  
GIULIA GETTI
Keyword(s):  
Leishmania Major ◽  
Fluorescent Protein ◽  
Protein A ◽  
Host Cells ◽  
Mammalian Host ◽  
Disease Pathogenesis ◽  
Compound Libraries ◽  
Green Fluorescent

SUMMARYGreen fluorescent protein (GFP)-parasite transfectants have been widely used as a tool for studying disease pathogenesis in several protozoan models and their application in drug screening assays has increased rapidly. In the past decade, the expression of GFP has been established in severalLeishmaniaspecies, mostly forin vitrostudies. The current work reports generation of four transgenic parasites constitutively expressing GFP (Leishmania mexicana, Leishmania aethiopica, Leishmania tropicaandLeishmania major) and their validation as a representative model of infection. This is the first report where stable expression of GFP has been achieved inL. aethiopicaandL. tropica. Integration of GFP was accomplished through homologous recombination of the expression construct, pRib1.2αNEOαGFP downstream of the 18S rRNA promoter in all species. A homogeneous and high level expression of GFP was detected in both the promastigote and the intracellular amastigote stages. All transgenic species showed the same growth pattern, ability to infect mammalian host cells and sensitivity to reference drugs as their wild type counterparts. All four transgenicLeishmaniaare confirmed as models forin vitroand possiblyin vivoinfections and represent an ideal tool for medium throughput testing of compound libraries.

scholarly journals Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

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.

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

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.

scholarly journals The Great Capacity on Promoting Melanogenesis of Three Compatible Components in Vernonia anthelmintica (L.) Willd.

2021 ◽  
Vol 22 (8) ◽  
pp. 4073
Author(s):  
Yifan Lai ◽  
Qingyuan Feng ◽  
Rui Zhang ◽  
Jing Shang ◽  
Hui Zhong
Keyword(s):  
Herbal Medicine ◽  
Caffeic Acid ◽  
Fluorescent Protein ◽  
Traditional Chinese Herbal Medicine ◽  
Expression Of Genes ◽  
Proliferation And Differentiation ◽  
Green Fluorescent ◽  
Vernonia Anthelmintica

To investigate a possible methodology of exploiting herbal medicine and design polytherapy for the treatment of skin depigmentation disorder, we have made use of Vernonia anthelmintica (L.) Willd., a traditional Chinese herbal medicine that has been proven to be effective in treating vitiligo. Here, we report that the extract of Vernonia anthelmintica (L.) Willd. effectively enhances melanogenesis responses in B16F10. In its compound library, we found three ingredients (butin, caffeic acid and luteolin) also have the activity of promoting melanogenesis in vivo and in vitro. They can reduce the accumulation of ROS induced by hydrogen peroxide and inflammatory response induced by sublethal concentrations of copper sulfate in wild type and green fluorescent protein (GFP)-labeled leukocytes zebrafish larvae. The overall objective of the present study aims to identify which compatibility proportions of the medicines may be more effective in promoting pigmentation. We utilized the D-optimal response surface methodology to optimize the ratio among three molecules. Combining three indicators of promoting melanogenesis, anti-inflammatory and antioxidant capacities, we get the best effect of butin, caffeic acid and luteolin at the ratio (butin:caffeic acid:luteolin = 7.38:28.30:64.32) on zebrafish. Moreover, the effect of melanin content recovery in the best combination is stronger than that of the monomer, which suggests that the three compounds have a synergistic effect on inducing melanogenesis. After simply verifying the result, we performed in situ hybridization on whole-mount zebrafish embryos to further explore the effects of multi-drugs combination on the proliferation and differentiation of melanocytes and the expression of genes (tyr, mitfa, dct, kit) related to melanin synthesis. In conclusion, the above three compatible compounds can significantly enhance melanogenesis and improve depigmentation in vivo.

scholarly journals Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain

2021 ◽  
Vol 30 ◽  
pp. 096368972097821
Author(s):  
Andrea Tenorio-Mina ◽  
Daniel Cortés ◽  
Joel Esquivel-Estudillo ◽  
Adolfo López-Ornelas ◽  
Alejandro Cabrera-Wrooman ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Neural Induction ◽  
Human Keratinocytes ◽  
Differentiation Potential ◽  
Neuronal Proteins ◽  
Green Fluorescent

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins βIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

scholarly journals 20-HETE-mediated cytotoxicity and apoptosis in ischemic kidney epithelial cells

2008 ◽  
Vol 294 (3) ◽  
pp. F562-F570 ◽  
Author(s):  
Vani Nilakantan ◽  
Cheryl Maenpaa ◽  
Guangfu Jia ◽  
Richard J. Roman ◽  
Frank Park
Keyword(s):  
Caspase 3 ◽  
Fluorescent Protein ◽  
Ischemia Reperfusion ◽  
Atp Depletion ◽  
Cellular Damage ◽  
Apoptotic Pathway ◽  
Injury Model ◽  
Green Fluorescent

20-HETE, a metabolite of arachidonic acid, has been implicated as a mediator of free radical formation and tissue death following ischemia-reperfusion (IR) injury in the brain and heart. The present study examined the role of this pathway in a simulated IR renal injury model in vitro. Modified self-inactivating lentiviral vectors were generated to stably overexpress murine Cyp4a12 following transduction into LLC-PK1 cells (LLC-Cyp4a12). We compared the survival of control and transduced LLC-PK1 cells following 4 h of ATP depletion and 2 h of recovery in serum-free medium. ATP depletion-recovery of LLC-Cyp4a12 cells resulted in a significantly higher LDH release ( P < 0.05) compared with LLC-enhanced green fluorescent protein (EGFP) cells. Treatment with the SOD mimetic MnTMPyP (100 μM) resulted in decreased cytotoxicity in LLC-Cyp4a12 cells. The selective 20-HETE inhibitor HET-0016 (10 μM) also inhibited cytotoxicity significantly ( P < 0.05) in LLC-Cyp4a12 cells. Dihydroethidium fluorescence showed that superoxide levels were increased to the same degree in LLC-EGFP and LLC-Cyp4a12 cells after ATP depletion-recovery compared with control cells and that this increase was inhibited by MnTMPyP. There was a significant increase ( P < 0.05) of caspase-3 cleavage, an effector protease of the apoptotic pathway, in the LLC-Cyp4a12 vs. LLC-EGFP cells ( P < 0.05). This was abolished in the presence of HET-0016 ( P < 0.05) or MnTMPyP ( P < 0.01). These results demonstrate that 20-HETE overexpression can significantly exacerbate the cellular damage that is associated with renal IR injury and that the programmed cell death is mediated by activation of caspase-3 and is partially dependent on enhanced CYP4A generation of free radicals.

Atelocollagen Sponge as a Stem Cell Implantation Scaffold for the Treatment of Scarred Vocal Folds

2010 ◽  
Vol 119 (11) ◽  
pp. 805-810 ◽  
Author(s):  
Satoshi Ohno ◽  
Shigeru Hirano ◽  
Ichiro Tateya ◽  
Shin-Ichi Kanemaru ◽  
Hiroo Umeda ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cells ◽  
Fluorescent Protein ◽  
In Vitro Study ◽  
Vocal Folds ◽  
Green Fluorescent ◽  
Cell Implantation

Objectives: Treatment of vocal fold scarring remains a therapeutic challenge. Our group previously reported the efficacy of treating injured vocal folds by implantation of bone marrow—derived stromal cells containing mesenchymal stem cells. Appropriate scaffolding is necessary for the stem cell implant to achieve optimal results. Terudermis is an atelocollagen sponge derived from calf dermis. It has large pores that permit cellular entry and is degraded in vivo. These characteristics suggest that this material may be a good candidate for use as scaffolding for implantation of cells. The present in vitro study investigated the feasibility of using Terudermis as such a scaffold. Methods: Bone marrow—derived stromal cells were obtained from GFP (green fluorescent protein) mouse femurs. The cells were seeded into Terudermis and incubated for 5 days. Their survival, proliferation, and expression of extracellular matrix were examined. Results: Bone marrow—derived stromal cells adhered to Terudermis and underwent significant proliferation. Immunohistochemical examination demonstrated that adherent cells were positive for expression of vimentin, desmin, fibronectin, and fsp1 and negative for beta III tubulin. These findings indicate that these cells were mesodermal cells and attached to the atelocollagen fibers biologically. Conclusions: The data suggest that Terudermis may have potential as stem cell implantation scaffolding for the treatment of scarred vocal folds.

In vitro and in vivo evaluation of a green fluorescent protein-HSV1-TK dual-function pet reporter gene

2001 ◽  
Vol 44 (S1) ◽  
pp. S339-S341
Author(s):  
K. E. Luker ◽  
G. D. Luker ◽  
C. M. Pica ◽  
J. L. Dahlheimer ◽  
T. J. Fahrner ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Reporter Gene ◽  
Fluorescent Protein ◽  
Dual Function ◽  
In Vivo Evaluation ◽  
Green Fluorescent

scholarly journals Targeting Mucosal Sites by Polymeric Immunoglobulin Receptor-directed Peptides

2002 ◽  
Vol 196 (4) ◽  
pp. 551-555 ◽  
Author(s):  
Kendra D. White ◽  
J. Donald Capra
Keyword(s):  
Epithelial Cells ◽  
Fluorescent Protein ◽  
Secretory Component ◽  
In Vivo Studies ◽  
Polymeric Immunoglobulin Receptor ◽  
Immunoglobulin Receptor ◽  
Potential Binding Site ◽  
Green Fluorescent

Polymeric immunoglobulins provide first line humoral defense at mucosal surfaces to which they are specifically transported by the polymeric immunoglobulin receptor (pIgR) on mucosal and glandular epithelial cells. Previous studies from our laboratory suggested that amino acids 402–410 of the Cα3 domain of dimeric IgA (dIgA) represented a potential binding site for the pIgR. Here by binding human secretory component to overlapping decapeptides of Cα3, we confirm these residues and also uncover an additional site. Furthermore, we show that the Cα3 motif appears to be sufficient to direct transport of green fluorescent protein through the pIgR-specific cellular transcytosis system. An alternative approach identified phage peptides, selected from a library by the in vitro Madin Darby Canine Kidney transcytosis assay, for pIgR-mediated transport through epithelial cells. Some transcytosis-selected peptides map to the same 402–410 pIgR-binding Cα3 site. Further in vivo studies document that at least one of these peptides is transported in a rat model measuring hepatic bile transport. In addition to identifying small peptides that are both bound and transported by the pIgR, this study provides evidence that the pIgR-mediated mucosal secretion system may represent a means of targeting small molecule therapeutics and genes to mucosal epithelial cells.

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