ALBERT

Notes: Site-directed mutations involving selected amino acids of Escherichia coli single-stranded DNA-binding protein (SSB) were tested for their in vivo functionality when introduced into a chromosomal ssb deletion strain on a plasmid. All mutants complemented the ssb deletion for viability when present on a pSC101 derivative. The generation time with ssbW54S doubled in comparison to the ssb* control, and both the ssbW54S- and ssbH55K-containing strains exhibited temperature sensitivity. ssbH55K, ssbW54S, ssbW88T, and ssbH55Y (ssb-1) strains displayed reduced survival to ultraviolet irradiation, while ssbW40T and ssbF60L strains were comparable to the ssb+ control strain. This study represents the first investigation of the in vivo properties of ssb mutations constructed for in vitro analysis of DNA binding by SSB.

Notes: Plasma melatonin synthesis in juvenile Atlantic salmon Salmo salar decreased with increasing light intensity. Melatonin profiles reflected accurately the photoperiod under which the salmon were maintained. Groups maintained at 12°C showed significantly higher (P 〈 0·01) levels of dark phase plasma melatonin compared with the groups maintained at 4°C.

Notes: There is increasing public, governmental and commercial interest in the welfare of intensively farmed fish and stocking density has been highlighted as an area of particular concern. Here we draw scientific attention and debate to this emerging research field by reviewing the evidence for effects of density on rainbow trout. Although no explicit reference to ‘welfare’ has been made, there are 43 studies which have examined the effects of density on production and physiological parameters of rainbow trout. Increasing stocking density does not appear to cause prolonged crowding stress in rainbow trout. However, commonly reported effects of increasing density are reductions in food conversion efficiency, nutritional condition and growth, and an increase in fin erosion. Such changes are indicative of a reduced welfare status—although the magnitude of the effects has tended to be dependent upon study-specific conditions. Systematic observations on large scale commercial farms are therefore required, rather than extrapolation of these mainly small-scale experimental findings. There is dispute as to the cause of the observed effects of increasing density, with water quality deterioration and/or an increase in aggressive behaviour being variously proposed. Both causes can theoretically generate the observed effects of increasing density, and the relative contribution of the two causes may depend upon the specific conditions. However, documentation of the relationship between density and the effects of aggressive behaviour at relevant commercial densities is lacking. Consequently only inferential evidence exists that aggressive behaviour generates the observed effects of increasing density, whereas there is direct experimental evidence that water quality degradation is responsible. Nevertheless, there are contradictory recommendations in the literature for key water quality parameters to ensure adequate welfare status. The potential for welfare to be detrimentally affected by non-aggressive behavioural interactions (abrasion, collision, obstruction) and low densities (due to excessive aggressive behaviour and a poor feeding response) have been largely overlooked. Legislation directly limiting stocking density is likely to be unworkable, and a more practical option might be to prescribe acceptable levels of water quality, health, nutritional condition and behavioural indicators.

Notes: An endophytic streptomycete (NRRL 30566) is described and partially characterized from a fern-leaved grevillea (Grevillea pteridifolia) tree growing in the Northern Territory of Australia. This endophytic streptomycete produces, in culture, novel antibiotics – the kakadumycins. Methods are outlined for the production and chemical characterization of kakadumycin A and related compounds. This antibiotic is structurally related to a quinoxaline antibiotic, echinomycin. Each contains, by virtue of their amino acid compositions, alanine, serine and an unknown amino acid. Other biological, spectral and chromatographic differences between these two compounds occur and are given. Kakadumycin A has wide spectrum antibiotic activity, especially against Gram-positive bacteria, and it generally displays better bioactivity than echinomycin. For instance, against Bacillus anthracis strains, kakadumycin A has minimum inhibitory concentrations of 0.2–0.3 μg ml−1 in contrast to echinomycin at 1.0–1.2 μg ml−1. Both echinomycin and kakadumycin A have impressive activity against the malarial parasite Plasmodium falciparum with LD50s in the range of 7–10 ng ml−1. In macromolecular synthesis assays both kakadumycin A and echinomycin have similar effects on the inhibition of RNA synthesis. It appears that the endophytic Streptomyces sp. offer some promise for the discovery of novel antibiotics with pharmacological potential.

Notes: Single-chain antibody fragments against the cyanobacterial hepatotoxin microcystin-LR were isolated from a naive human phage display library and expressed in Escherichia coli. In competition enzyme-linked immunosorbent assay (ELISA), the most sensitive antibody clone selected from the library detected free microcystin-LR with an IC50 value of 4 μM. It was found to cross react with three other microcystin variants – microcystin-RR, microcystin-LW and microcystin-LF – and detected microcystins in extracts of the cyanobacterium Microcystis aeruginosa, found to contain the toxins by high-performance liquid chromatography (HPLC). The quantification of microcystins in these extracts by ELISA and HPLC showed good correlation. Although the antibody isolated in this study was considerably less sensitive than the polyclonal and monoclonal antibodies already available for microcystin detection, phage display technology represents a cheaper, more rapid alternative for the production of anti-microcystin antibodies than the methods currently in use.

Notes: Antibody phage display libraries (Griffin and Tomlinson I) displaying antibody genes and maintained and amplified in Escherichia coli were used to isolate antibodies to the hapten target microcystin LR (1000 Da) conjugated to either bovine serum albumin or keyhole limpet haemocyanin. In competition enzyme-linked immunosorbent assay, bacterially expressed antibodies selected via the Griffin library showed at least 300 times greater sensitivity than those isolated from the Tomlinson library, for free microcystin. Bacterially expressed phage antibody libraries provide a rapid and relatively easy route for the selection of monoclonal antibodies specific for even the most difficult of antigenic targets such as free haptens.

Notes: Abstract The four species of Bordetella differed in their ability to grow at 37°C in membrane-filtered tracheobronchial washings (TBW) from seven vertebrate species, including their natural hosts. From washed inocula of approximately 2×103 colony-forming units per ml (cfu ml−1), Bordetella bronchiseptica and B. avium grew much better than the other two bordetellae and yielded stationary-phase cultures containing 108−109 cfu ml−1 in most of the TBW samples. These counts were only moderately lower than those attained in CL medium which contains about a 450-times higher concentration of amino acids. B. bronchiseptica and B. avium also grew to a limited extent in phosphate-buffered saline without nutrient supplements. B. parapertussis grew in TBW from man, sheep, rabbit, mouse and chicken, but not in TBW from a dog and a horse or in PBS. B. pertussis grew well in CL medium, but not in PBS or in any of 13 samples of TBW from the seven vertebrate species, which included three samples of lung lavage fluid from human patients. Analysis of the TBW samples for known Bordetella nutrients revealed concentrations of amino acids and nicotinic acid averaging 0.35 mM and 0.56 μg ml− respectively.

Notes: Filamentous microbial mats from three aphotic sulfidic springs in Lower Kane Cave, Wyoming, were assessed with regard to bacterial diversity, community structure, and ecosystem function using a 16S rDNA-based phylogenetic approach combined with elemental content and stable carbon isotope ratio analyses. The most prevalent mat morphotype consisted of white filament bundles, with low C:N ratios (3.5–5.4) and high sulfur content (16.1–51.2%). White filament bundles and two other mat morphotypes had organic carbon isotope values (mean δ13C =−34.7‰, 1σ= 3.6) consistent with chemolithoautotrophic carbon fixation from a dissolved inorganic carbon reservoir (cave water, mean δ13C =−7.4‰ for two springs, n= 8). Bacterial diversity was low overall in the clone libraries, and the most abundant taxonomic group was affiliated with the “Epsilonproteobacteria” (68%), with other bacterial sequences affiliated with Gammaproteobacteria (12.2%), Betaproteobacteria (11.7%), Deltaproteobacteria (0.8%), and the Acidobacterium (5.6%) and Bacteriodetes/Chlorobi (1.7%) divisions. Six distinct epsilonproteobacterial taxonomic groups were identified from the microbial mats. Epsilonproteobacterial and bacterial group abundances and community structure shifted from the spring orifices downstream, corresponding to changes in dissolved sulfide and oxygen concentrations and metabolic requirements of certain bacterial groups. Most of the clone sequences for epsilonproteobacterial groups were retrieved from areas with high sulfide and low oxygen concentrations, whereas Thiothrix spp. and Thiobacillus spp. had higher retrieved clone abundances where conditions of low sulfide and high oxygen concentrations were measured. Genetic and metabolic diversity among the “Epsilonproteobacteria” maximizes overall cave ecosystem function, and these organisms play a significant role in providing chemolithoautotrophic energy to the otherwise nutrient-poor cave habitat. Our results demonstrate that sulfur cycling supports subsurface ecosystems through chemolithoautotrophy and expand the evolutionary and ecological views of “Epsilonproteobacteria” in terrestrial habitats.

Notes: Abstract Bordetella bronchiseptica grew from small inocula, and retained viability for at least 24 weeks, in unsupplemented lakewater or phosphate-buffered saline. From washed inocula of around 103 colony-forming units/ml, there was growth at both 10°C and 37°C to give 106–107 colony-forming units/ml. At 10°C, these counts were maintained with little diminution up to week 24 when observations ceased. In the tests at 37°C, two of three strains tested showed similar retention of viability. These results suggest that B. bronchiseptica may exist as hitherto unsuspected reservoirs of infection in freshwater habitats.

Notes: Abstract Tumour necrosis factor-α (TNF-α) and lymphotoxin (LT or TNF-β) are cytokines, best known for their cytotoxic or cytostatic effects on some tumour cells. They are structurally related, compete for a common receptor, and are potent inducers of similar biological responses. TNF-α and LT appear to have distinct three-dimensional structures because they differ greatly in their sensitivity to various proteases and chemical agents, and antibodies raised against one cytokine do not cross-react with the other cytokine. The closely linked TNF-α and LT genes are independently regulated since many cell types produce only TNF-α or LT. Expression of the TNF-α gene can be controlled either at the transcriptional or at the post-transcriptional level. In some cell types, TNF-α and LT induce qualitatively or quantitatively different biological responses, and LT can antagonize the action of TNF-α. The disparate biological activities of TNF-α and LT may be related to their different interactions with a common receptor. It is possible that TNF-α and LT have different physiological roles.