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L-myo-Inositol 1-phosphate synthase (I-1-P synthase) catalyses the primary reaction for the synthesis of inositol in a variety of prokaryotes, eukaryotes and in the chloroplasts of algae and higher plants. Inositol is a precursor of essential macromolecules like membrane phospholipids, GPI anchor proteins and lipophosphoglycans, which play a determinant role in the pathogenesis of protozoan parasites such as Leishmania and Entamoeba. However, there is no report of I-1-P synthase or its gene from these organisms. The gene INO1 coding for this enzyme was first cloned from Saccharomyces cerevisiae and subsequently from several plants. Using molecular cloning techniques we have isolated and characterised the INO1 gene coding for the enzyme I-1-P synthase from Entamoeba histolytica. Simultaneously, we have purified and characterised the native enzyme from E. histolytica trophozoites and the cloned gene product from Escherichia coli. The gene product and the purified enzyme were both shown to be recognised by a heterologous anti-I-1-P synthase antibody from the phytoflagellate Euglena gracilis. Phylogenetic analysis of I-1-P synthase sequences from different eukaryotes suggest that it is highly conserved across species and the origin of this enzyme precedes the evolutionary divergence of modern eukaryotes.
Reactive oxygen species (ROS) generated in aerobic metabolism and oxidative stress lead to macromolecules damage, such as to proteins, lipids, and DNA, which can be eliminated by the redox buffer mycothiol (AcCys-GlcN-Ins, MSH). Myo-inositol-phosphate synthase (Ino-1) catalyzes the first committed step in the synthesis of MSH, thus playing a critical role in the growth of the organism. Although Ino-1s have been systematically studied in eukaryotes, their physiological and biochemical functions remain largely unknown in bacteria. In this study, we report that Ino-1 plays an important role in oxidative stress resistance in the gram-positive Actinobacteria Corynebacterium glutamicum. Deletion of the ino-1 gene resulted in a decrease in cell viability, an increase in ROS production, and the aggravation of protein carbonylation levels under various stress conditions. The physiological roles of Ino-1 in the resistance to oxidative stresses were corroborated by the absence of MSH in the Δino-1 mutant. In addition, we found that the homologous expression of Ino-1 in C. glutamicum yielded a functionally active protein, while when expressed in Escherichia coliBL21(DE3), it lacked measurable activity. An examination of the molecular mass (Mr) suggested that Ino-1 expressed in E. coliBL21(DE3) was not folded in a catalytically competent conformation. Together, the results unequivocally showed that Ino-1 was important for the mediation of oxidative resistance by C. glutamicum.
Myo-inositol-1-phosphate synthase (MIPS, EC 5.5.1.4) plays important roles in plant growth and development, stress responses, and cellular signal transduction. MIPS genes were found preferably expressed during fiber cell initiation and early fast elongation in upland cotton (Gossypium hirsutum), however, current understanding of the function and regulatory mechanism of MIPS genes to involve in cotton fiber cell growth is limited. Here, by genome-wide analysis, we identified four GhMIPS genes anchoring onto four chromosomes in G. hirsutum and analyzed their phylogenetic relationship, evolutionary dynamics, gene structure and motif distribution, which indicates that MIPS genes are highly conserved from prokaryotes to green plants, with further exon-intron structure analysis showing more diverse in Brassicales plants. Of the four GhMIPS members, based on the significant accumulated expression of GhMIPS1D at the early stage of fiber fast elongating development, thereby, the GhMIPS1D was selected to investigate the function of participating in plant development and cell growth, with ectopic expression in the loss-of-function Arabidopsis mips1 mutants. The results showed that GhMIPS1D is a functional gene to fully compensate the abnormal phenotypes of the deformed cotyledon, dwarfed plants, increased inflorescence branches, and reduced primary root lengths in Arabidopsis mips1 mutants. Furthermore, shortened root cells were recovered and normal root cells were significantly promoted by ectopic expression of GhMIPS1D in Arabidopsis mips1 mutant and wild-type plants respectively. These results serve as a foundation for understanding the MIPS family genes in cotton, and suggest that GhMIPS1D may function as a positive regulator for plant cell elongation.
myo-inositol (MI) is an essential growth factor, nutritional source, and important precursor for many derivatives like D-chiro-inositol. In this study, attempts were made to achieve the "green biosynthesis" of MI in a model photosynthetic cyanobacterium Synechocystis sp. PCC 6803. First, several genes encoding myo-inositol-1-phosphate synthases and myo-inositol-1-monophosphatase, catalyzing the first or the second step of MI synthesis, were introduced, respectively, into Synechocystis. The results showed that the engineered strain carrying myo-inositol-1-phosphate synthase gene from Saccharomyces cerevisiae was able to produce MI at 0.97 mg L-1. Second, the combined overexpression of genes related to the two catalyzing processes increased the production up to 1.42 mg L-1. Third, to re-direct more cellular carbon flux into MI synthesis, an inducible small RNA regulatory tool, based on MicC-Hfq, was utilized to control the competing pathways of MI biosynthesis, resulting in MI production of ∼7.93 mg L-1. Finally, by optimizing the cultivation condition via supplying bicarbonate to enhance carbon fixation, a final MI production up to 12.72 mg L-1 was achieved, representing a ∼12-fold increase compared with the initial MI-producing strain. This study provides a light-driven green synthetic strategy for MI directly from CO2 in cyanobacterial chassis and represents a renewable alternative that may deserve further optimization in the future.
A rare stereoisomer of inositol, scyllo-inositol, is a therapeutic agent that has shown potential efficacy in preventing Alzheimer's disease. Mycobacterium tuberculosis ino1 encoding myo-inositol-1-phosphate (MI1P) synthase (MI1PS) was introduced into Bacillus subtilis to convert glucose-6-phosphate (G6P) into MI1P. We found that inactivation of pbuE elevated intracellular concentrations of NAD+·NADH as an essential cofactor of MI1PS and was required to activate MI1PS. MI1P thus produced was dephosphorylated into myo-inositol by an intrinsic inositol monophosphatase, YktC, which was subsequently isomerized into scyllo-inositol via a previously established artificial pathway involving two inositol dehydrogenases, IolG and IolW. In addition, both glcP and glcK were overexpressed to feed more G6P and accelerate scyllo-inositol production. Consequently, a B. subtilis cell factory was demonstrated to produce 2 g L-1 scyllo-inositol from 20 g L-1 glucose. This cell factory provides an inexpensive way to produce scyllo-inositol, which will help us to challenge the growing problem of Alzheimer's disease in our aging society.
The physiology of Prunus fruit ripening is a complex and not completely understood process. To improve this knowledge, postharvest behavior during the shelf-life period at the transcriptomic level has been studied using high-throughput sequencing analysis (RNA-Seq). Monitoring of fruits has been analyzed after different ethylene regulator treatments, including 1-MCP (ethylene-inhibitor) and Ethrel (ethylene-precursor) in two contrasting selected apricot (Prunus armeniaca L.) and Japanese plum (P. salicina L.) cultivars, 'Goldrich' and 'Santa Rosa'. KEEG and protein-protein interaction network analysis unveiled that the most significant metabolic pathways involved in the ripening process were photosynthesis and plant hormone signal transduction. In addition, previously discovered genes linked to fruit ripening, such as pectinesterase or auxin-responsive protein, have been confirmed as the main genes involved in this process. Genes encoding pectinesterase in the pentose and glucuronate interconversions pathway were the most overexpressed in both species, being upregulated by Ethrel. On the other hand, auxin-responsive protein IAA and aquaporin PIP were both upregulated by 1-MCP in 'Goldrich' and 'Santa Rosa', respectively. Results also showed the upregulation of chitinase and glutaredoxin 3 after Ethrel treatment in 'Goldrich' and 'Santa Rosa', respectively, while photosystem I subunit V psaG (photosynthesis) was upregulated after 1-MCP in both species. Furthermore, the overexpression of genes encoding GDP-L-galactose and ferredoxin in the ascorbate and aldarate metabolism and photosynthesis pathways caused by 1-MCP favored antioxidant activity and therefore slowed down the fruit senescence process.
Glucaric acid is a high-value-added chemical that can be used in various fields. Because chemical oxidation of glucose to produce glucaric acid is not environmentally friendly, microbial production has attracted increasing interest recently. Biological pathways to synthesize glucaric acid from glucose in both Escherichia coli and Saccharomyces cerevisiae by co-expression of genes encoding myo-inositol-1-phosphate synthase (Ino1), myo-inositol oxygenase (MIOX), and uronate dehydrogenase (Udh) have been constructed. However, low activity and instability of MIOX from Mus musculus was proved to be the bottleneck in this pathway.
d-pinitol is the most commonly accumulated sugar alcohol in the Leguminosae family and has been observed to increase significantly in response to abiotic stress. While previous studies have identified genes involved in d-pinitol synthesis, no study has investigated transcript expression in planta. The present study quantified the expression of several genes involved in d-pinitol synthesis in different plant tissues and investigated the accumulation of d-pinitol, myo-inositol and other metabolites in response to a progressive soil drought in soybean (Glycine max). Expression of myo-inositol 1-phosphate synthase (INPS), the gene responsible for the conversion of glucose-6-phosphate to myo-inositol-1-phosphate, was significantly up regulated in response to a water deficit for the first two sampling weeks. Expression of myo-inositol O-methyl transferase (IMT1), the gene responsible for the conversion of myo-inositol into d-ononitol was only up regulated in stems at sampling week 3. Assessment of metabolites showed significant changes in their concentration in leaves and stems. d-Pinitol concentration was significantly higher in all organs sampled from water deficit plants for all three sampling weeks. In contrast, myo-inositol, had significantly lower concentrations in leaf samples despite up regulation of INPS suggesting the transcriptionally regulated flux of carbon through the myo-inositol pool is important during water deficit.
European canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Reverse transcription quantitative real-time PCR (RT-qPCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate RT-qPCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.
Drought is a widespread limiting factor in coffee plants. It affects plant development, fruit production, bean development and consequently beverage quality. Genetic diversity for drought tolerance exists within the coffee genus. However, the molecular mechanisms underlying the adaptation of coffee plants to drought are largely unknown. In this study, we compared the molecular responses to drought in two commercial cultivars (IAPAR59, drought-tolerant and Rubi, drought-susceptible) of Coffea arabica grown in the field under control (irrigation) and drought conditions using the pyrosequencing of RNA extracted from shoot apices and analysing the expression of 38 candidate genes.
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