Polymorphisms of the CYP2D6 gene increase susceptibility to ankylosing spondylitis

Ankylosing spondylitis (AS) is a common and highly familial rheumatic disorder. The sibling recurrence risk ratio for the disease is 63 and heritability assessed in twins > 90%. Although MHC genes, including HLA-B27, contribute only 20-50% of the genetic risk for the disease, no non-MHC gene has yet been convincingly demonstrated to influence either susceptibility to the disease or its phenotypic expression. Previous linkage and association studies have suggested the presence of a susceptibility gene for AS close to, or within, the cytochrome P450 2D6 gene (CYP2D6, debrisoquine hydroxylase) located at chromosome 22q13.1. We performed a linkage study of chromosome 22 in 200 families with AS affected sibling-pairs. Association of alleles of the CYP2D6 gene was examined by both case-control and within-family means. For case-control studies, 617 unrelated individuals with AS (361 probands from sibling-pair and parent-case trio families and 256 unrelated non-familial sporadic cases) and 402 healthy ethnically matched controls were employed. For within-family association studies, 361 families including 161 parent-case trios and 200 affected sibling-pair families were employed. Homozygosity for poor metabolizer alleles was found to be associated with AS. Heterozygosity for the most frequent poor metabolizer allele (CYP2D6*4) was not associated with increased susceptibility to AS. Significant within-family association of CYP2D6*4 alleles and AS was demonstrated. Weak linkage was also demonstrated between CYP2D6 and AS. We postulate that altered metabolism of a natural toxin or antigen by the CYP2D6 gene may increase susceptibility to AS.

Studies of water and electrolyte movement from oral rehydration solutions (rice- and glucose-based) across a normal and secreting gut using a dual isotope tracer technique in a rat perfusion model

Aims: To establish a model to measure bidirectional flow of water from a glucose oral rehydration solution (G-ORS) and a newly developed rice-based oral rehydration solution (R-ORS) using a dual isotope tracer technique in a rat perfusion model. To measure net water, sodium and potassium absorption from the ORS. Methods: In viva steady-state perfusion studies were carried out in normal and secreting (induced by cholera toxin) rat small intestine (n = 11 in each group). To determine bidirectional flow of water from the ORS the animals were initially labelled with tritium, and deuterium was added to the perfusion solution. Sequential perfusate and blood samples were collected after attainment of steady-state conditions and analysed for water and electrolyte content. Results: There was a significant increase in net water absorption from the R-ORS compared to the G-ORS in both the normal (P < 0.02) and secreting intestine (P < 0.05). Water efflux was significantly reduced in the R-ORS group compared to the G-ORS group in both the normal (P < 0.01) and the secreting intestine (P < 0.01). There was an increase in sodium absorption in the R-ORS group compared to the G-ORS. The G-ORS produced a significantly greater blood glucose level at 75 min compared to the R-ORS (P < 0.03) in the secreting intestine. Conclusions: This study demonstrates the improved water absorption from a rice-based ORS in both the normal and secreting intestine. Evidence that the absorption of water may be influenced by the osmolality of the ORS was also demonstrated.

The effect of pH on the unfolding pathway and stability of ribosome-inactivating protein abrin-II

The effect of pH on the unfolding pathway acid the stability of the toxic protein abrin-II have been studied by increasing denaturant concentrations of guanidine hydrochloride and by monitoring the change in 8,1-anilino naphthalene sulfonic acid (ANS) fluorescence upon binding to the hydrophobic sites of the protein. Intrinsic protein fluorescence, far and near UV-circular dichroism (CD) spectroscopy and ANS binding studies reveal that the unfolding of abrin-II occurs through two intermediates at pH 7.2 and one intermediate at pH 4.5. At pH 7.2, the two subunits A and B of abrin-II unfold sequentially. The native protein is more stable at pH 4.5 than at pH 7.2. However, the stability of the abrin-II A-subunit is not affected by a change in pH. These observations may assist in an understanding of the physiologically relevant transmembrane translocation of the toxin.

Topological mimicry and epitope duplication in the guanylyl cyclase C receptor

Guanylyl cyclase C (GCC) is the receptor for the gastrointestinal hormones, guanylin, and uroguanylin, in addition to the bacterial heat-stable enterotoxins, which are one of the major causes of watery diarrhea the world over. GCC is expressed in intestinal cells, colorectal tumor tissue and tumors originating from metastasis of the colorectal carcinoma. We have earlier generated a monoclonal antibody to human GCC, GCC:B10, which was useful for the immunohistochemical localization of the receptor in the rat intestine (Nandi A et al., 1997, J Cell Biochem 66:500-511), and identified its epitope to a 63-amino acid stretch in the intracellular domain of GCC. In view of the potential that this antibody has for the identification of colorectal tumors, we have characterized the epitope for GCC:B10 in this study. Overlapping peptide synthesis indicated that the epitope was contained in the sequence HIPPENIFPLE. This sequence was unique to GCC, and despite a short stretch of homology with serum amyloid protein and pertussis toxin, no cross reactivity was detected. The core epitope was delineated using a random hexameric phage display library, and two categories of sequences were identified, containing either a single, or two adjacent proline residues. No sequence identified by phage display was identical to the epitope present in GCC, indicating that phage sequences represented mimotopes of the native epitope. Alignment of these sequences with HIPPENIFPLE suggested duplication of the recognition motif, which was confirmed by peptide synthesis. These studies allowed us not only to define the requirements of epitope recognition by GCC:B10 monoclonal antibody, but also to describe a novel means of epitope recognition involving topological mimicry and probable duplication of the cognate epitope in the native guanylyl cyclase C receptor sequence.

Expression of the extracellular domain of the human heat-stable enterotoxin receptor in Escherichia coli and generation of neutralizing antibodies

The entire extracellular domain of the human heat-stable enterotoxin (ST) receptor as well as a truncated N-terminal domain were cloned as glutathione S-transferase fusion proteins and expressed in Escherichia coli. The recombinant fusion proteins were purified from both the cytosol and the inclusion body fractions by selective detergent extraction followed by glutathione-agarose affinity chromatography. The purified protein, corresponding to the entire extracellular domain, bound the stable toxin peptide with an affinity comparable to that of the native receptor characterized from the human colonic T84 cell line. No binding was observed with the N-terminal truncated fragment of the receptor under similar conditions, Polyclonal antibodies were raised to the entire extracellular domain fusion protein as well as the truncated extracellular domain fusion protein, and the antibodies were purified by affinity chromatography. Addition of the purified antibodies to T84 cells inhibited ST binding and abolished ST-mediated cGMP production, indicating that critical epitopes involved in ligand interaction are present in the N-terminal fragment of the receptor, Purified antibodies recognized a single protein of M(r) 160,000 Da on Western blotting with T84 membranes, corresponding to a size of the native glycosylated receptor in T84 cells. These studies are the first report of the expression, purification, and characterization of any member of the guanylyl cyclase family of receptors in E. coli and show that binding of the toxin to the extracellular domain of the receptor is possible in the absence of any posttranslational modifications such as glycosylation. The recombinant fusion proteins as well as the antibodies that we have generated could serve as useful tools in the identification of critical residues of the extracellular domain involved in ligand interaction.

Functional correlation of bacterial LuxS with their quaternary associations: interface analysis of the structure networks

Background The genome of a wide variety of prokaryotes contains the luxS gene homologue, which encodes for the protein S-ribosylhomocysteinelyase (LuxS). This protein is responsible for the production of the quorum sensing molecule, AI-2 and has been implicated in a variety of functions such as flagellar motility, metabolic regulation, toxin production and even in pathogenicity. A high structural similarity is present in the LuxS structures determined from a few species. In this study, we have modelled the structures from several other species and have investigated their dimer interfaces. We have attempted to correlate the interface features of LuxS with the phenotypic nature of the organisms. Results The protein structure networks (PSN) are constructed and graph theoretical analysis is performed on the structures obtained from X-ray crystallography and on the modelled ones. The interfaces, which are known to contain the active site, are characterized from the PSNs of these homodimeric proteins. The key features presented by the protein interfaces are investigated for the classification of the proteins in relation to their function. From our analysis, structural interface motifs are identified for each class in our dataset, which showed distinctly different pattern at the interface of LuxS for the probiotics and some extremophiles. Our analysis also reveals potential sites of mutation and geometric patterns at the interface that was not evident from conventional sequence alignment studies. Conclusion The structure network approach employed in this study for the analysis of dimeric interfaces in LuxS has brought out certain structural details at the side-chain interaction level, which were elusive from the conventional structure comparison methods. The results from this study provide a better understanding of the relation between the luxS gene and its functional role in the prokaryotes. This study also makes it possible to explore the potential direction towards the design of inhibitors of LuxS and thus towards a wide range of antimicrobials.

Effective Use in Livestock Feeds of Mouldy and Weather-damaged Grain Containing Mycotoxins-Case Histories and Economic Assessments Pertaining to Pig and Poultry Industries of Queensland

Mould growth in field crops or stored grain reduces starch and lipid content, with consequent increases in fibre, and an overall reduction in digestible energy; palatability is often adversely affected. If these factors are allowed for, and mycotoxin concentrations are low, there are sound economic reasons for using this cheaper grain. Mycotoxins are common in stock feed but their effects on animal productivity are usually slight because either the concentration is too low or the animal is tolerant to the toxin. In Australia, aflatoxins occur in peanut by-products and in maize and sorghum if the grain is moist when stored. Zearalenone is found in maize and in sorghum and wheat in wetter regions. Nivalenol and deoxynivalenol are found in maize and wheat but at concentrations that rarely affect pigs, with chickens and cattle being even more tolerant. Other mycotoxins including cyclopiazonic acid, T-2 toxin, cytochalasins and tenuazonic acid are produced by Australian fungi in culture but are not found to be significant grain contaminants. Extremely mouldy sorghum containing Alternaria and Fusarium mycotoxins decreased feed conversion in pigs and chickens by up to 14%. However, E moniliforme- and Diplodia maydis-infected maize produced only slight reductions in feed intake by pigs and Ustilago- infected barley produced no ill effects. Use of these grains would substantially increase profits if the grain can be purchased cheaply.

The mycotoxins 4-deoxynivalenol, zearalenone and aflatoxin in weather-damaged wheat harvested 1983-1985 in south-east Queensland

A survey for various mycotoxins was carried out on samples of all wheat delivered to nine storage and marketing depots in south-eastern Queensland, selected as most likely to receive mycotoxin-contaminated grain. All wheat was surveyed during 1983, when the degree of weather damage was high. Samples of the poorest grade of wheat from these depots were also surveyed in 1984 and 1985. The surveys included all regions where head scab of wheat caused by Fusariurn graminearurn Schwabe Group 2 had been reported to occur at significant levels. 4-Deoxynivalenol was detected in nearly all pooled samples representing bulk wheat at concentrations ranging from traces of <0.01 up to 1.7 mg kg-1. The highest concentration of zearlenone detected in a pooled wheat sample was 0.04 mg kg-1. In a few samples representing individual wheat deliveries and with up to 2.8% by weight of pink grains, 4-deoxynivalenol concentrations ranged up to 11.7 mg kg-' and zearalenone up to 0.43 mg kg-l. Aflatoxins B,, B2, G1 and G2 were detected in only one pooled sample of wheat, at a total aflatoxin concentration of 0.003 mg kg-'. Ochratoxin A, sterigmatocystin and T-2 toxin were not detected. Higher concentrations of mycotoxins were found in the poorer grades of wheat.

Mycotoxins and Fungal Damage in Maize Harvested during 1982 in Far North Queensland

A survey for mycotoxins and fungal damage in maize (Zea mays L.) grown during 1982 in Far North Queensland is reported. This season had a rainfall distribution which was typical for the reglon. The 293 samples examined came from 11 1 farms in eight maize-growing districts. The samples were first subjected to rapid screening tests for fungal damage. Aflatoxins B1, B2, G1, G2 ochratoxin A, T-2 toxin, and sterigmatocystin were not detected, but zearalenone was found in 85% of the samples. The concentrations of zearalenone were correlated with the extent of Gibberella zeae cob rot as indicated by the proportion (up to 2%) of kernels in each sample having a reddish-purple discoloration. In four samples the zearalenone concentration exceeded 1 mg kg-1, but the mean ¦ s.d. (n = 293) concentration in all samples was 0.17 ¦ 0.225 mg kg-1. Concentrations were highest in districts with the highest rainfall during the period of maize growth.

Phenotypic and genotypic characterisation of Pasteurella multocida isolated from pigs at slaughter in New Zealand

AIMS: To examine pigs at slaughter in New Zealand for the presence of Pasteurella multocida, and to determine for isolates, their biochemical profi les, somatic and capsular types, and the presence or absence of the HSB and toxA genes, associated with haemorrhagic septicaemia (HS) and progressive atrophic rhinitis (PAR), respectively. METHODS: Swabs from 173 lungs, 158 palatine tonsils and 82 nasal passages of pigs at two abattoirs in New Zealand were cultured for P. multocida using conventional techniques, and isolated colonies were subjected to biochemical tests for identi- fi cation of biovars. Somatic serotyping was conducted using an agar gel immunodiffusion (AGID) test. Polymerase chain reaction (PCR) assays were used to confi rm phenotypic identifi cation of colonies using species-specifi c primers, capsule type using serogroup-specifi c primers and multiplex PCR, and to test for the presence of HSB and toxA genes. RESULTS: Pasteurella multocida was isolated from 11/173 (6.4%) lung, 32/158 (20.2%) palatine tonsil and 5/82 (6.1 %) nasal swab samples, a total of 48 isolates from 413 samples (11.6%). Isolation rates per farm ranged from 1–53% of tissue samples collected from pigs 5–6 months of age. On phenotypic characterisation, isolates were allocated to seven main biovars, viz 1, 2, 3, 5, 9, 12, and a dulcitol-negative variant of Biovar 8, the majority (30/48) being Biovar 3. Of the 42 isolates for which somatic serotyping was conducted, 10% were Serovar 1, 79% were Serovar 3, 2% were Serovar 6,1, 2% were Serovar 12, and 7% could not be typed. All 48 isolates were confi rmed as P. multocida using a species-specifi c PCR. In the capsular multiplex PCR, 92% of isolates were Capsular (Cap) type A, 2% were Cap D, and 6% could not be typed. None of the samples were positive for the HSB or toxA genes. CONCLUSION: Serovars or capsular types of P. multocida associated with HS or PAR in pigs were not detected. Establishment of species-specifi c, capsular and toxin PCR assays allowed the rapid screening of isolates of P. multocida, while serotyping provided an additional tool for epidemiological and tracing purposes.

Non-target impacts of poison baiting for predator control in Australia

1. Mammalian predators are controlled by poison baiting in many parts of the world, often to alleviate their impacts on agriculture or the environment. Although predator control can have substantial benefits, the poisons used may also be potentially harmful to other wildlife. 2. Impacts on non-target species must be minimized, but can be difficult to predict or quantify. Species and individuals vary in their sensitivity to toxins and their propensity to consume poison baits, while populations vary in their resilience. Wildlife populations can accrue benefits from predator control, which outweigh the occasional deaths of non-target animals. We review recent advances in Australia, providing a framework for assessing non-target effects of poisoning operations and for developing techniques to minimize such effects. We also emphasize that weak or circumstantial evidence of non-target effects can be misleading. 3. Weak evidence that poison baiting presents a potential risk to non-target species comes from measuring the sensitivity of species to the toxin in the laboratory. More convincing evidence may be obtained by quantifying susceptibility in the field. This requires detailed information on the propensity of animals to locate and consume poison baits, as well as the likelihood of mortality if baits are consumed. Still stronger evidence may be obtained if predator baiting causes non-target mortality in the field (with toxin detected by post-mortem examination). Conclusive proof of a negative impact on populations of non-target species can be obtained only if any observed non-target mortality is followed by sustained reductions in population density. 4. Such proof is difficult to obtain and the possibility of a population-level impact cannot be reliably confirmed or dismissed without rigorous trials. In the absence of conclusive evidence, wildlife managers should adopt a precautionary approach which seeks to minimize potential risk to non-target individuals, while clarifying population-level effects through continued research.

The impacts and management of foxes Vulpes vulpes in Australia

1. The successful introduction of the red fox Vulpes vulpes into Australia in the 1870s has had dramatic and deleterious impacts on both native fauna and agricultural production. Historical accounts detail how the arrival of foxes in many areas coincided with the local demise of native fauna. Recent analyses suggest that native fauna can be successfully reintroduced to their former ranges only if foxes have been controlled, and several replicated removal experiments have confirmed that foxes are the major agents of extirpation of native fauna. Predation is the primary cause of losses, but competition and transmission of disease may be important for some species. 2. In agricultural landscapes, fox predation on lambs can cause losses of 1–30%; variation is due to flock size, health and management, as well as differences in the timing and duration of lambing and the density of foxes. 3. Fox control measures include trapping, shooting, den fumigation and exclusion fencing; baiting using the toxin 1080 is the most commonly employed method. Depending on the baiting strategy, habitat and area covered, baiting can reduce fox activity by 50–97%. We review patterns of baiting in a large sheep-grazing region in central New South Wales, and propose guidelines to increase landholder awareness of baiting strategies, to concentrate and coordinate bait use, and to maximize the cost-effectiveness of baiting programs. 4. The variable reduction in fox density within the baited area, together with the ability of the fox to recolonize rapidly, suggest that current baiting practices in eastern Australia are often ineffective, and that reforms are required. These might include increasing landholder awareness and involvement in group control programs, and the use of more efficient broadscale techniques, such as aerial baiting.

LC/MS/MS analysis of the daphnane orthoester simplexin in poisonous Pimelea species of Australian rangelands.

Liquid chromatography/mass spectrometry (MS)/MS was used to analyse toxins in P. trichostachia, P. simplex subsp. continua, P. simplex subsp. continua and P. elongata samples (flowers, seeds, branches, main stem, leaves and roots) collected from various locations in Queensland, Saskatchewan and New South Wales, Australia. Simplexin was the major analyte in all taxa, with varying minor levels of huratoxin. Simplexin levels in P. trichostachia and P. elongata were higher (580 and 540 mg/kg in flowering foliage, respectively) than in P. simplex (255 mg/kg). Levels of huratoxin were higher in P. simplex (relative to simplexin) than in P. trichostachia or P. elongata. P. simplex flower heads and roots contained similar simplexin levels, with very small amounts of toxins detected in branches, stems and leaves. In P. trichostachia, simplexin levels were high in flower heads but low in the the other plant parts. The simplexin levels in aerial parts were generally higher from the pre-flowering to the flowering stage, decreasing towards the post-flowering stage; similar trends were recorded for P.elongata samples collected from a site near Bollon and P. trichostachia samples collected from a site near Jericho (both sites in Queensland). The simplexin concentration in roots was much less variable. Flowers and seeds had much higher simplexin levels than the foliage. The breakdown of the toxin in litter was more rapid compared to seeds under the same weathering conditions. Unlike the results from the litter samples, no significant decrease occurred in seed samples after 18 months of exposure.

Development of sustainable management for Pimelea and pimelea poisoning

Toxic Pimelea species (desert riceflower) are naturally occurring species found throughout beef cattle regions of Queensland, New South Wales, South Australia and the Northern Territory. Three species of Pimelea (simplex, elongata, and trichostachya) are poisonous to livestock and potentially fatal to cattle, with serious economic consequences through the loss of production, stock deaths and the costs of agistment. A better understanding of the ecology of the plant/disease is required to develop best practice to manage Pimelea in cattle-producing areas. Development of a chemical assay for the toxin (simplexin) is a key component of the current research project enabling toxin levels to be related to stage of plant growth, environmental and climatic factors.

Rab8 and Rab8-interacting proteins as players in cell polarization

Rab8 and its interacting proteins as regulators of cell polarization During the development of a multi-cellular organism, progenitor cells have to divide and migrate appropriately as well as organize their differentiation with one another, in order to produce a viable embryo. To divide, differentiate and migrate cells have to undergo polarization, a process where internal and external components such as actin, microtubules and adhesion receptors are reorganized to produce a cell that is asymmetric, with functionally different surfaces. Also in the adult organism there is a continuous need for these processes, as cells need to migrate in response to tissue damage and to fight infection. Improper regulation of cell proliferation and migration can conversely lead to disease such as cancer. GTP-binding proteins function as molecular switches by cycling between a GTP-bound (active) conformation and a GDP-bound (inactive) conformation. The Ras super-family of small GTPases are found in all eukaryotic cells. They can be functionally divided into five subfamilies. The Ras family members mainly regulate gene expression, controlling cell proliferation and differentiation. Ras was in fact the first human oncogene to be characterized, and as much as 30% of all human tumors may be directly or indirectly caused by mutations of Ras molecules The Rho family members mainly regulate cytoskeletal reorganization. Arf proteins are known to regulate vesicle budding and Rab proteins regulate vesicular transport. Ran regulates nuclear transport as well as microtubule organization during mitosis. The focus of the thesis of Katarina Hattula, is on Rab8, a small GTPase of the Rab family. Activated Rab8 has previously been shown to induce the formation of new surface extensions, reorganizing both actin and microtubules, and to have a role in directed membrane transport to cell surfaces. However, the exact membrane route it regulates has remained elusive. In the thesis three novel interactors of Rab8 are presented. Rabin8 is a Rab8-specific GEF that localizes to vesicles where it presumably recruits and activates its target Rab8. Its expression in cells leads to remodelling of actin and the formation of polarized cell surface domains. Optineurin, known to be associated with a leading cause of blindness in humans (open-angle glaucoma), is shown to interact specifically with GTP-bound Rab8. Rab8 binds to an amino-terminal region and interestingly, the Huntingtin protein binds a carboxy-terminal region of optineurin. (Aberrant Huntingtin protein is known to be the cause Huntington s disease in humans.) Co-expression of Huntingtin and optineurin enhanced the recruitment of Huntingtin to Rab8-positive vesicular structures. Furthermore, optineurin promoted cell polarization in a similar way to Rab8. A third novel interactor of Rab8 presented in this thesis is JFC1, a member of the synaptogamin-like protein (Slp) family. JFC1 interacts with Rab8 specifically in its GTP-bound form, co-localizes with endogenous Rab8 on tubular and vesicular structures, and is probably involved in controlling Rab8 membrane dynamics. Rab8 is in this thesis work clearly shown to have a strong effect on cell shape. Blocking Rab8 activity by expression of Rab8 RNAi, or by expressing the dominant negative Rab8 (T22N) mutant leads to loss of cell polarity. Conversely, cells expressing the constitutively active Rab8 (Q67L) mutant exhibit a strongly polarized phenotype. Experiments in live cells show that Rab8 is associated with macropinosomes generated at ruffling areas of the membrane. These macropinosomes fuse with or transform into tubules that move toward the cell centre, from where they are recycled back to the leading edge to participate in protrusion formation. The biogenesis of these tubules is shown to be dependent on both actin and microtubule dynamics. The Rab8-specific membrane route studied contained several markers known to be internalized and recycled (1 integrin, transferrin, transferrin receptor, cholera toxin B subunit (CTxB), and major histocompatibility complex class I protein (MHCI)). Co-expression studies revealed that Rab8 localization overlaps with that of Rab11 and Arf6. Rab8 is furthermore clearly functionally linked to Arf6. The data presented in this thesis strongly suggests a role for Rab8 as a regulator for a recycling compartment, which is involved in providing structural and regulatory components to the leading edge to participate in protrusion formation.