scholarly journals A bacterial assay for rapid screening of IAA catabolic enzymes

2019 ◽  
Author(s):  
Brunoni Federica ◽  
Collani Silvio ◽  
Šimura Jan ◽  
Schmid Markus ◽  
Bellini Catherine ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Transport Processes ◽  
Rapid Screening ◽  
Supernatant Fraction ◽  
Enzymatic Reactions ◽  
Activity Measurements ◽  
Glucosyl Ester

AbstractBackgroundPlants rely on concentration gradients of the native auxin, indole-3-acetic acid (IAA), to modulate plant growth and development. Both metabolic and transport processes participate in the dynamic regulation of IAA homeostasis. Free IAA levels can be reduced by inactivation mechanisms, such as conjugation and degradation. IAA can be conjugated via ester linkage to glucose, or via amide linkage to amino acids, and degraded via oxidation. Members of the UDP glucosyl transferase (UGT) family catalyze the conversion of IAA to indole-3-acetyl-1-glucosyl ester (IAGlc); by contrast, IAA is irreversibly converted to indole-3-acetyl-L-aspartic acid (IAAsp) and indole-3-acetyl glutamic acid (IAGlu) by Group II of the GRETCHEN HAGEN3 (GH3) family of acyl amido synthetases. DIOXYGENASE OF AUXIN OXIDATION (DAO) irreversibly oxidizes IAA to oxindole-3-acetic acid (oxIAA) and, in turn, oxIAA can be further glucosylated to oxindole-3-acetyl-1-glucosyl ester (oxIAGlc) by UGTs. These metabolic pathways have been identified based on mutant analyses, in vitro activity measurements, and in planta feeding assays. In vitro assays for studying protein activity are based on expressing Arabidopsis enzymes in a recombinant form in bacteria or yeast followed by recombinant protein purification. However, the need to extract and purify the recombinant proteins represents a major obstacle when performing in vitro assays.ResultsIn this work we report a rapid, reproducible and cheap method to screen the enzymatic activity of recombinant proteins that are known to inactivate IAA. The enzymatic reactions are carried out directly in bacteria that express the recombinant protein. The enzymatic products can be measured by direct injection of a small supernatant fraction from the bacterial culture on ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UHPLC-ESI-MS/MS). Experimental procedures were optimized for testing the activity of different classes of IAA-modifying enzymes without the need to purify recombinant protein.ConclusionsThis new method represents an alternative to existing in vitro assays. It can be applied to the analysis of IAA metabolites that are produced upon supplementation of substrate to engineered bacterial cultures and can be used for a rapid screening of orthologous candidate genes from non-model species.

scholarly journals A bacterial assay for rapid screening of IAA catabolic enzymes

Plant Methods ◽  
2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Federica Brunoni ◽  
Silvio Collani ◽  
Jan Šimura ◽  
Markus Schmid ◽  
Catherine Bellini ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Transport Processes ◽  
Rapid Screening ◽  
In Vitro Assays ◽  
Supernatant Fraction ◽  
Activity Measurements ◽  
Glucosyl Ester

Abstract Background Plants rely on concentration gradients of the native auxin, indole-3-acetic acid (IAA), to modulate plant growth and development. Both metabolic and transport processes participate in the dynamic regulation of IAA homeostasis. Free IAA levels can be reduced by inactivation mechanisms, such as conjugation and degradation. IAA can be conjugated via ester linkage to glucose, or via amide linkage to amino acids, and degraded via oxidation. Members of the UDP glucosyl transferase (UGT) family catalyze the conversion of IAA to indole-3-acetyl-1-glucosyl ester (IAGlc); by contrast, IAA is irreversibly converted to indole-3-acetyl-l-aspartic acid (IAAsp) and indole-3-acetyl glutamic acid (IAGlu) by Group II of the GRETCHEN HAGEN3 (GH3) family of acyl amido synthetases. Dioxygenase for auxin oxidation (DAO) irreversibly oxidizes IAA to oxindole-3-acetic acid (oxIAA) and, in turn, oxIAA can be further glucosylated to oxindole-3-acetyl-1-glucosyl ester (oxIAGlc) by UGTs. These metabolic pathways have been identified based on mutant analyses, in vitro activity measurements, and in planta feeding assays. In vitro assays for studying protein activity are based on producing Arabidopsis enzymes in a recombinant form in bacteria or yeast followed by recombinant protein purification. However, the need to extract and purify the recombinant proteins represents a major obstacle when performing in vitro assays. Results In this work we report a rapid, reproducible and cheap method to screen the enzymatic activity of recombinant proteins that are known to inactivate IAA. The enzymatic reactions are carried out directly in bacteria that produce the recombinant protein. The enzymatic products can be measured by direct injection of a small supernatant fraction from the bacterial culture on ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UHPLC–ESI-MS/MS). Experimental procedures were optimized for testing the activity of different classes of IAA-modifying enzymes without the need to purify recombinant protein. Conclusions This new method represents an alternative to existing in vitro assays. It can be applied to the analysis of IAA metabolites that are produced upon supplementation of substrate to engineered bacterial cultures and can be used for a rapid screening of orthologous candidate genes from non-model species.

Callus Induction and Plant Regeneration in Rauvolfia Serpentina (L.) Benth Ex. Kurz.

2009 ◽  
Vol 24 ◽  
pp. 82-88 ◽  
Author(s):  
Saraswoti Aryal ◽  
Sanu Devi Joshi
Keyword(s):  
Acetic Acid ◽  
Callus Induction ◽  
Coconut Milk ◽  
Ms Medium ◽  
Rauvolfia Serpentina ◽  
History Museum ◽  
Short Period ◽  
Murashige Skoog ◽  
Callus Tissue Culture

Rauvolfia serpentina (L.) ex. Kurz is an important medicinal plant. Callus induction and regeneration was studied from stem explant of in-vitro grown plant of Rauvolfia serpentina(L.) Benth. ex Kurz (Apocynaceae) on Murashige Skoog (1962) medium supplemented with 1mg/l 2,4-Dichlorophenocy acetic acid (2,4-D) and 1mg/l Kinetin (Kn). Vigorous growth of callus occurs after 4 weeks of culture. Callus was sub-cultured on Murashige and Skoog (MS) medium supplemented with different concentration of 2, 4-D (0.5-3.0 mg/l) and 10% coconut milk. Regeneration of plantlets occurred on MS medium containing 3 mg/1 of 2, 4-D and 10% coconut milk. These plantlets were rooted on MS medium supplemented with 1 mg/l IAA .The regenerated plantlets were able to grow on soil after short period ofacclimatization. Key words: Explant; In-vitro culture; MS medium;  2, 4 Dichlorophenoxy acetic acid; Kinetin; Callus; Tissue culture; Coconut milk. Journal of Natural History Museum Vol. 24, 2009 Page: 82-88

Advancement Of Changed Pulsincap Medicate Conveyance Arrangement Of Metronidazole For Tranquilize Focusing

2020 ◽  
Vol 2 (05) ◽  
pp. I-IV
Author(s):  
Roshni Jha ◽  
Anjali Minj
Keyword(s):  
Acetic Acid ◽  
Sodium Alginate ◽  
Guar Gum

A changed Pulsincap measurements type of metronidazole was created to target tranquilize discharge in the colon. Groups of hard gelatin cases were treated with formaldehyde keeping the tops in that capacity. Metronidazole pellets arranged by expulsion spheronization technique were consolidated into these particular container shells and stopped with polymers guar gum, hydroxypropylmethylcellulose 10K, carboxymethylcellulose sodium and sodium alginate independently at fixations 20 mg, 30 mg and 40 mg. The filled cases were totally covered with 5% cellulose acetic acid derivation phthalate to forestall variable gastric purging. All the definitions were tested to decide sedate substance and the capacity of the adjusted Pulsincap to give colon-explicit medication conveyance was surveyed by in vitro tranquilize discharge concentrates in cushion pH 1.2 for 2 h, pH 7.4 (reproduced intestinal liquid) for 3 h and pH 6.8 (animated colonic liquid) for 7 h. The outcomes showed that critical medication discharge happened simply after 5 h from the beginning of analysis.

In vitro Lipolysis as a Tool for the Establishment of IVIVC for Lipid-Based Drug Delivery Systems

2019 ◽  
Vol 16 (8) ◽  
pp. 688-697
Author(s):  
Ravinder Verma ◽  
Deepak Kaushik
Keyword(s):  
Drug Delivery ◽  
Ph Value ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Rapid Screening ◽  
Resonance Spectroscopy ◽  
Fed State ◽  
In Vitro Lipolysis ◽  
Ph Stat

: In vitro lipolysis has emerged as a powerful tool in the development of in vitro in vivo correlation for Lipid-based Drug Delivery System (LbDDS). In vitro lipolysis possesses the ability to mimic the assimilation of LbDDS in the human biological system. The digestion medium for in vitro lipolysis commonly contains an aqueous buffer media, bile salts, phospholipids and sodium chloride. The concentrations of these compounds are defined by the physiological conditions prevailing in the fasted or fed state. The pH of the medium is monitored by a pH-sensitive electrode connected to a computercontrolled pH-stat device capable of maintaining a predefined pH value via titration with sodium hydroxide. Copenhagen, Monash and Jerusalem are used as different models for in vitro lipolysis studies. The most common approach used in evaluating the kinetics of lipolysis of emulsion-based encapsulation systems is the pH-stat titration technique. This is widely used in both the nutritional and the pharmacological research fields as a rapid screening tool. Analytical tools for the assessment of in vitro lipolysis include HPLC, GC, HPTLC, SEM, Cryo TEM, Electron paramagnetic resonance spectroscopy, Raman spectroscopy and Nanoparticle Tracking Analysis (NTA) for the characterization of the lipids and colloidal phases after digestion of lipids. Various researches have been carried out for the establishment of IVIVC by using in vitro lipolysis models. The current publication also presents an updated review of various researches in the field of in vitro lipolysis.

Novel Compound from Flowers of Moringa oleifera Active Against Multi- Drug Resistant Gram-negative Bacilli

2020 ◽  
Vol 20 (1) ◽  
pp. 69-75
Author(s):  
Santi M. Mandal ◽  
Subhanil Chakraborty ◽  
Santanu Sahoo ◽  
Smritikona Pyne ◽  
Samaresh Ghosh ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Moringa Oleifera ◽  
Aqueous Acetic Acid ◽  
Phytochemical Analysis ◽  
Hydroxyl Groups ◽  
Drug Resistant ◽  
Gram Negative ◽  
Isolated Compound ◽  
Conventional Antibiotics

Background: The need for suitable antibacterial agents effective against Multi-drug resistant Gram-negative bacteria is acknowledged globally. The present study was designed to evaluate the possible antibacterial potential of an extracted compound from edible flowers of Moringa oleifera. Methods: Five different solvents were used for preparing dried flower extracts. The most effective extract was subjected to fractionation and further isolation of the active compound with the highest antibacterial effect was obtained using TLC, Column Chromatography and reverse phase- HPLC. Approaches were made for characterization of the isolated compound using FTIR, NMR and Mass spectrometry. Antibacterial activity was evaluated according to the CLSI guidelines. Results: One fraction of aqueous acetic acid extract of M. oleifera flower was found highly effective and more potent than conventional antibiotics of different classes against Multi-drug resistant Gram-negative bacilli (MDR-GNB) when compared. The phytochemical analysis of the isolated compound revealed the presence of hydrogen-bonded amine and hydroxyl groups attributable to unsaturated amides. Conclusion: The present study provided data indicating a potential for use of the flowers extract of M. oleifera in the fight against infections caused by lethal MDR-GNB. Recommendations: Aqueous acetic acid flower extract of M. oleifera is effective, in-vitro, against Gram-negative bacilli. This finding may open a scope in pharmaceutics for the development of new classes of antibiotics.

scholarly journals Characterization of Plant Growth-Promoting Traits and Inoculation Effects on Triticum durum of Actinomycetes Isolates under Salt Stress Conditions

Soil Systems ◽  
2021 ◽  
Vol 5 (2) ◽  
pp. 26
Author(s):  
Rihab Djebaili ◽  
Marika Pellegrini ◽  
Massimiliano Rossi ◽  
Cinzia Forni ◽  
Maria Smati ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Salt Stress ◽  
Plant Growth ◽  
Durum Wheat ◽  
Growth And Development ◽  
Indole Acetic Acid ◽  
Greenhouse Experiment ◽  
Hydrocyanic Acid ◽  
In Vitro Assays

This study aimed to characterize the halotolerant capability, in vitro, of selected actinomycetes strains and to evaluate their competence in promoting halo stress tolerance in durum wheat in a greenhouse experiment. Fourteen isolates were tested for phosphate solubilization, indole acetic acid, hydrocyanic acid, and ammonia production under different salt concentrations (i.e., 0, 0.25, 0.5, 0.75, 1, 1.25, and 1.5 M NaCl). The presence of 1-aminocyclopropane-1-carboxylate deaminase activity was also investigated. Salinity tolerance was evaluated in durum wheat through plant growth and development parameters: shoot and root length, dry and ash-free dry weight, and the total chlorophyll content, as well as proline accumulation. In vitro assays have shown that the strains can solubilize inorganic phosphate and produce indole acetic acid, hydrocyanic acid, and ammonia under different salt concentrations. Most of the strains (86%) had 1-aminocyclopropane-1-carboxylate deaminase activity, with significant amounts of α-ketobutyric acid. In the greenhouse experiment, inoculation with actinomycetes strains improved the morpho-biochemical parameters of durum wheat plants, which also recorded significantly higher content of chlorophylls and proline than those uninoculated, both under normal and stressed conditions. Our results suggest that inoculation of halotolerant actinomycetes can mitigate the negative effects of salt stress and allow normal growth and development of durum wheat plants.

scholarly journals Pilot Study of the Effects of Polyphenols from Chestnut Involucre on Methane Production, Volatile Fatty Acids, and Ammonia Concentration during In Vitro Rumen Fermentation

Animals ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 108
Author(s):  
Yichong Wang ◽  
Sijiong Yu ◽  
Yang Li ◽  
Shuang Zhang ◽  
Xiaolong Qi ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Acetic Acid ◽  
Propionic Acid ◽  
Acid Content ◽  
Volatile Fatty Acids ◽  
Rumen Fermentation ◽  
Gas Production ◽  
Quadratic Response ◽  
In Vitro Rumen Fermentation

Nutritional strategies can be employed to mitigate greenhouse emissions from ruminants. This article investigates the effects of polyphenols extracted from the involucres of Castanea mollissima Blume (PICB) on in vitro rumen fermentation. Three healthy Angus bulls (350 ± 50 kg), with permanent rumen fistula, were used as the donors of rumen fluids. A basic diet was supplemented with five doses of PICB (0%–0.5% dry matter (DM)), replicated thrice for each dose. Volatile fatty acids (VFAs), ammonia nitrogen concentration (NH3-N), and methane (CH4) yield were measured after 24 h of in vitro fermentation, and gas production was monitored for 96 h. The trial was carried out over three runs. The results showed that the addition of PICB significantly reduced NH3-N (p < 0.05) compared to control. The 0.1%–0.4% PICB significantly decreased acetic acid content (p < 0.05). Addition of 0.2% and 0.3% PICB significantly increased the propionic acid content (p < 0.05) and reduced the acetic acid/propionic acid ratio, CH4 content, and yield (p < 0.05). A highly significant quadratic response was shown, with increasing PICB levels for all the parameters abovementioned (p < 0.01). The increases in PICB concentration resulted in a highly significant linear and quadratic response by 96-h dynamic fermentation parameters (p < 0.01). Our results indicate that 0.2% PICB had the best effect on in-vitro rumen fermentation efficiency and reduced greenhouse gas production.

scholarly journals A molecularly defined skin test reagent for the diagnosis of bovine tuberculosis compatible with vaccination against Johne’s Disease

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sonya Middleton ◽  
Sabine Steinbach ◽  
Michael Coad ◽  
Kevina McGill ◽  
Colm Brady ◽  
...  
Keyword(s):  
Skin Test ◽  
Recombinant Proteins ◽  
Bovine Tuberculosis ◽  
Synthetic Peptides ◽  
Blood Test ◽  
High Specificity ◽  
Active Components ◽  
Interferon Gamma Release Assays ◽  
Skin Responses

AbstractTuberculin Purified Protein Derivatives (PPDs) exhibit multiple limitations: they are crude extracts from mycobacterial cultures with largely unknown active components; their production depends on culture of mycobacteria requiring expensive BCL3 production facilities; and their potency depends on the technically demanding guinea pig assay. To overcome these limitations, we developed a molecularly defined tuberculin (MDT) by adding further antigens to our prototype reagent composed of ESAT-6, CFP-10 and Rv3615c (DIVA skin test, DST). In vitro screening using PBMC from infected and uninfected cattle shortlisted four antigens from a literature-based list of 18 to formulate the MDT. These four antigens plus the previously identified Rv3020c protein, produced as recombinant proteins or overlapping synthetic peptides, were formulated together with the three DST antigens into the MDT to test cattle experimentally and naturally infected with M. bovis, uninfected cattle and MAP vaccinated calves. We demonstrated significant increases in MDT-induced skin responses compared to DST in infected animals, whilst maintaining high specificity in unvaccinated or MAP vaccinated calves. Further, MDT can also be applied in in vitro blood-based interferon-gamma release assays. Thus, MDT promises to be a robust diagnostic skin and blood test reagent overcoming some of the limitations of PPDs and warrants full validation.

scholarly journals Analgesic and Anti-Inflammatory Effects of Aucklandia lappa Root Extracts on Acetic Acid-Induced Writhing in Mice and Monosodium Iodoacetate-Induced Osteoarthritis in Rats

Plants ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 42
Author(s):  
Hee-Geun Jo ◽  
Geon-Yeong Lee ◽  
Chae Yun Baek ◽  
Ho Sueb Song ◽  
Donghun Lee
Keyword(s):  
Acetic Acid ◽  
Weight Bearing ◽  
Bone Diseases ◽  
Joint Disease ◽  
Root Extracts ◽  
Raw264.7 Macrophages ◽  
Age Related ◽  
Monosodium Iodoacetate ◽  
Anti Inflammatory

Osteoarthritis (OA) is an age-related joint disease and one of the most common degenerative bone diseases among elderly people. The currently used therapeutic strategies relying on nonsteroidal anti-inflammatory drugs (NSAIDs) and steroids for OA are often associated with gastrointestinal, cardiovascular, and kidney disorders, despite being proven effective. Aucklandia lappa is a well-known traditional medicine. The root of A. lappa root has several bioactive compounds and has been in use as a natural remedy for bone diseases and other health conditions. We evaluated the A. lappa root extracts on OA progression as a natural therapeutic agent. A. lappa substantially reduced writhing numbers in mice induced with acetic acid. Monosodium iodoacetate (MIA) was injected into the rats through their knee joints of rats to induce experimental OA, which shows similar pathological characteristics to OA in human. A. lappa substantially reduced the MIA-induced weight-bearing of hind limb and reversed the cartilage erosion in MIA rats. IL-1β, a representative inflammatory mediator in OA, was also markedly decreased by A. lappa in the serum of MIA rats. In vitro, A. lappa lowered the secretion of NO and suppressed the IL-1β, COX-2, IL-6, and iNOS production in RAW264.7 macrophages activated with LPS. Based on its analgesic and anti-inflammatory effects, A. lappa could be a potential remedial agent against OA.

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