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Biomedical subjects

P Freestone

Publications and source records attributed to P Freestone.

15 recordsLinked to original sources
PubMed2000

Effects of calcium and calcium chelators on growth and morphology of Escherichia coli L-form NC-7.

Growth of a wall-less, L-form of Escherichia coli specifically requires calcium, and in its absence, cells ceased dividing, became spherical, swelled, developed large vacuoles, and eventually lysed. The key cell division protein, FtsZ, was present in the L-form at a concentration five times less than that in the parental strain. One interpretation of these results is that the L-form possesses an enzoskeleton partly regulated by calcium.

Bacterial Proteins↗
PubMed1999

The effects of multiple metal contamination on ectomycorrhizal Scots pine (Pinus sylvestris) seedlings.

Experiments were conducted to investigate the effects of single and multiple metal contamination (Cd, Pb, Zn, Sb, Cu) on Scots pine seedlings colonised by ectomycorrhizal (ECM) fungi from natural soil inoculum. Seedlings were grown in either contaminated field soil from the site of a chemical accident, soils amended with five metals contaminating the site, or in soil from an uncontaminated control site. Although contaminated and metal-amended soil significantly inhibited root and shoot growth of the Scots pine seedlings, total root tip density was not affected. Of the five metals tested in amended soils, Cd was the most toxic to ECM Scots pine. Field-contaminated soil had a toxic effect on ECM fungi associated with Scots pine seedlings and caused shifts in ECM species composition on ECM seedlings. When compared to soils amended with only one metal, soils amended with a combination of all five metals tested had lower relative toxicity and less accumulation of Pb, Zn and Sb into seedlings. This would indicate that the toxicity of multiple metal contamination cannot be predicted from the individual toxicity of the metals investigated.

Journal Article↗
PubMed1998

Tyrosine phosphorylation in Escherichia coli.

The phosphorylation on tyrosine of a protein in Escherichia coli both in vivo and in vitro was revealed by recognition by anti-phosphotyrosine antibodies, labelling with [gamma-32P]ATP, and phosphoamino acid analysis. This protein, which we name TypA, is the product of the o591 reading frame as revealed by N-terminal sequencing and antibody cross-reactivity. Inactivation of typA altered the patterns of protein synthesis during both exponential growth and carbon starvation. These alterations included the disappearance of an acidic isoform of both the universal stress protein UspA and carbon starvation protein Csp15, and increased synthesis of the histone-like protein H-NS. The sequence of TypA from strain K-12 differs from that of an enteropathogenic strain in six amino acid residues and the protein is three residues shorter. We propose that TypA interacts with global regulatory networks and that its phosphorylation may be relevant to pathogenesis.

Adenosine Triphosphate↗
PubMed1998

Rapid growth mutants of Escherichia coli.

If rapid growth (rap) mutants of Escherichia coli could be obtained, these might prove a valuable contribution to fields as diverse as growth rate control, biotechnology and the regulation of the bacterial cell cycle. To obtain rap mutants, a dnaQ mutator strain was grown for four and a half days continuously in batch culture. At the end of the selection period, there was no significant change in growth rate. The result means that selecting rap mutants may require an alternative strategy and a number of such alternatives are discussed.

Bacteriological Techniques↗
PubMed1998

Protein phosphorylation in Escherichia coli L. form NC-7.

Wall-less L-forms of Escherichia coli constitute an interesting, and relatively underused, model system for numerous studies of bacterial physiology including the cell cycle, intracellular structure and protein phosphorylation. Total extracts of the L-form revealed a pattern of protein phosphorylation similar to that of an enteropathogenic strain but very different from its parental K-12 strain. In particular, the L-form extract revealed phosphorylation on tyrosine of a protein important in pathogenesis, TypA, and calcium-specific phosphorylation of a 40 kDa protein. Two new phosphoproteins were identified in the L-form as the DNA-binding protein Dps, and YfiD, a protein of 14 kDa with homology to pyruvate formate-lyase and a region containing a tRNA cluster in bacteriophage T5.

Amino Acid Sequence↗
PubMed1997

The universal stress protein, UspA, of Escherichia coli is phosphorylated in response to stasis.

Transcriptional induction of the uspA gene of Escherichia coli occurs whenever conditions cause growth arrest and cells deficient in UspA survive poorly in stationary phase. We demonstrate that the product of uspA is a serine and threonine phosphoprotein. In vivo, three isoforms of UspA were detected, two of which were phosphorylated as determined by alkaline phosphatase treatment; in vitro, phosphorylation with [gamma-32P]ATP yielded two radioactive UspA isoforms. The phosphorylated isoforms were barely visible in growing cells but one increased during starvation conditions causing growth arrest. This phosphorylation is dependent on the o591 gene, which encodes an autophosphorylating tyrosine phosphoprotein and which is involved in the synthesis or modification of six other proteins. In vitro, UspA undergoes a rapid and dynamic autophosphorylation, as shown by chase experiments with GTP or ATP as phosphate donors.

Amino Acid Sequence↗
PubMed1997

Artefactual cleavage of E coli H-NS by OmpT.

In the bacterium Escherichia coli, H-NS-(H1, H1a) is a heat-stable protein with a molecular mass of 15.5 kDa involved in nucleoid organisation and gene regulation linked to certain signal transduction pathways. We have shown that, following addition of preparations of everted inner membrane vesicles, heat-stable cleavage products of approximately 10 kDa of H-NS are formed in vitro from newly synthesised, radio-labelled H-NS and from purified H-NS. The 15.5 kDa protein and its cleavage products were also recovered from a minicell system. These results raised the possibility that cleavage of H-NS is physiologically significant. However, the cleavage of H-NS observed appears to occur during cell breakage and to depend on the method of protein extraction and the presence of the outer membrane protease, OmpT. Nevertheless, the results indicate that H-NS may contain at least two separate domains with cleavage occurring between these domains at a preferred OmpT site. Failure to take account of H-NS cleavage in sample preparation and analysis can lead to serious underestimation of H-NS levels.

Artifacts↗
PubMed1996

Elements of a unifying theory of biology.

To discover a unifying theory of biology, it is necessary first to believe in its existence and second to seek its elements. Such a theory would explain the regulation of the cell cycle, differentiation and the origin of life. Some elements of the theory may be obtained by considering both eukaryotic and prokaryotic cell cycles. These elements include cytoskeletal proteins, calcium, cyclins, protein kinase C, phosphorylation, transcriptional sensing, autocatalytic gene expression and the physical properties of lipids. Other more exotic candidate elements include the dynamic enzoskeleton, ATP generation, mechanotransduction, the piezoelectric effect and resonance. Bringing these disparate elements together--and discovering others--will require extensive collaborations between specialists from different sciences. This can only be achieved within the context of an integrated approach to biology.

Animals↗
PubMed1996

Second-generation rodenticides and polecats (Mustela putorius) in Britain.

In Britain, polecats Mustela putorius hunt around farm buildings, especially in winter, and, as a result, may be secondarily exposed to rodenticides by eating contaminated prey. This paper reports the first survey of second-generation rodenticides in polecats. Twenty-nine adult polecats which had been killed either accidentally on roads (24) and in traps (4), or had died of an unknown cause (1) were collected during 1992-1994. The livers of 24 animals and the stomach walls of the remaining five, for which the livers were not available, were analysed for difenacoum, bromadiolone, brodifacoum and flocoumafen. In total, rodenticide residues were detected in 31% of the polecats analysed. Residues were found in seven of the 24 livers (29%) and in two of the five stomachs analysed (40%). Difenacoum was detected most frequently (28% of animals), and was the only rodenticide in the stomach, while bromadiolone and brodifacoum were detected in only 10% and 3% of polecats, respectively. Flocoumafen was not detected in any animals. More than one rodenticide occurred in the livers of two animals; one contained difenacoum and bromadiolone, the other also contained brodifacoum. There was no sex bias in the proportion of animals containing rodenticides. Animals with detectable residues came from more than one county and were collected only during January-April in each year.

Journal Article↗
PubMed1995

Characterization of eukaryotic-like kinase activity in Escherichia coli using the gene-protein database.

The gene-protein database was used to obtain the two-dimensional polyacrylamide gel coordinates of proteins phosphorylated in extracts of Escherichia coli including those phosphorylated by eukaryotic-like kinase activities. These suggest that the phosphoproteins correspond to, or co-migrate with, the product of an open reading frame at 1.3 min (Orf80), Enzyme 1 of the phosphoenolpyruvate-dependent phosphotransferase system (PtsI), the tRNA synthetase for histidine (HisS), and proteins involved in the response to carbon starvation and quinone treatment.

Bacterial Proteins↗
PubMed1995

Identification of phosphoproteins in Escherichia coli.

The substrates of ion- and lipid-stimulated protein kinase activity in extracts of Escherichia coli were purified by chromatography. Subsequent N-terminal sequencing suggests that these substrates include the following: a novel 80 kDa protein co-purifying with RNA polymerase but partially homologous to elongation factor G; a protein with an apparent molecular weight of 65 kDa identified as the ribosomal protein S1; and a 32 kDa protein identified as succinyl CoA synthetase, a key enzyme in the tricarboxylic acid cycle. The phosphorylation of these three proteins was markedly stimulated by the addition of manganese, and occurred on threonine, serine or tyrosine residues as indicated by the stability of the phosphoresidues during acid treatment. In addition, a calcium-stimulated protein of 70 kDa was identified as the heat-shock protein DnaK, and a 17 kDa lipid-stimulated phosphoprotein as nucleotide diphosphate kinase.

Adenosine Triphosphate↗
PubMed1990

Rodenticides in British barn owls.

Out of 145 Barn Owls found dead through accidents (66%), starvation (32%), shooting (2%) and poisoning (<1%), 10% contained residues of rodenticides, difenacoum or brodifacoum, in their livers. Difenacoum was in the range 0.005-0.106 microg g(-1) fresh weight, and brodifacoum was in the range 0.019-0.515 microg g(-1). Minimum levels of detection were about 0.005 microg g(-1) for both chemicals. Mice fed for 1 day on food containing difenacoum and brodifacoum died after 2-11 days. Within these mice residues were present at greater concentration in the liver than in the rest of the carcass. The mean mass of residue in a whole 35g mouse was estimated at 10.17 microg (range 4.73-20.65 microg) for difenacoum and 15.36 microg (range 8.07-26.55) for brodifacoum. Such poisoned mice were fed to Barn Owls for successive periods of 1, 3 and 6 days. All six owls fed on difenacoum-dosed mice survived all three treatments, in which up to an estimated 101.7 microg of difenacoum was consumed, and the coagulation times of their blood returned to near normal in less than 5-23 days. Four of the six owls fed on brodifacoum-dosed mice died 6-17 days after the 1-day treatment, but the survivors also survived the 3-day and 6-day treatments. Those that died had each eaten 3 mice, with a combined weight of about 105g and a total brodifacoum content of about 46.07 microg, which was equivalent to a dose of 0.150-0.182 mg kg(-1) of owl body weight. After death these owls had 0.63-1.25 micro g(-1) of brodifacoum in their livers. Blood from the survivors would not coagulate at 9 days post-treatment, but did so at 16 days in one bird and between 38 and 78 days in the other. It is concluded that: (1) Barn Owls in Britain are now widely exposed to second-generation rodenticides; (2) not all owls exposed to these chemicals are likely to receive a lethal dose; (3) brodifacoum is more toxic to owls than difenacoum; and (4) while there is yet no evidence that rodenticides have had any appreciable effect on Barn Owl populations in Britain, further monitoring of residue levels and population trends in desirable.

Journal Article↗
PubMed1990

Trends in organochlorine and mercury levels in Gannet eggs.

Levels of DDE, HEOD, PCBs and mercury were measured in Gannet eggs from several colonies around Britain during 1971-1987. At two colonies (Ailsa Craig and Bass Rock) sampled throughout this period, organochlorine levels declined between the early 1970s and 1983, and then increased again. The trends at other colonies, sampled over shorter periods, were generally consistent with those at Ailsa Craig and Bass Rock. Trends in mercury levels were more variable, and increases were observed at four colonies and declines at two. Some shell-thinning occurred in association with DDE contamination. However, levels of this chemical were too low to affect breeding success in the years concerned. The same was probably true of the other chemicals.

Journal Article↗