High-level genotypic variation and antibiotic sensitivity among Escherichia coli O157 strains isolated from two Scottish beef cattle farms

Escherichia coli O157:H7 is a human pathogen that is carried and transmitted by cattle. Scotland is known to have one of the highest rates of E. coli O157 human infections in the world. Two hundred ninety-three isolates were obtained from naturally infected cattle and the environment on two farms in the Scottish Highlands. The isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI restriction endonuclease enzyme, and 19 different variations in patterns were found. There was considerable genomic diversity within the E. coli O157 population on the two farms. The PFGE pattern of one of the observed subtypes matched exactly with that of a strain obtained from a Scottish patient with hemolytic-uremic syndrome. To examine the stability of an individual E. coli O157 strain, continuous subculturing of a strain was performed 110 times. No variation from the original PFGE pattern was observed. We found three indistinguishable subtypes of E. coli O157 on both study farms, suggesting common sources of infection. We also examined the antibiotic resistance of the isolated strains. Phenotypic studies demonstrated resistance of the strains to sulfamethoxazole (100%), chloramphenicol (3.07%), and at a lower rate, other antibiotics, indicating the preservation of antibiotic sensitivity in a rapidly changing population of E. coli O157.     

Rapid detection of `rare' actinomycetes in environmental samples

Natural products continue to be a useful source of new leads for the pharmaceutical industry. Actinomycetes are prolific producers of natural products and one strategy to increase the possibility of discovering novel chemical entities is to screen actinomycetes considered 'rare' in the environment and previously under-represented in natural product screening collections. We describe a method using bacteriophage as a marker to detect these actinomycetes in environmental samples. This method allows samples to be pre-screened for the presence of target actinomycetes before lengthy isolation programmes are undertaken.     

Visibility of the impact of environmental noise: a response to Kaitala and Ranta

Based on simulation modelling, Kaitala and Ranta (2001 Proc. R. Soc. Lond. B 268, 1769-1774) have argued that detecting the statistical relationships between environmental variability and population fluctuations will be difficult. However, their study was limited in that only one pattern of density dependence and one detection method were used. Here, we show that their conclusion is in part a consequence of their choice of population model and in part a consequence of using relatively weak or inappropriate statistical methods. Other patterns of density dependence respond differently to environmental fluctuations, and the impact of the disturbance on these is clearly visible using their methods. For some patterns of population dynamics, environmental impacts are more readily detectable by correlating running-average environmental conditions with the population time-series or by correlating the first differences of the population time-series with environmental noise. When more appropriate statistical methods are used, environmental forcing is detectable in the majority of cases used by Kaitala and Ranta. The interplay between environmental stochasticity and density-dependent population growth means that there is no single best method to detect the influence of environmental forcing, even when population dynamics are approximately linear. But environmental forcing will often be detectable, contrary to Kaitala and Ranta's assertions.     

Development of the signalling pathways associated with sperm capacitation during epididymal maturation

As spermatozoa mature within the epididymis they acquire the potential for capacitation and ultimately fertilization. In biochemical terms, the former is reflected in the progressive activation of a signal transduction pathway characterized by cAMP-mediated induction of phosphotyrosine expression on the sperm tail. In this study, we have examined the cellular mechanisms controlling this maturational event. Caput epididymal spermatozoa exhibited tyrosine phosphorylation on the sperm head that was largely unresponsive to cAMP and not significantly impaired by removal of extracellular HCO(3) (-). In contrast, caudal epididymal spermatozoa exhibited low levels of phosphorylation on the sperm head, yet responded dramatically to cAMP by phosphorylating a new set of proteins on the sperm tail via mechanisms that were highly dependent on extracellular HCO(3) (-). The impact of extracellular HCO(3) (-) depletion on caudal cells was not associated with a significant change in the redox regulation of cAMP but could be fully reversed by buffering the intracellular pH with N-Tris[Hydroxymethyl]methyl-3-amino-propanesulfonic acid (TAPS). The pattern of tyrosine phosphorylation was also profoundly influenced by the presence or absence of added extracellular calcium. In the presence of this cation, only caudal spermatozoa could respond to increased extracellular cAMP with tyrosine phosphorylation of the sperm tail. However, in calcium-depleted medium, this difference completely disappeared. Under these conditions, caput and caudal spermatozoa were equally competent to exhibit phosphotyrosine expression on the sperm tail in response to cAMP. These results emphasize the pivotal role played by calcium and HCO(3) (-) in modulating the changes in tyrosine phosphorylation observed during epididymal maturation.     

The speed of soil carbon throughput in an upland grassland is increased by liming

In situ (13)C pulse labelling was used to measure the temporal and spatial carbon flow through an upland grassland. The label was delivered as (13)C-CO(2) to vegetation in three replicate plots in each of two treatments: control and lime addition. Harvests occurred over a two month period and samples were taken along transects away from the label delivery area. The (13)C concentration of shoot, root, bulk soil, and soil-respired CO(2) was measured. There was no difference in the biomass and (13)C concentration of shoot and root material for the control and lime treatments meaning that the amount of (13)C-CO(2) assimilated by the vegetation and translocated below ground was the same in both treatments. The (13)C concentration of the bulk soil was lower in the lime treatment than in the control and, conversely, the (13)C concentration of the soil-respired CO(2) was higher in the lime. Unlike the difference in bulk soil (13)C concentration between treatments, the difference in the (13)C concentration of the soil-respired CO(2) was obvious only at the delivery site and primarily within 1 d after labelling. An observed increase in the abundance of mycorrhizal fungi in the lime treatment was a possible cause for this faster carbon throughput. The potential key role of mycorrhizas in the soil carbon cycle is discussed. The importance of a better understanding of soil processes, especially biological ones, in relation to the global carbon cycle and environmental change is highlighted.     

Murid herpesvirus 4 strain 68 M2 protein is a B-cell-associated antigen important for latency but not lymphocytosis

This work describes analyses of the function of the murid herpesvirus 4 strain 68 (MHV-68) M2 gene. A frameshift mutation was made in the M2 open reading frame that caused premature termination of translation of M2 after amino acid residue 90. The M2 mutant showed no defect in productive replication in vitro or in lungs after infection of mice. Likewise, the characteristic transient increase in spleen cell number, Vbeta4 T-cell-receptor-positive CD8(+) T-cell mononucleosis, and establishment of latency were unaffected. However, the M2 mutant virus was defective in its ability to cause the transient sharp rise in latently infected cells normally seen in the spleen after infection of mice. We also demonstrate that expression of M2 is restricted to B cells in the spleen and that M2 encodes a 30-kDa protein localizing predominantly in the cytoplasm and plasma membrane of B cells.     

Tyrosine phosphorylation of HSP-90 during mammalian sperm capacitation

The process of sperm capacitation is correlated with activation of a signal transduction pathway leading to protein tyrosine phosphorylation. Whereas phosphotyrosine expression is an essential prerequisite for fertilization, the proteins that are phosphorylated during capacitation have not yet been identified. In the present study, we observed that a major target of this signaling pathway is the molecular chaperone protein, heat shock protein (HSP)-86, a member of the HSP-90 family of HSPs. We used cross-immunoprecipitation experiments to confirm the tyrosine phosphorylation of HSP-86, a process that is not inhibited by the ansamycin antibiotic, geldanamycin. The general significance of these findings was confirmed by studies in which HSP-90 was also found to be tyrosine phosphorylated in human and rat spermatozoa when incubated under conditions that support capacitation. To our knowledge, these results represent the first report of a protein that undergoes tyrosine phosphorylation during mouse sperm capacitation and the first study implicating molecular chaperones in the processes by which mammalian spermatozoa gain the ability to fertilize the oocyte.     

Forced evolution of a herbicide detoxifying glutathione transferase

Plant Tau class glutathione transferases (GSTUs) detoxify diphenylether herbicides such as fluorodifen, determining their selectivity in crops and weeds. Using reconstructive PCR, a series of mutant GSTUs were generated from in vitro recombination and mutagenesis of the maize sequences ZmGSTU1 and ZmGSTU2 (with the prefix Zm designating Zea mays L.). A screen of 5000 mutant GSTUs identified seven enzymes with enhanced fluorodifen detoxifying activity. The best performing enhanced fluorodifen detoxifying mutant (EFD) had activity 19-fold higher than the parent enzymes, with a single point mutation conferring this enhancement. Further mutagenesis of this residue generated an EFD with a 29-fold higher catalytic efficiency toward fluorodifen as compared with the parents but with unaltered catalysis toward other substrates. When expressed in Arabidopsis thaliana, the optimized EFD, but not the parent enzymes, conferred enhanced tolerance to fluorodifen. Molecular modeling predicts that the serendipitous mutation giving the improvement in detoxification is due to the removal of an unfavorable interaction together with the introduction of a favorable change in conformation of residues 107-119, which contribute to herbicide binding.     

Adrenomedullin influences magnocellular and parvocellular neurons of paraventricular nucleus via separate mechanisms

We previously reported that adrenomedullin (AM) decreases blood pressure following microinjection into the paraventricular nucleus of the hypothalamus (PVN) of the rat. With the use of whole cell recordings in rat hypothalamic slice preparations, we characterized the effects of AM on electrophysiologically identified PVN neurons and described the membrane events underlying such actions. AM hyperpolarized magnocellular (type I) neurons in a dose-dependent manner, a response associated with an increase in the frequency and amplitude of inhibitory postsynaptic potentials. Blockade of action potentials with tetrodotoxin (TTX) abolished AM effects on membrane potential and synaptic activity in magnocellular neurons, suggesting direct actions on inhibitory interneurons. Furthermore, blockade of inhibitory synaptic transmission with the GABA(A) receptor antagonist bicuculline methiodide also abolished AM effects on membrane potential in magnocellular neurons. In contrast, parvocellular (type II) neurons depolarized following AM receptor activation. AM effects on parvocellular neurons were dose dependent and were maintained in the presence of TTX, indicating direct effects on this population of neurons. Voltage-clamp recordings from parvocellular neurons showed AM enhances a nonselective cationic conductance, suggesting a potential mechanism through which AM influences membrane potential. These observations show clear population-specific actions of AM on separate identified groups of PVN neurons. Such effects on magnocellular neurons likely contribute to the hypotensive actions of this peptide in PVN. Although the effects on parvocellular neurons may also contribute to such cardiovascular effects of AM, it is more likely that actions on this population of PVN neurons underlie the previously demonstrated activational effects of AM on the hypothalamic-pituitary-adrenal axis.