Increased transcript levels and kinetic function of pyruvate kinase during severe dehydration in aestivating African clawed frogs, Xenopus laevis.

The African clawed frog, Xenopus laevis, can withstand extremely arid conditions through aestivation, resulting in dehydration and urea accumulation. Aestivating X. laevis reduce their metabolic rate, and rely on anaerobic glycolysis to meet reduced ATP demands. The present study investigated how severe dehydration affected the transcript levels, kinetic profile, and phosphorylation state of the key glycolytic enzyme pyruvate kinase (PK) in the liver and skeletal muscle of X. laevis. Compared to control frogs, severely dehydrated frogs showed an increase in the transcript abundance of both liver and muscle isoforms of PK. While the kinetics of muscle PK did not differ between dehydrated and control frogs, PK from the liver of dehydrated frogs had a lower Km for phosphoenolpyruvate (PEP) (38%), a lower Ka for fructose-1,6-bisphosphate (F1,6P2) (32%), and a greater activation of PK via F1,6P2 (1.56-fold). PK from dehydrated frogs also had a lower phosphorylation-state (25%) in comparison to the enzyme from control frogs in the liver. Experimental manipulation of the phosphorylation-state of liver PK taken from control frogs by endogenous protein phosphatases resulted in decreased phosphorylation, and a similar kinetic profile as seen in dehydrated frogs. The physiological consequence of dehydration-induced PK modification appears to adjust PK function to remain active during a metabolically depressed state. This study provides evidence for the maintenance of PK activity through elevated mRNA levels and a dephosphorylation event which activates frog liver PK in the dehydrated state in order to facilitate the production of ATP via anaerobic glycolysis.

Efficient Malic Acid Production in Escherichia coli Using a Synthetic Scaffold Protein Complex.

Recently, malic acid has gained attention due to its potential application in food, pharmaceutical, and medical industries. In this study, the synthetic scaffold complex strategy was employed between the two key enzymes pyruvate kinase (PykF) and malic enzyme (SfcA); SH3 ligand was attached to PykF, and the SH3 domain was attached to the C-terminus of ScfA. Synthetic scaffold systems can organize enzymes spatially and temporally to increase the local concentration of intermediates. In a flask culture, the recombinant strain harboring scaffold complex produced a maximum concentration of 5.72 g/L malic acid from 10 g/L glucose. The malic acid production was significantly increased 2.1-fold from the initial culture period. Finally, malic acid production was elevated to 30.2 g in a 5 L bioreactor from recombinant strain XL-1 blue.

Pyruvate kinase M2 (PKM2) expression correlates with prognosis in solid cancers: a meta-analysis.

Pyruvate kinase M2 (PKM2) is the key enzyme in the Warburg effect and plays a central role in cancer cell metabolic reprogramming. Recently, quite a few studies have investigated the correlation between PKM2 expression and prognosis in multiple cancer patients, but results were inconsistent. We therefore performed a meta-analysis to explore the prognostic value of PKM2 expression in patients with solid cancer. Here twenty-seven individual studies from 25 publications with a total of 4796 cases were included to explore the association between PKM2 and overall survival (OS) or disease-free survival (DFS)/ progression-free survival (PFS)/ recurrent-free survival (RFS) in subjects with solid cancer. Pooled analysis showed that high levels of PKM2 was significantly associated with a poorer overall survival (HR = 1.73; 95%CI = 1.48-2.03) and DFS/ PFS/ RFS (HR = 1.90; 95%CI = 1.39-2.59) irrespective of cancer types. Different analysis models (univariate or multivariate models), sample-sizes (≤100 or >100), and methods for data collection (direct extraction or indirect extraction) had no impact on the negative prognostic effect of PKM2 over-expression. Nevertheless, stratified by cancer type, high-expression of PKM2 was associated with an unfavorable OS in breast cancer, esophageal squamous carcinoma, hepatocellular carcinoma and gallbladder cancer; whereas was not correlated with a worse OS in pancreatic cancer and gastric cancer. In conclusion, over-expression of PKM2 is associated with poor prognosis in most solid cancers and it might be a potentially useful biomarker for predicting cancer prognosis in future clinical applications.

Dietary Macronutrient Composition Directs ChREBP Isoform Expression and Glucose Metabolism in Mice.

Carbohydrate response element binding protein (ChREBP) is a lipogenic transcription factor that is thought to be involved in the development of hepatic steatosis and insulin resistance. Increased ChREBP expression in liver results in increased hepatic steatosis, and the isoform ChREBPß in adipose tissue can predict insulin sensitivity in obese humans. As ChREBP is activated by glucose, it was postulated that the composition of diet would regulate ChREBP isoform expression in metabolically relevant tissues. We compared the effects of diets with high complex carbohydrate, high fat, or a normal chow on ChREBP expression and metabolic parameters in C57BL/6 mice. We found that diets high in fat decrease ChREBP expression in adipose tissue, but isocaloric diets high in carbohydrate have no effect. Interestingly, this decrease in adipose ChREBP was associated with increased inflammatory markers. In the same animals a high carbohydrate diet induced a robust increase in hepatic ChREBPß expression (≈2-fold; p = 0.0002), but little detectable change in the more abundant ChREBPα transcript. This change was accompanied by increased expression of target genes liver pyruvate kinase (p<0.0001), acetyl-CoA carboxylase (p = 0.0191) and stearoyl-CoA desaturase-1 (p = 0.0045). This increase in ChREBP expression was associated with increased hepatic steatosis, despite no changes in body weight or body fat when compared to chow-fed mice. Unexpectedly, mice fed a high carbohydrate diet displayed enhanced sensitivity to exogenous insulin, despite having mild glucose intolerance and increased liver steatosis. In summary, we have shown the composition of diet can selectively regulate ChREBP isoform expression in a tissue specific manner. Furthermore, we have shown a high complex carbohydrate diet selectively increases hepatic ChREBPß expression, which associates with hepatic steatosis but not insulin resistance. In contrast, a high fat diet reduces adipose ChREBP, which associates with inflammation and insulin resistance.

[Influence of γ-Irradiated Seeds on the Enzyme Activity in Barley Seedlings].

Influence of γ-irradiation of barley seeds (Nur variety) at the doses of 8-50 Gy on catalase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and guaiacol peroxidase activities was studied in the seedlings on the 3, 5 and 7 days after germination. It has been shown that activities of the studied enzymes increase in the dose range that causes the growth stimulation in the seedlings (16-20 Gy).

Gemfibrozil not fenofibrate decreases systemic glucose level via PPARα.

BACKGROUND: Concurrence of high glucose or diabetes in patients with dyslipidemia is presenting major challenges for clinicians. Although sporadically reported, a rational basis for the use of fibrates for the treatment of dyslipidemia with concurrent metabolic syndrome has not been established. METHODS: In this study, wild-type (WT) and Ppara-null (KO) mice were fed a serial gemfibrozil- and fenofibrate-containing diet under the same experimental conditions for 14 days. Glucose level in the blood, glycogen storage in the liver tissues, and the potential toxic responses were assayed. Genes involved in glucose metabolism were determined by quantitative polymerase chain reaction analysis. RESULTS: Both the blood glucose level and the glycogen content in the liver were down-regulated by gemfibrozil but not by fenofibrate in WT mice, in a dose-dependent manner. This decrement did not occur in KO mice for either fibrate agent. Secondary regulation on the transcription of pyruvate kinase, and gluconolactonase were observed following gemfibrozil treatment, which was differential between WT mice and KO mice. CONCLUSIONS: Gemfibrozil, not fenofibrate, down-regulates systemic glucose level and glycogen storage in the liver dependent on PPARα, suggesting its potential value for treatment of dyslipidemia with concurrent diabetes or high glucose levels.

Tanshinone â ¡A inhibits human esophageal cancer cell growth through miR-122-mediated PKM2 down-regulation.

Pyruvate kinase M2 (PKM2) plays a pivotal role in the growth, survival and metabolic reprogramming of cancer cells. Here, we presented for the first time that tanshinone ⅡA inhibited human esophagus cancer cell growth through miR-122-mediated PKM2 down-regulation pathway. Tanshinone ⅡA inhibited cell proliferation and induced cell cycle arrest in S phase in human Ec109 cells. As expected, tanshinone ⅡA down-regulated PKM2 mRNA and protein expression in Ec109 cells. Given these findings, we further investigated microRNAs regulation of PKM2 and confirmed miR-122 for targeting PKM2. Moreover, we found that tanshinone ⅡA-induced up-regulation of miR-122 expression inhibited PKM2 expression in Ec109 cells. Meanwhile, tanshinone ⅡA inhibited proliferation through miR122-medated PKM2 down-regulation. It was demonstrated that the anticancer activity of tanshinone ⅡA was targeted at metabolic regulation of miR-122/PKM2 in human esophagus cancer cells. Taken together, our results revealed tanshinone ⅡA targeting at PKM2-mediated metabolic reprogramming play an important role in inhibition of esophageal cancer cell growth.

Pyruvate Kinase M2 and Lactate Dehydrogenase A Are Overexpressed in Pancreatic Cancer and Correlate with Poor Outcome.

Pancreatic cancer has a 5-year survival rate of less than 4%. Despite advances in diagnostic technology, pancreatic cancer continues to be diagnosed at a late and incurable stage. Accurate biomarkers for early diagnosis and to predict treatment response are urgently needed. Since alteration of glucose metabolism is one of the hallmarks of cancer cells, we proposed that pyruvate kinase type M2 (M2PK) and lactate dehydrogenase A (LDHA) enzymes could represent novel diagnostic markers and potential therapeutic targets in pancreatic cancer. In 266 tissue sections from normal pancreas, pancreatic cystic neoplasms, pancreatic intraepithelial neoplasia (PanIN) and cancer, we evaluated the expression of PKM2, LDHA, Ki-67 and CD8+ by immunohistochemistry and correlated these markers with clinicopathological characteristics and patient survival. PKM2 and LDHA expression was also assessed by Western blot in 10 human pancreatic cancer cell lines. PKM2 expression increased progressively from cyst through PanIN to cancer, whereas LDHA was overexpressed throughout the carcinogenic process. All but one cell line showed high expression of both proteins. Patients with strong PKM2 and LDHA expression had significantly worse survival than those with weak PKM2 and/or LDHA expression (7.0 months vs. 27.9 months, respectively, p = 0.003, log rank test). The expression of both PKM2 and LDHA correlated directly with Ki-67 expression, and inversely with intratumoral CD8+ cell count. PKM2 was significantly overexpressed in poorly differentiated tumours and both PKM2 and LDHA were overexpressed in larger tumours. Multivariable analysis showed that combined expression of PKM2 and LDHA was an independent poor prognostic marker for survival. In conclusion, our results demonstrate a high expression pattern of two major glycolytic enzymes during pancreatic carcinogenesis, with increased expression in aggressive tumours and a significant adverse effect on survival.

M-Type Pyruvate Kinase Isoforms and Lactate Dehydrogenase A in the Mammalian Retina: Metabolic Implications.

PURPOSE: Like cancer cells, photoreceptor cells produce lactate aerobically, requiring lactate dehydrogenase A (LDH-A). Cancer cells also use glycolytic intermediates for biosynthesis. The molecular switch controlling glycolytic flow is thought to be an isoenzyme of pyruvate kinase (PKM2). Here, we determined the expression and localization of PKM2 and LDH-A in mammalian retina and make comparisons with the brain. METHODS: Single- and double-labeling immunohistochemistry for PKM2, pyruvate kinase M1 (PKM1), and LDH-A were performed using retinal sections from C57BL/6 mice, Sprague-Dawley rats, rabbits, marmosets, and humans. Pyruvate kinase M1 and PKM2 mRNA and protein expression levels were quantified in rodent retina and brain by using qPCR and immunoblotting. The quaternary forms of PKM2 in rat retina were also determined. RESULTS: Pyruvate kinase M2 was present in some glial cells and rod and cone photoreceptors in the retina of all species but was exclusively localized to glia in the brain. Pyruvate kinase M1 was confined to neurons in the retina and brain. Lactate dehydrogenase A was principally found in photoreceptors and inner portion of the avascular rabbit retina. Western blotting and qPCR confirmed high levels of PKM2 and LDH-A in the retina. There was a 6- to 9-fold greater expression of PKM2 mRNA in the rodent retina than in the brain. Both the dimeric (inactive, biosynthesis-driving form) and the active tetrameric (glycolytic-driving) forms of PKM2 were present in retina but not in brain. CONCLUSIONS: Mammalian photoreceptors contain dimeric and tetrameric PKM2 and LDH-A. This is consistent with the ability to switch between energy production and biosynthesis like a proliferating tissue, possibly due to demands of opsin synthesis.

Expression of pyruvate kinase M2 in human colorectal cancer and its prognostic value.

Reprogrammed metabolism is a hallmark of cancer cells. Pyruvate kinase isozyme type M2 (PKM2), which is frequently up-regulated in multiple human malignancies, has been demonstrated to play a critical function in glucose metabolism, gene transcription and tumorigenesis. However, limited knowledge is known about the expression pattern and prognostic value of PKM2 in colorectal cancer (CRC). In this study, we first observed that the mRNA level of PKM2 is commonly up-regulated in CRC tissues compared with their normal counterparts as demonstrated by data derived from Oncomine database. Similar results were also found in 32 paired CRC tumor and non-tumor specimens in our cohort and 4 CRC cell lines. Furthermore, by a large scale of immunohistochemical analysis in a tissue microarray containing 345 cases of CRC specimens, we demonstrated that the protein expression of PKM2 expression is up-regulated in 79.4% (274/345) samples detected and elevated PKM2 expression is closely correlated with enhanced TNM stage and higher serum CEA level. Meanwhile, Kaplan-Meier survival analysis showed that CRC patients with a higher PKM2 expression have a poorer clinical outcome than those with a lower PKM2 expression. Multivariate Cox regression analysis revealed that PKM2 and TNM stage are two independent prognostic factors for overall survival rate of CRC patients. Taken together, our studies reveal the prognostic value of PKM2 in CRC and support that PKM2 may act as a molecular target for CRC treatment.

Alteration of O-GlcNAcylation affects serine phosphorylation and regulates gene expression and activity of pyruvate kinase M2 in colorectal cancer cells.

O-GlcNAcylation is a dynamic post-translational modification that has extensive crosstalk with phosphorylation either at the same or adjacent sites of various proteins. We have previously reported that O-GlcNAcylation level was increased in primary breast and colorectal cancer, but the interplay of the two modifications remains unclear. Therefore, we explored crosstalk of the modifications by RNA interference against O-GlcNAc transferase (OGT) in colorectal cancer cells. Two-dimensional immunoblotting and mass spectrometric analysis showed that the levels of O-GlcNAc and serine phosphorylation of many proteins including serine hydroxymethyltransferase, cytokeratin-8, pyruvate kinase M2 (PKM2), heterogeneous nuclear ribonucleoprotein L, and lamin-B1, were reduced in siOGT cells compared to siScramble cells. In HT29 cells, immunoprecipitated PKM2 revealed decreased O-GlcNAc and serine phosphorylation levels after siOGT knockdown, but increased levels after treatment with Thiamet-G, an inhibitor of O-GlcNAcase (OGA). In addition, when global O-GlcNAcylation was enhanced by treating cells with Thiamet-G, PKM2 expression level was upregulated, but PKM2-specific activity was decreased. On the other hand, in OGT knockdown cells, PKM2 expression level was downregulated, but PKM2-specific activity was increased. Moreover, the metastatic colorectal cancer cells, SW620, had more O-GlcNAc-PKM2 and showed lower PKM2-specific activity compared to the non-metastatic colorectal cancer SW480 cells. These results suggested roles of O-GlcNAcylation in modulating serine phosphorylation, as well as in regulating PKM2 activity and expression. Interfering levels of O-GlcNAcylation of PKM2 may be a novel target in controlling cancer metabolism and tumorigenesis of colorectal cancer.

KSHV induces aerobic glycolysis and angiogenesis through HIF-1-dependent upregulation of pyruvate kinase 2 in Kaposi's sarcoma.

Kaposi's sarcoma (KS) is a vascular neoplasm caused by infection of endothelial or endothelial precursor cells with the Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8). Research efforts have focused on defining the molecular events explaining how KSHV promotes pathological angiogenesis and KS tumor formation. mTOR/HIF-1 is a fundamental pathway driving these processes through the upregulation of angiogenic and inflammatory proteins, including VEGF, ANGPTL4, and ANGPT2. Interestingly, HIF-1 has also been implicated in the upregulation of metabolic genes associated with aerobic glycolysis and the growth of solid tumors. However, whether HIF-1 plays a role in regulating cell metabolism in KS remains unexplored. Here, we show that the HIF-1 metabolic effector, pyruvate kinase 2 (PKM2), is upregulated upon KSHV infection of endothelial cells and is necessary to maintain aerobic glycolysis in infected cells. We further demonstrate that PKM2 regulates KS angiogenic phenotype by acting as a coactivator of HIF-1 and increasing the levels of HIF-1 angiogenic factors, including VEGF. Indeed, inhibition of PKM2 expression blocked endothelial cell migration and differentiation and the angiogenic potential of KSHV-infected cells. We also investigated whether PKM2 regulates the angiogenic dysregulation induced by the KSHV-encoded G protein-coupled receptor (vGPCR), a viral oncogene that promotes Kaposi's sarcomagenesis through the upregulation of HIF angiogenic factors. Interestingly, we found that PKM2 controls vGPCR-induced VEGF paracrine secretion and vGPCR oncogenesis. Our findings provide a molecular mechanism for how HIF-1 dysregulation fuels both angiogenesis and tumor metabolism in KS and support further investigations on therapeutic approaches targeting HIF-1 and PKM2 for KS treatment.

Natural compounds regulate glycolysis in hypoxic tumor microenvironment.

In the early twentieth century, Otto Heinrich Warburg described an elevated rate of glycolysis occurring in cancer cells, even in the presence of atmospheric oxygen (the Warburg effect). Recently it became a therapeutically interesting strategy and is considered as an emerging hallmark of cancer. Hypoxia inducible factor-1 (HIF-1) is one of the key transcription factors that play major roles in tumor glycolysis and could directly trigger Warburg effect. Thus, how to inhibit HIF-1-depended Warburg effect to assist the cancer therapy is becoming a hot issue in cancer research. In fact, HIF-1 upregulates the glucose transporters (GLUT) and induces the expression of glycolytic enzymes, such as hexokinase, pyruvate kinase, and lactate dehydrogenase. So small molecules of natural origin used as GLUT, hexokinase, or pyruvate kinase isoform M2 inhibitors could represent a major challenge in the field of cancer treatment. These compounds aim to suppress tumor hypoxia induced glycolysis process to suppress the cell energy metabolism or enhance the susceptibility of tumor cells to radio- and chemotherapy. In this review, we highlight the role of natural compounds in regulating tumor glycolysis, with a main focus on the glycolysis under hypoxic tumor microenvironment.

Differential abundance of sarcoplasmic proteome explains animal effect on beef Longissimus lumborum color stability.

The sarcoplasmic proteome of beef Longissimus lumborum demonstrating animal-to-animal variation in color stability was examined to correlate proteome profile with color. Longissimus lumborum (36 h post-mortem) muscles were obtained from 73 beef carcasses, aged for 13 days, and fabricated to 2.5-cm steaks. One steak was allotted to retail display, and another was immediately vacuum packaged and frozen at -80°C. Aerobically packaged steaks were stored under display, and color was evaluated on days 0 and 11. The steaks were ranked based on redness and color stability on day 11, and ten color-stable and ten color-labile carcasses were identified. Sarcoplasmic proteome of frozen steaks from the selected carcasses was analyzed. Nine proteins were differentially abundant in color-stable and color-labile steaks. Three glycolytic enzymes (phosphoglucomutase-1, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase M2) were over-abundant in color-stable steaks and positively correlated (P<0.05) to redness and color stability. These results indicated that animal variations in proteome contribute to differences in beef color.

Expression, purification and characterization of Solanum tuberosum recombinant cytosolic pyruvate kinase.

The cDNA encoding for a Solanum tuberosum cytosolic pyruvate kinase 1 (PKc1) highly expressed in tuber tissue was cloned in the bacterial expression vector pProEX HTc. The construct carried a hexahistidine tag in N-terminal position to facilitate purification of the recombinant protein. Production of high levels of soluble recombinant PKc1 in Escherichia coli was only possible when using a co-expression strategy with the chaperones GroES-GroEL. Purification of the protein by Ni(2 +) chelation chromatography yielded a single protein with an apparent molecular mass of 58kDa and a specific activity of 34unitsmg(-1) protein. The recombinant enzyme had an optimum pH between 6 and 7. It was relatively heat stable as it retained 80% of its activity after 2min at 75°C. Hyperbolic saturation kinetics were observed with ADP and UDP whereas sigmoidal saturation was observed during analysis of phosphoenolpyruvate binding. Among possible effectors tested, aspartate and glutamate had no effect on enzyme activity, whereas α-ketoglutarate and citrate were the most potent inhibitors. When tested on phosphoenolpyruvate saturation kinetics, these latter compounds increased S0.5. These findings suggest that S. tuberosum PKc1 is subject to a strong control by respiratory metabolism exerted via citrate and other tricarboxylic acid cycle intermediates.

Cancer metabolism and mass spectrometry-based proteomics.

Cancer metabolism has been extensively investigated by various tools, and the fact of diverse metabolic reprogramming in cancer cells has been gradually unveiled. In this review, we discuss some contributions in cancer metabolism by general proteomic analysis and post-translational modification analysis using mass spectrometry (MS) technique. Instead of following one or several metabolic enzymes/pathways, the current MS approach can quickly identify a large number of proteins and compare their expression levels in different samples, providing a potentially comprehensive picture of cancer metabolism. The MS analyses from pancreatic cancer cells support a hypothesis that hypoxia promotes cells in solid tumor to reprogram metabolic pathways in order to minimize the oxygen consumption. The oxidative stress in pancreatic cancer cells is lower than that in normal duct cells, and the cancer cells adaptively express less antioxidant proteins, contrary to claims that oxidative stress is higher in cancer cells. Separately, the MS analyses confirm that pyruvate kinase isoform 2 (PKM2) can be detected in both cancer and normal cells, disagreeing with report that tumor cells express exclusively PKM2. In addition, MS analyses from pancreatic cancer cells demonstrate that lactate dehydrogenase-B is significantly upregulated in pancreatic cancer cells, whereas previous reports show that lactate dehydrogenase-A is overexpressed and is responsible for lactate production in cancer cells. Lastly, the result from MS analysis suggests that the glutaminolysis in pancreatic cancer cells is different from that observed in glioblastoma cells.

Adaptations of energy metabolism during cerebellar neurogenesis are co-opted in medulloblastoma.

Recent studies show that metabolic patterns typical of cancer cells, including aerobic glycolysis and increased lipogenesis, are not unique to malignancy, but rather originate in physiologic development. In the postnatal brain, where sufficient oxygen for energy metabolism is scrupulously maintained, neural progenitors nevertheless metabolize glucose to lactate and prioritize lipid synthesis over fatty acid oxidation. Medulloblastoma, a cancer of neural progenitors that is the most common malignant brain tumor in children, recapitulates the metabolic phenotype of brain progenitor cells. During the physiologic proliferation of neural progenitors, metabolic enzymes generally associated with malignancy, including Hexokinase 2 (Hk2) and Pyruvate kinase M2 (PkM2) configure energy metabolism to support growth. In these non-malignant cells, expression of Hk2 and PkM2 is driven by transcriptional regulators that are typically identified as oncogenes, including N-myc. Importantly, N-myc continues to drive Hk2 and PkM2 in medulloblastoma. Similarly E2F transcription factors and PPARγ function in both progenitors and medulloblastoma to optimize energy metabolism to support proliferation. These findings show that the "metabolic transformation" that is a hallmark of cancer is not specifically limited to cancer. Rather, metabolic transformation represents a co-opting of developmental programs integral to physiologic growth. Despite their physiologic origins, the molecular mechanisms that mediate metabolic transformation may nevertheless present ideal targets for novel anti-tumor therapy.

Activation of the carbohydrate response element binding protein (ChREBP) in response to anoxia in the turtle Trachemys scripta elegans.

BACKGROUND: ChREBP (carbohydrate response element binding protein) is a glucose-responsive transcription factor that is known to be an important regulator of glycolytic and lipogenic genes in response to glucose. We hypothesized that activation of ChREBP could be relevant to anoxia survival by the anoxia-tolerant turtle, Trachemys scripta elegans. METHODS: Expression of ChREBP in response to 5 and 20h of anoxia was examined using RT-PCR and Western immunoblotting. In addition, subcellular localization and DNA-binding activity of ChREBP protein were assessed and transcript levels of liver pyruvate kinase (LPK), a downstream gene under ChREBP control were quantified using RT-PCR. RESULTS: ChREBP was anoxia-responsive in kidney and liver, with transcript levels increasing by 1.2-1.8 fold in response to anoxia and protein levels increasing by 1.8-1.9 fold. Enhanced nuclear presence under anoxia was also observed in both tissues by 2.2-2.8 fold. A 4.2 fold increase in DNA binding activity of ChREBP was also observed in liver in response to 5h of anoxia. In addition, transcript levels of LPK increased by 2.1 fold in response to 5h of anoxia in the liver. CONCLUSIONS: The results suggest that activation of ChREBP in response to anoxia might be a crucial factor for anoxia survival in turtle liver by contributing to elevated glycolytic flux in the initial phases of oxygen limitation. GENERAL SIGNIFICANCE: This study provides the first demonstration of activation of ChREBP in response to anoxia in a natural model of anoxia tolerance, further improving our understanding of the molecular nature of anoxia tolerance.

Comparison of fecal pyruvate kinase isoform M2 and calprotectin in assessment of pediatric inflammatory bowel disease severity and activity.

AIMS: Accurate assessment of inflammatory bowel disease (IBD) activity is the cornerstone of effective therapy. Fecal M2 isoform of pyruvate kinase (M2-PK) and fecal calprotectin (FC) are noninvasive markers of mucosal inflammation in IBD. The aim of this study was to compare performance of M2-PK and FC in assessment of pediatric ulcerative colitis (UC) and Crohn's disease (CD) severity and activity. MATERIALS AND METHODS: 121 patients with IBD, including 75 with UC and 46 with CD were recruited. Control group consisted of 35 healthy children (HS). Patients were assigned to groups depending on disease severity and activity. M2-PK and calprotectin concentration were determined in stool samples using ELISA. Areas under receiver operating characteristic curves (AUC) for FC and M2-PK with cut-off level at which M2-PK specificity was matching FC specificity were calculated and compared. RESULTS: Performance of M2-PK at identifying patients with IBD, UC and CD among HS was inferior to FC. The differences in AUC were respectively: -0.10 (95% confidence interval [CI] [-0.13-(-0.06)], p<0.0001), -0.14 (95% CI [-0.19-(-0.09)], p<0.0001) and -0.03 (95% CI [-0.05-(-0.001)], p<0.02). M2-PK was inferior to FC in discriminating patients with mild UC from those with HS (AUC difference -0.23, 95% CI [-0.31-(-0.15)], p<0.0001). CONCLUSIONS: FC reflects pediatric IBD severity and activity better than M2-PK. This difference is particularly pronounced when identifying patients with mild UC and UC in remission.

Differential expression of the proteome of myeloid dendritic cells in severe aplastic anemia.

Severe aplastic anemia (SAA) is a syndrome of severe bone marrow failure with high mortality. Our previous studies have demonstrated that both immature and activated DC1 increased in the bone marrow of SAA patients, and the balance of DC1 subsets shifted the stable form to active one, which might promote Th0 cells to polarize to Th1 cells and cause the over-function of T lymphocytes and hematopoiesis failure in SAA. So we assumed myeloid dendritic cells (mDCs) may be the key immune cells that cause destruction of hematopoietic cells in SAA, but the mechanism of activation of mDCs is unclear. Here, we investigated the proteome of mDCs in SAA patients to further explore the pathogenesis of SAA and the possible antigen that leads to immune activation in SAA. mDCs from 12 SAA patients, 12 remission patients and 12 controls were sorted by flow cytometry and examined by two-dimensional gel electrophoresis and mass spectrometry. Intensity changes of 41 spots were detected with statistical significance. Nine of the 41 spots were identified by MALDI-TOF/TOF tandem mass spectrometry. Changes in protein expression levels were found in the SAA group. These changes reveal that abnormal expression of cofilin, glucose-6-phosphate dehydrogenase and pyruvate kinase enzyme M2 in mDCs from SAA patients may be the reason for mDC hyperfunction.