scholarly journals Moderate and intensive mechanical loading differentially modulate the phenotype of tendon stem/progenitor cells in vivo

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0242640
Author(s):  
Jianying Zhang ◽  
Daibang Nie ◽  
Kelly Williamson ◽  
Arthur McDowell ◽  
MaCalus V. Hogan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Cell Morphology ◽  
Fluorescent Protein ◽  
Treadmill Running ◽  
Whole Body ◽  
Tendon Cells ◽  
Elevated Expression ◽  
Green Fluorescent

To examine the differential mechanobiological responses of specific resident tendon cells, we developed an in vivo model of whole-body irradiation followed by injection of either tendon stem/progenitor cells (TSCs) expressing green fluorescent protein (GFP-TSCs) or mature tenocytes expressing GFP (GFP-TNCs) into the patellar tendons of wild type C57 mice. Injected mice were subjected to short term (3 weeks) treadmill running, specifically moderate treadmill running (MTR) and intensive treadmill running (ITR). In MTR mice, both GFP-TSC and GFP-TNC injected tendons maintained normal cell morphology with elevated expression of tendon related markers collagen I and tenomodulin. In ITR mice injected with GFP-TNCs, cells also maintained an elongated shape similar to the shape found in normal/untreated control mice, as well as elevated expression of tendon related markers. However, ITR mice injected with GFP-TSCs showed abnormal changes, such as cell morphology transitioning to a round shape, elevated chondrogenic differentiation, and increased gene expression of non-tenocyte related genes LPL, Runx-2, and SOX-9. Increased gene expression data was supported by immunostaining showing elevated expression of SOX-9, Runx-2, and PPARγ. This study provides evidence that while MTR maintains tendon homeostasis by promoting the differentiation of TSCs into TNCs, ITR causes the onset of tendinopathy development by inducing non-tenocyte differentiation of TSCs, which may eventually lead to the formation of non-tendinous tissues in tendon tissue after long term mechanical overloading conditions on the tendon.

scholarly journals Moderate and intensive mechanical loading differentially modulate the phenotype of tendon stem/progenitor cells in vivo

2020 ◽  
Author(s):  
Jianying Zhang ◽  
Daibang Nie ◽  
Kelly Williamson ◽  
Arthur McDowell ◽  
MaCalus V. Hogan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Cell Morphology ◽  
Fluorescent Protein ◽  
Treadmill Running ◽  
Whole Body ◽  
Tendon Cells ◽  
Elevated Expression ◽  
Green Fluorescent

ABSTRACTTo examine the differential mechanobiological responses of specific resident tendon cells, we developed an in vivo model of whole-body irradiation followed by injection of either tendon stem/progenitor cells (TSCs) expressing green fluorescent protein (GFP-TSCs) or mature tenocytes expressing GFP (GFP-TNCs) into the patellar tendons of wild type C57 mice. Injected mice were subjected to moderate treadmill running (MTR) and intensive treadmill running (ITR). In MTR mice, both GFP-TSC and GFP-TNC injected tendons maintained normal cell morphology with elevated expression of tendon related markers collagen I and tenomodulin. In ITR mice injected with GFP-TNCs, cells also maintained an elongated shape similar to the shape found in normal/untreated control mice, as well as elevated expression of tendon related markers. However, ITR mice injected with GFP-TSCs showed typical tendinopathic changes, such as cell morphology transitioning to a round shape, elevated chondrogenic differentiation, and increased gene expression of non-tenocyte related genes LPL, Runx-2, and SOX-9. Increased gene expression data was supported by immunostaining showing elevated expression of SOX-9, Runx-2, and PPARγ. This study provides evidence that while MTR maintains tendon homeostasis by promoting the differentiation of TSCs into tenocytes, ITR may cause the development of tendinopathy by inducing non-tenocyte differentiation of TSCs, thus leading to the presence of non-tendinous tissues in tendon.

Cryptococcus neoformans Differential Gene Expression Detected In Vitro and In Vivo with Green Fluorescent Protein

1999 ◽  
Vol 67 (4) ◽  
pp. 1812-1820
Author(s):  
Maurizio del Poeta ◽  
Dena L. Toffaletti ◽  
Thomas H. Rude ◽  
Sara D. Sparks ◽  
Joseph Heitman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Cryptococcus Neoformans ◽  
Differential Gene Expression ◽  
Fluorescent Protein ◽  
Differential Gene ◽  
Green Fluorescent

scholarly journals Use of Green Fluorescent Protein To Assess Urease Gene Expression by Uropathogenic Proteus mirabilis during Experimental Ascending Urinary Tract Infection

1998 ◽  
Vol 66 (1) ◽  
pp. 330-335 ◽  
Author(s):  
Hui Zhao ◽  
Richard B. Thompson ◽  
Virginia Lockatell ◽  
David E. Johnson ◽  
Harry L. T. Mobley
Keyword(s):  
Gene Expression ◽  
Urinary Tract Infection ◽  
Green Fluorescent Protein ◽  
Urinary Tract ◽  
Tract Infection ◽  
Fluorescence Intensity ◽  
Proteus Mirabilis ◽  
Fluorescent Protein ◽  
Green Fluorescent

ABSTRACT Proteus mirabilis, a cause of complicated urinary tract infection, expresses urease when exposed to urea. While it is recognized that the positive transcriptional activator UreR induces gene expression, the levels of expression of the enzyme during experimental infection are not known. To investigate in vivo expression of P. mirabilis urease, the gene encoding green fluorescent protein (GFP) was used to construct reporter fusions. Translational fusions of urease accessory gene ureD, which is preceded by a urea-inducible promoter, were made withgfp (modified to express S65T/V68L/S72A [B. P. Cormack et al. Gene 173:33–38, 1996]). Constructs were confirmed by sequencing of the fusion junctions. UreD-GFP fusion protein was induced by urea in both Escherichia coli DH5α and P. mirabilis HI4320. By using Western blotting with antiserum raised against GFP, expression level was shown to correlate with urea concentration (tested from 0 to 500 mM), with highest induction at 200 to 500 mM urea. Fluorescent E. coli and P. mirabilis bacteria were observed by fluorescence microscopy following urea induction, and the fluorescence intensity of GFP in cell lysates was measured by spectrophotofluorimetry. P. mirabilis HI4320 carrying the UreD-GFP fusion plasmid was transurethrally inoculated into the bladders of CBA mice. One week postchallenge, fluorescent bacteria were detected in thin sections of both bladder and kidney samples; the fluorescence intensity of bacteria in bladder tissue was higher than that in the kidney. Kidneys were primarily infected with single-cell-form fluorescent bacteria, while aggregated bacterial clusters were observed in the bladder. Elongated swarmer cells were only rarely observed. These observations demonstrate that urease is expressed in vivo and that using GFP as a reporter protein is a viable approach to investigate in vivo expression ofP. mirabilis virulence genes in experimental urinary tract infection.

scholarly journals Flow Cytometric Testing of Green Fluorescent Protein-Tagged Lactobacillus rhamnosus GG for Response to Defensins

2006 ◽  
Vol 72 (7) ◽  
pp. 4923-4930 ◽  
Author(s):  
Sigrid C. J. De Keersmaecker ◽  
Kristien Braeken ◽  
Tine L. A. Verhoeven ◽  
Mónica Perea Vélez ◽  
Sarah Lebeer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Heterologous Gene Expression ◽  
Lactobacillus Rhamnosus ◽  
General Interest ◽  
Green Fluorescent ◽  
And Behavior ◽  
Nice System

ABSTRACT Lactobacillus rhamnosus GG is of general interest as a probiotic. Although L. rhamnosus GG is often used in clinical trials, there are few genetic tools to further determine its mode of action or to develop it as a vehicle for heterologous gene expression in therapy. Therefore, we developed a reproducible, efficient electroporation procedure for L. rhamnosus GG. The best transformation efficiency obtained was 104 transformants per μg of DNA. We validated this protocol by tagging L. rhamnosus GG with green fluorescent protein (GFP) using the nisin-controlled expression (NICE) system. Parameters for overexpression were optimized, which allowed expression of gfp in L. rhamnosus GG upon induction with nisin. The GFP+ strain can be used to monitor the survival and behavior of L. rhamnosus GG in vivo. Moreover, implementation of the NICE system as a gene expression switch in L. rhamnosus GG opens up possibilities for improving and expanding the performance of this strain. The GFP-labeled strain was used to demonstrate that L. rhamnosus GG is sensitive to human beta-defensin-2 but not to human beta-defensin-1.

scholarly journals The Tubulin-Like RepX Protein Encoded by the pXO1 Plasmid Forms Polymers In Vivo in Bacillus anthracis

2009 ◽  
Vol 191 (8) ◽  
pp. 2493-2500 ◽  
Author(s):  
Parvez Akhtar ◽  
Syam P. Anand ◽  
Simon C. Watkins ◽  
Saleem A. Khan
Keyword(s):  
Bacillus Anthracis ◽  
Cell Morphology ◽  
Fluorescent Protein ◽  
Host Cells ◽  
E Coli ◽  
Green Fluorescent ◽  
Related Proteins ◽  
Polymeric Structures

ABSTRACT Bacillus anthracis contains two megaplasmids, pXO1 and pXO2, that are critical for its pathogenesis. Stable inheritance of pXO1 in B. anthracis is dependent upon the tubulin/FtsZ-like RepX protein encoded by this plasmid. Previously, we have shown that RepX undergoes GTP-dependent polymerization in vitro. However, the polymerization properties and localization pattern of RepX in vivo are not known. Here, we utilize a RepX-green fluorescent protein (GFP) fusion to show that RepX forms foci and three distinct forms of polymeric structures in B. anthracis in vivo, namely straight, curved, and helical filaments. Polymerization of RepX-GFP as well as the nature of polymers formed were dependent upon concentration of the protein inside the B. anthracis cells. RepX predominantly localized as polymers that were parallel to the length of the cell. RepX also formed polymers in Escherichia coli in the absence of other pXO1-encoded products, showing that in vivo polymerization is an inherent property of the protein and does not require either the pXO1 plasmid or proteins unique to B. anthracis. Overexpression of RepX did not affect the cell morphology of B. anthracis cells, whereas it drastically distorted the cell morphology of E. coli host cells. We discuss the significance of our observations in view of the plasmid-specific functions that have been proposed for RepX and related proteins encoded by several megaplasmids found in members of the Bacillus cereus group of bacteria.

scholarly journals Real-Time In Vivo Green Fluorescent Protein Imaging of a Murine Leishmaniasis Model as a New Tool for Leishmania Vaccine and Drug Discovery

2008 ◽  
Vol 15 (12) ◽  
pp. 1764-1770 ◽  
Author(s):  
Sanjay R. Mehta ◽  
Robert Huang ◽  
Meng Yang ◽  
Xing-Quan Zhang ◽  
Bala Kolli ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Real Time ◽  
Fluorescent Protein ◽  
Therapeutic Vaccine ◽  
Model Systems ◽  
Whole Body ◽  
Time Data ◽  
Green Fluorescent ◽  
Thickness Measurements

ABSTRACT Leishmania species are obligate intracellular protozoan parasites that cause a broad spectrum of clinical diseases in mammalian hosts. The most frequently used approach to quantify parasites in murine model systems is based on thickness measurements of the footpad or ear after experimental infection. To overcome the limitations of this method, we used a Leishmania mutant episomally transfected with enhanced green fluorescent protein, enabling in vivo real-time whole-body fluorescence imaging, to follow the progression of Leishmania infection in parasitized tissues. Fluorescence correlated with the number of Leishmania parasites in the tissue and demonstrated the real-time efficacy of a therapeutic vaccine. This approach provides several substantial advantages over currently available animal model systems for the in vivo study of immunopathogenesis, prevention, and therapy of leishmaniasis. These include improvements in sensitivity and the ability to acquire real-time data on progression and spread of the infection.

scholarly journals Karyopherin α2: a control step of glucose-sensitive gene expression in hepatic cells

2002 ◽  
Vol 364 (1) ◽  
pp. 201-209 ◽  
Author(s):  
Ghislaine GUILLEMAIN ◽  
Maria J. MUÑOZ-ALONSO ◽  
Aurélia CASSANY ◽  
Martine LOIZEAU ◽  
Anne-Marie FAUSSAT ◽  
...  
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Pyruvate Kinase ◽  
Glucose Transporter ◽  
Fluorescent Protein ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Mrna Accumulation ◽  
Green Fluorescent

Glucose is required for an efficient expression of the glucose transporter GLUT2 and other genes. We have shown previously that the intracytoplasmic loop of GLUT2 can divert a signal, resulting in the stimulation of glucose-sensitive gene transcription. In the present study, by interaction with the GLUT2 loop, we have cloned the rat karyopherin α2, a receptor involved in nuclear import. The specificity of the binding was restricted to GLUT2, and not GLUT1 or GLUT4, and to karyopherin α2, not α1. When rendered irreversible by a cross-linking agent, this transitory interaction was detected in vivo in hepatocytes. A role for karyopherin α2 in the transcription of two glucose-sensitive genes was investigated by transfection of native and inactive green fluorescent protein—karyopherin α2 in GLUT2-expressing hepatoma cells. The amount of inactive karyopherin α2 receptor reduced, in a dose-dependent manner, the GLUT2 and liver pyruvate kinase mRNA levels by competition with endogenous active receptor. In contrast, the overexpression of karyopherin α2 did not significantly stimulate GLUT2 and liver pyruvate kinase mRNA accumulation in green fluorescent protein-sorted cells. The present study suggests that, in concert with glucose metabolism, karyopherin α2 transmits a signal to the nucleus to regulate glucose-sensitive gene expression. The transitory tethering of karyopherin α2 to GLUT2 at the plasma membrane might indicate that the receptor can load the cargo to be imported locally.

scholarly journals Analysis of Green Fluorescent Protein Expression in Transgenic Rats for Tracking Transplanted Neural Stem/Progenitor Cells

2005 ◽  
Vol 53 (10) ◽  
pp. 1215-1226 ◽  
Author(s):  
Andrea J. Mothe ◽  
Iris Kulbatski ◽  
Rita L. van Bendegem ◽  
Linda Lee ◽  
Eiji Kobayashi ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Green Fluorescent Protein ◽  
Progenitor Cells ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Rat Strain ◽  
Direct Fluorescence ◽  
Green Fluorescent

Green fluorescent protein (GFP) expression was evaluated in tissues of different transgenic rodents—Sprague-Dawley (SD) rat strain [SD-Tg(GFP)Bal], W rat strain [Wistar-TgN(CAG-GFP)184ys], and M mouse strain [Tg(GFPU)5Nagy/J]—by direct fluorescence of native GFP expression and by immunohistochemistry. The constitutively expressing GFP transgenic strains showed tissue-specific differences in GFP expression, and GFP immunohistochemistry amplified the fluorescent signal. The fluorescence of stem/progenitor cells cultured as neurospheres from the ependymal region of the adult spinal cord from the GFP SD and W rat strains was assessed in vitro. After transplantation of the cells into wildtype spinal cord, the ability to track the grafted cells was evaluated in vivo. Cultured stem/progenitor cells from the SD strain required GFP immunostaining to be visualized. Likewise, after transplantation of SD cells into the spinal cord, immunohistochemical amplification of the GFP signal was required for detection. In contrast, GFP expression of stem/progenitor cells generated from the W strain was readily detected by direct fluorescence both in vitro and in vivo without the need for immunohistochemical amplification. The cultured stem/progenitor cells transplanted into the spinal cord survived for at least 49 days after transplantation, and continued to express GFP, demonstrating stable expression of the GFP transgene in vivo.

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