Feasibility study for the development of certified reference materials for specific migration testing. Part 2: estimation of diffusion parameters and comparison of experimental and predicted data.

This paper describes the second part of a project whose main objective was to develop the know-how to produce certified reference materials (CRMs) for specific migration testing. Certification parameters discussed are the diffusion coefficient, D(P), the respective polymer-specific coefficient, A(P), of the migrant polymer combinations and the partitioning coefficient, K(P,F), describing the partitioning of the migrant between the polymer and a food simulant. The parameters were determined for 16 preliminary candidate CRMs. Each parameter was determined by one laboratory. The six materials most suitable as reference materials were selected and the parameters then determined by four laboratories. The coefficients resulting from this small-scale interlaboratory comparison study can be regarded as the most reliable values available to date. These coefficients were applied for a comparison of experimental and predicted migration data. The experimental migration data arose from the same project and were determined by one laboratory for the first 16 materials and subsequently by four laboratories for the six materials selected in the second phase. Overall, experimental and predicted migration data fit together quite well. Roughly half of the predicted data were within +/-10%; almost all predicted data were within +/-40% compared with the experimental data.

Feasibility study for the development of certified reference materials for specific migration testing. Part 1: initial migrant concentration and specific migration.

The paper describes a project with the main objective of developing the know how to produce certified reference materials (CRMs) for specific migration testing. Certification parameters discussed are the initial concentration of the migrant in the polymer (C(P),0) and the specific migration into a food simulant under certain temperature/time conditions. Sixteen preliminary candidate CRMs were defined and produced. The most important polymers (low- and high-density polyethylene (LDPE and HDPE), polypropylene (PP), polystyrene (PS), polyethylene terephtalate (PET), plasticized polyvinyl chloride (PVC), rigid PVC, polyamides (PA)) and additives as well as monomers representing different physicochemical properties as target substances for migration were chosen. The stability and homogeneity of the migrants in the materials were tested and methods for the determination of the certification parameters were developed and validated. > From the 16 materials produced, the six most suitable CRM candidates (LDPE//Irganox 1076/Irgafos 168, LDPE//1,4-diphenyl-1,3-butadiene (DPBD), HDPE//Chimassorb 81/Uvitex OB, PP homo//Irganox 1076/Irgafos 168, HIPS, 1% mineral oil//styrene, PA 6//caprolactam) were selected. The feasibility of CRM production for the six candidate materials was demonstrated and a trial certification exercise was performed with participation of all four partner laboratories. All six materials showed suitable properties for future production as certified reference materials.

Specific stimulation of peripheral blood mononuclear cells from patients with acute myocarditis by peptide-bound flavin adenine dinucleotide (FAD), a naturally occurring autologous hapten.

The tryptic FAD-peptide carrying the flavin in 8alpha-(N3)histidyl linkage as natural hapten was isolated by HPLC from the bacterial enzyme 6-hydroxy-d-nicotine oxidase. The same flavin protein linkage is found in the mitochondrial succinate dehydrogenase flavoprotein subunit, the predominant flavoprotein with covalently bound FAD in mitochondria of cardiomyocytes. Peripheral blood mononuclear cells (PBMC) were isolated from four patients with acute myocarditis, seven patients with dilated cardiomyopathy (DCM) and from four healthy control individuals. The response of PBMC to the FAD-peptide was evaluated by measuring proliferation ([3H]-dThd incorporation) and cytokine secretion [interferon (IFN)-gamma]. PBMC from all patients with acute myocarditis showed positive responses to the FAD-peptide, in contrast to PBMC from patients with DCM or control individuals. Following the recovery of the patients from the acute inflammation of the heart, PBMC no longer exhibited a proliferation response to the FAD-peptide. A chemically synthesized FAD-free peptide with identical amino acid sequence induced no response of PBMC. The results are consistent with a recall response by activated T cells, specific for the normally cryptic mitochondrial flavin-hapten, which may be liberated following cardiomyocyte destruction during the inflammation of the heart.

Analysis of mitochondrial antigens reveals inner membrane succinate dehydrogenase flavoprotein subunit as autoantigen to antibodies in anti-M7 sera.

The role of mitochondrial proteins as antigens to antibodies of anti-M7 sera was analysed by flavin fluorescence, one- and two-dimensional Western blots and blue native gel electrophoresis. Flavin fluorescence of succinate dehydrogenase (SucDH, complex II of the respiratory chain) of rat liver inner mitochondrial membranes correlated with the immunoreactivity of a representative anti-M7 myocarditis serum. Antigens of isolated bovine heart mitochondria reacting with antibodies of myocarditis serum on two-dimensional Western blots were identified by MALDI-TOF and NanoESI mass spectrometry as myosin heavy chain beta and as dihydrolipoamide dehydrogenase of the mitochondrial 2-oxoacid dehydrogenase complexes. The SucDH-flavoprotein was not resolved as a discrete protein spot on two-dimensional polyacrylamide gels. However, separation of the rat liver inner mitochondrial membrane complexes by blue native gel electrophoresis followed by Western blotting, and Western blots of purified Escherichia coli SucDH complex revealed that anti-M7 sera contained antibodies directed against the SucDH-flavoprotein subunit.

Screening for organotin compounds in European landfill leachates.

As industrial chemicals, organotin compounds are predominantly applied as polyvinyl chloride (PVC) stabilizers and biocides. They are widely encountered in environmental samples and may be introduced into landfills by disposal of municipal solid waste or sewage sludge. In the present study, leachate samples were obtained from several sanitary landfill sites in Sweden, Italy, and Germany. These samples were analyzed by means of a highly sensitive and species-selective method for methyltin, butyltin, and octyltin species. In total, twelve samples from eight different landfill sites at various ages were investigated. The findings of all target compounds range between less than the limit of detection at 0.1 microg/L and, at maximum, 4 microg/L. Only octyltin compounds can be attributed to PVC products with any certainty, whereas for methyltin and butyltin compounds alternative and less distinct sources exist. Organotin compounds are subject to microbial transformation, such as dealkylation and methylation processes. Consequently, caution should be exercised when attributing findings to potential sources and deriving any predicted environmental concentrations.

Gene cluster on pAO1 of Arthrobacter nicotinovorans involved in degradation of the plant alkaloid nicotine: cloning, purification, and characterization of 2,6-dihydroxypyridine 3-hydroxylase.

A 27,690-bp gene cluster involved in the degradation of the plant alkaloid nicotine was characterized from the plasmid pAO1 of Arthrobacter nicotinovorans. The genes of the heterotrimeric, molybdopterin cofactor (MoCo)-, flavin adenine dinucleotide (FAD)-, and [Fe-S] cluster-dependent 6-hydroxypseudooxynicotine (ketone) dehydrogenase (KDH) were identified within this cluster. The gene of the large MoCo subunit of KDH was located 4,266 bp from the FAD and [Fe-S] cluster subunit genes. Deduced functions of proteins encoded by open reading frames (ORFs) of the cluster were correlated to individual steps in nicotine degradation. The gene for 2,6-dihydroxypyridine 3-hydroxylase was cloned and expressed in Escherichia coli. The purified homodimeric enzyme of 90 kDa contained 2 mol of tightly bound FAD per mol of dimer. Enzyme activity was strictly NADH-dependent and specific for 2,6-dihydroxypyridine. 2,3-Dihydroxypyridine and 2,6-dimethoxypyridine acted as irreversible inhibitors. Additional ORFs were shown to encode hypothetical proteins presumably required for holoenzyme assembly, interaction with the cell membrane, and transcriptional regulation, including a MobA homologue predicted to be specific for the synthesis of the molybdopterin cytidine dinucleotide cofactor.

Coxsackievirus B3 infection induces anti-flavoprotein antibodies in mice.

Enteroviruses, the most common cause of acute myocarditis, are also supposed aetiological agents of dilated cardiomyopathy. Autoantibodies (anti-M7; Klein & Berg, Clin Exp Immunol 1990; 58:283-92) directed against flavoproteins with covalently bound flavin (alphaFp-Ab; Otto et al., Clin Exp Immunol 1998; 111:541-2) are detected in up to 30% of sera of patients with myocarditis and idiopathic dilated cardiomyopathy (IDCM). Mice inoculated with a myocarditic variant of coxsackievirus B3 (CVB3) were employed to study the occurrence of serum alphaFp-Ab following viral infection. The presence of alphaFp-Ab was analysed by Western blotting with the flavoprotein antigens 6-hydroxy-D-nicotine oxidase (6HDNO) and sarcosine oxidase (SaO). Of 10 sera from CVB3-infected mice, five showed a strong reaction with both antigens. The sera were reactive also to the mitochondrial covalently flavinylated proteins dimethylglycine dehydrogenase and sarcosine dehydrogenase. Sera of non-infected mice did not react with these antigens. A 6HDNO mutant protein with non-covalently bound FAD no longer reacted on Western blots with sera of CVB3-infected mice. Preincubation with FAD abolished or reduced the reaction of the sera with the 6HDNO antigen. At 2 weeks p.i. the alphaFp-Ab were of the IgM and IgG isotypes, at 7 and 9 weeks p.i. of the IgG isotype. The sera of CVB3-infected mice reproduced closely the antigenic specificity of the anti-M7 sera of patients, lending further support to the role of coxsackieviruses in the pathogenesis of IDCM.

Association of betaine-homocysteine S-methyltransferase with microtubules.

In mammals, betaine of the mitochondrial matrix is used in the cytosol by betaine-homocysteine S-methyltransferase for methionine synthesis. The resulting dimethylglycine is shuttled back into the mitochondrial matrix for further degradation. Nanospray tandem mass spectrometry and N-terminal amino acid sequencing of microtubule-associated proteins from rat liver tubulin revealed that betaine-homocysteine S-methyltransferase is microtubule associated. This was confirmed by confocal laser scanning microscopy of HepG2 cells labeled with betaine-homocysteine S-methyltransferase- and alpha-tubulin-specific monoclonal antibodies. The association of betaine-homocysteine S-methyltransferase with the cytoskeleton may functionally integrate the mitochondrial and cytoplasmic compartments of choline degradation.

A protein factor of rat liver mitochondrial matrix involved in flavinylation of dimethylglycine dehydrogenase.

The involvement of rat liver mitochondria in the flavinylation of the mitochondrial matrix flavoenzyme dimethylglycine dehydrogenase (Me2GlyDH) has been investigated. Me2GlyDH was synthesized as an apoenzyme in the rabbit reticulocyte lysate (RL) transcription/translation system and its flavinylation was monitored by virtue of the trypsin resistance of the holoenzyme. The rate of holoenzyme formation in the presence of FAD was stimulated with increasing efficiency by the addition of solubilized mitoplasts, mitochondrial matrix and DEAE-purified matrix fraction. Apo-Me2GlyDH was also converted into holoenzyme when the solubilized mitoplasts were supplemented with FMN and ATP. This observation is consistent with the existence of a mitochondrial FAD synthetase generating the FAD needed for holoenzyme formation from its precursors. Holoenzyme formation in the presence of FAD increased linearly with the concentration of matrix protein in the assay, and depended on the amount of externally added Me2GlyDH with saturation characteristics. These findings suggest the presence of a protein factor in the mitochondrial matrix which stimulates Me2GlyDH flavinylation. This factor was different from both mitochondrial heat shock protein (Hsp)70, as shown by immunodepletion experiments, and mitochondrial Hsp60, as demonstrated by the capability of a DEAE-purified matrix fraction devoid of Hsp60 to accelerate flavinylation of both RL translated and purified Me2GlyDH.

Anti-mitochondrial flavoprotein autoantibodies of patients with myocarditis and dilated cardiomyopathy (anti-M7): interaction with flavin-carrying proteins, effect of vitamin B2 and epitope mapping.

Vitamin B2 and flavin cofactors are transported tightly bound to immunoglobulin in human serum. We reasoned that anti-mitochondrial flavoprotein autoantibodies (alpha Fp-AB) present in the serum of patients with myocarditis and cardiomyopathy of unknown aetiology may form immunoglobulin aggregates with these serum proteins. However, immunodiffusion and Western blot assays demonstrated that the flavin-carrying proteins were not recognized by alpha Fp-AB. Apparently the flavin moiety in the native protein conformation was inaccessible to alpha Fp-AB. This conclusion was supported by the absence of an immunoreaction between the riboflavin-binding protein from egg white and alpha FP-AB. Intravenous application of vitamin B2 to rabbits immunized with 6-hydroxy-D-nicotine oxidase, a bacterial protein carrying covalently attached FAD, did not neutralize alpha Fp-AB which had been raised in the serum of the animals. FAD-carrying peptides generated from 6-hydroxy-D-nicotine oxidase by trypsin and chymotrypsin treatment were not recognized by the alpha Fp-AB, but those generated by endopeptidase Lys were. This demonstrates that the epitope recognized by alpha Fp-AB comprises, besides the flavin moiety, protein secondary structure elements.

Surface-promoted replication and exponential amplification of DNA analogues.

Self-replicating chemical systems have been designed and studied to identify the minimal requirements for molecular replication, to translate the principle into synthetic supramolecular systems and to derive a better understanding of the scope and limitations of self-organization processes that are believed to be relevant to the origin of life on Earth. Current implementations make use of oligonucleotide analogues, peptides, and other molecules as templates and are based either on autocatalytic, cross-catalytic, or collectively catalytic pathways for template formation. A common problem of these systems is product inhibition, leading to parabolic instead of exponential amplification. The latter is the dynamic prerequisite for selection in the darwinian sense. We here describe an iterative, stepwise procedure for chemical replication which permits an exponential increase in the concentration of oligonucleotide analogues. The procedure employs the surface of a solid support and is called SPREAD (surface-promoted replication and exponential amplification of DNA analogues). Copies are synthesized from precursor fragments by chemical ligation on immobilized templates, and then liberated and immobilized to become new templates. The process is repeated iteratively. The role of the support is to separate complementary templates which would form stable duplexes in solution. SPREAD combines the advantages of solid-phase chemistry with chemical replication, and can be further developed for the non-enzymatic and enzymatic amplification of RNA, peptides and other templates as well as for studies of in vitro evolution and competition in artificial chemical systems. Similar processes may also have played a role in the origin of life on Earth, because the earliest replication systems may have proliferated by spreading on mineral surfaces.

Molecular cloning and tissue distribution of rat sarcosine dehydrogenase.

Sarcosine dehydrogenase (SarDH) is a mitochondrial flavoenzyme involved in the oxidative degradation of choline to glycine. The absence of SarDH activity in humans is genetically transmitted and is the cause of an amino acid metabolism disorder called sarcosinemia. Tryptic fragments of the purified enzyme from rat liver were subjected to Edman degradation and the sequences obtained were used to clone the cDNA encoding the full length protein. The deduced amino acid sequence of SarDH shares an overall similarity of 47% with dimethylglycine dehydrogenase (Me2GlyDH), another flavoenzyme involved in the mitochondrial choline catabolism with a similar FAD-binding domain. Covalent binding of FAD to SarDH was demonstrated by the observation of strong fluorescence at 530 nm under excitation at 450 nm of the enzyme immunoprecipitated under denaturing conditions from liver extracts. The localization of SarDH immunoreactivity in the mitochondrial matrix was confirmed by Western-blot analysis of purified mitochondrial fractions. Finally, the tissue distribution of SarDH was investigated by Northern-blot analysis of total RNA and Western-blot analysis of total protein from several rat tissues. A strong expression in the liver, but also in the lung, pancreas, kidney, thymus, and oviduct was observed. We therefore suggest that the enzymes of the choline catabolism pathway are important also for metabolism in nonhepatic tissues.

The conformational change induced by FAD in covalently flavinylated 6-hydroxy-D-nicotine oxidase does not require (8alpha)FAD-(N3)histidyl bond formation.

The contribution of (8alpha)-(N3)histidyl bond formation to the conformation of covalently flavinylated proteins was investigated by trypsin treatment of wild type and mutant versions of a model enzyme, 6-hydroxy-D-nicotine oxidase (6-HDNO) of Arthrobacter nicotinovorans. In the absence of FAD, apo-6-HDNO exhibited a conformation exposing a protease accessible site. Holoenzyme formation through FAD-attachment to His71 induced a conformational change in the protein that shielded the trypsin recognition site. This conformational change, however, did not require FAD-histidyl bond formation since trypsin resistance was also exhibited by a 6-HDNO.Cys71 mutant protein which was unable to bind FAD covalently. Replacement of Arg67, an amino acid residue supposed to be essential in flavinylation, by Ala rendered the protein protease sensitive as did replacement of Pro73 by Ala. These amino acids apparently play an essential role in stabilizing the native protein conformation. The inability to reach the native conformation also prevented FAD attachment, indicating that a specific conformation of the protein is a prerequisite for FAD-histidyl bond formation. Deletion of Phe448 and Arg449 from the 458 amino acid residues-containing enzyme resulted in complete protease sensitivity, demonstrating that flavinylation takes place posttranslationally.

Anti-mitochondrial antibodies in patients with dilated cardiomyopathy (anti-M7) are directed against flavoenzymes with covalently bound FAD.

Anti-mitochondrial antibodies (anti-M7) in sera from patients with dilated cardiomyopathy and myocarditis recognize, besides mitochondrial antigens, bacterial sarcosine dehydrogenase. The common target antigen was identified as the covalently bound FAD of mitochondrial and bacterial flavoenzymes. Thus, anti-M7-positive serum reacted on Western blots exclusively with covalently flavinylated enzymes. The antigenic specificity of anti-M7 sera was reproduced by an antiserum raised in rabbits with 6-hydroxy-D-nicotine oxidase. The heart mitochondrial membrane antigen recognized by anti-M7 serum was identified as the flavoprotein subunit of succinate dehydrogenase, the antigens in rat liver mitochondrial matrix as the flavoenzymes dimethylglycine dehydrogenase and sarcosine dehydrogenase. Anti-M7 serum contained a specific anti-flavoenzyme antibody fraction. Nanomolar concentrations of FAD and riboflavin inhibited the immune reaction on Western blots and in ELISA, and incubation with FAD-agarose depleted the anti-M7 activity of the serum. N-terminally deleted dimethylglycine dehydrogenase proteins were only immunoprecipitated by anti-M7 sera when the FAD was covalently incorporated. An affinity constant (KD) of 10(-8) M was established for the anti-flavoenzyme antibodies by competitive ELISA. Of patients with cardiomyopathy and myocarditis, 36% and 25%, respectively, were anti-flavoenzyme-positive by Western blot and ELISA, but only two of 15 patients with other heart diseases and none of 50 healthy controls.

IS1473, a putative insertion sequence identified in the plasmid pAO1 from Arthrobacter nicotinovorans: isolation, characterization, and distribution among Arthrobacter species.

A putative insertion sequence (IS1473) has been cloned and sequenced. The 1087-bp element was found between the moaA and the ndhA genes in the upstream region of the nicotine dehydrogenase (ndh) operon in the 160-kb pAO1 plasmid of Arthrobacter nicotinovorans. It is flanked by an imperfect repeat of 33 bp and carries two overlapping open reading frames which, by programmed -1 translational frameshifting, may produce a transposase of 36.735 Da with a pI = 10. 18. The deduced protein is similar to the transposases IS481 and IS1002 from Bordetella and IS476 from Xanthomonas campestris, all members of the IS3 family. The putative insertion element was found as a single copy in the pAO1 plasmid and absent on the chromosome of the A. nicotinovorans genome. Similar sequences were detected by hybridization on total DNA from Arthrobacter globiformis, Arthrobacter ramosus, and Arthrobacter ureafaciens.

Molybdate-uptake genes and molybdopterin-biosynthesis genes on a bacterial plasmid--characterization of MoeA as a filament-forming protein with adenosinetriphosphatase activity.

A gene cluster consisting of homologs to Escherichia coli moaA, moeA, moaC and moaE, which encode enzymes involved in the biosynthesis of molybdopterin cofactor (MoCo), and to modA, modB and modC, which encode a high-affinity molybdate transporter, were identified on pAO1 of Arthrobacter nicotinovorans near genes of molybdopterin-dependent enzymes involved in nicotine degradation. This gene arrangement suggests a coordinated expression of the MoCo-dependent and the MoCo-biosynthesis genes and shows that catabolic plasmids may carry the transport and biosynthetic machinery for the synthesis of the cofactors needed for the functioning of the enzymes they encode. pAO1 MoeA functionally complemented E. coli moeA mutants. The overexpressed and purified protein, of molecular mass 44,500 Da, associated into high-molecular-mass complexes and spontaneously formed gels at concentrations above 1 mg/ml. Transmission electron microscopy and atomic force microscopy revealed that MoeA forms fibrilar structures. In the presence of Mg2+ MoeA exhibited ATPase activity (0.020 pmol ATP x pmol protein(-1) x min(-1)). ATP, ADP or AMP induced the disassembly of the MoeA fibers into aggregates. pAO1 MoeA shows 39% identity to the C-terminal domain of the rat neuroprotein gephyrin. Like gephyrin it binds to neurotubulin, but binds with preference to tubulin dimers.

Covalent attachment of FAD derivatives to a fusion protein consisting of 6-hydroxy-D-nicotine oxidase and a mitochondrial presequence. Folding, enzyme activity, and import of the modified protein into yeast mitochondria.

Autoflavinylation of 6-hydroxy-D-nicotine oxidase (6-HDNO) was successfully employed to modify the protein covalently with FAD derivatives. The model compounds N6-(2-aminoethyl)-FAD and N6-(6-carboxyhexyl)-FAD were spontaneously bound to a fusion protein consisting of the mitochondrial targeting sequence of Neurospora crassa F0-ATPase subunit 9 (Su9) attached to 6-HDNO. When translated in the rabbit reticulocyte lysate, Su9-6-HDNO was in the trypsin-sensitive apoenzyme form; when translated in the presence of flavins it adopted a trypsin-resistant conformation characteristic of the 6-HDNO holoenzyme. With flavin derivatives, Su9-6-HDNO exhibited approximately 50% of the 6-HDNO activity observed with FAD. The covalently modified Su9-6-HDNO was imported into Saccharomyces cerevisiae mitochondria with an efficiency equal to that of the apoenzyme. Apparently the increase in size and charge of the FAD moiety did not hamper translocation across the mitochondrial membranes. Yeast mutant ssc1-2 mitochondria deficient in mtHsp70 unfoldase activity imported the flavinylated Su9-6-HDNO protein. In mutant ssc1-3 mitochondria deficient in both mtHsp70 unfoldase and translocase activity Su9-6-HDNO was trapped as translocation intermediate; the Su9 presequence was passed to the matrix where it was proteolytically cleaved by the mitochondrial processing peptidase; (MPP); the translocation-arrested 6-HDNO moiety adopted a trypsin-sensitive conformation. Our results indicate that unfolding of the FAD-stabilized flavin-binding domain of 6-HDNO in passage through the mitochondrial general insertion pore does not require the activity of mtHsp70.

MoaA of Arthrobacter nicotinovorans pAO1 involved in Mo-pterin cofactor synthesis is an Fe-S protein.

MoaA, involved in an early step in the biosynthesis of the molybdopterin cofactor (MoCo), has not yet been characterized biochemically and the reaction it catalyzes is unknown. We overexpressed MoaA from pAO1 of Arthrobacter nicotinovorans in Escherichia coli as a N-terminal fusion with either glutathione-S-transferase or a 6-histidine tag. The pAO1 encoded MoaA as well as the fusion proteins functionally complement E. coli moaA mutants. Here we show that purified MoaA contains approximately 4 microM Fe and approximately 3 microM acid-labile S/microM protein. EPR spectroscopy revealed a predominant signal at g(av) = 2.01, indicative of a [3Fe-xS] cluster.

The design of an alternative, covalently flavinylated 6-hydroxy-D-nicotine oxidase by replacing the FAD-binding histidine by cysteine and reconstitution of the holoenzyme with 8-(methylsulfonyl)FAD.

The cofactor of several flavoenzymes is autocatalytically bound to the polypeptide via a histidyl(N3)-(8alpha)-FAD linkage which makes the generation of apoenzyme difficult. We introduced an alternative covalent protein-FAD bond at the active site of 6-hydroxy-D-nicotine oxidase (6HDNO) by replacing the FAD-binding histidine with cysteine. The resulting mutant enzyme was expressed with noncovalently attached cofactor. Incubation with 8-(methylsulfonyl)FAD, and less efficiently with 8-chloro-FAD, resulted in the spontaneous replacement of the noncovalently bound FAD by the flavin derivative and the formation of an 8-(N-acetylcysteinyl)FAD linkage. The flavinylated 6HDNO.cys exhibited close to wild-type activity levels. This strategy may be generally applicable to the attachment of artificially designed flavin derivatives to the active site of covalently flavinylated enzymes.