Growth rate-dependent changes in Escherichia coli membrane structure and protein leakage

The phospholipid and fatty acid content of the Escherichia coli membrane were investigated during continuous cultivation. At low growth rates, there was an increase in cardiolipin produced at the expense of phosphatidylethanolamine. Phosphatidylglycerol had a maximum at a growth rate of 0.3 h(-1). The amount of cyclic fatty acids was markedly increased at lower growth rates, while there was an evident minimum at 0.3 h(-1). This was also the case for saturated fatty acids. At this point, the unsaturated fatty acids had a maximum depending mainly on changes in cis-vaccenic acid. The mechanical strength towards sonication and osmotic shock/enzymatic treatment showed that the cells were more rigid at low dilution rates. However, this was accompanied by a higher cell lysis, a reduced capacity for total and specific protein production and a lower yield of cells. The amount of lipid A in the medium (endotoxin) was constant and negligible at all growth rates. The leakage of periplasmic protein to the medium had an optimum at 0.3 h(-1), resulting in a transport of 20% of the total recombinant product. It is argued that this constitutes the point of highest membrane fluidity and thus an increase possibility for protein transport.     

An amperometric enzyme-linked immunosensor for Nitrobacter

A new amperometric enzyme-linked immunoassay for specific enumeration of Nitrobacter has been developed. This assay uses an electrode made of glassy carbon, on which the immunological reaction is carried out. The method is based on a competitive immunoassay principle, utilising monoclonal primary antibody and alkaline-phosphatase-labelled secondary antibody. The enzyme substrate 5-bromo-4-chloro-3-indolyl phosphate generates an electroactive product which is amperometrically detected. The effects of different parameters on the performance of the sensor have been studied. Quantitative detection of Nitrobacter using the immunosensor has been compared to a previously developed enzyme-linked immunosorbent assay showing compatible results. In addition, the overall assay time can be shortened with this new sensor. A detection limit of approximately 3 × 10⁶ Nitrobacter cells/ml was obtained. Received: 27 May 1998 / Received revision: 28 August 1998 / Accepted: 28 August 1998     

Genetic markers, genealogies and biogeographic patterns in the cladocera

Cladoceran crustaceans are an important component of zooplankton in a wide range of freshwater habitats. Although the ecological characteristics of several cladoceran species have been well studied, biogeographical studies have been hampered by problematic taxonomic affiliations. However, recently developed molecular techniques, provide a powerful tool to subject aquatic taxa to comparative analyses. Here we highlight recent molecular approaches in aquatic ecology by presenting a simple method of DNA preparation and PCR amplification of the mitochondrial DNA (16S rDNA) in species from nine different families within the cladocera. On a broad taxonomic scale, sequence analysis of this mtDNA fragment has been used to produce the first molecular based phylogeny of the cladocera. This analysis clustered the cladoceran families in a fashion similar to that suggested by previous systematic classifications. In a more detailed analysis of the family Daphniidae, nuclear randomly amplified polymorphic DNA (RAPD), mitochondrial restriction fragment length polymorphism (RFLP) and morphological analyses were combined to identify species and interspecific hybrids within the Daphnia galeata species complex across 50 lakes in 13 European countries and one lake in Africa. The study revealed interspecific hybridization and backcrossing between some taxa (D. cucullata and D. galeata) to be widespread, and species and hybrids to frequently occur in sympatry. Genetic, as well as morphological information, suggests the occurrence of D. hyalina outside the Holarctic.     

Mechanisms for the emergence of catecholamine-sensitive adenylate cyclase and beta-adrenergic receptors in cultured hepatocytes. Dependence on protein and RNA synthesis and suppression by isoproterenol

Adult male rat hepatocytes, which normally respond poorly to beta-adrenergic agents, acquire such responsiveness during primary monolayer culture. We here show that the rise in catecholamine-sensitive adenylate cyclase activity in hepatocytes in vitro is closely paralleled by an increase in the ability to bind the beta-adrenoceptor ligand [125I]cyanopindolol. The emergence of beta-adrenergic responsiveness did not require cell attachment or serum. Addition of dexamethasone, insulin, thyroxine or dihydrotestosterone to the cultures, singly or in combination, did not prevent the augmented beta-adrenergic responsiveness. The increase in catecholamine-sensitive adenylate cyclase activity and [125I]cyanopindolol binding could be blocked by cycloheximide or actinomycin D. Exposure of the cultures to isoproterenol at 3-hourly intervals led to a dose-dependent suppression of the rise in isoproterenol-responsive adenylate cyclase and prevented the increase in beta-adrenoceptor binding.     

Cytoplasmic Male Sterility in Nicotiana, Restoration of Fertility, and the Nucleolus. II. N. DEBNEYI Cytoplasm

Previously, it was shown that a fragment chromosome, apparently derived from the Nicotiana repanda chromosomal complement, restores to normal the morphology and fertility of the abortive and feminized anthers produced by plants that possess the N. tabacum genome in cytoplasm from N. repanda. Furthermore, that restorer chromosome organizes the nucleolus and inhibits the nucleolus-forming activity of the nucleolar organizers of N. tabacum chromosomes present in the same cells, particularly in pollen mother cells. To determine whether these relations are basic or only coincidental, restorer chromosomes for other cytoplasms are now being investigated. The present paper describes a study of a chromosome, presumably derived from N. debneyi, with partial restoring power. Acting in the cytoplasm of N. debneyi, it directs formation of morphologically normal anthers, without, however, restoring pollen fertility. We find that this chromosome also has a functioning nucleolar organizer, but only slightly inhibits the nucleolus-forming capacity of N. tabacum chromosomes. The suggestion of a relationship between the nucleolar apparatus and restoration of normal anthers is thus strengthened by the observation that restorers are found on nucleolus-forming chromosomes from two very distinct Nicotiana species, as well as in several comparable cases cited from the Triticinae. The manner in which the nucleolus, or its organizer, may direct defeminization and restoration of anther morphology is not known; suggestions were offered in the preceding paper in this series (Gerstel, Burns and Burk 1978).     

Microtubules and cyclic amp in human leukocytes: on the order of things

We have shown previously that the β-adrenergic agonist isoproterenol (2μM) and the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) produce a much greater increase in cyclic AMP in human leukocytes that have been pretreated with colchicine (or with other agents that affect microtubule assembly) than in control leukocytes. The effects of colchicines were both time- and dose-dependant. These and other data suggested that the generation of cyclic AMP is normally restricted by an intact system of cytoplasmic microtubules. If so, then the same time and dose dependencies might apply to other colchicines-induced changes in leukocyte function. We have now assayed the distribution of concanavalin A (Con A)-receptor complexes on the leukocyte membrane, taking into account that leukocytes competent to assemble microtubules show a uniform distribution of surface- bound Con A whereas microtubule-deficient cells accumulate Con A in surface caps. We have found that the effect of colchicine on capping is also both time- and dose dependent, and that the dose-response relationships conform to those required to increase cyclic AMP levels. These findings provide further evidence that both colchicine-induced Con-A capping and colchicine- induced cyclic AMP generation depend upon the relaxation of constraints normally imposed by cytoplasmic microtubules upon the plasma membrane, which limit, respectively, lateral mobility of the lectin-receptor complexes, and expression of hormone-sensitive adenylate cyclase. Moreover, colchicine-induced Con-A cap formation is not affected even by very large changes in leukocyte cyclic AMP levels. Thus, elevated cyclic AMP levels do not appear to promote the dissolution of microtubules; rather, the dissolution of microtubules permits the generation of increased amounts of cyclic AMP.     

A modular treatment of molecular traffic through the active site of cholinesterase

We present a model for the molecular traffic of ligands, substrates, and products through the active site of cholinesterases (ChEs). First, we describe a common treatment of the diffusion to a buried active site of cationic and neutral species. We then explain the specificity of ChEs for cationic ligands and substrates by introducing two additional components to this common treatment. The first module is a surface trap for cationic species at the entrance to the active-site gorge that operates through local, short-range electrostatic interactions and is independent of ionic strength. The second module is an ionic-strength-dependent steering mechanism generated by long-range electrostatic interactions arising from the overall distribution of charges in ChEs. Our calculations show that diffusion of charged ligands relative to neutral isosteric analogs is enhanced approximately 10-fold by the surface trap, while electrostatic steering contributes only a 1.5- to 2-fold rate enhancement at physiological salt concentration. We model clearance of cationic products from the active-site gorge as analogous to the escape of a particle from a one-dimensional well in the presence of a linear electrostatic potential. We evaluate the potential inside the gorge and provide evidence that while contributing to the steering of cationic species toward the active site, it does not appreciably retard their clearance. This optimal fine-tuning of global and local electrostatic interactions endows ChEs with maximum catalytic efficiency and specificity for a positively charged substrate, while at the same time not hindering clearance of the positively charged products.     

Awareness reactions and avoidance responses to sound in juvenile Atlantic salmon, Salmo salar L.

The possibility of using intense sound as a deterrent for juvenile Atlantic salmon ( Sulmo salar L. ) was studied by recording both physiological awareness reactions in an acoustic tube and behavioural avoidance responses in a pool. The measured awareness reactions consisted of decreased heart rate and breathing movements. Three criteria for the awareness reaction were used to compare the effect of different frequencies between 5 and 150 Hz: (i) threshold for spontaneous awareness reactions relative to the hearing thresholds, (ii) magnitude of the change in heart rate, (iii) degree of habituation to sound. After these criteria the lowest frequencies (5–10 Hz) were most effective in eliciting an awareness reaction from the fish. The responses of freely swimming fish to 10 and 150 Hz sounds were studied in an artificial pool. Juvenile salmon showed avoidance responses to 10 Hz stimulation at intensities 10–15 dB above the threshold for spontaneous awareness reactions measured in the acoustic tube. The 150 Hz sound failed to evoke avoidance responses, even at a level 30 dB above the threshold for spontaneous awareness reactions.     

Application of an amperometric immunosensor for the enumeration of Nitrobacter in activated sludge

A competitive immunosensor using a monoclonal antibody has been developed for the enumeration of Nitrobacter in activated sludge and other environmental samples. Its cross-reactivity was tested against a number of bacterial strains and isolates. All strains of the nitrite-oxidising genera Nitrobacter and Nitrococcus reacted strongly with the monoclonal antibody. The nitrite-oxidising Nitrospira moscoviensis, as well as the ammonia oxidising bacteria and the heterotrophic bacteria tested, did not show any affinity towards the antibody in the immunosensor. The numbers of Nitrobacter were analysed in sludge samples from several wastewater treatment plants in Sweden. Detectable amounts were found in all samples. This study shows the adequacy of using this immunosensor for the enumeration of Nitrobacter in natural environments. Received: 4 October 1999 / Received revision: 20 February 2000 / Accepted: 25 February 2000