Laminar disorganisation of mitral cells in the olfactory bulb does not affect topographic targeting of primary olfactory axons

Primary olfactory neurons expressing the same odorant receptor protein typically project to topographically fixed olfactory bulb sites. While cell adhesion molecules and odorant receptors have been implicated in guidance of primary olfactory axons. the postsynaptic mitral cells may also have a role in final target selection. We have examined the effect of disorganisation of the mitral cell soma layer in mutant mice heterozygous for the beta-subunit of platelet activating factor acetylhydrolase (Lis1(-/+)) on the targeting of primary olfactory axons. Lis1(-/+) mice display abnormal lamination of neurons in the olfactory bulb. Lis1(-/+) mice were crossed with the P2-IRES-tau:LacZ line of transgenic mice that selectively expresses beta-galactosidase in primary olfactory neurons expressing the P2 odorant receptor. LacZ histochemistry revealed blue-stained P2 axons that targeted topographically fixed glomeruli in these mice in a manner similar to that observed in the parent P2-IRES-tau:LacZ line. Thus, despite the aberrant organisation of postsynaptic mitral cells in Lis1(-/+) mice, primary olfactory axons continued to converge and form glomeruli at correct sites in the olfactory bulb. Next we examined whether challenging primary olfactory axons in adult Lis(-/+) mice with regeneration would affect their ability to converge and form glomeruli. Following partial chemical ablation of the olfactory neuroepithelium with dichlobenil, primary olfactory neurons die and are replaced by newly differentiating neurons that project axons to the olfactory bulb where they converge and form glomeruli. Despite the aberrant mitral cell layer in Lis(-/+) mice. primary olfactory axons continued to converge and form glomeruli during regeneration. Together these results demonstrate that the convergence of primary olfactory axons during development and regeneration is not affected by gross perturbations to the lamination of the mitral cell layer. Thus, these results support evidence from other studies indicating that mitral cells do not play a major role in the convergence and targeting of primary olfactory axons in the olfactory bulb. (C) 2002 Elsevier Science B.V. All rights reserved.

Possible reasons for variation in Ixodes holocyclus toxicity

Tick paralysis caused by Ixodes holocyclus affects an estimated 20,000 domestic animals each year along the eastern coast of Australia (Stone 1988). Animals are presented with clinical signs ranging from mild paresis to ascending flaccid paralysis and varying degrees of respiratory and cardiac compromise. Mortality rates are significantly increased in animals presented with respiratory compromise compared to those animals without respiratory compromise, regardless of the degree of flaccid paralysis (Atwell et al 2001). Anecdotal evidence of ticks (collected from different sites in northern New South Wales) causing different clinical signs and mortality in hyper-immune dogs used for serum production, suggests that there may be a variation in toxin production and toxin content between individual ticks (Warne, N 2002 pers comm). This literature review suggests that two possible contributing factors to toxin variation may be the genetic variation within the I. holocyclus species and the variation in the host's response to tick feeding.

Neonatal ethanol exposure reduces AMPA but not NMDA receptor levels in the rat neocortex

Fetal alcohol syndrome (FAS) is the leading cause of mental retardation in western society. We investigated possible changes in glutamate receptor levels in neonatal animals following ethanol exposure using radioligand binding and western blot analysis. We used a vapor chamber to administer ethanol to neonatal Wistar rats 3 h a day from postnatal day (PND) 4-9. A separation control group was separated from their mothers for the same time and duration as the vapor treatment, while a normal control group was left to develop normally. Daily ethanol administrations resulted in decreased brain weight and body weight, as well as microencephaly (decreased brain:body weight ratio). Neither the affinity nor maximum binding of [H-3]MK-801 (dizoclipine maleate) in the cortex of PND10 rats differed between treatment groups. Western blot analysis also failed to reveal any changes in NMDAR1, NMDAR2A, or NMDAR2B receptor levels. In contrast, the AMPA receptor subunit GluR1 was greatly reduced in vapor-treated pups compared with control pups, as revealed by western blot analysis. A similar reduction was found in westerns with an antibody recognizing the GluR2 and 4 subunits. These results indicate that ethanol reduces AMPA rather than NMDA receptors in the developing neocortex, possibly by blocking NMDA receptors during development. (C) 2002 Elsevier Science B.V. All rights reserved.

Comparison between human and animal isolates of Shiga toxin-producing Escherichia coli O157 from Australia

There is very little human disease associated with enterohaemorrhagic Escherichia coli O157 in Australia even though these organisms are present in the animal population. A group of Australian isolates of E. coli O157:H7 and O157:H- from human and animal sources were tested for the presence of virulence markers and compared by XbaI DNA macrorestriction analysis using pulsed-field gel electrophoresis (PFGE). Each of 102 isolates tested contained the gene eae which encodes the E. coli attaching and effacing factor and all but one carried the enterohaemolysin gene, ehxA, found on the EHEC plasmid. The most common Shiga toxin gene carried was stx(2c), either alone (16%) or in combination with stx(1) (74%) or stx(2) (3%) PFGE grouped the isolates based on H serotype and some clusters were source specific. Australian E. coli O157:H7 and H- isolates from human, animal and meat sources carry all the virulence markers associated with EHEC disease in humans therefore other factors must be responsible for the low rates of human infection in Australia.

Horizontal transmission of Shiga toxin-producing Escherichia coli within groups of dairy calves

To examine the dissemination of Shiga-toxigenic Escherichia coli (STEC) within cattle groups, dairy calves on two farms utilizing different calf-rearing practices were exposed to a traceable STEC strain. Test strain dissemination differed significantly between farms, with a higher prevalence being associated with group penning. Pen floors and calf hides may be the main environmental mechanisms of transmission. Dairy calf husbandry represents a control point for reducing on-farm STEC prevalence.

The synthesis and structure of an n-terminal dodecanoic acid conjugate of a-conotoxin MII

The alpha-conotoxin MII is a 16 amino acid long peptide toxin isolated from the marine snail, Conus magus. This toxin has been found to be a highly selective and potent inhibitor of neuronal nicotinic acetylcholine receptors of the subtype alpha3beta2. To improve the bioavailability of this peptide, we have coupled to the N-terminus of conotoxin MII, 2-amino-D,L-dodecanoic acid (Laa) creating a lipidic linear peptide which was then successfully oxidised to produce the correctly folded conotoxin MII construct.

Epidemiology and strain characteristics of Echinococcus granulosus in the Benghazi area of eastern Libya

The incidence of surgically confirmed cystic echinococcosis in eastern Libya was estimated to be at least 4.2 cases/100,000, with significantly more female cases than male. The prevalences of infection with Echinococcus granulosus among 1087 sheep, 881 goats, 428 camels and 614 cattle from the same region, determined postmortem in abattoirs, were 20%, 3.4%, 13.6% and 11%, respectively. Infection in the livestock was age-dependent and, generally, the female animals were more often infected than the male. The measurements of rostellar hooks on protoscoleces collected from sheep and cattle were similar but significantly different from the corresponding measurements of parasites of human or camel origin. However, when a portion of the cytochrome c-oxidase subunit I (coxl) gene from each of 30 protoscolex samples from Libya (12 from cattle, three from humans, five from camels and 10 from sheep) was sequenced, the sequences were all found to be identical to that published for the common sheep strain of E. granulosus.

Analysis of toxin profiles in three different fish species causing ciguatera fish poisoning in Guadeloupe, French West Indies

A grey snapper (Lutjanus griseus), a grouper (Serranidae) and a blackjack (Caranx lugubris) were implicated in three different ciguatera poisonings in Guadeloupe, French West Indies. A mouse bioassay indicated toxicity for each specimens: 0.5-1, greater than or equal to 1 and > 1 M Ug g(-1), respectively. After purification by gel filtration chromatography, the samples were analysed by high-performance liquid chromatography coupled to mass spectrometry (LC-MS). The toxin profiles differ from one fish to another. C-CTX-1 was detected at 0.24, 0.90 and 13.8 ng g(-1) flesh in the snapper, grouper and jack, respectively. It contributed only to part of the whole toxicity determined by the mouse bioassay. Other toxins identified were C-CTX-2 (a C-CTX-1 epimer), three additional isomers of C-CTX-1 or -2, and five ciguatoxin congeners (C-CTX-1127, C-CTX-1143 and its isomer C-CTX-1143a, and C-CTX-1157 and its isomer C-CTX-1157b). Putative hydroxy-polyether-like compounds were also detected in the flesh of the grouper with [M+ + H](+) ions at m/z 851.51, 857.50, 875.51, 875.49 and 895.54 Da. Some of these compounds have the same mass range as some known dinoflagellate toxins. In conclusion, this study confirms the usefulness of LC-MS analysis to determine the ciguatoxins levels and the toxin profile in fish flesh hazardous to humans.

Inhibitors of COP-mediated Transport and Cholera Toxin Action Inhibit Simian Virus 40 Infection

Simian virus 40 (SV40) is a nonenveloped virus that has been shown to pass from surface caveolae to the endoplasmic reticulum in an apparently novel infectious entry pathway. We now show that the initial entry step is blocked by brefeldin A and by incubation at 20degreesC. Subsequent to the entry step, the virus reaches a domain of the rough endoplasmic reticulum by an unknown pathway. This intracellular trafficking pathway is also brefeldin A sensitive. Infection is strongly inhibited by expression of GTP-restricted ADP-ribosylation factor 1 (Arf1) and Sar1 mutants and by microinjection of antibodies to betaCOP. In addition, we demonstrate a potent inhibition of SV40 infection by the dipeptide N-benzoyl-oxycarbonyl-Gly-Phe-amide, which also inhibits late events in cholera toxin action. Our results identify novel inhibitors of SV40 infection and show that SV40 requires COPI- and COPII-dependent transport steps for successful infection.

Solution Structures of the cis- and trans-Pro30 Isomers of a Novel 38-Residue Toxin from the Venom of Hadronyche Infensa sp. that Contains a Cystine-Knot Motif within Its Four Disulfide Bonds

The primary sequence and three-dimensional structure of a novel peptide toxin isolated from the Australian funnel-web spider Hadronyche infensa sp. is reported. ACTX-HI:OB4219 contains 38 amino acids, including eight-cysteine residues that form four disulfide bonds. The connectivities of these disulfide bonds were previously unknown but have been unambiguously determined in this study. Three of these disulfide bonds are arranged in an inhibitor cystine-knot (ICK) motif, which is observed in a range of other disulfide-rich peptide toxins. The motif incorporates an embedded ring in the structure formed by two of the disulfides and their connecting backbone segments penetrated by a third disulfide bond. Using NMR spectroscopy, we determined that despite the isolation of a single native homologous product by RP-HPLC, ACTX-HI:OB4219 possesses two equally populated conformers in solution. These two conformers were determined to arise from cis/trans isomerization of the bond preceding Pro30. Full assignment of the NMR spectra for both conformers allowed for the calculation of their structures, revealing, the presence of a triple-stranded antiparallel sheet consistent with the inhibitor cystine-knot (ICK) motif.

Adapting immunity with subunit vaccines: case studies with group A Streptococcus and malaria

Although vaccines have widely been regarded as the most cost-effective way to improve public health, for some organisms new technological advances in vaccine design and delivery, incurring additional developmental costs, will be essential. These organisms are typically those for which natural immunity is either slow to develop or does not develop at all. Clearly, such organisms have evolved strategies to evade immune responses and innovative approaches will be required to induce a type of immune response which is both different to that which develops naturally and is effective. This article describes some approaches to develop vaccines for two such organisms (malaria parasites and Streptococcus pyogenes (group A Streptococcus)) that are associated with widespread mortality and morbidity, mostly in the poorest countries of the world. At this stage, the challenges are primarily scientific, but if these hurdles are surmounted then the challenges will become financial ones - developing much needed vaccines for people least able to afford them. (C) 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Osteonectin/SPARC induction by ectopic beta(3) integrin in human radial growth phase primary melanoma cells

Expression of the beta(3) integrin subunit in melanoma in situ has been found to correlate with tumor thickness, the ability to invade and metastasize, and poor prognosis. Transition from the radial growth phase (RGP) to the vertical growth phase (VGP) is a critical step in melanoma progression and survival and is distinguished by the expression of beta(3), integrin. The molecular pathways that operate in melanoma cells associated with invasion and metastasis were examined by ectopic induction of the beta(3), integrin subunit in RGP SBcl2 and WM1552C melanoma cells, which converts these cells to a VGP phenotype. We used cDNA representational difference analysis subtractive hybridization between beta(3)-Positive and -negative melanoma cells to assess gene expression profile changes accompanying RGP to VGP transition. Fourteen fragments from known genes including osteonectin (also known as SPARC and BM-40) were identified after three rounds of representational difference analysis. Induction of osteonectin was confirmed by Northern and Western blot analysis and immunohistochemistry and correlated in organotypic cultures with the beta(3)-induced progression from RGP to VGP melanoma. Expression of osteonectin was also associated with reduced adhesion to vitronectin, but not to fibronectin. Osteonectin expression was not blocked when melanoma cells were cultured with anti-alpha(v)beta(3) LM609 mAb, mitogen-activated protein kinase, or protein kinase C inhibitors, indicating that other signaling pathway(s) operate through a(v)beta(3) integrin during conversion from RGP to VGP.