Isolation of 3 Flavonoids from Mentha longifolia (L.) Hudson subsp. longifolia and Determination of Their Genotoxic Potentials by Using the E. coli WP2 Test System

2011 ◽  
Vol 76 (9) ◽  
pp. T212-T217 ◽  
Author(s):  
Ozlem Baris ◽  
Mehmet Karadayi ◽  
Derya Yanmis ◽  
Zuhal Guvenalp ◽  
Tugba Bal ◽  
...  
Keyword(s):  
Test System ◽  
E Coli ◽  
Mentha Longifolia

Concentration determination of chromatin in unstained resin-embedded sections by means of Z-contrast in STEM

1990 ◽  
Vol 48 (2) ◽  
pp. 186-187
Author(s):  
M. Haider ◽  
B. Bohrmann
Keyword(s):  
Energy Loss ◽  
Freeze Substitution ◽  
Biological Macromolecules ◽  
Local Concentration ◽  
E Coli ◽  
Concentration Determination ◽  
Phosphorous Content ◽  
Z Contrast ◽  
Electron Energy Loss

The technique of Z-contrast in STEM offers the possibility to determine the local concentration of macromolecules like lipids, proteins or DNA. Contrast formation depends on the atomic composition of the particular structure. In the case of DNA, its phosphorous content discriminates it from other biological macromolecules. In our studies, sections of E. coli, the dinoflagellate Amphidinium carterae and Euglena spec. cells were used which were obtained by cryofixation followed by freeze-substitution into acetone with 3% glutaraldehyde. The samples were then embedded either in Lowicryl HM20 at low temperature or in Epon at high temperature. Sections were coated on both sides with 30Å carbon.The DF- and the inelastic image have been recorded simultaneously with a Cryo-STEM. This Cryo-STEM is equipped with a highly dispersive Electron Energy Loss Spectrometer. With this instrument pure Z-contrast can be achieved either with a Filtered DF-image divided by the inelastic image or, as is used in this paper, by dividing the conventional DF-image by an inelastic image which has been recorded with an inelastic detector whose response is dependent on the total energy loss of the inelastically scattered electrons.

Immunogold labeling of CMP-KDO synthetase produced by recombinant E. Coli cells

1987 ◽  
Vol 45 ◽  
pp. 924-925
Author(s):  
F. A. Durum ◽  
R. G. Goldman ◽  
T. J. Bolling ◽  
M. F. Miller
Keyword(s):  
Inclusion Bodies ◽  
Large Scale ◽  
Recombinant Dna ◽  
Test System ◽  
Cell Protein ◽  
Gram Negative Bacteria ◽  
Cytoplasmic Inclusions ◽  
Isolation And Purification ◽  
E Coli ◽  
Low Concentrations

CMP-KDO synthetase (CKS) is an enzyme which plays a key role in the synthesis of LPS, an outer membrane component unique to gram negative bacteria. CKS activates KDO to CMP-KDO for incorporation into LPS. The enzyme is normally present in low concentrations (0.02% of total cell protein) which makes it difficult to perform large scale isolation and purification. Recently, the gene for CKS from E. coli was cloned and various recombinant DNA constructs overproducing CKS several thousandfold (unpublished data) were derived. Interestingly, no cytoplasmic inclusions of overproduced CKS were observed by EM (Fig. 1) which is in contrast to other reports of large proteinaceous inclusion bodies in various overproducing recombinant strains. The present immunocytochemical study was undertaken to localize CKS in these cells.Immune labeling conditions were first optimized using a previously described cell-free test system. Briefly, this involves soaking small blocks of polymerized bovine serum albumin in purified CKS antigen and subjecting them to various fixation, embedding and immunochemical conditions.

Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

2018 ◽  
Vol 40 (4) ◽  
Author(s):  
Dang Thi Ngoc Ha ◽  
Le Thi Thu Hong ◽  
Truong Nam Hai
Keyword(s):  
Recombinant Antibody ◽  
Blood Type ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Blood Group Antigens ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Scfv ◽  
Single Chain Variable Fragments

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody. 

Identification of N-Substituted Triazolo-azetidines as Novel Antibacterials using pDualrep2 HTS Platform

2019 ◽  
Vol 22 (5) ◽  
pp. 346-354
Author(s):  
Yan A. Ivanenkov ◽  
Renat S. Yamidanov ◽  
Ilya A. Osterman ◽  
Petr V. Sergiev ◽  
Vladimir A. Aladinskiy ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Large Scale ◽  
Underlying Mechanism ◽  
Luciferase Assay ◽  
Reporter System ◽  
E Coli ◽  
Starting Point ◽  
High Concentrations ◽  
Mic Value

Aim and Objective: Antibiotic resistance is a serious constraint to the development of new effective antibacterials. Therefore, the discovery of the new antibacterials remains one of the main challenges in modern medicinal chemistry. This study was undertaken to identify novel molecules with antibacterial activity. Materials and Methods: Using our unique double-reporter system, in-house large-scale HTS campaign was conducted for the identification of antibacterial potency of small-molecule compounds. The construction allows us to visually assess the underlying mechanism of action. After the initial HTS and rescreen procedure, luciferase assay, C14-test, determination of MIC value and PrestoBlue test were carried out. Results: HTS rounds and rescreen campaign have revealed the antibacterial activity of a series of Nsubstituted triazolo-azetidines and their isosteric derivatives that has not been reported previously. Primary hit-molecule demonstrated a MIC value of 12.5 µg/mL against E. coli Δ tolC with signs of translation blockage and no SOS-response. Translation inhibition (26%, luciferase assay) was achieved at high concentrations up to 160 µg/mL, while no activity was found using C14-test. The compound did not demonstrate cytotoxicity in the PrestoBlue assay against a panel of eukaryotic cells. Within a series of direct structural analogues bearing the same or bioisosteric scaffold, compound 2 was found to have an improved antibacterial potency (MIC=6.25 µg/mL) close to Erythromycin (MIC=2.5-5 µg/mL) against the same strain. In contrast to the parent hit, this compound was more active and selective, and provided a robust IP position. Conclusion: N-substituted triazolo-azetidine scaffold may be used as a versatile starting point for the development of novel active and selective antibacterial compounds.

scholarly journals ANTIMICROBIAL AND ANTIOXIDANT EFFECT OF NATURAL EXTRACTS FROM LEAVES, ROOT, STEM AND FLOWERS OF BACCHARIS LATIFOLIA FROM ECUADOR

2020 ◽  
pp. 78-84
Author(s):  
FAVIAN BAYAS-MOREJON ◽  
ANGELICA TIGRE ◽  
RIVELINO RAMON ◽  
DANILO YANEZ
Keyword(s):  
Antimicrobial Activity ◽  
Antioxidant Activity ◽  
Potassium Phosphate ◽  
Natural Antioxidants ◽  
Butylated Hydroxytoluene ◽  
Penicillin G ◽  
Diffusion Method ◽  
E Coli ◽  
Antibacterial And Antioxidant Activity

Objective: The increase in chronic and degenerative diseases and the use of synthetic antioxidants such as (butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT)) are being restricted because they can be considered carcinogenic. Therefore, there is a growing interest in the search for natural antioxidants, especially from plants, due to their content in different bioactive compounds, such as antioxidants and antimicrobials. To evaluate the antibacterial and antioxidant activity of Baccharislatifolia extracts. Methods: For the determination of the antimicrobial activity of extracts of leaves, root, stem and flowers of Baccharislatifolia (Bl), the disk plate diffusion method was used, the strains of Listeria, Salmonella and E. coli were studied; antibiotics Penicillin G and Ciprofloxacin were the controls. For the antioxidant activity, a solution of H2O2 (Abs at 230 nm) was prepared in Potassium Phosphate Monobasic-Sodium Hydroxide buffer. Results: The antimicrobial activity against Listeria and Salmonella, showed that the extracts of leaves and flowers were more effective with inhibition zones>15 mm and>20 mm respectively. In front of E. coli, the extracts of flowers and stem were the best with zones>7.0 mm. Antibiotics studied inhibited the development of Listeria and Salmonella. However, E. coli isolates were resistant. In the antioxidant activity, the flower extract of Bl in 60 mg/ml presents a higher effect with 47.25%. Conclusion: Bl extracts from leaves and flowers were more efficient both in their antimicrobial and antioxidant capacity.

Determination of genotoxic and antigenotoxic properties of essential oil from Ferula orientalis L. using Ames/Salmonella and E. coli WP2 bacterial test systems

2012 ◽  
Vol 30 (8) ◽  
pp. 714-723 ◽  
Author(s):  
Hakan Ozkan ◽  
Derya Yanmis ◽  
Mehmet Karadayi ◽  
Tugba Bal ◽  
Ozlem Baris ◽  
...  
Keyword(s):  
Essential Oil ◽  
E Coli ◽  
Test Systems

Determination of methane yield of cellulose using different experimental setups

2014 ◽  
Vol 70 (4) ◽  
pp. 599-604 ◽  
Author(s):  
Bing Wang ◽  
Ivo Achu Nges ◽  
Mihaela Nistor ◽  
Jing Liu
Keyword(s):  
Water Column ◽  
Test System ◽  
Methane Yield ◽  
Biochemical Methane Potential ◽  
Methane Potential ◽  
Bmp Test ◽  
Potential Test

In this work, biochemical methane potential (BMP) tests with cellulose as a model substrate were performed with the aid of three manually operated or conventional experimental setups (based on manometer, water column and gas bag) and one automated apparatus specially designed for analysis of BMP. The methane yields were 340 ± 18, 354 ± 13, 345 ± 15 and 366 ± 5 ml CH4/g VS obtained from experimental setups with manometer, water column, gas bag, and automatic methane potential test system, which corresponded to a biodegradability of 82, 85, 83 and 88% respectively. The results demonstrated that the methane yields of cellulose obtained from conventional and automatic experimental setups were comparable; however, the methane yield obtained from the automated apparatus showed greater precision. Moreover, conventional setups for the BMP test were more time- and labour-intensive compared with the automated apparatus.

scholarly journals ELISA- and Activity Assay-Based Quantification of BMP-2 Released In Vitro Can Be Biased by Solubility in “Physiological” Buffers and an Interfering Effect of Chitosan

Pharmaceutics ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 582
Author(s):  
Julius Sundermann ◽  
Steffen Sydow ◽  
Laura Burmeister ◽  
Andrea Hoffmann ◽  
Henning Menzel ◽  
...  
Keyword(s):  
Matrix Effects ◽  
In Vitro Release ◽  
Tween 20 ◽  
Activity Assay ◽  
E Coli ◽  
Reversible Aggregation ◽  
Aggregation Tendency ◽  
Bio Assay

Chitosan nanogel-coated polycaprolactone (PCL) fiber mat-based implant prototypes with tailored release of bone morphogenic protein 2 (BMP-2) are a promising approach to achieve implant-mediated bone regeneration. In order to ensure reliable in vitro release results, the robustness of a commercially available ELISA for E. coli-derived BMP-2 and the parallel determination of BMP-2 recovery using a quantitative biological activity assay were investigated within a common release setup, with special reference to solubility and matrix effects. Without bovine serum albumin and Tween 20 as solubilizing additives to release media buffed at physiological pH, BMP‑2 recoveries after release were notably reduced. In contrast, the addition of chitosan to release samples caused an excessive recovery. A possible explanation for these effects is the reversible aggregation tendency of BMP-2, which might be influenced by an interaction with chitosan. The interfering effects highlighted in this study are of great importance for bio-assay-based BMP-2 quantification, especially in the context of pharmaceutical release experiments.

Potential Public Health Significance of Non-Escherichia coli Coliforms in Food

1979 ◽  
Vol 42 (2) ◽  
pp. 161-163 ◽  
Author(s):  
ROBERT M. TWEDT ◽  
BRENDA K. BOUTIN
Keyword(s):  
Public Health ◽  
Escherichia Coli ◽  
Diarrheal Disease ◽  
Health Hazard ◽  
High Yield ◽  
Potential Health ◽  
E Coli ◽  
Potential Health Hazard ◽  
Health Significance

Several coliform species other than Escherichia coli are often associated with and possibly responsible for acute and chronic diarrheal disease. Recent evidence suggests that non-Escherichia coli coliforms may be capable of colonizing the human intestine and producing enterotoxin(s) in high-yield. Whether these organisms are newly capable of causing disease because of infestation with extrachromosomal factors mediating pathogenicity or simply because of inherent pathogenic capabilities that have gone unrecognized, they pose a potential health hazard. Food, medical, and public health microbiologists should be aware that the non-E. coli coliforms contaminating foods may be potential enteropathogens. This possibility may make determination of their pathogenic capabilities even more important than identification of their taxonomic characteristics.

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