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Biomedical subjects

P Knight

Publications and source records attributed to P Knight.

At least 19 recordsLinked to original sources
PubMed2006

cDNA cloning and substrate specificity of equine tryptase, a possible mediator in equine heaves.

BACKGROUND: Mast cell mediators are believed to play a central role in inflammatory lung disorders such as human allergic and occupational asthma. Equine heaves is characterized by reversible neutrophilic airway inflammation and airway obstruction, primarily due to bronchospasm and mucus hypersecretion, following exposure of susceptible horses to organic stable dusts. As such, heaves shares many similarities with human occupational dust-induced asthma and therefore it is proposed that mast cells may also be implicated in the pathogenesis of heaves. Tryptase, a mast cell-specific proteinase, can be used as an indicator of biological mast cell activity. OBJECTIVE: The aim of this study was to determine the cDNA sequence of equine tryptase and to investigate its substrate specificity in order to rationalize its enzymatic activity. METHODS: RT-PCR cloning was used to sequence equine tryptase. Substrate specificity of equine tryptase was investigated using arginine and lysine containing substrates. RESULTS: The cDNA and deduced amino acid (Aa) sequences for equine tryptase shared strong identity with other tryptases. Unusually for a trypsin-like proteinase however, equine tryptase has alanine at residue 216, rather than glycine, which confers increased arginine substrate specificity in vitro and may restrict fibrinogenolysis in vivo. CONCLUSION: Cloning and sequencing of the mast cell proteinase equine tryptase will allow molecular probing of its expression in the lung of control and heaves-affected horses. Further work is warranted to determine the biological relevance of the unique alanine 216 substitution in the molecular sequence of the equine tryptase substrate-binding pocket.

Amino Acid Sequence↗
PubMed2004

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Anesthesia, General↗
PubMed1999

Novel selective compounds for the investigation of imidazoline receptors.

Over several years our group has sought to synthesize and identify selective ligands for imidazoline (I) receptors, in particular the I2 binding site. As a consequence, [3H]2-(2-benzofuranyl)-2-imidazoline (2BFI) has proved extremely useful for binding and autoradiographic studies. More recently we have synthesized a BU series of compounds and examined these for their affinities for both I1 and I2 binding sites. BU224 (2-(4,5-dihydroimidaz-2-yl)-quinoline) shows high affinity for I2 receptors with a Ki of 2.1 nM. BU226 (2-(4,5-dihydroimidaz-2-yl)-isoquinoline) demonstrated slightly higher affinity (Ki 1.4 nM) for I2 receptors, but overall BU224 displayed greater selectivity for I2 over I1 receptors (832-fold) than BU226 (380-fold). Both compounds showed low (microM) affinity for alpha 2-adrenoceptors. Given BU224's ability to cross the blood brain barrier, we predict that its in vivo effects are likely to be mediated via I2 receptors. Brain dialysis revealed BU224 to dose dependently (0-20 mg/kg i.p.) elevate basal noradrenaline in rat frontal cortex and basal dopamine in striatum. In a rat model of opiate withdrawal, behavioral studies showed that BU224 (10 mg/kg, s.c.) was able to reduce acute weight loss and diarrhea, but not the number of wet dog shakes associated with the withdrawal syndrome.

Animals↗
PubMed1999

Implications for space radiation environment models from CREAM & CREDO measurements over half a solar cycle.

Flight data obtained between 1990 and 1997 from the Cosmic Radiation Environment Monitors CREAM & CREDO carried on UoSAT-3, Space Shuttle, STRV-1a (Space Technology Research Vehicle) and APEX (Advanced Photovoltaic and Electronics Experiment Spacecraft) provide coverage over half a solar cycle. The modulation of cosmic rays and evolution of the South Atlantic Anomaly are observed, the former comprising a factor of three increase at high latitudes and the latter a general increase accompanied by a north-westward drift. Comparison of particle fluxes and linear energy transfer (LET) spectra is made with improved environment & radiation transport calculations which account for shield distributions and secondary particles. While there is an encouraging convergence between predictions and observations, significant improvements are still required, particularly in the treatment of locally produced secondary particles. Solar-particle events during this time period have LET spectra significantly below the October 1989 event which has been proposed as a worst case model.

Atlantic Ocean↗
PubMed1999

An investigation of the components of best nursing practice in the care of acutely ill hospitalized older patients with coincidental dementia: a multi-method design.

The main aim of this study was to examine, from the consumer's perspective, the components of best nursing practice in the care of acutely ill hospitalized elderly people who coincidentally suffered from dementia. Due to the complexity of the problem under investigation a triangulated design was formulated. This design comprised survey methodology, audit and critical incident technique. Data derived from each of these approaches were analysed first in isolation and subsequently combined at a conceptual level. The sample comprised 213 people aged 65 years and older, who were consecutive admissions from two acute medical and two acute care of elderly people wards. Sixty-six per cent of these individuals were found to suffer from cognitive impairment, 40% experiencing severe problems. The documentary survey revealed that patients with normal or severely abnormal cognitive functioning were more likely to have this noted than those with mild-moderate levels of impairment. No evidence of formal assessment was located and only 15% of these patients had items included in their nursing care plans associated with their chronic confusion. A sub-sample of 41 chronically confused people and their main hospital visitor were invited to participate in critical incident interviews. These conversations led to the identification of four major themes which reflected the most valued aspects of the nursing care which had been received or observed. The relationship between documentary care scores and the proportion of positive descriptions of care within each of the four themes was explored. The findings indicate that acute nursing care is deemed only to be at its best when it is delivered in tandem with dementia care. Discrete areas for practice development are identified and standards to guide nursing care are proposed. In addition to the practice development issues raised by the work, the methodological lessons further advance the practice of multi-method research.

Acute Disease↗
PubMed1999

Evaluation of recombinant sporozoite antigen SPAG-1 as a vaccine candidate against Theileria annulata by the use of different delivery systems.

The major sporozoite surface antigen of Theileria annulata (SPAG-1) is a candidate for inclusion in a subunit vaccine. In this paper we summarize the results of 4 vaccination experiments using recombinant SPAG-1 expressed in different systems and presented in different adjuvants. The antigen has been presented as either a C terminal 108 amino acid peptide (called SR1) expressed as both beta-galactosidase and hepatitis B core antigen fusions or as a full-length form expressed as a GST fusion with an N terminal His6 tag. We used different adjuvants, namely Freund's, saponin, ISCOMs and a proprietary adjuvant supplied by SmithKline Beecham, which we call SKBA. The data point to the conclusion that SPAG-1 can elicit partial protection and is therefore suitable for inclusion in an eventual multicomponent subunit vaccine.

Animals↗
PubMed1998

Histochemical and ultrastructural modification of mucosal mast cell granules in parasitized mice lacking the beta-chymase, mouse mast cell protease-1.

The soluble beta-chymases mouse mast cell protease-1 (mMCP-1) and rat mast cell protease-II are predominantly expressed by intestinal mucosal mast cells (IMMCs) and may promote mucosal epithelial permeability when released during intestinal allergic hypersensitivity responses. To study the function of these chymases, we generated mice with a homozygous null mutation of the mMCP-1 gene and investigated their response to infection with the intestinal nematode Nippostrongylus brasiliensis. Whereas mMCP-2, -4, and -5 were transcribed normally, there was no transcription of the mMCP-1 gene in null (-/-) mice, nor was mature mMCP-1 protein detected in (-/-) jejunal mucosa. In contrast, levels of mMCP-1 in wild-type (+/+) jejunal mucosa increased 200- to 350-fold from 0.66 microg mMCP-1/g wet weight in uninfected mice to 129 and 229 microg/g wet weight on days 8 and 10 of infection, respectively. The kinetics of IMMC recruitment differed in -/- mice compared with +/+ controls on days 8 (P < 0.05) and 10 (P < 0.03) of infection. The IMMCs in infected -/- mice stained poorly, if at all, for esterase with naphthol AS-D chloroacetate compared with the intense staining observed in +/+ controls. Ultrastructurally, the prominent crystal intragranular structures that are found in intraepithelial +/+ IMMCs were absent from -/- IMMCs. These data show that disruption of the mMCP-1 gene leads to profound histochemical and ultrastructural changes in IMMC granules.

Animals↗
PubMed1998

Intrasound vibration in the early diagnosis of scaphoid fracture.

A prospective trial was designed to assess the sensitivity and specificity of intrasound vibration for the early detection of scaphoid fracture. We replicated the method described by Finkenburg et al. (J Hand Surg 1993;18A:4-7) in an attempt to corroborate their results. We found the test to be 73% sensitive and 51% specific. Because the test was not 100% sensitive, as claimed by Finkenburg et al., we discontinued the use of this device in our hospital.

Binomial Distribution↗
PubMed1998

Updated measurements from CREAM & CREDO & implications for environment & shielding models.

Flight data obtained between 1995 and 1997 from the Cosmic Radiation Environment Monitors CREAM & CREDO carried on UoSat-3, Space Shuttle, STRV-1a (Space Technology Research Vehicle) and APEX (Advanced Photovoltaic and Electronics Experiment Spacecraft) have been added to the dataset affording coverage since 1990. The modulation of cosmic rays and evolution of the South Atlantic Anomaly are observed, the former comprising a factor three increase at high latitudes and the latter a general increase accompanied by a westward drift. Comparison of particle fluxes and linear energy transfer spectra is made with improved environment & radiation transport calculations which account for shield distributions and secondary particles. While there is an encouraging convergence between predictions and observations, significant improvements are still required, particularly in the treatrnent of locally produced secondary particles.

Atlantic Ocean↗
PubMed1997

X-ray intensifying screen visible light detection meter.

A light meter has been designed and built for the purpose of measuring the light emitted from an intensifying screen during x-ray irradiation. The meter uses a photodiode detector with a minimal drift amplification system. The meter repeatability was better than 0.5% and was found to be linear. A significant x-ray induced signal was recorded during measurement which needed to be subtracted from readings to deduce the intensification screen light output. The energy response of four screen types was subsequently measured.

Radiometry↗
PubMed1996

The structure of the head-tail junction of the myosin molecule.

An atomic model of the junction between the two heads and tail of a myosin molecule has been created by attaching a scallop regulatory domain to the end of each of the two alpha-helical strands of a model of the scallop alpha-helical coiled coil. The C-terminal alpha-helix of the heavy chain of each regulatory domain was superposed over the corresponding sequence in the coiled coil. In the structure created, the two heads lie alongside one another with their bases in contact but remarkably without steric clash. The principal interactions between the two heads are between the regulatory light chains and there are also head-tail interactions between each regulatory light chain and its heavy chain partner in the coiled coil. The invariant proline residues cause the heavy chains to flare to form the fork. The direction of the turn at the WQW sequence within the regulatory domain causes the long alpha-helix of the heavy chains within the head to continue the sense of the supercoil. With the bases of heads interacting, motion of the heads could still occur by a flexing of the coiled coil close to the heads and by a flexing and twisting of the long alpha-helices in the head. The model accounts for some of the conserved sequence features in myosins from different sources and provides a structural basis for understanding the head-head interactions in regulated myosin. Using the C alpha atoms of subfragment 1 we have also constructed a model with two complete heads. The clockwise curvature of the heads when the model is viewed end-on towards the tail accounts for the most common appearance of myosin molecules in electron micrographs. These models are predicated on the assumption that the entire heptad sequence of the heavy chains forms a coiled coil. Previous evidence from electron micrographs of myosin molecules that this was not the case can be explained by the foreshortening of the tail close to the heads.

Amino Acid Sequence↗
PubMed1996

Conservation of neutralizing determinants between the sporozoite surface antigens of Theileria annulata and Theileria parva.

The sporozoite surface antigens SPAG-1 of Theileria annulata and p67 of Theileria parva are postulated to contain determinants necessary for host cell invasion and/or recognition and are both being considered as candidates for inclusion in subunit vaccines. Preliminary data suggest that these are related molecules. In this paper we describe the investigation of the relationship between these sporozoite antigens further by analysis of the immunological cross-reactivity using Mabs and sera raised against each antigen. The cross-reactions were examined by carrying out Western blots, IFA tests, and in vitro sporozoite neutralization assays. In addition, sequence comparison data which clearly establish that these surface antigens are encoded by related genes are presented. The regions of SPAG-1 identified as containing cross-reactive epitopes recognized by p67 antiserum correlated to regions of high predicted homology between p67 and SPAG-1, which are located at their respective N- and C-termini. Furthermore, p67 and SPAG-1 were found to contain cross-reactive determinants responsible for neutralization of sporozoite infectivity in vitro, and at least some of these were located in the C-termini of both molecules. The relevance of these findings to the possible roles for these molecules in host cell invasion is discussed.

Amino Acid Sequence↗
PubMed1995

The value of endorectal ultrasonography in the follow-up of intracavitary radiation treated early rectal cancer.

In selected patients with early rectal cancer, intracavitary radiation is a successful treatment. Endorectal ultrasound has proved an accurate method for staging and selecting such cancers for treatment. The value of endorectal ultrasound in the follow-up of patients with intracavitary radiation has not been previously assessed. Between 1989 and 1991, 30 patients treated at the Hamilton Regional Cancer Centre with intracavitary radiation were assessed by endorectal ultrasound. The mean age was 65 +/- 12 years with a range of 37-78 years. There were 17 males and 13 females. All patients were treated with curative intent. The dose of radiation administered was 8963 +/- 1506 cGy over 3.5 +/- 0.7 fractions. No patient received supplemental iridium implantation. Thirty-seven endorectal ultrasounds were carried out in 30 of the intracavitary radiation treated patients. Clinical findings (digital, sigmoidoscopic, and histology) were compared with the radiologist's interpretation of the endorectal ultrasounds. Using a 2 x 2 table accepting the clinical findings as the 'Gold Standard', the sensitivity of endorectal ultrasound was 71%, the specificity 61%, the positive predictive value 53%, the negative predictive value 78% with an overall accuracy of 75%. We conclude that endorectal ultrasound in the routine follow-up of patients treated with intracavitary radiotherapy for carcinoma of the rectum is questionable.

Adult↗
PubMed1995

The influence of age on the management of anal cancer.

The standard treatment for anal cancer is combination chemo-radiotherapy. Management decisions such as radical chemotherapy, resective surgery for poor response or relapse are frequently modified by age-associated comorbid factors. Between 1980 and 1990, our regional cancer center serving a population of 1.8 million saw 78 patients with squamous carcinoma of the anus. We have compared patients who were younger than 65 years (n = 38) with those older than 65 years (n = 38). The mean +/- standard deviation age for the whole cohort was 65 +/- 12 years, with a ratio of 2 females to each male presenting. Fewer of the elderly age group had major surgery (26% vs. 42%) (p = 0.03), and fewer suffered no toxicity (42% vs. 26%) (p = 0.03). However, 61% of the under-65-year age group are alive disease-free vs. 26% of the elderly group (p = 0.03). Similarly, only 18% of the under-65-year group died with disease compared with 37% of the elderly group (b = 0.03). For the series as a whole, the crude mortality was 42%, with 27% dying of their disease. The stage distribution, and the amount of radiotherapy or chemotherapy administered was not age-specific, but younger patients had more surgery and suffered more toxicity, with a greater proportion remaining alive and disease-free, and fewer dying of their disease. These data suggest that a more aggressive multi-modality approach in the elderly may improve disease response and survival.

Age Factors↗
PubMed1994

Polymorphism of SPAG-1, a candidate antigen for inclusion in a sub-unit vaccine against Theileria annulata.

SPAG-1, a Theileria annulata sporozoite surface antigen, is a vaccine candidate. Data is presented, based on the clonal segregation of SPAG-1 associated RFLPs, showing that this antigen is encoded by a single copy gene. We have cloned and sequenced a full-length genomic copy of the SPAG-1 gene and a comparison of this with a previously published SPAG-1 cDNA sequence demonstrates a high degree of polymorphism. We infer that these sequences represent two distinct allelic SPAG-1 variants. The deduced polypeptides show an overall identity of 92% with the most variable stretch (60% identity) occurring towards the middle of the molecule. The N and C termini are more conserved with identities of 92% and 97% respectively. The elastin receptor ligand, VGVAPG, present 3 times in the protein sequence derived from the cDNA is not found in that deduced from the genomic copy. Evidence for 2 further SPAG-1 alleles was obtained from PCR based sequences using macroschizont clones containing different SPAG-1 associated RFLPs. In summary we have shown the existence of at least 4 highly polymorphic SPAG-1 alleles. The implications of such polymorphism between and within distinct geographical isolates for the development of a SPAG-1 based subunit vaccine is discussed.

Alleles↗