A sensitive fluorographic method for the detection of nopaline and octopine synthase activities in Brassica crown-gall tissues

1986 ◽  
Vol 64 (2) ◽  
pp. 126-132 ◽  
Author(s):  
Larry A. Holbrook ◽  
Margaret Haffner ◽  
Brian L. A. Miki
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Crown Gall ◽  
Brassica Species ◽  
Paper Electrophoresis ◽  
In Vitro Assay ◽  
In Vitro Assays ◽  
Tissue Samples ◽  
Vitro Assay ◽  
Ti Plasmids

L-[guanido-14C]Arginine has been used as a precursor for nopaline and octopine syntheses in the in vitro assay for opine synthase activities. Fluorographic detection of [14C]nopaline and [14C]octopine was optimized following their separation from other labelled compounds by paper electrophoresis. Compared with detection by phenanthrenequinone, fluorography was more specific for the guanidine compounds and severalfold more sensitive. Alternate precursors such as α-[14C]ketoglutarate or [14C]pyruvate were poor substitutes for [14C]arginine because they generated compounds that comigrated with opines. Four nonopine radioactive spots recurred in the in vitro assays. These have not been identified but have been distinguished from both octopine and nopaline. This technique has provided clear evidence for transformation of Brassica species by Ti plasmids of Agrobacterium tumefaciens and will be valuable in the analysis of extremely small tissue samples.

scholarly journals In Vitro Characterization of the Enzyme Properties of the Phospholipid N-Methyltransferase PmtA from Agrobacterium tumefaciens

2009 ◽  
Vol 191 (7) ◽  
pp. 2033-2041 ◽  
Author(s):  
Meriyem Aktas ◽  
Franz Narberhaus
Keyword(s):  
Agrobacterium Tumefaciens ◽  
In Vitro Assay ◽  
Enzyme Properties ◽  
Vitro Assay ◽  
Plant Infection ◽  
In Vitro Characterization ◽  
End Products ◽  
And Control

ABSTRACT Agrobacterium tumefaciens requires phosphatidylcholine (PC) in its membranes for plant infection. The phospholipid N-methyltransferase PmtA catalyzes all three transmethylation reactions of phosphatidylethanolamine (PE) to PC via the intermediates monomethylphosphatidylethanolamine (MMPE) and dimethylphosphatidylethanolamine (DMPE). The enzyme uses S-adenosylmethionine (SAM) as the methyl donor, converting it to S-adenosylhomocysteine (SAH). Little is known about the activity of bacterial Pmt enzymes, since PC biosynthesis in prokaryotes is rare. In this article, we present the purification and in vitro characterization of A. tumefaciens PmtA, which is a monomeric protein. It binds to PE, the intermediates MMPE and DMPE, the end product PC, and phosphatidylglycerol (PG) and phosphatidylinositol. Binding of the phospholipid substrates precedes binding of SAM. We used a coupled in vitro assay system to demonstrate the enzymatic activity of PmtA and to show that PmtA is inhibited by the end products PC and SAH and the antibiotic sinefungin. The presence of PG stimulates PmtA activity. Our study provides insights into the catalysis and control of a bacterial phospholipid N-methyltransferase.

Assessment of Inhibition of Bovine Hepatic Cytochrome P450 by 43 Commercial Bovine Medicines Using a Combination of In Vitro Assays and Pharmacokinetic Data from the Literature

2020 ◽  
Vol 13 (2) ◽  
pp. 123-131
Author(s):  
Steven X. Hu ◽  
Chase A. Mazur ◽  
Kenneth L. Feenstra
Keyword(s):  
Liver Microsomes ◽  
In Vitro Assay ◽  
In Vitro Assays ◽  
Pharmacokinetic Studies ◽  
Pharmacokinetic Data ◽  
Vitro Assay ◽  
Administration Routes ◽  
Ic50 Values

Background: There has been a lack of information about the inhibition of bovine medicines on bovine hepatic CYP450 at their commercial doses and dosing routes. Objective: The aim of this work was to assess the inhibition of 43 bovine medicines on bovine hepatic CYP450 using a combination of in vitro assay and Cmax values from pharmacokinetic studies with their commercial doses and dosing routes in the literature. Methods: Those drugs were first evaluated through a single point inhibitory assay at 3 μM in bovine liver microsomes for six specific CYP450 metabolisms, phenacetin o-deethylation, coumarin 7- hydroxylation, tolbutamide 4-hydroxylation, bufuralol 1-hydroxylation, chlorzoxazone 6-hydroxylation and midazolam 1’-hydroxylation. When the inhibition was greater than 20% in the assay, IC50 values were then determined. The potential in vivo bovine hepatic CYP450 inhibition by those drugs was assessed using a combination of the IC50 values and in vivo Cmax values from pharmacokinetic studies at their commercial doses and administration routes in the literature. Results: Fifteen bovine medicines or metabolites showed in vitro inhibition on one or more bovine hepatic CYP450 metabolisms with different IC50 values. Desfuroylceftiour (active metabolite of ceftiofur), nitroxinil and flunixin have the potential to inhibit one of the bovine hepatic CYP450 isoforms in vivo at their commercial doses and administration routes. The rest of the bovine medicines had low risks of in vivo bovine hepatic CYP450 inhibition. Conclusion: This combination of in vitro assay and in vivo Cmax data provides a good approach to assess the inhibition of bovine medicines on bovine hepatic CYP450.

scholarly journals In Vitro Assays for Phototoxicity

1998 ◽  
Vol 17 (5) ◽  
pp. 567-570
Author(s):  
A. Kornhauser ◽  
R. R. Wei ◽  
W. G. Warner
Keyword(s):  
In Vitro Assay ◽  
In Vitro Assays ◽  
Vitro Assay ◽  
Cellular Targets ◽  
Photooxidative Damage ◽  
Us Government ◽  
Promising Area ◽  
Rna And Dna

This paper summarizes a few in vitro methods to assess photodamage in cells irradiated with UV of various wavelengths in the presence of a number of photo-sensitizers. A single in vitro assay for phototoxicity (photoirritation) is not likely to be predictive because of different mechanisms of phototoxicity and diverse cellular targets for injury. A number of methods have to be combined to provide a better prediction of these phenomena. Measurement of mechanistically relevant biomarkers also represents a promising area of in vitro testing for phototoxicity, and it is also briefly reviewed in this paper. Photodynamic sensitizers, representing a large class of phototoxic agents, can now be identified by sensitive measurement of photooxidative damage to cellular RNA and DNA. Currently, US government agencies have not identified a single in vitro assay for phototoxicity which would be acceptable for replacing an in vivo assay for regulatory purposes.

scholarly journals Synthesis and “in Vitro” Trypanocidal Activity Evaluation of Some Organo-iron Compounds.

2002 ◽  
Vol 8 (6) ◽  
pp. 329-332 ◽  
Author(s):  
Máfircio L. A. e Silva ◽  
Alberto F. Neto ◽  
Silvia A. Cardoso ◽  
Sérgio Albuquerque ◽  
Joseph Miller
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
In Vitro Assay ◽  
In Vitro Assays ◽  
Ferrocene Derivatives ◽  
Iron Compounds ◽  
Vitro Assay ◽  
Trypanocidal Activity ◽  
Activity Evaluation

Eight organo-iron ferrocene derivatives and arenocenium salts were prepared and evaluated by “in vitro” assay against one strain of Trypanosoma cruzi (Y). Six of the eight organo-iron compounds assayed, piperazinium diferrocenoate 1, η6-(o-xylene)-η5-(cyclopentadienyl) Iron(II) hexafluorophosphate 3, η6 -(mesitylene)-η5 -(cyclopentadienyl) iron(II) hexafluorphosphate 5, η6 -(durene)-η5 -(cyclopentadienyl) iron(II) hexafluorphosphate 6, η6 -(ρ-chlorotoluene)-η5 -(cyclopentadienyl) Iron(II) hexafluorphosphate 7 and η6 -(chlorobenzene)-η5 -(cyclopentadienyl) iron(II) picrate 8 , were poorly active in the “in vitro” assays. Only two compounds 1,1'–(N-pyperidinocarbonyl) ferrocene 2(IC50=2.4    μg/mL) and η6-(o-xylene)-η5(cyclopentadienyl) iron(II) picrate 4(IC50=12.08    μg/mL), were more active. Thus, some of the compounds are promising to be used against Chagas' disease as a prophylactic agents.

scholarly journals Evaluation of Iron Nutritional Quality of Two Amaranth Species sing Hemoglobin Repletion in Rats

HortScience ◽  
1995 ◽  
Vol 30 (4) ◽  
pp. 811A-811
Author(s):  
Anusuya Rangarajan ◽  
Wanda Chenoweth ◽  
John F. Kelly ◽  
Karen Agee
Keyword(s):  
Nutritional Quality ◽  
In Vitro Assay ◽  
Total Iron ◽  
In Vitro Assays ◽  
Sprague Dawley ◽  
Vitro Assay ◽  
Sprague Dawley Rats ◽  
Amaranth Species

Studies have been underway to evaluate the genetic variation in iron nutritional quality of the green leafy vegetable Amaranthus. Initial screening of 35 lines of amaranth from 12 species indicated wide variation in total iron, and small, but significant, differences in bioavailable iron, as determined by an in vitro assay. To verify if the differences in bioavailable iron detected by the in vitro assay were biologically significant, two lines of amaranth, A. tricolor Ames 5113 and A. hypochondriacus Ames 2171, were evaluated using a hemoglobin repletion assay in rats. Weanling Sprague-Dawley rats were made anemic by feeding an ironfree casein-based diet for 4 weeks. The anemic animals were fed treatment diets in which all Fe was provided by the amaranth lines. Hemoglobin levels were measured at the start and end of the treatment period to determine bioavailability. Although A. tricolor contained a higher concentration of total iron (670 ppm), the bioavailability of this iron to rats was lower than from the A. hypochondnacus line (total Fe = 210 ppm). Similar amounts of either amaranth line added to the diet produced similar changes in hemoglobin, although total iron concentrations were significantly different, confirming results observed with in vitro assays.

In Vitro Assay of Platelet Adhesiveness with Washed and Tanned Human Red Cells

1968 ◽  
Vol 20 (03/04) ◽  
pp. 384-396 ◽  
Author(s):  
G Zbinden ◽  
S Tomlin
Keyword(s):  
Platelet Adhesion ◽  
Sodium Citrate ◽  
Blood Platelets ◽  
In Vitro Assay ◽  
Red Cells ◽  
Platelet Adhesiveness ◽  
Vitro Assay ◽  
In Vitro System ◽  
Human Red Cells

SummaryAn in vitro system is described in which adhesion of blood platelets to washed and tannic acid-treated red cells was assayed quantitatively by microscopic observation. ADP, epinephrine and TAME produced a reversible increase in platelet adhesiveness which was antagonized by AMP. With Evans blue, polyanetholsulfonate, phthalanilide NSC 38280, thrombin and heparin at concentrations above 1-4 u/ml the increase was irreversible. The ADP-induced increase in adhesiveness was inhibited by sodium citrate, EDTA, AMP, ATP and N-ethylmaleimide. EDTA, AMP and the SH-blocker N-ethylmaleimide also reduced spontaneous platelet adhesion to red cells. No significant effects were observed with adenosine, phenprocoumon, 5-HT, phthalanilide NSC 57155, various estrogens, progestogens and fatty acids, acetylsalicylic acid and similarly acting agents, hydroxylamine, glucose and KCN. The method may be useful for the screening of thrombogenic and antithrombotic properties of drugs.

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