Plasma metabolic profile in COPD patients: effects of exercise and endurance training

The study examines plasma metabolic profiles of patients with chronic obstructive pulmonary disease (COPD) to prove whether the disease influences metabolism at rest and after endurance training. This is based on the hypothesis that metabolome levels should reflect impaired skeletal muscle bioenergetics in COPD. The study aims to test this hypothesis by evaluating plasma metabolic profiles in COPD patients before and after 8?weeks of endurance exercise training. We studied blood samples from 18 COPD patients and 12 healthy subjects. Pre- and post-training blood plasma samples at rest and after constant-work rate exercise (CWRE) at 70% of pre-training Watts peak were analyzed by ¹H-nuclear magnetic resonance spectroscopy to assess metabolite profiles. The two groups presented training-induced physiological changes in the VO₂ peak and in blood lactate levels (P?<?0.01 each). Before training, the two groups also showed differences in metabolic profiles at rest (P?<?0.05). Levels of valine (r?=?0.51, P?<?0.01), alanine (r?=?0.45, P?<?0.05) and isoleucine (r?=?0.51, P?<?0.01) were positively associated with body composition (Fat Free Mass Index). While training showed a significant impact on the metabolic profile in healthy subjects (P?<?0.001), with changes in levels of amino acids, creatine, succinate, pyruvate, glucose and lactate (P?<?0.05 each), no equivalent training-induced effects were seen in COPD patients in whom only lactate decreased (P?<?0.05). This study shows that plasma metabolic profiling contributes to the phenotypic characterization of COPD patients.     

Physiological and Metabolic Effects of 5-Aminolevulinic Acid for Mitigating Salinity Stress in Creeping Bentgrass

The objectives of this study were to determine whether foliar application of a chlorophyll precursor, 5-aminolevulinic acid (ALA), could mitigate salinity stress damages in perennial grass species by regulating photosynthetic activities, ion content, antioxidant metabolism, or metabolite accumulation. A salinity-sensitive perennial grass species, creeping bentgrass (Agrostis stolonifera), was irrigated daily with 200 mM NaCl for 28 d, which were foliar sprayed with water or ALA (0.5 mg L⁻¹) weekly during the experiment in growth chamber. Foliar application of ALA was effective in mitigating physiological damage resulting from salinity stress, as manifested by increased turf quality, shoot growth rate, leaf relative water content, chlorophyll content, net photosynthetic rate, stomatal conductance and transpiration rate. Foliar application of ALA also alleviated membrane damages, as shown by lower membrane electrolyte leakage and lipid peroxidation, which was associated with increases in the activities of antioxidant enzymes. Leaf content of Na⁺ was reduced and the ratio of K⁺/Na⁺ was increased with ALA application under salinity stress. The positive effects of ALA for salinity tolerance were also associated with the accumulation of organic acids (α-ketoglutaric acid, succinic acid, and malic acid), amino acids (alanine, 5-oxoproline, aspartic acid, and γ -aminobutyric acid), and sugars (glucose, fructose, galactose, lyxose, allose, xylose, sucrose, and maltose). ALA-mitigation of physiological damages by salinity could be due to suppression of Na⁺ accumulation and enhanced physiological and metabolic activities related to photosynthesis, respiration, osmotic regulation, and antioxidant defense.     

Freeze and Thaw of CD4+CD25+Foxp3+ Regulatory T Cells Results in Loss of CD62L Expression and a Reduced Capacity to Protect against Graft-versus-Host Disease

The adoptive transfer of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) in murine models of allogeneic hematopoietic cell transplantation (HCT) has been shown to protect recipient mice from lethal acute graft-versus-host disease (GVHD) and this approach is being actively investigated in human clinical trials. Here, we examined the effects of cryopreservation on Tregs. We found that freeze and thaw of murine and human Tregs is associated with reduced expression of L-selectin (CD62L), which was previously established to be an important factor that contributes to the in vivo protective effects of Tregs. Frozen and thawed murine Tregs showed a reduced capacity to bind to the CD62L binding partner MADCAM1 in vitro as well as an impaired homing to secondary lymphoid organs in vivo. Upon adoptive transfer frozen and thawed Tregs failed to protect against lethal GVHD compared with fresh Tregs in a murine model of allogeneic HCT across major histocompatibility barriers. In summary, the direct administration of adoptively transferred frozen and thawed Tregs adversely affects their immunosuppressive potential which is an important factor to consider in the clinical implementation of Treg immunotherapies.     

Bile acid sequestration reduces plasma glucose levels in db/db mice by increasing its metabolic clearance rate

Aims/Hypothesis

Bile acid sequestrants (BAS) reduce plasma glucose levels in type II diabetics and in murine models of diabetes but the mechanism herein is unknown. We hypothesized that sequestrant-induced changes in hepatic glucose metabolism would underlie reduced plasma glucose levels. Therefore, in vivo glucose metabolism was assessed in db/db mice on and off BAS using tracer methodology.

Methods

Lean and diabetic db/db mice were treated with 2% (wt/wt in diet) Colesevelam HCl (BAS) for 2 weeks. Parameters of in vivo glucose metabolism were assessed by infusing [U-¹³C]-glucose, [2-¹³C]-glycerol, [1-²H]-galactose and paracetamol for 6 hours, followed by mass isotopologue distribution analysis, and related to metabolic parameters as well as gene expression patterns.

Results

Compared to lean mice, db/db mice displayed an almost 3-fold lower metabolic clearance rate of glucose (p = 0.0001), a ∼300% increased glucokinase flux (p = 0.001) and a ∼200% increased total hepatic glucose production rate (p = 0.0002). BAS treatment increased glucose metabolic clearance rate by ∼37% but had no effects on glucokinase flux nor total hepatic or endogenous glucose production. Strikingly, BAS-treated db/db mice displayed reduced long-chain acylcarnitine content in skeletal muscle (p = 0.0317) but not in liver (p = 0.189). Unexpectedly, BAS treatment increased hepatic FGF21 mRNA expression 2-fold in lean mice (p = 0.030) and 3-fold in db/db mice (p = 0.002).

Conclusions/Interpretation

BAS induced plasma glucose lowering in db/db mice by increasing metabolic clearance rate of glucose in peripheral tissues, which coincided with decreased skeletal muscle long-chain acylcarnitine content.     

Lack of the Lysosomal Membrane Protein,GLMP, in Mice Results in Metabolic Dysregulation in Liver

Ablation of glycosylated lysosomal membrane protein (GLMP, formerly known as NCU-G1) has been shown to cause chronic liver injury which progresses into liver fibrosis in mice. Both lysosomal dysfunction and chronic liver injury can cause metabolic dysregulation. Glmp^gt/gt mice (formerly known as Ncu-g1^gt/gtmice) were studied between 3 weeks and 9 months of age. Body weight gain and feed efficiency of Glmp^gt/gt mice were comparable to wild type siblings, only at the age of 9 months the Glmp^gt/gt siblings had significantly reduced body weight. Reduced size of epididymal fat pads was accompanied by hepatosplenomegaly in Glmp^gt/gt mice. Blood analysis revealed reduced levels of blood glucose, circulating triacylglycerol and non-esterified fatty acids in Glmp^gt/gt mice. Increased flux of glucose, increased de novo lipogenesis and lipid accumulation were detected in Glmp^gt/gt primary hepatocytes, as well as elevated triacylglycerol levels in Glmp^gt/gt liver homogenates, compared to hepatocytes and liver from wild type mice. Gene expression analysis showed an increased expression of genes involved in fatty acid uptake and lipogenesis in Glmp^gt/gt liver compared to wild type. Our findings are in agreement with the metabolic alterations observed in other mouse models lacking lysosomal proteins, and with alterations characteristic for advanced chronic liver injury.     

Macrophage polarization state affects lipid composition and the channeling of exogenous fatty acids into endogenous lipid pools

Adipose-tissue-resident macrophages (ATMs) maintain metabolic homeostasis but also contribute to obesity-induced adipose tissue inflammation and metabolic dysfunction. Central to these contrasting effects of ATMs on metabolic homeostasis is the interaction of macrophages with fatty acids. Fatty acid levels are increased within adipose tissue in various pathological and physiological conditions, but appear to initiate inflammatory responses only upon interaction with particular macrophage subsets within obese adipose tissue. The molecular basis underlying these divergent outcomes is likely due to phenotypic differences between ATM subsets, although how macrophage polarization state influences the metabolism of exogenous fatty acids is relatively unknown. Herein, using stable isotope-labeled and nonlabeled fatty acids in combination with mass spectrometry lipidomics, we show marked differences in the utilization of exogenous fatty acids within inflammatory macrophages (M1 macrophages) and macrophages involved in tissue homeostasis (M2 macrophages). Specifically, the accumulation of exogenous fatty acids within triacylglycerols and cholesterol esters is significantly higher in M1 macrophages, while there is an increased enrichment of exogenous fatty acids within glycerophospholipids, ether lipids, and sphingolipids in M2 macrophages. Finally, we show that functionally distinct ATM populations in vivo have distinct lipid compositions. Collectively, this study identifies new aspects of the metabolic reprogramming that occur in distinct macrophage polarization states. The channeling of exogenous fatty acids into particular lipid synthetic pathways may contribute to the sensitivity/resistance of macrophage subsets to the inflammatory effects of increased environmental fatty acid levels.     

Suppression of Murine Retrovirus Polypeptide Termination: Effect of Amber Suppressor tRNA on the Cell-Free Translation of Rauscher Murine Leukemia Virus, Moloney Murine Leukemia Virus, and Moloney Murine Sarcoma Virus 124 RNA

The effect of suppressor tRNA's on the cell-free translation of several leukemia and sarcoma virus RNAs was examined. Yeast amber suppressor tRNA (amber tRNA) enhanced the synthesis of the Rauscher murine leukemia virus and clone 1 Moloney murine leukemia virus Pr200^gag-pol polypeptides by 10- to 45-fold, but at the same time depressed the synthesis of Rauscher murine leukemia virus Pr65^gag and Moloney murine leukemia virus Pr63^gag. Under suppressor-minus conditions, Moloney murine leukemia virus Pr70^gag was present as a closely spaced doublet. Amber tRNA stimulated the synthesis of the “upper” Moloney murine leukemia virus Pr70^gag polypeptide. Yeast ochre suppressor tRNA appeared to be ineffective. Quantitative analyses of the kinetics of viral precursor polypeptide accumulation in the presence of amber tRNA showed that during linear protein synthesis, the increase in accumulated Moloney murine leukemia virus Pr200^gag-pol coincided closely with the molar loss of Pr63^gag. Enhancement of Pr200^gag-pol and Pr70^gag by amber tRNA persisted in the presence of pactamycin, a drug which blocks the initiation of protein synthesis, thus arguing for the addition of amino acids to the C terminus of Pr63^gag as the mechanism behind the amber tRNA effect. Moloney murine sarcoma virus 124 30S RNA was translated into four major polypeptides, Pr63^gag, P42, P38, and P23. In the presence of amber tRNA, a new polypeptide, Pr67^gag, appeared, whereas Pr63^gag synthesis was decreased. Quantitative estimates indicated that for every 1 mol of Pr67^gag which appeared, 1 mol of Pr63^gag was lost.     

Cross-species Binding Analyses of Mouse and Human Neonatal Fc Receptor Show Dramatic Differences in Immunoglobulin G and Albumin Binding

The neonatal Fc receptor (FcRn) regulates the serum half-life of both IgG and albumin through a pH-dependent mechanism that involves salvage from intracellular degradation. Therapeutics and diagnostics built on IgG, Fc, and albumin fusions are frequently evaluated in rodents regarding biodistribution and pharmacokinetics. Thus, it is important to address cross-species ligand reactivity with FcRn, because in vivo testing of such molecules is done in the presence of competing murine ligands, both in wild type (WT) and human FcRn (hFcRn) transgenic mice. Here, binding studies were performed in vitro using enzyme-linked immunosorbent assay and surface plasmon resonance with recombinant soluble forms of human (shFcRn^WT) and mouse (smFcRn^WT) receptors. No binding of albumin from either species was observed at physiological pH to either receptor. At acidic pH, a 100-fold difference in binding affinity was observed. Specifically, smFcRn^WT bound human serum albumin with a K_D of ∼90 μm, whereas shFcRn^WT bound mouse serum albumin with a K_D of 0.8 μm. shFcRn^WT ignored mouse IgG1, and smFcRn^WT bound strongly to human IgG1. The latter pair also interacted at physiological pH with calculated affinity in the micromolar range. In all cases, binding of albumin and IgG from either species to both receptors were additive. Cross-species albumin binding differences could partly be explained by non-conserved amino acids found within the α2-domain of the receptor. Such distinct cross-species FcRn binding differences must be taken into consideration when IgG- and albumin-based therapeutics and diagnostics are evaluated in rodents for their pharmacokinetics.     

Monoiodo-d-tyrosine,an artificial amino acid radiopharmaceutical for selective measurement of membrane amino acid transport in the pancreas

Using ¹¹C-labeled natural amino acids, the functional diagnosis of tissue metabolism has been actively studied. Our interest has been focused on developing a clinically available ¹²³I-labeled artificial amino acid with a single metabolic function. For this study, [¹²³I]3-iodo-d-tyrosine ([¹²³I]d-MIT) was selected. In vitro and in vivo studies using ¹²⁵I-labeled d-MIT indicated that it showed a high pancreatic accumulation, selective affinity for membrane active transport systems, and was stable against enzymatic deiodination. A canine scintigraphic study using ¹²³I-labeled d-MIT and kinetic analysis showed that it behaved as an “artificial amino acid” radiopharmaceutical with selective membrane amino acid transport affinity in the pancreas.     

Induction of SerpinB2 and Th1/Th2 Modulation by SerpinB2 during Lentiviral Infections In Vivo

SerpinB2, also known as plasminogen activator inhibitor type 2, is a major product of activated monocytes/macrophages and is often strongly induced during infection and inflammation; however, its physiological function remains somewhat elusive. Herein we show that SerpinB2 is induced in peripheral blood mononuclear cells following infection of pigtail macaques with CCR5-utilizing (macrophage-tropic) SIV_mac239, but not the rapidly pathogenic CXCR4-utilizing (T cell-tropic) SHIV_mn229. To investigate the role of SerpinB2 in lentiviral infections, SerpinB2^−/− mice were infected with EcoHIV, a chimeric HIV in which HIV gp120 has been replaced with gp80 from ecotropic murine leukemia virus. EcoHIV infected SerpinB2^−/− mice produced significantly lower anti-gag IgG1 antibody titres than infected SerpinB2^+/+ mice, and showed slightly delayed clearance of EcoHIV. Analyses of published microarray studies showed significantly higher levels of SerpinB2 mRNA in monocytes from HIV-1 infected patients when compared with uninfected controls, as well as a significant negative correlation between SerpinB2 and T-bet mRNA levels in peripheral blood mononuclear cells. These data illustrate that SerpinB2 can be induced by lentiviral infection in vivo and support the emerging notion that a physiological role of SerpinB2 is modulation of Th1/Th2 responses.     

Aminoacylation of Plasmodium falciparum tRNAAsn and Insights in the Synthesis of Asparagine Repeats

Genome sequencing revealed an extreme AT-rich genome and a profusion of asparagine repeats associated with low complexity regions (LCRs) in proteins of the malarial parasite Plasmodium falciparum. Despite their abundance, the function of these LCRs remains unclear. Because they occur in almost all families of plasmodial proteins, the occurrence of LCRs cannot be associated with any specific metabolic pathway; yet their accumulation must have given selective advantages to the parasite. Translation of these asparagine-rich LCRs demands extraordinarily high amounts of asparaginylated tRNA^Asn. However, unlike other organisms, Plasmodium codon bias is not correlated to tRNA gene copy number. Here, we studied tRNA^Asn accumulation as well as the catalytic capacities of the asparaginyl-tRNA synthetase of the parasite in vitro. We observed that asparaginylation in this parasite can be considered standard, which is expected to limit the availability of asparaginylated tRNA^Asn in the cell and, in turn, slow down the ribosomal translation rate when decoding asparagine repeats. This observation strengthens our earlier hypothesis considering that asparagine rich sequences act as “tRNA sponges” and help cotranslational folding of parasite proteins. However, it also raises many questions about the mechanistic aspects of the synthesis of asparagine repeats and about their implications in the global control of protein expression throughout Plasmodium life cycle.     

RanBP2 modulates Cox11 and hexokinase I activities and haploinsufficiency of RanBP2 causes deficits in glucose metabolism

The Ran-binding protein 2 (RanBP2) is a large multimodular and pleiotropic protein. Several molecular partners with distinct functions interacting specifically with selective modules of RanBP2 have been identified. Yet, the significance of these interactions with RanBP2 and the genetic and physiological role(s) of RanBP2 in a whole-animal model remain elusive. Here, we report the identification of two novel partners of RanBP2 and a novel physiological role of RanBP2 in a mouse model. RanBP2 associates in vitro and in vivo and colocalizes with the mitochondrial metallochaperone, Cox11, and the pacemaker of glycolysis, hexokinase type I (HKI) via its leucine-rich domain. The leucine-rich domain of RanBP2 also exhibits strong chaperone activity toward intermediate and mature folding species of Cox11 supporting a chaperone role of RanBP2 in the cytosol during Cox11 biogenesis. Cox11 partially colocalizes with HKI, thus supporting additional and distinct roles in cell function. Cox11 is a strong inhibitor of HKI, and RanBP2 suppresses the inhibitory activity of Cox11 over HKI. To probe the physiological role of RanBP2 and its role in HKI function, a mouse model harboring a genetically disrupted RanBP2 locus was generated. RanBP2^−/− are embryonically lethal, and haploinsufficiency of RanBP2 in an inbred strain causes a pronounced decrease of HKI and ATP levels selectively in the central nervous system. Inbred RanBP2^+/− mice also exhibit deficits in growth rates and glucose catabolism without impairment of glucose uptake and gluconeogenesis. These phenotypes are accompanied by a decrease in the electrophysiological responses of photosensory and postreceptoral neurons. Hence, RanBP2 and its partners emerge as critical modulators of neuronal HKI, glucose catabolism, energy homeostasis, and targets for metabolic, aging disorders and allied neuropathies.     

Surface localized and extracellular Glyceraldehyde-3-phosphate dehydrogenase of Bacillus anthracis is a plasminogen binding protein

The role of anchorless proteins on the surface of most pathogenic microorganisms has long been studied in context to their interactions with multiple host proteins, facilitating the dissemination of pathogen within the host tissues. In order to gain more insights into anthrax pathogenesis, we hereby report the presence of a prominent moonlighting enzyme, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on the surface and in the extracellular medium of Bacillus anthracis. Out of the three heterologously expressed recombinant isoforms, rGapA (334 amino acids in native form; GapA) showed a significant NAD⁺ mediated GAPDH activity, whereas rGapB (342 amino acids in native form; GapB) showed a slight activity with NADP⁺. The rGapN (479 amino acids in native form; GapN) was enzymatically inactive with either NAD⁺ or NADP⁺. GapA was ascertained to be present in the extracellular medium and on the surface of B. anthracis. On the other hand, GapN was absent from both the surface and extracellular medium, whereas GapB was scarcely present on the surface of B. anthracis. Human plasminogen predominantly interacted with the rGapA isoform at physiological concentrations and the interaction was found to be lysine dependent. Immunization with rGapA resulted in a significant protection upon challenge with Bacillus anthracis in the murine model.     

Aluminium induces changes in organic acids metabolism in Coffea arabica suspension cells with differential Al-tolerance

The primary Al-tolerance mechanism in plants involves exudation and/or accumulation of specific organic acid species, which form non-phytotoxic complexes with Al³⁺ under physiological conditions. An evaluation was done of the role of organic acids in the tolerance mechanism of a cell suspension line of coffee Coffea arabica that exhibits Al-tolerance (LAMt) but for which the metabolic tolerance mechanism remains unknown. Significant differences existed in malate dehydrogenase and citrate synthase activities (key enzymes in organic acids metabolism) between protein extracts (day 7 of culture cycle) of the L2 (Al-sensitive) and LAMt (Al-tolerant) cells when cell suspensions were treated with 100 μM AlCl₃. HPLC analysis showed that the suspension cells of both lines exudate malate when incubated in a minimal solution but that exudation was not enhanced by treatment with AlCl₃ (100 μM). This is the first study demonstrating that plant Al-tolerance may be associated with down-regulation of malate dehydrogenase and citrate synthase activities.     

Anthrax lethal toxin suppresses murine cardiomyocyte contractile function and intracellular Ca2+ handling via a NADPH oxidase-dependent mechanism

Objectives

Anthrax infection is associated with devastating cardiovascular sequelae, suggesting unfavorable cardiovascular effects of toxins originated from Bacillus anthracis namely lethal and edema toxins. This study was designed to examine the direct effect of lethal toxins on cardiomyocyte contractile and intracellular Ca²⁺ properties.

Methods

Murine cardiomyocyte contractile function and intracellular Ca²⁺ handling were evaluated including peak shortening (PS), maximal velocity of shortening/ relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), intracellular Ca²⁺ rise measured as fura-2 fluorescent intensity (ΔFFI), and intracellular Ca²⁺ decay rate. Stress signaling and Ca²⁺ regulatory proteins were assessed using Western blot analysis.

Results

In vitro exposure to a lethal toxin (0.05 – 50 nM) elicited a concentration-dependent depression on cardiomyocyte contractile and intracellular Ca²⁺ properties (PS, ± dL/dt, ΔFFI), along with prolonged duration of contraction and intracellular Ca²⁺ decay, the effects of which were nullified by the NADPH oxidase inhibitor apocynin. The lethal toxin significantly enhanced superoxide production and cell death, which were reversed by apocynin. In vivo lethal toxin exposure exerted similar time-dependent cardiomyocyte mechanical and intracellular Ca²⁺ responses. Stress signaling cascades including MEK1/2, p38, ERK and JNK were unaffected by in vitro lethal toxins whereas they were significantly altered by in vivo lethal toxins. Ca²⁺ regulatory proteins SERCA2a and phospholamban were also differentially regulated by in vitro and in vivo lethal toxins. Autophagy was drastically triggered although ER stress was minimally affected following lethal toxin exposure.

Conclusions

Our findings indicate that lethal toxins directly compromised murine cardiomyocyte contractile function and intracellular Ca²⁺ through a NADPH oxidase-dependent mechanism.     

Further observations on uptake of S35-labelled sulfate by renal tissue in vitro

Additional studies have been made of the accumulation of S³⁵ by renal cortical tissue incubated in media containing radiosulfate. This process was found to occur in several mammalian species in addition to the rat, but was not observed as a significant occurrence in three species of lower vertebrates. In the case of rat renal tissue, S³⁵ uptake was found to be sensitive to the pH and osmolar concentration of the medium. The character of the anions present in conjunction with K⁺ affected it as well. Various factors known to be related to in vitro accumulative processes, as well as to renal sulfate reabsorption by the intact dog, were tested on rat kidney cortex to assess the effect on radiosulfate uptake. In general, all substances tested (amino acids, metabolic intermediates, ATP, metabolic inhibitors, competitive inhibitors for PAH accumulation in vitro) were found to lessen S³⁵ uptake, or to be without effect upon it. The one striking exception was phlorhizin, which enhanced markedly S³⁵ uptake in vitro, as it does sulfate reabsorption in vivo. Some implications of these findings have been discussed.     

Correlation of Versican Expression,Accumulation, and Degradation during Embryonic Development by Quantitative Immunohistochemistry

Versican, a chondroitin sulfate proteoglycan, is important in embryonic development, and disruption of the versican gene is embryonically lethal in the mouse. Although several studies show that versican is increased in various organs during development, a focused quantitative study on versican expression and distribution during lung and central nervous system development in the mouse has not previously been performed. We tracked changes in versican (Vcan) gene expression and in the accumulation and degradation of versican. Vcan expression and quantitative immunohistochemistry performed from embryonic day (E) 11.5 to E15.5 showed peak Vcan expression at E13.5 in the lungs and brain. Quantitative mRNA analysis and versican immunohistochemistry showed differences in the expression of the versican isoforms in the embryonic lung and head. The expression of Vcan mRNA and accumulation of versican in tissues was complementary. Immunohistochemistry demonstrated co-localization of versican accumulation and degradation, suggesting distinct roles of versican deposition and degradation in embryogenesis. Very little versican mRNA or protein was found in the lungs of 12- to 16-week-old mice but versican accumulation was significantly increased in mice with Pseudomonas aeruginosa lung infection. These data suggest that versican plays an important role in fundamental, overlapping cellular processes in lung development and infection.     

Induction of a Stringent Metabolic Response in Intracellular Stages of Leishmania mexicana Leads to Increased Dependence on Mitochondrial Metabolism

Leishmania parasites alternate between extracellular promastigote stages in the insect vector and an obligate intracellular amastigote stage that proliferates within the phagolysosomal compartment of macrophages in the mammalian host. Most enzymes involved in Leishmania central carbon metabolism are constitutively expressed and stage-specific changes in energy metabolism remain poorly defined. Using ¹³C-stable isotope resolved metabolomics and ²H₂O labelling, we show that amastigote differentiation is associated with reduction in growth rate and induction of a distinct stringent metabolic state. This state is characterized by a global decrease in the uptake and utilization of glucose and amino acids, a reduced secretion of organic acids and increased fatty acid β-oxidation. Isotopomer analysis showed that catabolism of hexose and fatty acids provide C4 dicarboxylic acids (succinate/malate) and acetyl-CoA for the synthesis of glutamate via a compartmentalized mitochondrial tricarboxylic acid (TCA) cycle. In vitro cultivated and intracellular amastigotes are acutely sensitive to inhibitors of mitochondrial aconitase and glutamine synthetase, indicating that these anabolic pathways are essential for intracellular growth and virulence. Lesion-derived amastigotes exhibit a similar metabolism to in vitro differentiated amastigotes, indicating that this stringent response is coupled to differentiation signals rather than exogenous nutrient levels. Induction of a stringent metabolic response may facilitate amastigote survival in a nutrient-poor intracellular niche and underlie the increased dependence of this stage on hexose and mitochondrial metabolism.     

Salt Stress Control of Intracellular Solutes in Streptomycetes Indigenous to Saline Soils

Actinomycetes were isolated from a number of saline and saline-sodic California soils. From these isolates, two species of Streptomyces (S. griseus and S. californicus) were selected to assess their physiological response to salinity. NaCl was more inhibitory to growth rates and specific growth yields than were equivalent concentrations of KCl. Intracellular concentrations of the free amino acid pool increased in response to salt stress. Whereas the neutral free amino acids proline, glutamine, and alanine accumulated as salinity increased, concentrations of the acidic free amino acids glutamate and aspartate were reduced. Accumulation of free amino acids by streptomycetes under salt stress suggests a response typical of procaryotes, although the specific amino acids involved differ from those associated with other gram-positive bacteria. Above a salinity threshold of about 0.75 M (−3.8 MPa), there was little further intracellular accumulation of free amino acids, whereas accumulation of K⁺ salts sharply increased.     

Schistosoma co-infection protects against brain pathology but does not prevent severe disease and death in a murine model of cerebral malaria

Co-infections of helminths and malaria parasites are common in human populations in most endemic areas. It has been suggested that concomitant helminth infections inhibit the control of malaria parasitemia but down-modulate severe malarial disease. We tested this hypothesis using a murine co-infection model of schistosomiasis and cerebral malaria. C57BL/6 mice were infected with Schistosoma mansoni and 8-9 weeks later, when Schistosoma infection was patent, mice were co-infected with Plasmodium berghei ANKA strain. We found that a concomitant Schistosoma infection increased parasitemia at the beginning of the P. berghei infection. It did not protect against P. berghei-induced weight loss and hypothermia, and P. berghei-mono-infected as well as S. mansoni-P. berghei-co-infected animals showed a high case fatality between days 6 and 8 of malarial infection. However, co-infection significantly reduced P. berghei-induced brain pathology. Over 40% of the S. mansoni-P. berghei-co-infected animals that died during this period were completely protected against haemorrhaging, plugging of blood vessels and infiltration, indicating that mortality in these animals was not related to cerebral disease. Schistosoma mansoni-P. berghei-co-infected mice had elevated plasma concentrations of IL-5 and IL-13 and on day 6 lower levels of IFN-γ, IL-10, monocyte chemoattractant protein-1 (MCP-1) and monokine induced by IFN-γ (MIG) than P. berghei-mono-infected mice. We conclude that in P. berghei infections, disease and early death are caused by distinct pathogenic mechanisms, which develop in parallel and are differentially influenced by the immune response to S. mansoni. This might explain why, in co-infected mice, death could be induced in the absence of brain pathology.