The influence of matrix type, diurnal rhythm and sample collection and processing on the measurement of plasma beta-amyloid isoforms using the INNO-BIA plasma Abeta forms multiplex assay.

OBJECTIVE: The aim of the study was to determine the extent to which plasma matrix types, diurnal rhythm and sample collection and processing procedures contribute to overall variability of measurements with the INNO-BIA plasma Abeta forms assay. METHODS: Plasma samples from healthy volunteers were collected at BARC-CRI. Analyte concentrations from various plasma matrix types (EDTA, heparin, fluoride) were compared to serum after collection of blood in commercial plastic and glass tubes. Sample processing variables including time and temperature before and after centrifugation, centrifugal force and plasma dilution factor were also investigated. Diurnal variability in plasma Abeta isoforms was determined in 29 healthy volunteers by analysis of EDTA plasma specimens serially collected over 24 hours and stored frozen following oral administration of a placebo treatment. All plasma samples from a given individual and experiment were analyzed in a single analytical run. RESULTS: Highest Abeta levels were obtained using EDTA-plasma samples (in contrast to serum, heparin, citrate, or fluoride). Addition of aprotinin to EDTA plasma had no effect on Abeta peptide recovery. The elapsed time and temperature exposure, before and after sample processing affects the recovery of Abeta isoforms. Analyte recovery was not significantly affected by the presence of platelets in plasma samples. At the subject level, analysis of serially collected EDTA plasma specimens from healthy volunteers revealed no evidence of diurnal variation in any of the Abeta isoforms investigated and results from samples collected on a monthly basis showed only very limited intra-individual variation. CONCLUSIONS: Optimal recovery of Abeta peptides was obtained from blood drawn into EDTA tubes and processed within 4 h. Plasma that was refrigerated after separation and analysed within 4 h gave comparable results to samples immediately processed and frozen at -70 degrees C.

Identification of a thiosulfate utilization gene cluster from the green phototrophic bacterium Chlorobium limicola.

Chlorobium is an autotrophic, green phototrophic bacterium which uses reduced sulfur compounds to fix carbon dioxide in the light. The pathways for the oxidation of sulfide, sulfur, and thiosulfate have not been characterized with certainty for any species of bacteria. However, soluble cytochrome c-551 and flavocytochrome c (FCSD) have previously been implicated in the oxidation of thiosulfate and sulfide on the basis of enzyme assays in Chlorobium. We have now made a number of observations relating to the oxidation of reduced sulfur compounds. (1) Western analysis shows that soluble cytochrome c-551 in Chlorobium limicola is regulated by thiosulfate, consistent with a role in the utilization of thiosulfate. (2) A membrane-bound flavocytochrome c-sulfide dehydrogenase (which is normally a soluble protein in other species) is constitutive and not regulated by sulfide as expected for an obligately autotrophic species dependent upon sulfide. (3) We have cloned the cytochrome c-551 gene from C. limicola and have found seven other genes, which are also presumably involved in sulfur metabolism and located near that for cytochrome c-551 (SoxA). These include genes for a flavocytochrome c flavoprotein homologue (SoxF2), a nucleotidase homologue (SoxB), four small proteins (including SoxX, SoxY, and SoxZ), and a thiol-disulfide interchange protein homologue (SoxW). (4) We have established that the constitutively expressed FCSD genes (soxEF1) are located elsewhere in the genome. (5) Through a database search, we have found that the eight thiosulfate utilization genes are clustered in the same order in the Chlorobium tepidum genome (www.tigr.org). Similar thiosulfate utilization gene clusters occur in at least six other bacterial species but may additionally include genes for rhodanese and sulfite dehydrogenase.

A novel system for heterologous expression of flavocytochrome c in phototrophic bacteria using the Allochromatium vinosum rbcA promoter.

Flavocytochrome c-sulfide dehydrogenase (FCSD), an enzyme that catalyzes the reversible conversion of sulfide to elemental sulfur in vitro, is common to bacteria that utilize reduced sulfur compounds as electron donors in the process of carbon dioxide fixation. FCSD is a heterodimer containing two different cofactors, a flavin (FAD) and one or two heme c groups, located on the separate protein subunits. Efforts to produce the holoproteins of the soluble Allochromatium vinosum FCSD and the membrane-bound Ectothiorhodospira vacuolata protein in Escherichia coli using several expression systems were unsuccessful. Although all systems used were able to export the recombinant FCSDs to the periplasm, the proteins did not incorporate heme. In order to develop a new expression system involving photosynthetic hosts (Rhodobacter capsulatus, Rhodobacter sphaeroides and Ect. vacuolata), plasmid mobilisation from E. coli donors was studied. In the search for efficient promoters for such hosts, a system was developed combining the broad-host-range plasmid pGV910 and the promoter of the A. vinosum RuBisCo gene, rbcA. Conjugation was used to enable transfer from the expression plasmid of E. coli into Rba. capsulatus, Rba. sphaeroides strains and into Ect. vacuolata. Both Rhodobacter hosts were able to transcribe the genes coding for FCSD from the rbcA promoter and to produce detectable amounts of recombinant FCSD holoprotein. Western blots showed that the best production was obtained from cells grown photosynthetically on malate or acetate with sulfide. This system may prove to be of general use for the production of recombinant c-type cytochromes in homologous or related host systems.

A membrane-bound flavocytochrome c-sulfide dehydrogenase from the purple phototrophic sulfur bacterium Ectothiorhodospira vacuolata.

The amino acid sequence of Ectothiorhodospira vacuolata cytochrome c-552, isolated from membranes with n-butanol, shows that it is a protein of 77 amino acid residues with a molecular mass of 9,041 Da. It is closely related to the cytochrome subunit of Chlorobium limicola f. sp. thiosulfatophilum flavocytochrome c-sulfide dehydrogenase (FCSD), having 49% identity. These data allowed isolation of a 5.5-kb subgenomic clone which contains the cytochrome gene and an adjacent flavoprotein gene as in other species which have an FCSD. The cytochrome subunit has a signal peptide with a normal cleavage site, but the flavoprotein subunit has a signal sequence which suggests that the mature protein has an N-terminal cysteine, characteristic of a diacyl glycerol-modified lipoprotein. The membrane localization of FCSD was confirmed by Western blotting with antibodies raised against Chromatium vinosum FCSD. When aligned according to the three-dimensional structure of Chromatium FCSD, all but one of the side chains near the flavin are conserved. These include the Cys 42 flavin adenine dinucleotide binding site; the Cys 161-Cys 337 disulfide; Glu 167, which modulates the reactivity with sulfite; and aromatic residues which may function as charge transfer acceptors from the flavin-sulfite adduct (C. vinosum numbering). The genetic context of FCSD is different from that in other species in that flanking genes are not conserved. The transcript is only large enough to encode the two FCSD subunits. Furthermore, Northern hybridization showed that the production of E. vacuolata FCSD mRNA is regulated by sulfide. All cultures that contained sulfide in the medium had elevated levels of FCSD RNA compared with cells grown on organics (acetate, malate, or succinate) or thiosulfate alone, consistent with the role of FCSD in sulfide oxidation.

Structure and characterization of Ectothiorhodospira vacuolata cytochrome b(558), a prokaryotic homologue of cytochrome b(5).

A soluble cytochrome b(558) from the purple phototropic bacterium Ectothiorhodospira vacuolata was completely sequenced by a combination of automated Edman degradation and mass spectrometry. The protein, with a measured mass of 10,094.7 Da, contains 90 residues and binds a single protoheme. Unexpectedly, the sequence shows homology to eukaryotic cytochromes b(5). As no prokaryotic homologue had been reported so far, we developed a protocol for the expression, purification, and crystallization of recombinant cytochrome b(558). The structure was solved by molecular replacement to a resolution of 1.65 A. It shows that cytochrome b(558) is indeed the first bacterial cytochrome b(5) to be characterized and differs from its eukaryotic counterparts by the presence of a disulfide bridge and a four-residue insertion in front of the sixth ligand (histidine). Eukaryotes contain a variety of b(5) homologues, including soluble and membrane-bound multifunctional proteins as well as multidomain enzymes such as sulfite oxidase, fatty-acid desaturase, nitrate reductase, and lactate dehydrogenase. A search of the Mycobacterium tuberculosis genome showed that a previously unidentified gene encodes a fatty-acid desaturase with an N-terminal b(5) domain. Thus, it may provide another example of a bacterial b(5) homologue.

Two new aminopeptidases from Ochrobactrum anthropi active on D-alanyl-p-nitroanilide.

Two new enzymes which hydrolyse D-alanyl-p-nitroanilide have been detected in Ochrobactrum anthropi LMG7991 extracts. The first enzyme, DmpB, was purified to homogeneity and found to be homologous to the Dap protein produced by O. anthropi SCRC C1-38 (ATCC49237). The second enzyme, DmpA, exhibits a similar substrate profile when tested on p-nitroanilide derivatives of glycine and L/D-alanine, but the amounts produced by the Ochrobactrum strain were not sufficient to allow complete purification. Interestingly, the DmpA preparation also exhibited an L-aminopeptidase activity on the tripeptide L-Ala-Gly-Gly but it was not possible to be certain that the same protein was responsible for both p-nitroanilide and peptide hydrolysing activities. The gene encoding the DmpA protein was cloned and sequenced. The deduced protein sequence exhibits varying degrees of similarity with those corresponding to several open reading frames found in the genomes of other prokaryotic organisms, including Mycobacteria. None of these gene products has been isolated or characterised, but a tentative relationship can be proposed with the NylC amidase from Flavobacterium sp. K172.