Synthesis Of Myo-inositol Research Articles

Metabolic adjustments of blood-stage Plasmodium falciparum in response to sublethal pyrazoleamide exposure

Due to the recurring loss of antimalarial drugs to resistance, there is a need for novel targets, drugs, and combination therapies to ensure the availability of current and future countermeasures. Pyrazoleamides belong to a novel class of antimalarial drugs that disrupt sodium ion homeostasis, although the exact consequences of this disruption in Plasmodium falciparum remain under investigation. In vitro experiments demonstrated that parasites carrying mutations in the metabolic enzyme PfATP4 develop resistance to pyrazoleamide compounds. However, the underlying mechanisms that allow mutant parasites to evade pyrazoleamide treatment are unclear. Here, we first performed experiments to identify the sublethal dose of a pyrazoleamide compound (PA21A092) that caused a significant reduction in growth over one intraerythrocytic developmental cycle (IDC). At this drug concentration, we collected transcriptomic and metabolomic data at multiple time points during the IDC to quantify gene- and metabolite-level alterations in the treated parasites. To probe the effects of pyrazoleamide treatment on parasite metabolism, we coupled the time-resolved omics data with a metabolic network model of P. falciparum. We found that the drug-treated parasites adjusted carbohydrate metabolism to enhance synthesis of myoinositol—a precursor for phosphatidylinositol biosynthesis. This metabolic adaptation caused a decrease in metabolite flux through the pentose phosphate pathway, causing a decreased rate of RNA synthesis and an increase in oxidative stress. Our model analyses suggest that downstream consequences of enhanced myoinositol synthesis may underlie adjustments that could lead to resistance emergence in P. falciparum exposed to a sublethal dose of a pyrazoleamide drug.

In-silico studies on Myo inositol-1-phosphate synthase of Leishmania donovani in search of anti-leishmaniasis

Myo-inositol is one of the vital nutritional requirements for the Leishmania parasites’ survival and virulence in the mammalian host. . Myo-inositol-1-phosphate synthase (MIPS) is responsible for the synthesis of myo-inositol in Leishmania, which plays a vital role in Leishmania’s virulence to mammalian hosts. Earlier studies suggest MIP synthase as a potential drug target against which valproate was used as a drug. So, MIP synthase can be used as a target for anti-leishmanial drugs, and its inhibition may help in preventing leishmaniasis. The present study aims to identify valproate's potent analogs as drugs against MIP synthase of L. donovani (Ld-MIPS) with minimum side effects and toxicity to host. In this study, the three-dimensional structure of Ld-MIPS was built, followed by active site prediction. Ligand-based virtual screening was done using hybrid similarity recognition methods. The best 123 valproate analogs were filtered based on their quantitative structure activity relationship (QSAR) properties and were docked against Ld-MIPS using FlexX, PyRx and iGEMDOCK software. The topmost five ligands were selected for molecular dynamics simulation and pharmacokinetic analysis based on the docking score. Simulation studies up to 30 ns revealed that all five lead molecules bound with Ld-MIPS throughout MD simulation and there was no variation in their backbone. All the chosen inhibitors exhibited good pharmacokinetics/ADMET predictions with an excellent absorption profile, metabolism, oral bioavailability, solubility, excretion, and minimal toxicity, suggesting that these inhibitors may further be developed as anti-leishmaniasis drugs to prevent the spread of leishmaniasis. Communicated by Ramaswamy H. Sarma

Diminished Myoinositol in Ventromedial Prefrontal Cortex Modulates the Endophenotype of Impulsivity.

Maladaptive impulsivity manifests in a variety of disorders, including attention-deficit hyperactivity disorder (ADHD), depression, and substance use disorder. However, the etiological mechanisms of impulsivity remain poorly understood. In the present study, we used in-vivo proton magnetic resonance spectroscopy (1H-MRS) to investigate neurometabolite content in the prefrontal cortex (PFC) and striatum of rats exhibiting low- versus high-impulsive (LI, HI) behavior on a visual attentional task. We validated our 1H-MRS findings using regionally resolved ex-vivo mass spectroscopy, transcriptomics, and site-directed RNA interference in the ventromedial PFC. We report a significant reduction in myoinositol levels in the PFC but not the striatum of HI rats compared with LI rats. Reduced myoinositol content was localized to the infralimbic (IL) cortex, where significant reductions in transcript levels of key proteins involved in the synthesis and recycling of myoinositol (IMPase1) were also present. Knockdown of IMPase1in the IL cortex increased impulsivity in nonimpulsive rats when the demand on inhibitory response control was increased. We conclude that diminished myoinositol levels in ventromedial PFC causally mediate a specific form of impulsivity linked to vulnerability for stimulant addiction in rodents. Myoinositol and related signaling substrates may thus offer novel opportunities for treating neuropsychiatric disorders comorbid with impulsive symptomology.

Co-suppression of AtMIPS demonstrates cooperation of MIPS1, MIPS2 and MIPS3 in maintaining myo-inositol synthesis.

Co-suppressed MIPS2 transgenic lines allow bypass of the embryo lethal phenotype of the previously published triple knock-out and demonstrate the effects of MIPS on later stages of development. Regulation of inositol production is of interest broadly for its effects on plant growth and development. The enzyme L-myo-inositol 1-phosphate synthase (MIPS, also known as IPS) isomerizes D-glucose-6-P to D-inositol 3-P, and this is the rate-limiting step in inositol production. In Arabidopsis thaliana, the MIPS enzyme is encoded by three different genes, (AtMIPS1, AtMIPS2 and AtMIPS3), each of which has been shown to produce proteins with biochemically similar properties but differential expression patterns. Here, we report phenotypic and biochemical effects of MIPS co-suppression. We show that some plants engineered to overexpress MIPS2 in fact show reduced expression of AtMIPS1, AtMIPS2 and AtMIPS3, and show altered vegetative phenotype, reduced size and root length, and delayed flowering. Additionally, these plants show reduced inositol, increased glucose levels, and alteration of other metabolites. Our results suggest that the three AtMIPS genes work together to impact the overall synthesis of myo-inositol and overall inositol homeostasis.

Altering carbon allocation in hybrid poplar (Populus alba×grandidentata) impacts cell wall growth and development.

Galactinol synthase is a pivotal enzyme involved in the synthesis of the raffinose family of oligosaccharides (RFOs) that function as transport carbohydrates in the phloem, as storage compounds in sink tissues and as soluble metabolites that combat both abiotic and biotic stress in several plant species. Hybrid poplar (Populus alba × grandidentata) overexpressing the Arabidopsis thaliana GolS3 (AtGolS3) gene showed clear effects on development; the extreme overexpressing lines were stunted and had cell wall traits characteristic of tension wood, whereas lines with only moderate up‐regulation grew normally and had moderately altered secondary cell wall composition and ultrastructure. Stem cross‐sections of the developing xylem revealed a significant increase in the number of vessels, as well as the clear presence of a G‐layer in the fibres. Furthermore, AtGolS3‐OE lines possessed higher cellulose and lower lignin contents, an increase in cellulose crystallinity, and significantly altered hemicellulose‐derived carbohydrates, notably manifested by their mannose and xylose contents. In addition, the transgenic plants displayed elevated xylem starch content. Transcriptome interrogation of the transgenic plants showed a significant up‐regulation of genes involved in the synthesis of myo‐inositol, along with genes involved in sucrose degradation. The results suggest that the overexpression of GolS and its product galactinol may serve as a molecular signal that initiates metabolic changes, culminating in a change in cell wall development and potentially the formation of tension wood.

Useful access to enantiomerically pure protected inositols from carbohydrates: the aldohexos-5-uloses route.

The intramolecular aldol condensation of aldohexos-5-ulose derivatives of the D-xylo and L-ribo stereoseries has been studied. Only one of the four possible inososes was isolated from both stereoseries in reasonable yields (30–38%). The results obtained, together with the previous findings for the L-arabino and L-lyxo stereoseries, allowed for the rationalisation of a mechanism of the reaction based on open-transition-state models and electron-withdrawing inductive effects. Complementary reductions of the intermediate inososes were possible by changing the reaction conditions, and two isomeric inositol derivatives were obtained with complete stereoselection from each inosose. The presented approach permits us to control the configuration of three out of the six stereocentres of the inositol frame and gives access to seven of the nine inositols. Noteworthy, for the D-xylo derivative, the two-step sequence (condensation followed by reduction with NaBH(OAc)3) represents the biomimetic synthesis of myo-inositol. Furthermore, the sugar-based pathway leads directly to enantiomerically pure selectively protected inositols and does not require any desymmetrisation procedure which is needed when myo-inositol and other achiral precursors are employed as starting materials. As an example of application of the method, the indirect selective protection of secondary inositols’ hydroxy functions, by placing specific protecting groups on the aldohexos-5-ulose precursor has been presented.

Osmoadaptative Strategy and Its Molecular Signature in Obligately Halophilic Heterotrophic Protists.

Halophilic microbes living in hypersaline environments must counteract the detrimental effects of low water activity and salt interference. Some halophilic prokaryotes equilibrate their intracellular osmotic strength with the extracellular milieu by importing inorganic solutes, mainly potassium. These “salt-in” organisms characteristically have proteins that are highly enriched with acidic and hydrophilic residues. In contrast, “salt-out” halophiles accumulate large amounts of organic solutes like amino acids, sugars and polyols, and lack a strong signature of halophilicity in the amino acid composition of cytoplasmic proteins. Studies to date have examined halophilic prokaryotes, yeasts, or algae, thus virtually nothing is known about the molecular adaptations of the other eukaryotic microbes, that is, heterotrophic protists (protozoa), that also thrive in hypersaline habitats. We conducted transcriptomic investigations to unravel the molecular adaptations of two obligately halophilic protists, Halocafeteria seosinensis and Pharyngomonas kirbyi. Their predicted cytoplasmic proteomes showed increased hydrophilicity compared with marine protists. Furthermore, analysis of reconstructed ancestral sequences suggested that, relative to mesophiles, proteins in halophilic protists have undergone fewer substitutions from hydrophilic to hydrophobic residues since divergence from their closest relatives. These results suggest that these halophilic protists have a higher intracellular salt content than marine protists. However, absence of the acidic signature of salt-in microbes suggests that Haloc. seosinensis and P. kirbyi utilize organic osmolytes to maintain osmotic equilibrium. We detected increased expression of enzymes involved in synthesis and transport of organic osmolytes, namely hydroxyectoine and myo-inositol, at maximal salt concentration for growth in Haloc. seosinensis, suggesting possible candidates for these inferred organic osmolytes.

Characterization of the inositol monophosphatase gene family in Arabidopsis.

Synthesis of myo-inositol is crucial in multicellular eukaryotes for production of phosphatidylinositol and inositol phosphate signaling molecules. The myo-inositol monophosphatase (IMP) enzyme is required for the synthesis of myo-inositol, breakdown of inositol (1,4,5)-trisphosphate, a second messenger involved in Ca2+ signaling, and synthesis of L-galactose, a precursor of ascorbic acid. Two myo-inositol monophosphatase -like (IMPL) genes in Arabidopsis encode chloroplast proteins with homology to the prokaryotic IMPs and one of these, IMPL2, can complement a bacterial histidinol 1-phosphate phosphatase mutant defective in histidine synthesis, indicating an important role for IMPL2 in amino acid synthesis. To delineate how this small gene family functions in inositol synthesis and metabolism, we sought to compare recombinant enzyme activities, expression patterns, and impact of genetic loss-of-function mutations for each. Our data show that purified IMPL2 protein is an active histidinol-phosphate phosphatase enzyme in contrast to the IMPL1 enzyme, which has the ability to hydrolyze D-galactose 1-phosphate, and D-myo-inositol 1-phosphate, a breakdown product of D-inositol (1,4,5) trisphosphate. Expression studies indicated that all three genes are expressed in multiple tissues, however, IMPL1 expression is restricted to above-ground tissues only. Identification and characterization of impl1 and impl2 mutants revealed no viable mutants for IMPL1, while two different impl2 mutants were identified and shown to be severely compromised in growth, which can be rescued by histidine. Analyses of metabolite levels in impl2 and complemented mutants reveals impl2 mutant growth is impacted by alterations in the histidine biosynthesis pathway, but does not impact myo-inositol synthesis. Together, these data indicate that IMPL2 functions in the histidine biosynthetic pathway, while IMP and IMPL1 catalyze the hydrolysis of inositol- and galactose-phosphates in the plant cell.

Molecular cloning and characterization of a cDNA encoding kiwifruit l-myo-inositol-1-phosphate synthase, a key gene of inositol formation

L-myo-inositol-1-phosphate synthase (MIPS; EC 5.5.1.4) is the key enzyme involved in de novo synthesis of myo-inositol, leading to numerous cellular functions. We isolated an open reading frame of Actinidia deliciosa MIPS (AdMIPS), which is 1,533 bp long and codes for 510 amino acids, with a predicted molecular weight of 56.3 kDa. Sequence analysis revealed its high similarity with MIPS proteins from other organisms. Gene expression and enzyme activity were highest in flower and young fruit. Transcription of AdMIPS was also detected in other tissues. Moderate drought drastically induced expression in the leaves whereas salinity stress induced transcription and enzyme activity in the leaves, phloem, and roots with different degrees. However, a longer period of saline exposure suppressed both expression and enzyme activity in all sampled tissues, indicating that AdMIPS is salt-sensitive.

Mutations in the Arabidopsis Homolog of LST8/GβL, a Partner of the Target of Rapamycin Kinase, Impair Plant Growth, Flowering, and Metabolic Adaptation to Long Days

The conserved Target of Rapamycin (TOR) kinase forms high molecular mass complexes and is a major regulator of cellular adaptations to environmental cues. The Lethal with Sec Thirteen 8/G protein β subunit-like (LST8/GβL) protein is a member of the TOR complexes, and two putative LST8 genes are present in Arabidopsis thaliana, of which only one (LST8-1) is significantly expressed. The Arabidopsis LST8-1 protein is able to complement yeast lst8 mutations and interacts with the TOR kinase. Mutations in the LST8-1 gene resulted in reduced vegetative growth and apical dominance with abnormal development of flowers. Mutant plants were also highly sensitive to long days and accumulated, like TOR RNA interference lines, higher amounts of starch and amino acids, including proline and glutamine, while showing reduced concentrations of inositol and raffinose. Accordingly, transcriptomic and enzymatic analyses revealed a higher expression of genes involved in nitrate assimilation when lst8-1 mutants were shifted to long days. The transcriptome of lst8-1 mutants in long days was found to share similarities with that of a myo-inositol 1 phosphate synthase mutant that is also sensitive to the extension of the light period. It thus appears that the LST8-1 protein has an important role in regulating amino acid accumulation and the synthesis of myo-inositol and raffinose during plant adaptation to long days.

Comparative Expression and Cellular Localization of Myo-inositol Phosphate Synthase (MIPS) in the Wild Type and in an EMS Mutant During Common Bean (Phaseolus vulgaris L.) Seed Development

In plants, the myo-inositol-1-phosphate synthase (EC 5.5.1.4; MIPS) is an evolutionarily conserved enzyme protein and catalyzes the synthesis of myo-inositol, which is a central molecule required for cell metabolism and plant growth. A full-length cDNA encoding common bean (Phaseolus vulgaris L.) MIPS (designated Pv_BAT93 MIPS) was isolated from seed of P. vulgaris (cv, BAT93) by rapid amplification of cDNA ends. The cDNA of Pv_BAT93 MIPS comprised 1,873 bp, which encodes 510 amino acids. The Pv_BAT93 MIPS gene was highly homologous with those of other plant species, such as P. vulgaris (cv, Taylor’s Horticultural), Arabidopsis thaliana, Medicago sativa and Glycine max. DNA blot analysis indicated that at least three copies of MIPS are present in the common bean genome. RT-PCR analysis indicated that the Pv_BAT93 MIPS transcripts existed in developing seeds and other common bean tissues, including leaves, flowers, and cotyledons but showed lower levels expression in roots and stems. Real-time quantitative PCR showed that Pv_BAT93 MIPS gene is differentially expressed during common bean seed development. In situ hybridization of developing seeds in the wild type and in the ethyl methanesulfonate mutant showed that expression of Pv_BAT93 MIPS was identified in the embryo tissues, from the globular to late cotyledon stages, confirming a possible involvement of Pv_BAT93 MIPS in common bean seed development.

D-myo-Inositol-3-Phosphate Affects Phosphatidylinositol-Mediated Endomembrane Function inArabidopsisand Is Essential for Auxin-Regulated Embryogenesis

In animal cells, myo-inositol is an important regulatory molecule in several physiological and biochemical processes, including signal transduction and membrane biogenesis. However, the fundamental biological functions of myo-inositol are still far from clear in plants. Here, we report the genetic characterization of three Arabidopsis thaliana genes encoding D-myo-inositol-3-phosphate synthase (MIPS), which catalyzes the rate-limiting step in de novo synthesis of myo-inositol. Each of the three MIPS genes rescued the yeast ino1 mutant, which is defective in yeast MIPS gene INO1, and they had different dynamic expression patterns during Arabidopsis embryo development. Although single mips mutants showed no obvious phenotypes, the mips1 mips2 double mutant and the mips1 mips2 mips3 triple mutant were embryo lethal, whereas the mips1 mips3 and mips1 mips2⁺/⁻ double mutants had abnormal embryos. The mips phenotypes resembled those of auxin mutants. Indeed, the double and triple mips mutants displayed abnormal expression patterns of DR5:green fluorescent protein, an auxin-responsive fusion protein, and they had altered PIN1 subcellular localization. Also, membrane trafficking was affected in mips1 mips3. Interestingly, overexpression of PHOSPHATIDYLINOSITOL SYNTHASE2, which converts myo-inositol to membrane phosphatidylinositol (PtdIns), largely rescued the cotyledon and endomembrane defects in mips1 mips3. We conclude that myo-inositol serves as the main substrate for synthesizing PtdIns and phosphatidylinositides, which are essential for endomembrane structure and trafficking and thus for auxin-regulated embryogenesis.

Differential methylation of the gene encoding myo -inositol 3-phosphate synthase ( Isyna1 ) in rat tissues

Myo-inositol levels are frequently altered in several brain disorders. Myo-inositol 3-phosphate synthase, encoded by the Isyna1 gene, catalyzes the synthesis of myo-inositol in cells. Very little is known about the mechanisms regulating Isyna1 expression in brain and other tissues. In this study, we have examined the role of DNA methylation in regulating Isyna1 expression in rat tissues. Transfection analysis using in vitro methylated promoter constructs, Southern blot analysis of genomic DNA from various tissues digested with a methylation-sensitive enzyme and CpG methylation profiling of genomic DNA from different tissues were used to determine differential methylation of Isyna1 in tissues. Transfection analysis using plasmids harboring mutated CpG residues in the 5'-upstream region of Isyna1 was used to identify critical residues mediating promoter activity. The -700 bp to -500 bp region (region 1) of Isyna1 exhibited increased methylation in brain cortex compared with other tissues; it also exhibited sex-specific methylation differences between matched male and female brain cortices. Mutation analysis identified one CpG residue in region 1 necessary for promoter activity in neuronal cells. A tissue-specific differentially methylated region (T-DMR) was found to be localized between +450 bp and +650 bp (region 3). This DMR was comparatively highly methylated in spleen, moderately methylated in brain cortex and poorly methylated in testis, consistent with mRNA levels observed in these tissues. Rat Isyna1 exhibits tissue-specific DNA methylation. Brain DNA was uniquely methylated in the 5'-upstream region and displayed gender specificity. A T-DMR was identified within the gene body of Isyna1. These findings suggest that Isyna1 is regulated, in part, by DNA methylation and that significant alterations in methylation patterns during development could have a major impact on inositol phosphate synthase expression in later life.

TheArabidopsis thaliana Myo-Inositol 1-Phosphate Synthase1 Gene Is Required forMyo-inositol Synthesis and Suppression of Cell Death

l-myo-inositol 1-phosphate synthase (MIPS; EC 5.5.1.4) catalyzes the rate-limiting step in the synthesis of myo-inositol, a critical compound in the cell. Plants contain multiple MIPS genes, which encode highly similar enzymes. We characterized the expression patterns of the three MIPS genes in Arabidopsis thaliana and found that MIPS1 is expressed in most cell types and developmental stages, while MIPS2 and MIPS3 are mainly restricted to vascular or related tissues. MIPS1, but not MIPS2 or MIPS3, is required for seed development, for physiological responses to salt and abscisic acid, and to suppress cell death. Specifically, a loss in MIPS1 resulted in smaller plants with curly leaves and spontaneous production of lesions. The mips1 mutants have lower myo-inositol, ascorbic acid, and phosphatidylinositol levels, while basal levels of inositol (1,4,5)P(3) are not altered in mips1 mutants. Furthermore, mips1 mutants exhibited elevated levels of ceramides, sphingolipid precursors associated with cell death, and were complemented by a MIPS1-green fluorescent protein (GFP) fusion construct. MIPS1-, MIPS2-, and MIPS3-GFP each localized to the cytoplasm. Thus, MIPS1 has a significant impact on myo-inositol levels that is critical for maintaining levels of ascorbic acid, phosphatidylinositol, and ceramides that regulate growth, development, and cell death.

VTC4 Is a Bifunctional Enzyme That Affects Myoinositol and Ascorbate Biosynthesis in Plants

Myoinositol synthesis and catabolism are crucial in many multiceullar eukaryotes for the production of phosphatidylinositol signaling molecules, glycerophosphoinositide membrane anchors, cell wall pectic noncellulosic polysaccharides, and several other molecules including ascorbate. Myoinositol monophosphatase (IMP) is a major enzyme required for the synthesis of myoinositol and the breakdown of myoinositol (1,4,5)trisphosphate, a potent second messenger involved in many biological activities. It has been shown that the VTC4 enzyme from kiwifruit (Actinidia deliciosa) has similarity to IMP and can hydrolyze l-galactose 1-phosphate (l-Gal 1-P), suggesting that this enzyme may be bifunctional and linked with two potential pathways of plant ascorbate synthesis. We describe here the kinetic comparison of the Arabidopsis (Arabidopsis thaliana) recombinant VTC4 with d-myoinositol 3-phosphate (d-Ins 3-P) and l-Gal 1-P. Purified VTC4 has only a small difference in the V(max)/K(m) for l-Gal 1-P as compared with d-Ins 3-P and can utilize other related substrates. Inhibition by either Ca(2+) or Li(+), known to disrupt cell signaling, was the same with both l-Gal 1-P and d-Ins 3-P. To determine whether the VTC4 gene impacts myoinositol synthesis in Arabidopsis, we isolated T-DNA knockout lines of VTC4 that exhibit small perturbations in abscisic acid, salt, and cold responses. Analysis of metabolite levels in vtc4 mutants showed that less myoinositol and ascorbate accumulate in these mutants. Therefore, VTC4 is a bifunctional enzyme that impacts both myoinositol and ascorbate synthesis pathways.

Barley (Hordeum vulgare L.) inositol monophosphatase: gene structure and enzyme characteristics

The cellular myo-inositol (Ins) pool is important to many metabolic and signaling pathways in plants. Ins monophosphatase (IMPase; EC 3.1.3.25) activity is essential for the de novo synthesis of myo-Inositol (Ins), and for recycling of Ins in Ins(1,4,5)P3. However, proteins encoded by at least one family of IMP genes also have L-galactose-1-P phosphatase activity important to ascorbic acid synthesis, indicating a bifunctionality that links these two branches of carbon metabolism. As part of research into the regulation of Ins synthesis and supply during seed development, the barley IMP-1 gene and gene products were studied. The 1.4 kb barley IMP-1 promoter contains one low temperature response element (RE), two heat shock REs, one gibberellin and two auxin REs, and five sugar REs. Barley IMP-1 is expressed in all tissues assayed, and expression levels were not greatly altered by abiotic stress treatments. Reduced use of Ins for Ins P6 synthesis in developing seed of barley low phytic acid (lpa) mutants results in Ins accumulation, and IMP-1 expression is reduced in proportion to the increase in Ins level. The barley recombinant enzyme had a lower Km, indicating higher affinity, for D/L-Ins(3)P1 (Km = 9.7 microM) as compared with reported Km (Ins P1) values for other eukaryotic IMPases (43-330 microM) or with a reported Km (L-Gal-1P) of 150 microM for a kiwifruit (Actinidia deliciosa) enzyme. These and other data indicate that the barley IMP-1 gene is regulated at least in part in response to Ins metabolic needs, and that the enzyme it encodes displays catalytic properties well suited for a role in Ins synthesis, in addition to other roles as an L-gal-1-P phosphatase important to ascorbate synthesis, or as an IMPase important to Ins(1,4,5)P3 signal recycling.

Localization of myo-inositol phosphate synthase (GmMIPS-1) during the early stages of soybean seed development

As a precursor to a large variety of compounds, myo-inositol is a central molecule required for cell metabolism and plant growth. The de novo synthesis of myo-inositol requires the activity of the enzyme D-myo-inositol-3-phosphate synthase (MIPS). MIPS cDNAs encoding one or more isoforms have been cloned from a number of species, nevertheless, little is known about the regulation of MIPS expression in developing seed. Seed-specific expression of a soybean isoform (GmMIPS-1) has been demonstrated, but tissue-specific localization during embryo development has not been reported. Using immunolocalization techniques, a specialized area of GmMIPS-1 expression was identified in the outer integumentary layer during early soybean seed development. In addition, localization data provided evidence that MIPS was associated with oxalate crystal idioblasts.

Whole-Genome Expression Profiling Defines the HrpL Regulon of Pseudomonas syringae pv. tomato DC3000, Allows de novo Reconstruction of the Hrp cis Element, and Identifies Novel Coregulated Genes

Pseudomonas syringae pv. tomato DC3000 is a model pathogen of tomato and Arabidopsis that uses a hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS) to deliver virulence effector proteins into host cells. Expression of the Hrp system and many effector genes is activated by the HrpL alternative sigma factor. Here, an open reading frame-specific whole-genome microarray was constructed for DC3000 and used to comprehensively identify genes that are differentially expressed in wild-type and deltahrpL strains. Among the genes whose differential regulation was statistically significant, 119 were upregulated and 76 were downregulated in the wild-type compared with the deltahrpL strain. Hierarchical clustering revealed a subset of eight genes that were upregulated particularly rapidly. Gibbs sampling of regions upstream of HrpL-activated operons revealed the Hrp promoter as the only identifiable regulatory motif and supported an iterative refinement involving real-time polymerase chain reaction testing of additional HrpL-activated genes and refinements in a hidden Markov model that can be used to predict Hrp promoters in P. syringae strains. This iterative bioinformatic-experimental approach to a comprehensive analysis of the HrpL regulon revealed a mix of genes controlled by HrpL, including those encoding most type III effectors, twin-arginine transport (TAT) substrates, other regulatory proteins, and proteins involved in the synthesis or metabolism of phytohormones, phytotoxins, and myo-inositol. This analysis provides an extensively verified, robust method for predicting Hrp promoters in P. syringae genomes, and it supports subsequent identification of effectors and other factors that likely are important to the host-specific virulence of P. syringae.

Synthesis of the Trypanosoma cruzi LPPG Heptasaccharyl myo-Inositol

Synthesis of the heptasaccharyl myo-inositol found in Trypanosoma cruzi lipopeptidophosphoglycan was accomplished using a convergent assembly of three building blocks. The target compound is the first complete 2-aminoethyl phosphonic acid substituted glycan related to the glycosylphosphatidylinositol anchor family to be synthesized. The order of assembly enables synthesis of phosphoinositol oligosaccharides related to other glycosylinositolphospholipids in Tr. cruzi, the protozoan parasite causing Chagas' disease, which is endemic in South America.

The myo-inositol-1-phosphate synthase gene is essential in Trypanosoma brucei

The de novo synthesis of myo-inositol occurs via a two-step process: first, glucose 6-phosphate is converted into inositol 1-phosphate by an INO1 (myo-inositol-1-phosphate synthase; EC 5.5.1.4); then, it is dephosphorylated by an inositol monophosphatase. The myo-inositol can then be incorporated into PI (phosphatidylinositol), which is utilized in a variety of cellular functions, including the biosynthesis of GPI (glycosylphosphatidylinositol) anchors. A putative INO1 was identified in the Trypanosoma brucei genome database and, by recombinant expression in Escherichia coli, was shown to be a catalytically active INO1. To investigate the importance of INO1, we created a conditional knockout, which, under non-permissive conditions, showed that INO1 is an essential gene in bloodstream form T. brucei and that the de novo synthesized myo-inositol is used for the formation of PI and GPI anchors.