Microbiological degradation of the herbicide dicamba

Summary Pseudomonas paucimobilis was isolated from a consortium which was capable of degrading dicamba (3,6-dichloro-2-methoxybenzoic acid) as the sole source of carbon. The degradation of dicamba byP. paucimobilis and the consortium was examined over a range of substrate concentration, temperature, and pH. In the concentration range of 100–2000 mg dicamba L^–1 (0.5–9.0 mM), the degradation was accompanied by a stoichiometric release of 2 mol of Cl^– per mol of dicamba degraded. The cultures had an optimum pH 6.5–7.0 for dicamba degradation. Growth studies at 10°C, 20°C, and 30°C yielded activation energy values in the range of 19–36 kcal mol^–1 and an average Q₁₀ value of 4.0. Compared with the pure cultureP. paucimobilis, the consortium was more active at the lower temperature.     

An assessment of some improved techniques for estimating the abundance (frequency) of sedentary organisms

The traditional sampling method for estimating frequency (the number of sub-quadrats containing a basal part of the organisms) is compared, using both computer simulations and direct comparison in the field, to two new methods that use a compound series of variable-sized concentric sub-quadrats. Both the new frequency-score and the new importance-score methods are closer approximations of density than is the standard frequency method, and the estimates produced by both of the new methods are less affected by the choice of sub-quadrat size and the spatial distribution (dispersion) of the organisms (i.e. clumping and regularity). Thus, the two nested-quadrat methods appear to ameliorate the usual frequency limitations associated with sub-quadrat size and organism dispersion, by the use of a range of different sub-quadrat sizes. This is important in community studies, where the component species may show a wide range of densities and dispersions. Both of the new methods are easily employed in the field. The importance-score method involves no more sampling effort than does standard qualitative (presence-absence) sampling, and it can therefore be used to sample a larger quadrat area than would normally be used for frequency sampling. This makes the method much more cost-effective as a means of estimating abundance, and it allows a greater number of the rarer species to be included in the sampling. The frequency-score method is more time-consuming, but it is capable of detecting more subtle community patterns. This means that it is particularly useful for the study of species-poor communities or where small variations in composition need to be detected.     

The status and ecology of a Juniperus excelsa subsp. polycarpos woodland in the northern mountains of Oman

Juniperus excelsa subsp. polycarpos (K. Koch) Takhtajan is found in mountain areas from Turkey through to India and as an isolated population on Jebel Akhadar in the northern mountains of Oman. Juniperus is one of the dominant plant species in these mountains and a major landscape feature of several proposed National Nature and Scenic Reserves and of Hayl Juwari, a wooded valley at 2250 m altitude proposed as a Botanical Site of Special Interest. Above 2400 m altitude the Juniperus woodlands generally appear to be regenerating and in good condition, both on exposed slopes and in wadis and sheltered gullies, whereas below 2400 m most stands are in poor condition and exhibit few signs of regeneration. If the apparently poor condition of the lower altitude woodlands is due to any long term change in climatic conditions, both tree status and regeneration would be poorer in relatively more xeric habitats. To test this prediction we have carried out a detailed survey of the status and ecology of a 32 ha area of Hayl Juwari, and analysed differences in tree status and regeneration between wadis (relatively more nesic sites) and non-wadi areas (relatively more xeric). Approximately one third of the trees are dead, and an analysis of the height, condition, regeneration, female cone production, preferred germination sites and spatial distribution of trees indicates the importance of topography, hydrology and microclimate for growth. However, although there are relatively greater numbers of dead and poor-condition trees in the more xeric non-wadi habitat, there is no unequivocal evidence that the present distribution of small, sexually immature trees in both habitats could not form a pattern of larger, sexually mature trees similar to that seen today. We speculate, however, that the climate at this altitude may be marginal for the survival of a J. excelsa subsp. polycarpos woodland and that even small increases in climatic stress could imperil the woodland's present status.     

Continuous measurement of changes in intracellular calcium concentration in mouse splenic T cells attached to a glass substrate

Mitogen- and isoproterenol-induced changes of [Ca²⁺]_i in T cells attached to a glass substrate were examined. Murine (C57BL/6) splenic T cells were attached to coverslips or 35-mm dishes (MatTek) precoated with Cell Tak® (3.5 µg/cm²). The cells were then loaded with fluorescent dye (2 µg/ml of fura2-AM or fluo3-AM) and changes in [Ca²⁺]_i in a population of cells (using a spectrofluorometer) or in single cells (using a confocal microscope) were measured during continuous superfusion. Population measurements of [Ca²⁺]_i demonstrated that concanavalin A (Con A, 2 or 5 µg/ml) caused an increase in [Ca²⁺]_i that rose to a peak and then declined to a steady state. The concentration-response relationship (0.05–5 µg/ml) had an EC₅₀ of 0.3 µg/ml. Isoproterenol decreased the Con A-induced elevation of steady state [Ca²⁺]_i. In single cell studies, the increase in [Ca²⁺]_i in response to Con A typically occurred in about 50% of the cells in a microscope field, and the delay before activation varied among cells. Taken together, these data demonstrate that Cell Tak® can be used to attach T cells to glass coverslips and will be useful for the study of signaling mechanisms in T cells.     

Application of the multiple antigenic peptides (MAP) strategy to the production of prophormone convertases antibodies: Synthesis,characterization and use of 8-branched immunogenic peptides

Antiserum against an N-terminal sequence of murine prohormone convertase-1 (mPC1) incorporating the sequence immediatley following the junction between the putative pro-region and the active enzyme was obtained. This was accomplished using the multiple antigenic peptide (MAP) approach whereupon an 8-branched polylysine core to which are grafted multiple copies of a 16 amino acid peptide representing the N-terminal sequence of mPC1 (positions 84–99) was synthesized by solid-phase Fmoc chemistry. The ensuing peptide was purified and fully characterized by RP-HPLC, ¹H-NMR, amino acid composition, peptide sequencing and ion-spray mass spectrometry. The immunological properties of the resulting antibodies in detecting recombinant PC1 in both crude and purified preparations were compared with antibodies raised against a similar N-terminal segment of PC1 but using the conventioanl method of peptide–carrier protein conjugation and also developed against a C-terminal fusion protein of PC1. Our data indicate that the MAP antibody was as efficient as both the amino and carboxy-terminal antibodies in qualitative as well as quantitative analysis of PC1 encoded protein by radioimmunoassay. Following an identical approach, antibodies against other prohormone convertases like furin, PC5/6 and PACE4 were also developed and subsequently applied to a number of biochemical and immunological studies. In each case, the ease of preparation and high immunogenicity of the MAP approach were confirmed and reside in the simplicity and rapidity with which a potent and useful antiserum is obtained.     

Expression and purification strategies for the production of single-chain antibody and T-cell receptor fragments inE. coli

This work describes protocols for the production of single-chain antibody and T-cell receptor fragments inE. coli. A choice of methods is given for the purification of the recombinant fragments that rely on the use of either immunoaffinity or metal chelate affinity chromatography. The TCR fragments may have to be denatured and refolded before the fragments attain their proper conformation.     

Eicosanoids and their role in immune modulation in fish—a brief overview

Eicosanoids have been demonstrated to play a central role in immune regulation in mammals brought about by their direct effects on cells such as macrophages and lymphocytes or by their indirect effects via cytokines. Studies have shown that fish mononuclear phagocytes, granulocytes and thrombocytes synthesize and release both cyclooxygenase- and lipoxygenase-derived products such as prostaglandin E₂, leukotriene B₄ and lipoxin A₄. Whether lymphocytes have the ability to generate leukotrienes and lipoxins is still unclear but they do appear to have 12-lipoxygenase activity that leads to the generation of 12-hydroxy fatty acid derivatives. As in mammals, leukotriene and lipoxin biosynthesis requires the presence of a 5-lipoxygenase activating protein-like molecule that is sensitive to the action of the specific inhibitor, MK-886. The prostaglandin-generating ability of trout macrophages can be altered by incubation with lipopolysaccharide suggesting the possible presence of an inducible cyclooxygenase activity. Prostaglandins have been found to suppress the mitogen-induced proliferation of trout leucocytes and the generation of humoral antibody and plasma cells both in vivo and in vitro. The lipoxygenase products, leukotriene B₄ and lipoxin A₄ have more variable effects ranging from inhibition to stimulation depending on the assay system employed. Overall, there is clear evidence that eicosanoids play a role in immune regulation in fish in a similar way to that reported in mammals.     

Kinetics of ligand binding to a cluster of membrane-associated receptors

The process of ligand binding to a cluster of membrane-associated receptors is examined theoretically. The theoretical model proposed involves the diffusion of ligands from the solution to the disc-like cluster of receptors on the surface of the spherical cell. When the ligand hits the internal part of the disc-like cluster, it begins to move laterally until it leaves the disc through its outer surface or is bound by one of the receptors inside the disc. If the ligand leaves the cluster, it returns to the solution and hits the disc again after a certain period, etc. According to our model the transition from a diffusion-limited to a reaction-limited process of binding is determined by the dimensionless parameter Dt _c/a ², where D is the lateral diffusion coefficient,t _c is the characteristic time of reaction, anda is the radius of the disc-like cluster. The forward rate constantk _f turns out to be a function of . Comparing the results of our calculations ofk _f with some experimental data we found that agreement is achieved at high , i.e. the process of ligand binding by clustered receptors is predominantly reaction-limited.     

Age,Experience, and the Response of Streamside Salamander Hatchlings to Chemical Cues from Predatory Sunfish

Previous work has shown that streamside salamander larvae (Ambystoma barbouri; Ambystomatidae) exhibit an adaptive ‘sink to the bottom’ response to chemical cues from predatory green sunfish (Lepomis cyanellus; Centrarchidae), that is, larvae sink to the bottom more quickly (thus minimizing exposure time to sunfish predation) when they are dropped into water with sunfish chemicals (as compared to Ashless controls). Here, we examined this anti-predator behaviour in early hatchlings and the effects of age and experience on subsequent expression of this behaviour. Hatchlings responded significantly to fish chemical cues within the first 18 h after hatching. Age did not significantly influence this response, i.e. regardless of age (1, 7, or 14 days after hatching) larvae showed a significant response during their first exposure to fish chemical cues. Experience also did not significantly influence the larval response to fish chemicals i.e., repeated exposures over 2 weeks did not significantly influence the magnitude of the response. Finally, comparisons of 3 siblingships detected significant variation among siblingships that might reflect genetic variation in this behaviour.     

A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements.

We have identified and characterized a new orphan member of the nuclear hormone receptor superfamily, called MB67, which is predominantly expressed in liver. MB67 binds and transactivates the retinoic acid response elements that control expression of the retinoic acid receptor beta 2 and alcohol dehydrogenase 3 genes, both of which consist of a direct repeat hexamers related to the consensus AGGTCA, separated by 5 bp. MB67 binds these elements as a heterodimer with the 9-cis-retinoic acid receptor, RXR. However, MB67 does not bind or activate other retinoic acid response elements with alternative hexamer arrangements or any of several other wild-type and synthetic hormone response elements examined. The transactivation of retinoic acid response elements by MB67 is weaker than that conferred by the retinoic acid receptors but does not require the presence of all-trans retinoic acid, 9-cis-retinoic acid, or any exogenously added ligand. We propose that MB67 plays an important role in the complex network of proteins that govern response to retinoic acid and its metabolites.