Inverse regulation of biofilm formation and swarming motility by Pseudomonas aeruginosa PA14

We previously reported that SadB, a protein of unknown function, is required for an early step in biofilm formation by the opportunistic pathogen Pseudomonas aeruginosa. Here we report that a mutation in sadB also results in increased swarming compared to the wild-type strain. Our data are consistent with a model in which SadB inversely regulates biofilm formation and swarming motility via its ability both to modulate flagellar reversals in a viscosity-dependent fashion and to influence the production of the Pel exopolysaccharide. We also show that SadB is required to properly modulate flagellar reversal rates via chemotaxis cluster IV (CheIV cluster). Mutational analyses of two components of the CheIV cluster, the methyl-accepting chemotaxis protein PilJ and the PilJ demethylase ChpB, support a model wherein this chemotaxis cluster participates in the inverse regulation of biofilm formation and swarming motility. Epistasis analysis indicates that SadB functions upstream of the CheIV cluster. We propose that P. aeruginosa utilizes a SadB-dependent, chemotaxis-like regulatory pathway to inversely regulate two key surface behaviors, biofilm formation and swarming motility.     

Restoration of Themeda australis swards suppresses soil nitrate and enhances ecological resistance to invasion by exotic annuals

Understanding processes that underlie ecological resistance to weed invasion is critical for sustainable restoration of invaded plant communities. Experimental studies have demonstrated that invasive nitrophilic annuals can be controlled by addition of carbon to reduce soil nitrate concentrations, sometimes leading to enhanced establishment of native plants. However, effects of carbon supplements on soil nitrate are temporary, and the longer-term value of carbon supplementation as a restoration tool is dependent on the resistance of the re-established ecosystem to repeat invasion. We investigated whether re-established swards of the tussock grass Themeda australis (R.Br.) Stapf (a natural understorey dominant in mesic grassy woodlands of SE Australia) could suppress soil nitrate concentrations, and through this or other means, could impart ongoing resistance to exotic invasion in restored woodlands. In a remnant invaded by exotic annuals, we applied three plot treatments (carbon supplements, annual spring burns and untreated control) and two seed treatments (± Themeda seed) in a replicated, factorial design. Within 3 years, successful establishment of Themeda swards on burnt and carbon-supplemented plots was associated with a reduction in soil nitrate to levels comparable with non-invaded, Themeda-dominated reference sites in the region (<3 mg/kg), and significantly reduced exotic cover compared with unseeded plots. By contrast, on plots not seeded with Themeda, soil nitrate increased after cessation of carbon addition and exotic cover returned to levels comparable with untreated control plots, despite a high cover of other native perennial grasses. Few persistent effects of carbon supplements or spring burning on soil nutrients were evident 9–19 months after cessation of these treatments. Results suggest that Themeda is a keystone species that regulates nitrate cycling, thereby imparting ecological resistance to invasion by nitrophilic annuals.     

Subcellular fractionation and distribution of cholinergic binding sites in fetal human brain

Conventional subcellular fractionation techniques have been applied to human fetal brain (13–15 weeks gestation) and the fractions have been characterized by assaying for marker enzymes, cholinergic binding sites and electron microscopy. Fractionation of the homogenate resulted in a nuclear pellet (P₁), a crude mitochrondrial pellet (P₂) and a supernatant (S₂). Further resolution of the P₂ fraction by density gradient centrifugation resulted in two bands at the gradient interfaces and a pellet. The P₂ and subsequently the P₂B fraction contained intact plasma membrane profiles as judged by the predominance of adenylate cyclase activity and the presence of occluded lactate dehydrogenase which constituted over 70% of the total activity in these fractions. Morphological examination of the gradient fractions revealed that the P₂B fraction contains membrane bound structures which resembie synaptosomes prepared from neonatal rat brain. These structures have a granular matrix in which mitochondria and frequently, neurofilaments were observed. Very few synaptic vesicles were present and there was no evidence for post synaptic attachments. The cholinergic markers choline acetyltransferase, acetylcholinesterase and receptor sites defined by quinuclidinyl benzilate and -bungarotoxin binding were enriched in fractions P₂ and P₂B which contained the bulk of nerve ending particles. This enriched preparation of fetal synaptosomes may be valuable for functional studies on pre-synaptic terminals in developing brain.Special Issue dedicated to Prof. Eduardo De Robertis.     

THE INCORPORATION OF [1-14C]ACETATE INTO UNESTERIFIED FATTY ACIDS IN RAT CEREBRAL CORTEX IN VIVO

—The incorporation of [1-¹⁴C]acetate into unesterified fatty acids and into the fatty acids of neutral glycerides and of phospholipids has been measured in rat cerebral cortex in vivo. The most rapid incorporation is seen in the unesterified fatty acids which have a turnover time of 5-6 min. It is suggested that unesterified fatty acids are precursors to neutral glycerides and phospholipids rather than being derived from them by lipase activity.     

Lateralization in Paridae: comparison of a storing and a non-storing species on a one-trial associative memory task

Summary We present evidence of a difference between a storing and non-storing species in lateralization and transfer of spatial memory processing. Using the technique of monocular occlusion, we compared the performance of a food-storing species, the marsh tit (Parus palustris), with a closely related species that does not store food, the blue tit (P. caeruleus), on a task which relies on one-trial learning for the spatial location of hidden food items. In this one-trial associative learning task, the birds had to return to sites in phase II of a trial where they had been allowed to eat some, but not all, of a piece of peanut in phase I. In the first experiment, in which the birds did not wear eye caps, marsh tits required fewer looks in phase II to find the hidden peanut than blue tits. By categorising the sites as seeded, unseeded or not visited in phase I, further analysis suggested that the two species also differ in the way they behave towards the 3 types of site. Marsh tits appear to distinguish between seeded and other sites, irrespective of whether the other sites were known to be empty (unseeded) or not (not visited); whereas blue tits preferentially returned to those sites that have been visited in phase I, irrespective of whether they contained a seed or not. In the second experiment, all birds wore an eye cap on the left or right eye for phase I and II of each trial. For both species, it was demonstrated that although the visual systems fed by both the right and left eye are involved in short-term storage, the right eye system is associated also with long-term storage. Thus, lateralization was found in both blue tits and marsh tits. However, unilateral transfer of these memories was found only in marsh tits, suggesting that there may be a difference between storers and non-storers in the mechanism of memory processing.     

Lateralization and unilateral transfer of spatial memory in marsh tits

Summary The results reported in this paper demonstrate lateralization and transfer of spatial memory processing in an adult, food-storig bird. The technique of monocular occlusion was used to investigate lateralization and memory transfer in food-storing marsh tits (Parus palustris) for two tasks, food-storing and one-trial associative learning, which rely on one-trial learning for the spatial location of hidden food items. In the food-storing task, marsh tits had to return to the sites where they had previously stored a seed; in the one-trial associative learning task, the birds had to return to sites where they had been allowed to eat some, but not all, of a piece of peanut. For both spatial memory tasks, it was demonstrated that although the visual systems fed by both eyes are involved in short-term storage, the right eye system is associated with long-term storage, and that memories are transferred from the left to the right eye system between 3 and 24 h after memory formation.     

BIOSITE: a program for the interactive comparison of aligned homologous protein sequences

A program, BIOSITE, providing for the interactive visual comparisonof aligned homologous amino-acid sequences is presented, includingan example of its application. The program allows for two typesof comparison sequence to be generated: an ‘identity’sequence and a ‘difference’ sequence. These maybe used on subsets of sequences and in further comparisons toidentify candidate sites involved in a distinct functional property.The program should prove a useful tool for biologists engagedin understanding sequence—function relationships.     

Nicotinic Modulation of [3H]Dopamine Release from Striatal Synaptosomes: Pharmacological Characterisation

Presynaptic nicotinic acetylcholine receptors on striatal nerve terminals modulate the release of dopamine. We have compared the effects of a number of nicotinic agonists and antagonists on a perfused synaptosome preparation preloaded with [3H]dopamine. (-)-Nicotine, acetylcholine, and the nicotinic agonists cytisine and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), at micromolar concentrations, stimulated the release of [3H]dopamine from striatal nerve terminals. Carbamylcholine was a much weaker agonist. The actions of (-)-nicotine, cytisine, and DMPP were inhibited by low concentrations of the nicotinic antagonists dihydro-beta-erythroidine, mecamylamine, pempidine, and neosurugatoxin; alpha-bungarotoxin was without effect, and extending the time of exposure to this toxin resulted in only very modest inhibition. This pharmacology points to a specific nicotinic receptor mechanism that is clearly distinct from that at the neuromuscular junction. Atropine failed to antagonise the effects of acetylcholine and carbamylcholine, suggesting that no muscarinic component is involved. The nicotinic receptor ligands (-)-[3H]nicotine and 125I-alpha-bungarotoxin bound to specific sites enriched in the synaptosome preparation. Drugs tested on the perfused synaptosomes were examined for their ability to interact with these two ligand binding sites in brain membranes. The differential sensitivity to the neurotoxins alpha-bungarotoxin and neosurugatoxin of the 125I-alpha-bungarotoxin and (-)-[3H]nicotine binding sites, respectively, leads to a tentative correlation of the (-)-[3H]nicotine site with the presynaptic nicotinic receptor on striatal nerve terminals.     

Sequence and functional expression of a single alpha subunit of an insect nicotinic acetylcholine receptor.

We report the isolation and sequence of a cDNA clone that encodes a locust (Schistocerca gregaria) nervous system nicotinic acetylcholine receptor (AChR) subunit (alpha L1). The calculated molecular weight of the unglycosylated polypeptide, which contains in the proposed extracellular domain two adjacent cysteine residues which are characteristic of alpha (ligand binding) subunits, is 60,641 daltons. Injection into Xenopus oocytes, of RNA synthesized from this clone in vitro, results in expression of functional nicotinic receptors in the oocyte membrane. In these, nicotine opens a cation channel; the receptors are blocked by both alpha-bungarotoxin (alpha-Bgt) and kappa-bungarotoxin (kappa-Bgt). Reversible block of the expressed insect AChR by mecamylamine, d-tubocurarine, tetraethylammonium, bicuculline and strychnine has also been observed. These data are entirely consistent with previously reported electrophysiological studies on in vivo insect nicotinic receptors and also with biochemical studies on an alpha-Bgt affinity purified locust AChR. Thus, a functional receptor exhibiting the characteristic pharmacology of an in vivo insect nicotinic AChR can be expressed in Xenopus oocytes by injection with a single subunit RNA.     

Comparison of PCR-based marker systems for the analysis of genetic relationships in cultivated potato

The application of AFLPs, RAPDs and SSRs to examine genetic relationships in the primary northwestern European cultivated potato gene pool was investigated. Sixteen potato cultivars were genotyped using five AFLP primer combinations, 14 RAPD primers, and 17 database-derived SSR primer pairs. All three approaches successfully discriminated between the 16 cultivars using a minimum of one assay. Similarity matrices produced for each marker type on the basis of Nei and Li coefficients showed low correlations when compared with different statistical tests. Dendrograms were produced from these data for each marker system. The usefulness of each system was examined in terms of number of loci revealed (effective multiplex ratio, or EMR) and the amount of polymorphism detected (diversity index, or DI). AFLPs had the highest EMR, and SSRs the highest DI. A single parameter, marker index (MI), which is the product of DI and EMR, was used to evaluate the overall utility of each marker system. The use of these PCR-based marker systems in potato improvement and statutory applications is discussed.Abbreviations: PCR, polymerase chain reaction; AFLP, amplified fragment length polymorphism; RAPD, randomly amplified polymorphic DNA; DNA, deoxyribonucleic acid; EMR, effective multiplex ratio; DI, diversity index; MI, marker index; RFLP, restriction fragment length polymorphism.