3-O-Methyl-D-Glucose Blunts Cold Ischemia Damage in Kidney via Inhibiting Ferroptosis.

BACKGROUND: The glucose derivative 3-O-methyl-D-glucose (OMG) is used as a cryoprotectant in freezing cells. However, its protective role and the related mechanism in static cold storage (CS) of organs are unknown. The present study aimed to investigate the effect of OMG on cod ischemia damage in cold preservation of donor kidney. METHODS: Pretreatment of OMG on kidney was performed in an isolated renal cold storage model in rats. LDH activity in renal efflux was used to evaluate the cellular damage. Indicators including iron levels, mitochondrial damage, MDA level, and cellular apoptosis were measured. Kidney quality was assessed via a kidney transplantation (KTx) model in rats. The grafted animals were followed up for 7 days. Ischemia reperfusion (I/R) injury and inflammatory response were assessed by biochemical and histological analyses. RESULTS: OMG pretreatment alleviated prolonged CS-induced renal damage as evidenced by reduced LDH activities and tubular apoptosis. Kidney with pCS has significantly increased iron, MDA, and TUNEL+ cells, implying the increased ferroptosis, which has been partly inhibited by OMG. OMG pretreatment has improved the renal function (p <0.05) and prolonged the 7-day survival of the grafting recipients after KTx, as compared to the control group. OMG has significantly decreased inflammation and tubular damage after KTx, as evidenced by CD3-positive cells and TUNEL-positive cells. CONCLUSION: Our study demonstrated that OMG protected kidney against the prolonged cold ischemia-caused injuries through inhibiting ferroptosis. Our results suggested that OMG might have potential clinical application in cold preservation of donor kidney.

Regulation of GLUT4 activity in myotubes by 3-O-methyl-d-glucose.

The rate of glucose influx to skeletal muscles is determined primarily by the number of functional units of glucose transporter-4 (GLUT4) in the myotube plasma membrane. The abundance of GLUT4 in the plasma membrane is tightly regulated by insulin or contractile activity, which employ distinct pathways to translocate GLUT4-rich vesicles from intracellular compartments. Various studies have indicated that GLUT4 intrinsic activity is also regulated by conformational changes and/or interactions with membrane components and intracellular proteins in the vicinity of the plasma membrane. Here we show that the non-metabolizable glucose analog 3-O-methyl-d-glucose (MeGlc) augmented the rate of hexose transport into myotubes by increasing GLUT4 intrinsic activity without altering the content of the transporter in the plasma membrane. This effect was not a consequence of ATP depletion or hyperosmolar stress and did not involve Akt/PKB or AMPK signal transduction pathways. MeGlc reduced the inhibitory potency (increased Ki) of indinavir, a selective inhibitor of GLUT4, in a dose-dependent manner. Kinetic analyses indicate that MeGlc induced changes in GLUT4 or GLUT4 complexes within the plasma membrane, which enhanced the hexose transport activity and reduced the potency of indinavir inhibition. Finally, we present a simple kinetic analysis for screening and discovering low molecular weight compounds that augment GLUT4 activity.

Cryopreservation of human spermatozoa with minimal non-permeable cryoprotectant.

Cryopreservation of human spermatozoa is a commonly used technique in assisted reproduction, however freezing low concentrations of sperm while maintaining adequate post-thaw motility remains a challenge. In an effort to optimize post-thaw motility yields, low volumes of human sperm were frozen in polyimide-coated fused silica micro-capillaries using 0.065 M, 0.125 M, 0.25 M, or 0.5 M trehalose as the only cryoprotectant. Micro-capillaries were either initially incubated in liquid nitrogen vapor before plunging into liquid nitrogen, or directly plunged into liquid nitrogen. Post thaw sperm counts and motility were estimated. Spermatozoa that were initially incubated in liquid nitrogen vapor had greater post thaw motility than those plunged immediately into liquid nitrogen independent of trehalose concentration. The protective effect of 0.125 M d-glucose, 3-O-methyl-d-glucopyranose, trehalose, sucrose, raffinose, or stachyose were evaluated individually. Trehalose and sucrose were the most effective cryoprotectants, recovering 69.0% and 68.9% of initial sperm motility, respectively.

Glucose promotes its own metabolism by acting on the cell-surface glucose-sensing receptor T1R3.

A homodimer of taste type 1 receptor 3 (T1R3) functions as a sweet taste-sensing receptor in pancreatic ß-cells. This receptor is activated by various sweet molecules including sugars such as glucose. To determine the role of this receptor in glucose-induced insulin secretion, we addressed whether or not this receptor modulates glucose metabolism in MIN6 cells. We measured changes in intracellular ATP ([ATP]i) in MIN6 cells expressing luciferase. Sucralose, an agonist of T1R3, induced immediate and sustained elevation of [ATP]i in the presence of 5.5 mM glucose. The effect of sucralose was dose-dependent and, at 5 mM, was greater than that induced by 25 mM glucose. In contrast, carbachol, GLP-1 or high concentration of potassium did not reproduce the sucralose action. Sucralose facilitated the increase in [ATP]i induced by a mitochondrial fuel methylsuccinate, and potentiated glucose-induced elevation of [ATP]i. Administration of a non-metabolizable glucose analogue, 3-O-methylglucose, which acts as an agonist of T1R3, induced a small and transient increase in [ATP]i. 3-O-Methylglucose augmented elevation of [ATP]i induced by methylsuccinate, and also enhanced glucose-induced increase in [ATP]i. Knock down of T1R3 by using shRNA attenuated [ATP]i-response to high concentration of glucose and also reduced the glucose-induced insulin secretion. These results indicate that activation of the homodimer of T1R3 facilitates the metabolic pathway in mitochondria and augments ATP production. The results obtained by using 3-O-methylglucose suggest that glucose, by acting on the homodimer of T1R3, promotes its own metabolism.

D-glucose­ and 3-O-methyl-D-glucose-induced upregulation of selected genes in rat hepatocytes and INS1E cells: re­evaluation of the possible role of hexose phosphorylation.

The biochemical events involved in the upregulation of selected glucose­responsive genes by 3­O­methyl­D­glucose (3­MG) remain to be elucidated. The present study mainly aimed to re­evaluate the possible role of 3­MG phosphorylation in the upregulation of the thioredoxin interacting protein (TXNIP) and liver pyruvate kinase (LPK) genes in rat hepatocytes and INS1E cells. TXNIP and LPK transcription was assessed in rat liver and INS1E cells exposed to a rise in D­glucose concentration, 2­deoxy­D­glucose (2­DG), 3­MG and, when required, D­mannoheptulose. The phosphorylation of D­[U­14C]glucose and 3­O­[14C]methyl­D­glucose (14C-labeled 3-MG) was measured in rat liver, INS1E cell and rat pancreatic islet homogenates. The utilization of D­[5­3H]glucose by intact INS1E cells was also measured. In rat hepatocytes, a rise in the D­glucose concentration increased the TXNIP/hypoxanthine­guanine phosphoribosyl transferase (HPRT) and LPK/HPRT ratios, while 2­DG and 3­MG also increased the TXNIP/HPRT ratio, but not the LPK/HPRT ratio. In INS1E cells, the TXNIP/HPRT and LPK/HPRT ratios were increased in response to the addition of D­glucose, 2­DG and 3­MG. Furthermore, D­mannoheptulose abolished the response to D­glucose and 2­DG, but not to 3­MG, in these cells. Liver cell homogenates catalyzed the phosphorylation of 3­MG to a modest extent, whilst INS1E and rat pancreatic islet cell homogenates did not. Moreover, 3­MG marginally decreased D­glucose phosphorylation in INS1E cell homogenates but not in liver cell homogenates. D­[5­3H]glucose utilization by intact INS1E cells was decreased by 2­DG, but not by 3­MG. These findings reinforce the view that the upregulation of the TXNIP and LPK genes induced by 3­MG is not attributable to its phosphorylation or any favorable effect on D­glucose metabolism.

Real-time analysis of intracellular glucose and calcium in pancreatic beta cells by fluorescence microscopy.

Glucose is the physiological stimulus for insulin secretion in pancreatic beta cells. The uptake and phosphorylation of glucose initiate and control downstream pathways, resulting in insulin secretion. However, the temporal coordination of these events in beta cells is not fully understood. The recent development of the FLII(12)Pglu-700µ-δ6 glucose nanosensor facilitates real-time analysis of intracellular glucose within a broad concentration range. Using this fluorescence-based technique, we show the shift in intracellular glucose concentration upon external supply and removal in primary mouse beta cells with high resolution. Glucose influx, efflux, and metabolism rates were calculated from the time-dependent plots. Comparison of insulin-producing cells with different expression levels of glucose transporters and phosphorylating enzymes showed that a high glucose influx rate correlated with GLUT2 expression, but was largely also sustainable by high GLUT1 expression. In contrast, in cells not expressing the glucose sensor enzyme glucokinase glucose metabolism was slow. We found no evidence of oscillations of the intracellular glucose concentration in beta cells. Concomitant real-time analysis of glucose and calcium dynamics using FLII(12)Pglu-700µ-δ6 and fura-2-acetoxymethyl-ester determined a glucose threshold of 4mM for the [Ca(2+)](i) increase in beta cells. Indeed, a glucose concentration of 7mM had to be reached to evoke large amplitude [Ca(2+)](i) oscillations. The K(ATP) channel closing agent glibenclamide was not able to induce large amplitude [Ca(2+)](i) oscillations in the absence of glucose. Our findings suggest that glucose has to reach a threshold to evoke the [Ca(2+)](i) increase and subsequently initiate [Ca(2+)](i) oscillations in a K(ATP) channel independent manner.

Preservation of mouse sperm by convective drying and storing in 3-O-methyl-D-glucose.

With the fast advancement in the genetics and bio-medical fields, the vast number of valuable transgenic and rare genetic mouse models need to be preserved. Preservation of mouse sperm by convective drying and subsequent storing at above freezing temperatures could dramatically reduce the cost and facilitate shipping. Mouse sperm were convectively dried under nitrogen gas in the Na-EGTA solution containing 100 mmol/L 3-O-methyl-D-glucose and stored in LiCl sorption jars (Relative Humidity, RH, 12%) at 4°C and 22°C for up to one year. The functionality of these sperm samples after storage was tested by intracytoplasmic injection into mouse oocytes. The percentages of blastocysts produced from sperm stored at 4°C for 1, 2, 3, 6, and 12 months were 62.6%, 53.4%, 39.6%, 33.3%, and 30.4%, respectively, while those stored at 22°C for 1, 2, and 3 months were 28.8%, 26.6%, and 12.2%, respectively. Transfer of 38 two- to four-cell embryos from sperm stored at 4°C for 1 year produced two live pups while 59 two- to four-cell embryos from sperm stored at 22°C for 3 months also produced two live pups. Although all the pups looked healthy at 3 weeks of age, normality of offspring produced using convectively dried sperm needs further investigation. The percentages of blastocyst from sperm stored in the higher relative humidity conditions of NaBr and MgCl(2) jars and driest condition of P(2)O(5) jars at 4°C and 22°C were all lower. A simple method of mouse sperm preservation is demonstrated. Three-O-methyl-D-glucose, a metabolically inactive derivative of glucose, offers significant protection for dried mouse sperm at above freezing temperatures without the need for poration of cell membrane.

Regulation of AtSUS2 and AtSUS3 by glucose and the transcription factor LEC2 in different tissues and at different stages of Arabidopsis seed development.

Sucrose synthase (SUS) is a key enzyme of carbon metabolism in heterotrophic tissues of plants. The Arabidopsis genome contains six SUS genes. Two members of this family, namely AtSUS2 (At5g49190) and AtSUS3 (At4g02280) are strongly and differentially expressed in Arabidopsis seed. Expression analysis was carried out using SUS:promoter-GUS fusion lines in a wild-type genetic background or in a mutant carrying a lesion in the transcription factor LEAFY COTYLEDON 2 (LEC2; At1g28300). The accumulation patterns of mRNA, protein, and SUS activity were altered in the lec2 mutant during seed development 9-18 days after flowering. This indicates that LEC2 acts epistatically on the expression of AtSUS2 and AtSUS3. It appears that LEC2 is required for cotyledon-specific expression of both SUS genes but it is not responsible for expression in the radicle tip during embryo development. The AtSUS2 promoter was induced in planta by feeding of glucose but less so by sucrose and trehalose. Non-phosphorylable glucose analogs such as 3-O-methyl-glucose and 2-deoxyglucose also caused an induction, suggesting that sugar signaling proceeds by a hexokinase-independent pathway, possibly involving hexose sensing. Analysis of transgenic lines carrying of truncated versions of the AtSUS2:promoter fused to Beta-glucuronidase activity revealed an internal 421 bp region that was responsible for expression in seeds. Bioinformatic sequence analysis revealed regulatory cis-elements putatively responsible for the spatio-temporal pattern of AtSUS2 expression such as the SEF3 (aaccca) and W-box (ttgact) motifs. These findings are discussed in relation to the roles played by AtSUS2, AtSUS3 and LEC2 in the biosynthesis of seed storage products in Arabidopsis.

The administration of nonmetabolizable glucose analogues fails to suppress the development of glycogen autophagy in newborn rat hepatocytes.

The effects of parenteral administration of glucose, 3-methylglucose (3MG), or 2-deoxyglucose (2DG) on the glycogen autophagy were studied in the newborn rat liver using electron microscopy and biochemical methods. The administration of glucose resulted in hyperglycemia and prevented the mobilization of hepatocytic glycogen. It also prevented the development of autophagic vacuoles in general and inhibited the glycogen-degrading activity of acid α-1,4-glucosidase. The nonphosphorylated and not further metabolized glucose analog 3MG also produced hyperglycemia, but increased acid glucosidase. Pretreating the newborns with the ß-adrenergic blocker propranolol inhibited the effects of 3MG. The phosphorylated but not fully metabolized glucose analog 2DG produced similar effects. The administration of xylitol to the newborns already treated with 2DG, suppressed acid glucosidase. The results of this and our previous studies suggest that glucose must be metabolized beyond its phosphorylation step to inhibit acid glucosidase activity.

Structure-based design of a periplasmic binding protein antagonist that prevents domain closure.

Many receptors undergo ligand-induced conformational changes to initiate signal transduction. Periplasmic binding proteins (PBPs) are bacterial receptors that exhibit dramatic conformational changes upon ligand binding. These proteins mediate a wide variety of fundamental processes including transport, chemotaxis, and quorum sensing. Despite the importance of these receptors, no PBP antagonists have been identified and characterized. In this study, we identify 3-O-methyl-d-glucose as an antagonist of glucose/galactose-binding protein and demonstrate that it inhibits glucose chemotaxis in E. coli. Using small-angle X-ray scattering and X-ray crystallography, we show that this antagonist acts as a wedge. It prevents the large-scale domain closure that gives rise to the active signaling state. Guided by these results and the structures of open and closed glucose/galactose-binding protein, we designed and synthesized an antagonist composed of two linked glucose residues. These findings provide a blueprint for the design of new bacterial PBP inhibitors. Given the key role of PBPs in microbial physiology, we anticipate that PBP antagonists will have widespread uses as probes and antimicrobial agents.

Thioredoxin-interacting protein: an oxidative stress-related gene is upregulated by glucose in human prostate carcinoma cells.

Thioredoxin-interacting protein (TXNIP), also known as vitamin-D(3) upregulated protein 1, interacts with reduced thioredoxin. This protein modulates the cellular redox state and plays a role in stress-induced cellular apoptosis. This study examined TXNIP gene expression in prostate cancer cells. In vitro studies by immunoblot assay have shown that elevated glucose levels (1-15 mM) upregulate TXNIP gene expression two- to fourfold in human prostate carcinoma cells (LNCaP) and hepatocellular carcinoma cells (HepG2). Transient gene expression assays reveal that the promoter activity of the TXNIP gene is upregulated by glucose, 3-O-methylglucose, and maltose, but not by mannitol. These results suggest that glucose and 3-O-methylglucose induce TXNIP expression through both glucose metabolism-dependent and -independent pathways. Cotransfection of a plasmid expression carbohydrate response element-binding protein (ChREBP) with a TXNIP reporter vector into LNCaP cells dramatically enhances reporter activity in a low glucose (1 mM) condition. The effects of glucose are apparently mediated in a region located -341 to -324 bp upstream of the translational starting point of the TXNIP gene as indicated by 5'-deletion and site-directed mutagenesis reporter assays. Mutation of the putative carbohydrate response element (ChoRE) from CACGAGGGCAGCACGAG to TTTGAGGGCAGCACGAG abolishes glucose upregulation of TXNIP promoter activity. The present study demonstrates that TXNIP is transcription induced in both LNCaP and HepG2 cells in an increased glucose metabolism-dependent or -independent response, and a putative glucose regulatory system including ChREBP and ChoRE is needed for glucose-induced TXNIP gene in human prostate carcinoma cells.

Regulation of avilamycin biosynthesis in Streptomyces viridochromogenes: effects of glucose, ammonium ion, and inorganic phosphate.

Effects of glucose, ammonium ions and phosphate on avilamycin biosynthesis in Streptomyces viridochromogenes AS4.126 were investigated. Twenty grams per liter of glucose, 10 mmol/L ammonium ions, and 10 mmol/L phosphate in the basal medium stimulated avilamycin biosynthesis. When the concentrations of glucose, ammonium ions, and phosphate in the basal medium exceeded 20 g/L, 10 mmol/L, and 10 mmol/L, respectively, avilamycin biosynthesis greatly decreased. When 20 g/L glucose was added at 32 h, avilamycin yield decreased by 70.2%. Avilamycin biosynthesis hardly continued when 2-deoxy-glucose was added into the basal medium at 32 h. There was little influence on avilamycin biosynthesis with the addition of the 3-methyl-glucose (20 g/L) at 32 h. In the presence of excess (NH4)2SO4 (20 mmol/L), the activities of valine dehydrogenase and glucose-6-phosphate dehydrogenase were depressed 47.7 and 58.3%, respectively, of that of the control at 48 h. The activity of succinate dehydrogenase increased 49.5% compared to the control at 48 h. The intracellular adenosine triphosphate level and 6-phosphate glucose content of S. viridochromogenes were 128 and 129%, respectively, of that of the control at 48 h, with the addition of the 40 mmol/L of KH2PO4. As a result, high concentrations of glucose, ammonium ions, and inorganic phosphate all led to the absence of the precursors for avilamycin biosynthesis and affected antibiotic synthesis.

Metabolism-independent sugar effects on gene transcription: the role of 3-O-methylglucose.

Glucose effects on cellular functions such as gene expression require, in general, glucose metabolism at least to glucose-6-phosphate (G-6-P). However, the example of thioredoxin-interacting protein (TXNIP), a glucose-regulated gene involved in the cellular redox state and pancreatic beta cell apoptosis, demonstrates that this rule may not always apply. We found that aside form glucose, the nonmetabolizable sugars 2-deoxyglucose, which is still converted to G-6-P as well as 3-O-methylglucose (3-MG), which cannot be phosphorylated by glucokinase, stimulate TXNIP expression. In contrast, incubation of INS-1 beta cells with equimolar amounts (25 mM) of l-glucose or mannitol had no effect on TXNIP expression as measured by real-time RT-PCR, eliminating the possibility of an osmotic effect. Also, glucose uptake into the cell is critical because phloretin, an inhibitor of glucose transporter 2, blunted the glucose effects. Moreover, the 3-MG effect was not restricted to a cell line and was observed in 293 cells and primary human islets. Incubation of INS-1 cells with 30mM mannoheptulose, an inhibitor of glucose metabolism, blunted all glucose-induced gene expression but left the 3-MG effects unaltered. Using transient transfection studies and deletion constructs of the human TXNIP promoter, we found that the effects of glucose and 3-MG were dependent on the same region of the TXNIP promoter containing an E-box repeat carbohydrate response element (ChoRE). Thus, these findings provide the first evidence for regulation of gene expression by 3-MG, which is independent of glucose metabolism and suggest that glucose and 3-MG regulate transcription by two distinct pathways converging at a common ChoRE.

Glucose induces anion conductance and cytosol-to-membrane transposition of ICln in INS-1E rat insulinoma cells.

The metabolic coupling of insulin secretion by pancreatic beta cells is mediated by membrane depolarization due to increased glucose-driven ATP production and closure of K(ATP) channels. Alternative pathways may involve the activation of anion channels by cell swelling upon glucose uptake. In INS-1E insulinoma cells superfusion with an isotonic solution containing 20 mM glucose or a 30% hypotonic solution leads to the activation of a chloride conductance with biophysical and pharmacological properties of anion currents activated in many other cell types during regulatory volume decrease (RVD), i.e. outward rectification, inactivation at positive membrane potentials and block by anion channel inhibitors like NPPB, DIDS, 4-hydroxytamoxifen and extracellular ATP. The current is not inhibited by tolbutamide and remains activated for at least 10 min when reducing the extracellular glucose concentration from 20 mM to 5 mM, but inactivates back to control levels when cells are exposed to a 20% hypertonic extracellular solution containing 20 mM glucose. This chloride current can likewise be induced by 20 mM 3-Omethylglucose, which is taken up but not metabolized by the cells, suggesting that cellular sugar uptake is involved in current activation. Fluorescence resonance energy transfer (FRET) experiments show that chloride current activation by 20 mM glucose and glucose-induced cell swelling are accompanied by a significant, transient redistribution of the membrane associated fraction of ICln, a multifunctional 'connector hub' protein involved in cell volume regulation and generation of RVD currents.

Nonmetabolizable glucose compounds impart cryotolerance to primary rat hepatocytes.

We herein report a novel method for the cryopreservation of hepatocytes using a non-metabolizable glucose derivative in an attempt to mimic the natural cryoprotective adaptations observed in freeze-tolerant frogs. Primary rat hepatocytes were loaded with 3-O-methyl glucose (3OMG) through endogenous glucose transporters without evident toxicity. The 3OMG-loaded hepatocytes were then frozen in a controlled rate freezer down to -80 degrees C and stored in liquid nitrogen at -196 degrees C. Hepatocytes cryopreserved with a relatively small amount of intracellular 3OMG (<0.2 M) showed high post-thaw viability and maintained long-term hepatospecific functions, including synthesis, metabolism, and detoxification. Metabolite uptake and secretion rates were also largely preserved in the cryopreserved hepatocytes. This is the first study to demonstrate the use of the non-metabolizable glucose derivative 3OMG in hepatocyte cryopreservation.

Nateglinide, a non-sulfonylurea rapid insulin secretagogue, increases pancreatic islet blood flow in rats.

We studied whether the rapid hypoglycemic action of nateglinide is associated with an increase in islet blood flow. Islet blood flow was measured using the two-colour microsphere method. Orally administered nateglinide with glucose acutely increased islet blood flow to levels greater than those after glucose alone or tolbutamide with glucose in conscious Sprague-Dawley rats (percent increase at 10 min after oral administration; nateglinide+glucose, 125+/-25%; glucose, 33+/-11%, p<0.001; tolbutamide+glucose, 42+/-23%, p<0.01). Nateglinide administered with non-metabolisable 3-O-methylglucose also increased islet blood flow (61+/-17%). The stimulated islet blood flow significantly correlated with serum insulin levels. N(G)-monomethyl-L-arginine, a nitric oxide synthase inhibitor, completely inhibited the increase in islet blood flow induced by nateglinide with glucose. Intravenously administered nateglinide did not significantly affect the already increased islet blood flow in diabetic Otsuka Long-Evans Tokushima Fatty rats. Our results indicated that nateglinide acutely increased islet blood flow at least in part through a nitric oxide-dependent mechanism.

Defining the pHi-hyperthermia sensitivity relationship for the RIF-1 tumor in vivo: a 31P MR spectroscopy study.

This study quantifies the enhancement of the therapeutic efficacy of hyperthermia resulting from an acutely acidified and accurately monitored intracellular pH (pHi) in a mouse tumor model in vivo. Metabolic manipulation of the physiology of RIF-1 tumor (subcutaneous, on the hind flanks of female C3H/HeJ mice) achieved by i.p. bolus injection of glucose (glycolytic tumor acidification) or 3-O-methylglucose (non-glycolytic tumor acidification) was monitored by 31P magnetic resonance (31P MR) prior to, during and up to 1 h after localized hyperthermia. The pre-hyperthermia 31P MR-observable metabolic parameter that correlates most strongly with thermal sensitivity is pHi. Thermal sensitivity increases linearly with decreasing pHi regardless of the mechanism (glycolytic or non-glycolytic) of metabolic manipulation. The quantitative relationship is described by log10(SF)/EQ43=0.0079 pHi,preHT -0.0606 (R=0.63, P<0.0001), where EQ43 is the thermal heat dose delivered to the tumor (in units of equivalent minutes at 42.5 degrees C), pHi,preHT is the intracellular pH immediately prior to hyperthermia, and SF is the surviving fraction. The therapeutic enhancement is not as dramatic as expected based upon previously reported in vitro studies but is generally consistent with other in vivo studies. The method still represents a viable strategy for enhancing the therapeutic efficacy of hyperthermia, especially when used in combination with other therapeutic modalities.

Identification of more than 200 glucose-responsive Arabidopsis genes none of which responds to 3-O-methylglucose or 6-deoxyglucose.

The response of some plant genes to glucose analogues 3-O-methylglucose (3OMG) or 6-deoxyglucose (6DOG) has been cited as evidence for metabolism-independent glucose signalling. To analyse such signalling using a genetic approach, we sought to identify Arabidopsis glucose-responsive genes which also respond to 3OMG and 6DOG in seedlings. Microarray analysis of gene expression in glucose-treated seedlings and RT-PCR analysis of glucose-treated leaf sections identified more than 200 glucose-responsive genes, but none responded to 3OMG or 6DOG. These data together with other published data on individual genes fail to identify any Arabidopsis sugar-responsive genes which also respond to 3OMG or 6DOG.

Cryptosporidium parvum: effect of multi-drug reversing agents on the expression and function of ATP-binding cassette transporters.

In the present study, the gene expression of three multidrug resistance (MDR) and resistance-associated protein (MRP) transport proteins or efflux pumps was characterized and the phenotypic evidence for such pumps was demonstrated in cultured Madin-Darby canine kidney (MDCK) cells. A gradient for the fluorescent probe calcein was established between parasite and host cell suggestive of a parasite extrusion pump at the parasite-host interface. This gradient was decreased in a glucose-free medium containing 2-deoxyglucose or 3-O-methylglucose, by probenecid, and by the isoflavonoid, narigenin, suggesting that the calcein extrusion was energy-dependent and involved an MRP-like pump. While neither MDR or MRP inhibiters significantly affected transcript levels of any of the ABC transporters, transcript levels of the Cryptosporidium parvum ABC protein (CpABC1), an MRP transporter, were consistently expressed 4 logs higher than either CpABC3 or CpABC2, suggesting a prominent role in the intracellular stages of the parasite.

In plants, 3-o-methylglucose is phosphorylated by hexokinase but not perceived as a sugar.

In plants, sugars are the main respiratory substrates and important signaling molecules in the regulation of carbon metabolism. Sugar signaling studies suggested that sugar sensing involves several key components, among them hexokinase (HXK). Although the sensing mechanism of HXK is unknown, several experiments support the hypothesis that hexose phosphorylation is a determining factor. Glucose (Glc) analogs transported into cells but not phosphorylated are frequently used to test this hypothesis, among them 3-O-methyl-Glc (3-OMG). The aim of the present work was to investigate the effects and fate of 3-OMG in heterotrophic plant cells. Measurements of respiration rates, protein and metabolite contents, and protease activities and amounts showed that 3-OMG is not a respiratory substrate and does not contribute to biosynthesis. Proteolysis and lipolysis are induced in 3-OMG-fed maize (Zea mays L. cv DEA) roots in the same way as in sugar-starved organs. However, contrary to the generally accepted idea, phosphorous and carbon nuclear magnetic resonance experiments and enzymatic assays prove that 3-OMG is phosphorylated to 3-OMG-6-phosphate, which accumulates in the cells. Insofar as plant HXK is involved in sugar sensing, these findings are discussed on the basis of the kinetic properties because the catalytic efficiency of HXK isolated from maize root tips is five orders of magnitude lower for 3-OMG than for Glc and Man.