scholarly journals In Vitro Assay of Translation Inhibition by Trichothecenes Using a Commercially Available System

Toxins ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 696
Author(s):  
Takahito Toyotome ◽  
Katsuhiko Kamei
Keyword(s):  
Protein Synthesis ◽  
Secondary Metabolites ◽  
Operation Time ◽  
In Vitro Assay ◽  
Ease Of Use ◽  
Vitro Assay ◽  
Vast Number ◽  
Translation Inhibition ◽  
Short Operation Time

Trichothecenes are a family of major secondary metabolites produced by some common filamentous fungi, including plant pathogenic and entomopathogenic fungi. It may be considered difficult to conduct a comparison between the toxicities of trichothecenes with consideration of different conditions and cell lines. In the current study, we developed an in vitro assay based on a commercially available system to estimate the translation inhibition, that is, the main toxicity, of trichothecenes. The assay was applied to estimate the inhibition of protein synthesis by trichothecenes. Initially, we examined the assay using trichothecene dissolved in water followed by an assessment of trichothecene solutions dissolved in acetonitrile. The obtained data showed that the assay tolerated the small amount of acetonitrile. The assay examined in this study has the advantages of a short operation time (one day), ease of use, and data stability, as it is a non-cell-based assay whose components are commercially available. It is expected that this assay will contribute to the evaluation of the toxicity of a vast number of trichothecenes.

‘Unmasking’ of stored maternal mRNAs and the activation of protein synthesis at fertilization in sea urchins

Development ◽  
1991 ◽  
Vol 111 (2) ◽  
pp. 623-633 ◽  
Author(s):  
L.C. Kelso-Winemiller ◽  
M.M. Winkler
Keyword(s):  
Protein Synthesis ◽  
Sea Urchins ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Translational Machinery ◽  
Maternal Mrnas ◽  
Additional Support ◽  
And Control

The isolation and in vitro assay of maternal mRNPs has led to differing conclusions as to whether maternal mRNAs in sea urchin eggs are in a repressed or ‘masked’ form. To circumvent the problems involved with in vitro approaches, we have used an in vivo assay to determine if the availability of mRNA and/or components of the translational machinery are limiting protein synthesis in the unfertilized egg. This assay involves the use of a protein synthesis elongation inhibitor to create a situation in the egg in which there is excess translational machinery available to bind mRNA. Eggs were fertilized and the rate of entry into polysomes of individual mRNAs was measured in inhibitor-treated and control embryos using 32P-labeled cDNA probes. The fraction of ribosomes in polysomes and the polysome size were also determined. The results from this in vivo approach provide strong evidence for the coactivation of both mRNAs and components of the translational machinery following fertilization. The average polysome size increases from 7.5 ribosomes per message in 15 min embryos to approximately 10.8 ribosomes in 2 h embryos. This result gives additional support to the idea that translational machinery, as well as mRNA, is activated following fertilization. We also found that individual mRNAs are recruited into polysomes with different kinetics, and that the fraction of an mRNA in polysomes in the unfertilized egg correlates with the rate at which that mRNA is recruited into polysomes following fertilization.

scholarly journals The Effect of Various pH Medium on the Secondary Metabollites Production from Trichoderma harzianum T10 to Control Damping Off on Cucumber Seedlings

2020 ◽  
Vol 3 (2) ◽  
pp. 65
Author(s):  
Nur Chalimah ◽  
Loekas Soesanto ◽  
Woro Sri Suharti
Keyword(s):  
Secondary Metabolites ◽  
Trichoderma Harzianum ◽  
Disease Incidence ◽  
In Vitro Assay ◽  
Cucumber Seedling ◽  
Damping Off ◽  
In Planta ◽  
Vitro Assay ◽  
Cucumber Seedlings

Damping-off is one of the main diseases in cucumber seedlings caused by Pythium sp. Secondary metabolites of Trichoderma harzianum T10 can conduct the control of the disease. The pH of the medium influences the production of secondary metabolites. The research aimed to determine the effective pH medium on production of T. harzianum T10 secondary metabolites, and the effect of the T. harzianum T10 secondary metabolites application in damping-off disease control also to the growth of cucumber seedling. The research was consist of two steps; 1) in vitro assay with various pH levels 5; 3; 3.5; 4; 4.5; 5.5; 6; 6.5; and 7, 2) In planta treatments consisted of control, fungicide (Mancozeb), secondary metabolites in pH 5 and 5.5 with the concentration of 5, 10 and 15% each. The research showed that: 1) the effective pH medium for the production of T. harzianum T10 secondary metabolites was 5 and 5.5. 2) application of the T. harzianum T10 secondary metabolites on pH 5 and 5.5 with a concentration of 5, 10, and 15% could decrease the disease incidence and support cucumber seedling growth.

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.

Angiogenic Effect of Pravastatin Alone and with Sera from Healthy and Complicated Pregnancies Studied by in vitro Vasculogenesis/Angiogenesis Assay

2021 ◽  
pp. 1-9
Author(s):  
Anita Virtanen ◽  
Outi Huttala ◽  
Kati Tihtonen ◽  
Tarja Toimela ◽  
Tuula Heinonen ◽  
...  
Keyword(s):  
Direct Effect ◽  
Late Onset ◽  
Maternal Serum ◽  
In Vitro Assay ◽  
Serum Samples ◽  
Therapeutic Concentration ◽  
Tubule Formation ◽  
Vitro Assay ◽  
Angiogenesis Assay

<b><i>Objective:</i></b> To determine the direct effect of pravastatin on angiogenesis and to study the interaction between pravastatin and maternal sera from women with early- or late-onset pre-eclampsia (PE), intrauterine growth restriction, or healthy pregnancy. <b><i>Methods:</i></b> We collected 5 maternal serum samples from each group. The effect of pravastatin on angiogenesis was assessed with and without maternal sera by quantifying tubule formation in a human-based in vitro assay. Pravastatin was added at 20, 1,000, and 8,000 ng/mL concentrations. Concentrations of angiogenic and inflammatory biomarkers in serum and in test medium after supplementation of serum alone and with pravastatin (1,000 ng/mL) were measured. <b><i>Results:</i></b> Therapeutic concentration of pravastatin (20 ng/mL) did not have significant direct effect on angiogenesis, but the highest concentrations inhibited angiogenesis. Pravastatin did not change the levels of biomarkers in the test media. There were no changes in angiogenesis when therapeutic dose of pravastatin was added with maternal sera, but there was a trend to wide individual variation towards enhanced angiogenesis, particularly in the early-onset PE group. <b><i>Conclusions:</i></b> At therapeutic concentration, pravastatin alone or with maternal sera has no significant effect on angiogenesis, but at high concentrations the effect seems to be anti-angiogenic estimated by in vitro assay.

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