Membrane organization of photosystem I complexes in the most abundant phototroph on Earth.

Prochlorococcus is a major contributor to primary production, and globally the most abundant photosynthetic genus of picocyanobacteria because it can adapt to highly stratified low-nutrient conditions that are characteristic of the surface ocean. Here, we examine the structural adaptations of the photosynthetic thylakoid membrane that enable different Prochlorococcus ecotypes to occupy high-light, low-light and nutrient-poor ecological niches. We used atomic force microscopy to image the different photosystem I (PSI) membrane architectures of the MED4 (high-light) Prochlorococcus ecotype grown under high-light and low-light conditions in addition to the MIT9313 (low-light) and SS120 (low-light) Prochlorococcus ecotypes grown under low-light conditions. Mass spectrometry quantified the relative abundance of PSI, photosystem II (PSII) and cytochrome b6f complexes and the various Pcb proteins in the thylakoid membrane. Atomic force microscopy topographs and structural modelling revealed a series of specialized PSI configurations, each adapted to the environmental niche occupied by a particular ecotype. MED4 PSI domains were loosely packed in the thylakoid membrane, whereas PSI in the low-light MIT9313 is organized into a tightly packed pseudo-hexagonal lattice that maximizes harvesting and trapping of light. There are approximately equal levels of PSI and PSII in MED4 and MIT9313, but nearly twofold more PSII than PSI in SS120, which also has a lower content of cytochrome b6f complexes. SS120 has a different tactic to cope with low-light levels, and SS120 thylakoids contained hundreds of closely packed Pcb-PSI supercomplexes that economize on the extra iron and nitrogen required to assemble PSI-only domains. Thus, the abundance and widespread distribution of Prochlorococcus reflect the strategies that various ecotypes employ for adapting to limitations in light and nutrient levels.

Methylene-linked bis-phenylbenzimidazoles - a new scaffold to target telomeric DNA/RNA hybrid duplex.

We report a series of novel methylene-linked bis-phenylbenzimidazoles intercalators that stabilize telomeric DNA/RNA hybrid (tDRH) structures by up to 7.2 °C at a 1 µM ligand concentration while having negligible affinity for DNA/DNA duplexes, although with a low affinity for quadruplex DNA. We have used molecular modelling studies to rationalize this selectivity, concluding that the methylene spacer between the terminal benzimidazole and phenylene moieties plays a key role in facilitating the bis-intercalating process. This scaffold may be used to develop chemical tools or new therapeutics to selectively target the telomeric DNA/RNA duplex without affecting normal genomic DNA.

Hot, humid air decontamination of a C-130 aircraft contaminated with spores of two acrystalliferous Bacillus thuringiensis strains, surrogates for Bacillus anthracis.

AIM: To develop test methods and evaluate survival of Bacillus thuringiensis kurstaki cry(-) HD-1 and B. thuringiensis Al Hakam spores after exposure to hot, humid air inside of a C-130 aircraft. METHODS AND RESULTS: Bacillus thuringiensis spores were either pre-inoculated on 1 × 2 or 2 × 2 cm substrates or aerosolized inside the cargo hold of a C-130 and allowed to dry. Dirty, complex surfaces (10 × 10 cm) swabbed after spore dispersal showed a deposition of 8-10 log10 m(-2) through the entire cargo hold. After hot, humid air decontamination at 75-80°C, 70-90% relative humidity for 7 days, 87 of 98 test swabs covering 0·98 m(2) , showed complete spore inactivation. There was a total of 1·67 log10 live CFU detected in 11 of the test swabs. Spore inactivation in the 98 test swabs was measured at 7·06 log10 m(-2) . CONCLUSIONS: Laboratory test methods for hot, humid air decontamination were scaled for a large-scale aircraft field test. The C-130 field test demonstrated that hot, humid air can be successfully used to decontaminate an aircraft. SIGNIFICANCE AND IMPACT OF THE STUDY: Transition of a new technology from research and development to acquisition at a Technology Readiness Level 7 is unprecedented.

Genetic diversity among Botulinum Neurotoxin-producing clostridial strains.

Clostridium botulinum is a taxonomic designation for many diverse anaerobic spore-forming rod-shaped bacteria that have the common property of producing botulinum neurotoxins (BoNTs). The BoNTs are exoneurotoxins that can cause severe paralysis and death in humans and other animal species. A collection of 174 C. botulinum strains was examined by amplified fragment length polymorphism (AFLP) analysis and by sequencing of the 16S rRNA gene and BoNT genes to examine the genetic diversity within this species. This collection contained representatives of each of the seven different serotypes of botulinum neurotoxins (BoNT/A to BoNT/G). Analysis of the16S rRNA gene sequences confirmed previous identifications of at least four distinct genomic backgrounds (groups I to IV), each of which has independently acquired one or more BoNT genes through horizontal gene transfer. AFLP analysis provided higher resolution and could be used to further subdivide the four groups into subgroups. Sequencing of the BoNT genes from multiple strains of serotypes A, B, and E confirmed significant sequence variation within each serotype. Four distinct lineages within each of the BoNT A and B serotypes and five distinct lineages of serotype E strains were identified. The nucleotide sequences of the seven toxin genes of the serotypes were compared and showed various degrees of interrelatedness and recombination, as was previously noted for the nontoxic nonhemagglutinin gene, which is linked to the BoNT gene. These analyses contribute to the understanding of the evolution and phylogeny within this species and assist in the development of improved diagnostics and therapeutics for the treatment of botulism.

Fluorescent Amplified Fragment Length Polymorphism Analysis of Norwegian Bacillus cereus and Bacillus thuringiensis Soil Isolates.

We examined 154 Norwegian B. cereus and B. thuringiensis soil isolates (collected from five different locations), 8 B. cereus and 2 B. thuringiensis reference strains, and 2 Bacillus anthracis strains by using fluorescent amplified fragment length polymorphism (AFLP). We employed a novel fragment identification approach based on a hierarchical agglomerative clustering routine that identifies fragments in an automated fashion. No method is free of error, and we identified the major sources so that experiments can be designed to minimize its effect. Phylogenetic analysis of the fluorescent AFLP results reveals five genetic groups in these group 1 bacilli. The ATCC reference strains were restricted to two of the genetic groups, clearly not representative of the diversity in these bacteria. Both B. anthracis strains analyzed were closely related and affiliated with a B. cereus milk isolate (ATCC 4342) and a B. cereus human pathogenic strain (periodontitis). Across the entire study, pathogenic strains, including B. anthracis, were more closely related to one another than to the environmental isolates. Eight strains representing the five distinct phylogenetic clusters were further analyzed by comparison of their 16S rRNA gene sequences to confirm the phylogenetic status of these groups. This analysis was consistent with the AFLP analysis, although of much lower resolution. The innovation of automated genotype analysis by using a replicated and statistical approach to fragment identification will allow very large sample analyses in the future.

Frication noise modulated by voicing, as revealed by pitch-scaled decomposition.

A decomposition algorithm that uses a pitch-scaled harmonic filter was evaluated using synthetic signals and applied to mixed-source speech, spoken by three subjects, to separate the voiced and unvoiced parts. Pulsing of the noise component was observed in voiced frication, which was analyzed by complex demodulation of the signal envelope. The timing of the pulsation, represented by the phase of the anharmonic modulation coefficient, showed a step change during a vowel-fricative transition corresponding to the change in location of the noise source within the vocal tract. Analysis of fricatives [see text] demonstrated a relationship between steady-state phase and place, and f0 glides confirmed that the main cause was a place-dependent delay.

Multiple-locus variable-number tandem repeat analysis reveals genetic relationships within Bacillus anthracis.

Bacillus anthracis is one of the most genetically homogeneous pathogens described, making strain discrimination particularly difficult. In this paper, we present a novel molecular typing system based on rapidly evolving variable-number tandem repeat (VNTR) loci. Multiple-locus VNTR analysis (MLVA) uses the combined power of multiple alleles at several marker loci. In our system, fluorescently labeled PCR primers are used to produce PCR amplification products from eight VNTR regions in the B. anthracis genome. These are detected and their sizes are determined using an ABI377 automated DNA sequencer. Five of these eight loci were discovered by sequence characterization of molecular markers (vrrC(1), vrrC(2), vrrB(1), vrrB(2), and CG3), two were discovered by searching complete plasmid nucleotide sequences (pXO1-aat and pXO2-at), and one was known previously (vrrA). MLVA characterization of 426 B. anthracis isolates identified 89 distinct genotypes. VNTR markers frequently identified multiple alleles (from two to nine), with Nei's diversity values between 0.3 and 0.8. Unweighted pair-group method arithmetic average cluster analysis identified six genetically distinct groups that appear to be derived from clones. Some of these clones show worldwide distribution, while others are restricted to particular geographic regions. Human commerce doubtlessly has contributed to the dispersal of particular clones in ancient and modern times.

Diversity in a variable-number tandem repeat from Yersinia pestis.

We have identified a tetranucleotide repeat sequence, (CAAA)(N), in the genome of Yersinia pestis, the causative agent of plague. This variable-number tandem repeat (VNTR) region has nine alleles and great diversity (calculated as 1 minus the sum of the squared allele frequencies) (diversity value, 0.82) within a set of 35 diverse Y. pestis strains. In contrast, the nucleotide sequence of the lcrV (low-calcium-response) gene differed only slightly among these strains, having a haplotype diversity value of 0.17. Replicated cultures, phenotypic variants of particular strains, and extensively cultured replicates within strains did not differ in VNTR allele type. Thus, while a high mutation rate must contribute to the great diversity of this locus, alleles appear stable under routine laboratory culture conditions. The classic three plague biovars did not have single identifying alleles, although there were allelic biases within biovar categories. The antiqua biovar was the most diverse, with four alleles observed in 5 strains, while the orientalis and mediaevalis biovars exhibited five alleles in 21 strains and three alleles in 8 strains, respectively. The CAAA VNTR is located immediately adjacent to the transcriptional promoters for flanking open reading frames and may affect their activity. This VNTR marker may provide a high-resolution tool for epidemiological analyses of plague.

Sequence and organization of pXO1, the large Bacillus anthracis plasmid harboring the anthrax toxin genes.

The Bacillus anthracis Sterne plasmid pXO1 was sequenced by random, "shotgun" cloning. A circular sequence of 181,654 bp was generated. One hundred forty-three open reading frames (ORFs) were predicted using GeneMark and GeneMark.hmm, comprising only 61% (110,817 bp) of the pXO1 DNA sequence. The overall guanine-plus-cytosine content of the plasmid is 32.5%. The most recognizable feature of the plasmid is a "pathogenicity island," defined by a 44.8-kb region that is bordered by inverted IS1627 elements at each end. This region contains the three toxin genes (cya, lef, and pagA), regulatory elements controlling the toxin genes, three germination response genes, and 19 additional ORFs. Nearly 70% of the ORFs on pXO1 do not have significant similarity to sequences available in open databases. Absent from the pXO1 sequence are homologs to genes that are typically required to drive theta replication and to maintain stability of large plasmids in Bacillus spp. Among the ORFs with a high degree of similarity to known sequences are a collection of putative transposases, resolvases, and integrases, suggesting an evolution involving lateral movement of DNA among species. Among the remaining ORFs, there are three sequences that may encode enzymes responsible for the synthesis of a polysaccharide capsule usually associated with serotype-specific virulent streptococci.

Molecular diversity in Bacillus anthracis.

Molecular typing of Bacillus anthracis has been extremely difficult due to the lack of polymorphic DNA markers. We have identified nine novel variable number tandemly repeated loci from previously known amplified fragment length polymorphism markers or from the DNA sequence. In combination with the previously known vrrA locus, these markers provide discrimination power to genetically characterize B. anthracis isolates. The variable number tandem repeat (VNTR) loci are found in both gene coding (genic) and non-coding (non-genic) regions. The genic differences are 'in frame' and result in additions or deletion of amino acids to the predicted proteins. Due the rarity of molecular differences, the VNTR changes represent a significant portion of the genetic variation found within B. anthracis. This variation could represent an important adaptive mechanism. Marker similarity and differences among diverse isolates have identified seven major diversity groups that may represent the only world-wide B. anthracis clones. The lineages reconstructed using these data may reflect the dispersal and evolution of this pathogen.

Genetic comparison of Bacillus anthracis and its close relatives using amplified fragment length polymorphism and polymerase chain reaction analysis.

Amplified fragment length polymorphism (AFLP) analysis allows a rapid, relatively simple analysis of a large portion of a microbial genome, providing information about the species and its phylogenetic relationship to other microbes (Vos et al. 1995). The method simply surveys the genome for length and sequence polymorphisms. The AFLP pattern identified can be used for comparison to the genomes of other species. Unlike other methods, it does not rely on analysis of a single genetic locus that may bias the interpretation of results and does not require any prior knowledge of the targeted organism. Moreover, a standard set of reagents can be applied to any species without using species-specific information or molecular probes. We are using AFLP analysis to rapidly identify different bacterial species. A comparison of AFLP profiles generated from a large battery of Bacillus anthracis strains shows very little variability among different isolates (Keim et al. 1997). By contrast, there is a significant difference between AFLP profiles generated for any B. anthracis strain and even the most closely related Bacillus species. Sufficient variability is apparent among all known microbial species to allow phylogenetic analysis based on large numbers of genetically unlinked loci. These striking differences among AFLP profiles allow unambiguous identification of previously identified species and phylogenetic placement of newly characterized isolates relative to known species based on a large number of independent genetic loci. Data generated thus far show that the method provides phylogenetic analyses that are consistent with other widely accepted phylogenetic methods. However, AFLP analysis provides a more detailed analysis of the targets and samples a much larger portion of the genome. Consequently, it provides an inexpensive, rapid means of characterizing microbial isolates to further differentiate among strains and closely related microbial species. Such information cannot be rapidly generated by other means. AFLP sample analysis quickly generates a very large amount of molecular information about microbial genomes. However, this information cannot be analysed rapidly using manual methods. We are developing a large archive of electronic AFLP signatures that is being used to identify isolates collected from medical, veterinary, forensic and environmental samples. We are also developing the computational packages necessary to rapidly and unambiguously analyse the AFLP profiles and conduct a phylogenetic comparison of these data relative to information already in our database. We will use this archive and the associated algorithms to determine the species identity of previously uncharacterized isolates and place them phylogenetically relative to other microbes based on their AFLP signatures. This study provides significant new information about microbes with environmental, veterinary and medical significance. This information can be used in further studies to understand the relationships among these species and the factors that distinguish them from one another. It should also allow the identification of unique factors that contribute to important microbial traits, including pathogenicity and virulence. We are also using AFLP data to identify, isolate and sequence DNA fragments that are unique to particular microbial species and strains. The fragment patterns and sequence information provide insights into the complexity and organization of bacterial genomes relative to one another. They also provide the information necessary for the development of species-specific polymerase chain reaction primers that can be used to interrogate complex samples for the presence of B. anthracis, other microbial pathogens or their remnants.

Disruption of anthrax toxin binding with the use of human antibodies and competitive inhibitors.

The protective antigen (PA83) of Bacillus anthracis is integral to the mechanism of anthrax toxicity. We have isolated a human single-chain Fv antibody fragment (scFv) that blocks binding of a fluorescently tagged protective antigen (PA) moiety to cell surface receptors. Several phage-displayed scFv were isolated from a naive library biopanned against PA83. Soluble, monomeric scFv were characterized for affinity and screened for their capacity to disrupt receptor-mediated binding of PA. Four unique scFv bound to PA83, as determined by surface plasmon resonance, the tightest binder exhibiting a Kd of 50 nM. Two scFv had similar affinities for natural PA83 and a novel, recombinant, 32-kDa carboxy-terminal PA fragment (PA32). Binding of scFv to green fluorescent protein fused to the amino-terminal 32-kDa fragment of B. anthracis edema factor, EGFP-EF32, was used to confirm specificity. Fusion of EGFP to PA32 facilitated development of a novel flow cytometric assay that showed that one of the scFv disrupted PA receptor binding. This method can now be used as a rapid assay for small molecule inhibitors of PA binding to cell receptors. The combined data presented suggest the potential utility of human scFv as prophylactics against anthrax poisoning. Moreover, recombinant PA32 may also be useful as a therapeutic agent to compete with anthrax toxins for cellular receptors during active infection.

Genetic diversity in the protective antigen gene of Bacillus anthracis.

Bacillus anthracis is a gram-positive spore-forming bacterium that causes the disease anthrax. The anthrax toxin contains three components, including the protective antigen (PA), which binds to eucaryotic cell surface receptors and mediates the transport of toxins into the cell. In this study, the entire 2,294-nucleotide protective antigen gene (pag) was sequenced from 26 of the most diverse B. anthracis strains to identify potential variation in the toxin and to further our understanding of B. anthracis evolution. Five point mutations, three synonymous and two missense, were identified. These differences correspond to six different haploid types, which translate into three different amino acid sequences. The two amino acid changes were shown to be located in an area near a highly antigenic region critical to lethal factor binding. Nested primers were used to amplify and sequence this same region of pag from necropsy samples taken from victims of the 1979 Sverdlovsk incident. This investigation uncovered five different alleles among the strains present in the tissues, including two not seen in the 26-sample survey. One of these two alleles included a novel missense mutation, again located just adjacent to the highly antigenic region. Phylogenetic (cladistic) analysis of the pag corresponded with previous strain grouping based on chromosomal variation, suggesting that plasmid evolution in B. anthracis has occurred with little or no horizontal transfer between the different strains.

Origin of dimeric structure in the ribonuclease superfamily.

To enable application of postgenomic evolutionary approaches to understand the divergence of behavior and function in ribonucleases (RNases), the impact of divergent sequence on the divergence of tertiary and quaternary structure is analyzed in bovine pancreatic and seminal ribonucleases, which differ by 23 amino acids. In a crystal, seminal RNase is a homodimer joined by two "antiparallel" intersubunit disulfide bonds between Cys-31 from one subunit and Cys-32' from the other and having composite active sites arising from the "swap" of residues 1-20 from each subunit. Specialized Edman degradation techniques have completed the structural characterization of the dimer in solution, new cross-linking methods have been developed to assess the swap, and sequence determinants of quaternary structure have been explored by protein engineering using the reconstructed evolutionary history of the protein family as a guide. A single Cys at either position 32 (the first to be introduced during the divergent evolution of the family) or 31 converts monomeric RNase A into a dimer. Even with an additional Phe at position 31, another residue introduced early in the seminal lineage, swap is minimal. A hydrophobic contact formed by Leu-28, however, also introduced early in the seminal lineage, increases the amount of "antiparallel" connectivity of the two subunits and facilitates swapping of residues 1-20. Efficient swapping requires addition of a Pro at position 19, a residue also introduced early in the divergent evolution of the seminal RNase gene. Additional cysteines required for dimer formation are found to slow refolding of the protein through formation of incorrect disulfide bonds, suggesting a paradox in the biosynthesis of the protein. Further studies showed that the dimeric form of seminal RNase known in the crystal is not the only form in vivo, where a substantial amount of heterodimer is known. These data complete the acquisition of the background needed to understand the evolution of new structure, behavior, and function in the seminal RNase family of proteins.

Development and testing of a bacterial biosensor for toluene-based environmental contaminants.

A bacterial biosensor for benzene, toluene, and similar compounds has been constructed, characterized, and field tested on contaminated water and soil. The biosensor is based on a plasmid incorporating the transcriptional activator xylR from the TOL plasmid of Pseudomonas putida mt-2. The XylR protein binds a subset of toluene-like compounds and activates transcription at its promoter, Pu. A reporter plasmid was constructed by placing the luc gene for firefly luciferase under the control of XylR and Pu. When Escherichia coli cells were transformed with this plasmid vector, luminescence from the cells was induced in the presence of benzene, toluene, xylenes, and similar molecules. Accurate concentration dependencies of luminescence were obtained and exhibited K1/2 values ranging from 39.0 +/- 3.8 microM for 3-xylene to 2,690 +/- 160 microM for 3-methylbenzylalcohol (means +/- standard deviations). The luminescence response was specific for only toluene-like molecules that bind to and activate XylR. The biosensor cells were field tested on deep aquifer water, for which contaminant levels were known, and were able to accurately detect toluene derivative contamination in this water. The biosensor cells were also shown to detect BETX (benzene, toluene, and xylene) contamination in soil samples. These results demonstrate the capability of such a bacterial biosensor to accurately measure environmental contaminants and suggest a potential for its inexpensive application in field-ready assays.

PCR analysis of tissue samples from the 1979 Sverdlovsk anthrax victims: the presence of multiple Bacillus anthracis strains in different victims.

An outbreak of human anthrax occurred in Sverdlovsk, Union of Soviet Socialists Republic (now Ekaterinburg, Russia) in April 1979. Officials attributed this to consumption of contaminated meat, but Western governments believed it resulted from inhalation of spores accidentally released from a nearby military research facility. Tissue samples from 11 victims were obtained and methods of efficiently extracting high-quality total DNA from these samples were developed. Extracted DNA was analyzed by using PCR to determine whether it contained Bacillus anthracis-specific sequences. Double PCR using "nested primers" increased sensitivity of the assay significantly. Tissue samples from 11 persons who died during the epidemic were examined. Results demonstrated that the entire complement of B. anthracis toxin and capsular antigen genes required for pathogenicity were present in tissues from each of these victims. Tissue from a vaccination site contained primarily nucleic acids from a live vaccine, although traces of genes from the infecting organisms were also present. PCR analysis using primers that detect the vrrA gene variable region on the B. anthracis chromosome demonstrated that at least four of the five known strain categories defined by this region were present in the tissue samples. Only one category is found in a single B. anthracis strain.

Characterization of the variable-number tandem repeats in vrrA from different Bacillus anthracis isolates.

PCR analysis of 198 Bacillus anthracis isolates revealed a variable region of DNA sequence differing in length among the isolates. Five polymorphisms differed by the presence of two to six copies of the 12-bp tandem repeat 5'-CAATATCAACAA-3'. This variable-number tandem repeat (VNTR) region is located within a larger sequence containing one complete open reading frame that encodes a putative 30-kDa protein. Length variation did not change the reading frame of the encoded protein and only changed the copy number of a 4-amino-acid sequence (QYQQ) from 2 to 6. The structure of the VNTR region suggests that these multiple repeats are generated by recombination or polymerase slippage. Protein structures predicted from the reverse-translated DNA sequence suggest that any structural changes in the encoded protein are confined to the region encoded by the VNTR sequence. Copy number differences in the VNTR region were used to define five different B. anthracis alleles. Characterization of 198 isolates revealed allele frequencies of 6.1, 17.7, 59.6, 5.6, and 11.1% sequentially from shorter to longer alleles. The high degree of polymorphism in the VNTR region provides a criterion for assigning isolates to five allelic categories. There is a correlation between categories and geographic distribution. Such molecular markers can be used to monitor the epidemiology of anthrax outbreaks in domestic and native herbivore populations.

Protease digestion studies of an equilibrium intermediate in the unfolding of creatine kinase.

Protease digestion experiments have been used to characterize the structure of an equilibrium intermediate in the unfolding of creatine kinase (CK) by low concentrations (0.625 M) of guanidine hydrochloride (GdnHCl). Eighteen of the major products of digestion by trypsin, chymotrypsin and endoproteinase Glu-C have been identified by microsequencing after separation by SDS/PAGE and electroblotting on poly(vinylidene difluoride) membranes. The C-terminal portion (Gly215 to Lys380) was much more resistant to digestion than the N-terminal portion (Pro1 to Gly133), although the area most sensitive to proteolysis was in the middle of the CK sequence (Arg134 to Arg214). These experiments are consistent with the two-domain model for the CK monomer. The structure of the intermediate is proposed to consist of a folded C-terminal domain and a partly folded N-terminal domain separated by an unfolded central linker. Protease susceptibility is clustered within two N-terminal regions and one central region. These regions are evidently exposed as a result of the partial unfolding and/or separation of the N-terminal domain. Further evidence for the structure of this intermediate comes from gel filtration studies. Treatment of CK with 0.625 M GdnHCl resulted in slow aggregation at 37 degrees C, but not at 12 degrees C, a phenomenon previously reported for phosphoglycerate kinase. The aggregation did not occur at higher GdnHCl concentrations and was unaffected by a reducing agent. It is proposed that aggregation is a consequence of non-specific interactions between hydrophobic regions, possibly domain/domain interfaces, which become exposed in the intermediate.

Subcellular Location of O-Acetylserine Sulfhydrylase Isoenzymes in Cell Cultures and Plant Tissues of Datura innoxia Mill.

O-Acetylserine sulfhydrylase (OASS; EC 4.2.99.8) catalyzes the formation of L-cysteine from O-acetylserine and inorganic sulfide. Three OASS isoenzymes that differ in molecular mass and subunit structure are present in shoot and root tissues and in cadmium-resistant and cadmium-susceptible cell cultures of Datura innoxia Mill. Different OASS forms predominate in leaves, roots, and suspension-cell cultures. To determine the subcellular location of the OASS isoenzymes, purified mitochondria, chloroplasts, and cytosolic fractions from protoplasts were obtained. The isoenzymes are compartmentalized in D. innoxia cells, with a different isoenzyme predominant in the chloroplast, cytosol, and mitochondria, suggesting that they serve different functions in the plant cell. The chloroplast form is most abundant in green leaves and leaf protoplasts. The cytosolic form is most abundant in roots and cell cultures. A mitochondrial form is abundant in cell cultures, but is a minor form in leaves or roots. Cadmium-tolerant cell cultures contain 1.8 times as much constitutive OASS activity as the wild-type cell line, and 2.9 times more than the cadmium-hypersensitive cell line. This may facilitate rapid production of glutathione and metal-binding phytochelatins when these cultures are exposed to cadmium.

Identification of trimetazidine metabolites in human urine and plasma.

1. The objective was to use modern mass spectrometric techniques to update current information on the metabolism of trimetazidine in human subjects found by previous studies. 2. Urine and plasma samples were taken from four healthy human volunteers taking part in a larger kinetic study. Each subject received an oral dose of 80-mg trimetazidine daily for 4 days. 3. Identification and quantitation of trimetazidine and its metabolites in urine and plasma were achieved using modern liquid chromatography-mass spectrometric methods. 4. The major drug-related component observed in urine and plasma was unchanged trimetazidine. In addition to the parent drug, 10 metabolites were detected in urine in concentrations ranging from 0.008 (0.01% dose) to 1.094 micrograms.ml-1 (1.4% dose). Metabolic profiles following acute and chronic doses of trimetazidine were qualitatively similar.