Summer Meeting 2013: growth and physiology of bifidobacteria.

Bifidobacteria are a minor fraction of the human colon microbiota with interesting properties for carbohydrate degradation. Monosaccharides such as glucose and fructose are degraded through the bifid shunt, a dedicated pathway involving phosphoketolase activity. Its stoechiometry learns that three moles of acetate and two moles of lactate are produced per two moles of glucose or fructose that are degraded. However, deviations from this 3 : 2 ratio occur, depending on the rate of substrate consumption. Slower growth rates favour the production of acetate and pyruvate catabolites (such as formate) at the cost of lactate. Interestingly, bifidobacteria are capable to degrade inulin-type fructans (ITF) (oligofructose and inulin) and arabinoxylan-oligosaccharides (AXOS). Beta-fructofuranosidase activity enables bifidobacteria to degrade ITF. However, this property is strain-dependent. Some strains consume both fructose and oligofructose, with different preferences and degradation rates. Small oligosaccharides (degree of polymerization or DP of 2-7) are taken up, in a sequential order, indicating intracellular degradation and as such giving these bacteria a competitive advantage towards other inulin-type fructan degraders such as lactobacilli, bacteroides and roseburias. Other strains consume long fractions of oligofructose and inulin. Exceptionally, oligosaccharides with a DP of up to 20 (long-chain inulin) are consumed by specific strains. Also, the degradation of AXOS by α-arabinofuranosidase and ß-xylosidase is strain-dependent. Particular strains consume the arabinose substituents, whether or not together with a consumption of the xylose backbones of AXOS, either up to xylotetraose or higher and either extra- or intracellularly. The production of high amounts of acetate that accompanies inulin-type fructan degradation by bifidobacteria cross-feeds other colon bacteria involved in the production of butyrate. However, bifidobacterial strain-dependent differences in prebiotic degradation indicate the existence of niche-specific adaptations and hence mechanisms to avoid competition among each other and to favour coexistence with other colon bacteria.

Reduced activity of AMP-activated protein kinase protects against genetic models of motor neuron disease.

A growing body of research indicates that amyotrophic lateral sclerosis (ALS) patients and mouse models of ALS exhibit metabolic dysfunction. A subpopulation of ALS patients possesses higher levels of resting energy expenditure and lower fat-free mass compared to healthy controls. Similarly, two mutant copper zinc superoxide dismutase 1 (mSOD1) mouse models of familial ALS possess a hypermetabolic phenotype. The pathophysiological relevance of the bioenergetic defects observed in ALS remains largely elusive. AMP-activated protein kinase (AMPK) is a key sensor of cellular energy status and thus might be activated in various models of ALS. Here, we report that AMPK activity is increased in spinal cord cultures expressing mSOD1, as well as in spinal cord lysates from mSOD1 mice. Reducing AMPK activity either pharmacologically or genetically prevents mSOD1-induced motor neuron death in vitro. To investigate the role of AMPK in vivo, we used Caenorhabditis elegans models of motor neuron disease. C. elegans engineered to express human mSOD1 (G85R) in neurons develops locomotor dysfunction and severe fecundity defects when compared to transgenic worms expressing human wild-type SOD1. Genetic reduction of aak-2, the ortholog of the AMPK α2 catalytic subunit in nematodes, improved locomotor behavior and fecundity in G85R animals. Similar observations were made with nematodes engineered to express mutant tat-activating regulatory (TAR) DNA-binding protein of 43 kDa molecular weight. Altogether, these data suggest that bioenergetic abnormalities are likely to be pathophysiologically relevant to motor neuron disease.

Manipulating thyroid status alters endoplasmic reticulum calcium homeostasis in rat cerebellum.

Thyroid-related hormones regulate the efficiency and expression of sarco-endoplasmic reticulum calcium ATPases in cardiac and skeletal muscle. However, little is known about the relationship between thyroid hormones and calcium (Ca2+) homeostasis in the brain. It is hypothesized that manipulating rat thyroid hormone levels would induce significant brain Ca2+ adaptations consistent with clinical findings. Adult male Sprague-Dawley rats were assigned to one of three treatment groups for 28 days: control, hypothyroid (6-n-propyl-2-thiouracil (PTU), an inhibitor of thyroxine (T4) synthesis), and hyperthyroid (T4). Throughout, rats were given weekly behavioral tests. Ca2+ accumulation decreased in the cerebellum in both hyper- and hypothyroid animals. This was specific to different ER pools of calcium with regional heterogeneity in the response to thyroid hormone manipulation. Behavioral tasks demonstrated sensitivity to thyroid manipulation, and corresponded to alterations in calcium homeostasis. Ca2+ accumulation heterogeneity in chronic hyper- and hypothyroid animals potentially explains clinical manifestations of altered thyroid status.

Succinate dehydrogenase and fumarate hydratase: linking mitochondrial dysfunction and cancer.

The phenomenon of enhanced glycolysis in tumours has been acknowledged for decades, but biochemical evidence to explain it is only just beginning to emerge. A significant hint as to the triggers and advantages of enhanced glycolysis in tumours was supplied by the recent discovery that succinate dehydrogenase (SDH) and fumarate hydratase (FH) are tumour suppressors and which associated, for the first time, mitochondrial enzymes and their dysfunction with tumorigenesis. Further steps forward showed that the substrates of SDH and FH, succinate and fumarate, respectively, can mediate a 'metabolic signalling' pathway. Succinate or fumarate, which accumulate in mitochondria owing to the inactivation of SDH or FH, leak out to the cytosol, where they inhibit a family of prolyl hydroxylase enzymes (PHDs). Depending on the PHD inhibited, two newly recognized pathways that support tumour maintenance may ensue: affected cells become resistant to certain apoptotic signals and/or activate a pseudohypoxic response that enhances glycolysis and is conveyed by hypoxia-inducible factor.

Identification of 50 kDa snake venom proteins which specifically inhibit platelet adhesion to collagen.

Of 32 snake venoms tested, the crude venoms of four (B.atrox, B.jararaca, A.halys blomhoffi and C.basiliscus) showed strong inhibitory activity in an assay of platelet adhesion to collagen. Active 50 kDa proteins were purified to homogeneity from each venom and found to be rich in cysteine on amino acid analysis. A monoclonal antibody raised against the purified B. atrox protein crossreacted strongly with the 50 kDa proteins from B.jararaca and A.halys blomhoffi and weakly with the protein from C.basiliscus, indicating that all four proteins possess a similar epitope. The proteins inhibited platelet aggregation induced by collagen but not by other agonists.

ADP stimulates IP3 formation in human platelets.

Aspirinated human platelets labeled with 32PO4 showed a 1.7-fold increase in [32P]IP3 when stimulated with ADP. ADP-stimulated mobilization of internal Ca2+ and phosphorylation of myosin were enhanced in the presence of extracellular Ca2+ but the increase in IP3 was not significantly affected by external Ca2+. The Ca2+ ionophore, ionomycin, elevated internal Ca2+ and induced myosin phosphorylation without a detectable change in IP3. These results indicate that the mechanism of ADP stimulation of human platelets is similar to that of other platelet agonists and supports the theory that IP3 functions to liberate internal Ca2+.

Inositol trisphosphate does not release Ca2+ from permeabilized cardiac myocytes and sarcoplasmic reticulum.

The possibility that inositol 1,4,5-trisphosphate (IP3) may act as a Ca2+-mobilizing second messenger in cardiac muscle in a manner analogous to its actions in other cell types has been examined using saponin-permeabilized myocytes and isolated cardiac sarcoplasmic reticulum. Myocytes permeabilized in the presence of MgATP2- sequestered Ca2+ to a level of about 200 nM, similar to the cytosolic free Ca2+ concentration of intact cells, but addition of IP3 was ineffective in causing Ca2+ release from intracellular stores. Similarly, IP3 (up to 50 microM) was unable to inhibit Ca2+ uptake or cause Ca2+ release from isolated canine cardiac sarcoplasmic reticulum vesicles in the presence of either EGTA or sodium vanadate. These results indicate that IP3 is unlikely to mediate mobilization of intracellular Ca2+ stores in myocardial cells.

Neutrophil elastase potentiates cathepsin G-induced platelet activation.

We have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10-60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidative damage to mitochondrial DNA and activity of mitochondrial enzymes in chronic active lesions of multiple sclerosis.

Soluble products of activated immune cells include reactive oxygen species (ROS) and nitric oxide (NO) with a high potential to induce biochemical modifications and degenerative changes in areas of inflammation in the central nervous system (CNS). Previously, we demonstrated an increased production of ROS by activated mononuclear cells (MNC) of patients with multiple sclerosis (MS) compared to those of controls, and development of oxidative damage to total DNA in association with inflammation in chronic active plaques. The current study aimed to determine whether mitochondrial (mt)DNA is affected by oxidative damage, and whether oxidative damage to mitochondrial macromolecules (including mtDNA) is associated with a decline in the activity of mitochondrial enzyme complexes. Using molecular and biochemical methods we demonstrate a trend for impaired NADH dehydrogenase (DH) activity and a possible compensatory increase in complex IV activity in association with oxidative damage to mtDNA in chronic active plaques. Immunohistochemistry confirms the increase of oxidative damage to DNA predominantly located in the cytoplasmic compartment of cells in chronic active plaques. These observations suggest that oxidative damage to macromolecules develops in association with inflammation in the CNS, and may contribute to a decline of energy metabolism in affected cells. As observed in neurodegenerative diseases of non-inflammatory origin, decreased ATP synthesis can ultimately lead to cell death or degeneration. Therefore, elucidation of this pathway in MS deserves further studies which may identify neuroprotective strategies to prevent tissue degeneration and the associated clinical disability.

Mitochondrial activity in Pompe's disease.

Mitochondrial oxidative metabolism was examined in two infants with Pompe's disease. The clinical diagnosis was confirmed by the demonstration of intralysosomal glycogen accumulation and a deficiency of acid alpha-D-glucosidase in muscle biopsies. Light and electron microscopy studies demonstrated a normal number of mitochondria with normal ultrastructure. Spectrophotometric measurements revealed that the specific activities of citrate synthase and the partial reactions of electron transport were markedly elevated in the skeletal muscle homogenates prepared from both infants with Pompe's disease when calculated as micromoles per minute per gram wet weight of tissue. However, when respiratory chain enzyme activities were expressed relative to citrate synthase as a marker mitochondrial enzyme, a different pattern emerged, in which all Pompe muscle respiratory enzymes, except complex IV, were decreased relative to control subjects. These observations demonstrate that caution should be exercised when analyzing and interpreting data obtained from tissue homogenates in general and, in particular, in those prepared from tissues in which the wet weight of tissue may be altered, for example, by pathologic accumulation of carbohydrate or lipid.

Distribution of oligodendrocyte loss and mitochondrial toxicity in the cuprizone-induced experimental demyelination model.

Cuprizone is a copper-chelating mitochondrial toxin that causes oligodendrocyte apoptosis and demyelination preferentially in the corpus callosum (CC) and the superior cerebellar peduncles, but not in the spinal cord (SC) of C57BL/6 mice. Here we aimed to determine the activities of copper-containing enzymes in correlation with the distribution of demyelination during exposure to cuprizone. The study revealed mitochondrial complex IV and superoxide dismutase activity alterations in both the pathology-affected CC and the non-affected SC. This observation raises the possibility that regionally different subcellular molecular interactions lead to the selective oligodendrocyte loss induced by the nonselective mitochondrial toxin, cuprizone.

Mitochondrial energetic and AKT status mediate metabolic effects and apoptosis of metformin in human leukemic cells.

Previous reports demonstrate that metformin, an anti-diabetic drug, can decrease the risk of cancer and inhibit cancer cell growth. However, its mechanism in cancer cells is still unknown. Metformin significantly blocks cell cycle and inhibits cell proliferation and colony formation of leukemic cells. However, the apoptotic response to metformin varies. Furthermore, daily treatment with metformin induces apoptosis and reduces tumor growth in vivo. While metformin induces early and transient activation of AMPK, inhibition of AMPKα1/2 does not abrogate anti-proliferative or pro-apoptotic effects of metformin. Metformin decreases electron transport chain complex I activity, oxygen consumption and mitochondrial ATP synthesis, while stimulating glycolysis for ATP and lactate production, pentose phosphate pathway for purine biosynthesis, fatty acid metabolism, as well as anaplerotic and mitochondrial gene expression. Importantly, leukemic cells with high basal AKT phosphorylation, glucose consumption or glycolysis exhibit a markedly reduced induction of the Pasteur effect in response to metformin and are resistant to metformin-induced apoptosis. Accordingly, glucose starvation or treatment with deoxyglucose or an AKT inhibitor induces sensitivity to metformin. Overall, metformin elicits reprogramming of intermediary metabolism leading to inhibition of cell proliferation in all leukemic cells and apoptosis only in leukemic cells responding to metformin with AKT phosphorylation and a strong Pasteur effect.

Reactivating HIF prolyl hydroxylases under hypoxia results in metabolic catastrophe and cell death.

Cells exposed to low-oxygen conditions (hypoxia) alter their metabolism to survive. This response, although vital during development and high-altitude survival, is now known to be a major factor in the selection of cells with a transformed metabolic phenotype during tumorigenesis. It is thought that hypoxia-selected cells have increased invasive capacity and resistance to both chemo- and radiotherapies, and therefore represent an attractive target for antitumor therapy. Hypoxia inducible factors (HIFs) are responsible for the majority of gene expression changes under hypoxia, and are themselves controlled by the oxygen-sensing HIF prolyl hydroxylases (PHDs). It was previously shown that mutations in succinate dehydrogenase lead to the inactivation PHDs under normoxic conditions, which can be overcome by treatment with alpha-ketoglutarate derivatives. Given that solid tumors contain large regions of hypoxia, the reactivation of PHDs in these conditions could induce metabolic catastrophe and therefore prove an effective antitumor therapy. In this report we demonstrate that derivatized alpha-ketoglutarate can be used as a strategy for maintaining PHD activity under hypoxia. By increasing intracellular alpha-ketoglutarate and activating PHDs we trigger PHD-dependent reversal of HIF1 activation, and PHD-dependent hypoxic cell death. We also show that derivatized alpha-ketoglutarate can permeate multiple layers of cells, reducing HIF1alpha levels and its target genes in vivo.

Cathepsin g activates platelets in the presence of plasma and stimulates phosphatidic Acid formation and lysosomal enzyme release.

We have previously demonstrated that cathepsin G is a strong platelet agonist. However, the ability of cathepsin G to function in this capacity in vivo has remained speculative because the enzyme might be expected to be rapidly neutralized by the high concentration of circulating plasma antiproteases. To examine the physiological significance of cathepsin G as a paracrine mediator, indo-1 and (14)C-5-hydroxytryptamine-loaded platelets were incubated with autologous unloaded neutrophils specifically activated by addition of fMet-Leu-Phe. FMet-Leu-Phe induced substantial increases in cytosolic calcium and 5-hydroxytryptamine release even in the presence of increasing amounts of citrated plasma, indicating that cathepsin G can stimulate platelets under conditions similar to those that may be encountered in vivo. Platelet stimulation was abolished by addition of α1-antichymotrypsin, demonstrating that cathepsin G was the neutrophil mediator responsible for cell activation. Having obtained evidence that cathepsin G can function in the presence of plasma, we measured its ability to hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdIns4,5-P2) and generate phosphatidic acid (PtdA) in aspirin-treated platelets. Our previous observations suggested that cathepsin G stimulates phospholipase C since the protease induces an elevation in [Ca(2+)]i in the presence of exogenous EGTA. Within 10 s of stimulation cathepsin G induced a transient loss in [(32)P]-PtdIns4,5-P2 and a concurrent increase in [(32)P]-PtdA. [(32)P]-PtdA formation was increased over 15-fold in a concentration-dependent manner by cathepsin G. We also determined that cathepsin G induces the release of the lysosomal enzyme ß-N-acetyl-glucosaminidase. Both the increase in PtdA and the release of ß-hexoseaminidase were comparable to responses elicited by thrombin. These results provide additional evidence that cathepsin G is a strong platelet agonist, support the conclusion that cathepsin G stimulates phospholipase C, and clearly suggest that cathepsin G can function as an agonist in vivo.

Neutrophil-platelet interactions in inflammation.

Inflammation is a multicomponent system that involves a network of cellular crosstalk and control. Many different cell types, including neutrophils and platelets, participate as both sources and targets of biological mediators that are generated or released in acute and chronic inflammatory states. Owing to the complex nature of inflammation, the magnitude as well as the spatial and temporal characteristics of the responses are likely to vary with the type, concentration, and duration of the inflammatory stimulus. Despite the potential variations in responses to diverse stimuli, a feature common to and responsible for the major characteristics of inflammation (heat, pain, redness, swelling) is proteases. In the early stages of inflammation, the neutrophil is the predominant cell to infiltrate the tissue, and the extent of inflammatory injury has been shown to be directly dependent on the extent of neutrophil infiltration. Since both cathepsin G and elastase are neutral serine proteases present in large amounts in azurophilic granules and are known to affect platelet function, it is thus likely that these neutrophil enzymes are important contributing factors to inflammatory reactions in general and to neutrophil-platelet interactions specifically.

Cathepsin G and thrombin: evidence for two different platelet receptors.

Neutrophil cathepsin G and thrombin, the only platelet agonists that are proteases, exhibit a mandatory requirement for catalytic activity to induce platelet aggregation and signal transduction. The thrombin receptor is a G-protein-coupled receptor which undergoes proteolysis to generate a tethered ligand that causes self-activation. Since cathepsin G strongly resembles thrombin in its ability to activate platelets, we have attempted to determine whether cathepsin G and thrombin function through the same or different receptors. Evidence that thrombin and cathepsin G act at different receptors was as follows: (a) an antibody directed against the thrombin receptor blocked thrombin-induced but not cathepsin G-induced platelet responses; (b) human fibroblasts responded to thrombin and to a synthetic thrombin receptor peptide (comprising residues 42-55 of the thrombin receptor) by exhibiting an elevation in cytosolic Ca2+ concentration but did not respond to cathepsin G; and (c) platelets pretreated with neutrophil elastase failed to respond to thrombin but responded when rechallenged by cathepsin G. Thrombin and cathepsin G exhibit heterologous desensitization that is potentiated by okadaic acid and is attenuated by staurosporine, indicating that phosphorylation of serine/threonine residues is important for desensitization and that protein kinase C may be involved. Since catalytic activity of cathepsin G is required for platelet stimulation, it is probable that platelet activation by cathepsin G requires receptor proteolysis and that a tethered ligand mechanism is involved, suggesting that platelets may possess a family of protease receptors.

Respiration capacity of mitochondria isolated from unfertilized and fertilized sea urchin eggs.

Mitochondria were isolated from unfertilized and fertilized eggs of the sea urchin, Strongylocentrotus purpuratus. Both preparations exhibited coupled adenosine 5'-diphosphate (ADP)-dependent) oxidation of flavin and pyridine-linked substrates and both yielded the expected P:O ratios with these substrates. Highest respiratory control indices (greater than 4.0) were observed when succinate or pyruvate + malate were used as substrates. Mitochondria from unfertilized and fertilized eggs exhibited sensitivity to respiratory and phosphorylation inhibitors and uncouplers and both preparations exhibited cross-over points at sites I, II and III of the respiratory chain. Low-temperature difference spectra revealed a normal complement of cytochromes c, b and aa3, although cytochrome c from unfertilized eggs appears to be more subject to extraction during the course of mitochondrial isolation than does cytochrome c from fertilized eggs. An unidentified pigment absorbing at approx. 570 nm was visible in low-temperature spectra of unfertilized eggs and unfertilized egg mitochondria.

Cathepsin G binding to human platelets. Evidence for a specific receptor.

We have shown previously that purified human neutrophil cathepsin G is a strong platelet agonist. We now demonstrate that cathepsin G exhibits saturable, reversible binding to human platelets which is characteristic of binding to a specific receptor. At room temperature, cathepsin G displayed apparent positive co-operativity of binding, as indicated by sigmoidal binding curves and a Hill coefficient greater than unity. By contrast, binding curves conducted with native enzyme at 0.5 degrees C displayed a much smaller degree of sigmoidicity, and binding studies performed with phenylmethanesulphonyl fluoride-treated enzyme at 22-25 degrees C exhibited hyperbolic binding curves. The concentrations of cathepsin G required to give half-saturation (S0.5) with inhibitor-treated enzyme or with native enzyme at either room temperature or 0.5 degrees C were all similar, suggesting that sigmoidal binding curves did not result from an alteration in the affinity of the binding sites for cathepsin G. However, platelets bound approximately twice as many molecules of native enzyme as molecules of phenylmethanesulphonyl fluoride-treated cathepsin G per cell. From these observations it can be inferred that the apparent positive co-operativity may in part reflect the exposure of binding sites due to the proteolytic activity of cathepsin G. However, this conclusion is not supported by experiments conducted with subsaturating cathepsin G concentrations, which demonstrated that ligand binding did not show an expected increase at longer time intervals. Measurement of Ca2+ mobilization and cathepsin G binding in the same platelet suspensions demonstrated that elevations in cytosolic free Ca2+ concentration had achieved near-maximal levels in the presence of 15 micrograms of cathepsin G/ml, whereas maximal binding was observed at approx. 35 micrograms/ml, indicating that only a fraction of the total binding sites need be occupied to elicit platelet activation. Pretreatment of platelets with forskolin or phorbol 12-myristate 13-acetate (PMA) decreased cathepsin G binding by approx. 60% and 40% respectively, indicating that the receptor may be desensitized or down-regulated by phosphorylation due to protein kinases. Since forskolin and PMA could diminish receptor availability by activating negative feedback mechanisms, inhibition of negative signal-transduction pathways could conversely play a role in the up-regulation of cathepsin G binding. In any event, these results show that cathepsin G is an agonist that must bind to platelets to initiate processes associated with cell activation, and suggest a role for cathepsin G in platelet function.