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P Spooner

Publications and source records attributed to P Spooner.

10 recordsLinked to original sources
PubMed2001

Molecular and immunological characterisation of Theileria parva stocks which are components of the 'Muguga cocktail' used for vaccination against East Coast fever in cattle.

The 'Muguga cocktail' which is composed of three Theileria parva stocks Muguga, Kiambu 5 and Serengeti-transformed has been used extensively for live vaccination against East Coast fever in cattle in eastern, central and southern Africa. Herein we describe the molecular characterisation of the T. parva vaccine stocks using three techniques, an indirect fluorescent antibody test with a panel of anti-schizont monoclonal antibodies (MAb), Southern blotting with four T. parva repetitive DNA probes and polymerase chain reaction (PCR)-based assays detecting polymorphism within four single copy loci encoding antigen genes. The Muguga and Serengeti-transformed stocks exhibited no obvious differences in their reactivity with the panel of MAbs, whereas Kiambu 5 differed with several MAbs. Kiambu 5 DNA was very distinct from the Muguga and Serengeti-transformed isolates in the hybridisation pattern with all four nucleic acid probes, whereas Muguga and Serengeti-transformed isolates exhibited minor differences and could not be discriminated with one of the probes. PCR amplification in combination with restriction fragment length polymorphism analysis indicated that Kiambu 5 was also markedly divergent from the Muguga and Serengeti-transformed stocks within two of the four antigen coding genes. The T. parva Serengeti-transformed stock did not contain a 130 base pair insert within the p67 sporozoite antigen gene, which has been observed previously in most T. parva parasites isolated from buffalo, and could not be discriminated from T. parva Muguga at any of the four single copy loci. Collectively the data indicate that two of the cocktail components T. parva Serengeti-transformed and Muguga are genetically closely related, while the third component Kiambu 5 is quite distinct. Based on the findings, there may be a need to include only one of the T. parva Muguga and Serengeti-transformed components in the immunising cocktail. The study demonstrates the value of molecular characterisation data for monitoring of live vaccines.

Animals↗
PubMed2000

Microsequence heterogeneity and expression of the LSU rRNA genes within the two single copy ribosomal transcription units of Theileria parva.

The nucleotide sequences of the large subunit ribosomal RNA coding genes within the two single copy ribosomal DNA transcription units of a cloned Theileria parva isolate from a buffalo were determined. The two LSU rRNA coding units differed by 11 nucleotide substitutions and two deletions of 1 and 6 bp, all located in the 5' end of the LSU coding region. We also observed microsequence heterogeneity between the two buffalo parasite LSU sequences and the previously determined LSU rRNA gene of a T. parva parasite isolated from cattle. At all positions which were variable between the two LSU rRNA coding sequences of the buffalo-derived parasite, either unit 1 or unit 2 matched the LSU rRNA coding sequence of the cattle-derived T. parva parasite in a mosaic pattern. Synthetic oligonucleotides specific for LSU units 1 and 2 of the buffalo T. parva were developed, and used to assay expression of the two units. Both units were expressed in the intra-lymphocytic schizont stage of T. parva. A 2.5-10-fold excess of rRNA derived from LSU unit 1, compared with unit 2, was observed in the schizont stage, the difference being attributable to variation in the level of expression of unit 2. Theileria represents the third genus of Sporozoan protozoa, in addition to Plasmodium and Babesia, exhibiting rRNA coding genes, which are divergent in sequence between different transcription units.

Animals↗
PubMed2000

31P-CP-MAS NMR studies on TPP+ bound to the ion-coupled multidrug transport protein EmrE.

The binding of tetraphenylphosphonium (TPP+) to EmrE, a membrane-bound, 110 residue Escherichia coli multidrug transport protein, has been observed by 31P cross-polarisation-magic-angle spinning nuclear magnetic resonance spectroscopy (CP-MAS NMR). EmrE has been reconstituted into dimyristoyl phosphatidylcholine bilayers. CP-MAS could selectively distinguish binding of TPP+ to EmrE in the fluid membrane. A population of bound ligand appears shifted 4 ppm to lower frequency compared to free ligand in solution, which suggests a rather direct and specific type of interaction of the ligand with the protein. This is also supported by the observed restricted motion of the bound ligand. The observation of another weakly bound substrate population arises from ligand binding to negatively charged residues in the protein loop regions.

Amino Acid Sequence↗
PubMed1997

A distance measurement between specific sites on the cytoplasmic surface of bovine rhodopsin in rod outer segment disk membranes.

Structural information on mammalian integral membrane proteins is scarce. As part of work on an alternative approach to the structure of bovine rhodopsin, a method was devised to obtain an intramolecular distance between two specific sites on rhodopsin while in the rod outer segment disk membrane. In this report, the distance between the rhodopsin kinase phosphorylation site(s) on the carboxyl terminal and the top of the third transmembrane helix was measured on native rhodopsin. Rhodopsin was labeled with a nuclear spin label (31P) by limited phosphorylation with rhodopsin kinase. Major phosphorylation occurs at serines 343 and 338 on the carboxyl terminal. The phosphorylated rhodopsin was then specifically labeled on cysteine 140 with an electron spin label. Magic angle spinning 31P-nuclear magnetic resonance revealed the resonance arising from the phosphorylated protein. The enhancement of the transverse relaxation of this resonance by the paramagnetic spin label was observed. The strength of this perturbation was used to determine the through-space distance between the phosphorylation site(s) and the spin label position. A distance of 18 +/- 3 A was obtained.

Animals↗
PubMed1995

Theileria: improved species discrimination using oligonucleotides derived from large subunit ribosomal RNA sequences.

Partial nucleotide sequences of large subunit ribosomal RNA (LSU rRNA) coding genes of Theileria parva and T. taurotragi were determined. Alignment of the T. parva and T. taurotragi sequences revealed species-specific regions within the LSU rRNA genes. A major species-variable region, located between the first and second conserved secondary structure domains of LSU rRNA, was identified. The sequence of the major species-variable region was determined for T. annulata, T. buffeli, and an unclassified Theileria species isolated from buffalo. Species-specific oligonucleotides complementary to LSU rRNA sequences were designed for five species of Theileria. The oligonucleotide probes discriminated between Theileria species when hybridized to amplified parasite LSU rRNA genes or directly to parasite rRNA. Three species-specific oligonucleotides derived from nonoverlapping sequences were developed for each of T. parva and T. taurotragi. A simplified procedure was developed in which T. parva- and T. taurotragi-specific oligonucleotides were used to discriminate the two species under nonstringent hybridization and washing conditions. Use of the nonstringent conditions also increased the sensitivity of target detection. Comparison of Theileria species-specific oligonucleotides derived from large subunit and small subunit rRNA genes demonstrated that, for closely related species, probes derived from LSU rRNA sequences had improved specificity.

Animals↗
PubMed1993

The antiviral peptide carbobenzoxy-D-phenylalanyl-L-phenylalanylglycine changes the average conformation of phospholipids in membranes.

The influence of the antiviral peptide, carbobenzoxy-D-phenylalanyl-L-phenylalanylglycine (ZfFG), on the average conformation of phosphatidylcholine in hydrated bilayers was investigated with multinuclear solid state magnetic resonance (NMR). Phosphatidylcholine was specifically deuterated (separately) in the choline N-methyls, the alpha and beta positions of the choline, the C2 carbon of the acyl chains, and at all the carbons of the acyl chains of the phosphatidylcholine. Phosphatidylcholine was also synthesized with the carbonyl carbons of the ester bonds between the glycerol and the hydrocarbon chains enriched in 13C. 2H NMR of the phosphatidylcholine perdeuterated in the acyl chains showed a loss of intensity from the deuteriums with the largest quadrupole splitting in the presence of ZfFG, while the remainder of the powder pattern was largely unaffected. The phosphatidylcholine specifically deuterated at the C2 carbon (representative of the C-D bonds giving rise to the largest quadrupole splittings) showed the same loss of intensity suggesting changes in the phospholipid conformation and conformational dynamics near the glycerol. Analysis of the powder patterns in the 13C NMR spectrum of phosphatidylcholine labeled with 13C in the carbonyl carbons revealed a significant change in the average orientation of the sn-1 carbonyl due to the presence of the ZfFG and no change in the sn-2 carbonyl orientation. Changes in the headgroup conformation, as detected by 2H NMR of the deuteriums in the alpha and beta methylenes of the choline headgroup and 31P NMR of the phosphate segment, reflected the electrostatic nature of the interaction of the carboxyl of ZfFG with phosphatidylcholine bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1993

Discrimination between six species of Theileria using oligonucleotide probes which detect small subunit ribosomal RNA sequences.

The complete small subunit ribosomal RNA (srRNA) gene of Theileria parva was cloned and sequenced. Two primers were designed which permitted the specific amplification of part of the Theileria srRNA gene from Theileria-infected cell line samples which were predominantly (> 95%) bovine DNA. The sequence of the central (variable) region of the srRNA genes of T. annulata, T. taurotragi, T. mutants and two unidentified parasites referred to as Theileria sp. (buffalo) and Theileria sp. (Marula) were obtained. An alignment of the sequences was generated from which 6 oligonucleotide probes, corresponding to species-specific regions, were designed. These probes were demonstrated to provide unequivocal identification of each of the 6 species either by direct detection of parasite srRNA or by hybridization to amplified parasite srRNA genes. The probes were not able to distinguish buffalo-derived T. parva, the causal agent of Corridor disease, from cattle-derived T. parva, the causal agent of East Coast fever.

Animals↗
PubMedDate not supplied

Field observations on influenza vaccination among horses in Britain, 1971-1976.

The observations reported in this paper were obtained as part of a long term surveillance programme designed to monitor the efficacy of influenza vaccines and study the prevalence of influenza and other respiratory viruses among horses in Britain. Inactivated influenza vaccines were found to be effective in protecting horses from disease caused by influenza A/equine-1 but were less successful in protecting horses against influenza A/equine-2. The paper presents the clinical, epidemiological virological and serological findings obtained between 1971 and 1976.

Animals↗