In vitro gene expression and mRNA translocation from transformed walnut (Juglans regia) rootstocks expressing DsRED fluorescent protein to wild-type scions

2017 ◽  
Vol 36 (6) ◽  
pp. 877-885 ◽  
Author(s):  
Xiaochen Liu ◽  
Sriema L. Walawage ◽  
Charles A. Leslie ◽  
Abhaya M. Dandekar ◽  
David M. Tricoli ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Juglans Regia ◽  
Wild Type

scholarly journals IDH mutation status is associated with distinct vascular gene expression signatures in lower-grade gliomas

2018 ◽  
Vol 20 (11) ◽  
pp. 1505-1516 ◽  
Author(s):  
Lei Zhang ◽  
Liqun He ◽  
Roberta Lugano ◽  
Kenney Roodakker ◽  
Michael Bergqvist ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Lower Grade ◽  
Idh Mutation ◽  
Wild Type ◽  
Who Grade ◽  
Mutation Status ◽  
Grade Ii ◽  
Grade Ii Glioma

Abstract Background Vascular gene expression patterns in lower-grade gliomas (LGGs; diffuse World Health Organization [WHO] grades II–III gliomas) have not been thoroughly investigated. The aim of this study was to molecularly characterize LGG vessels and determine if tumor isocitrate dehydrogenase (IDH) mutation status affects vascular phenotype. Methods Gene expression was analyzed using an in-house dataset derived from microdissected vessels and total tumor samples from human glioma in combination with expression data from 289 LGG samples available in the database of The Cancer Genome Atlas. Vascular protein expression was examined by immunohistochemistry in human brain tumor tissue microarrays (TMAs) representing WHO grades II–IV gliomas and nonmalignant brain samples. Regulation of gene expression was examined in primary endothelial cells in vitro. Results Gene expression analysis of WHO grade II glioma indicated an intermediate stage of vascular abnormality, less severe than that of glioblastoma vessels but distinct from normal vessels. Enhanced expression of laminin subunit alpha 4 (LAMA4) and angiopoietin 2 (ANGPT2) in WHO grade II glioma was confirmed by staining of human TMAs. IDH wild-type LGGs displayed a specific angiogenic gene expression signature, including upregulation of ANGPT2 and serpin family H (SERPINH1), connected to enhanced endothelial cell migration and matrix remodeling. Transcription factor analysis indicated increased transforming growth factor beta (TGFβ) and hypoxia signaling in IDH wild-type LGGs. A subset of genes specifically induced in IDH wild-type LGG vessels was upregulated by stimulation of endothelial cells with TGFβ2, vascular endothelial growth factor, or cobalt chloride in vitro. Conclusion IDH wild-type LGG vessels are molecularly distinct from the vasculature of IDH-mutated LGGs. TGFβ and hypoxia-related signaling pathways may be potential targets for anti-angiogenic therapy of IDH wild-type LGG.

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 Protein kinase D inhibits plasma membrane Na+/H+exchanger activity

1999 ◽  
Vol 277 (6) ◽  
pp. C1202-C1209 ◽  
Author(s):  
Robert S. Haworth ◽  
James Sinnett-Smith ◽  
Enrique Rozengurt ◽  
Metin Avkiran
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Protein Kinase D ◽  
Wild Type ◽  
Ph Indicator

The regulation of plasma membrane Na+/H+exchanger (NHE) activity by protein kinase D (PKD), a novel protein kinase C- and phorbol ester-regulated kinase, was investigated. To determine the effect of PKD on NHE activity in vivo, intracellular pH (pHi) measurements were made in COS-7 cells by microepifluorescence using the pH indicator cSNARF-1. Cells were transfected with empty vector (control), wild-type PKD, or its kinase-deficient mutant PKD-K618M, together with green fluorescent protein (GFP). NHE activity, as reflected by the rate of acid efflux ( J H), was determined in single GFP-positive cells following intracellular acidification. Overexpression of wild-type PKD had no significant effect on J H(3.48 ± 0.25 vs. 3.78 ± 0.24 mM/min in control at pHi 7.0). In contrast, overexpression of PKD-K618M increased J H (5.31 ± 0.57 mM/min at pHi 7.0; P < 0.05 vs. control). Transfection with these constructs produced similar effects also in A-10 cells, indicating that native PKD may have an inhibitory effect on NHE in both cell types, which is relieved by a dominant-negative action of PKD-K618M. Exposure of COS-7 cells to phorbol ester significantly increased J H in control cells but failed to do so in cells overexpressing either wild-type PKD (due to inhibition by the overexpressed PKD) or PKD-K618M (because basal J Hwas already near maximal). A fusion protein containing the cytosolic regulatory domain (amino acids 637–815) of NHE1 (the ubiquitous NHE isoform) was phosphorylated in vitro by wild-type PKD, but with low stoichiometry. These data suggest that PKD inhibits NHE activity, probably through an indirect mechanism, and represents a novel pathway in the regulation of the exchanger.

scholarly journals Modulation of Virulence by Two Acidified Nitrite-Responsive Loci of Salmonella enterica Serovar Typhimurium

2003 ◽  
Vol 71 (6) ◽  
pp. 3196-3205 ◽  
Author(s):  
Charles C. Kim ◽  
Denise Monack ◽  
Stanley Falkow
Keyword(s):  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Dependent Manner ◽  
Wild Type ◽  
Independent Manner ◽  
Inducible Promoters ◽  
Serovar Typhimurium ◽  
Bacterial Genes ◽  
Green Fluorescent

ABSTRACT Two acidified nitrite-inducible genes of Salmonella enterica serovar Typhimurium were identified with a green fluorescent protein-based promoter-trap screen. The nitrite-inducible promoters were located upstream of loci that we designated nipAB and nipC, which correspond to hcp-hcr (hybrid cluster protein) of Escherichia coli and norA of Alcaligenes eutrophus, respectively. Maximal induction of the promoters by nitrite was dependent on pH. The nipAB promoter was regulated by oxygen in an Fnr-dependent manner. The nipC promoter was also regulated by oxygen but in an Fnr-independent manner. The promoters were upregulated in activated RAW264.7 macrophage-like cells, which produce NO via the inducible nitric oxide synthase (iNOS), and the induction was inhibited by aminoguanidine, an inhibitor of iNOS. Although the nipAB and nipC mutants displayed no defects under a variety of in vitro conditions or in tissue culture infections, they exhibited lower oral 50% lethal doses (LD50s) than did the wild type in C57BL/6J mouse infections. The lower LD50s reflected an unexpected increased ability of small inoculating doses of the mutant bacteria to cause lethal infection 2 to 3 weeks after challenge, compared to a similar challenge dose of wild-type bacteria. We conclude that these genes are regulated by physiological nitrogen oxides and that the absence of these bacterial genes in some way diminishes the ability of mice to clear a low dose infection.

An enhanced GFP reporter system to monitor gene expression in Borrelia burgdorferi

Microbiology ◽  
2003 ◽  
Vol 149 (7) ◽  
pp. 1819-1828 ◽  
Author(s):  
James A. Carroll ◽  
Philip E. Stewart ◽  
Patricia Rosa ◽  
Abdallah F. Elias ◽  
Claude F. Garon
Keyword(s):  
Gene Expression ◽  
Borrelia Burgdorferi ◽  
Fluorescent Protein ◽  
Regulation Of Gene Expression ◽  
Epifluorescence Microscopy ◽  
Reporter System ◽  
Environmental Signals ◽  
Gfp Reporter ◽  
Green Fluorescent

Borrelia burgdorferi regulates genes in response to a number of environmental signals such as temperature and pH. A green fluorescent protein (GFP) reporter system using the ospC, ospA and flaB promoters from B. burgdorferi B31 was introduced into infectious clonal isolates of strains B31 and N40 to monitor and compare gene expression in response to pH and temperature in vitro. GFP could be assayed by epifluorescence microscopy, immunoblotting or spectrofluorometry and was an accurate reporter of target gene expression. It was determined that only 179 bp 5′ of ospC was sufficient to regulate the reporter gfp in vitro in response to pH and temperature in B. burgdorferi B31. The loss of linear plasmid (lp) 25, lp28-1, lp36 and lp56 had no effect on the ability of B. burgdorferi B31 to regulate ospC in response to pH or temperature. The amount of OspC in N40 transformants was unaffected by changes in pH or temperature of the culture medium. This suggests that regulation of gene expression in response to pH and temperature may vary between these two B. burgdorferi strains.

scholarly journals Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila

2017 ◽  
Vol 85 (5) ◽  
Author(s):  
Yoon-Suk Kang ◽  
James E. Kirby
Keyword(s):  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Life Cycles ◽  
Wild Type ◽  
Intracellular Replication ◽  
Content Type ◽  
Type Iv ◽  
Reporter Systems ◽  
Brucella Neotomae

ABSTRACT We established a new Brucella neotomae in vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus, B. melitensis, and B. suis, B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila, we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila. Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitro Brucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection.

scholarly journals Location of sequences in polyomavirus DNA that are required for early gene expression in vivo and in vitro.

1984 ◽  
Vol 4 (12) ◽  
pp. 2594-2609 ◽  
Author(s):  
C R Mueller ◽  
A M Mes-Masson ◽  
M Bouvier ◽  
J A Hassell
Keyword(s):  
Gene Expression ◽  
Thymidine Kinase ◽  
Dna Sequences ◽  
Viral Genome ◽  
Simian Virus 40 ◽  
Early Gene ◽  
Wild Type ◽  
Transcription In Vitro

To define the DNA sequences required for the expression of the polyomavirus early transcription unit, we cloned part of the viral genome in a plasmid vector, isolated mutants bearing lesions introduced in vitro within DNA sequences upstream of the transcriptional start site, and measured the capacity of these various mutant genomes to transform cells and to function as templates for transcription in vitro by comparison with wild-type DNA. One set of mutants bore 5' unidirectional deletions beginning at position -810 and extending downstream to position +4. Another set of mutants bore 3' undirectional deletions starting at position +4 and progressing upstream to position -311. The last set of mutants bore internal deletions between positions -810 and +4. Analyses of the properties of these mutant DNAs led us to conclude that the region between positions -403 and -311 includes an enhancer of gene expression. Deletion of this area from the viral genome reduced gene expression in vivo to 1 to 2% of wild-type levels, as measured by transformation assays. Moreover, this region increased the frequency of transformation of thymidine kinase-negative Rat-2 cells by the herpes simplex virus thymidine kinase (tk) gene from 5- to 20-fold. This occurred only if the polyomavirus sequences were covalently linked to the tk gene and then occurred independently of their orientation or position relative to the tk gene. A second transcriptional element is located downstream of the enhancer between positions -311 and -213. This element together with the enhancer was sufficient to bring about transformation of Rat-1 cells at nearly wild-type frequencies, and together these elements constitute the minimal sequences required for gene expression in vivo. The sequences making up the second element may be functionally duplicated downstream of position -165 (between positions -165 and -60). This was revealed by the characterization of mutant genomes with deletions between positions -349 and -60. The role of these redundant elements is not known; however, they may be analogous to the 21-base-pair repeats of simian virus 40. Finally, sequences between positions -57 and -1 were required for accurate and efficient transcription in vitro. However, this DNA stretch, which includes the TATA box and major transcriptional start sites, was not absolutely required for gene expression in vivo. We conclude that the polyomavirus promoter comprises multiple functional elements which are distributed across a DNA stretch of about 400 base pairs.

scholarly journals Phosphoprotein Enriched in Astrocytes-15 kDa Expression Inhibits Astrocyte Migration by a Protein Kinase Cδ-dependent Mechanism

2006 ◽  
Vol 17 (12) ◽  
pp. 5141-5152 ◽  
Author(s):  
François Renault-Mihara ◽  
Frédéric Beuvon ◽  
Xavier Iturrioz ◽  
Brigitte Canton ◽  
Sophie De Bouard ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Fluorescent Protein ◽  
Constitutive Expression ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Bryostatin 1 ◽  
Calcium Calmodulin Dependent ◽  
Mitogen Activated Protein ◽  
Green Fluorescent

Phosphoprotein enriched in astrocytes-15 kDa (PEA-15), a phosphoprotein enriched in astrocytes, inhibits both apoptosis and proliferation in normal and cancerous cells. Here, analysis of PEA-15 expression in glioblastoma organotypic cultures revealed low levels of PEA-15 in tumor cells migrating away from the explants, regardless of the expression levels in the originating explants. Because glioblastomas are highly invasive primary brain tumors that can originate from astrocytes, we explored the involvement of PEA-15 in the control of astrocyte migration. PEA-15−/− astrocytes presented an enhanced motility in vitro compared with their wild-type counterparts. Accordingly, NIH-3T3 cells transfected by green fluorescent protein-PEA-15 displayed a reduced migration. Reexpression of PEA-15 restored PEA-15−/− astrocyte motility to wild-type levels. Pharmacological manipulations excluded a participation of extracellular signal-regulated kinase/mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt, and calcium/calmodulin-dependent protein kinase II in this effect of PEA-15. In contrast, treatment by bisindolylmaleimide, Gö6976, and rottlerin, and chronic application of phorbol 12-myristate 13-acetate and/or bryostatin-1 indicated that PKCδ mediated PEA-15 inhibition of astrocyte migration. PEA-15−/− astrocytes constitutively expressed a 40-kDa form of PKCδ that was down-regulated upon PEA-15 reexpression. Together, these data reveal a new function for PEA-15 in the inhibitory control of astrocyte motility through a PKCδ-dependent pathway involving the constitutive expression of a catalytic fragment of PKCδ.

scholarly journals Thyroid Hormone-Dependent Gene Expression in Differentiated Embryonic Stem Cells and Embryonal Carcinoma Cells: Identification of Novel Thyroid Hormone Target Genes by Deoxyribonucleic Acid Microarray Analysis

Endocrinology ◽  
2005 ◽  
Vol 146 (2) ◽  
pp. 776-783 ◽  
Author(s):  
Yan-Yun Liu ◽  
Gregory A. Brent
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Thyroid Hormone ◽  
Early Development ◽  
Target Genes ◽  
Es Cells ◽  
Embryonic Stem ◽  
Wild Type ◽  
Ec Cells

Abstract T3 is required for normal early development, but relatively few T3-responsive target genes have been identified. In general, in vitro stem cell differentiation techniques stimulate a wide range of developmental programs, including thyroid hormone receptor (TR) pathways. We developed several in vitro stem cell models to more specifically identify TR-mediated gene expression in early development. We found that embryonic carcinoma (EC) cells have reduced T3 nuclear binding capacity and only modestly express the known T3 target genes, neurogranin (RC3) and Ca2+/calmodulin-dependent protein kinase IV (CaMKIV), in response to T3. Full T3 induction in transient transfection of EC cells was restored with cotransfection of a TR expression vector. We, therefore, performed gene expression profiles in wild-type embryonic stem (ES) cells compared with expression in cells with deficient (EC) or mutant TR (TRα P398H mutant ES cells), to identify T3 target genes. T3 stimulation of wild-type ES cells altered mRNA expression of 610 known genes (26% of those studied), although only approximately 60 genes (1%) met criteria for direct T3 stimulation based on the magnitude of induction and requirement for the presence of TR. We selected five candidate T3 target genes, neurexophilin 2, spermatid perinuclear RNA-binding protein (SPNR), kallikrein-binding protein (KBP), prostate-specific membrane antigen (PSMA), and synaptotagmin II, for more detailed study. T3 responsiveness of these genes was evaluated in both in vitro endogenous gene expression and in vivo mouse model systems. These genes identified in a novel stem cell system, including those induced and repressed in response to T3, may mediate thyroid hormone actions in early development.

scholarly journals A simple method for ex vivo honey bee cell culture capable of in vitro gene expression analysis

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257770
Author(s):  
Kazuyo Watanabe ◽  
Mikio Yoshiyama ◽  
Gaku Akiduki ◽  
Kakeru Yokoi ◽  
Hiroko Hoshida ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Culture ◽  
Honey Bee ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Plasmid Vector ◽  
Simple Method

Cultured cells are a very powerful tool for investigating biological events in vitro; therefore, cell lines have been established not only in model insect species, but also in non-model species. However, there are few reports on the establishment of stable cell lines and development of systems to introduce genes into the cultured cells of the honey bee (Apis mellifera). We describe a simple ex vivo cell culture system for the honey bee. Hemocyte cells obtained from third and fourth instar larvae were cultured in commercial Grace’s insect medium or MGM-450 insect medium for more than two weeks maintaining a normal morphology without deterioration. After an expression plasmid vector bearing the enhanced green fluorescent protein (egfp) gene driven by the immediate early 2 (IE2) viral promoter was transfected into cells, EGFP fluorescence was detected in cells for more than one week from one day after transfection. Furthermore, double-stranded RNA corresponding to a part of the egfp gene was successfully introduced into cells and interfered with egfp gene expression. A convenient and reproducible method for an ex vivo cell culture that is fully practicable for gene expression assays was established for the honey bee.

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