Free Myo-inositol Research Articles

Effects of increasing dose of a hybrid bacterial 6-phytase on apparent total tract nutrient digestibility, release of free myoinositol, and retention of calcium and phosphorus, and growth performance of pigs

The objective of this study was to investigate the effects of increasing doses of a hybrid bacterial 6-phytase on apparent total tract digestibility (ATTD) of energy and nutrients, plasma level of free myoinositol, retention of calcium (Ca) and phosphorus (P), and growth performance of growing pigs fed Ca and P deficient diets. A total of 15 barrows [21.5 ± 1.4 kg BW; Duroc × (Landrace × Yorkshire)] were allotted to a triplicated 5 × 5 Latin square design with 5 dietary treatments and 5 periods per each square. Pigs were individually housed in a metabolism crate. Five dietary treatments consisted of 5 levels of phytase (0, 500, 1000, 1500, and 2000 FTU/kg) supplemented to a basal diet containing nutrients meeting the requirement except for Ca and P. The basal diet was deficient in total Ca and P by 2.2 and 1.7 g/kg below the recommendations from NRC, respectively. Data were analyzed using orthogonal polynomial contrasts using Proc MIXED of SAS and the optimal phytase levels were determined by a broken line analysis using Proc NLMIXED. Increasing phytase supplementation increased (linear: P < 0.01; quadratic: P < 0.01) ATTD of Ca and P and retention of Ca and P. Increasing phytase supplementation increased (linear: P < 0.05; quadratic: P < 0.05) myoinositol contents in plasma. Based on broken line analyses, ATTD of Ca and retention of Ca were increased until phytase supplementation were increased from 0 to 668 ± 65 FTU/kg (P < 0.01) and 0 to 668 ± 69 FTU/kg (P < 0.01), respectively. The ATTD of P and retention of P were increased until phytase supplementation contents were increased from 0 to 780 ± 86 FTU/kg (P < 0.01) and 0 to 773 ± 93 FTU/kg (P < 0.01), respectively. Myoinositol contents in plasma were increased (11.4 to 22.8 µmol/L) until phytase supplementation were increased from 0 to 705 ± 212 FTU/kg (P < 0.05). Increasing phytase supplementation linearly increased (P < 0.05) average daily gain and tended to linearly increase (P = 0.053) gain to feed ratio. In conclusion, supplementation of a hybrid bacterial 6-phytase improved ATTD of Ca and P, retention of Ca and P, complete hydrolysis of phytate, and growth performance of pigs compared with pigs fed a Ca and P deficient basal diet. Supplementation of a hybrid bacterial 6-phytase at a range of 650 to 800 FTU/kg feeds provided the optimal improvement of Ca, P, and phytate utilization.

Bioprospecting for enzymes in bryophytes: Extraction of L-Myo-inositol-1-phosphate synthase from Sphagnum junghuhnianum Doz. et Molk. and its characterization

L-myo-inositol-1-phosphate synthase (MIPS) and its product, free myo-inositol have been isolated from different bryophytes. MIPS was purified for the first time from the moss, Sphagnum junghuhnianum to 58.67 fold over its homogenate fraction with about 32.86 % recovery. D-glucose-6-phosphate was the exclusive substrate of the enzyme without which there was no enzyme activity and the deduction of NH4Cl, ME and NAD+ substantially reduced the activity of the enzyme. The Km for D-glucose-6-phosphate and NAD+were 1.81 mM and 0.25 mM, while the Vmax for the same were 1.42 mM and 1.12 mM respectively. The molecular weight of the enzyme was assessed to be approximately 174 kDa. The activity of the enzyme increased with the increase in the duration of incubation time for up to 90 min and with the increase in protein concentration for up to 300 µg. The pH and temperature maxima were pH 7.0 and at 30 °C, respectively, but a significant activity was observed at 10 °C also. NH4Cl substantially stimulated the enzyme activity while K+ and Ca2+ also raised the activity slightly . Li+ greatly inhibited the activity. Inhibitory activity was also shown by Cd2+, Mn2+, Zn2+and Hg2+ of which Hg2+ showed the maximum inhibition. Interestingly, the Sphagnum junghuhnianum MIPS showed the characteristics of both Class-II and Class-III aldolase.

272 Effects of Increasing Dose of a Novel Hybrid Bacterial 6-Phytase on Apparent Total Tract Nutrient Digestibility, Release of Free Myoinositol, and Retention of Calcium and Phosphorus in Pigs

Abstract The objective of this study was to investigate the effects of increasing dose of a novel hybrid bacterial 6-phytase on apparent total tract digestibility (ATTD) of energy and nutrients, plasma concentrations of free myoinositol, and retention of calcium (Ca) and phosphorus (P). A total of 15 barrows (21.5 ± 1.4 kg BW) were allotted to a triplicate 5 × 5 Latin square design with 5 dietary treatments and 5 periods per each square. Pigs were individually housed in a metabolism crate. Five dietary treatments consisted of 5 levels of phytase-supplemented diets with increasing phytase units (0, 500, 1,000, 1,500, and 2,000 FTU/kg). Diets contained nutrients meeting the requirement except for Ca and P. Degree of deficient Ca and P content in the NC diet were 0.22 and 0.17%, respectively. Each period lasted 10 days consisting of a 5-d adaptation period and 5-d collection period for total collection. Data were analyzed using orthogonal polynomial contrasts using the Proc MIXED and optimal phytase levels were determined using the NLMIXED of SAS. The increasing phytase supplementation linearly increased (P &amp;lt; 0.05) average daily gain (651 to 695 g/d) and tended to linearly increase (P = 0.072) gain to feed ratio (0.53 to 0.56). Increasing phytase supplementation linearly increased (P &amp;lt; 0.01) ATTD of Ca and P and retention of Ca and P and also showed quadratic effects (P &amp;lt; 0.01). Increasing phytase supplementation linearly increased (P &amp;lt; 0.01) myoinositol content in plasma and also showed quadratic effects (P &amp;lt; 0.05). Broken line analyses showed that ATTD of Ca (P &amp;lt; 0.01) and retention of Ca (P &amp;lt; 0.01) were increased until phytase supplementation were increased (79.4 to 92.1% and 77.4 to 91.4%) from 0 to 671 FTU/kg (SE = 67; P &amp;lt; 0.01) and 0 to 668 FTU/kg (SE = 72; P &amp;lt; 0.01), respectively. Broken line analyses showed that ATTD of P (P &amp;lt; 0.01) and retention of P (P &amp;lt; 0.01) were increased (59.0 to 80.1% and 55.7 to 78.2%) until phytase contents were increased from 0 to 783 FTU/kg (SE = 91; P &amp;lt; 0.01) and 0 to 776 FTU/kg (SE = 76; P &amp;lt; 0.01), respectively. The broken line analyses showed that myoinositol contents in plasma (P &amp;lt; 0.01) were increased (13.2 to 23.2 µmol/L) until phytase supplementation were increased from 0 to 624 FTU/kg (SE = 174; P &amp;lt; 0.01). In conclusion, increasing phytase supplementation up to 2,000 FTU/kg feed improved ATTD of Ca and P, retention of Ca and P, complete hydrolysis of phytate, and growth performance of pigs compared with a P-deficient diet. Phytase supplementation at a range of 600 to 800 FTU/kg feeds provided the optimal improvement of Ca, P, and phytate utilization.

High impacts of cultivar and home-cooking practice on the content of free myo-inositol, a bioavailable health-promoting cyclitol, in sweet potato.

Free myo-inositol is a bioavailable form of a cyclitol having various health-promoting activities. The impact of cultivar and home-cooking practice on the content of free myo-inositol in sweet potatoes (12 cultivars grown in 2 different locations) was studied. A GC-MS/MS method following in situ trimethylsilylation was established and validated to determine free myo-inositol. The established analytical method was sensitive, precise, and accurate. It was found that free myo-inositol content in sweet potato varied greatly (sevenfolds) with cultivar, ranging from 377.1 to 2628.3mg/kg dw. A cultivar Poongwon-mi was found to be an exceptionally rich source of free myo-inositol (2628.3mg/kg dw). Home-cooking practice markedly increased free myo-inositol content (maximum 240%). Baking showed the highest impact on the increase in free myo-inositol, followed by steaming, microwave cooking, and boiling, in decreasing order. This represents the first report of the remarkably high impact of cultivar and home-cooking practice on the free myo-inositol content in sweet potato. PRACTICAL APPLICATION: The free myo-inositol content in sweet potato varied greatly with the cultivars. Poongwon-mi contained a surprisingly high content of free myo-inositol. Home-cooking dramatically increased the free myo-inositol content.

Salinity endurance of marine macro Rhodophycean algae with special emphasis on myo-inositol biosynthesis: An enzymological analysis from Halymenia venusta Børgesen

Altered salinity is one the most important perils encountered by marine plants inclusive of algae. Under hyper saline condition plants accumulate several stress relieving osmolytes including myo-inositol, the most widespread cyclitol in plants. The present communication reports the occurrence of myo-inositol biosynthesis in six different Rhodophycean seaweeds growing under stressful intertidal habitats of the Okha coast (Gujarat, India), on the basis of a study conducted on two marker enzymes of myo-inositol biosysnthesis [L-myo-inositol-1-phosphate synthase and D/L-myo-inositol-1-phosphate phosphatise]. Both enzymes were partially purified from Halymenia venusta to about 27 and 39 folds respectively over the homogenate following low-speed centrifugation, 30-75% ammonium sulphate fractionation, successive chromatography through DEAE-cellulose / CM-Cellulose, Sephadex G-200 and BioGel 0.5m / UltrogelAcA 34 columns. The temperature and pH optima for both the enzymes were similar and were recorded to be 350C and 7.5 respectively. For MIPS, D-glucose-6-phosphate and NAD were the exclusive substrate and coenzyme respectively and D/L-MIP was the sole substrate for MIPP. The Km values for D-glucsoe-6-phosphate and β-NAD were recorded to be 3.599 mM and 0.2366 mM respectively, while the Km value for D-MIP was found to be 0.4070 mM. Monovalent cations K+ had slight stimulatory, Li+ was strong inhibitory for both the enzymes. Divalent cations Ca2+ exhibited slight stimulatory and Cd2+ reduced MIPS and MIPP activities. MIPP was stimulated by Mg2+. Cu2+ and Hg2+ were strong inhibitors of both the enzymes. A steady and proportionate increase in the content of free myo-inositol was observed along with elevated levels of recorded salinity.

Biosynthesis of Myo-Inositol in Chloroplasts of Salinity-Stressed Marine Macro Alga Ulva lactuca

AbstractThe present communication reports enhanced myo-inositol biosynthesis under natural conditions in Ulva lactuca Linn. based on the study conducted on its two prime enzymes [L-myo-inositol-1-phosphate synthase (MIPS) and myo-inositol-1-phosphate phosphatase (MIPP)] involved in myo-inositol biosynthesis. The two key enzymes obtained from chloroplastidial sources were partially purified to about 49- and 58-fold, respectively, over the homogenate following low speed centrifugation, high speed centrifugation, 0–80% ammonium sulphate precipitation and successive chromatography using ion exchange, gel filtration and molecular sieve packed columns. MIPS preparations specifically utilized D-glucose-6-phosphate and NAD as its exclusive substrate and coenzyme, while MIPP preparations used D/L-myo-inositol -1- phosphate as its principal substrate. Using non-linear regression kinetics method, the Km values of MIPS for G-6-P and NAD were calculated to be 2.6340 mM and 0.1271 mM, while the Km value of MIPP for D-MIP was recorded to be 0.02128 mM. Both enzymes were remarkably active within a temperature range of 20–40°C, and the optimum pH for both enzymes were found to be 7.5. Different cations and organic modifiers exhibited variable effects on the activity of both enzymes. The content of free myo-inositol was found to increase proportionately with the increase of surface salinity of the Chilika Lagoon, Odisha, India.

Influence of phytase or myo-inositol supplements on performance and phytate degradation products in the crop, ileum, and blood of broiler chickens

The objective of this study was to investigate the effects of supplementation with free myo-inositol (MI) or graded levels of phytase on inositol phosphate (InsP) degradation, concentrations of MI in the digestive tract and blood, bone mineralization, and prececal digestibility of amino acids (AA). Ross 308 broiler hatchlings were allocated to 40 pens with 11 birds each and assigned to one of 5 treatments. The birds were fed a starter diet until d 11 and a grower diet from d 11 to d 22. All diets were based on wheat, soybean meal, and corn. Birds were fed a control diet, calculated to contain adequate levels of all nutrients without (C) or with MI supplementation (C+MI), or one of 3 experimental diets that differed in phytase level (modified E. coli-derived 6-phytase; Phy500, Phy1500, or Phy3000 FTU/kg), with P and Ca levels adapted to the recommendations of the phytase supplier for a phytase level of 500 FTU/kg. The gain:feed ratio (G:F) was increased by MI or phytase in the starter+grower phase by 0.02 g/g. Prececal P and Ca digestibility, P and Ca concentration in blood serum, and tibia ash weight did not differ among treatments (P > 0.05). MI supplementation led to the highest MI concentration in the crop, ileum, and blood plasma across treatments. Phytase supplementation increased MI concentrations in the crop and ileum digesta in a dose-dependent manner and in plasma without any dose effect (P > 0.05). Prececal digestibility of some AA was increased by phytase. These outcomes indicate that MI might have been a relevant cause for the increase in G:F. Therefore, it is likely that the release of MI after complete dephosphorylation of phytate is one of the beneficial effects of phytase, along with the release of P and improvement in digestibility of other nutrients. Simultaneously, MI seems to have no diminishing effects on InsP degradation.

Survey of Inositol in Infant Formula

Results of free and bound myo-inositol in infant formula (IF) are presented. Inositol was analyzed by HILIC ultraperformance liquid chromatography coupled with mass spectrometer. The levels of free myo-inositol in 27 Australian and 4 EU originated IF samples were 300-600 mg/kg of powder or 1.6-3.1 mg/100 kJ. The amount of bound inositol in lipid fraction of IF was, on average, 10% of free myo-inositol.

Determination of Myo-Inositol in Infant, Pediatric, and Adult Formulas by Liquid Chromatography-Pulsed Amperometric Detection with Column Switching: Collaborative Study, Final Action 2011.18.

AOAC First Action Method 2011.18, Myo-Inositol (Free and Bound as Phosphatidylinositol) in Infant and Pediatric Formulas and Adult Nutritionals, was collaboratively studied. With this method free myo-inositol and phosphatidylinositol bound myo-inositol are extracted using two different sample preparation procedures, separated by ion chromatography using a combination of Dionex Carbo Pac PA1 and MA1 columns with column switching, and detected with pulsed amperometry using a gold electrode. Free myo-inositol is extracted from samples with dilute hydrochloric acid and water. Phosphatidylinositol is extracted from samples with chloroform and separated from other fats with silica SPE cartridges. Myo-inositol is then released from the glycerol backbone with concentrated acetic and hydrochloric acids at 120°C. During this collaborative study, nine laboratories from five different countries analyzed blind duplicates of nine infant and pediatric nutritional formulas for both free and phosphatidylinositol bound myo-inositol, and one additional laboratory only completed the free myo-inositol analyses. The method demonstrated acceptable repeatability and reproducibility and met the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) Standard Method Performance Requirements (SMPRs®) for free myo-inositol plus phosphatidylinositol bound myo-inositol for all the matrixes analyzed. SMPRs for repeatability were ≤5% RSD at myo-inositol concentrations of 2-68 mg/100 g ready-to-feed (RTF) liquid. SMPRs for reproducibility were ≤8% RSD in products with myo-inositol concentrations ranging from 2 to 68 mg/100 g RTF liquid. During this collaborative study, repeatability RSDs ranged from 0.51 to 3.22%, and RSDs ranged from 2.66 to 7.55% for free myo-inositol plus phosphatidylinositol bound myo-inositol.

Fast separation and determination of free myo-inositol by hydrophilic liquid chromatography

A fast liquid chromatography method for separation and determination of myo-inositol is reported. Determination of the biologically important isomer of inositols, myo-inositol, was optimized to avoid overlapping to possible interferents according to European Pharmacopoeia (glycerol, d-mannitol) and saccharose. The method in HILIC mode is extremely selective to other carbohydrates which allows to separate myo-inositol from allo- and d-chiro-inositol with resolution 12.3 and 5.2, resp. and this way it enables to separate myo-inostiol from contingent carbohydrates present in a sample matrix. Retention time of myo-inositol was 12min at 10°C, though higher temperatures (25°C or 40°C) or higher water content in the mobile phase could speed up the separation and determination to four minutes. LOD of the method was 9mg/L at 10°C, and 5mg/L at 25°C, resp.

Determination of Free and Total Myo-Inositol in Infant Formula and Adult/Pediatric Nutritional Formula by High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection, Including a Novel Total Extraction Using Microwave-Assisted Acid Hydrolysis and Enzymatic Treatment: First Action 2012.12

After an assessment of data generated from a single-laboratory validation study published in J. AOAC Int. 95, 1469-1478 (2012), a method for determining total myo-inositol in infant formula and adult/ pediatric nutritional formula by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), including extraction by using microwave-assisted acid hydrolysis and enzymatic treatment was presented for consideration by AOAC during the AOAC Annual Meeting held in Las Vegas, NV, from September 30 to October 3, 2012. The Expert Review Panel on Infant Formula and Adult Nutritionals concluded that the method met the criteria set by the standard method performance requirements (SMPRs) for the determination of free myo-inositol and approved the method as AOAC Official First Action. The method also determines total myo-inositol, but includes bound sources that the SMPRs exclude. The method involves using HPAEC-PAD for free myo-inositol and a total myo-inositol determination by two different techniques. The first technique uses the conventional acid hydrolysis with 6 h incubation in an autoclave. The second uses a microwave-assisted acid hydrolysis with enzymatic treatment that decreases the extraction time.

Phytases and &lt;i&gt;myo&lt;/i&gt;-inositol modulate performance, bone mineralization and alter lipid fractions in the serum of broilers

Under conditions simulating intestinal digestion in broilers, phytase B was found to free myo-inositol from low non-phytate phospho- rus maize-soyabean meal-based and wheat-soyabean meal-based diets, whereas both 3- and 6-phytases A accelerated myo-inositol release only in the presence of phytase B. In the feeding experiment, myo-inositol at 1 g · kg -1 increased the feed intake and body weight gain of broilers fed both types of feeds. Phytase B at 1300 acid phosphatase units (AcPU) · kg -1 enhanced feed consumption, body weight gain, and tibia mineralization, whereas phytase B mixed with 6-phytase A at 500 phytase units (FTU) · kg -1 produced further en- hancements in performance and bone mineralization. After 6 weeks, broilers fed the maize-based diets supplemented with both phytase B and 6-phytase A had increased serum concentrations of triglycerides and HDL-cholesterol, as well as an enhanced HDL/total cholesterol ratio. The increases in this ratio cannot be attributed to the release of phosphorus or to the generation of myo- inositol by the supplemented enzymes, but can only be explained as effects of intermediate products of phytate dephosphorylation.

Tumour cells can employ extracellular Ins(1,2,3,4,5,6)P6 and multiple inositol-polyphosphate phosphatase 1 (MINPP1) dephosphorylation to improve their proliferation

InsP(6) [Ins(1,2,3,4,5,6)P6; phytate] is the most abundant inositol phosphate in mammalian cells with cytosolic/nuclear concentrations of up to 50 μM. We noticed that InsP6 in culture medium at a concentration of ≤50 μM significantly stimulates H1299 tumour cell growth, whereas larger concentrations of InsP6 inhibit growth. A detailed study of the fate of 30 μM InsP6 added to H199 cells revealed a major fraction of InsP6 initially precipitates as cell-surface metal complexes, but becomes slowly re-solubilized by extracellular dephosphorylation first to InsP3 isomers and subsequently to free myo-inositol. The precipitated metal-InsP6 complex is endocytosed in a receptor-independent but intact-glycocalyx-dependent manner and appears in lysosomes, where it is immediately dephosphorylated to Ins(1,2,4,5,6)P5 and very slowly to free inositol. By RNA knockdown, we identified secreted and lysosome targeted MINPP1 (multiple inositol-polyphosphate phosphatase 1), the mammalian 3-phytase, to be essentially involved both in extracellular and in lysosomal InsP6 dephosphorylation. The results of the present study indicate that tumour cells employ this enzyme to utilize the micronutrients myo-inositol and metal-phosphate when encountering extracellular InsP6 and thus to enhance their growth potential.

Analysis of Free and Total Myo-Inositol in Foods, Feeds, and Infant Formula by High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection, Including a Novel Total Extraction Using Microwave-Assisted Acid Hydrolysis and Enzymatic Treatment

A method for the analysis of free and total myo-inositol in foods, feeds, and infant formulas has been developed and validated using high-performance anion exchange chromatography with pulsed amperometric detection. The option of a free myo-inositol determination or a complete total myo-inositol determination from main bound sources can be achieved. These sources include phytates, lower'phosphorylated forms, and phosphatidylinositol. This approach gives the option for subtraction of myo-inositol from nonbioavailable sources when it is quantified using other methods if a total bioavailable myo-inositol result is desired for nutritional labeling of a product. The free analysis was validated in a milk-based infant formula, giving RSD(R) of 2.29% and RSD, of 2.06%. A mean recovery of 97.9% was achieved from various spike levels of myo-inositol. Certified National Institute of Standards and Technology reference material verified the method's compatibility and specificity. Two different total analyses were validated in a soy-based infant formula and compared. One technique involved using a conventional acid hydrolysis with autoclave incubation for 6 h, while the other used a novel technique of microwave-assisted acid hydrolysis with enzymatic treatment that can minimize extraction to 1 day. The autoclave analysis had RSD(R) of 2.08% and RSDr of 1.55%, along with a mean spike recovery of 102.1% at various myo-inositol spike levels. The microwave/enzyme total analysis had RSD(R) of 4.34% and RSD, of 4.70%, along with a mean spike recovery of 104.2% at various spike levels of myo-inositol. Main sources of myo-inositol including phytic acid and phosphatidylinositol were tested with both total analyses. Mean recoveries of phytic acid and phosphatidylinositol through the autoclave total analysis were 90.4 and 98.3%, respectively. Mean spike recoveries for these same sources in soy- based infant formula through the microwave/enzyme total analysis were 97.2 and 96.3%, respectively. Comparison of soy-based infant formula and corn grain samples with high levels of these main sources showed in similar results, indicating both total analyses are acceptable for use. An additional glycerol kinase step was also developed to remove glycerol from the chromatographic elution window of myoinositol in samples with high levels of glycerol.

Determination of Myo-Inositol (Free and Bound as Phosphatidylinositol) in Infant Formula and Adult Nutritionals by Liquid Chromatography/Pulsed Amperometry with Column Switching: First Action 2011.18

Myo-inositol is a 6-carbon cyclic polyalcohol also known as meso-inositol, meat sugar, inosite, and i-inositol. It occurs in nature in both free (myo-inositol) and bound (inositol phosphates and phosphatidylinositol) forms. For the determination of free myo-inositol, samples are mixed with dilute hydrochloric acid to extract myo-inositol and precipitate proteins, diluted with water, and filtered. For the determination of myo-inositol bound as phosphatidylinositol, samples are extracted with chloroform, isolated from other fats with silica SPE cartridges, and hydrolyzed with concentrated acid to free myo-inositol. Prepared samples are first injected onto a Dionex CarboPac PA1 column, which separates myo-inositol from other late-eluting carbohydrates. After column switching, myo-inositol is further separated on a CarboPac MA1 column using a 0.12% sodium hydroxide mobile phase; strongly retained carbohydrates are eluted from the PA1 column with a 3% sodium hydroxide mobile phase. Eluant from the CarboPac MA1 analytical column passes through an electrochemical detector cell where myo-inositol is detected by pulsed amperometry using a gold electrode. The method showed appropriate performance characteristics versus selected established standard method performance requirement parameters for the determination of myo-inositol: linear response; repeatability (RSDr) of 2%; and intermediate precision (RSDir) of 2.5%. Instrument LOD and LOQ were 0.0004 and 0.0013 mg/100 mL, respectively, and correspond to a free myo-inositol quantitation limit of 0.026 mg/100 g and a phosphatidylinositol quantitation limit of 0.016 mg/100 g. Correlation with the reference microbiological assay was good. The proposed method has been accepted by the Expert Review Panel as an AOAC First Action Method, suitable for the routine determination of myo-inositol in infant formula and adult nutritionals.

血中FREE MYO-INOSITOLと腎機能について

血中FREE MYO-INOSITOLと腎機能について

Myo‐inositol oxygenase genes are involved in the development of syncytia induced by Heterodera schachtii in Arabidopsis roots

* In plants, UDP-glucuronic acid is synthesized by the oxidation of UDP-glucose by UDP-glucose dehydrogenase or the oxygenation of free myo-inositol by myo-inositol oxygenase (MIOX). In Arabidopsis, myo-inositol oxygenase is encoded by four genes. Transcriptome analysis of syncytia induced by the cyst nematode Heterodera schachtii in Arabidopsis roots revealed that MIOX genes are among the most strongly upregulated genes. * We have used beta-glucuronidase (GUS) analysis, in situ reverse transcription polymerase chain reaction (RT-PCR), and real-time RT-PCR to study the expression of all four MIOX genes in syncytia induced by H. schachtii in Arabidopsis roots. All these methods showed that MIOX genes are strongly induced in syncytia. GeneChip data were analysed for the expression of genes related to the MIOX pathway (mapman). * Two complementary double mutants were used to study the importance of MIOX genes. Results of the infection assay with double mutants in two combinations (Deltamiox1+2, Deltamiox4+5) showed a significant reduction (P < 0.05) in the number of females per plant when compared with the wild-type. Furthermore, syncytia in double mutants were significantly smaller than in wild-type plants. * Our data demonstrate an important role of the MIOX genes for syncytium development and for the development of female nematodes.

L-myo-InositoL-1- phosphate synthase from bryophytes: purification and characterization of the enzyme from Lunularia cruciata (L.) Dum

L-myo-inositol-1-phosphate synthase (MIPS; EC: 5.5.1.4) catalyzes the conversion of D-glucose-6-phosphate to 1L-myo-inositol-1-phosphate, the rate limiting step in the biosynthesis of all inositol containing compounds. Myo-inositol and its derivatives are implicated in membrane biogenesis, cell signaling, salinity stress tolerance and a number of other metabolic reactions in different organisms. This enzyme has been reported from a number of bacteria, fungi, plants and animals. In the present study some bryophytes available in the Eastern Himalaya have been screened for free myo-inositol content. It is seen that Bryum corinatum, a bryopsid shows the highest content of free myo-inositol among the species screened. Subsequently , the enzyme MIPS has been partially purified to the tune of about 70 fold with approximately 18% recovery form the reproductive part bearing gametophytes of Lunularia cruciata. The L. cruciata synthase specifically utilized D-glucose-6-phosphate and NAD+ as its substrate and co-factor respectively. The optimum pH shown was 7.0 while the temperature maximum was at 30ºC. The enzyme activity was slightly stimulated by Mg2+ and Ca2+; remarkably stimulated by NH4+; slightly inhibited by Mn2+; highly inhibited by Cu2+, Zn2+ and Hg2+. The Km values for D-glucose-6-phosphate and NAD+ was found to be 0.80 and 0. 034 mM respectively while the Vmax values were 2.8 and 1.21 mM for D-glucose-6-phosphate and NAD+ respectively.

Homozygote inositol transporter knockout mice show a lithium‐like phenotype

Lithium inhibits inositol monophosphatase and also reduces inositol transporter function. To determine if one or more of these mechanisms might underlie the behavioral effects of lithium, we studied inositol transporter knockout mice. We previously reported that heterozygous knockout mice with reduction of 15-37% in brain inositol had no abnormalities of pilocarpine sensitivity or antidepressant-like behavior in the Porsolt forced swim test. We now report on studies of homozygous inositol transporter knockout mice. Homozygote knockout mice were rescued by 2% inositol supplementation to the drinking water of the dam mice through pregnancy and lactation. Genotyping was carried out by polymerase chain reaction followed by agarose electrophoresis. Brain free myo-inositol levels were determined gas-chromatographically. Motor activity and coordination were assessed by the rotarod test. Behavior of the mice was studied in lithium-pilocarpine seizure models for lithium action and in the Porsolt forced swim test model for depression. In homozygote knockout mice, free inositol levels were reduced by 55% in the frontal cortex and by 60% in the hippocampus. There were no differences in weight or motor coordination by the rotarod test. They behaved similarly to lithium-treated animals in the model of pilocarpine seizures and in the Porsolt forced swimming test model of depression. Reduction of brain inositol more than 15-37% may be required to elicit lithium-like neurobehavioral effects.

Dimerization of inositol monophosphatase Mycobacterium tuberculosis SuhB is not constitutive, but induced by binding of the activator Mg2+.

BackgroundThe cell wall of Mycobacterium tuberculosis contains a wide range of phosphatidyl inositol-based glycolipids that play critical structural roles and, in part, govern pathogen-host interactions. Synthesis of phosphatidyl inositol is dependent on free myo-inositol, generated through dephosphorylation of myo-inositol-1-phosphate by inositol monophosphatase (IMPase). Human IMPase, the putative target of lithium therapy, has been studied extensively, but the function of four IMPase-like genes in M. tuberculosis is unclear.ResultsWe determined the crystal structure, to 2.6 Å resolution, of the IMPase M. tuberculosis SuhB in the apo form, and analysed self-assembly by analytical ultracentrifugation. Contrary to the paradigm of constitutive dimerization of IMPases, SuhB is predominantly monomeric in the absence of the physiological activator Mg2+, in spite of a conserved fold and apparent dimerization in the crystal. However, Mg2+ concentrations that result in enzymatic activation of SuhB decisively promote dimerization, with the inhibitor Li+ amplifying the effect of Mg2+, but failing to induce dimerization on its own.ConclusionThe correlation of Mg2+-driven enzymatic activity with dimerization suggests that catalytic activity is linked to the dimer form. Current models of lithium inhibition of IMPases posit that Li+ competes for one of three catalytic Mg2+ sites in the active site, stabilized by a mobile loop at the dimer interface. Our data suggest that Mg2+/Li+-induced ordering of this loop may promote dimerization by expanding the dimer interface of SuhB. The dynamic nature of the monomer-dimer equilibrium may also explain the extended concentration range over which Mg2+ maintains SuhB activity.