scholarly journals The Biosynthetic Pathway of Major Avenanthramides in Oat

Metabolites ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 163 ◽  
Author(s):  
Zhiyong Li ◽  
Yi Chen ◽  
Dauenpen Meesapyodsuk ◽  
Xiao Qiu
Keyword(s):  
Biosynthetic Pathway ◽  
Molecular Mechanisms ◽  
Functional Characterization ◽  
Acyl Donor ◽  
Acyl Acceptor ◽  
Coa Ligase ◽  
Health Promoting ◽  
Hydroxyanthranilic Acid ◽  
Coa Thioesters

Avenanthramides are a group of N-cinnamoylanthranilic acids, with health-promoting properties mainly found in oat (Avena sativa L.). However, the biosynthetic mechanism for the main three types of avenanthramides (Avn-A, Avn-B and Avn-C) is not completely understood. In the present study, we report molecular identification and functional characterization of three different types of genes from oat encoding 4-coumarate-CoA ligase (4CL), hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) and a caffeoyl-CoA O-methyltransferase (CCoAOMT) enzymes, all involved in the biosynthesis of these avenanthramides. In vitro enzymatic assays using the proteins expressed in Escherichia coli showed that oat 4CL could convert p-coumaric acid, caffeic acid and ferulic acid to their CoA thioesters. Oat HHTs were only responsible for the biosynthesis of Avn-A and Avn-C using hydroxyanthranilic acid as an acyl acceptor and p-coumaroyl-CoA and caffeoyl-CoA as an acyl donor, respectively. Avn-B was synthesized by a CCoAOMT enzyme through the methylation of Avn-C. Collectively, these results have elucidated the molecular mechanisms for the biosynthesis of three major avenanthramides in vitro and paved the way for metabolic engineering of the biosynthetic pathway in heterologous systems to produce nutraceutically important compounds and make possible genetic improvement of this nutritional trait in oat through marker-assisted breeding.

scholarly journals Biochemical and Functional Characterization of Anthocyanidin Reductase (ANR) from Mangifera indica L.

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2876 ◽  
Author(s):  
Lin Tan ◽  
Mei Wang ◽  
Youfa Kang ◽  
Farrukh Azeem ◽  
Zhaoxi Zhou ◽  
...  
Keyword(s):  
Enzyme Assay ◽  
Molecular Mechanisms ◽  
Mangifera Indica ◽  
Functional Characterization ◽  
Citrate Buffer ◽  
Anthocyanidin Reductase ◽  
High Amino Acid ◽  
Optimum Ph

Mango (Mangifera indica L.) is abundant in proanthocyanidins (PAs) that are important for human health and plant response to abiotic stresses. However, the molecular mechanisms involved in PA biosynthesis still need to be elucidated. Anthocyanidin reductase (ANR) catalyzes a key step in PA biosynthesis. In this study, three ANR cDNAs (MiANR1-1,1-2,1-3) were isolated from mango, and expressed in Escherichia coli. In vitro enzyme assay showed MiANR proteins convert cyanidin to their corresponding flavan-3-ols, such as (−)-catechin and (−)-epicatechin. Despite high amino acid similarity, the recombinant ANR proteins exhibited differences in enzyme kinetics and cosubstrate preference. MiANR1-2 and MiANR1-3 have the same optimum pH of 4.0 in citrate buffer, while the optimum pH for MiANR1-1 is pH 3.0 in phosphate buffer. MiANR1-1 does not use either NADPH or NADH as co-substrate while MiANR1-2/1-3 use only NADPH as co-substrate. MiANR1-2 has the highest Km and Vmax for cyanidin, followed by MiANR1-3 and MiANR1-1. The overexpression of MiANRs in ban mutant reconstructed the biosynthetic pathway of PAs in the seed coat. These data demonstrate MiANRs can form the ANR pathway, leading to the formation of two types of isomeric flavan-3-ols and PAs in mango.

scholarly journals Curcumin: Could This Compound Be Useful in Pregnancy and Pregnancy-Related Complications?

Nutrients ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 3179 ◽  
Author(s):  
Tiziana Filardi ◽  
Rosaria Varì ◽  
Elisabetta Ferretti ◽  
Alessandra Zicari ◽  
Susanna Morano ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Curcuma Longa ◽  
Growth Disorders ◽  
Beneficial Effects ◽  
Toxic Agents ◽  
Health Promoting ◽  
Short And Long Term ◽  
The Impact ◽  
In Pregnancy

Curcumin, the main polyphenol contained in turmeric root (Curcuma longa), has played a significant role in medicine for centuries. The growing interest in plant-derived substances has led to increased consumption of them also in pregnancy. The pleiotropic and multi-targeting actions of curcumin have made it very attractive as a health-promoting compound. In spite of the beneficial effects observed in various chronic diseases in humans, limited and fragmentary information is currently available about curcumin’s effects on pregnancy and pregnancy-related complications. It is known that immune-metabolic alterations occurring during pregnancy have consequences on both maternal and fetal tissues, leading to short- and long-term complications. The reported anti-inflammatory, antioxidant, antitoxicant, neuroprotective, immunomodulatory, antiapoptotic, antiangiogenic, anti-hypertensive, and antidiabetic properties of curcumin appear to be encouraging, not only for the management of pregnancy-related disorders, including gestational diabetes mellitus (GDM), preeclampsia (PE), depression, preterm birth, and fetal growth disorders but also to contrast damage induced by natural and chemical toxic agents. The current review summarizes the latest data, mostly obtained from animal models and in vitro studies, on the impact of curcumin on the molecular mechanisms involved in pregnancy pathophysiology, with the aim to shed light on the possible beneficial and/or adverse effects of curcumin on pregnancy outcomes.

scholarly journals Molecular Mechanisms of Pollen-Pistil Interactions in Interspecific Crossing Barriers in the Tomato Family

2000 ◽  
Author(s):  
Yoram Eyal ◽  
Sheila McCormick
Keyword(s):  
Pollen Germination ◽  
Molecular Mechanisms ◽  
Functional Characterization ◽  
Dominant Negative ◽  
Peptide Sequence ◽  
Peptide Ligand ◽  
Hybrid Breakdown ◽  
Degenerate Primers

During the evolutionary process of speciation in plants, naturally occurring barriers to reproduction have developed that affect the transfer of genes within and between related species. These barriers can occur at several different levels beginning with pollination-barriers and ending with hybrid-breakdown. The interaction between pollen and pistils presents one of the major barriers to intra- and inter-specific crosses and is the focus of this research project. Our long-term goal in this research proposal was defined to resolve questions on recognition and communication during pollen-pistil interactions in the extended tomato family. In this context, this work was initiated and planned to study the potential involvement of tomato pollen-specific receptor-like kinases (RLK's) in the interaction between pollen and pistils. By special permission from BARD the objectives of this research were extended to include studies on pollen-pistil interactions and pollination barriers in horticultural crops with an emphasis on citrus. Functional characterization of 2 pollen-specific RLK's from tomato was carried out. The data shows that both encode functional kinases that were active as recombinant proteins. One of the kinases was shown to accumulate mainly after pollen germination and to be phosphorylated in-vitro in pollen membranes as well as in-vivo. The presence of style extract resulted in dephosphorylation of the RLK, although no species specificity was observed. This data implies a role for at least one RLK in pollination events following pollen germination. However, a transgenic plant analysis of the RLK's comprising overexpression, dominant-negative and anti-sense constructs failed to provide answers on their role in pollination. While genetic effects on some of the plants were observed in both the Israeli and American labs, no clear functional answers were obtained. An alternative approach to addressing function was pursued by screening for an artificial ligand for the receptor domain using a peptide phage display library. An enriched peptide sequence was obtained and will be used to design a peptide-ligand to be tested for its effect o pollen germination and tube growth. Self-incompatibility (SI) in citrus was studied on 3 varieties of pummelo. SI was observed using fluorescence microscopy in each of the 3 varieties and compatibility relations between varieties was determined. An initial screen for an S-RNase SI mechanism yielded only a cDNA homologous to the group of S-like RNases, suggesting that SI results from an as yet unknown mechanism. 2D gel electrophoresis was applied to compare pollen and style profiles of different compatibility groups. A "polymorphic" protein band from style extracts was observed, isolated and micro-sequenced. Degenerate primers designed based on the peptide sequence date will be used to isolate the relevant genes i order to study their potential involvement in SI. A study on SI in the apple cultivar Top red was initiated. SI was found, as previously shown, to be complete thus requiring a compatible pollinator variety. A new S-RNase allele was discovered fro Top red styles and was found to be highly homologous to pear S-RNases, suggesting that evolution of these genes pre-dated speciation into apples and pears but not to other Rosaceae species. The new allele provides molecular-genetic tools to determine potential pollinators for the variety Top red as well as a tool to break-down SI in this important variety.

scholarly journals 3-Hydroxyanthralinic acid metabolism controls the hepatic SREBP/lipoprotein axis, inhibits inflammasome activation in macrophages, and decreases atherosclerosis in Ldlr−/− mice

2019 ◽  
Vol 116 (12) ◽  
pp. 1948-1957 ◽  
Author(s):  
Martin Berg ◽  
Konstantinos A Polyzos ◽  
Hanna Agardh ◽  
Roland Baumgartner ◽  
Maria J Forteza ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Plasma Lipids ◽  
Nuclear Translocation ◽  
Vascular Inflammation ◽  
Kynurenine Pathway ◽  
Chronic Inflammatory Disease ◽  
Inflammasome Activation ◽  
Mrna Levels ◽  
Hydroxyanthranilic Acid

Abstract Aims Atherosclerosis is a chronic inflammatory disease involving immunological and metabolic processes. Metabolism of tryptophan (Trp) via the kynurenine pathway has shown immunomodulatory properties and the ability to modulate atherosclerosis. We identified 3-hydroxyanthranilic acid (3-HAA) as a key metabolite of Trp modulating vascular inflammation and lipid metabolism. The molecular mechanisms driven by 3-HAA in atherosclerosis have not been completely elucidated. In this study, we investigated whether two major signalling pathways, activation of SREBPs and inflammasome, are associated with the 3-HAA-dependent regulation of lipoprotein synthesis and inflammation in the atherogenesis process. Moreover, we examined whether inhibition of endogenous 3-HAA degradation affects hyperlipidaemia and plaque formation. Methods and results In vitro, we showed that 3-HAA reduces SREBP-2 expression and nuclear translocation and apolipoprotein B secretion in HepG2 cell cultures, and inhibits inflammasome activation and IL-1β production by macrophages. Using Ldlr−/− mice, we showed that inhibition of 3-HAA 3,4-dioxygenase (HAAO), which increases the endogenous levels of 3-HAA, decreases plasma lipids and atherosclerosis. Notably, HAAO inhibition led to decreased hepatic SREBP-2 mRNA levels and lipid accumulation, and improved liver pathology scores. Conclusions We show that the activity of SREBP-2 and the inflammasome can be regulated by 3-HAA metabolism. Moreover, our study highlights that targeting HAAO is a promising strategy to prevent and treat hypercholesterolaemia and atherosclerosis.

Health-Improving and Disease-Preventing Potential of Camel Milk Against Chronic Diseases and Autism

2020 ◽  
pp. 155-184
Author(s):  
Mo'ez Al-Islam Ezzat Faris ◽  
Hadeel Ghazzawi
Keyword(s):  
Chronic Diseases ◽  
Molecular Mechanisms ◽  
Experimental Studies ◽  
Nutritional Composition ◽  
Camel Milk ◽  
Bowel Diseases ◽  
Liver And Kidney ◽  
Health Promoting

Camel milk has been used as part of the human diet since ancient times. This chapter tries to elaborate the different aspects of nutraceutical functional properties of camel milk, focusing on the nutritional composition, presence of bioactive zoochemicals and peptides, antioxidant nutrients (vitamin C), and health rendering properties of this unique milk. Recent research has identified camel milk as a prophylactic and therapeutic functional food due to its noticeable content of essential macronutrients, namely bioactive functional proteins and peptides, along with its considerable content of essential micronutrients. Indeed, the presence of this unique mixture has shown to be promising contributors to the management and prevention of chronic diseases, such as cancer, diabetes, liver and kidney, metabolic syndrome, inflammatory bowel diseases in adults, and autism. In vivo, in vitro, and epidemiological and experimental studies were reviewed, and molecular mechanisms were highlighted for better understanding of the health-promoting, disease-preventing potential of camel milk.

scholarly journals PRKACA Somatic Mutations Are Rare Findings in Aldosterone-Producing Adenomas

2016 ◽  
Vol 101 (8) ◽  
pp. 3010-3017 ◽  
Author(s):  
Yara Rhayem ◽  
Luis G. Perez-Rivas ◽  
Anna Dietz ◽  
Kerstin Bathon ◽  
Christian Gebhard ◽  
...  
Keyword(s):  
Cushing’S Syndrome ◽  
Primary Aldosteronism ◽  
Molecular Mechanisms ◽  
Somatic Mutations ◽  
Functional Characterization ◽  
Cushing's Syndrome ◽  
Antihypertensive Medications ◽  
Unilateral Adrenalectomy ◽  
Vitro Analysis

Context: Somatic mutations have been found causative for endocrine autonomy in aldosterone-producing adenomas (APAs). Whereas mutations of PRKACA (catalytic subunit of protein kinase A) have been identified in cortisol-producing adenomas, the presence of PRKACA variants in APAs is unknown, especially in those that display cosecretion of cortisol. Objective: The objective of the study was to investigate PRKACA somatic variants identified in APA cases. Design: Identification of PRKACA somatic variants in APAs by whole-exome sequencing followed by in vitro analysis of the enzymatic activity of PRKACA variants and functional characterization by double immunofluorescence of CYP11B2 and CYP11B1 expression in the corresponding tumor tissues. Setting and Patients: APA tissues were collected from 122 patients who underwent unilateral adrenalectomy for primary aldosteronism between 2005 and 2015 at a single institution. Results: PRKACA somatic mutations were identified in two APA cases (1.6%). One APA carried a newly identified p.His88Asp variant, whereas in a second case, a p.Leu206Arg mutation was found, previously described only in cortisol-producing adenomas with overt Cushing's syndrome. Functional analysis showed that the p.His88Asp variant was not associated with gain of function. Although CYP11B2 was strongly expressed in the p.His88Asp-mutated APA, the p.Leu206Arg carrying APA predominantly expressed CYP11B1. Accordingly, biochemical Cushing's syndrome was present only in the patient with the p.Leu206Arg mutation. After adrenalectomy, both patients improved with a reduced number of antihypertensive medications and normalized serum potassium levels. Conclusions: We describe for the first time PRKACA mutations as rare findings associated with unilateral primary aldosteronism. As cortisol cosecretion occurs in a subgroup of APAs, other molecular mechanisms are likely to exist.

scholarly journals Hepatic Differentiation of Murine Disease-Specific Induced Pluripotent Stem Cells Allows Disease ModellingIn Vitro

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Reto Eggenschwiler ◽  
Komal Loya ◽  
Malte Sgodda ◽  
Francoise André ◽  
Tobias Cantz
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Molecular Mechanisms ◽  
Functional Characterization ◽  
Hepatic Differentiation ◽  
Fumarylacetoacetate Hydrolase ◽  
Induced Pluripotent ◽  
Disease Specific

Direct reprogramming of somatic cells into pluripotent cells by retrovirus-mediated expression of OCT4, SOX2, KLF4, and C-MYC is a promising approach to derive disease-specific induced pluripotent stem cells (iPSCs). In this study, we focused on three murine models for metabolic liver disorders: the copper storage disorder Wilson's disease (toxic-milk mice), tyrosinemia type 1 (fumarylacetoacetate-hydrolase deficiency, FAH−/−mice), and alpha1-antitrypsin deficiency (PiZ mice). Colonies of iPSCs emerged 2-3 weeks after transduction of fibroblasts, prepared from each mouse strain, and were maintained as individual iPSC lines. RT-PCR and immunofluorescence analyses demonstrated the expression of endogenous pluripotency markers. Hepatic precursor cells could be derived from these disease-specific iPSCs applying anin vitrodifferentiation protocol and could be visualized after transduction of a lentiviral albumin-GFP reporter construct. Functional characterization of these cells allowed the recapitulation of the disease phenotype for further studies of underlying molecular mechanisms of the respective disease.

scholarly journals Functional Characterization of the Cleavage Specificity of the Sapovirus Chymotrypsin-Like Protease

2008 ◽  
Vol 82 (16) ◽  
pp. 8085-8093 ◽  
Author(s):  
Ivonne Robel ◽  
Julia Gebhardt ◽  
Jeroen R. Mesters ◽  
Alexander Gorbalenya ◽  
Bruno Coutard ◽  
...  
Keyword(s):  
High Performance ◽  
Molecular Mechanisms ◽  
Rna Virus ◽  
Functional Characterization ◽  
Mass Spectrometry Analysis ◽  
Viral Protease ◽  
Spectrometry Analysis ◽  
Cleavage Specificity ◽  
Scissile Bond

ABSTRACT Sapovirus is a positive-stranded RNA virus with a translational strategy based on processing of a polyprotein precursor by a chymotrypsin-like protease. So far, the molecular mechanisms regulating cleavage specificity of the viral protease are poorly understood. In this study, the catalytic activities and substrate specificities of the predicted forms of the viral protease, the 3C-like protease (NS6) and the 3CD-like protease-polymerase (NS6-7), were examined in vitro. The purified NS6 and NS6-7 were able to cleave synthetic peptides (15 to 17 residues) displaying the cleavage sites of the sapovirus polyprotein, both NS6 and NS6-7 proteins being active forms of the viral protease. High-performance liquid chromatography and subsequent mass spectrometry analysis of digested products showed a specific trans cleavage of peptides bearing Gln-Gly, Gln-Ala, Glu-Gly, Glu-Pro, or Glu-Lys at the scissile bond. In contrast, peptides bearing Glu-Ala or Gln-Asp at the scissile bond (NS4-NS5 and NS5-NS6, or NS6-NS7 junctions, respectively) were resistant to trans cleavage by NS6 or NS6-7 proteins, whereas cis cleavage of the Glu-Ala scissile bond of the NS5-NS6 junction was evidenced. Interestingly, the presence of a Phe at position P4 overruled the resistance to trans cleavage of the Glu-Ala junction (NS5-NS6), whereas substitutions at the P1 and P2′ positions altered the cleavage efficiency. The differential cleavage observed is supported by a model of the substrate-binding site of the sapovirus protease, indicating that the P4, P1, and P2′ positions in the substrate modulate the cleavage specificity and efficiency of the sapovirus chymotrypsin-like protease.

scholarly journals In vitro reconstitution of indolmycin biosynthesis reveals the molecular basis of oxazolinone assembly

2015 ◽  
Vol 112 (9) ◽  
pp. 2717-2722 ◽  
Author(s):  
Yi-Ling Du ◽  
Lona M. Alkhalaf ◽  
Katherine S. Ryan
Keyword(s):  
Molecular Basis ◽  
Biosynthetic Pathway ◽  
Therapeutic Agents ◽  
Trna Synthetase ◽  
Ring Assembly ◽  
Coa Ligase ◽  
Biochemical Assays ◽  
In Vitro Reconstitution

The bacterial tryptophanyl–tRNA synthetase inhibitor indolmycin features a unique oxazolinone heterocycle whose biogenetic origins have remained obscure for over 50 years. Here we identify and characterize the indolmycin biosynthetic pathway, using systematic in vivo gene inactivation, in vitro biochemical assays, and total enzymatic synthesis. Our work reveals that a phenylacetate–CoA ligase-like enzyme Ind3 catalyzes an unusual ATP-dependent condensation of indolmycenic acid and dehydroarginine, driving oxazolinone ring assembly. We find that Ind6, which also has chaperone-like properties, acts as a gatekeeper to direct the outcome of this reaction. With Ind6 present, the normal pathway ensues. Without Ind6, the pathway derails to an unusual shunt product. Our work reveals the complete pathway for indolmycin formation and sets the stage for using genetic and chemoenzymatic methods to generate indolmycin derivatives as potential therapeutic agents.

scholarly journals Functional characterization and discovery of modulators of SbMATE, the agronomically important aluminium tolerance transporter from Sorghum bicolor

2017 ◽  
Author(s):  
Rupak Doshi ◽  
Aaron P. McGrath ◽  
Miguel Piñeros ◽  
Paul Szewczyk ◽  
Denisse M. Garza ◽  
...  
Keyword(s):  
Sorghum Bicolor ◽  
Molecular Mechanisms ◽  
Arable Land ◽  
Functional Characterization ◽  
Monovalent Cation ◽  
Root Tip ◽  
Aluminium Tolerance ◽  
Transporter Protein ◽  
Heterologous Expression Systems

ABSTRACTAbout 50% of the world’s arable land is strongly acidic (soil pH < 5). The low pH of these soils solubilizes root-toxic ionic aluminium (Al3+) species from clay minerals, driving the evolution of various counteractive adaptations in cultivated crops. The food crop Sorghum bicolor, for example, upregulates the membrane-embedded transporter protein SbMATE in its roots. SbMATE mediates efflux of the anionic form of the organic acid, citrate, into the soil rhizosphere, chelating Al3+ ions and thereby imparting Al-resistance based on excluding Al+3 from the growing root tip. Here, we use electrophysiological, radiolabeled, and fluorescence-based transport assays in two heterologous expression systems to establish a broad substrate recognition profile of SbMATE, showing the transport of 14C- citrate anion, as well as the organic monovalent cation, ethidium, but not the divalent ethidium-derivative, propidium. The transport cycle is proton and/or sodium-driven, and shares certain molecular mechanisms with bacterial MATE-family transporters. We further complement our transport assays by directly measuring substrate binding to detergent-purified SbMATE protein. Finally, we use the functionally-folded, purified membrane protein as an antigen to discover high-affinity, native conformation-binding and transport function-altering nanobodies using an animal-free, mRNA/cDNA display technology. Our results demonstrate the utility of using Pichia pastoris as an efficient eukaryotic host to express large quantities of functional plant transporter proteins for in vitro characterization. The nanobody discovery approach is applicable to other low immunogenic plant proteins.

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