Scale-up issues for in situ anaerobic tetrachloroethene bioremediation

For the full scale implementation of in situ anaerobic bioremediation of tetrachloroethene (PCE) in groundwater, the following issues must be addressed: which organic substrates at which concentration would be most effective in promoting dechlorination and are economical; how far the substrate, electron acceptor, and nutrients can be transported in the aquifer; and the placement of delivery and recovery wells for distributing these amendments. In a microcosm study, almost all of the tested inexpensive substrates supported reductive dechlorination of PCE through vinyl chloride (VC) under methanogenic conditions. A minimum of about 60 mg L⁻¹ of organic carbon was needed to dechlorinate 23 μM PCE with a single feeding. In a second microcosm study dechlorination stopped at 1,2-dichloroethene (DCE) in microcosms fed higher concentrations of several substrates. At the highest concentrations the substrates inhibited DCE production. Three field tracer tests were conducted to evaluate methods to distribute the amendments across the aquifer. The natural groundwater gradient is not sufficient to distribute substrate evenly. Groundwater injection at 60 times the natural flux rate increased the distribution of substrate. A mixing strategy of cross-gradient injection further increased the distribution of the substrate. Ammonia-nitrogen, sulfate, and phosphate were retarded relative to the substrate and inorganic tracer. Received 30 October 1995/ Accepted in revised form 07 June 1996     

Survival of Desulfotomaculum spores from estuarine sediments after serial autoclaving and high-temperature exposure

Bacterial spores are widespread in marine sediments, including those of thermophilic, sulphate-reducing bacteria, which have a high minimum growth temperature making it unlikely that they grow in situ. These Desulfotomaculum spp. are thought to be from hot environments and are distributed by ocean currents. Their cells and spores upper temperature limit for survival is unknown, as is whether they can survive repeated high-temperature exposure that might occur in hydrothermal systems. This was investigated by incubating estuarine sediments significantly above (40–80 °C) maximum in situ temperatures (∼23 °C), and with and without prior triple autoclaving. Sulphate reduction occurred at 40–60 °C and at 60 °C was unaffected by autoclaving. Desulfotomaculum sp. C1A60 was isolated and was most closely related to the thermophilic D. kuznetsovii^T (∼96% 16S rRNA gene sequence identity). Cultures of Desulfotomaculum sp. C1A60, D. kuznetsovii^Tand D. geothermicum B2T survived triple autoclaving while other related Desulfotomaculum spp. did not, although they did survive pasteurisation. Desulfotomaculum sp. C1A60 and D. kuznetsovii cultures also survived more extreme autoclaving (C1A60, 130 °C for 15 min; D. kuznetsovii, 135 °C for 15 min, maximum of 154 °C reached) and high-temperature conditions in an oil bath (C1A60, 130° for 30 min, D. kuznetsovii 140 °C for 15 min). Desulfotomaculum sp. C1A60 with either spores or predominantly vegetative cells demonstrated that surviving triple autoclaving was due to spores. Spores also had very high culturability compared with vegetative cells (∼30 × higher). Combined extreme temperature survival and high culturability of some thermophilic Desulfotomaculum spp. make them very effective colonisers of hot environments, which is consistent with their presence in subsurface geothermal waters and petroleum reservoirs.     

MAP2 IHC detection: a marker of antigenicity in CNS tissues

Immunohistochemistry (IHC) is used to detect antibody-specific antigens in tissues; the results depend on the ability of the primary antibodies to bind to their antigens. Therefore, results depend on the quality of preservation of the specimen. Many investigators have overcome the deleterious effects of over-fixation on the binding of primary antibodies to specimen antigens using IHC, but if the specimen is under-fixed or fixation is delayed, false negative results could be obtained despite certified laboratory practices. Microtubule-associated protein 2 (MAP2) is an abundant microtubule-associate protein that participates in the outgrowth of neuronal processes and synaptic plasticity; it is localized primarily in cell bodies and dendrites of neurons. MAP2 immunolabeling has been reported to be absent in areas of the entorhinal cortex and hippocampus of Alzheimer’s disease brains that were co-localized with the dense-core type of amyloid plaques. It was hypothesized that the lack of MAP2 immunolabeling in these structures was due to the degradation of the MAP2 antigen by the neuronal proteases that were released as the neurons lysed leading to the formation of these plaques. Because MAP2 is sensitive to proteolysis, we hypothesized that changes in MAP2 immunolabeling may be correlated with the degree of fixation of central nervous system (CNS) tissues. We detected normal MAP2 immunolabeling in fixed rat brain tissues, but MAP2 immunolabeling was decreased or lost in unfixed and delayed-fixed rat brain tissues. By contrast, two ubiquitous CNS-specific markers, myelin basic protein and glial fibrillary acidic protein, were unaffected by the degree of fixation in the same tissues. Our observations suggest that preservation of various CNS-specific antigens differs with the degree of fixation and that the lack of MAP2 immunolabeling in the rat brain may indicate inadequate tissue fixation. We recommend applying MAP2 IHC for all CNS tissues as a pre-screen to assess the quality of the tissue preservation and to avoid potentially false negative IHC results.     

Mannan transglycosylase: a novel enzyme activity in cell walls of higher plants

Mannan transglycosylase is a novel cell wall enzyme activity acting on mannan-based plant polysaccharides in primary cell walls of monocotyledons and dicotyledons. The enzyme activity was detected by its ability to transfer galactoglucomannan (GGM) polysaccharides to tritium-labelled GGM-derived oligosaccharides generating tritium-labelled GGM polysaccharides. Mannan transglycosylase was found in a range of plant species and tissues. High levels of the enzyme activity were present in flowers of some kiwifruit (Actinidia) species and in ripe tomato (Solanum lycopersicum L.) fruit. Low levels were detected in mature green tomato fruit and activity increased during tomato fruit ripening up to the red ripe stage. Essentially all activity was found in the tomato skin and outermost 2 mm of tissue. Mannan transglycosylase activity in tomato skin and outer pericarp is specific for mannan-based plant polysaccharides, including GGM, galactomannan, glucomannan and mannan. The exact structural requirements for valid acceptors remain to be defined. Nevertheless, a mannose residue at the second position of the sugar chain and the absence of a galactose substituent on the fourth residue (counting from the non-reducing end) appear to be minimal requirements. Mannan-based polysaccharides in the plant cell wall may have a role analogous to that of xyloglucans, introducing flexibility and forming growth-restraining networks with cellulose. Thus mannan transglycosylase and xyloglucan endotransglycosylase, the only other known transglycosylase activity in plant cell walls, may both be involved in remodelling and refining the cellulose framework in developmental processes throughout the life of a plant.Abbreviations EBM Endo--mannanase - GGM galactoglucomannan - GGMO Galactoglucomannan-derived oligosaccharide - G₂M₅ Di-galactosyl mannopentaitol - M₂–M₅ Mannobiitol to mannopentaitol oligosaccharides - SK+OP Skin plus outer pericarp - XET Xyloglucan endotransglucosylase - XG Xyloglucan     

Association between chloroplast and mitochondrial lineages in oaks

Patterns of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) variation were studied in 378 populations of oak trees sampled throughout the southern half of France. Six cpDNA haplotypes detected in a previous European survey and three new cpDNA haplotypes were found in this region. Two mitochondrial polymorphisms detected earlier by restriction analysis of PCR-amplified fragments alone, or in combination with single-strand conformation polymorphism (SSCP), were compared with the cpDNA data. Sequencing revealed the nature of the two mitochondrial mutations: a single-base substitution and a 4-bp inversion associated with a 22-bp hairpin secondary structure. The single-base substitution was then analyzed by allele-specific amplification. Results for the two cytoplasmic genomes were combined, which allowed the identification of 12 cpDNA-mtDNA haplotypes. The 4-bp mtDNA inversion has appeared independently in different cpDNA lineages. Given the peculiar nature of this mtDNA mutation, we suggest that intramolecular recombination leading to repeated inversions of the 4-bp sequence (rather than paternal leakage of one of the two genomes) is responsible for this pattern. Furthermore, the geographic locations of the unusual cpDNA-mtDNA associations (due to the inversion) usually do not match the zones of contact between divergent haplotypes. In addition, in southern France, the groupings of populations based on the mtDNA substitution were strictly congruent with those based on cpDNA. Because many populations that are polymorphic for both cpDNA and mtDNA have remained in contact since postglacial recolonization in this area without producing any new combination of cytoplasms involving the mitochondrial substitution, we conclude that paternal leakage is not a significant factor at this timescale. Such results confirm and expand our earlier conclusions based on controlled crosses.      

Mannans in tomato fruit are not depolymerized during ripening despite the presence of endo-β-mannanase

Cell walls of tomato fruit contain hemicellulosic mannans that may fulfill a structural role. Two populations were purified from cell walls of red ripe tomato tissue and named galactoglucomannan-glucuronoxylan I and II (GGM-GX I and II), respectively. Both polysaccharides not only consisted of mannose, glucose and galactose, indicating the presence of GGM, but also contained xylose and glucuronic acid, indicating the presence of GX. Treatment of both polysaccharides with xylanase or endo-β-mannanase showed that the GX and the GGM were associated in a complex. The composition of GGM-GX II changed slightly during tomato ripening, but both GGM-GX I and II showed no change in molecular weight, indicating that they were not hydrolyzed during ripening. Ripe tomato fruit also possess an endo-β-mannanase, an enzyme that in vitro was capable of either hydrolyzing GGM-GX I and II (endo-β-mannanase activity), or transglycosylating them in the presence of mannan oligosaccharides (mannan transglycosylase activity). The lack of evidence for hydrolysis of these potential substrates in vivo suggests either that the enzyme and potential substrates are not accessible to each other for some reason, or that the main activity of endo-β-mannanase is not hydrolysis but transglycosylation, a reaction in which polysaccharide substrates and end-products are indistinguishable. Transglycosylation would remodel rather than weaken the cell wall and allow the fruit epidermis to possibly retain flexibility and plasticity to resist cracking and infection when the fruit is ripe.     

The molecular basis of color vision in colorful fish: Four Long Wave-Sensitive (LWS) opsins in guppies (<Emphasis Type="Italic">Poecilia reticulata</Emphasis>) are defined by amino acid substitutions at key functional sites

Background  

Comparisons of functionally important changes at the molecular level in model systems have identified key adaptations driving isolation and speciation. In cichlids, for example, long wavelength-sensitive (LWS) opsins appear to play a role in mate choice and male color variation within and among species. To test the hypothesis that the evolution of elaborate coloration in male guppies (Poecilia reticulata) is also associated with opsin gene diversity, we sequenced long wavelength-sensitive (LWS) opsin genes in six species of the family Poeciliidae.