scholarly journals Characterization and Application of a Glucose-Repressible Promoter in Francisella tularensis

2008 ◽  
Vol 74 (7) ◽  
pp. 2161-2170 ◽  
Author(s):  
Joseph Horzempa ◽  
Deanna M. Tarwacki ◽  
Paul E. Carlson ◽  
Cory M. Robinson ◽  
Gerard J. Nau
Keyword(s):  
Gene Expression ◽  
Francisella Tularensis ◽  
Gene Function ◽  
Fluorescent Protein ◽  
Genomic Sequence ◽  
Protein A ◽  
Hypothetical Protein ◽  
Protein Levels

ABSTRACT Francisella tularensis, the causative agent of tularemia, is a category A biodefense agent. The examination of gene function in this organism is limited due to the lack of available controllable promoters. Here, we identify a promoter element of F. tularensis LVS that is repressed by glucose (termed the Francisella glucose-repressible promoter, or FGRp), allowing the management of downstream gene expression. In bacteria cultured in medium lacking glucose, this promoter induced the expression of a red fluorescent protein allele, tdtomato. FGRp activity was used to produce antisense RNA of iglC, an important virulence factor, which severely reduced IglC protein levels. Cultivation in glucose-containing medium restored IglC levels, indicating the usefulness of this promoter for controlling both exogenous and chromosomal gene expression. Moreover, FGRp was shown to be active during the infection of human macrophages by using the fluorescence reporter. In this environment, the FGRp-mediated expression of antisense iglC by F. tularensis LVS resulted in reduced bacterial fitness, demonstrating the applicability of this promoter. An analysis of the genomic sequence indicated that this promoter region controls a gene, FTL_0580, encoding a hypothetical protein. A deletion analysis determined the critical sites essential for FGRp activity to be located within a 44-bp region. This is the first report of a conditional promoter and the use of antisense constructs in F. tularensis, valuable genetic tools for studying gene function both in vitro and in vivo.

scholarly journals Regulation of Translational Efficiency by Disparate5′-UTRs of PPARγSplice Variants

PPAR Research ◽  
2009 ◽  
Vol 2009 ◽  
pp. 1-8 ◽  
Author(s):  
Shawn McClelland ◽  
Roopali Shrivastava ◽  
Jheem D. Medh
Keyword(s):  
Secondary Structure ◽  
Posttranscriptional Regulation ◽  
Protein A ◽  
Firefly Luciferase ◽  
Translational Efficiency ◽  
Protein Levels ◽  
Gene Assay ◽  
Firefly Luciferase Gene

The PPAR-γgene encodes for at least 7 unique transcripts due to alternative splicing of five exons in the5′-untranslated region (UTR). The translated region is encoded by exons 1–6, which are identical in all isoforms. This study investigated the role of the5′-UTR in regulating the efficiency with which the message is translated to protein. A coupledin vitrotranscription-translation assay demonstrated that PPAR-γ1, -γ2, and -γ5 are efficiently translated, whereas PPAR-γ4 and -γ7 are poorly translated. Anin vivoreporter gene assay using each5′-UTR upstream of the firefly luciferase gene showed that the5′-UTRs for PPAR-γ1, -γ2, and -γ4 enhanced translation, whereas the5′-UTRs for PPAR-γ5 and -γ7 inhibited translation. Models of RNA secondary structure, obtained by the mfold software, were used to explain the mechanism of regulation by each5′-UTR. In general, it was found that the translational efficiency was inversely correlated with the stability of the mRNA secondary structure, the presence of base-pairing in the consensus Kozak sequence, the number of start codons in the5′-UTR, and the length of the5′-UTR. A better understanding of posttranscriptional regulation of translation will allow modulation of protein levels without altering transcription.

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.

255 THE ESTROGENIC EVALUATION OF PARABENS USING RAT UTERINE CALBINDIN-D9k

2010 ◽  
Vol 22 (1) ◽  
pp. 285
Author(s):  
T. T. B. Vo ◽  
E. B. Jeung
Keyword(s):  
Gene Expression ◽  
Adverse Effects ◽  
Treated Group ◽  
Environmental Chemicals ◽  
Female Rats ◽  
Protein Levels ◽  
Calbindin D9k ◽  
The Difference

In the current study, calbindin-D9k (CaBP-9k), a potent biomarker for screening estrogen-like environmental chemicals in vivo and in vitro, was adopted to examine the potential estrogen-like property of the following parabens: propyl-, isopropyl-, butyl-, and isobutyl-paraben. Immature female rats were administered for 3 days from postnatal day 14 to 16 with 17?-ethinylestradiol (EE, 1 mg/kg of body weight (BW) per day) or parabens (62.5, 250, and 1000 mg/kg of BW per day). In uterotrophic assays, significantly increased uterus weights were detected in the EE-treated group and in the groups treated with the greatest dose of isopropyl-, butyl- and isobutyl-paraben. In addition, these parabens induced uterine CaBP-9k mRNA and protein levels, whereas co-treatment of parabens and fulvestrant (Faslodex, formerly known as ICI 182, 780), a pure estrogen receptor (ER) antagonist, completely reversed the paraben-induced gene expression and increased uterine weights. To investigate the ER-mediated mechanism(s) by which parabens exert their effects, the expression level of ERα and progesterone receptor (PR) was analyzed. Exposure to EE or parabens caused a dramatic decrease in expression of both ER? mRNA and protein levels, whereas co-treatment with fulvestrant reversed these effects. These data showed the difference of CaBP-9k and ER? expression, suggesting that CaBP-9k might not express via ER? pathway. In the effect of parabens on CaBP-9k expression through PR mediation, a significantly increased expression of uterine PR gene, a well-known ER regulating gene, at both transcriptional and translational levels was indicated in the greatest dose of isopropyl- and butyl-paraben. These parabens induced PR gene expression that was completely blocked by fulvestrant. This result indicates that CaBP-9k expression might involve PR mediates in the estrogenic effect of paraben in immature rat uteri. Taken together, parabens exhibited an estrogen-like property in vivo, which might be mediated by a PR and/or ER? signaling pathway. In addition, our results expanded the current understanding of the potential adverse effects of parabens associated with their estrogen-like activities. Further investigation is needed to elucidate in greater detail the adverse effects of parabens in humans and wildlife.

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 Mutagenesis of the Rns regulator of enterotoxigenic Escherichia coli reveals roles for a linker sequence and two helix–turn–helix motifs

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2796-2806 ◽  
Author(s):  
Vivienne Mahon ◽  
Cyril J. Smyth ◽  
Stephen G. J. Smith
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Protein A ◽  
Diarrhoeal Disease ◽  
Enterotoxigenic Escherichia Coli ◽  
Insertion Mutagenesis

The pathogenesis of diarrhoeal disease due to human enterotoxigenic Escherichia coli absolutely requires the expression of fimbriae. The expression of CS1 fimbriae is positively regulated by the AraC-like protein Rns. AraC-like proteins are DNA-binding proteins that typically contain two helix–turn–helix (HTH) motifs. A program of pentapeptide insertion mutagenesis of the Rns protein was performed, and this revealed that both HTH motifs are required by Rns to positively regulate CS1 fimbrial gene expression. Intriguingly, a pentapeptide insertion after amino acid C102 reduced the ability of Rns to transactivate CS1 fimbrial expression. The structure of Rns in this vicinity (NACRS) was predicted to be disordered and thus might act as a flexible linker. This hypothesis was confirmed by deletion of this amino acid sequence from the Rns protein; a truncated protein that lacked this sequence was no longer functional. Strikingly, this sequence could be functionally substituted in vivo and in vitro by a flexible seven amino acid sequence from another E. coli AraC-like protein RhaS. Our data indicate that HTH motifs and a flexible sequence are required by Rns for maximal activation of fimbrial gene expression.

scholarly journals Investigation of exposure to lifestyle and environmental factors on cumulus-oocyte complex function

2021 ◽  
Author(s):  
◽  
Kelly Anne Campen
Keyword(s):  
Gene Expression ◽  
Oocyte Quality ◽  
Protein Levels ◽  
Time Interaction ◽  
Cumulus Oocyte Complex ◽  
In Vitro Exposure ◽  
In Vitro Effects ◽  
In Vivo Effects

<p>Pathways involved in bi-directional communication within the cumulus-oocyte complex (COC) include gap junction (GJ) communication, oocyte growth factor production, and glucose metabolism and are essential for oocyte health. Perturbation of these pathways may result in reduced oocyte quality due to altered COC function. Using rats as a model, in vitro effects of exposure to bisphenol A (BPA), caffeine, nicotine, ethanol, methylenedioxymeth- amphetamine (MDMA), or Δ⁹-tetrahydrocannabinol (THC) on COC function were investigated. Furthermore, MDMA was administered to rats to compare in vitro with in vivo effects.  The transfer of a fluorescent dye (calcein) from cumulus cells (CC) to the oocyte was used as a measure of GJ communication. Expression of CC-derived (Atr, Cx43, Cycs, Gfpt1, Pfkp) and oocyte-derived (Atr, Bmp15, Cx37, Gdf9) genes were measured using multiplex TaqMan quantitative PCR. Levels of CX43 and GDF9 proteins were quantified using Western blotting.  Optimisation of the GJ bioassay included the addition of phosphodiesterase inhibitors (rolipram and dipyridamole), and a 1 hour post-calcein incubation period to allow dye transfer. Quantification of gene expression in calcein-treated CC and oocytes was validated, enabling direct comparisons between GJ communication and gene expression.  To determine the in vitro effects, COC were incubated with test factors at high physiological concentrations over 25 hours. GJ communication decreased over time in control COC. This reduction was attenuated after exposure to BPA and nicotine, and partially by caffeine. Furthermore, exposure to ethanol maintained oocyte meiotic arrest, whereas MDMA and THC promoted meiotic resumption.  Oocyte-derived gene expression was mostly unaffected by in vitro exposure to the lifestyle and environmental factors, although a treatment x time interaction for Cx37 levels following nicotine exposure was observed. Of the CC-derived genes, Cx43 was the most sensitive where BPA, MDMA, and THC increased, and caffeine and ethanol decreased, expression. In CC, exposure to MDMA and THC increased Gfpt1 levels and exposure to MDMA resulted in a treatment x time interaction in Cycs and Pfkp expression.  In COC, caffeine increased CX43 protein levels after 1 hour. Nicotine initially reduced, but with time increased CX43 levels. Furthermore, CX43 levels decreased and increased after 25 hour exposures to ethanol and MDMA, respectively. GDF9 protein levels in COC exhibited wide within-treatment variation. Overall, BPA and caffeine reduced GDF9 levels after 1 hour whereas GDF9 levels were increased following exposure to BPA, caffeine, MDMA, and THC for 25 hours.  To determine in vivo effects, female rats were administered saline or 5 mg/kg/day MDMA for 3 days. COC from MDMA-treated rats had higher levels of CX43 protein but gene expression and meiotic reactivation were unaffected.  In conclusion, COC function was altered by in vitro exposure to BPA, caffeine, ethanol, nicotine, MDMA, and THC. Furthermore, in vivo exposure to MDMA elicits similar, albeit reduced, effects on COC function. A role for CC in protecting the oocyte from harmful contaminants is proposed. Perturbation of the bi-directional communication pathway is likely to influence oocyte quality due to alterations in nutrient availability and timing of follicular events, although these may not be associated with negative outcomes. This study provides evidence that exposure to lifestyle factors and environmental contaminants affect COC function.</p>

scholarly journals Satellite cell-mediated angiogenesis in vitro coincides with a functional hypoxia-inducible factor pathway

2009 ◽  
Vol 296 (6) ◽  
pp. C1321-C1328 ◽  
Author(s):  
R. P. Rhoads ◽  
R. M. Johnson ◽  
C. R. Rathbone ◽  
X. Liu ◽  
C. Temm-Grove ◽  
...  
Keyword(s):  
Gene Expression ◽  
Satellite Cell ◽  
Hypoxia Inducible Factor ◽  
Collagen Gel ◽  
Culture Conditions ◽  
Cell Physiology ◽  
Vegf Gene ◽  
Protein Levels

Muscle regeneration involves the coordination of myogenesis and revascularization to restore proper muscle function. Myogenesis is driven by resident stem cells termed satellite cells (SC), whereas angiogenesis arises from endothelial cells and perivascular cells of preexisting vascular segments and the collateral vasculature. Communication between myogenic and angiogenic cells seems plausible, especially given the number of growth factors produced by SC. To characterize these interactions, we developed an in vitro coculture model composed of rat skeletal muscle SC and microvascular fragments (MVF). In this system, isolated epididymal MVF suspended in collagen gel are cultured over a rat SC monolayer culture. In the presence of SC, MVF exhibit greater indices of angiogenesis than MVF cultured alone. A positive dose-dependent effect of SC conditioned medium (CM) on MVF growth was observed, suggesting that SC secrete soluble-acting growth factor(s). Next, we specifically blocked VEGF action in SC CM, and this was sufficient to abolish satellite cell-induced angiogenesis. Finally, hypoxia-inducible factor-1α (HIF-1α), a transcriptional regulator of VEGF gene expression, was found to be expressed in cultured SC and in putative SC in sections of in vivo stretch-injured rat muscle. Hypoxic culture conditions increased SC HIF-1α activity, which was positively associated with SC VEGF gene expression and protein levels. Collectively, these initial observations suggest that a heretofore unexplored aspect of satellite cell physiology is the initiation of a proangiogenic program.

Transketolase Activity but not Thiamine Membrane Transport Change in Response to Hyperglycaemia and Kidney Dysfunction

2017 ◽  
Vol 126 (04) ◽  
pp. 255-262 ◽  
Author(s):  
Katarína Chalásová ◽  
Lukáš Pácal ◽  
Anna Pleskačová ◽  
Lucia Knopfová ◽  
Jitka Řehořová ◽  
...  
Keyword(s):  
Gene Expression ◽  
Kidney Disease ◽  
Pentose Phosphate ◽  
Protective Mechanism ◽  
Protein Levels ◽  
Human Experiments ◽  
Key Enzyme ◽  
Vitro Analysis

Abstract Aim Pentose phosphate pathway (PPP) with key enzyme transketolase (TKT), represents a potentially ‘protective’ mechanism in hyperglycaemia. Diabetic kidney disease (DKD), a common complication of both type 1 and type 2 diabetes associated with significant morbidity and mortality, represents the most common cause of chronic kidney disease (CKD). We hypothesized that protective PPP action in diabetes and eventually even more severely in concomitant DKD might be compromised by limited intracellular availability of an active TKT cofactor thiamine diphosphate (TDP). Methods Effect of hyperglycaemia on gene expression and protein levels of key PPP loci was studied in vitro using human cell lines relevant to diabetes (HUVEC and HRGEC) and (together with measurement of TKT activity, plasma thiamine and erythrocyte TDP concentration) in vivo in diabetic vs. non-diabetic subjects with comparable renal function (n=83 in total). Results Hyperglycaemia significantly decreased protein levels of RFC-1, THTR1, THTR2 and TKT (P<0.05) in vitro. Analysis of blood samples from CKD patients with and without diabetes and from controls did not reveal any difference in gene expression and protein levels of thiamine transporters while TKT activity and TDP in erythrocytes gradually increased with decreasing kidney function being highest in patients with CKD3-4 of both diabetic and non-diabetic aetiology. Hyperglycaemia and uremic serum mimicking CKD in diabetes did not affect TKT activity in vitro (P<0.05). Conclusion Both in vitro and human experiments showed decrease or unchanged expression, respectively, of thiamine transporters induced by hyperglycaemia while TKT activity in parallel with intracellular TDP was increased in CKD patients with or without diabetes. Therefore, lack of adaptive increase of thiamine transmembrane transport allowing further increase of TKT activity might contribute to compromised PPP function in diabetes and CKD and to the development of glycotoxic injury.

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.

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