Myo-inositol Oxygenase Research Articles

Sodium-Glucose Co-Transporter 2 Inhibitors Correct Metabolic Maladaptation of Proximal Tubular Epithelial Cells in High-Glucose Conditions.

Glucose filtered in the glomerulus is actively reabsorbed by sodium-glucose co-transporter 2 (SGLT2) in proximal tubular epithelial cells (PTEC) and passively returned to the blood via glucose transporter 2 (GLUT2). Healthy PTEC rely primarily on fatty acid beta-oxidation (FAO) for energy. In phase III trials, SGLT2 inhibitors improved outcomes in diabetic kidney disease (DKD). Tubulointerstitial renal fibrosis due to altered metabolic reprogramming of PTEC might be at the root of the pathogenesis of DKD. Here, we investigated the molecular mechanism of SGLT2 inhibitors’ renoprotective effect by examining transcriptional activity of Spp1, which encodes osteopontin, a key mediator of tubulointerstitial renal fibrosis. With primary cultured PTEC from Spp1-enhanced green fluorescent protein knock-in mice, we proved that in high-glucose conditions, increased SGLT2- and GLUT-mediated glucose uptake is causatively involved in aberrant activation of the glycolytic pathway in PTEC, thereby increasing mitochondrial reactive oxygen species (ROS) formation and transcriptional activation of Spp1. FAO activation did not play a direct role in these processes, but elevated expression of a tubular-specific enzyme, myo-inositol oxygenase, was at least partly involved. Notably, canagliflozin blocked overexpression of myo-inositol oxygenase. In conclusion, SGLT2 inhibitors exerted renoprotective effects by inhibiting aberrant glycolytic metabolism and mitochondrial ROS formation in PTEC in high-glucose conditions.

Research Note: Jejunum phosphatases and systemic myo-inositol in broiler chickens fed without or with supplemented phytase

As a constituent of animal cells, myo-inositol (MI) has been hypothesized to be crucial in several metabolic and regulatory pathways. Recently, it was shown that dietary phytase contributes to release of MI from phytate in the poultry digestive tract, increasing its systemic concentrations. This study investigated the activities of phosphatases in the jejunum and systemic plasma MI concentration in broilers not supplemented or supplemented with phytase through analyses based on modifications from commercial enzyme activity kits. Three hundred sixty male Ross 308 broilers were randomly allocated to 24 pens (15 birds per pen) in 4 dietary groups. The positive control group was fed with an adequate basal diet. The negative control group (NC) was fed with a reduced level of P and Ca. Groups Phy1500 and Phy3000 were fed with the NC diet plus 1,500 or 3,000 FTU of phytase per kilogram of feed, respectively. One bird per pen was selected for the measurement of jejunal phosphatase activity; MI concentration in plasma, the liver, and the kidney; and key MI enzyme concentrations (liver inositol monophosphatase 1 [IMPase 1] and kidney myo-inositol oxygenase [MIOX]). Endogenous phytase and alkaline phosphatase activity as well as IMPase 1 and MIOX expression were not statistically different among the dietary groups. The supplementation of 1500 FTU of phytase per kilogram of feed resulted in increase of plasma (P < 0.001) and kidney (P < 0.05) but not liver MI concentrations. The results indicated that systemic MI might reflect MI released from dietary sources; however, it did not appear to change expression of enzymes related to endogenous MI synthesis in the liver and catabolism in the kidney. New and larger studies are necessary to reach stronger evidence on the effects of dietary phytase on intestinal and systemic MI concentrations in broilers.

Combinatorial synthetic pathway fine-tuning and cofactor regeneration for metabolic engineering of Escherichia coli significantly improve production of D-glucaric acid

D-glucaric acid (GA) has been identified as among promising biotechnological alternatives to oil-based chemicals. GA and its derivatives are widely used in food additives, dietary supplements, drugs, detergents, corrosion inhibitors and biodegradable materials. The increasing availability of a GA market is improving the cost-effectiveness and efficiency of various biosynthetic pathways. In this study, an engineered Escherichia coli strain GA10 was constructed by systematic metabolic engineering. This involved redirecting metabolic flux into the GA biosynthetic pathways, blocking the conversion pathways of d-glucuronic acid (GlcA) and GA into by-products, introducing an in situ NAD+ regeneration system and fine-tuning the activity of the key enzyme, myo-inositol oxygenase (Miox). Subsequently, the culture medium was optimized to achieve the best performance of the GA10 strain. GA was produced at 5.35 g/L (extracellular and intracellular), with a maximized yield of ∼0.46 mol/mol on d-glucose and glycerol, by batch fermentation. This work demonstrates efficient biosynthetic pathways of GA in E. coli by metabolic engineering and should accelerate the application of GA biosynthetic pathways in industrial processes.

Functional characterization of wheat myo-inositol oxygenase promoter under different abiotic stress conditions in Arabidopsis thaliana.

The production of wheat is severely affected by abiotic stresses such as cold, drought, salinity, and high temperature. Although constitutive promoters are frequently used to regulate the expression of alien genes, these may lead to undesirable side-effects in transgenic plants. Therefore, identification and characterization of an inducible promoter that can express transgene only when exposed to stresses are of great importance in the genetic engineering of crop plants. Previous studies have indicated the abiotic stress-responsive behavior of myo-inositol oxygenase (MIOX) gene in different plants. Here, we isolated the MIOX gene promoter from wheat (TaMIOX). The in-silico analysis revealed the presence of various abiotic stress-responsive cis-elements in the promoter region. The TaMIOX promoter was fused with the UidA reporter gene and transformed into Arabidopsis thaliana. The T3 single-copy homozygous lines were analyzed for GUS activity using histochemical and fluorometric assays. Transcript expression of TaMIOX::UidA was significantly up-regulated by heat (fivefold), cold (sevenfold), and drought (fivefold) stresses as compared to transgenic plants grown without stress-induced conditions. The CaMV35S::UidA plants showed very high GUS activity even in normal conditions. In contrast, the TaMIOX::UidA plants showed prominent GUS activity only in stress treatments (cold, heat, and drought), which suggests the inducible behavior of the TaMIOX promoter. The substrate myo-inositol feeding assay of TaMIOX::UidA plants showed lesser GUS activity as compared to plants treated in abiotic stress conditions. Results support that the TaMIOX promoter could be used as a potential candidate for conditional expression of the transgene in abiotic stress conditions.

Modulation of Renal Injury by Variable Expression of Myo-Inositol Oxygenase (MIOX) via Perturbation in Metabolic Sensors.

Obesity is associated with perturbations in cellular energy homeostasis and consequential renal injury leading to chronic renal disease (CKD). Myo-inositol oxygenase (MIOX), a tubular enzyme, alters redox balance and subsequent tubular injury in the settings of obesity. Mechanism(s) for such adverse changes remain enigmatic. Conceivably, MIOX accentuates renal injury via reducing expression/activity of metabolic sensors, which perturb mitochondrial dynamics and, if sustained, would ultimately contribute towards CKD. In this brief communication, we utilized MIOX-TG (Transgenic) and MIOXKO mice, and subjected them to high fat diet (HFD) administration. In addition, ob/ob and ob/MIOXKO mice of comparable age were used. Mice fed with HFD had increased MIOX expression and remarkable derangements in tubular injury biomarkers. Decreased expression of p-AMPKα (phospho AMP-activated protein kinase) in the tubules was also observed, and it was accentuated in MIOX-TG mice. Interestingly, ob/ob mice also had decreased p-AMPKα expression, which was restored in ob/MIOXKO mice. Parallel changes were observed in Sirt1/Sirt3 (silent mating type information regulation 2 homolog), and expression of other metabolic sensors, i.e., PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) and Yin Yang (YY-1). In vitro experiments with tubular cells subjected to palmitate-BSA and MIOX-siRNA had results in conformity with the in vivo observations. These findings link the biology of metabolic sensors to MIOX expression in impaired cellular energy homeostasis with exacerbation/amelioration of renal injury.

Sequence-based bioprospecting of myo-inositol oxygenase (Miox) reveals new homologues that increase glucaric acid production in Saccharomyces cerevisiae

myo-Inositol oxygenase (Miox) is a rate-limiting enzyme for glucaric acid production via microbial fermentation. The enzyme converts myo-inositol to glucuronate, which is further converted to glucaric acid, a natural compound with industrial uses that range from detergents to pharmaceutical synthesis to polymeric materials. More than 2,000 Miox sequences are available in the Uniprot database but only thirteen are classified as reviewed in Swiss-Prot (August 2019). In this study, sequence similarity networks were used to identify new homologues to be expressed in Saccharomyces cerevisiae for glucaric acid production. The expression of four homologues did not lead to product formation. Some of these enzymes may have a defective “dynamic lid” – a structural feature important to close the reaction site – which might explain the lack of activity. Thirty-one selected Miox sequences did allow for product formation, of which twenty-five were characterized for the first time. Expression of Talaromyces marneffei Miox led to the accumulation of 1.76 ± 0.33 g glucaric acid/L from 20 g glucose/L and 10 g/L myo-inositol. Specific glucaric acid titer with TmMiox increased 44 % compared to the often-used Arabidopsis thaliana variant AtMiox4 (0.258 vs. 0.179 g glucaric acid/g biomass). AtMiox4 activity decreased from 12.47 to 0.40 nmol/min/mg protein when cells exited exponential phase during growth on glucose, highlighting the importance of future research on Miox stability in order to further improve microbial production of glucaric acid.

Genome-wide analysis of Myo-inositol oxygenase gene family in tomato reveals their involvement in ascorbic acid accumulation

BackgroundAscorbic acid (Vitamin C, AsA) is an antioxidant metabolite involved in plant development and environmental stimuli. AsA biosynthesis has been well studied in plants, and MIOX is a critical enzyme in plants AsA biosynthesis pathway. However, Myo-inositol oxygenase (MIOX) gene family members and their involvement in AsA biosynthesis and response to abiotic stress remain unclear.ResultsIn this study, five tomato genes encoding MIOX proteins and possessing MIOX motifs were identified. Structural analysis and distribution mapping showed that 5 MIOX genes contain different intron/exon patterns and unevenly distributed among four chromosomes. Besides, expression analyses indicated the remarkable expression of SlMIOX genes in different plant tissues. Furthermore, transgenic lines were obtained by over-expression of the MIOX4 gene in tomato. The overexpression lines showed a significant increase in total ascorbate in leaves and red fruits compared to control. Expression analysis revealed that increased accumulation of AsA in MIOX4 overexpression lines is possible as a consequence of the multiple genes involved in AsA biosynthesis. Myo inositol (MI) feeding in leaf and fruit implied that the Myo-inositol pathway improved the AsA biosynthesis in leaves and fruits. MIOX4 overexpression lines exhibited a better light response, abiotic stress tolerance, and AsA biosynthesis capacity.ConclusionsThese results showed that MIOX4 transgenic lines contribute to AsA biosynthesis, evident as better light response and improved oxidative stress tolerance. This study provides the first comprehensive analysis of the MIOX gene family and their involvement in ascorbate biosynthesis in tomato.

Myo-inositol oxygenese activity in second trimester of pregnancy: altered myoinositol catabolism in gestational diabetes mellitus

Objective We aimed to determine myo-inositol oxygenase (MIOX) activity in gestational diabetes mellitus (GDM). Material and Methods The insulin, HbA1c, and MIOX levels of 80 pregnant women were analysed after 75 g OGTT. Group I included patients with no risk factor for GDM, Group II: patients with high risk for GDM, and Group III: GDM patients. Results Fasting plasma glucose and Homeostatic Model Assessment for Insulin Resistance index were significantly higher in GDM cases (Group 3). Regarding the MIOX levels, significantly higher levels were recorded at 0-h in Group 3 compared to Groups 1 and 2. Significant alteration in MIOX activity was found between 0- and 2-h in Group 3 compared to Groups 1 and 2 (p < .029). Conclusions MIOX levels were higher in GDM cases so, it may be have a role in myo-inositol catabolism.

Myo-inositol Oxygenase (MIOX) Overexpression Drives the Progression of Renal Tubulointerstitial Injury in Diabetes

Conceivably, upregulation of myo-inositol oxygenase (MIOX) is associated with altered cellular redox. Its promoter includes oxidant-response elements, and we also discovered binding sites for XBP1, a transcription factor of endoplasmic reticulum (ER) stress response. Previous studies indicate that MIOX’s upregulation in acute tubular injury is mediated by oxidant and ER stress. Here, we investigated whether hyperglycemia leads to accentuation of oxidant and ER stress while these boost each other’s activities, thereby augmenting tubulointerstitial injury/fibrosis. We generated MIOX-overexpressing transgenic (MIOX-TG) and MIOX knockout (MIOX-KO) mice. A diabetic state was induced by streptozotocin administration. Also, MIOX-KO were crossbred with Ins2Akita to generate Ins2Akita/KO mice. MIOX-TG mice had worsening renal functions with kidneys having increased oxidant/ER stress, as reflected by DCF/dihydroethidium staining, perturbed NAD-to-NADH and glutathione-to-glutathione disulfide ratios, increased NOX4 expression, apoptosis and its executionary molecules, accentuation of TGF-β signaling, Smads and XBP1 nuclear translocation, expression of GRP78 and XBP1 (ER stress markers), and accelerated tubulointerstitial fibrosis. These changes were not seen in MIOX-KO mice. Interestingly, such changes were remarkably reduced in Ins2Akita/KO mice and, likewise, in vitro experiments with XBP1 siRNA. These findings suggest that MIOX expression accentuates, while its deficiency shields kidneys from, tubulointerstitial injury by dampening oxidant and ER stress, which mutually enhance each other’s activity.

Transcriptomics analyses reveal that OsMIOX improves rice drought tolerance by regulating the expression of plant hormone and sugar related genes

Drought stress, as a key negative factor, could result in the reduction of yield and quality in rice. Myo-inositol oxygenase (MIOX), a unique monooxygenase, exerts an essential role in plant drought resistance. To investigate the function of the OsMIOX gene in drought process, a transcriptomic profile between OsMIOX-overexpressing (OE) plants and wild-type (WT) was performed by RNA-sequencing in rice. Under drought stress, a total of 23 million high-quality reads, assembled into 28,552 unigenes, were generated and 743 genes (534 up-regulated and 209 down-regulated) were significantly differentially expressed between OE plants and WT. These detected genes were mainly associated with plant hormones transduction and sugar metabolism. Interestingly, several transcription factors (TFs) were prominently identified in OE lines. A significant positive correlation between RNA-Seq data and qRT-PCR results was identified. In all, our results could partially provide relatively valuable clues in identifying candidate genes involved in drought-stress resistance and elucidating basis theory underlying the role of OsMIOX in drought resistance.

Effectiveness of myo-inositol oxygenase in the early diagnosis of experimental acute kidney injury.

The effectiveness of the myo-inositol oxygenase (MIOX) enzyme was investigated in the diagnosis of acute kidney injury (AKI). In total, 40 rats were divided into 5 groups (n = 8, for each group) while left kidney ischemia-reperfusion was implemented in groups 2, 3, 4 and 5. Group 1 was the control group. Group 2 underwent 1‑hour ischemia and 2‑hour reperfusion. Group 3 underwent 1‑hour ischemia and 4‑hour reperfusion. Group 4 underwent 2‑hour ischemia and 2‑hour reperfusion. Group 5 underwent 2‑hour ischemia and 4‑hour reperfusion. Serum creatinine and blood urea nitrogen levels in all ischemia-reperfusion groups were higher than in the control group (p<0.001). Serum MIOX level was higher in groups 2, 3 and 4 than in group 1 (p=0.002). Tissue MIOX level was lower in groups 2, 4, and 5 than in group 1 (p=0.039). Serum and tissue neutrophil gelatinase-associated lipocalin levels were not significantly different between the groups. The injury level in histopathologic examination was as follows: group 1<group 3<group 2=group 4<group 5. The serum MIOX level increases in the early stages of AKI, however, decreases subsequently. Therefore, the serum MIOX may be a potential promising biomarker in the early diagnosis of AKI (Tab. 5, Fig. 6, Ref. 30).

Myo-inositol oxygenase expression profile modulates pathogenic ferroptosis in the renal proximal tubule

Overexpression of myo-inositol oxygenase (MIOX), a proximal tubular enzyme, exacerbates cellular redox injury in acute kidney injury (AKI). Ferroptosis, a newly coined term associated with lipid hydroperoxidation, plays a critical role in the pathogenesis of AKI. Whether or not MIOX exacerbates tubular damage by accelerating ferroptosis in cisplatin-induced AKI remains elusive. Cisplatin-treated HK-2 cells exhibited notable cell death, which was reduced by ferroptosis inhibitors. Also, alterations in various ferroptosis metabolic sensors, including lipid hydroperoxidation, glutathione peroxidase 4 (GPX4) activity, NADPH and reduced glutathione (GSH) levels, and ferritinophagy, were observed. These perturbations were accentuated by MIOX overexpression, while ameliorated by MIOX knockdown. Likewise, cisplatin-treated CD1 mice exhibited tubular damage and derangement of renal physiological parameters, which were alleviated by ferrostatin-1, a ferroptosis inhibitor. To investigate the relevance of MIOX to ferroptosis, WT mice, MIOX-overexpressing transgenic (MIOX-Tg) mice, and MIOX-KO mice were subjected to cisplatin treatment. In comparison with cisplatin-treated WT mice, cisplatin-treated MIOX-Tg mice had more severe renal pathological changes and perturbations in ferroptosis metabolic sensors, which were minimal in cisplatin-treated MIOX-KO mice. In conclusion, these findings indicate that ferroptosis, an integral process in the pathogenesis of cisplatin-induced AKI, is modulated by the expression profile of MIOX.

Urinary myo-inositol is associated with the clinical outcome in focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) have similar initial histological findings; however, their prognoses are distinct. Therefore, it is of great importance to discriminate FSGS from MCD in the early phase of disease and predict clinical prognosis. A discovery set of 184 urine samples (61 healthy control, 80 MCD, and 43 FSGS) and a validation set of 61 urine samples (12 healthy control, 26 MCD, and 23 FSGS) were collected at the time of kidney biopsy. Metabolic profiles were examined using nuclear magnetic resonance spectroscopy. Of 70 urinary metabolites, myo-inositol was significantly higher in FSGS patients than in control patients (discovery set, 2.34-fold, P < 0.001; validation set, 2.35-fold, P = 0.008) and MCD patients (discovery set, 2.48-fold, P = 0.002; validation set, 1.69-fold, P = 0.042). Myo-inositol showed an inverse relationship with the initial estimated glomerular filtration rate (eGFR) and was associated with the plasma level of soluble urokinase-type plasminogen activator receptor in FSGS patients. Myo-inositol treatment ameliorated the decreased expression of ZO-1 and synaptopodin in an in vitro FSGS model, and as myo-inositol increased, myo-inositol oxygenase tissue expression decreased proportionally to eGFR. Furthermore, urinary myo-inositol exhibited an increase in the power to discriminate FSGS patients, and its addition could better predict the response to initial treatment. In conclusion, urinary myo-inositol may be an important indicator in the diagnosis and treatment of FSGS patients.

Mechanisms underlying the enhanced biomass and abiotic stress tolerance phenotype of an Arabidopsis MIOX over-expresser.

Myo‐inositol oxygenase (MIOX) is the first enzyme in the inositol route to ascorbate (L‐ascorbic acid, AsA, vitamin C). We have previously shown that Arabidopsis plants constitutively expressing MIOX have elevated foliar AsA content and displayed enhanced growth rate, biomass accumulation, and increased tolerance to multiple abiotic stresses. In this work, we used a combination of transcriptomics, chromatography, microscopy, and physiological measurements to gain a deeper understanding of the underlying mechanisms mediating the phenotype of the AtMIOX4 line. Transcriptomic analysis revealed increased expression of genes involved in auxin synthesis, hydrolysis, transport, and metabolism, which are supported by elevated auxin levels both in vitro and in vivo, and confirmed by assays demonstrating their effect on epidermal cell elongation in the AtMIOX4 over‐expressers. Additionally, we detected up‐regulation of transcripts involved in photosynthesis and this was validated by increased efficiency of the photosystem II and proton motive force. We also found increased expression of amylase leading to higher intracellular glucose levels. Multiple gene families conferring plants tolerance/expressed in response to cold, water limitation, and heat stresses were found to be elevated in the AtMIOX4 line. Interestingly, the high AsA plants also displayed up‐regulation of transcripts and hormones involved in defense including jasmonates, defensin, glucosinolates, and transcription factors that are known to be important for biotic stress tolerance. These results overall indicate that elevated levels of auxin and glucose, and enhanced photosynthetic efficiency in combination with up‐regulation of abiotic stresses response genes underly the higher growth rate and abiotic stresses tolerance phenotype of the AtMIOX4 over‐expressers.

Myo-inositol in the protection from cadmium-induced toxicity in mice kidney: An emerging nutraceutical challenge

Cadmium (Cd) induces functional and morphological changes in kidney. Therefore, the effects of a natural nutraceutical antioxidant, myo-inositol (MI), were evaluated in mice kidneys after Cd challenge. Twenty-eight C57 BL/6 J mice were divided into these groups: 0.9% NaCl; MI (360 mg/kg/day); CdCl2 (2 mg/kg/day) plus vehicle; CdCl2 (2 mg/kg/day) plus MI (360 mg/kg/day). After 14 days, kidneys were processed for structural, biochemical and morphometric evaluation. Treatment with CdCl2 increased urea nitrogen and creatinine in serum and augmented tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) expression. Furthermore, monocyte chemoattractant protein-1 (MCP-1), kidney injury molecule-1 (KIM-1) and myo-inositol oxygenase (MIOX) immunoreactivity, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells number were significantly higher than control and MI groups. Glutathione (GSH) content and glutathione peroxidase (GPx) activity were reduced and structural changes were evident. The treatment with MI significantly lowered urea nitrogen and creatinine levels, TNF-α and iNOS expression, MCP-1, KIM-1 and MIOX immunoreactivity and TUNEL positive cells number, increased GSH content and GPx activity and preserved kidney morphology. A protection of MI against Cd-induced damages in mice kidney was demonstrated, suggesting a strong antioxidant role of this nutraceutical against environmental Cd harmful effects on kidney lesions.

Measurement of myo-inositol oxygenase as an early biomarker in renal ischemia – reperfusion injury in rats

23rd IFCC-EFLM European Congress of Clinical Chemistry and Laboratory Medicine (EuroMedLab) -- MAY 19-23, 2019 -- Barcelona, SPAIN

Production of d-glucuronic acid from myo-inositol using Escherichia coli whole-cell biocatalyst overexpressing a novel myo-inositol oxygenase from Thermothelomyces thermophile

D-glucuronic acid (GlcUA) is an important intermediate with numerous applications in the food, cosmetics, and pharmaceutical industries. Its biological production routes which employ myo-inositol oxygenase (MIOX) as the key enzyme are attractive. In this study, five diverse MIOX-encoding genes, from Cryptococcus neoformans, Chaetomium thermophilum, Arabidopsis thaliana, Thermothelomyces thermophila, and Mus musculus were overexpressed in Escherichia coli, respectively. A novel MIOX from Thermothelomyces thermophila (TtMIOX) exhibited high specific activity, and efficiently converted myo-inositol to GlcUA. Meanwhile, the degradation of GlcUA was inhibited by inactivation of uxaC from the Escherichia coli genome. Finally, the BWΔuxaC whole-cell biocatalyst harboring TtMIOX resulted in the production of 106 g/L GlcUA within 12 h in a 1-L bioreactor, corresponding to a conversion of 91% and productivity of 8.83 g/L/h. This study provides a feasible method for the industrial production of GlcUA.

A New Perspective on the Effect of UV-B on l-Ascorbic Acid Metabolism in Cucumber Seedlings.

This study aimed to examine the effects of UV-B on AsA and gene expression in cucumber seedlings. Particular emphasis was placed on identifying genes that were responsive to UV-B to increase AsA levels and elucidate the key UV-B response pathway. We found that the activities of myo-inositol oxygenase (MIOX), galactono-1,4-lactone dehydrogenase (GLDH), ascorbate oxidase (AO), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR), and the transcript levels of CsMIOX1, CsGLDH, CsAO2, CsAO4, CsGR1, CsAPX5, and CsDHAR1 significantly increased with UV-B exposure. These observations indicate that UV-B induces the expression of genes involved in d-mannose/l-galactose and myo-inositol pathways and the ascorbate-glutathione system. Moreover, several genes related to the low and high UV-B fluence responses were considered. CsHY5 and CsMYB60 were involved with the low-fluence response and appeared to be responsive from 2 to 28 h. Together, these data show that these genes respond to UV-B to increase AsA levels through the low-fluence UV-B response pathway.

Myo-inositol oxygenase accentuates renal tubular injury initiated by endoplasmic reticulum stress.

Besides oxidant stress, endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of various metabolic disorders affecting the kidney. These two forms of stresses are not mutually exclusive to each other and may operate by a feedback loop in worsening the cellular injury. To attest to this contention, studies were performed to assess whether in such a setting, there is worsening of tubulointerstitial injury. We employed tunicamycin as a model of ER stress and used tubular cells and mice overexpressing myo-inositol oxygenase (MIOX), an enzyme involved in glycolytic events with excessive generation of ROS. Concomitant treatment of tunicamycin and transfection of cells with MIOX-pcDNA led to a marked generation of ROS, which was reduced by MIOX-siRNA. Likewise, an accentuated expression of ER stress sensors, GRP78, XBP1, and CHOP, was observed, which was reduced with MIOX-siRNA. These sensors were markedly elevated in MIOX-TG mice compared with WT treated with tunicamycin. This was accompanied with marked deterioration of tubular morphology, along with impairment of renal functions. Interestingly, minimal damage and elevation of ER stressors was observed in MIOX-KO mice. Downstream events that were more adversely affected in MIOX-TG mice included accentuated expression of proapoptogenic proteins, proinflammatory cytokines, and extracellular matrix constituents, although expression of these molecules was unaffected in MIOX-KO mice. Also, their tunicamycin-induced accentuated expression in tubular cells was notably reduced with MIOX-siRNA. These studies suggest that the biology of MIOX-induced oxidant stress and tunicamycin-induced ER stress are interlinked, and both of the events may feed into each other to amplify the tubulointerstitial injury.

Construction of a glucaric acid biosensor for screening myo-inositol oxygenase variants

Glucaric acid (GA), a top value-added chemical from biomass, has been widely used for prevention and control of diseases and the production of polymer materials. In GA biosynthesis pathway, the conversion of inositol to glucuronic acid that catalyzed by myo-inositol oxygenase is the limiting step. It is necessary to improve MIOX activity. In the present study, we constructed a high-throughput screening system through combing the concentration of GA with the green fluorescent protein fluorescence intensity. By applying this screening system, three positive variants (K59V/R60A, R171S and D276A) screened from the mutant library. In comparison, the recombinant strain Escherichia coli BL21(DE3)/MU-R171S accumulated more GA, 136.5% of that of the parent strain.