Cytokine network analysis of cerebrospinal fluid in myalgic encephalomyelitis/chronic fatigue syndrome.

Myalgic encephalomyelitis/chronic fatigue syndrome is an unexplained debilitating disorder that is frequently associated with cognitive and motor dysfunction. We analyzed cerebrospinal fluid from 32 cases, 40 subjects with multiple sclerosis and 19 normal subjects frequency-matched for age and sex using a 51-plex cytokine assay. Group-specific differences were found for the majority of analytes with an increase in cases of CCL11 (eotaxin), a chemokine involved in eosinophil recruitment. Network analysis revealed an inverse relationship between interleukin 1 receptor antagonist and colony-stimulating factor 1, colony-stimulating factor 2 and interleukin 17F, without effects on interleukin 1α or interleukin 1ß, suggesting a disturbance in interleukin 1 signaling. Our results indicate a markedly disturbed immune signature in the cerebrospinal fluid of cases that is consistent with immune activation in the central nervous system, and a shift toward an allergic or T helper type-2 pattern associated with autoimmunity.

Cytokines in the cerebrospinal fluids of patients with chronic fatigue syndrome/myalgic encephalomyelitis.

OBJECTIVES: Previous research has provided evidence for dysregulation in peripheral cytokines in patients with Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME). To date only one study has examined cytokines in cerebrospinal fluid (CSF) samples of CFS/ME patients. The purpose of this pilot study was to examine the role of cytokines in CSF of CFS/ME patients. METHODS: CSF was collected from 18 CFS/ME patients and 5 healthy controls. The CSF samples were examined for the expression of 27 cytokines (interleukin- (IL-) 1ß, IL-1ra, IL-2, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17, basic FGF, eotaxin, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1 (MCAF), MIP-1α, MIP-1ß, PDGF-BB, RANTES, TNF-α, and VEGF) using the Bio-Plex Human Cytokine 27-plex Assay. RESULTS: Of the 27 cytokines examined, only IL-10 was significantly reduced in the CFS/ME patients in comparison to the controls. CONCLUSIONS: This preliminary investigation suggests that perturbations in inflammatory cytokines in the CSF of CFS/ME patients may contribute to the neurological discrepancies observed in CFS/ME.

Reduced coenzyme F420: heterodisulfide oxidoreductase, a proton- translocating redox system in methanogenic bacteria.

Washed everted vesicles of the methanogenic bacterium strain Go1 were found to couple the F420H2-dependent heterodisulfide reduction with the transfer of protons across the membrane into the lumen of the everted vesicles. The transmembrane electrochemical potential of protons thereby generated was shown to be competent in driving ATP synthesis from ADP + Pi, exhibiting a stoichiometry of 2 H+ translocated or 0.4 ATP synthesized per F420H2 oxidized. This enzyme system exhibits the phenomenon of coupling and uncoupling and represents a different kind of electron transport chain with the heterodisulfide of 2-mercaptoethanesulfonate and 7-mercaptoheptanoylthreonine phosphate as terminal electron acceptor. The heterodisulfide and methane are formed in the methyl coenzyme M reductase reaction. The reducing equivalents are derived from reduced coenzyme F420, which represents an analogue of NADH + H+ in other respiratory chains. It is assumed that the proton-translocating oxidoreductase discovered in strain Go1 is of principal importance to all methanogenic bacteria not utilizing H2.

Biochemistry of coenzyme B12-dependent glycerol and diol dehydratases and organization of the encoding genes.

Glycerol and diol dehydratases exhibit a subunit composition of alpha 2 beta 2 gamma 2 and contain coenzyme B12 in the base-on form. The dehydratase reaction proceeds via a radical mechanism. The dehydratases are subject to reaction inactivation by the substrate glycerol which is caused by a cessation of the catalytic cycle because coenzyme B12 is not regenerated, instead 5'-deoxyadenosine and a catalytically inactive cobalamin are formed. The genetic organization of the dehydratase genes is quite similar in all organisms. Downstream of the dehydratase genes an open reading frame encoding a polypeptide of approximately 600 amino acids was identified which is apparently involved in the reactivation of suicide-inactivated enzyme.

The Na(+)-translocating methyltransferase complex from methanogenic archaea.

Methanogenic archaea are dependent on sodium ions for methane formation. A sodium ion-dependent step has been shown to be methyl transfer from N(5)-methyltetrahydromethanopterin to coenzyme M. This exergonic reaction (DeltaG degrees '=-30 kJ/mol) is catalyzed by a Na(+)-translocating membrane-associated multienzyme complex composed of eight different subunits, MtrA-H. Subunit MtrA harbors a cob(I)amide prosthetic group which is methylated and demethylated in the catalytic cycle, demethylation being sodium ion-dependent. Based on the finding that in the cob(II)amide oxidation state the corrinoid is bound in a base-off/His-on configuration it is proposed that methyl transfer from MtrA to coenzyme M is associated with a conformational change of the protein and that this change drives the electrogenic translocation of the sodium ions.

Cloning, sequencing and expression of the genes encoding the sodium translocating N5-methyltetrahydromethanopterin : coenzyme M methyltransferase of the methylotrophic archaeon Methanosarcina mazei Gö1.

The N5-methyltetrahydromethanopterin:coenzyme M methyltransferase of Methanosarcina mazei Gö1 is a membrane-associated, corrinoid-containing protein that uses a transmethylation reaction to drive an energy-conserving sodium ion pump. The eight open reading frames encoding the eight different subunits of the methyltransferase were identified and sequenced. All of these subunits are shown to be heterologously expressed in minicells of the Escherichia coli mutant DK6. Sequence comparisons with the methyltransferases of thermophilic and hypothermophilic methanogenic archaea are presented. The participation of the gene product of mtrD in sodium ion translocation as well as a consensus sequence of a corrinoid binding motif in MtrA are discussed.

Novel reactions involved in energy conservation by methanogenic archaea.

Methanogenic archaea of the order Methanosarcinales which utilize C(1) compounds such as methanol, methylamines or H(2)+CO(2), employ two novel membrane-bound electron transport systems generating an electrochemical proton gradient: the H(2):heterodisulfide oxidoreductase and the F(420)H(2):heterodisulfide oxidoreductase. The systems are composed of the heterodisulfide reductase and either a membrane-bound hydrogenase or a F(420)H(2) dehydrogenase which is functionally homologous to the proton-translocating NADH dehydrogenase. Cytochromes and the novel electron carrier methanophenazine are also involved. In addition, the methyl-H(4)MPT:HS-CoM methyltransferase is bioenergetically relevant. The enzyme couples methyl group transfer with the translocation of sodium ions and seems to be present in all methanogens. The proton-translocating systems with the participation of cytochromes and methanophenazine have been found so far only in the Methanosarcinales.

A methyl-CoM methylreductase system from methanogenic bacterium strain Gö 1 not requiring ATP for activity.

Crude inside-out vesicles from the methanogenic strain Gö1 were prepared via protoplasts. These vesicles catalyzed methane formation from methyl-CoM and H2 at a maximal rate of 35 nmol/min.mg protein. Methane formation by the vesicles did not depend on the addition of ATP. This was in contrast to conventionally prepared crude extracts from the same organism or from Methanosarcina barkeri which exhibited strict ATP dependence of methanogenesis. ATP analogues inhibited methanogenesis by extracts to a much higher extent than that by vesicles. Both, particulate and soluble components prepared from the crude vesicles by ultracentrifugation were necessary for ATP-independent methane formation from methyl-CoM and H2. Hydrogenase activity was mainly associated with the particulate fraction whereas methyl-CoM methylreductase could be assigned to the soluble fraction. The detergent sulfobetaine inhibited methane formation from methyl-CoM without affecting hydrogenase or titanium citrate-dependent methylreductase activities, indicating that an additional membraneous component is involved in methanogenesis for methyl-CoM and H2.

A sodium-stimulated ATP synthase in the acetogenic bacterium Acetobacterium woodii.

Experiments with resting cells of Acetobacterium woodii were performed to elucidate the coupling ion used by the ATP synthase. A. woodii synthesized ATP in response to an artificial delta pH, indicating the presence of a proton-translocating ATPase. On the other hand, a delta pNa, as well as a proton diffusion potential, could serve as a driving force for ATP synthesis with the latter strictly dependent on Na+. These results are indicative for the presence of a Na(+)-translocating ATP synthase in A. woodii.

The F420H2:heterodisulfide oxidoreductase system from Methanosarcina species. 2-Hydroxyphenazine mediates electron transfer from F420H2 dehydrogenase to heterodisulfide reductase.

F420H2-dependent CoB-S-S-CoM reduction as catalyzed by the F420H2:heterodisulfide oxidoreductase from Methanosarcina strains was observed in a defined system containing purified F420H2 dehydrogenase from Methanosarcina mazei Gö1, 2-hydroxyphenazine and purified heterodisulfide reductase from Methanosarcina thermophila. The process could be divided into two partial reactions: (1) reducing equivalents from F420H2 were transferred to 2-hydroxyphenazine by the F420H2 dehydrogenase with a Vmax value of 12 U/mg protein; (2) reduced 2-hydroxyphenazine acted as electron donor for CoB-S-S-CoM reduction as catalyzed by the heterodisulfide reductase. The specific activity was 14-16 U/mg protein at 37 degrees C and 60-70 U/mg protein at 60 degrees C. The partial reactions could be combined in the presence of both enzymes. Under these conditions reduced 2-hydroxyphenazine was rapidly oxidized by the heterodisulfide reductase thereby producing the electron acceptor for the F420H2 dehydrogenase. Above a concentration of 50 microM of 2-hydroxyphenazine, the specific activity of the latter enzyme reached the Vmax value. When other phenazines or quinone derivatives were used as electron carriers, the activity of F420H2-dependent CoB-S-S-CoM reduction was much lower than the rate obtained with 2-hydroxyphenazine. Thus, this water-soluble analogue of methanophenazine best mimics the natural electron acceptor methanophenazine in aqueous systems.

Phosphorylation of citrate lyase ligase in Clostridium sphenoides and regulation of anaerobic citrate metabolism in other bacteria.

Since anaerobic bacteria cannot take advantage of citrate oxidation through the reactions of the tricarboxylic acid cycle special enzymes are needed for its fermentation. The activity of citrate lyase (the key enzyme of the citrate fermentation pathway) is in most cases strictly controlled by acetylation/deacetylation and configurational changes. In order to efficiently regulate citrate metabolism the activity of various regulatory enzymes, that modulate citrate lyase activity, are in turn under stringent control. Covalent modification by phosphorylation/dephosphorylation and electron transport dependent processes are some of the regulatory mechanisms that are here involved. L-Glutamate, which signals the availability of citrate, plays a central role in the regulation of citrate metabolism by influencing the enzymes that are acting in a complex cascade system.

Growth temperature-dependent activity of glycerol dehydratase in Escherichia coli expressing the Citrobacter freundii dha regulon.

Using the cosmid pWE15, a genomic library of Citrobacter freundii DNA in Escherichia coli ECL707 was prepared and screened for glycerol utilization. Six out of approximately 3000 clones were positive. One clone, harboring the recombinant cosmid pRD1, expressed glycerol dehydratase in high activity when grown at 28 degrees C but not at 37 degrees C. The growth temperature had little effect on the activity of the other enzymes encoded by the dha regulon. When the glycerol-containing medium was supplemented with corrinoids, the recombinant E. coli strain produced 1,3-propanediol in high amounts at 28 degrees C.

Transfer and expression of the tetracycline resistance transposon Tn925 in Acetobacterium woodii.

The Enterococcus faecalis conjugative plasmid pCF10 was used to introduce Tn925 into Acetobacterium woodii by filter mating. Tetracycline resistance was transferred at frequencies of about 10(-6) per donor, but no plasmid DNA was found in the transconjugants. DNA hybridization analyses of HindIII-digested chromosomal DNA demonstrated the insertion of Tn925 at a variety of locations, whereas wild type DNA showed no hybridization at all. The transconjugants were used as donor in mating experiments with tetracycline-sensitive Bacillus subtilis. Transfer of tetracycline resistance was observed at frequencies of 10(-8) per recipient.

Properties and sequence of the coenzyme B12-dependent glycerol dehydratase of Clostridium pasteurianum.

The genes encoding coenzyme B12-dependent glycerol dehydratase of Clostridium pasteurianum were subcloned and expressed in Escherichia coli. The native molecular mass of the enzyme is 190,000 Da. The enzyme converts glycerol, 1,2-propanediol and 1,2-ethanediol to 3-hydroxypropionaldehyde, propionaldehyde and acetaldehyde, respectively, but glycerol is the preferred substrate. The nucleotide sequences of the dhaBCE genes encoding the three subunits of glycerol dehydratase and of orfZ whose function is unknown were determined. The deduced products of the dhaBCE genes with calculated molecular masses of 60,813, 19,549 and 16,722 Da, respectively, revealed high similarity to amino acid sequences of subunits of coenzyme B12-dependent glycerol and diol dehydratases from other organisms.

Biosynthesis of poly(4-hydroxybutyric acid) by recombinant strains of Escherichia coli.

The aim of this study was the production of the homopolyester poly(4-hydroxybutyric acid) (poly(4HB)) with recombinant strains of Escherichia coli. Wild-type strains and other widely used non-recombinant strains of E. coli are not able to produce polyhydroxyalkanoic acids (PHA) as storage compounds and cannot utilize 4-hydroxybutyric acid as sole carbon source. Accordingly, hybrid plasmids of pBluescript vectors were constructed which harbored the Alcaligenes eutrophus PHA synthase gene (phaC) and the Clostridium kluyveri orfZ putatively encoding a 4-hydroxybutyric acid-coenzyme A transferase. A 3.5-kb genomic SmaI/ApaI fragment from A. eutrophus, which comprises phaC, and a 1.8-kb genomic ApaI/EcoRI fragment from C kluyveri, which contained orfZ, were inserted into the SmaI and EcoRI sites of the vectors pKS- and pSK-, respectively. The two resulting plasmids pSKSE5.3 and pKSSE5.3 comprising phaC and orfZ colinear or antilinear to lacZ, respectively, were transformed into E. coli XL1-Blue. Recombinant strains synthesized the homopolyester poly(4HB), when the cells were cultivated in Luria-Bertani broth and if glucose and 4-hydroxybutyric acid were provided as carbon sources. If glucose was omitted, a copolyester of 3-hydroxybutyric acid and 4-hydroxybutyric acid was accumulated. The homopolyester poly(4HB) was also accumulated during cultivation of these strains in M9 mineral salts medium containing glucose plus 4-hydroxybutyric acid as carbon sources. Poly(4HB) could amount up to approximately 80% (w/w) of the cell dry matter if E. coli XL1-Blue harboring pKSSE5.3 was cultivated in M9 mineral salts medium and if the cultures were not sufficiently supplied with oxygen. 4HB was also incorporated into PHA if gamma-butyrolactone was used as carbon source. If levulinic acid, 4-hydroxyvaleric acid or gamma-valerolactone were used as carbon sources, only very low amounts of PHA were accumulated which did not contain 4-hydroxyalkanoic acids as constituents.

Glycerol conversion to 1,3-propanediol by Clostridium pasteurianum: cloning and expression of the gene encoding 1,3-propanediol dehydrogenase.

When grown on glycerol as sole carbon and energy source, cell extracts of Clostridium pasteurianum exhibited activities of glycerol dehydrogenase, dihydroxyacetone kinase, glycerol dehydratase and 1,3-propanediol dehydrogenase. The genes encoding the latter two enzymes were cloned by colony hybridization using the dhaT gene of Citrobacter freundii as a heterologous DNA probe and expressed in Escherichia coli. The native molecular mass of 1,3-propanediol dehydrogenase (DhaT) is 440,000 Da. The dhaT gene of C. pasteurianum was subcloned and its nucleotide sequence (1158 bp) was determined. The deduced gene product (41,776 Da) revealed high similarity to DhaT of C. freundii (80.5% identity; 89.8% similarity).

Identification of the syntrophic partners in a coculture coupling anaerobic methanol oxidation to Fe(III) reduction.

From enrichments with methanol and ferric pyrophosphate a coculture was isolated which coupled methanol oxidation to carbon dioxide with the reduction of Fe(III) to Fe(II). 16S rRNA gene analysis of the isolated syntrophic partners revealed 99.5% similarity to Clostridium sphenoides and 98.5% to Shewanella putrefaciens. Formation of Fe(II) coupled to methanol oxidation was confirmed by using strains of culture collections (C. sphenoides DSM 632 and S. putrefaciens DSM 9461). The importance of this process is discussed, also with respect to the anaerobic oxidation of methane.

The F420H2-dehydrogenase from Methanolobus tindarius: cloning of the ffd operon and expression of the genes in Escherichia coli.

The membrane-bound F420H2-dehydrogenase from the methylotrophic methanogen Methanolobus tindarius oxidizes reduced coenzyme F420 and feeds the electrons into an energy-conserving electron transport chain. Based on the N-terminal amino acid sequence of the 40-kDa subunit of F420H2-dehydrogenase the corresponding gene ffdB was detected in chromosomal DNA of M. tindarius. Sequence analysis, primer extension, and RT-PCR experiments indicated that ffdB is part of an operon harboring three additional open reading frames (ffdA, ffdC, ffdD). The corresponding mRNA transcript and transcription start sites were determined. All four genes could be heterologously expressed in Escherichia coli.

Membrane-bound F420H2-dependent heterodisulfide reduction in methanococcus voltae

Washed membranes prepared from H2+CO2- or formate-grown cells of Methanococcus voltae catalyzed the oxidation of coenzyme F420H2 and the reduction of the heterodisulfide (CoB-S-S-CoM) of 2-mercaptoethanesulfonate and 7-mercaptoheptanoylthreonine phosphate, which is the terminal electron acceptor of the methanogenic pathway. The reaction followed a 1:1 stoichiometry according to the equation: F420H2 + COB-S-S-CoM --> F420 + CoM-SH + CoB-SH. These findings indicate that the reaction depends on a membrane-bound F420H2-oxidizing enzyme and on the heterodisulfide reductase, which remains partly membrane-bound after cell lysis. To elucidate the nature of the F420H2-oxidizing protein, washed membranes were solubilized with detergent, and the enzyme was purified by sucrose density centrifugation, anion-exchange chromatography, and gel filtration. Several lines of evidence indicate that F420H2 oxidation is catalyzed by a membrane-associated F420-reducing hydrogenase. The purified protein catalyzed the H2-dependent reduction of methyl viologen and F420. The apparent molecular mass and the subunit composition (43, 37, and 27 kDa) are almost identical to those of the F420-reducing hydrogenase that has already been purified from Mc. voltae. Moreover, the N-terminus of the 37-kDa subunit is identical to the amino acid sequence deduced from the fruG gene of the operon encoding the selenium-containing F420-reducing hydrogenase from Mc. voltae. A distinct F420H2 dehydrogenase, which is present in methylotrophic methanogens, was not found in this organism.

Osmoadaptation in halophilic and alkaliphilic methanotrophs

By using (1)H- and (13)C-NMR spectroscopy, an accumulation of sucrose and two cyclic amino acids [ectoine (1,4,5, 6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) and 5-oxoproline (pyrrolidone carboxylic acid)] was detected in the halotolerant methanotrophs Methylobacter alcaliphilus 20Z and Methylobacter modestohalophilus 10S. The organic solute pool was found to increase upon raising the NaCl concentration. In M. alcaliphilus 20Z, the intracellular level of the total solutes was shown to be sufficient to balance the osmotic pressure of the medium, whereas in M. modestohalophilus 10S their content was several times lower. Additionally, phosphatidylglycerol and phosphatidylcholine were predominant cell phospholipids in salt-adapted M. alcaliphilus 20Z. However, no phosphatidylcholine was found in M. modestohalophilus 10S, and the portion of phosphatidylglycerol increased while phosphatidylethanolamine decreased upon elevated external NaCl concentrations. Regularly arranged glycoprotein surface layers (S-layers) of hexagonal and linear (p2) symmetry were observed on the outer cell walls of M. alcaliphilus 20Z and M. modestohalophilus 10S. The S-layer in M alcaliphilus 20Z consisting of tightly packed, cup-shaped subunits was lost during growth at pH 7.2 (the lowest possible pH) in the absence of NaCl. Hence, osmoadaptation in the methanotrophs studied involves structure/function alterations of cell envelopes and changes in the chemical composition of membranes as well as de novo synthesis of compatible solutes.