Inositol Transport Research Articles

Comparative Proteomic Analysis of Two Wild Soybean (Glycine soja) Genotypes Reveals Positive Regulation of Saline-Alkaline Stress Tolerance by Tonoplast Transporters.

Soil saline-alkalization is a significant constraint for soybean production. Owing to higher genetic diversity of wild soybean, we compared the proteomic landscape of saline-alkaline stress-tolerant (SWBY032) and stress-sensitive (SWLJ092) wild soybean (Glycine soja) strains under saline and saline-alkaline stress. Out of 346 differentially expressed proteins (DEPs) specifically involved in saline-alkaline stress, 159 and 133 DEPs were identified in only SWLJ092 and SWBY032, respectively. Functional annotations revealed that more ribosome proteins were downregulated in SWLJ092, whereas more membrane transporters were upregulated in SWBY032. Moreover, protein-protein interaction analysis of 133 DEPs revealed that 14 protein-synthesis- and 2 TCA-cycle-related DEPs might alter saline-alkaline tolerance by affecting protein synthesis and amino acid metabolism. Furthermore, we confirmed G. soja tonoplast intrinsic protein (GsTIP2-1 and GsTIP2-2), inositol transporter (GsINT1), sucrose transport protein (GsSUC4), and autoinhibited Ca2+-ATPase (GsACA11) as tonoplast transporters can synergistically improve saline-alkaline tolerance in soybean, possibly by relieving the inhibition of protein synthesis and amino acid metabolism. Overall, our findings provided a foundation for molecular breeding of a saline-alkaline stress-tolerant soybean.

A genome-wide association study of freezing tolerance in red clover (Trifolium pratense L.) germplasm of European origin.

Improvement of persistency is an important breeding goal in red clover (Trifolium pratense L.). In areas with cold winters, lack of persistency is often due to poor winter survival, of which low freezing tolerance (FT) is an important component. We conducted a genome wide association study (GWAS) to identify loci associated with freezing tolerance in a collection of 393 red clover accessions, mostly of European origin, and performed analyses of linkage disequilibrium and inbreeding. Accessions were genotyped as pools of individuals using genotyping-by-sequencing (pool-GBS), generating both single nucleotide polymorphism (SNP) and haplotype allele frequency data at accession level. Linkage disequilibrium was determined as a squared partial correlation between the allele frequencies of pairs of SNPs and found to decay at extremely short distances (< 1 kb). The level of inbreeding, inferred from the diagonal elements of a genomic relationship matrix, varied considerably between different groups of accessions, with the strongest inbreeding found among ecotypes from Iberia and Great Britain, and the least found among landraces. Considerable variation in FT was found, with LT50-values (temperature at which 50% of the plants are killed) ranging from -6.0°C to -11.5°C. SNP and haplotype-based GWAS identified eight and six loci significantly associated with FT (of which only one was shared), explaining 30% and 26% of the phenotypic variation, respectively. Ten of the loci were found within or at a short distance (<0.5 kb) from genes possibly involved in mechanisms affecting FT. These include a caffeoyl shikimate esterase, an inositol transporter, and other genes involved in signaling, transport, lignin synthesis and amino acid or carbohydrate metabolism. This study paves the way for a better understanding of the genetic control of FT and for the development of molecular tools for the improvement of this trait in red clover through genomics assisted breeding.

Exogenous myo-inositol increases salt tolerance and accelerates CAM induction in the early juvenile stage of the facultative halophyte Mesembryanthemum crystallinum but not in the late juvenile stage.

Mesembryanthemum crystallinum L. (ice plant) develops salt tolerance during the transition from the juvenile to the adult stage through progressive morphological, physiological, biochemical, and molecular changes. Myo-inositol is the precursor for the synthesis of compatible solute D-pinitol and promotes Na+ transport in ice plants. We previously showed that supplying myo-inositol to 9-day-old seedlings alleviates salt damage by coordinating the expression of genes involved in inositol synthesis and transport, affecting osmotic adjustment and the Na/K balance. In this study, we examined the effects of myo-inositol on physiological parameters and inositol-related gene expression in early- and late-stage juvenile plants. The addition of myo-inositol to salt-treated, hydroponically grown late juvenile plants had no significant effects on growth or photosynthesis. In contrast, supplying exogenous myo-inositol to salt-treated early juvenile plants increased leaf biomass, relative water content, and chlorophyll content and improved PSII activity and CO2 assimilation. The treatment combining high salt and myo-inositol synergistically induced the expression of myo-inositol phosphate synthase (INPS), myo-inositol O-methyltransferase (IMT), and inositol transporters (INTs), which modulated root-to-shoot Na/K ratio and increased leaf D-pinitol content. The results indicate that sufficient myo-inositol is a prerequisite for high salt tolerance in ice plant.

Genome-wide characterization of the inositol transporters gene family in Populus and functional characterization of PtINT1b in response to salt stress

Inositol transporters (INTs) can mediate the transmembrane transport of inositol, and play crucial roles in plant growth, development and stress resistance. However, the INT gene family in Populus has not been reported. Herein, nine INT genes were identified in the Populus trichocarpa genome and divided into three clades. Tandem duplication and whole-genome duplication events could induce the expansion of PtINT gene family. It was worth noting that PtINT1c* and 1d* formed by twice tandem gene duplication events of PtINT1b, but both had undergone partial structural loss during evolution. PtINT2_p1* and PtINT2_p2* might be originated from one INT2 gene by stop codon- and start codon-gain variants. Different members of PtINTs were localized to the plasma membrane or vacuolar membrane. PtINTs had diversified tissue expression profiles, and many members were significantly induced or suppressed after salt and drought treatments. PtINT1b was induced by drought and salinity stresses, and encoded a vacuolar inositol transporter. Overexpression of PtINT1b rendered the transgenic Arabidopsis plants more resistant to salt stress. In conclusion, this study provides valuable clues for future research on the function of PtINTs, and PtINT1b was identified as a candidate gene for genetic engineering to enhance salinity tolerance in plants.

Glucose and Inositol Transporters, SLC5A1 and SLC5A3, in Glioblastoma Cell Migration.

(1) Background: The recurrence of glioblastoma multiforme (GBM) is mainly due to invasion of the surrounding brain tissue, where organic solutes, including glucose and inositol, are abundant. Invasive cell migration has been linked to the aberrant expression of transmembrane solute-linked carriers (SLC). Here, we explore the role of glucose (SLC5A1) and inositol transporters (SLC5A3) in GBM cell migration. (2) Methods: Using immunofluorescence microscopy, we visualized the subcellular localization of SLC5A1 and SLC5A3 in two highly motile human GBM cell lines. We also employed wound-healing assays to examine the effect of SLC inhibition on GBM cell migration and examined the chemotactic potential of inositol. (3) Results: While GBM cell migration was significantly increased by extracellular inositol and glucose, it was strongly impaired by SLC transporter inhibition. In the GBM cell monolayers, both SLCs were exclusively detected in the migrating cells at the monolayer edge. In single GBM cells, both transporters were primarily localized at the leading edge of the lamellipodium. Interestingly, in GBM cells migrating via blebbing, SLC5A1 and SLC5A3 were predominantly detected in nascent and mature blebs, respectively. (4) Conclusion: We provide several lines of evidence for the involvement of SLC5A1 and SLC5A3 in GBM cell migration, thereby complementing the migration-associated transportome. Our findings suggest that SLC inhibition is a promising approach to GBM treatment.

Investigating Genetic Determinants of Plasma Inositol Status in Adult Humans

Myo-inositol (MI) is incorporated into numerous biomolecules, including phosphoinositides and inositol phosphates. Disturbance of inositol availability or metabolism is associated with various disorders, including neurological conditions and cancers, whereas supplemental MI has therapeutic potential in conditions such as depression, polycystic ovary syndrome, and congenital anomalies. Inositol status can be influenced by diet, synthesis, transport, utilization, and catabolism. We aimed to investigate potential genetic regulation of circulating MI status and to evaluate correlation of MI concentration with other metabolites. GC-MS was used to determine plasma MI concentration of >2000 healthy, young adults (aged 18-28 y) from the Trinity Student Study. Genotyping data were used to test association of plasma MI with single nucleotide polymorphisms (SNPs) in candidate genes, encoding inositol transporters and synthesizing enzymes, and test for genome-wide association. We evaluated potential correlation of plasma MI with d-chiro-inositol (DCI), glucose, and other metabolites by Spearman rank correlation. Mean plasma MI showed a small but significant difference between males and females (28.5 and 26.9 μM, respectively). Candidate gene analysis revealed several nominally significant associations with plasma MI, most notably for SLC5A11 (solute carrier family 5 member 11), encoding a sodium-coupled inositol transporter, also known as SMIT2 (sodium-dependent myo-inositol transporter 2). However, these did not survive correction for multiple testing. Subsequent testing for genome-wide association with plasma MI did not identify associations of genome-wide significance (P < 5 × 10-8). However, 8 SNPs exceeded the threshold for suggestive significant association with plasma MI concentration (P < 1 × 10-5), 3 of which were located within or close to genes: MTDH (metadherin), LAPTM4B (lysosomal protein transmembrane 4 β), and ZP2 (zona pellucida 2). We found significant positive correlation of plasma MI concentration with concentration of dci and several other biochemicals including glucose, methionine, betaine, sarcosine, and tryptophan. Our findings suggest potential for modulation of plasma MI in young adults by variation in SLC5A11, which is worthy of further investigation.

Metabolic engineering of Pichia pastoris for myo-inositol production by dynamic regulation of central metabolism

BackgroundThe methylotrophic budding yeast Pichia pastoris GS115 is a powerful expression system and hundreds of heterologous proteins have been successfully expressed in this strain. Recently, P. pastoris has also been exploited as an attractive cell factory for the production of high-value biochemicals due to Generally Recognized as Safe (GRAS) status and high growth rate of this yeast strain. However, appropriate regulation of metabolic flux distribution between cell growth and product biosynthesis is still a cumbersome task for achieving efficient biochemical production.ResultsIn this study, P. pastoris was exploited for high inositol production using an effective dynamic regulation strategy. Through enhancing native inositol biosynthesis pathway, knocking out inositol transporters, and slowing down carbon flux of glycolysis, an inositol-producing mutant was successfully developed and low inositol production of 0.71 g/L was obtained. The inositol production was further improved by 12.7% through introduction of heterologous inositol-3-phosphate synthase (IPS) and inositol monophosphatase (IMP) which catalyzed the rate-limiting steps for inositol biosynthesis. To control metabolic flux distribution between cell growth and inositol production, the promoters of glucose-6-phosphate dehydrogenase (ZWF), glucose-6-phosphate isomerase (PGI) and 6-phosphofructokinase (PFK1) genes were replaced with a glycerol inducible promoter. Consequently, the mutant strain could be switched from growth mode to production mode by supplementing glycerol and glucose sequentially, leading to an increase of about 4.9-fold in inositol formation. Ultimately, the dissolved oxygen condition in high-cell-density fermentation was optimized, resulting in a high production of 30.71 g/L inositol (~ 40-fold higher than the baseline strain).ConclusionsThe GRAS P. pastoris was engineered as an efficient inositol producer for the first time. Dynamic regulation of cell growth and inositol production was achieved via substrate-dependent modulation of glycolysis and pentose phosphate pathways and the highest inositol titer reported to date by a yeast cell factory was obtained. Results from this study provide valuable guidance for engineering of P. pastoris for the production of other high-value bioproducts.

Arabidopsis HOPS subunit VPS41 carries out plant-specific roles in vacuolar transport and vegetative growth.

The homotypic fusion and protein sorting (HOPS) complex is a conserved, multi-subunit tethering complex in eukaryotic cells. In yeast and mammalian cells, the HOPS subunit vacuolar protein sorting-associated protein 41 (VPS41) is recruited to late endosomes after Ras-related protein 7 (Rab7) activation and is essential for vacuole fusion. However, whether VPS41 plays conserved roles in plants is not clear. Here, we demonstrate that in the model plant Arabidopsis (Arabidopsis thaliana), VPS41 localizes to distinct condensates in root cells in addition to its reported localization at the tonoplast. The formation of condensates does not rely on the known upstream regulators but depends on VPS41 self-interaction and is essential for vegetative growth regulation. Genetic evidence indicates that VPS41 is required for both homotypic vacuole fusion and cargo sorting from the adaptor protein complex 3, Rab5, and Golgi-independent pathways but is dispensable for the Rab7 cargo inositol transporter 1. We also show that VPS41 has HOPS-independent functions in vacuolar transport. Taken together, our findings indicate that Arabidopsis VPS41 is a unique subunit of the HOPS complex that carries out plant-specific roles in both vacuolar transport and developmental regulation.

Myo‐inositol transport and metabolism participate in salt tolerance of halophyte ice plant seedlings

Myo-inositol and its metabolic derivatives such as pinitol, galactinol, and raffinose affect growth and development and are also involved in stress adaptation. Previous studies have identified myo-inositol transporters (INTs) as transporters of Na+ from root to shoot in the halophyte ice plant (Mesembryanthemum crystallinum). We found that the supply of myo-inositol could alleviate the dehydration effects of salt-stressed ice plant seedlings by decreasing the Na/K ratio in roots and increasing the Na/K ratio in shoots. Analyses of the uptake of exogenous myo-inositol revealed that ice plant seedlings contained intrinsic high-affinity transporters and inducible low-affinity uptake systems. The presence of Na+ facilitated both high- and low-affinity myo-inositol uptake. Six INT genes were identified from the ice plant transcriptome and named McINT1a, 1b, 2, 4a, 4b, and 4c, according to the classification of the Arabidopsis INT family. In seedlings treated with myo-inositol, salt, or myo-inositol plus salt, the expression patterns of all McINT members differed in shoot and root, which indicates organ-specific regulation of McINTs by salt and myo-inositol. The expression of McINT2, 4a, 4b, and 4c was induced by salt stress in shoot and root, but that of McINT1a and 1b was salt-induced only in shoot. The expression of pinitol biosynthesis gene IMT1 was induced by salt and myo-inositol, and their combination had a synergistic effect on the accumulation of pinitol. Supply of myo-inositol to salt-treated seedlings alleviated the detrimental effects by maintaining a low root Na/K ratio and providing precursors for the synthesis of compatible solute to maintain the osmotic balance.

Contrasting the effects of phytase and pure myo-inositol on the performance, digestibility, blood and egg yolk inositol levels and digestion physiology of laying hens

ABSRACT 1. An experiment was designed to compare the effects of supplementing laying hen diets with phytase and myo-inositol (inositol). 2. Five diets were formulated: high balanced protein (HBP – 840 mg of Dlys/hen/day), HBP with inositol (HBP+I – 0.16%), reduced balance protein (RBP – 672 mg of Dlys/hen/day), RBP with inositol (RBP+I – 0.16%) and RBP with phytase (RBP+P – 3000 FTU/kg). 3. Laying hen production, inositol concentrations, digestive tract morphology, amino acid digestibility and intestinal inositol transporters transcript abundance were evaluated. Data were analysed with a one-way ANOVA in SAS 9.4. Contrasts were used to assess the effect of protein, inositol, phytase and phytase vs. inositol. Differences were accepted when P ≤ 0.05. 4. No effect on hen-day egg production or feed efficiency was found. However, feed intake and the incidence of abnormally shaped eggs were 0.77 g/h/d and 0.17% higher, respectively, in inositol treatments. Inositol decreased egg specific gravity from 1.088 to 1.0865. 5. Inositol concentration in egg yolk was similar among HBP+I, RBP+I and RBP+P, and higher than for the HBP and RBP diet groups. Both gizzard and ileal digesta were enriched in inositol in all supplemented treatments, and phytase supplementation decreased the level of IP5 and IP6 in the gizzard and ileum. Generally, neither phytase or inositol affected amino acid digestibility. 6. Inositol increased transcript abundance of alkaline phosphatase in the ileum, while phytase upregulated duodenal alkaline phosphatase and SMIT1, jejunal SMIT2 and reduced ileal HMIT and SMIT1 abundance. 7. In conclusion, no effect of phytase or inositol was found for laying hen production performance or amino acid digestibility, but egg quality was reduced by inositol supplementation. Inositol concentration in egg yolk was similar among supplemented treatments.

Placental Inositol Reduced in Gestational Diabetes as Glucose Alters Inositol Transporters and IMPA1 Enzyme Expression.

Perturbed inositol physiology in insulin-resistant conditions has led to proposals of inositol supplementation for gestational diabetes (GDM) prevention, but placental inositol biology is poorly understood. Investigate associations of maternal glycemia with placental inositol content, determine glucose effects on placental expression of inositol enzymes and transporters, and examine relations with birthweight. Case-control study of placentae from term singleton pregnancies (GDM n = 24, non-GDM n = 26), and culture of another 9 placentae in different concentrations of glucose and myo-inositol for 48 hours. Placental inositol was quantified by the Megazyme assay. Relative expression of enzymes involved in myo-inositol metabolism and plasma membrane inositol transport was determined by quantitative RT-PCR and immunoblotting. Linear regression analyses were adjusted for maternal age, body mass index, ethnicity, gestational age, and sex. Placental inositol content was 17% lower in GDM compared with non-GDM. Higher maternal mid-gestation glycemia were associated with lower placental inositol. Increasing fasting glycemia was associated with lower protein levels of the myo-inositol synthesis enzyme, IMPA1, and the inositol transporters, SLC5A11 and SLC2A13, the expression of which also correlated with placental inositol content. In vitro, higher glucose concentrations reduced IMPA1 and SLC5A11 mRNA expression. Increasing fasting glycemia positively associated with customized birthweight percentile as expected in cases with low placental inositol, but this association was attenuated with high placental inositol. Glycemia-induced dysregulation of placental inositol synthesis and transport may be implicated in reduced placental inositol content in GDM, and this may in turn be permissive to accelerated fetal growth.

Genome-Wide Identification and Expression Profiling of Monosaccharide Transporter Genes Associated with High Harvest Index Values in Rapeseed (Brassica napus L.).

Sugars are important throughout a plant’s lifecycle. Monosaccharide transporters (MST) are essential sugar transporters that have been identified in many plants, but little is known about the evolution or functions of MST genes in rapeseed (Brassica napus). In this study, we identified 175 MST genes in B. napus, 87 in Brassica oleracea, and 83 in Brassica rapa. These genes were separated into the sugar transport protein (STP), polyol transporter (PLT), vacuolar glucose transporter (VGT), tonoplast monosaccharide transporter (TMT), inositol transporter (INT), plastidic glucose transporter (pGlcT), and ERD6-like subfamilies, respectively. Phylogenetic and syntenic analysis indicated that gene redundancy and gene elimination have commonly occurred in Brassica species during polyploidization. Changes in exon-intron structures during evolution likely resulted in the differences in coding regions, expression patterns, and functions seen among BnMST genes. In total, 31 differentially expressed genes (DEGs) were identified through RNA-seq among materials with high and low harvest index (HI) values, which were divided into two categories based on the qRT-PCR results, expressed more highly in source or sink organs. We finally identified four genes, including BnSTP5, BnSTP13, BnPLT5, and BnERD6-like14, which might be involved in monosaccharide uptake or unloading and further affect the HI of rapeseed. These findings provide fundamental information about MST genes in Brassica and reveal the importance of BnMST genes to high HI in B. napus.

Fungal kinases and transcription factors regulating brain infection in Cryptococcus neoformans

Cryptococcus neoformans causes fatal fungal meningoencephalitis. Here, we study the roles played by fungal kinases and transcription factors (TFs) in blood-brain barrier (BBB) crossing and brain infection in mice. We use a brain infectivity assay to screen signature-tagged mutagenesis (STM)-based libraries of mutants defective in kinases and TFs, generated in the C. neoformans H99 strain. We also monitor in vivo transcription profiles of kinases and TFs during host infection using NanoString technology. These analyses identify signalling components involved in BBB adhesion and crossing, or survival in the brain parenchyma. The TFs Pdr802, Hob1, and Sre1 are required for infection under all the conditions tested here. Hob1 controls the expression of several factors involved in brain infection, including inositol transporters, a metalloprotease, PDR802, and SRE1. However, Hob1 is dispensable for most cellular functions in Cryptococcus deuterogattii R265, a strain that does not target the brain during infection. Our results indicate that Hob1 is a master regulator of brain infectivity in C. neoformans.

TORC1 specifically inhibits microautophagy through ESCRT-0

Nutrient starvation induces the degradation of specific plasma membrane proteins through the multivesicular body (MVB) sorting pathway and of vacuolar membrane proteins through microautophagy. Both of these processes require the gateway protein Vps27, which recognizes ubiquitinated cargo proteins at phosphatidylinositol 3-phosphate-rich membranes as part of a heterodimeric complex coined endosomal sorting complex required for transport 0. The target of rapamycin complex 1 (TORC1), a nutrient-activated central regulator of cell growth, directly phosphorylates Vps27 to antagonize its function in microautophagy, but whether this also serves to restrain MVB sorting at endosomes is still an open question. Here, we show that TORC1 inhibits both the MVB pathway-driven turnover of the plasma membrane-resident high-affinity methionine permease Mup1 and the inositol transporter Itr1 and the microautophagy-dependent degradation of the vacuolar membrane-associated v-ATPase subunit Vph1. Using a Vps277D variant that mimics the TORC1-phosphorylated state of Vps27, we further show that cargo sorting of Vph1 at the vacuolar membrane, but not of Mup1 and Itr1 at endosomes, is sensitive to the TORC1-controlled modifications of Vps27. Thus, TORC1 specifically modulates microautophagy through phosphorylation of Vps27, but controls MVB sorting through alternative mechanisms.

Inositols in Insulin Signaling and Glucose Metabolism.

In the past decades, both the importance of inositol for human health and the complex interaction between glucose and inositol have been the subject of increasing consideration. Glucose has been shown to interfere with cellular transmembrane transport of inositol, inhibiting, among others, its intestinal absorption. Moreover, intracellular glucose is required for de novo biosynthesis of inositol through the inositol-3-phosphate synthase 1 pathway, while a few glucose-related metabolites, like sorbitol, reduce intracellular levels of inositol. Furthermore, inositol, via its major isomers myo-inositol and D-chiro-inositol, and probably some of its phosphate intermediate metabolites and correlated enzymes (like inositol hexakisphosphate kinase) participate in both insulin signaling and glucose metabolism by influencing distinct pathways. Indeed, clinical data support the beneficial effects exerted by inositol by reducing glycaemia levels and hyperinsulinemia and buffering negative effects of sustained insulin stimulation upon the adipose tissue and the endocrine system. Due to these multiple effects, myoIns has become a reliable treatment option, as opposed to hormonal stimulation, for insulin-resistant PCOS patients.

The Tonoplastic Inositol Transporter INT1 From Arabidopsis thaliana Impacts Cell Elongation in a Sucrose-Dependent Way.

The tonoplastic inositol transporter INT1 is the only known transport protein in Arabidopsis that facilitates myo-inositol import from the vacuole into the cytoplasm. Impairment of the release of vacuolar inositol by knockout of INT1 results in a severe inhibition of cell elongation in roots as well as in etiolated hypocotyls. Importantly, a more strongly reduced cell elongation was observed when sucrose was supplied in the growth medium, and this sucrose-dependent effect can be complemented by the addition of exogenous myo-inositol. Comparing int1 mutants (defective in transport) with mutants defective in myo-inositol biosynthesis (mips1 mutants) revealed that the sucrose-induced inhibition in cell elongation does not just depend on inositol depletion. Secondary effects as observed for altered availability of inositol in biosynthesis mutants, as disturbed membrane turnover, alterations in PIN protein localization or alterations in inositol-derived signaling molecules could be ruled out to be responsible for impairing the cell elongation in int1 mutants. Although the molecular mechanism remains to be elucidated, our data implicate a crucial role of INT1-transported myo-inositol in regulating cell elongation in a sucrose-dependent manner and underline recent reports of regulatory roles for sucrose and other carbohydrate intermediates as metabolic semaphores.

Tamoxifen inhibits the biosynthesis of inositolphosphorylceramide in Leishmania

Previous work from our group showed that tamoxifen, an oral drug that has been in use for the treatment of breast cancer for over 40 years, is active both in vitro and in vivo against several species of Leishmania, the etiological agent of leishmaniasis. Using a combination of metabolic labeling with [3H]-sphingosine and myo-[3H]-inositol, alkaline hydrolysis, HPTLC fractionations and mass spectrometry analyses, we observed a perturbation in the metabolism of inositolphosphorylceramides (IPCs) and phosphatidylinositols (PIs) after treatment of L. amazonensis promastigotes with tamoxifen, with a significant reduction in the biosynthesis of the major IPCs (composed of d16:1/18:0-IPC, t16:0/C18:0-IPC, d18:1/18:0-IPC and t16:0/20:0-IPC) and PIs (sn-1-O-(C18:0)alkyl -2-O-(C18:1)acylglycerol-3-HPO4-inositol and sn-1-O-(C18:0)acyl-2-O-(C18:1)acylglycerol-3-HPO4-inositol) species. Substrate saturation kinetics of myo-inositol uptake analyses indicated that inhibition of inositol transport or availability were not the main reasons for the reduced biosynthesis of IPC and PI observed in tamoxifen treated parasites. An in vitro enzymatic assay was used to show that tamoxifen was able to inhibit the Leishmania IPC synthase with an IC50 value of 8.48 μM (95% CI 7.68–9.37), suggesting that this enzyme is most likely one of the targets for this compound in the parasites.

Regulation of the inositol transporter Itr1p by hydrogen peroxide in Saccharomyces cerevisiae.

Myo-inositol is a precursor of several membrane phospholipids and sphingolipids and plays a key role in gene regulation in Saccharomyces cerevisiae (S. cerevisiae). Here, we tested whether H2O2 was affecting the levels of the inositol transporters and thus inositol uptake. In S. cerevisiae cells adapted to H2O2 Itr1-GFPp accumulated in the plasma membrane until 20min, concomitantly with an inhibition of its internalization. Exposure to H2O2 did not alter Itr2-GFPp cellular levels and induced only an 8% decrease at 10min in the plasma membrane. Therefore, decreased inositol intracellular levels are not caused by decreased levels of inositol transporters in the plasma membrane. However, results show that H2O2 adaptation affects Itr1p turnover and, consequently, H2O2-adapted yeast cells display an inositol transporter phenotype comparable to cells grown in the absence of inositol in growth medium, i.e. accumulation in the plasma membrane and decreased degradation.

Insights into aluminum-tolerance pathways in Stylosanthes as revealed by RNA-Seq analysis

Stylo has a great potential for Al3+ resistance in acidic soils through secretion of citrate from the roots. To get insight into the molecular mechanisms responsible, transcriptomic changes were investigated in the roots after treatment with T01 (−Al3+, pH6.0), T02 (−Al3+, pH4.3) and T03 (50 µM AlCl3, pH4.3). In total, 83,197 unigenes generated from 130,933 contigs were obtained. Of them, 282, 148 and 816 differentially expressed unigenes (DEGs) were revealed in T01_vs_T02, T02_vs_T03 and T01_vs_T03 comparison, respectively (FDR < 0.001, log2FC > 2). DEGs by Al3+ were related to G-proteins, diacyglycerol and inositol metabolism, calcium-signaling, transcription regulation, protein modification and transporters for detoxification of Al3+. Additionally, Al3+ facilitates citrate synthesis via modifying gene expression of pathways responsible for citrate metabolism. Overall, Al3+ resistance in stylo involves multiple strategies and enhancement of citrate anabolism. The Al3+ signal transmits through heterotrimeric G-proteins, phospholipase C, inositol triphosphate, diacylglycerol, Ca2+ and protein kinases, thereby activating transcription and anion channels in plasma membrane, and resulting in citrate secretion from stylo roots.

Effect of phytase on growth performance, phytate degradation and gene expression of myo- inositol transporters in the small intestine, liver and kidney of 21 day old broilers

An experiment was conducted to evaluate phytase supplementation on growth, phytate degradation, and the gene expression of myo-inositol transporters in 21-day old broilers. Ross 308, male broilers (n = 240) were assigned to one of four diets, with 10 pens/diet and six birds/pen from day one to 21. The diets consisted of a negative control (NC) formulated to meet or exceed Ross 308 nutrient requirements, with the exception of calcium (Ca) and available P (avP), which were reduced by 0.16 and 0.15%, respectively. The NC diet was supplemented with 0, 500, 1,500, or 4,500 units/kg of phytase (FTU) to create four experimental diets. On day 21, all birds per pen were euthanized to obtain digesta and tissue samples for phytate degradation and gene expression. Data were analyzed as an analysis of variance using the fit model platform in JMP v 13.0. The model included phytase and significant means were separated using orthogonal linear and quadratic contrasts. Phytase supplementation increased gain (linear, P < 0.05). Phytate (iP6; quadratic, P < 0.05), phytate ester (iP5, iP4, iP3; quadratic, P < 0.05), and inositol (linear, P < 0.05) concentration in the gizzard was influenced by phytase supplementation. Phytate concentration decreased (linear, P < 0.05), iP5 or iP4 concentration increased and then decreased (quadratic, P < 0.05), and inositol concentration increased (quadratic, P < 0.05) in the ileal digesta as phytase supplementation increased in the diet. There was a tendency for the gene expression of the H+-dependent myo-inositol transporter, HMIT, to increase (linear, P < 0.05) in the ileum as phytase dose increased. Gene expression of the sodium-dependent myo-inositol transporter, SMIT2, increased in the jejunum (quadratic, P < 0.05) as phytase dose increased. Intestinal alkaline phosphatase expression increased (linear, P < 0.05) in the ileum as phytase supplementation increased in the diet. The influence of phytase on phytate, phytate esters, and inositol may influence intestinal alkaline phosphatase activity and the gene expression of myo-inositol transporters in the small intestine.