Analysis of a microbial community oxidizing inorganic sulfide and mercaptans.

Successful treatment of refinery spent-sulfidic caustic (which results from the addition of sodium hydroxide solutions to petroleum refinery waste streams) was achieved in a bioreactor containing an enrichment culture immobilized in organic polymer beads with embedded powdered activated carbon (Bio-Sep). The aerobic enrichment culture had previously been selected using a gas mixture of hydrogen sulfide and methyl mercaptan (MeSH) as the sole carbon and energy sources. The starting cultures for the enrichment consisted of several different Thiobacilli spp. (T. thioparus, T. denitrificans, T. thiooxidans, and T. neopolitanus), as well as activated sludge from a refinery aerobic wastewater treatment system and sludge from an industrial anaerobic digester. Microscopic examination (light and SEM) of the beads and of microbial growth on the walls of the bioreactor revealed a great diversity of microorganisms. Further characterization was undertaken starting with culturable aerobic heterotrophic microorganisms (sequencing of PCR-amplified DNA coding for 16S rRNA, Gram staining) and by PCR amplification of DNA coding for 16S rRNA extracted directly from the cell mass, followed by the separation of the PCR products by DGGE (denaturing gradient gel electrophoresis). Eight prominent bands from the DGGE gel were sequenced and found to be closest to sequences of uncultured Cytophagales (3 bands), Gram-positive cocci (Micrococcineae), alpha proteobacteria (3 bands), and an unidentified beta proteobacterium. Culturable microbes included several genera of fungi as well as various Gram-positive and Gram-negative heterotrophic bacteria not seen in techniques using direct DNA extraction.

Value of serum-soluble intercellular adhesion molecule-1 for the noninvasive risk assessment of transplant coronary artery disease, posttransplant ischemic events, and cardiac graft failure.

BACKGROUND: Adhesion molecules on arterial endothelium have been implicated in spontaneous atherosclerosis and transplant coronary artery disease (CAD). We studied whether elevated serum-soluble intercellular adhesion molecule-1 (sICAM-1) during the immediate posttransplant period was a risk factor for CAD, posttransplant ischemic events, or cardiac graft failure. METHODS AND RESULTS: We initially studied serum sICAM-1 in a subset of 16 cardiac allograft recipients (5.5+/-0.7 samples per patient) to determine a cutoff point that best correlated with presence of arterial and arteriolar endothelial ICAM-1 in matching endomyocardial biopsies. The cutoff value was 308 ng/mL. Subsequently, we prospectively evaluated serum sICAM-1 in serial samples (5.3+/-0.1 per patient) obtained during the first 3 months after transplantation in a validation subset of 130 recipients and correlated early sICAM-1 levels with long-term outcome. Serum sICAM-1 >308 ng/mL correlated significantly with ICAM-1 on arterial and arteriolar endothelium (P:=0.02). Cardiac allograft recipients with serum sICAM-1 >308 ng/mL had 2.67 (95% CI, 1.28 to 5.59, P:=0.009) times greater risk of CAD and 3.63 (95% CI, 1.05 to 12.5, P:=0.04) times greater risk of graft failure. Recipients with sICAM-1 >308 ng/mL also developed more severe CAD (P:=0.009) and more ischemic events (P:=0.03) after transplantation. CONCLUSIONS: Serum sICAM-1 levels can be used to noninvasively assess risk of transplant CAD, posttransplant ischemic events, and cardiac graft failure.

Biotreatment of refinery spent-sulfidic caustic using an enrichment culture immobilized in a novel support matrix.

Sodium hydroxide solutions are used in petroleum refining to remove hydrogen sulfide (H2S) and mercaptans from various hydrocarbon streams. The resulting sulfide-laden waste stream is called spent-sulfidic caustic. An aerobic enrichment culture was previously developed using a gas mixture of H2S and methyl-mercaptan (MeSH) as the sole energy source. This culture has now been immobilized in a novel support matrix, DuPont BIO-SEP beads, and is used to bio-treat a refinery spent-sulfidic caustic containing both inorganic sulfide and mercaptans in a continuous flow, fluidized-bed column bioreactor. Complete oxidation of both inorganic and organic sulfur to sulfate was observed with no breakthrough of H2S and < 2 ppmv of MeSH produced in the bioreactor outlet gas. Excessive buildup of sulfate (> 12 g/L) in the bioreactor medium resulted in an upset condition evidenced by excessive MeSH breakthrough. Therefore, bioreactor performance was limited by the steady-state sulfate concentration. Further improvement in volumetric productivity of a bioreactor system based on this enrichment culture will be dependent on maintenance of sulfate concentrations below inhibitory levels.

Determining the physical limits of the Brassica S locus by recombinational analysis.

A genetic analysis was performed to study the frequency of recombination for intervals across the Brassica S locus region. No recombination was observed between the S locus glycoprotein gene and the S receptor kinase gene in the segregating populations that we analyzed. However, a number of recombination breakpoints in regions flanking these genes were identified, allowing the construction of an integrated genetic and physical map of the genomic region encompassing one S haplotype. We identified, based on the pollination phenotype of plants homozygous for recombinant S haplotypes, a 50-kb region that encompasses all specificity functions in the S haplotype that we analyzed. Mechanisms that might operate to preserve the tight linkage of self-incompatibility specificity genes within the S locus complex are discussed in light of the relatively uniform recombination frequencies that we observed across the S locus region and of the structural heteromorphisms that characterize different S haplotypes.

Comparative mapping of the Brassica S locus region and its homeolog in Arabidopsis. Implications for the evolution of mating systems in the Brassicaceae.

The crucifer family includes self-incompatible genera, such as Brassica, and self-fertile genera, such as Arabidopsis. To gain insight into mechanisms underlying the evolution of mating systems in this family, we used a selective comparative mapping approach between Brassica campestris plants homozygous for the S8 haplotype and Arabidopsis. Starting with markers flanking the self-incompatibility genes in Brassica, we identified the homeologous region in Arabidopsis as a previously uncharacterized segment of chromosome 1 in the immediate vicinity of the ethylene response gene ETR1. A total of 26 genomic and 21 cDNA markers derived from Arabidopsis yeast artificial and bacterial artificial chromosome clones were used to analyze this region in the two genomes. Approximately half of the cDNAs isolated from the region represent novel expressed sequence tags that do not match entries in the DNA and protein databases. The physical maps that we derived by using these markers as well as markers isolated from bacteriophage clones spanning the S8 haplotype revealed a high degree of synteny at the submegabase scale between the two homeologous regions. However, no sequences similar to the Brassica S locus genes that are known to be required for the self-incompatibility response were detected within this interval or other regions of the Arabidopsis genome. This observation is consistent with deletion of self-recognition genes as a mechanism for the evolution of autogamy in the Arabidopsis lineage.

A significant fraction of calcium transients in intact guinea pig ventricular myocytes is mediated by Na(+)-Ca2+ exchange.

Ca2+ mobilization elicited by simulation with brief pulses of high K+ were monitored with confocal laser scanned microscopy in intact, guinea pig cardiac myocytes loaded with the calcium indicator fluo-3. Single wavelength ratioing of fluorescence images obtained after prolonged integration times revealed non-uniformities of intracellular Ca2+ changes across the cell, suggesting the presence of significant spatial Ca2+ gradients. Treatment with 20 microM ryanodine, an inhibitor of Ca2+ release from the SR, and 10 microM verapamil, a calcium channel blocker, reduced by 42% and 76% respectively the changes in [Ca2+]i elicited by membrane depolarization. The overall spatial distribution of [Ca2+]i changes appeared unchanged. Ca2+ transients recorded in the presence of verapamil and ryanodine (about 20% of the size of control responses), diminished in the presence of 50 microM 2-4 Dichlorbenzamil (DCB) or 5 mM nickel, two relatively specific inhibitors of the Na+/Ca2+ exchange mechanism. Conversely, when the reversal potential of the Na+/Ca2+ exchange was shifted to negative potentials by lowering [NA+]o or by increasing [Na+]i by treatment with 20 microM monensin, the amplitude of these Ca2+ transients increased. Ca2+ transients elicited by membrane depolarization and largely mediated by reverse operation of Na(+)-Ca2+ exchange could be recorded in the presence of ryanodine, verapamil and monensin. These finding suggest that in intact guinea pig cardiac cells, Ca2+ influx through the Na+/Ca2+ exchange mechanism activated by a membrane depolarization in the physiological range can be sufficient to play a significant role in excitation-contraction coupling.