Inhibition of DNA gyrase activity in an in vitro transcription-translation system stimulates gyr A expression in a DNA concentration dependent manner

1990 ◽  
Vol 214 (2) ◽  
pp. 397-406 ◽  
Author(s):  
Maynard Carty ◽  
Rolf Menzel
Keyword(s):  
Dna Gyrase ◽  
In Vitro Transcription ◽  
Translation System ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Dna Concentration

scholarly journals A tridecamer DNA sequence supports human mitochondrial RNA 3'-end formation in vitro.

1988 ◽  
Vol 8 (10) ◽  
pp. 4502-4509 ◽  
Author(s):  
T W Christianson ◽  
D A Clayton
Keyword(s):  
Dna Sequence ◽  
Transcription Termination ◽  
In Vitro Transcription ◽  
Mitochondrial Rna ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Transcription System ◽  
Flanking Regions

Vertebrate mitochondrial genomes contain a putative transcription termination site at the boundary between the genes for 16S rRNA and leucyl-tRNA. We have described previously an in vitro transcription system from human cells with the capacity to generate RNA 3' ends with the same map positions as those synthesized in vivo. By assaying the ability of variously truncated templates to support 3'-end formation, we demonstrated that the tridecamer sequence 5'-TGGCAGAGCCCCGG-3', contained entirely within the gene for leucyl-tRNA, is necessary to direct accurate termination. When two tridecamer sequences and their immediate flanking regions were placed in tandem, termination occurred at both promoter-proximal and promoter-distal sites. Furthermore, termination was competitively inhibited, in a concentration-dependent manner, by DNA containing the tridecamer sequence. These results suggest a modest sequence requirement for transcription termination that is contingent on a factor capable of recognizing the presence of the tridecamer DNA sequence.

scholarly journals Cell-free Expression of Proton-Coupled Folate Transporter in the Presence of Nanodiscs

2021 ◽  
Author(s):  
Hoa Quynh Do ◽  
Carla M Bassil ◽  
Elizabeth I Andersen ◽  
Michaela Jansen
Keyword(s):  
Tumor Cells ◽  
Plasma Membranes ◽  
In Vitro Translation ◽  
Translation System ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
E Coli ◽  
Membrane Scaffold ◽  
The Impact

The Proton-Coupled Folate Transporter (PCFT) is a transmembrane transport protein that controls the absorption of dietary folates in the small intestine. PCFT also mediates uptake of chemotherapeutically used antifolates into tumor cells. PCFT has been identified within lipid rafts observed in phospholipid bilayers of plasma membranes, a micro environment that is altered in tumor cells. The present study aimed at investigating the impact of different lipids within Lipid-protein nanodiscs (LPNs), discoidal lipid structures stabilized by membrane scaffold proteins, to yield soluble PCFT expression in an E. coli lysate-based cell-free transcription/translation system. In the absence of detergents or lipids, we observed PCFT quantitatively as precipitate in this system. We then explored the ability of LPNs to support solubilized PCFT expression when present during in-vitro translation. LPNs consisted of either dimyristoyl phosphatidylcholine (DMPC), palmitoyl-oleoyl phosphatidylcholine (POPC), or dimyristoyl phosphatidylglycerol (DMPG). While POPC did not lead to soluble PCFT expression, both DMPG and DMPC supported PCFT translation directly into LPNs, the latter in a concentration dependent manner. The results obtained through this study provide insights into the lipid preferences of PCFT. Membrane-embedded or solubilized PCFT will enable further studies with diverse biophysical approaches to enhance the understanding of the structure and molecular mechanism of folate transport through PCFT.

scholarly journals Functional Analysis of the Heat Shock Regulator HrcA of Chlamydia trachomatis

2002 ◽  
Vol 184 (23) ◽  
pp. 6566-6571 ◽  
Author(s):  
Adam C. Wilson ◽  
Ming Tan
Keyword(s):  
Heat Shock ◽  
Chlamydia Trachomatis ◽  
In Vitro Transcription ◽  
Negative Regulator ◽  
Heat Shock Gene ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Topological State ◽  
Heat Shock Gene Expression

ABSTRACT HrcA is a regulator of bacterial heat shock gene expression that binds to a cis-acting DNA element called CIRCE. It has been proposed that HrcA and CIRCE function as a repressor-operator pair. We have purified recombinant HrcA from the pathogenic bacterium Chlamydia trachomatis and have shown that it is a DNA-binding protein that functions as a negative regulator of transcription. HrcA bound specifically to the CIRCE element in a concentration-dependent manner. HrcA repressed the in vitro transcription of a chlamydial heat shock promoter, and this repression was promoter specific. HrcA-mediated repression appears to be dependent on the topological state of the promoter, as repression on a supercoiled promoter template was greater than that on a linearized template. These results provide direct support for the role of HrcA as a transcriptional repressor in bacteria. This is the first report of the in vitro reconstitution of transcriptional regulation in Chlamydia.

A tridecamer DNA sequence supports human mitochondrial RNA 3'-end formation in vitro

1988 ◽  
Vol 8 (10) ◽  
pp. 4502-4509
Author(s):  
T W Christianson ◽  
D A Clayton
Keyword(s):  
Dna Sequence ◽  
Transcription Termination ◽  
In Vitro Transcription ◽  
Mitochondrial Rna ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Transcription System ◽  
Flanking Regions

Vertebrate mitochondrial genomes contain a putative transcription termination site at the boundary between the genes for 16S rRNA and leucyl-tRNA. We have described previously an in vitro transcription system from human cells with the capacity to generate RNA 3' ends with the same map positions as those synthesized in vivo. By assaying the ability of variously truncated templates to support 3'-end formation, we demonstrated that the tridecamer sequence 5'-TGGCAGAGCCCCGG-3', contained entirely within the gene for leucyl-tRNA, is necessary to direct accurate termination. When two tridecamer sequences and their immediate flanking regions were placed in tandem, termination occurred at both promoter-proximal and promoter-distal sites. Furthermore, termination was competitively inhibited, in a concentration-dependent manner, by DNA containing the tridecamer sequence. These results suggest a modest sequence requirement for transcription termination that is contingent on a factor capable of recognizing the presence of the tridecamer DNA sequence.

scholarly journals Cooperative Interaction of CI Protein Regulates Lysogeny of Lactobacillus casei by Bacteriophage A2

1999 ◽  
Vol 73 (5) ◽  
pp. 3920-3929 ◽  
Author(s):  
Pilar García ◽  
Victor Ladero ◽  
Juan C. Alonso ◽  
Juan E. Suárez
Keyword(s):  
Rna Polymerase ◽  
Lactobacillus Casei ◽  
In Vitro Transcription ◽  
Cooperative Interaction ◽  
Dependent Manner ◽  
Transcription Start ◽  
Concentration Dependent Manner ◽  
Negative Effect ◽  
Dna Fragment

ABSTRACT The temperate bacteriophage A2 forms stable lysogens inLactobacillus casei. The A2-encoded cI product (CI), which is responsible for maintaining the A2 prophage in the lysogenic state, has been purified. The CI protein, which is a monomer of 25.3 kDa in solution, specifically binds to a 153-bp DNA fragment that contains two divergent promoters, PL and PR. These promoters mediate transcription fromcI and a putative cro, respectively. Three similar, although not identical, 20-bp inverted repeated DNA segments (operator sites O1, O2, and O3) were found in this segment. CI selectively interacts with O1, which is placed downstream from the transcription start point of the cro gene, and with O2 and O3, which overlap with the −35 region of the two promoters. Using a heterologous RNA polymerase, we have determined the transcription start points of PL and PR. CI exerts a negative effect on the in vitro transcription of PR by repositioning the RNA polymerase in a concentration-dependent manner. CI, when bound to O1and O2, enhances the positioning of the RNA polymerase with the PL promoter. Our data indicate that the CI protein regulates the lytic and lysogenic pathways of the A2 phage.

scholarly journals Impact of nanodisc lipid composition on cell-free expression of proton-coupled folate transporter

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0253184
Author(s):  
Hoa Quynh Do ◽  
Carla M. Bassil ◽  
Elizabeth I. Andersen ◽  
Michaela Jansen
Keyword(s):  
Tumor Cells ◽  
Plasma Membranes ◽  
In Vitro Translation ◽  
Translation System ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Dimyristoyl Phosphatidylcholine ◽  
Membrane Scaffold ◽  
The Impact

The Proton-Coupled Folate Transporter (PCFT) is a transmembrane transport protein that controls the absorption of dietary folates in the small intestine. PCFT also mediates uptake of chemotherapeutically used antifolates into tumor cells. PCFT has been identified within lipid rafts observed in phospholipid bilayers of plasma membranes, a micro environment that is altered in tumor cells. The present study aimed at investigating the impact of different lipids within Lipid-protein nanodiscs (LPNs), discoidal lipid structures stabilized by membrane scaffold proteins, to yield soluble PCFT expression in an E. coli lysate-based cell-free transcription/translation system. In the absence of detergents or lipids, we observed PCFT quantitatively as precipitate in this system. We then explored the ability of LPNs to support solubilized PCFT expression when present during in-vitro translation. LPNs consisted of either dimyristoyl phosphatidylcholine (DMPC), palmitoyl-oleoyl phosphatidylcholine (POPC), or dimyristoyl phosphatidylglycerol (DMPG). While POPC did not lead to soluble PCFT expression, both DMPG and DMPC supported PCFT translation directly into LPNs, the latter in a concentration dependent manner. The results obtained through this study provide insights into the lipid preferences of PCFT. Membrane-embedded or solubilized PCFT will enable further studies with diverse biophysical approaches to enhance the understanding of the structure and molecular mechanism of folate transport through PCFT.

Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity

2019 ◽  
Vol 26 (7) ◽  
pp. 494-501 ◽  
Author(s):  
Sameer Suresh Bhagyawant ◽  
Dakshita Tanaji Narvekar ◽  
Neha Gupta ◽  
Amita Bhadkaria ◽  
Ajay Kumar Gautam ◽  
...  
Keyword(s):  
Biological Effects ◽  
Exchange Chromatography ◽  
Health Concern ◽  
Carbohydrate Binding ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ic50 Values ◽  
Chickpea Lectin

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

Improved Method for Obtaining of Arctigenin from Arctium Lappa L. and its Antiproliferative Effect on Human Hepatocarcinoma HepG2 Cells

2020 ◽  
Vol 16 (3) ◽  
pp. 358-362
Author(s):  
Renan S. Teixeira ◽  
Paulo H.D. Carvalho ◽  
Jair A.K. Aguiar ◽  
Valquíria P. Medeiros ◽  
Ademar A. Da Silva Filho ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Crude Extract ◽  
Hepg2 Cells ◽  
Liver Carcinoma ◽  
Adhesion Assay ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Arctium Lappa ◽  
Nih 3T3

Background: Arctigenin is a lignan found in Arctium lappa L. (Asteraceae) that displays anti-inflammatory activities. Previous studies showed that the crude extract of A. Lappa has antitumor activity in human liver carcinoma, lung and stomach cancer cells. The aim of this study was to obtain arctigenin from A. lappa L., as well as to evaluate its antiproliferative effects in cells of liver carcinoma (HepG2) and fibroblasts (NIH/3T3). Methods: Arctigenin was obtained from the hydrolysis of arctiin, which was isolated from the crude extract of A. lappa. The effects of arctigenin and arctiin on HepG2 cell viability and cell adhesion were analyzed by MTT method. Adhesion assay was also carried out to evaluate the antitumor activity. Results: Our results showed that the analytical process to obtain arctigenin was fast and easy. In vitro experiments showed that arctigenin (107-269 μM) decreased HepG2 cells viability and did not cause cytotoxicity on NIH/3T3 cells. Arctigenin (27-269 μM) demonstrated anti-adhesion in HepG2 cells in a concentration-dependent manner, when compared with control. Conclusion: These results suggest a promising pharmacological activity for arctigenin as an antiproliferative compound.

scholarly journals Human Neuronal Cell Lines as An In Vitro Toxicological Tool for the Evaluation of Novel Psychoactive Substances

2021 ◽  
Vol 22 (13) ◽  
pp. 6785
Author(s):  
Valeria Sogos ◽  
Paola Caria ◽  
Clara Porcedda ◽  
Rafaela Mostallino ◽  
Franca Piras ◽  
...  
Keyword(s):  
Cell Death ◽  
Neuronal Cell ◽  
In Vitro Study ◽  
Dependent Manner ◽  
Novel Psychoactive Substances ◽  
Psychoactive Substances ◽  
Concentration Dependent Manner ◽  
Mechanisms Of Toxicity ◽  
Neuronal Cell Lines

Novel psychoactive substances (NPS) are synthetic substances belonging to diverse groups, designed to mimic the effects of scheduled drugs, resulting in altered toxicity and potency. Up to now, information available on the pharmacology and toxicology of these new substances is very limited, posing a considerable challenge for prevention and treatment. The present in vitro study investigated the possible mechanisms of toxicity of two emerging NPS (i) 4′-methyl-alpha-pyrrolidinoexanophenone (3,4-MDPHP), a synthetic cathinone, and (ii) 2-chloro-4,5-methylenedioxymethamphetamine (2-Cl-4,5-MDMA), a phenethylamine. In addition, to apply our model to the class of synthetic opioids, we evaluated the toxicity of fentanyl, as a reference compound for this group of frequently abused substances. To this aim, the in vitro toxic effects of these three compounds were evaluated in dopaminergic-differentiated SH-SY5Y cells. Following 24 h of exposure, all compounds induced a loss of viability, and oxidative stress in a concentration-dependent manner. 2-Cl-4,5-MDMA activates apoptotic processes, while 3,4-MDPHP elicits cell death by necrosis. Fentanyl triggers cell death through both mechanisms. Increased expression levels of pro-apoptotic Bax and caspase 3 activity were observed following 2-Cl-4,5-MDMA and fentanyl, but not 3,4-MDPHP exposure, confirming the different modes of cell death.

Sign in / Sign up

Export Citation Format

Share Document