scholarly journals Method combining BAC film and positive staining for the characterization of DNA intermediates by dark-field electron microscopy

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Yann Benureau ◽  
Eliana Moreira Tavares ◽  
Ali-Akbar Muhammad ◽  
Sonia Baconnais ◽  
Eric Le Cam ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Dark Field ◽  
In Vitro Assays ◽  
Positive Staining ◽  
Synthetic Dna ◽  
Dark Field Imaging ◽  
Alkyl Ammonium

Abstract DNA intermediate structures are formed in all major pathways of DNA metabolism. Transmission electron microscopy (TEM) is a tool of choice to study their choreography and has led to major advances in the understanding of these mechanisms, particularly those of homologous recombination (HR) and replication. In this article, we describe specific TEM procedures dedicated to the structural characterization of DNA intermediates formed during these processes. These particular DNA species contain single-stranded DNA regions and/or branched structures, which require controlling both the DNA molecules spreading and their staining for subsequent visualization using dark-field imaging mode. Combining BAC (benzyl dimethyl alkyl ammonium chloride) film hyperphase with positive staining and dark-field TEM allows characterizing synthetic DNA substrates, joint molecules formed during not only in vitro assays mimicking HR, but also in vivo DNA intermediates.

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

scholarly journals RBP-L, a transcription factor related to RBP-Jkappa.

1997 ◽  
Vol 17 (5) ◽  
pp. 2679-2687 ◽  
Author(s):  
S Minoguchi ◽  
Y Taniguchi ◽  
H Kato ◽  
T Okazaki ◽  
L J Strobl ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Notch Receptor ◽  
In Vitro Assays ◽  
Functional Interaction ◽  
Protein Protein Interaction ◽  
Isolation And Characterization ◽  
Signalling Molecule

RBP-Jkappa is a sequence-specific DNA binding protein which plays a central role in signalling downstream of the Notch receptor by physically interacting with its intracellular region. Although at least four Notch genes exist in mammals, it is unknown whether each Notch requires a specific downstream signalling molecule. Here we report isolation and characterization of a mouse RBP-Jkappa-related gene named RBP-L that is expressed almost exclusively in lung, in contrast to the ubiquitous expression of RBP-Jkappa. For simplicity, we propose to call RBP-Jkappa RBP-J. The RBP-L protein bound to a DNA sequence almost identical to that of RBP-J. Surprisingly, RBP-L did not interact with any of the known four mouse Notch proteins. Although we found that RBP-L and EBNA-2 cooperated in transcriptional activation, they did not show significantly strong protein-protein interaction that can be detected by several in vivo and in vitro assays. This is again in contrast to physical association of RBP-J with EBNA-2. Several models to explain functional interaction between RBP-L and EBNA-2 are discussed.

scholarly journals Use of in vivo and in vitro assays for the characterization of mammalian excision repair and isolation of repair proteins

1990 ◽  
Vol 236 (2-3) ◽  
pp. 223-238 ◽  
Author(s):  
J.H.J. Hoeijmakers ◽  
A.P.M. Eker ◽  
R.D. Wood ◽  
P. Robins
Keyword(s):  
Excision Repair ◽  
In Vitro Assays

Immunolocalization and characterization of the low molecular weight antigen (4–5 kDa) ofToxoplasma gondiithat elicits an early IgM response upon primary infection

Parasitology ◽  
1994 ◽  
Vol 108 (2) ◽  
pp. 139-145 ◽  
Author(s):  
S. Tomavo ◽  
G. Couvreur ◽  
M. A. Leriche ◽  
A. Sadak ◽  
A. Achbarou ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Primary Infection ◽  
Low Molecular Weight ◽  
Immuno Electron Microscopy ◽  
Surface Localization

SUMMARYA striking feature of toxoplasmic seroconversion is the prominent and early IgM response to a low molecular weight antigen of 4–5 kDa. Two different monoclonal antibodies directed against the 4–5 kDa antigen have been generated and used to characterize this molecule. Using these monoclonal antibodies, we could demonstrate the surface localization of the lowMrantigen by immunofluorescence and immuno-electron microscopy assays. By immunoblotting, we observed that one of the monoclonal antibodies was unable to recognize the 4–5 kDa antigen in tachyzoites propagated in cell culture, indicating an epitope variability betweenToxoplasma gondiitachyzoites grownin vivoandin vitro. We discuss the implications of this latter finding in the design of diagnostic reagents.

scholarly journals Pharmacological characterization of repeated administration of the first generation abused synthetic cannabinoid CP47,497

2016 ◽  
Vol 27 (3) ◽  
Author(s):  
Travis W. Grim ◽  
Kimberly L. Samano ◽  
Bogna Ignatowska-Jankowska ◽  
Qing Tao ◽  
Laura J. Sim-Selly ◽  
...  
Keyword(s):  
G Proteins ◽  
First Generation ◽  
Repeated Administration ◽  
Synthetic Cannabinoid ◽  
In Vitro Assays ◽  
Pharmacological Effects ◽  
Pharmacological Characterization

AbstractA series of in vivo and in vitro assays were conducted to characterize the pharmacological effects of the first generation abused synthetic cannabinoid CP47,497, a racemic bicyclic cannabinoid that is similar in structure to the potent, high-efficacy synthetic cannabinoid CP55,940. CP47,497 was less efficacious than CP55,940 in activating G-proteins and dose-dependently produced common CB

Dark field imaging of rattle-type silica nanorattles coated gold nanoparticles <italic>in vitro</italic> and<italic> in vivo</italic>

2013 ◽  
Vol 58 (7) ◽  
pp. 531-536 ◽  
Author(s):  
LongFei TAN ◽  
FangQiong TANG ◽  
ChangHui FU ◽  
HuiYu LIU ◽  
Dong CHEN ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Dark Field ◽  
Dark Field Imaging ◽  
Field Imaging

In vitro anti-Pythium insidiosum activity of biogenic silver nanoparticles

2018 ◽  
Vol 57 (7) ◽  
pp. 858-863 ◽  
Author(s):  
Júlia de Souza Silveira Valente ◽  
Caroline Quintana Braga ◽  
Carolina Litchina Brasil ◽  
Cristiane Telles Baptista ◽  
Guilherme Fonseca Reis ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Walls ◽  
In Vitro Assays ◽  
Pythium Insidiosum ◽  
Biogenic Silver Nanoparticles ◽  
Transmission Electron ◽  
Cellular Wall ◽  
Scanning Electron

AbstractPythium insidiosum belongs to the phylum Oomycota. It is capable of infecting mammals causing a serious condition called pythiosis, which affects mainly horses in Brazil and humans in Thailand. The objective of the present study was to verify the in vitro anti-P. insidiosum activity of a biogenic silver nanoparticle (bio-AgNP) formulation. The in vitro assays were evaluated on P. insidiosum isolates (n = 38) following the M38-A2 protocol. Damage to the P. insidiosum hyphae ultrastructure was verified by means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Bio-AgNP inhibition concentrations on P. insidiosum isolates ranged from 0.06 to 0.47 μg/ml. It was observed through SEM that P. insidiosum hyphae treated showed surface roughness, as well as cell walls with multiple retraction areas, loss of continuity, and rupture in some areas. The TEM of treated hyphae did not differentiate organelle structures; also, the cellular wall was rarefied, showing wrinkled and partly ruptured borders. The bio-AgNP evaluated has excellent in vitro anti-P. insidiosum activity. However, further studies on its in vivo action are necessary as so to determine the possibility of its use in the treatment of the disease in affected hosts.

scholarly journals Virulence parameters in the characterization of strains of Entamoeba histolytica

1997 ◽  
Vol 39 (2) ◽  
pp. 65-70 ◽  
Author(s):  
Maria A GOMES ◽  
Maria N. MELO ◽  
Gil P.M. PENA ◽  
Edward F. SILVA
Keyword(s):  
Entamoeba Histolytica ◽  
Cytopathic Effect ◽  
Axenic Culture ◽  
Vero Cells ◽  
In Vitro Assays ◽  
Biological Assays ◽  
Restriction Fragment Pattern

Differences in virulence of strains of Entamoeba histolytica have long been detected by various experimental assays, both in vivo and in vitro. Discrepancies in the strains characterization have been arisen when different biological assays are compared. In order to evaluate different parameters of virulence in the strains characterization, five strains of E. histolytica, kept under axenic culture, were characterized in respect to their, capability to induce hamster liver abscess, erythrophagocytosis rate and cytopathic effect upon VERO cells. It was found significant correlation between in vitro biological assays, but not between in vivo and in vitro assays. Good correlation was found between cytopathic effect and the mean number of uptaken erythrocytes, but not with percentage of phagocytic amoebae, showing that great variability can be observed in the same assay, according to the variable chosen. It was not possible to correlate isoenzyme and restriction fragment pattern with virulence indexes since all studied strains presented pathogenic patterns. The discordant results observed in different virulence assays suggests that virulence itself may not the directly assessed. What is in fact assessed are different biological characteristics or functions of the parasite more than virulence itself. These characteristics or functions may be related or not with pathogenic mechanisms occurring in the development of invasive amoebic disease

scholarly journals Characterization of βN-Octadecanoyl-5-hydroxytryptamide Anti-Inflammatory Effect

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3709
Author(s):  
Thais Biondino Sardella Giorno ◽  
Fernanda Alves Lima ◽  
Ana Laura Macedo Brand ◽  
Camila Martins de Oliveira ◽  
Claudia Moraes Rezende ◽  
...  
Keyword(s):  
Cell Types ◽  
In Vitro Assays ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Protein Extravasation ◽  
Anti Inflammatory ◽  
Different Cell Types

Background: N-octadecanoyl-5-hydroxytryptamide (C18-5HT) is an amide that can be obtained by the coupling of serotonin and octadecanoic acid. This study aims to characterize the in vivo and in vitro anti-inflammatory activity of C18-5HT. Methods: A subcutaneous air pouch model (SAP) was used. The exudates were collected from SAP after carrageenan injection to assess cell migration and inflammatory mediators production. RAW 264.7 cells were used for in vitro assays. Results: C18-5HT significantly inhibited leukocyte migration into the SAP as well as nitric oxide (NO) and cytokines production and protein extravasation. We also observed an reduction in some cytokines and an increase in IL-10 production. Assays conducted with RAW 264.7 cells indicated that C18-5HT inhibited NO and cytokine produced. Conclusions: Taken together, our data suggest that C18-5HT presents a significant effect in different cell types (leukocytes collected from exudate, mainly polumorphonuclear leukocytes and cell culture macrophages) and is a promising compound for further studies for the development of a new anti-inflammatory drug.

scholarly journals In Vitro and In Vivo Evaluation of Silver Nanoparticles Phytosynthesized Using Raphanus sativus L. Waste Extracts

Materials ◽  
2021 ◽  
Vol 14 (8) ◽  
pp. 1845
Author(s):  
Camelia Ungureanu ◽  
Irina Fierascu ◽  
Radu Claudiu Fierascu ◽  
Teodora Costea ◽  
Sorin Marius Avramescu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Silver Nanoparticles ◽  
Raphanus Sativus ◽  
Venturia Inaequalis ◽  
In Vitro Assays ◽  
Podosphaera Leucotricha ◽  
Scavenger Activity ◽  
Raphanus Sativus L

The aim of the current paper is the development of phytosynthesized silver nanoparticles mediated by Raphanus sativus L. extracts obtained through two extraction methods (temperature and microwave) and to test their potential application for controlling apple crops pathogens. The phytosynthesized materials were characterized by X-ray diffraction, scanning electron microscopy, and transmission electron microscopy. All the materials were evaluated in terms of antioxidant and in vitro antimicrobial activity (against bacteria, molds, and yeast: Escherichia coli ATCC 8738, Staphylococcus aureus ATTC 25923, Pseudomonas aeruginosa ATCC 9027, Salmonella typhimurium ATCC 14028, Candida albicans ATCC 10231, Venturia inaequalis, Podosphaera leucotricha, Fusarium oxysporum ATCC 48112, Penicillium hirsutum ATCC 52323, and Aspergillus niger ATCC 15475). Considering the results obtained in the in vitro assays, formulations based on nanoparticles phytosynthesized using Raphanus sativus L. waste extracts (RS1N) were evaluated as potential antifungal agents for horticultural crops protection, against Venturia inaequalis and Podosphaera leucotricha through in vivo assays. For the DPPH assay, the inhibition (%) varied between 37.06% (for RS1N at 0.8 mg/mL concentration) and 83.72% (for RS1N at 7.2 mg/mL concentration) compared to 19.97% (for RS2N at 0.8 mg/mL) and only 28.91% (for RS2N at 7.2 mg/mL). Similar results were obtained for RS3N (85.42% inhibition at 7.2 mg/mL) compared with RS4N (21.76% inhibition at 7.2 mg/mL). Regarding the ABTS assay, the highest scavenger activity values were recorded for samples RS1N (91.43% at 1.6 mg/mL) and RS3N (96.62% at 1.6 mg/mL).

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