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Biomedical subjects

C J Thomas

Publications and source records attributed to C J Thomas.

At least 19 recordsLinked to original sources
PubMed2006

Real-time RT-PCR detection of Bovine Viral Diarrhoea virus in whole blood using an external RNA reference.

A novel two-step real-time RT-PCR assay using SYBR Green I was developed for the detection of acute Bovine Viral Diarrhoea virus (BVDV) infection in whole blood from cattle. During infection animals experience a characteristic transient leucopenia and the number of cells per volume of blood changes over time; so quantitation of viral load by reference to a cellular housekeeping gene is not ideal as this may hide significant animal to animal variation. Therefore, to facilitate comparison of different samples, an external RNA reference was used for normalisation whereby each sample was spiked with the RNA virus, Canine Enteric Coronavirus (CECov), prior to RNA extraction, for comparative purposes. Real-time RT-PCR was carried out with two primer sets designed to amplify either a 156 bp region of the BVDV 5'-UTR or a 280 bp region of the CECov nucleocapsid protein gene. Linearity and efficiency of the assay was established and the method assessed using samples from BVDV-challenged calves. Viral RNA was quantified on days 6 and 14 post-challenge by real-time RT-PCR. Infectious virus isolation by traditional cell culture was negative after day 7. This study demonstrates encouraging results for rapid, sensitive and reliable detection of acute BVDV infection and provides an alternative real-time RT-PCR method for use on whole blood samples or samples where suitable housekeeping genes are not available.

5' Untranslated Regions↗
PubMed2006

Differential effects of aging on fluid intake in response to hypovolemia, hypertonicity, and hormonal stimuli in Munich Wistar rats.

A significant proportion of aged humans may have impaired thirst and inadequate fluid intake after a period of fluid deprivation. We have studied the water drinking responses, relative to body weight, of Munich Wistar (MW) rats in response to osmotic, hypovolemic, dehydrational, and angiotensin (Ang)-related stimuli as they aged from 3 to 24 months. Young 3-months-old (m.o.) rats had the largest daily fluid intakes and drinking responses to hypertonic and dehydrational stimuli, suggesting that they have accentuated thirst in comparison with older age groups. There were no differences in daily fluid intake from 6-24 m.o.; however, drinking responses to i.p. injection of hypertonic 0.4 mol/liter NaCl gradually declined over this period so that in 24-m.o. rats the response was only half that of 6-m.o. rats. Water intake after 24-h water deprivation also declined gradually over 24 months. Drinking responses to hypovolemia induced by s.c. injection of colloid (polyethylene glycol) were unchanged in 6- to 15-m.o. rats, then declined precipitously in 18- to 24-m.o. rats. Drinking responses to s.c. Ang II or s.c. isoproterenol were not reduced in 24-m.o. rats, nor was the drinking associated with feeding. Therefore, there are specific impairments of water intake in response to hypertonicity and hypovolemia in aged MW rats, but Ang-related drinking is not reduced. Like aged humans, aged MW rats exhibit high plasma atrial natriuretic peptide levels and impaired cardiovascular reflexes that could contribute to the impairment of thirst with age.

Adrenergic beta-Agonists↗
PubMed2005

Immune responses to non-structural protein 3 (NS3) of bovine viral diarrhoea virus (BVDV) in NS3 DNA vaccinated and naturally infected cattle.

Immune responses to non-structural protein 3 (NS3) of bovine viral diarrhoea virus (BVDV) were investigated. cDNA encoding NS3 from type 1a BVDV was used to vaccinate five calves, another five calves remained unvaccinated. Three weeks after final vaccination animals were challenged intranasally with heterologous type 1a BVDV. Anti-NS3 antibodies were detected in only one animal post-vaccination. Partial protection from virus challenge was observed in the vaccinates. Virus was not isolated from nasal mucosa of two vaccinates, and virus clearance from nasal mucosa was faster in the vaccinates compared to the controls. While elevated rectal temperatures were evident in both groups 7 days post-challenge, the mean increase in the controls was twice that observed in the vaccinates. In conclusion, NS3 DNA vaccination induced humoral immunity in one calf, and prevented fever and virus establishment in the nasal mucosa in 2/5 calves, demonstrating the efficacy of NS3 vaccination, which may benefit future development of pestivirus and flavivirus vaccines.

Animals↗
PubMed2004

Structural plasticity of peanut lectin: an X-ray analysis involving variation in pH, ligand binding and crystal structure.

Until recently, it has only been possible to grow crystals of peanut lectin when complexed with sugar ligands. It is now shown that it is possible to grow peanut lectin crystals at acidic pH in the presence of oligopeptides corresponding to a loop in the lectin molecule. Crystals have also been prepared in the presence of these peptides as well as lactose. Low-pH crystal forms of the lectin-lactose complex similar to those obtained at neutral pH have also been grown. Thus, crystals of peanut lectin grown under different environmental conditions, at two pH values with and without sugar bound to the lectin, are now available. They have been used to explore the plasticity and hydration of the molecule. A detailed comparison between different structures shows that the lectin molecule is sturdy and that the effect of changes in pH, ligand binding and environment on it is small. The region involving the curved front beta-sheet and the loops around the second hydrophobic core is comparatively rigid. The back beta-sheet involved in quaternary association, which exhibits considerable variability, is substantially flexible, as is the sugar-binding region. The numbers of invariant water molecules in the hydration shell are small and they are mainly involved in metal coordination or in stabilizing unusual structural features. Small consistent movements occur in the combining site upon sugar binding, although the site is essentially preformed.

Binding Sites↗
PubMed2004

Effect of topography on the risk of malaria infection in the Usambara Mountains, Tanzania.

We investigated whether the risk of infection with malaria parasites was related to topography in the Usambara Mountains, Tanzania. Clinical surveys were carried out in seven villages, situated at altitudes from 300 m to 1650 m. Each village was mapped and incorporated into a Digital Terrain Model. Univariate analysis showed that the risk of splenomegaly declined with increasing altitude and with decreasing potential for water to accumulate. Logistic regression showed that altitude alone could correctly predict 73% of households where an occupant had an enlarged spleen or not. The inclusion of land where water is likely to accumulate within 400 m of each household increased the accuracy of the overall model slightly to 76%, but significantly improved predictions between 1000 m and 1200 m, where malaria is unstable, and likely to be epidemic. This novel approach illustrates how topography could help identify local areas prone to epidemics in the African highlands.

Adolescent↗
PubMed2003

Localized breeding of the Anopheles gambiae complex (Diptera: Culicidae) along the River Gambia, West Africa.

A study was undertaken to identify the major larval habitats of the Anopheles gambiae (Giles) complex in rural Gambia. Mosquito larvae and pupae were sampled along transects and in specific habitats in the central region of the country during the rainy seasons of 1996 and 1997. The sampling showed that the major breeding sites were located on the flooded alluvial soils bordering the river. The largest numbers of larvae were found during September, one month after the peak rains. Polymerase chain reaction analysis of specimens showed that Anopheles melas (Theobald) was the dominant species in the flooded areas (81.5%), followed by A. gambiae sensu stricto (Giles) (18.0%) and A. arabiensis (Patton) (0.5%). By sampling in specific habitats it was evident that A. arabiensis was mainly breeding in rain-fed rice fields along the edge of the alluvial soils. Anopheles melas and A. gambiae s.s. often coexisted but whereas A. melas were found in water with a salinity of up to 72% sea water (25.2 g NaCl l(-1)), A. gambiae s.s. only occurred in water with up to 30% sea water (10.5 g NaCl l(-1)). Anopheles melas larvae were found in association with plant communities dominated by sedges and grasses (Eleocharis sp., Paspalum sp., Sporobolus sp.) and sea-purslane Sesuvium portulacastrum (L.) and the presence of cattle hoof prints, whereas A. gambiae s.s. larvae mainly occurred in association with Paspalum sp. and Eleocharis sp. The study showed that even during the peak rainy season, breeding of the A. gambiae complex is almost entirely restricted to the extensive alluvial areas along the river.

Animals↗
PubMed2002

Generation of periodic waves by landscape features in cyclic predator-prey systems.

The vast majority of models for spatial dynamics of natural populations assume a homogeneous physical environment. However, in practice, dispersing organisms may encounter landscape features that significantly inhibit their movement. We use mathematical modelling to investigate the effect of such landscape features on cyclic predator-prey populations. We show that when appropriate boundary conditions are applied at the edge of the obstacle, a pattern of periodic travelling waves develops, moving out and away from the obstacle. Depending on the assumptions of the model, these waves can take the form of roughly circular 'target patterns' or spirals. This is, to our knowledge, a new mechanism for periodic-wave generation in ecological systems and our results suggest that it may apply quite generally not only to cyclic predator-prey interactions, but also to populations that oscillate for other reasons. In particular, we suggest that it may provide an explanation for the observed pattern of travelling waves in the densities of field voles (Microtus agrestis) in Kielder Forest (Scotland-England border) and of red grouse (Lagopus lagopus scoticus) on Kerloch Moor (northeast Scotland), which in both cases move orthogonally to any large-scale obstacles to movement. Moreover, given that such obstacles to movement are the rule rather than the exception in real-world environments, our results suggest that complex spatio-temporal patterns such as periodic travelling waves are likely to be much more common in the natural world than has previously been assumed.

Animals↗
PubMed2001

Mutational analysis at Asn-41 in peanut agglutinin. A residue critical for the binding of the tumor-associated Thomsen-Friedenreich antigen.

Peanut agglutinin is a clinically important lectin due to its application in the screening of mature and immature thymocytes as well as in the detection of cancerous malignancies. The basis for these applications is the remarkably strong affinity of the lectin for the tumor-associated Thomsen-Friedenreich antigen (T-antigen) and more so due to its ability to distinguish T-antigen from its cryptic forms. The crystal structure of the complex of peanut agglutinin with T-antigen reveals the basis of this specificity. Among the contacts involved in providing this specificity toward T-antigen is the water-mediated interaction between the side chain of Asn-41 and the carbonyl oxygen of the acetamido group of the second hexopyranose ring of the sugar molecule. Site-directed mutational changes were introduced at this residue with the objective of probing the role of this residue in T-antigen binding and possibly engineering an altered species with increased specificity for T-antigen. Of the three mutants tested, i.e. N41A, N41D, and N41Q, the last one shows improved potency for recognition of T-antigen. The affinities of the mutants can be readily explained on the basis of the crystal structure of the complex and simple modeling. In particular, the change of asparagine to glutamine could lead to a direct interaction of the side chain with the sugar while at the same time retaining the water bridge. This study strengthens the theory that in lectins the nonprimary contacts generally made through water bridges are involved in imparting exquisite specificity.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed2001

Thermodynamic analysis of the binding of galactose and poly-N-acetyllactosamine derivatives to human galectin-3.

Galectin-3, with a wide tissue distribution and marked developmental regulation, provides significant insights into the progression of various disease and developmental stages. Recognized by its specificity for galactose, a detailed characterization of its sugar binding ability has been investigated by isothermal titration calorimetry. The results presented here complement well with the earlier studies utilizing hapten inhibition assays. Among the various lactose derivatives studied, A-tetrasaccharide emerged with the highest affinity for binding to galectin-3 combining site. This blood group saccharide exhibited a binding affinity 37-fold higher and a 102 kJ/mol more favorable change in enthalpy over lactose at 280 K indicating the existence of additional subsites for both the alpha1-3-linked N-acetylgalactosamine at the non-reducing end and the alpha1-2-linked L-fucosyl residue. The thermodynamic parameters evaluated for other ligands substantiate further the carbohydrate recognition domain to be part of an extended binding site. Binding thermodynamics of galectin-3 with the galactose derivatives are essentially enthalpically driven and exhibit compensatory changes in DeltaH degrees and TDeltaS owing to solvent reorganization.

Antigens, Differentiation↗
PubMed2001

Kinetic and thermodynamic analysis of the interactions of 23-residue peptides with endotoxin.

Many naturally occurring peptides exhibit lipopolysaccharide binding properties. In this work we describe the endotoxin binding properties of a series of 23-residue peptides based on the sequence corresponding to the antisense strand of the magainin gene. Biochemical and biophysical characterization of these peptides reveals that they have the tendency to perturb both the inner and outer membranes of test pathogens. Structurally these peptides are amphiphilic and adopt helical conformations in membranes. Three of the seven peptides tested have high affinities for endotoxin that approach the values shown by polymyxin B, a cyclic cationic acylated decapeptide, which is used clinically in treating extreme cases of sepsis. The kinetic parameters obtained using stopped-flow methods and BIAcore analysis, when considered in conjunction with the isothermal titration calorimetry-derived thermodynamic parameters, allow us to highlight the key structural features essential for lipopolysaccharide (LPS) recognition by these peptides. The studies stress the role of ionic forces in the initial recognition of LPS. The fortification of the strength of these ionic charges increases affinity for LPS, whereas the hydrophobic residues involved in interactions are more amenable to disruptions in contiguity. Peptides that improve these features further are expected to perform better as endotoxin-neutralizing agents.

Amino Acid Sequence↗
PubMed2001

Crystal structures of the peanut lectin-lactose complex at acidic pH: retention of unusual quaternary structure, empty and carbohydrate bound combining sites, molecular mimicry and crystal packing directed by interactions at the combining site.

The crystal structures of a monoclinic and a triclinic form of the peanut lectin-lactose complex, grown at pH 4.6, have been determined. They contain two and one crystallographically independent tetramers, respectively. The unusual "open" quaternary structure of the lectin, observed in the orthorhombic complex grown in neutral pH, is retained at the acidic pH. The sugar molecule is bound to three of the eight subunits in the monoclinic crystals, whereas the combining sites in four are empty. The lectin-sugar interactions are almost the same at neutral and acidic pH. A comparison of the sugar-bound and free subunits indicates that the geometry of the combining site is relatively unaffected by ligand binding. The combining site of the eighth subunit in the monoclinic crystals is bound to a peptide stretch in a loop from a neighboring molecule. The same interaction exists in two subunits of the triclinic crystals, whereas density corresponding to sugar exists in the combining sites of the other two subunits. Solution studies show that oligopeptides with sequences corresponding to that in the loop bind to the lectin at acidic pH, but only with reduced affinity at neutral pH. The reverse is the case with the binding of lactose to the lectin. A comparison of the neutral and acidic pH crystal structures indicates that the molecular packing in the latter is directed to a substantial extent by the increased affinity of the peptide loop to the combining site at acidic pH.

Carbohydrates↗
PubMed2001

Expression of intron-containing GUS constructs is reduced due to activation of a cryptic 5' splice site.

An intron-containing beta-glucuronidase (GUS) gene has been used widely in promoter analyses and as a plant transformation marker. Maximal plant gene expression requires accurate and efficient removal of the intron from the expressed pre-mRNA transcripts by splicing. Detailed analysis of splicing of potato ST-LS1 and pea legumin introns from GUS constructs revealed the activation of a cryptic 5' splice site in the GUS coding sequence 4 nt upstream from the authentic intron 5' splice site. About 40% of transcripts utilised the cryptic 5' splice site in tobacco protoplasts, reducing the translational potential of expressed pre-mRNA. The same cryptic splicing event was evident in transgenic tobacco leaves but at reduced levels. Mutations that removed the cryptic 5' splice site are associated with a two-fold enhancement in GUS activity in tobacco protoplasts, highlighting the need for careful examination of introns and their sites of insertion into gene constructs to minimise variability in gene activity and maximise gene expression.

Glucuronidase↗
PubMed2001

Mapping distributions of chromosomal forms of Anopheles gambiae in West Africa using climate data.

The mosquito Anopheles gambiae Giles sensu stricto (Diptera: Culicidae), the principal vector of malaria in West Africa, comprises several chromosomal forms (e.g. Bissau, Forest, Mopti, Savanna) associated with climatic zones. Here we show how climate data can be used to map the geographical distribution of these chromosomal forms. The climate at 144 sites surveyed for mosquitoes in West Africa between 1971 and 92 was determined using computerized climate surfaces. Forest and Bissau forms occurred at relatively wet sites: median annual precipitation 1325 mm and 1438 mm, respectively, interquartile ranges (IQR) 1144-1858 mm and 1052-1825 mm), whilst the Mopti form was found at dry sites (annual 938 mm, IQR 713-1047 mm) and the Savanna form at sites intermediate between the wet and dry forms (annual 1067 mm, IQR 916-1279). Logistic regression analyses of the climate variables were carried out on a stratified random sample of half the sites. The resulting models correctly classified over 80% of the sites for presence or absence of each chromosomal form. When these models were tested against excluded sites they were also correct at over 80% of sites. The combined data produced models that were correct at over 86% of sites. Mean annual precipitation, evapotranspiration, minimum temperature and maximum temperature were the most important climate variables correlated with the distribution of these forms of An. gambiae. We used the logistic models to map the distribution of each chromosomal form within the reported range for An. gambiae s.s. in West Africa employing a geographical information system. Our maps indicate that each chromosomal form favours particular climate envelopes in well-defined ecoclimatic zones, although these forms are sympatric at the edges of their ranges. This study demonstrates that climate can be used to map the distribution of chromosomal forms of insects across large areas.

Africa, Western↗
PubMed2001

ANP, BNP, and CNP enhance bradycardic responses to cardiopulmonary chemoreceptor activation in conscious sheep.

We demonstrated previously that atrial natriuretic peptide (ANP) enhances reflex bradycardia to intravenous serotonin [5-hydroxytryptamine (5-HT)] (von Bezold-Jarisch reflex) in rats. To determine whether 1) ANP affects this cardiopulmonary vagal reflex in another species and 2) B-type (BNP) and C-type (CNP) natriuretic peptides share with ANP the ability to modulate this reflex, we used intravenous phenylbiguanide (PBG), a 5-HT(3) agonist, as the stimulus to evoke a von Bezold-Jarisch reflex (dose-related, reproducible bradycardia) in conscious adult sheep (n = 5). Three doses of PBG (13 +/- 3, 20 +/- 3, and 31 +/- 4 microg/kg) injected into the jugular vein caused reflex cardiac slowing of -7 +/- 1, -15 +/- 2, and -36 +/- 3 beats/min, respectively, under control conditions. These doses of PBG were repeated during infusions of ANP, BNP, or CNP (10 pmol. kg(-1). min(-1) iv), or vehicle (normal saline). Each of the natriuretic peptides significantly (P < 0.05) enhanced the sensitivity of bradycardic responses to PBG by 94 +/- 8% (ANP), 142 +/- 55% (BNP), and 61 +/- 16% (CNP). Thus not only did ANP sensitize cardiopulmonary chemoreceptor activation in a species with resting heart rate close to that in humans, but BNP and CNP also enhanced von Bezold-Jarisch reflex activity in conscious sheep.

Animals↗