Enolase Activity Research Articles

Isobavachalcone Exhibits Potent Antifungal Efficacy by Inhibiting Enolase Activity and Glycolysis in Candida albicans.

Invasive fungal diseases (IFDs) are becoming increasingly acknowledged as a significant concern linked to heightened rates of morbidity and mortality. Regrettably, the available antifungal therapies for managing IFDs are constrained. Emerging evidence indicates that enolase holds promise as a potential target protein for combating IFDs; however, there is currently a deficiency in antifungal medications specifically targeting enolase. This study establishes that isobavachalcone (IBC) exhibits noteworthy antifungal efficacy both in vitro and in vivo. Moreover, our study has demonstrated that IBC effectively targets Eno1 in Candida albicans (CaEno1), resulting in the suppression of the glycolytic pathway. Additionally, our research has indicated that IBC exhibits a higher affinity for CaEno1 compared to human Eno1 (hEno1), with the presence of isoprenoid in the side chain of IBC playing a crucial role in its ability to inhibit enolase activity. These findings contribute to the comprehension of antifungal approaches that target Eno1, identifying IBC as a potential inhibitor of Eno1 in human pathogenic fungi.

PROTEIN L‐ISOASPARTYL METHYLTRANSFERASE protects enolase dysfunction by repairing isoaspartyl‐induced damage and is positively implicated in agronomically important seed traits

SUMMARYThe protein‐repairing enzyme (PRE) PROTEIN L‐ISOASPARTYL METHYLTRANSFERASE (PIMT) influences seed vigor by repairing isoaspartyl‐mediated protein damage in seeds. However, PIMTs function in other seed traits, and the mechanisms by which PIMT affects such seed traits are still poorly understood. Herein, through molecular, biochemical, and genetic studies using overexpression and RNAi lines in Oryza sativa and Arabidopsis thaliana, we demonstrate that PIMT not only affects seed vigor but also affects seed size and weight by modulating enolase (ENO) activity. We have identified ENO2, a glycolytic enzyme, as a PIMT interacting protein through Y2H cDNA library screening, and this interaction was further validated by BiFC and co‐immunoprecipitation assay. We show that mutation or suppression of ENO2 expression results in reduced seed vigor, seed size, and weight. We also proved that ENO2 undergoes isoAsp modification that affects its activity in both in vivo and in vitro conditions. Further, using MS/MS analyses, amino acid residues that undergo isoAsp modification in ENO2 were identified. We also demonstrate that PIMT repairs such isoAsp modification in ENO2 protein, protecting its vital cellular functions during seed maturation and storage, and plays a vital role in regulating seed size, weight, and seed vigor. Taken together, our study identified ENO2 as a novel substrate of PIMT, and both ENO2 and PIMT in turn implicate in agronomically important seed traits.

Study on the role of tissue-specific and non-specific autoantibodies, matrix metalloproteinase-3 and neuron-specific enolase enzymes in the exacerbation of autoimmune thyroiditis

The aim of the study was to examine the involvement of tissue-specific and non-specific autoantibodies, matrix metalloproteinase-3 and neuron-specific enolase (NSE) enzymes in the development and exacerbation of autoimmune thyroiditis. Materials and methods. The study enrolled 170 patients with autoimmune thyroiditis (64 males and 106 females aged 18 to 64 years) to comprehensively examine their humoral immune response indicators (IgA, M, G), organ-specific (Ab-TG, Ab-TPO) and organ-non-specific antibodies (Ab-DNA), metalloproteinase-3 and NSE activity. The control group consisted of 65 individuals without thyroid pathologies or other autoimmune diseases, aged 20 to 65 years (26 males and 39 females). Results. The study has demonstrated changes in the levels of organ-specific and organ-nonspecific antibodies and statistically significantly increased metalloproteinase-3 activity in patients with autoimmune thyroiditis. Positive correlations have been found between elevated levels of IgG, Ab-TG, Ab-TPO, Ab-dsDNA and NSE activity. Negative correlations have been observed between NSE activity and IgA concentrations. Conclusions. Elevated titers of anti-DNA autoantibodies may indicate an aggravation of the autoimmune process due to cellular structure damage, resulting in gland dysfunction. The findings also suggest that metalloproteinase-3, a marker predicting thyroid tissue damage, may negatively impact the immune response induction, ultimately affecting the activity of neuron-specific enolase. The data have shown that studying biochemical indicators such as antinuclear antibodies (ANA), anti-DNA antibodies, metalloproteinase-3 and neurodegenerative indicators could provide informative markers to determine the nature of the disease development and worsening.

BCAT proteins: A metabolic link between Type II Diabetes and Alzheimer’s Disease

AbstractBackgroundAberrant energy pathways compounded by dysregulated cellular redox plays a pivotal role in the pathogenesis of Alzheimer’s disease (AD) but the underlying mechanisms are not clear. Clinical and epidemiological data show that those with Type II Diabetes Mellitus (T2DM) have a 65% increased risk of developing AD (Arvanitakis et al., 2004). Our group and others have shown that serum branched chain amino acids and the branched chain aminotransferase proteins (BCAT) are increased in subjects with AD and T2DM, which has been linked with dysregulated autophagy and metabolic reprogramming. Here, we explore how changes in cellular redox regulate and influence metabolic‐related pathology changes associated with AD.MethodsSHSY‐5Y cells were used as a neuronal model to observe the effects of BCATc dysregulation in neuronal metabolism. Cells were transfected with our BCATc overexpression constructs and metabolite load assessed using LC‐MS/MS. Activity of glycolytic enzymes GAPDH and enolase were assessed, and overall glycolytic rate characterised using a Seahorse XF analyser. Oxidative stress was measured using the HyPer‐7 molecular probe (Bousolov et al, 2004) and cells were visualised using confocal microscopy. Finally, Western blot analysis was performed to assess levels of BCATc in the presence of increasing glucose (+/‐ 100 nM insulin) and hydrogen peroxide treatments and to demonstrate changes in levels of β‐amyloid and hyperphosphorylated tau.ResultsHere we show that BCATc overexpression increases the level of glucose and fructose, whilst decreasing GAPDH, enolase activity and glycolytic rate (p < 0.05). There was an observable increase in hydrogen peroxide generation (p<0.001), while levels of amyloid precursor protein (P<0.01), β‐amyloid (p>0.05) and hyperphosphorylated tau (p<0.05) were significantly increased. In addition, levels of BCATc increased dose‐dependently following hydrogen peroxide treatments (p < 0.001) and decreased in the presence of excess glucose (+ 100 nM insulin) (p<0.05).ConclusionOur results demonstrate that the reprogramming of BCAT as a result of hallmarks of T2DM such as excess dietary‐derived nutrient signals and a concurrent increase in oxidative stress, are associated with aberrant processing and aggregation of β‐amyloid and tau protein and are likely to contribute to the pathophysiology of AD.

Inhibition by Ascorbate among Phosphofructokinase-1, Aldolase, Enolase, and Lactate Dehydrogenase in Rabbit Muscle

Background: These studies examined mutual protective relationships among rabbit muscle aldolase, enolase, phosphofructokinase-1 (PFK-1) and LDH from inhibitions by ascorbate (AA). It was proposed earlier that specific inhibitions of PFK-1 and LDH by AA faciltated glycogen storage in resting muscle by inhibiting glycolysis. Materials and Methods: The L-ascorbate (AA), L-ascorbyl dibutyrate (AADB), L-ascorbyl dipalmitate (AADP), L-ascorbyl palmitate (AAP), and L- ascorbyl stearate (AS) are shown in Figure 1 and were obtained from TCI and Alfa Aesar. Unless otherwise stated, all enzymes come from rabbit and all experimental temperatures were 25°C, pH 8.0. Results: Rabbit muscle enolase was examined for its protective effect on other rabbit muscle glycolytic enzymes against inhibitions by ascorbate (AA) and some AA-faty acid derivatives. The IC50 values of enolase by ascorbate (AA) and IC50 values of AA-fatty acid derivatives were compared to estimate inhibition potency. For example, ascorbyl dipalmitate (AADP) was 156 times more inhibitory to enolase than AA. It was previously shown that rabbit muscle aldolase prevented LDH activity loses due to AA inhibition and prevented PFK-1 activity losses due both to dilution and AA inhibition; enolase was found to have the same effects as aldolase. Additionally, PFK-1 prevented enolase and LDH inhibitions by AA. LDH did not prevent enolase or PFK-1 from inhibition by AA. LDH did stimulate enolase activity but not PFK-1 activity. Conclusion: The results suggest that interactions among glycolytic enzyme serve to mutually protect one another from activity losses. The inhibition properties of the AA-fatty acid derivatives are discussed in relation to their possible roles in cancer and diabetes.

Enolase, a cadmium resistance related protein from hyperaccumulator plant Phytolacca americana, increase the tolerance of Escherichia coli to cadmium stress

Phytolacca americana is a Cd hyperaccumulator plant that accumulates significant amounts of Cd in leaves, making it a valuable phytoremediation plant species. Our previous research found enolase (ENO) may play an important part in P. americana to cope with Cd stress. As a multifunctional enzyme, ENO was involved not only in glycolysis but also in the response of plants to various environmental stresses. However, there are few studies on the function of PaENO (P. americana enolase) in coping with Cd stress. In this study, the PaENO gene was isolated from P. americana, and the expression level of PaENO gene significantly increased after Cd treatment. The enzymatic activity analysis showed PaENO had typical ENO activity, and the 42-position serine was essential to the enzymatic activity of PaENO. The Cd resistance assay indicated the expression of PaENO remarkably enhanced the resistance of E. coli to Cd, which was achieved by reducing the Cd content in E. coli. Moreover, both the expression of inactive PaENO and PaMBP-1 (alternative translation product of PaENO) can improve the tolerance of E. coli to Cd. The results indicated PaENO may be alternatively translated into the transcription factor PaMBP-1 to participate in the response of P. americana to Cd stress.

Astaxanthin Ameliorates Diabetic Retinopathy in Swiss Albino Mice via Inhibitory Processes of Neuron-Specific Enolase Activity

Retinopathy is one of the most common complications of diabetes mellitus. Diabetic retinopathy (DR) occurs due to microvascular damage in retinal tissues provoked by high blood sugar levels. The available drugs for DR are limited. Astaxanthin (AST) has anti-hypertensive, anti-obesity, and anti-diabetic properties. However, the therapeutic effect of AST on DR remains elusive. The present study is designed to investigate the effects of AST on DR via inhibition of neuron-specific enolase (NSE) activity. DR was induced by the administration of streptozotocin (STZ, 35 mg/kg: intraperitoneal; and 20 μL of STZ: intravitreal) in mice. AST (10 and 20 mg/kg) was administered orally (p.o.) for 21 days. The DR associated visual changes were assessed at different time intervals via optokinetic motor response (OMR) and penta-maze (PM) tests. Blood glucose level as well as retinal catalase, lactate dehydrogenase (LDH), & neuron-specific enolase (NSE) were estimated. The reference drug i.e., dexamethasone (DEX, 10 mg/kg; p.o.) was administered for 21 days. The administration of AST showed significant ameliorative potential in DR. Hence, AST can be used as a natural medicine for the management of DR due to its potential antioxidant, anti-diabetic, and NSE inhibitory properties.

Riboregulation of Enolase 1 activity controls glycolysis and embryonic stem cell differentiation

Differentiating stem cells must coordinate their metabolism and fate trajectories. Here, we report that the catalytic activity of the glycolytic enzyme Enolase 1 (ENO1) is directly regulated by RNAs leading to metabolic rewiring in mouse embryonic stem cells (mESCs). We identify RNA ligands that specifically inhibit ENO1's enzymatic activity invitro and diminish glycolysis in cultured human cells and mESCs. Pharmacological inhibition or RNAi-mediated depletion of the protein deacetylase SIRT2 increases ENO1's acetylation and enhances its RNA binding. Similarly, induction of mESC differentiation leads to increased ENO1 acetylation, enhanced RNA binding, and inhibition of glycolysis. Stem cells expressing mutant forms of ENO1 that escape or hyper-activate this regulation display impaired germ layer differentiation. Our findings uncover acetylation-driven riboregulation of ENO1 as a physiological mechanism of glycolytic control and of the regulation of stem cell differentiation. Riboregulation may represent a more widespread principle of biological control.

Salvianolic Acid B Suppresses Non-Small-Cell Lung Cancer Metastasis through PKM2-Independent Metabolic Reprogramming.

Objective Salvianolic acid B (Sal B) has been demonstrated to be a potential chemoprevention agent for several cancers. Herein, we investigated the pharmacological function of Sal B on non-small-cell lung cancer (NSCLC) metastasis. Methods Two NSCLC cell lines (NCI-H2030 and NCI-H1650) were disposed of by 200 μM Sal B or 10 μM PKM2 agonist TEPP-46. Wound healing and transwell experiments were implemented for analyzing migratory and invasive capacities. Epithelial-to-mesenchymal transition (EMT) markers β-catenin and E-cadherin were measured via western blotting. Cellular bioenergetics were evaluated with glucose uptake, lactate production, enolase activity, cellular ATP levels, as well as seahorse-based oxygen consumption rate (OCR), extracellular acidification rate (ECAR) analysis. Metabolic reprogramming markers PKM2, LDHA, and GLUT1 were detected via western blotting and immunofluorescence. Results The results showed that Sal B disposal weakened the migration and invasion of NCI-H2030 and NCI-H1650 cells and inactivated the EMT process according to downregulation of β-catenin and upregulation of E-cadherin. Sal B-treated NSCLC cells displayed decreased glucose uptake, lactate production, enolase activity, cellular ATP levels, OCR, and ECAR, indicating a reduction in metabolic reprogramming. Additionally, Sal B downregulated the expression of PKM2, LDHA, and GLUT1. TEPP-46 may reverse the inhibitory effect of Sal B on metastasis as well as metabolic reprogramming. Conclusion Our findings provide evidence that Sal B enables to weaken NSCLC metastasis through PKM2-independent metabolic reprogramming, which sheds light on the promising therapeutic usage of Sal B in treating NSCLC.

Effects of low concentration fluoride released from fluoride-sustained-releasing composite resin on the bioactivity of Streptococcus mutans.

This study aimed to determine the effects of the low concentration of fluoride sustained-released from the composite resin on properties of Streptococcus mutans (S. mutans). The proliferation, adhesion, biofilm formation, and lactic acid production of the bacterium were investigated after the treatment with fluoride from the composite resin. The activity and expression of ATPase, glucosyltransferase (GTF), and enolase were also determined by colorimetric assay and western blot analysis. Fluoride from the sustained-releasing composite resin significantly inhibited biofilm formation, adhesion, and acid production of S. mutans but did not influence the growth and ATPase activity of the bacterial strain. Fluoride-sustained-releasing composite resin inhibited adhesion, biofilm formation, and acid production of S. mutans through the suppression of GTF expression as well as the expression and activity of enolase. These resultls suggest the clinical significance of fluoride-sustained-releasing composite resin.

Enolase of Staphylococcus lugdunensis Is a Surface-Exposed Moonlighting Protein That Binds to Extracellular Matrix and the Plasminogen/Plasmin System.

The coagulase-negative staphylococcal (CoNS) species Staphylococcus lugdunensis is unique in causing serious infections in humans that resemble those of Staphylococcus aureus rather than those of other CoNS species. The colonization and invasion of host tissue presupposes the presence of adherence factors, but only a few proteins mediating adhesion of S. lugdunensis to biotic surfaces are known yet. Here, we report on the functionality of the S. lugdunensis enolase (SlEno), which performs two distinct roles, first, as the metabolic enzyme of the glycolysis, and second, as an adherence factor to the extracellular matrix (ECM) of cells. Phylogenetic analyses of the SlEno confirmed their high conservation to enolases of other species and revealed a closer relationship to Staphylococcus epidermidis than to S. aureus. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry and Western blot experiments, we identified SlEno to be located in the cytoplasm as well as on the cell surface of S. lugdunensis. Recombinantly generated and surface-associated SlEno showed the usual enolase activity by catalyzing the conversion of 2-phosphoglycerate to phosphoenolpyruvate but, in addition, also displayed strong binding to immobilized laminin, fibronectin, fibrinogen, and collagen type IV in a dose-dependent manner. We also showed a strong binding of SlEno to plasminogen (Plg) and observed a tissue plasminogen activator (tPA)-dependent conversion of Plg to plasmin (Pln) whereby the Plg activation significantly increased in the presence of SlEno. This interaction might be dependent on lysines of the SlEno protein as binding to Plg was inhibited by ε-aminocaproic acid. Furthermore, the enhanced activation of the Plg/Pln system by SlEno enabled S. lugdunensis to migrate through a fibrin matrix. This migration was about 10-fold higher than without exogenously added SlEno. Finally, we observed a significantly higher clearance of S. lugdunensis by freshly prepared granulocytes and in the presence of anti-SlEno antibodies. In conclusion, these data demonstrate for the first time a moonlighting function of the S. lugdunensis enolase, which is an underrated virulence factor for colonization and invasion of tissues. Hence, SlEno might be a potential vaccine candidate to prevent severe infections caused by this pathogen.

Reduction of the canonical function of a glycolytic enzyme enolase triggers immune responses that further affect metabolism and growth in Arabidopsis.

Primary metabolism provides energy for growth and development as well as secondary metabolites for diverse environmental responses. Here we describe an unexpected consequence of disruption of a glycolytic enzyme enolase named LOW EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 2 (LOS2) in causing constitutive defense responses or autoimmunity in Arabidopsis thaliana. The autoimmunity in the los2 mutant is accompanied by a higher expression of about one-quarter of intracellular immune receptor nucleotide-binding leucine-rich repeat (NLR) genes in the genome and is partially dependent on one of these NLR genes. The LOS2 gene was hypothesized to produce an alternatively translated protein c-Myc Binding Protein (MBP-1) that functions as a transcriptional repressor. Complementation tests show that LOS2 executes its function in growth and immunity regulation through the canonical enolase activity but not the production of MBP-1. In addition, the autoimmunity in the los2 mutants leads to a higher accumulation of sugars and organic acids and a depletion of glycolytic metabolites. These findings indicate that LOS2 does not exert its function in immune responses through an alternatively translated protein MBP-1. Rather, they show that a perturbation of glycolysis from the reduction of the enolase activity results in activation of NLR-involved immune responses which further influences primary metabolism and plant growth, highlighting the complex interaction between primary metabolism and plant immunity.

Specific activation of glycolytic enzyme enolase 2 in BRAF V600E-mutated colorectal cancer.

The BRAF V600E mutation occurs in approximately 10% of patients with metastatic colorectal cancer (CRC) and constitutes a distinct subtype of the disease with extremely poor prognosis. To address this refractory disease, we investigated the unique metabolic gene profile of BRAF V600E‐mutated tumors via in silico analysis using a large‐scale clinical database. We found that BRAF V600E‐mutated tumors exhibited a specific metabolic gene expression signature, including some genes that are associated with poor prognosis in CRC. We discovered that BRAF V600E‐mutated tumors expressed high levels of glycolytic enzyme enolase 2 (ENO2), which is mainly expressed in neuronal tissues under physiological conditions. In vitro experiments using CRC cells demonstrated that BRAF V600E‐mutated cells exhibited enhanced dependency on ENO2 compared to BRAF wild‐type cancer cells and that knockdown of ENO2 led to the inhibition of proliferation and migration of BRAF V600E‐mutated cancer cells. Moreover, inhibition of ENO2 resulted in enhanced sensitivity to vemurafenib, a selective inhibitor of BRAF V600E. We identified AP‐1 transcription factor subunit (FOSL1) as being involved in the transcription of ENO2 in CRC cells. In addition, both MAPK and PI3K/Akt signaling were suppressed upon inhibition of ENO2, implying an additional oncogenic role of ENO2. These results suggest the crucial role of ENO2 in the progression of BRAF V600E‐mutated CRC and indicate the therapeutic implications of targeting this gene.

Moonlighting in Bacillus subtilis: The Small Proteins SR1P and SR7P Regulate the Moonlighting Activity of Glyceraldehyde 3-Phosphate Dehydrogenase A (GapA) and Enolase in RNA Degradation.

Moonlighting proteins are proteins with more than one function. During the past 25 years, they have been found to be rather widespread in bacteria. In Bacillus subtilis, moonlighting has been disclosed to occur via DNA, protein or RNA binding or protein phosphorylation. In addition, two metabolic enzymes, enolase and phosphofructokinase, were localized in the degradosome-like network (DLN) where they were thought to be scaffolding components. The DLN comprises the major endoribonuclease RNase Y, 3′-5′ exoribonuclease PnpA, endo/5′-3′ exoribonucleases J1/J2 and helicase CshA. We have ascertained that the metabolic enzyme GapA is an additional component of the DLN. In addition, we identified two small proteins that bind scaffolding components of the degradosome: SR1P encoded by the dual-function sRNA SR1 binds GapA, promotes the GapA-RNase J1 interaction and increases the RNase J1 activity. SR7P encoded by the dual-function antisense RNA SR7 binds to enolase thereby enhancing the enzymatic activity of enolase bound RNase Y. We discuss the role of small proteins in modulating the activity of two moonlighting proteins.

Influence of Water Polarization Caused by Phonon Resonance on Catalytic Activity of Enolase.

Enolase is a conservative protein. Its cellular enzymatic activity catalyzes the conversion of 2-phospho-d-glycerate (2-PGA) to a phosphoenolpyruvate (PEP) product in the glycolysis pathway. This enzyme also has a multifunctional nature participating in several biological processes. This work aims to determine the effect of water polarization on the catalytic activity of enolase. The experiments have been set based on the concept that water, a polar dielectric, may undergo the phenomenon of electric polarization, decreasing its configurational and vibrational entropy. Prior to the reaction, the 2-PGA substrate was incubated for 5 h in the glass cuvette with an attached chip-inductor. The latter device was designed to transfer quantum information about a given quantum state from the quantum state generator to water by a phonon resonance. Then, such substrate samples preincubated with the chip-inductor were removed every hour in a separate quartz cuvette with the enzyme to determine its catalytic activity. The influence of the chip-inductor on the preincubated substrate resulted in an increase in the catalytic activity of enolase by 30% compared to the control substrate, not preincubated with the chip-inductor. This suggests that the catalytic activity of the enzyme is augmented when the substrate was primed by chip-inductors. In another kind of experiment, wherein enolase was exposed to methylglyoxal modification, the catalytic activity of the enzyme dropped to 71.7%, while the same enzyme glycated with methylglyoxal primed by chip-inductors restored its activity by 8.4%. This shows the protective effect of chip-inductors on enolase activity despite the harmful effect of methylglyoxal on the protein.

LncRNA RNCR2 facilitates cell proliferation and epithelial-mesenchymal transition in melanoma through HK2-mediated Warburg effect via targeting miR-495-3p.

Melanoma is a potentially lethal skin cancer with a high death rate. LncRNAs were reported to be implicated in melanoma progression. However, the function and mechanisms of lncRNA RNCR2 in melanoma are little known. In this study, RNCR2, miR-495-3p, and HK2 expression levels were measured in melanoma tissue specimens and cell lines by qPCR. EdU and CCK-8 assays were performed to assess cell proliferation. Enolase activity, ATP level, lactate production, and glucose consumption measurement kits were used to evaluate the glycolysis of tumor cells. Immunofluorescence and western blot were used to detect the expression of epithelial-mesenchymal transition (EMT) and glycolysis-related proteins. Luciferase reporter assay was applied to confirm the target relationships. The role of RNCR2 in tumorigenesis was examined using murine xenograft models. LncRNA RNCR2 was upregulated in melanoma tissues and cell lines. Cell function detection showed that RNCR2 knockdown remarkably inhibited cell proliferation and EMT via glycolysis, as well as reduced the growth of a tumor. Mechanically, RNCR2 was confirmed to bind to miR-495-3p and positively regulated HK2 expression level, and the miR-495-3p level was negatively correlated with RNCR2 or HK2 in melanoma tissues. Further, miR-495-3p downregulation or HK2 upregulation partially reversed RNCR2 knockdown-induced inhibition of melanoma cell growth, EMT, and glycolysis. Collectively, RNCR2 might be an oncogenic lncRNA to promote tumor cell glycolysis and accelerate tumor growth via the miR-495-3p/HK2 axis, providing a promising treatment target for melanoma.

ENO2 Affects the Seed Size and Weight by Adjusting Cytokinin Content and Forming ENO2-bZIP75 Complex in Arabidopsis thaliana.

Arabidopsis thaliana ENO2 (AtENO2) encodes two proteins AtENO2 (enolase) and AtMBP-1 (c-Myc binding protein 1-like). The loss of AtENO2 function causes the constitutive developmental defects which are correlated with reduced enolase activity, but not AtMBP-1 transcript abundance. However, the regulation mechanism of AtENO2 on the seed properties is still not clear. In this study, we found that the mutation of AtENO2 reduced the seed size and weight. The level of glucose in seed was significantly elevated but that of starch was decreased in AtENO2 mutants compared to WT plants. We also found that AtENO2 mutation reduced the content of cytokinin which resulted in smaller cotyledons. The RNA-seq data showed that there were 1892 differentially expressed genes and secondary metabolic pathways were significantly enriched. Instead of AtMBP-1, AtENO2 protein interacted with AtbZIP75 which may mediate the secondary metabolism. Therefore, ENO2 alters the size and weight of seeds which is not only regulated by the content of cytokinin and secondary metabolism, but may be affected by the interaction of ENO2 and bZIP57. These results are helpful to understand the novel function of AtENO2 which provide a foundation for further exploration of the key candidate genes for crop breeding.

Molecular and biochemical characterization of enolase from Dermanyssus gallinae.

Enolase, a multifunctional glycolytic enzyme, is known to act as a plasminogen receptor in many species, involved in the pivotal processes such as motility, adhesion, invasion, growth, and differentiation of the parasites. Knowledge on the function of enolase from Dermanyssus gallinae is very limited. Here we report on the molecular cloning, enzymatic activity, tissue distribution and plasminogen binding activity of enolase from D. gallinae (DgENO). The full-length of cDNA was 1305bp, specifying a peptide of 434 amino acids. Bioinformatics analysis showed that DgENO was highly conserved compared with a range of organisms, indicating the potentially similar functions in D. gallinae. A recombinant DgENO (rDgENO) protein was produced and characterized, it catalyzed the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate, the optimal pH was 7.5. Polyclonal antibodies were generated in mice and western blotting indicated that antiserum specifically recognized the native enolase in the somatic extracts from D. gallinae. Immunohistochemical staining of mite sections revealed that the distribution of DgENO was ubiquitous with high level in salivary gland, mite digestive tissues and fat bodies in D. gallinae. Expression level of DgENO was observed mostly in engorged adult mites. Moreover, ELISA binding assay showed that rDgENO could bind plasminogen, and lysine analog ε-aminocaproic acid significantly inhibited this binding activity, indicating that D. gallinae enolase is a receptor of plasminogen. The present study provided foundation for understanding of the biological functions of DgENO and its application in development of vaccines against D. gallinae.

Sugar Metabolism of Scardovia wiggsiae, a Novel Caries-Associated Bacterium.

Scardovia wiggsiae has been detected from caries in children and adolescents and has been suggested to be a caries-associated microorganism. To investigate the cariogenic potential of S. wiggsiae, we examined carbohydrate metabolism and acid productivity, the fluoride sensitivity of carbohydrate metabolism and the mechanism by which fluoride inhibits carbohydrate metabolism, and the acid sensitivity of carbohydrate metabolism in this bacterium. S. wiggsiae metabolized glucose and reduced the environmental pH to 3.5. It mainly produced acetic acid from glucose, together with small amounts of lactic and formic acid. The 50% inhibitory concentration of fluoride for acid production was 8.0 mM at pH 7.0 and 1.5 mM at pH 5.5, which were much higher than those of representative caries-associated bacteria, such as Streptococcus mutans. Metabolomic profiles showed the accumulation of 3-phosphoglycerate and a marked reduction in the pyruvate concentration in the presence of fluoride, suggesting that fluoride inhibits the latter half of glycolysis, including enolase activity. Enolase activity was inhibited by fluoride in S. wiggsiae, but it was more fluoride-tolerant than the enolase activity of S. mutans. Unlike in S. mutans, lactic acid did not inhibit acid production by S. wiggsiae at acidic pH. These results indicate that S. wiggsiae exhibits high acid production and tolerance to fluoride and lactic acid. S. wiggsiae possesses a unique metabolic pathway, the F6PPK shunt, which might allow it to avoid the lactate-formate pathway, including fluoride-sensitive enolase activity, and enable metabolic flow to the fluoride-tolerant acetate pathway. The fluoride tolerance of S. wiggsiae’s enolase activity also increases the fluoride tolerance of its carbohydrate metabolism. The lactic acid tolerance of S. wiggsiae’s acid production might result in S. wiggsiae having high acidogenic and aciduric potential and make it ecologically competitive in acidic environments, such as caries lesions, where lactic acid predominates.

Total flavones of Rhododendron simsii Planch flower protect rat hippocampal neuron from hypoxia-reoxygenation injury via activation of BKCa channel.

To study the effects of total flavones of Rhododendra simsii Planch flower (TFR) on hypoxia/reoxygenation (H/R) injury in rat hippocampal neurons and its underlying mechanism. Model of H/R was established in newborn rat primary cultured hippocampal neuron. Lactate dehydrogenase (LDH) and neuron-specific enolase (NSE) activity as well as malondialdehyde (MDA) content in cultured supernatants of the neurons were examined. Methyl thiazolyl tetrazolium assay and Hoechst33258 staining were, respectively, used to detect cell viability and apoptosis of neurons. Protein expression and current of BKCa channel were assessed by using Western blotting and whole-cell patch-clamp methods, respectively. In the ranges of 3.7-300mg/l, TFR significantly inhibited H/R-induced decrease of neuronal viability and increases of LDH, NSE and MDA in the supernatants as well as apoptosis; TFR 33.3, 100 and 300mg/l markedly increased current of BKCa channel rather than the BKCa channel protein expression in the neurons. Total flavones of R.simsii Planch flower had a protective effect against H/R injury in rat hippocampal neuron, and activation of BKCa channel may contribute to the neuroprotection.