Further evidence in favour of a carbanion mechanism for glycolate oxidase.

The flavoenzyme glycolate oxidase oxidizes glycolic acid to glyoxylate and the latter, more slowly, to oxalate. It is a member of an FMN-dependent enzyme family that oxidizes l-2-hydroxy acids to keto acids. There has been a controversy concerning the chemical mechanism of substrate oxidation by these enzymes. Do they proceed by hydride transfer, as observed for NAD-dependent enzymes, or by initial formation of a carbanion that transfers the electrons to the flavin? The present work describes a comparison of the reactivity of glycolate, lactate and trifluorolactate with recombinant human glycolate oxidase, by means of rapid-kinetics experiments in anaerobiosis. We show that trifluorolactate is a substrate for glycolate oxidase, whereas it is known as an inhibitor for NAD-dependent enzymes, as is trifluoroethanol for NAD-dependent alcohol dehydrogenases. Unexpectedly, it was observed that, once reduced, a flavin transfers an electron to an oxidized flavin, so that the end species is a flavin semiquinone, whatever the substrate. This phenomenon has not previously been described for a glycolate oxidase. Altogether, considering that another member of this flavoenzyme family (flavocytochrome b2 , a lactate dehydrogenase) has also been shown to oxidize trifluorolactate (Lederer F et al. (2016) Biochim Biophys Acta 1864, 1215-21), this work provides another important piece of evidence which is hardly compatible with a hydride transfer mechanism for this flavoenzyme family.

Flavocytochrome b2 of the Methylotrophic Yeast Ogataea polymorpha: Construction of Overproducers, Purification, and Bioanalytical Application.

Flavocytochrome b2 (EC 1.1.2.3; L-lactate cytochrome: c oxidoreductase, FC b2) from the thermotolerant methylotrophic yeast Ogataea polymorpha is a thermostable enzyme-prospective for a highly selective L-lactate analysis in the medicine, nutrition sector, and quality control of commercial products. Here we describe the construction of FC b2 producers by overexpression of the CYB2 gene O. polymorpha, encoding FC b2, under the control of a strong alcohol oxidase promoter in the frame of plasmid for multicopy integration with the next transformation of recipient strain O. polymorpha C-105 (gcr1 catX) impaired in the glucose repression and devoid of catalase activity. The selected recombinant strain O. polymorpha "tr1" (gcr1 catX CYB2), characterized by eightfold increased FC b2 activity compared to the initial strain, was used as a source of the enzyme. For purification of FC b2 a new method of affinity chromatography was developed and purified preparations of the enzyme were used for the construction of the highly selective enzymatic kits and amperometric biosensor for L-lactate analysis in human liquids and foods.

Functional Assembly of Caenorhabditis elegans Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity.

Among seven homologs of cytochrome b561 in a model organism C. elegans, Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expressed in Pichia pastoris cells, purified, and reconstituted into a phospholipid bilayer nanodisc. The reconstituted Cecytb-2 in nanodisc environments was extremely stable and more reducible with ascorbate than in a detergent-micelle state. We confirmed the ferric reductase activity of Cecytb-2 by analyzing the oxidation of ferrous heme upon addition of ferric substrate under anaerobic conditions, where clear and saturable dependencies on the substrate concentrations following the Michaelis-Menten equation were observed. Further, we confirmed that the ferric substrate was converted to a ferrous state by using a nitroso-PSAP assay. Importantly, we observed that the ferric reductase activity of Cecytb-2 became enhanced in the phospholipid bilayer nanodisc.

ctDNA detected by ddPCR reveals changes in tumour load in metastatic malignant melanoma treated with bevacizumab.

Bevacizumab is included in an increasing number of clinical trials. To find biomarkers to predict and monitor treatment response, cancer and angiogenesis relevant mutations in tumour and circulating tumour DNA (ctDNA) were investigated in 26 metastatic melanoma patients treated with bevacizumab. Patients with >1% BRAF/NRAS ctDNA at treatment start had significantly decreased progression free survival (PFS) and overall survival (OS) (PFS: p = 0.019, median 54 vs 774 days, OS: p = 0.026, median 209 vs 1064 days). Patients with >1% BRAF/NRAS ctDNA during treatment showed similar results (PFS: p = 0.002, OS: p = 0.003). ≤1% BRAF/NRAS ctDNA and normal lactate dehydrogenase (LDH) levels both significantly predicted increased response to treatment, but BRAF/NRAS ctDNA was better at predicting response compared to LDH at treatment start (OR 16.94, p = 0.032 vs OR 4.57, p = 0.190), and at predicting PFS (HR 6.76, p = 0.002) and OS (HR 6.78, p = 0.002) during therapy. ctDNA BRAF p.V600D/E/K and NRAS p.G12V/p.Q61K/L/R were better biomarkers for response prediction than TERT promoter mutations (OR 1.50, p = 0.657). Next generation sequencing showed that all patients with ≥2 mutations in angiogenesis-relevant genes had progressive disease, but did not reveal other biomarkers identifying responders. To conclude, ctDNA and LDH are useful biomarkers for both monitoring and predicting response to bevacizumab.

Culture-based analysis of fungi in leaves after the primary and secondary fermentation processes during Ishizuchi-kurocha production and lactate assimilation of P. kudriavzevii.

Ishizuchi-kurocha is a Japanese traditional fermented tea that is produced by primary aerobic and secondary fermentation steps. The secondary fermentation step of Ishizuchi-kurocha is mainly mediated through lactic acid bacteria. Here, we performed quantitative analyses of the culturable fungal communities at each step and identified several morphologically representative fungal isolates. While filamentous fungi (median, 3.2 × 107 CFU/g sample) and yeasts (median, 3.7 × 107 CFU/g) were both detected after the primary fermentation step, only yeasts (median, 1.6 × 107 CFU/g) were detected in the end of the secondary fermentation step, suggesting that the fungal community in tea leaves are dramatically changed between the two steps. Pichia kudriavzevii and Pichia manshurica, the prevalent fungal species at the end of the secondary fermentation step, grew well in exudate from the secondary fermentation step. P. kudriavzevii also grew well in media containing d- or l-lactate as the sole carbon source. The growth of the disruptant of cyb2A encoding a cytochrome b2 lactate dehydrogenase in P. kudriavzevii was severely impaired on medium supplemented with l-lactate, but not d-lactate, suggesting that Cyb2Ap plays a crucial role in the use of l-lactate, and P. kudriavzevii efficiently uses both l- and d-lactate as carbon sources. Thus, lactate assimilation seems to be a key phenotype to become a prevalent species in the secondary fermentation step, and Cyb2Ap has a pivotal role in l-lactate metabolism in P. kudriavzevii. Further understanding and engineering of P. kudriavzevii and P. manshurica will contribute to the control of lactic acid bacteria fermentation during the fermented tea production and also to other industrial uses.

Closing the gap: yeast electron-transferring flavoprotein links the oxidation of d-lactate and d-α-hydroxyglutarate to energy production via the respiratory chain.

Electron-transferring flavoproteins (ETFs) have been found in all kingdoms of life, mostly assisting in shuttling electrons to the respiratory chain for ATP production. While the human (h) ETF has been studied in great detail, very little is known about the biochemical properties of the homologous protein in the model organism Saccharomyces cerevisiae (yETF). In view of the absence of client dehydrogenases, for example, the acyl-CoA dehydrogenases involved in the ß-oxidation of fatty acids, d-lactate dehydrogenase 2 (Dld2) appeared to be the only relevant enzyme that is serviced by yETF for electron transfer to the mitochondrial electron transport chain. However, this hypothesis was never tested experimentally. Here, we report the biochemical properties of yETF and Dld2 as well as the electron transfer reaction between the two proteins. Our study revealed that Dld2 oxidizes d-α-hydroxyglutarate more efficiently than d-lactate exhibiting kcatapp /KMapp values of 1200 ± 300 m-1 ·s-1 and 11 ± 2 m-1 ·s-1 , respectively. As expected, substrate-reduced Dld2 very slowly reacted with oxygen or the artificial electron acceptor 2,6-dichlorophenol indophenol. However, photoreduced Dld2 was rapidly reoxidized by oxygen, suggesting that the reaction products, that is, α-ketoglutarate and pyruvate, 'lock' the reduced enzyme in an unreactive state. Interestingly, however, we could demonstrate that substrate-reduced Dld2 rapidly transfers electrons to yETF. Therefore, we conclude that the formation of a product-reduced Dld2 complex suppresses electron transfer to dioxygen but favors the rapid reduction in yETF, thus preventing the loss of electrons and the generation of reactive oxygen species.

Neuroprotective effect of l-serine against white matter demyelination by harnessing and modulating inflammation in mice.

Demyelination in white matter is the end product of numerous pathological processes. This study was designed to evaluate the neuroprotective effect of l-serine and the underlying mechanisms against the demyelinating injury of white matter. A model of focal demyelinating lesions (FDL) was established using the two-point stereotactic injection of 0.25% lysophosphatidylcholine (LPC, 10 µg per point) into the corpus callosum of mice. Mice were then intraperitoneally injected with one of three doses of l-serine (114, 342, or 1026 mg/kg) 2 h after FDL, and then twice daily for the next five days. Behavior tests and histological analysis were assessed for up to twenty-eight days post-FDL induction. Electron microscopy was used for ultrastructural investigation. In vitro, we applied primary co-cultures of microglia and oligodendrocytes for oxygen glucose deprivation (OGD). After establishing FDL, l-serine treatment: 1) improved spatial learning, memory and cognitive ability in mice, and relieved anxiety for 4 weeks post-FDL induction; 2) reduced abnormally dephosphorylated neurofilament proteins, increased myelin basic protein, and preserved anatomic myelinated axons; 3) inhibited microglia activation and reduced the release of inflammatory factors; 4) promoted recruitment and proliferation of oligodendrocyte progenitor cells, and the efficiency of subsequent remyelination on day twenty-eight post-FDL induction. In vitro experiments, showed that l-serine not only directly protected against oligodendrocytes from OGD damage, but also provided an indirect protective effect by regulating microglia. In our study, l-serine offered long-lasting behavioral and oligodendrocyte protection and promoted remyelination. Therefore, l-serine may be an effective clinical treatment aganist white matter injury.

Translational misreading, amino acid misincorporation and misinterpretations. The case of the flavocytochrome b2 H373Q variant.

Amino acid misincorporation during protein synthesis occurs naturally at a low level. Protein sequence errors, depending on the level and the nature of the misincorporation, can have various consequences. When site-directed mutagenesis is used as a tool for understanding the role of a side chain in enzyme catalysis, misincorporation in a variant with intrinsically low activity may lead to misinterpretations concerning the enzyme mechanism. We report here one more example of such a problem, dealing with flavocytochrome b2 (Fcb2), a lactate dehydrogenase, member of a family of FMN-dependent L-2-hydroxy acid oxidizing enzymes. Two papers have described the properties of the Fcb2 catalytic base H373Q variant, each one using a different expression system with the same base change for the mutation. The two papers found similar apparent kinetic parameters. But the first one demonstrated the existence of a low level of histidine misincorporation, which led to an important correction of the variant residual activity (Gaume et al. (1995) Biochimie, 77, 621). The second paper did not investigate the possibility of a misincorporation (Tsai et al. (2007) Biochemistry, 46, 7844). The two papers had different mechanistic conclusions. We show here that in this case the misincorporation does not depend on the expression system. We bring the proof that Tsai et al. (2007) were led to an erroneous mechanistic conclusion for having missed the phenomenon as well as for having misinterpreted the crystal structure of the variant. This work is another illustration of the caution one should exercise when characterizing enzyme variants with low activity.

Trifluorosubstrates as mechanistic probes for an FMN-dependent l-2-hydroxy acid-oxidizing enzyme.

A controversy exists with respect to the mechanism of l-2-hydroxy acid oxidation by members of a family of FMN-dependent enzymes. A so-called carbanion mechanism was initially proposed, in which the active site histidine abstracts the substrate α-hydrogen as a proton, followed by electron transfer from the carbanion to the flavin. But an alternative mechanism was not incompatible with some results, a mechanism in which the active site histidine instead picks up the substrate hydroxyl proton and a hydride transfer occurs. Even though more recent experiments ruling out such a mechanism were published (Rao & Lederer (1999) Protein Science 7, 1531-1537), a few authors have subsequently interpreted their results with variant enzymes in terms of a hydride transfer. In the present work, we analyse the reactivity of trifluorolactate, a substrate analogue, with the flavocytochrome b2 (Fcb2) flavodehydrogenase domain, compared to its reactivity with an NAD-dependent lactate dehydrogenase (LDH), for which this compound is known to be an inhibitor (Pogolotti & Rupley (1973) Biochem. Biophys. Res. Commun, 55, 1214-1219). Indeed, electron attraction by the three fluorine atoms should make difficult the removal of the α-H as a hydride. We also analyse the reactivity of trifluoropyruvate with the FMN- and NAD-dependent enzymes. The results substantiate a different effect of the fluorine substituents on the two enzymes compared to their normal substrates. In the discussion we analyse the conclusions of recent papers advocating a hydride transfer mechanism for the family of l-2-hydroxy acid oxidizing FMN-dependent enzymes.

Evidence That Does Not Support Pyruvate Kinase M2 (PKM2)-catalyzed Reaction as a Rate-limiting Step in Cancer Cell Glycolysis.

It has been recognized that the rate-limiting function of pyruvate kinase M2 (PKM2) in glycolysis plays an important role in distributing glycolytic intermediates for anabolic and catabolic purposes in cancer cells. However, after analysis of the catalytic capacity of PKM2 relative to other glycolytic enzymes, the regulation range of PKM2 activity, metabolic flux control, and thermodynamics, we suggest that the PKM2-catalyzed reaction is not a rate-limiting step in cancer cell glycolysis. Hexokinase and phosphofructokinase 1 (PFK1), the first and third enzyme along the pathway, are rate-limiting enzymes that limit the overall glycolytic rate, whereas PKM2 and lactate dehydrogenase, the last two enzymes in the pathway, are for the fast removal of upstream intermediates to prevent the obstruction of the pathway. The argument is in accordance with the catalytic capacity of glycolytic enzymes, regulation range of enzyme activities, metabolic flux control, and thermodynamics.

Saccharomyces cerevisiae Forms D-2-Hydroxyglutarate and Couples Its Degradation to D-Lactate Formation via a Cytosolic Transhydrogenase.

The D or L form of 2-hydroxyglutarate (2HG) accumulates in certain rare neurometabolic disorders, and high D-2-hydroxyglutarate (D-2HG) levels are also found in several types of cancer. Although 2HG has been detected in Saccharomyces cerevisiae, its metabolism in yeast has remained largely unexplored. Here, we show that S. cerevisiae actively forms the D enantiomer of 2HG. Accordingly, the S. cerevisiae genome encodes two homologs of the human D-2HG dehydrogenase: Dld2, which, as its human homolog, is a mitochondrial protein, and the cytosolic protein Dld3. Intriguingly, we found that a dld3Δ knock-out strain accumulates millimolar levels of D-2HG, whereas a dld2Δ knock-out strain displayed only very moderate increases in D-2HG. Recombinant Dld2 and Dld3, both currently annotated as D-lactate dehydrogenases, efficiently oxidized D-2HG to α-ketoglutarate. Depletion of D-lactate levels in the dld3Δ, but not in the dld2Δ mutant, led to the discovery of a new type of enzymatic activity, carried by Dld3, to convert D-2HG to α-ketoglutarate, namely an FAD-dependent transhydrogenase activity using pyruvate as a hydrogen acceptor. We also provide evidence that Ser3 and Ser33, which are primarily known for oxidizing 3-phosphoglycerate in the main serine biosynthesis pathway, in addition reduce α-ketoglutarate to D-2HG using NADH and represent major intracellular sources of D-2HG in yeast. Based on our observations, we propose that D-2HG is mainly formed and degraded in the cytosol of S. cerevisiae cells in a process that couples D-2HG metabolism to the shuttling of reducing equivalents from cytosolic NADH to the mitochondrial respiratory chain via the D-lactate dehydrogenase Dld1.

Suppression of MAPK attenuates neuronal cell death induced by activated glia-conditioned medium in alpha-synuclein overexpressing SH-SY5Y cells.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disease with characteristics and symptoms that are well defined. Nevertheless, its aetiology remains unknown. PD is characterized by the presence of Lewy bodies inside neurons. α-Synuclein (α-syn) is a soluble protein present in the pre-synaptic terminal of neurons. Evidence suggests that α-syn has a fundamental role in PD pathogenesis, given that it is an important component of Lewy bodies localized in the dopaminergic neurons of PD patients. METHODS: In the present study, we investigated the influence of wild type (WT) and A30P α-syn overexpression on neuroblastoma SH-SY5Y toxicity induced by the conditioned medium (CM) from primary cultures of glia challenged with lipopolysaccharide (LPS) from Escherichia coli. RESULTS: We observed that SH-SY5Y cells transduced with α-syn (WT or A30P) and treated with CM from LPS-activated glia cells show evidence of cell death, which is not reverted by NF-κB inhibition by sodium salicylate or by blockage of P50 (NF-κB subunit). Furthermore, the expression of A30P α-syn in neuroblastoma SH-SY5Y decreases the cell death triggered by the CM of activated glia versus WT α-syn or control group. This effect of A30P α-syn may be due to the low MAPK42/44 phosphorylation. This finding is substantiated by MEK1 inhibition by PD98059, decreasing LDH release by CM in SH-SY5Y cells. CONCLUSION: Our results suggest that SH-SY5Y cells transduced with α-syn (WT or A30P) and treated with CM from LPS-activated glia cells show cell death, which is not reverted by NF-κB blockage. Additionally, the expression of A30P α-syn on neuroblastoma SH-SY5Y leads to decreased cell death triggered by the CM of activated glia, when compared to WT α-syn or control group. The mechanism underlying this process remains to be completely elucidated, but the present data suggest that MAPK42/44 phosphorylation plays an important role in this process. PROSPERO: CRD42015020829.

L-Lactate-selective microbial sensor based on flavocytochrome b2-enriched yeast cells using recombinant and nanotechnology approaches.

In the recent years, nanotechnology is the most developing branch due to a wide variety of potential applications in biomedical, biotechnological and agriculture fields. The binding nanoparticles with various biological molecules makes them attractive candidates for using in sensor technologies. The particularly actual is obtaining the bionanomembranes based on biocatalytic elements with improved sensing characteristics. The aim of this investigation is to study the properties of microbial L-lactate-selective sensor based on using the recombinant Hansenula polymorpha yeast cells overproducing flavocytochrome b2 (FC b2), as well as additionally enriched by the enzyme bound with gold nanoparticles (FC b2-nAu). Although, the high permeability of the living cells to nanoparticles is being intensively studied (mostly for delivery of drugs), the idea of using both recombinant technology and nanotechnology to increase the amount of the target enzyme in the biosensing layer is really novel. The FC b2-nAu-enriched living and permeabilized yeast cells were used for construction of a bioselective membrane of microbial L-lactate-selective amperometric biosensor. Phenazine methosulphate was served as a free defusing electron transfer mediator which provides effective electron transfer from the reduced enzyme to the electrode surface. It was shown that the output to L-lactate of FC b2-nAu-enriched permeabilized yeast cells is 2.5-fold higher when compared to the control cells. The obtained results confirm that additional enrichment of the recombinant yeast cell by the enzyme bound with nanoparticles improves the analytical parameters of microbial sensor.

GLYCOLATE OXIDASE3, a Glycolate Oxidase Homolog of Yeast l-Lactate Cytochrome c Oxidoreductase, Supports l-Lactate Oxidation in Roots of Arabidopsis.

In roots of Arabidopsis (Arabidopsis thaliana), l-lactate is generated by the reduction of pyruvate via l-lactate dehydrogenase, but this enzyme does not efficiently catalyze the reverse reaction. Here, we identify the Arabidopsis glycolate oxidase (GOX) paralogs GOX1, GOX2, and GOX3 as putative l-lactate-metabolizing enzymes based on their homology to CYB2, the l-lactate cytochrome c oxidoreductase from the yeast Saccharomyces cerevisiae. We found that GOX3 uses l-lactate with a similar efficiency to glycolate; in contrast, the photorespiratory isoforms GOX1 and GOX2, which share similar enzymatic properties, use glycolate with much higher efficiencies than l-lactate. The key factor making GOX3 more efficient with l-lactate than GOX1 and GOX2 is a 5- to 10-fold lower Km for the substrate. Consequently, only GOX3 can efficiently metabolize l-lactate at low intracellular concentrations. Isotope tracer experiments as well as substrate toxicity tests using GOX3 loss-of-function and overexpressor plants indicate that l-lactate is metabolized in vivo by GOX3. Moreover, GOX3 rescues the lethal growth phenotype of a yeast strain lacking CYB2, which cannot grow on l-lactate as a sole carbon source. GOX3 is predominantly present in roots and mature to aging leaves but is largely absent from young photosynthetic leaves, indicating that it plays a role predominantly in heterotrophic rather than autotrophic tissues, at least under standard growth conditions. In roots of plants grown under normoxic conditions, loss of function of GOX3 induces metabolic rearrangements that mirror wild-type responses under hypoxia. Thus, we identified GOX3 as the enzyme that metabolizes l-lactate to pyruvate in vivo and hypothesize that it may ensure the sustainment of low levels of l-lactate after its formation under normoxia.

Intracellular zinc is a critical intermediate in the excitotoxic cascade.

Excessive and sustained exposure to glutamate leads to injurious elevations of cytosolic calcium ([Ca(2+)]i), generation of reactive oxygen and nitrogen species (ROS, RNS), mitochondrial failure, mobilization of intracellular zinc ([Zn(2+)]i), and, ultimately, neuronal death. The relative contribution and temporal dynamics of the activation of these processes to promote the full development of excitotoxicity are still not completely understood. In this study, we exploited the unique features of nNOS positive neurons [nNOS (+)], a striatal subpopulation that is constitutively spared from NMDAR-dependent insults, and dissected NMDAR-driven [Ca(2+)]i, [Zn(2+)]i, ROS, and mitochondrial changes occurring in these neurons and the overall population of nNOS (-) striatal neurons. Comparing the two populations and employing pharmacological, biochemical, and single-cell imaging techniques, we show that [Zn(2+)]i mobilization acts as a critical intermediate in the cascade that links NMDAR-mediated ROS overproduction, mitochondrial failure, and [Ca(2+)]i deregulation to the production of neuronal damage. Results of this study may also provide the rationale for aiming at therapeutic agents that favor Zn(2+) homeostasis for the treatment of acute or chronic neurological conditions associated with excitotoxicity.

FTY720 attenuates excitotoxicity and neuroinflammation.

BACKGROUND: FTY720 (fingolimod, Gilenya™), a structural analog of sphingosine-1-phosphate (S1P), is the first oral drug approved for treatment the relapsing-remitting form of multiple sclerosis (MS), and its efficacy has been related to induced lymphopenia and consequent immunosuppression via modulation of S1P1 receptors (S1P1R). However, due to its lipophilic nature, FTY720 crosses the blood brain barrier (BBB) and could act directly on neural cells. In this study, we investigated the effectiveness of FTY720 as a neuroprotective agent using in vitro and in vivo models of excitotoxic neuronal death and examined if FTY720 exerts a direct action on neurons, or/and an indirect modulation of inflammation-mediated neurodegeneration as a possible mechanism of neuroprotection. METHODS: Primary neuronal and organotypic cortical cultures were treated with N-methyl-D-aspartic acid (NMDA) to induce excitotoxic cell death (measured by lactate dehydrogenase (LDH) assay or propidium iodide uptake, respectively). The effects of FTY720 treatment (10, 100 and 1,000 nM) on neuronal survival were examined. As an in vivo model of neuronal death and inflammation, we used intracerebroventricular (icv) administration of kainic acid (KA; 0.5 µg/2 µl) in Sprague-Dawley rats. FTY720 was applied icv (1 µg/2 µl), together with KA, plus intraperitoneally (ip; 1 mg/kg) 24 h before, and daily, until sacrifice 3 days after icv. Rats were evaluated for neurological score, neuronal loss in CA3 hippocampal region and activation of microglia at the lesion site. In addition, we tested FTY720 as a modulator of microglia responses using microglial cell cultures activated with lipopolysaccharide (LPS) and its effects in stress signalling pathways using western blotting for p38 and JNK1/2 mitogen-activated protein kinases (MAPKs). RESULTS: FTY720 was able to reduce excitotoxic neuronal death in vitro. Moreover, in vivo repeated FTY720 administration attenuated KA-induced neurodegeneration and microgliosis at the CA3 lesion site. Furthermore, FTY720 negatively modulates p38 MAPK in LPS-activated microglia, whereas it had no effect on JNK1/2 activation. CONCLUSIONS: These data support a role for FTY720 as a neuroprotective agent against excitotoxin-induced neuronal death and as a negative modulator of neuroinflammation by targeting the p38 MAPK stress signalling pathway in microglia.

NAD-Independent L-Lactate Dehydrogenase Required for L-Lactate Utilization in Pseudomonas stutzeri A1501.

UNLABELLED: NAD-independent L-lactate dehydrogenases (l-iLDHs) play important roles in L-lactate utilization of different organisms. All of the previously reported L-iLDHs were flavoproteins that catalyze the oxidation of L-lactate by the flavin mononucleotide (FMN)-dependent mechanism. Based on comparative genomic analysis, a gene cluster with three genes (lldA, lldB, and lldC) encoding a novel type of L-iLDH was identified in Pseudomonas stutzeri A1501. When the gene cluster was expressed in Escherichia coli, distinctive L-iLDH activity was detected. The expressed L-iLDH was purified by ammonium sulfate precipitation, ion-exchange chromatography, and affinity chromatography. SDS-PAGE and successive matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the purified L-iLDH indicated that it is a complex of LldA, LldB, and LldC (encoded by lldA, lldB, and lldC, respectively). Purified L-iLDH (LldABC) is a dimer of three subunits (LldA, LldB, and LldC), and the ratio between LldA, LldB, and LldC is 1:1:1. Different from the FMN-containing L-iLDH, absorption spectra and elemental analysis suggested that LldABC might use the iron-sulfur cluster for the L-lactate oxidation. LldABC has narrow substrate specificity, and only L-lactate and DL-2-hydrobutyrate were rapidly oxidized. Mg(2+) could activate L-iLDH activity effectively (6.6-fold). Steady-state kinetics indicated a ping-pong mechanism of LldABC for the L-lactate oxidation. Based on the gene knockout results, LldABC was confirmed to be required for the L-lactate metabolism of P. stutzeri A1501. LldABC is the first purified and characterized L-iLDH with different subunits that uses the iron-sulfur cluster as the cofactor. IMPORTANCE: Providing new insights into the diversity of microbial lactate utilization could assist in the production of valuable chemicals and understanding microbial pathogenesis. An NAD-independent L-lactate dehydrogenase (L-iLDH) encoded by the gene cluster lldABC is indispensable for the L-lactate metabolism in Pseudomonas stutzeri A1501. This novel type of enzyme was purified and characterized in this study. Different from the well-characterized FMN-containing L-iLDH in other microbes, LldABC in P. stutzeri A1501 is a dimer of three subunits (LldA, LldB, and LldC) and uses the iron-sulfur cluster as a cofactor.

Comparative study of changes in energy metabolism in rat cardiomyocytes in postinfarction cardiosclerosis and diabetes mellitus.

Cardiomyocyte energy metabolism in experimental unfolding postinfarction cardiosclerosis and diabetes mellitus was studied. Postinfarction cardiosclerosis formed 6 weeks after coronary artery occlusion. Diabetes mellitus was induced by intraperitoneal injection of streptozotocin (60 mg/kg). The rate of oxygen consumption in postinfarction cardiosclerosis and diabetes increased by 3.4 and 4.2 times, respectively. Stimulation of mitochondrial respiration (ATP, palmitic acid) significantly increased oxygen consumption in animals with postinfarction cardiosclerosis and significantly reduced this process in diabetes. The content of LDH and SDH in the myocardium of animals with diabetes and postinfarction cardiosclerosis was significantly below the control. Hence, the development of postinfarction cardiosclerosis and diabetes mellitus were characterized by reduced generation of ATP in anaerobic and aerobic pathways and oxidative phosphorylation in cardiomyocytes.

Flavocytochrome b2-based enzymatic method of L-lactate assay in food products.

L-lactate, a key metabolite of the anaerobic glycolytic pathway, plays an important role as a biomarker in medicine, in the nutritional sector and food quality control. For these reasons, there is a need for very specific, sensitive, and simple analytical methods for the accurate L-lactate measuring. A new highly selective enzymatic method for L-lactate determination based on the use of flavocytochrome b 2 (EC 1.1.2.3; FC b 2) isolated from the recombinant strain of the yeast Hansenula polymorpha has been developed. A proposed enzymatic method exploits an enzymatic oxidation of L-lactate to pyruvate coupled with nitrotetrazolium blue (NTZB) reduction to a colored product, formazan. The maximal absorption peak of the colored product is near λ = 525 nm and the linear range is observed in the interval 0.005-0.14 mM of L-lactate. The main advantages of the proposed method when compared to the LDH-based routine approaches are a higher sensitivity (2.0 µM of L-lactate), simple procedure of analysis, usage of inexpensive, nontoxic reagents, and small amount of the enzyme. Enzymatic oxidation of L-lactate catalyzed by flavocytochrome b 2 and coupled with formazan production from nitrotetrazolium blue was shown to be used for L-lactate assay in food samples. A high correlation between results of the proposed method and reference ones proves the possibility to use flavocytochrome b 2-catalysed reaction for enzymatic measurement of L-lactate in biotechnology and food chemistry.

PI3K-Akt signaling activates mTOR-mediated epileptogenesis in organotypic hippocampal culture model of post-traumatic epilepsy.

mTOR is activated in epilepsy, but the mechanisms of mTOR activation in post-traumatic epileptogenesis are unknown. It is also not clear whether mTOR inhibition has an anti-epileptogenic, or merely anticonvulsive effect. The rat hippocampal organotypic culture model of post-traumatic epilepsy was used to study the effects of long-term (four weeks) inhibition of signaling pathways that interact with mTOR. Ictal activity was quantified by measurement of lactate production and electrical recordings, and cell death was quantified with lactate dehydrogenase (LDH) release measurements and Nissl-stained neuron counts. Lactate and LDH measurements were well correlated with electrographic activity and neuron counts, respectively. Inhibition of PI3K and Akt prevented activation of mTOR, and was as effective as inhibition of mTOR in reducing ictal activity and cell death. A dual inhibitor of PI3K and mTOR, NVP-BEZ235, was also effective. Inhibition of mTOR with rapamycin reduced axon sprouting. Late start of rapamycin treatment was effective in reducing epileptic activity and cell death, while early termination of rapamycin treatment did not result in increased epileptic activity or cell death. The conclusions of the study are as follows: (1) the organotypic hippocampal culture model of post-traumatic epilepsy comprises a rapid assay of anti-epileptogenic and neuroprotective activities and, in this model (2) mTOR activation depends on PI3K-Akt signaling, and (3) transient inhibition of mTOR has sustained effects on epilepsy.