scholarly journals IMP dehydrogenase from Pneumocystis carinii as a potential drug target.

1997 ◽  
Vol 41 (1) ◽  
pp. 40-48 ◽  
Author(s):  
M J O'Gara ◽  
C H Lee ◽  
G A Weinberg ◽  
J M Nott ◽  
S F Queener
Keyword(s):  
Mycophenolic Acid ◽  
Expression System ◽  
Pneumocystis Carinii ◽  
Specific Inhibitor ◽  
Coli Strain ◽  
Northern Hybridization ◽  
Imp Dehydrogenase ◽  
E Coli ◽  
The Mean

Mycophenolic acid, a specific inhibitor of IMP dehydrogenase (IMPDH; EC 1.1.1.205), is a potent inhibitor of Pneumocystis carinii growth in culture, suggesting that IMPDH may be a sensitive target for chemotherapy in this organism. The IMPDH gene was cloned as a first step to characterizing the enzyme and developing selective inhibitors. A 1.3-kb fragment containing a portion of the P. carinii IMPDH gene was amplified by PCR with two degenerate oligonucleotides based on conserved sequences in IMPDH from humans and four different microorganisms. Northern hybridization analysis showed the P. carinii IMPDH mRNA to be approximately 1.6 kb. The entire cDNA encoding P. carinii IMPDH was isolated and cloned. The deduced amino acid sequence of P. carinii IMPDH shared homology with bacterial (31 to 38%), protozoal (48 to 59%), mammalian (60 to 62%), and fungal (62%) IMPDH enzymes. The IMPDH cDNA was expressed by using a T7 expression system in an IMPDH-deficient strain of Escherichia coli (strain S phi 1101). E. coli S phi 1101 cells containing the P. carinii IMPDH gene were able to grow on medium lacking guanine, implying that the protein expressed in vivo was functional. Extracts of these E. coli cells contained IMPDH activity that had an apparent Km for IMP of 21.7 +/- 0.3 microM and an apparent Km for NAD of 314 +/- 84 microM (mean +/- standard error of the mean; n = 3), and the activity was inhibited by mycophenolic acid (50% inhibitory concentration, 24 microM; n = 2).

scholarly journals A Methylation-Directed, Synthetic Pap Switch Based on Self-Complementary Regulatory DNA in an All-E. Coli Cell-Free Expression System

2020 ◽  
Author(s):  
Emanuel Worst ◽  
oemer Kurt ◽  
Marc Finkler ◽  
Marc Schenkelberger ◽  
Vincent Noireaux ◽  
...  
Keyword(s):  
Expression System ◽  
Regulatory Region ◽  
Phase Variation ◽  
Coli Strain ◽  
Coli Cell ◽  
Free Expression ◽  
E Coli ◽  
Regulatory Dna ◽  
Cell Free Expression System

<p>Pyelonephritis-associated pili (pap) enable migration of the uropathogenic Escherichia coli strain (UPEC) through the urinary tract. UPEC can switch between a stable 'ON phase' where the corresponding pap genes are expressed and a stable 'OFF phase' where their transcription is repressed. Hereditary, alternate DNA methylation of only two GATC motives within the regulatory region stabilizes the respective phase over many generations. The underlying molecular mechanism is only partly understood. Previous investigations suggest that in vivo phase-variation stability results from cooperative action of the transcriptional regulators Lrp and PapI. Here, we use an E. coli cell-free expression system to study the function of pap regulatory region based on a specially designed, synthetic construct flanked by two reporter genes encoding fluorescent proteins for simple readout. Based on our observations we suggest that Lrp and the conformation of the self-complementary regulatory DNA play a strong role in the regulation of phase-variation. Our work not only contributes to better understand the phase variation mechanism, but it represents a successful start for engineering stable, hereditary and strong expression control based on methylation.</p>

scholarly journals A Methylation-Directed, Synthetic Pap Switch Based on Self-Complementary Regulatory DNA in an All-E. Coli Cell-Free Expression System

2020 ◽  
Author(s):  
Emanuel Worst ◽  
oemer Kurt ◽  
Marc Finkler ◽  
Marc Schenkelberger ◽  
Vincent Noireaux ◽  
...  
Keyword(s):  
Expression System ◽  
Regulatory Region ◽  
Phase Variation ◽  
Coli Strain ◽  
Coli Cell ◽  
Free Expression ◽  
E Coli ◽  
Regulatory Dna ◽  
Cell Free Expression System

<p>Pyelonephritis-associated pili (pap) enable migration of the uropathogenic Escherichia coli strain (UPEC) through the urinary tract. UPEC can switch between a stable 'ON phase' where the corresponding pap genes are expressed and a stable 'OFF phase' where their transcription is repressed. Hereditary, alternate DNA methylation of only two GATC motives within the regulatory region stabilizes the respective phase over many generations. The underlying molecular mechanism is only partly understood. Previous investigations suggest that in vivo phase-variation stability results from cooperative action of the transcriptional regulators Lrp and PapI. Here, we use an E. coli cell-free expression system to study the function of pap regulatory region based on a specially designed, synthetic construct flanked by two reporter genes encoding fluorescent proteins for simple readout. Based on our observations we suggest that Lrp and the conformation of the self-complementary regulatory DNA play a strong role in the regulation of phase-variation. Our work not only contributes to better understand the phase variation mechanism, but it represents a successful start for engineering stable, hereditary and strong expression control based on methylation.</p>

scholarly journals In Vivo Modulation of a DnaJ Homolog, CbpA, by CbpM

2007 ◽  
Vol 189 (9) ◽  
pp. 3635-3638 ◽  
Author(s):  
Matthew R. Chenoweth ◽  
Nancy Trun ◽  
Sue Wickner
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Stationary Phase ◽  
Specific Inhibitor ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
E Coli ◽  
Hsp70 Chaperone

ABSTRACT CbpA, an Escherichia coli DnaJ homolog, can function as a cochaperone for the DnaK/Hsp70 chaperone system, and its in vitro activity can be modulated by CbpM. We discovered that CbpM specifically inhibits the in vivo activity of CbpA, preventing it from functioning in cell growth and division. Furthermore, we have shown that CbpM interacts with CbpA in vivo during stationary phase, suggesting that the inhibition of activity is a result of the interaction. These results reveal that the activity of the E. coli DnaK system can be regulated in vivo by a specific inhibitor.

scholarly journals Expression of novel fusion antiviral proteins Ricin A Chain-Pokeweed Antiviral Proteins (RTA-PAPs) in Escherichia coli and their inhibition of protein synthesis and of hepatitis B virus in vitro

2018 ◽  
Author(s):  
Yasser Hassan ◽  
Sherry Ogg ◽  
Hui Ge
Keyword(s):  
Protein Synthesis ◽  
Expression System ◽  
Therapeutic Index ◽  
Antiviral Protein ◽  
E Coli ◽  
Protein Isoform ◽  
Antiviral Proteins ◽  
A Chain

AbstractRicin A chain (RTA) and Pokeweed antiviral proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential antiviral value of novel fusion proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral protein isoform 1 from seeds (RTA-PAPS1) was produced in E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and protein synthesis inhibitory activity assayed by comparison to the production of a control protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded protein was bioactive with 50% protein synthesis inhibitory concentration (IC50) of 0.06nM (3.63ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03nM (1.82ng/ml) and a therapeutic index of >21818. The fusion protein was further optimized using in silico tools, produced in E. coli in vivo expression system, purified by three-step process from soluble lysate and protein synthesis inhibition activity assayed. Results showed that the optimized protein RTA mutant-Pokeweed antiviral protein isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function activity on protein synthesis inhibition with an IC50 of 0.03nM (1.82ng/ml). Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant antiviral activity against HBV in vitro with a high therapeutic index and, thus, meriting further development as potential antiviral agents against chronic HBV infection.

Design, Synthesis, Docking and Biological Evaluation of Novel 4-hydroxy Coumarin Derivatives

2020 ◽  
Vol 16 ◽  
Author(s):  
N. Ramalakshmi ◽  
S.R. Chitra ◽  
P. Manimegalai ◽  
S. Arunkumar
Keyword(s):  
Antibacterial Activity ◽  
Docking Studies ◽  
Biological Evaluation ◽  
Coli Strain ◽  
Coumarin Derivatives ◽  
In Vivo Evaluation ◽  
E Coli ◽  
Tract Infections ◽  
Multiple Drugs

Background: Hospital acquired (HA) infections are caused due to E. coliwhich is resistant to multiple drugs particularly to fluroquinolone class of drugs. Urinary tract infections (UTI) affects people in the community and in hospitals. 150 million people per annum are suffering from UTI worldwide. Methods: In this present study we designed 36 novel coumarin derivatives, also we predicted pharmacokinetic and toxicity parameters. Docking studies were also carriedand all the compounds were evaluated for antibacterial activity againstresistant quinolone E. coli strain ATCC 25922. It was interesting to note thatthe introduction of electron withdrawing group on the aromatic ringresulted in compounds with increased antibacterial activity which is observed in compound 6 (with4-nitro substitution), compound 23(chloro) and compound 30(chloro, nitro). Results: From the MIC results, was observed that compounds 6, 23 and 30 showed higher activity with 0.5µg/ml, 0. 12 µg/ml, 0.5 µg/mlrespectively. Docking studies were performed with the activesite of DNA gyrase (PDB ID: 4CKK ).The maximum binding energy was found to be -10.7Kcal/mol. Conclusion: From the study it was found that 3 compounds were potentially active against quinolone resistant E. coli strains. This study can be further extended for in vivo evaluation.

scholarly journals Amplification of the IMP dehydrogenase gene in Chinese hamster cells resistant to mycophenolic acid.

1987 ◽  
Vol 7 (9) ◽  
pp. 3328-3331 ◽  
Author(s):  
F R Collart ◽  
E Huberman
Keyword(s):  
Mycophenolic Acid ◽  
Cdna Probe ◽  
Chinese Hamster ◽  
Specific Inhibitor ◽  
Fold Increase ◽  
Immunoblot Analysis ◽  
Expression Library ◽  
Imp Dehydrogenase ◽  
V79 Cell ◽  
Resistant Cells

The regulation of IMP dehydrogenase (IMPDH) was analyzed in Chinese hamster V79 cell variants that exhibit different degrees of resistance to the cytotoxic effect of mycophenolic acid, a specific inhibitor of IMPDH. Western blot (immunoblot) analysis with an IMPDH antiserum revealed a 14- to 27-fold increase in the amount of enzyme in the mycophenolic acid-resistant cells. The antiserum was also used to screen for a phage containing the IMPDH cDNA sequence from a lambda gt11 expression library. Northern blot (RNA blot) analyses of total cellular and poly(A)+ RNA showed that an IMPDH cDNA probe hybridized to a 2.2-kilobase transcript, the amount of which was associated with increased resistance. Southern blotting with the probe indicated an amplification of the IMPDH gene in the mycophenolic acid-resistant cells. Our findings suggest that the acquired mycophenolic acid resistance of the V79 cell variants is associated with increases in the amount and activity of IMPDH and the number of IMPDH gene copies.

scholarly journals Leakage of Microbial Endotoxin through the Implant-Abutment Interface in Oral Implants: An In Vitro Study

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Rhoodie Garrana ◽  
Govindrau Mohangi ◽  
Paulo Malo ◽  
Miguel Nobre
Keyword(s):  
In Vitro Study ◽  
Endotoxin Level ◽  
Oral Implants ◽  
E Coli ◽  
Implant Abutment ◽  
The Mean ◽  
The Impact ◽  
External Connections

Background. Endotoxin initiates osteoclastic activity resulting in bone loss. Endotoxin leakage through implant abutment connections negatively influences peri-implant bone levels.Objectives. (i) To determine if endotoxin can traverse different implant-abutment connection (IAC) designs; (ii) to quantify the amount of endotoxins traversing the IAC; (iii) to compare the in vitro comportments of different IACs.Materials and Methods. Twenty-seven IACs were inoculated withE. coliendotoxin. Six of the twenty-seven IACs were external connections from one system (Southern Implants) and the remaining twenty-one IACs were made up of seven internal IAC types from four different implant companies (Straumann, Ankylos, and Neodent, Southern Implants).Results. Of the 27 IACs tested, all 6 external IACs leaked measurable amounts of endotoxin. Of the remaining 21 internal IACs, 9 IACs did not show measurable leakage whilst the remaining 12 IACs leaked varying amounts. The mean log endotoxin level was significantly higher for the external compared to internal types (p=0.015).Conclusion. Within the parameters of this study, we can conclude that endotoxin leakage is dependent on the design of the IAC. Straumann Synocta, Straumann Cross-fit, and Ankylos displayed the best performances of all IACs tested with undetectable leakage after 7 days. Each of these IACs incorporated a morse-like component in their design. Speculation still exists over the impact of IAC endotoxin leakage on peri-implant tissues in vivo; hence, further investigations are required to further explore this.

scholarly journals THE EFFECTS OF CRUDE RECOMBINANT VIRAL PROTEIN VACCINES AGAINST GROUPER SLEEPY DISEASE IRIDOVIRUS (GSDIV) ON HUMPBACK GROUPER (Cromileptes altivelis)

2015 ◽  
Vol 10 (2) ◽  
pp. 163
Author(s):  
Ketut Mahardika ◽  
Indah Mastuti
Keyword(s):  
Capsid Protein ◽  
Expression System ◽  
Survival Rates ◽  
Coli Strain ◽  
Sea Bream ◽  
Recombinant Bacteria ◽  
Gene Target ◽  
E Coli ◽  
Pi Index ◽  
Potential Vaccine

Infection of Megalocytivirus cause serious mass mortality in marine fish in South East Asian countries. The aim of this study was to produce recombinant of GSDIV capsid protein and its protection to humpback grouper Cromileptes altivelis against grouper sleepy disease iridovirus (GSDIV). A major capsid protein (MCP) was selected for use as a crude subunit vaccines. This gene target (MCP) was inserted to the protein expression system vector of pET SUMO and cloned in cells bacteria Escherichia coli strain BL-21. The MCP was succeded to be induced using 1 mM of IPTG. Results of protein analysis using MALDI TOF-TOF indicated that the MCP has measurement of 49.566 kDa with PI index of 6.00, and contained 453 amino acids. BLAST homology analysis exhibited that the amino acid sequence of the MCP showed high similarity with MCP of Red Sea Bream Iridovirus (RSIV). E. coli expressing MCP protein was inactivated using 0.03% formalin overnight and washed using PBS. The inactivated E. coli as a crude subunit vaccine was then injected intramuscularly to humpback grouper juveniles. Subsequently, the juveniles were challenged tested with GSDIV. The juveniles vaccinated with the MCP recombinant bacteria showed significantly higher survival rates than control those vaccinated with PBS. Thus, the MCP fusion protein is considered as a potential vaccine against GSDIV infections in grouper.

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