Developmental changes in presynaptic muscarinic modulation of excitatory and inhibitory neurotransmission in rat piriform cortex in vitro: relevance to epileptiform bursting susceptibility

Suppression of depolarizing postsynaptic potentials and isolated GABA-A receptor-mediated fast inhibitory postsynaptic potentials by the muscarinic acetylcholine receptor agonist, oxotremorine-M (10 microM), was investigated in adult and immature (P14-P30) rat piriform cortical (PC) slices using intracellular recording. Depolarizing postsynaptic potentials evoked by layers II-III stimulation underwent concentration-dependent inhibition in oxotremorine-M that was most likely presynaptic and M2 muscarinic acetylcholine receptor-mediated in immature, but M1-mediated in adult (P40-P80) slices; percentage inhibition was smaller in immature than in adult piriform cortex. In contrast, compared with adults, layer Ia-evoked depolarizing postsynaptic potentials in immature piriform cortex slices in oxotremorine-M, showed a prolonged multiphasic depolarization with superimposed fast transients and spikes, and an increased 'all-or-nothing' character. Isolated N-methyl-d-aspartate receptor-mediated layer Ia depolarizing postsynaptic potentials (although significantly larger in immature slices) were however, unaffected by oxotremorine-M, but blocked by dl-2-amino-5-phosphonovaleric acid. Fast inhibitory postsynaptic potentials evoked by layer Ib or layers II-III-fiber stimulation in immature slices were significantly smaller than in adults, despite similar estimated mean reversal potentials ( approximately -69 and -70 mV respectively). In oxotremorine-M, only layer Ib-fast inhibitory postsynaptic potentials were suppressed; suppression was again most likely presynaptic M2-mediated in immature slices, but M1-mediated in adults. The degree of fast inhibitory postsynaptic potential suppression was however, greater in immature than in adult piriform cortex. Our results demonstrate some important physiological and pharmacological differences between excitatory and inhibitory synaptic systems in adult and immature piriform cortex that could contribute toward the increased susceptibility of this region to muscarinic agonist-induced epileptiform activity in immature brain slices.

Antifungal activity of Bacillus subtilis filtrate to control Fusarium oxysporum f.sp. lentis, the causal organism of lentil vascular wilt

In dual cultures, the supernatant filtrate of the biological control agent Bacillus subtilis was evaluated against (Fusarium oxysporum f.sp. lentis) the causal organism of lentil vascular wilt. The antagonistic activity was evaluated as percent reduction of fungal growth (certainly due, in part, to the antifungal metabolites produced by the antagonistic bacterium). The in-vitro experiments showed that B. subtilis filtrate, whether solid or liquid media, had a strong inhibiting activity on the spore germination and mycelial growth of F. oxysporum f. sp. lentis. In a glasshouse experiment, soil was drenched with B. subtilis filtrate at 30 ml/kg (vol/wt) around seedlings of a susceptible lentil line (ILL 4605). In this treatment there was only 31% mortality compared with 100% kill of plants in the control treatment (P≤0.05).

Susceptibility of different insect pupae to the bacterial symbiont, Xenorhabdus nematophila, isolated from the entomopathogenic nematode Steinernema carpocapsae

Cells of the bacterial symbiont Xenorhabdus nematophila from the entomopathogenic nematode, Steinernema carpocapsae entered the pupae of Plutella xylostella after 15 minutes treatment with suspensions containing the bacterial cells. Secretions of Xenorhabdus nematophila, in either broth or water, were found lethal to the pupae of P. xylostella when applied in moist sand. The bacterial symbiont Xenorhabdus nematophila was found lethal to the pupae of greater wax moth (Galleria mellonella), beet armyworm (Spodoptera exigua), diamondback moth (Plutella xylostella) and black vine weevil (Otiorhynchus sulcatus) in the absence of the nematode vector and the cells of X. nematophila entered the haemocoele of the pupae.

Antifungal activity of bacterium symbiotically associated with Steinernema abbasi towards Fusarium oxysporum

Fusarium oxysporum f.sp. lycopersici (Fol) is the causal agent of the Fusarium wilt disease of tomato. Soil fumigant (mainly methyl bromide) applications are in use for its control. With the increasing environmental awareness, biological control methods are under investigation for their effectiveness, including the use of antagonists. Pseudomonas oryzihabitans (=Flavimonas oryzihabitans), a symbiont of the entomopathogenic nematode Steinernema abbasi was investigated as an antagonism of a Fol isolate in two laboratory and two glasshouse experiments. Bacteria and cell-free filtrate antifungal activity were tested both in dual cultures and in broth culture. In pot experiments, suspensions of bacteria in five concentrations (106, 105, 104, 103 and 102 cells/ml) were tested for their ability to control the pathogen at 25±3°C. In all tests the bacterium significantly inhibited the growth of Fol mycelium in vitro. Similar results were obtained when the bacterium was also tested against Fusarium oxysporum f.sp. radicis lycopersici and against Rhizoctonia solani. Moreover, when it was introduced into the soil, it was able to suppress the Fusarium wilt of tomato.

Susceptibility to the anticoagulants bromadiolone and coumatetralyl in wild Norway rats (Rattus norvegicus) from the UK and Germany

A new blood clotting response test was used to determine the susceptibility, to coumatetralyl and bromadiolone, of laboratory strains of Norway rat from Germany and the UK (Hampshire), and wild rats trapped on farms in Wales (UK) and Westphalia (Germany). Resistance factors were calculated in relation to the CD strain of Norway rat. An outbred strain of wild rats, raised from rats trapped in Germany, was found to be more susceptible to coumatetralyl by a factor of 0.5-0.6 compared to the CD strain. Homozygous and heterozygous animals of a strain of resistant rats from Westphalia were cross-resistant to coumatetralyl and bromadiolone, with a higher resistance factor for bromadiolone than that found in both UK strains. Our results show that the degree of altered susceptibility and resistance varies between strains of wild rat and between resistance foci. Some wild rat strains may be more susceptible than laboratory rat strains. Even in a well-established resistance area, it may be difficult to find infestations with resistance high enough to suspect control problems with bromadiolone, even after decades of use of this compound.

Thioquinolobactin, a pseudomonas siderophore with antifungal and anti-pythium activity

Under conditions of iron limitation Pseudomonas fluorescens ATCC 17400 produces two siderophores, pyoverdine, and a second siderophore quinolobactin, which itself results from the hydrolysis of the unstable molecule 8-hydroxy-4-methoxy-2-quinoline thiocarboxylic acid (thioquinolobactin). Pseudomonas fluorescens ATCC 17400 also displays a strong in vitro antagonism against the Oomycete Pythium, which is repressed by iron, suggesting the involvement of a siderophore(s). While a pyoverdine-negative mutant retains most of its antagonism, a thioquinolobactin-negative mutant only slowed-down Pythium growth, and a double pyoverdine-, thioquinolobactin-negative mutant, which does not produce any siderophore, totally lost its antagonism against Pythium. The siderophore thioquinolobactin could be purified and identified from spent medium and showed anti-Pythium activity, but it was quickly hydrolysed to quinolobactin, which we showed has no antimicrobial activity. Analysis of antagonism-affected transposon mutants revealed that genes involved in haem biosynthesis and sulfur assimilation are important for the production of thioquinolobactin and the expression of antagonism.

Evidence for complex multigenic inheritance of radiation AML susceptibility in mice revealed using a surrogate phenotypic assay

The mapping of genes which affect individual cancer risk is an important but complex challenge. A surrogate assay of susceptibility to radiation-induced acute myeloid leukaemia (AML) in the mouse based on chromosomal radiosensitivity has been developed and validated. This assay was applied to the mapping of radiation-induced AML risk modifier loci by association with microsatellite markers. A region on chromosome (chr) 18 with strong association is identified and confirmed by backcross analysis. Additional loci on chrs 8 and 13 show significant association. A key candidate gene Rbbp8 on chr18 is identified. Rbbp8 is shown to be upregulated in response to X-irradiation in the AML sensitive CBA strain but not AML resistant C57BL/6 strain. This study demonstrates the strength of utilizing surrogate endpoints of cancer susceptibility in the mapping of mouse loci and identifies additional loci that may affect radiation cancer risk.

Changes in in vitro susceptibility of influenza A H3N2 viruses to a neuraminidase inhibitor drug during evolution in the human host

Objectives: Influenza A H3N2 viruses isolated recently have characteristic receptor binding properties that may decrease susceptibility to neuraminidase inhibitor drugs. A panel of clinical isolates and recombinant viruses generated by reverse genetics were characterized and tested for susceptibility to zanamivir. Methods: Plaque reduction assays and neuraminidase enzyme inhibition assays were used to assess susceptibility to zanamivir. Receptor binding properties of the viruses were characterized by differential agglutination of red blood cells (RBCs) from different species. Sequence analysis of the haemagglutinin (HA) and neuraminidase (NA) genes was carried out. Results: Characterization of a panel of H3N2 clinical isolates from 1968 to 2000 showed a gradual decrease in agglutination of chicken and guinea pig RBCs over time, although all isolates could agglutinate turkey RBCs equally. Sequence analysis of the HA and NA genes identified mutations in conserved residues of the HA1 receptor binding site, in particular Leu-226 --> Ile-226/Val-226, and modification of potential glycosylation site motifs. This may be indicative of changes in virus binding to sialic acid (SA) receptors in recent years. Although recent isolates had reduced susceptibility to zanamivir in MDCK cell based plaque reduction assays, no difference was found in an NA enzyme-inhibition assay. Assays with recombinant isogenic viruses showed that the recent HA, but not the NA, conferred reduced susceptibility to zanamivir. Conclusion: This study demonstrates that recent clinical isolates of influenza A H3N2 virus no longer agglutinate chicken RBCs, but despite significant receptor binding changes as a result of changes in HA, there was little variation in sensitivity of the NA to zanamivir.

Plant- and marine-derived n-3 polyunsaturated fatty acids have differential effects on fasting and postprandial blood lipid concentrations and on the susceptibility of LDL to oxidative modification in moderately hyperlipidemic subjects

Background: Dietary a-linolenic acid (ALA) can be converted to long-chain n-3 polyunsaturated fatty acids (PUFAs) in humans and may reproduce some of the beneficial effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on cardiovascular disease risk factors. Objective: This study aimed to compare the effects of increased dietary intakes of ALA and EPA+DHA on a range of atherogenic risk factors. Design: This was a placebo-controlled, parallel study involving 150 moderately hyperlipidemic subjects randomly assigned to 1 of 5 interventions: 0.8 or 1.7 g EPA+DHA/d, 4.5 or 9.5 g ALA/d, or an n-6 PUFA control for 6 mo. Fatty acids were incorporated into 25 g of fat spread and 3 capsules to be consumed daily. Results: The change in fasting or postprandial lipid, glucose, or insulin concentrations or in blood pressure was not significantly different after any of the n-3 PUFA interventions compared with the n-6 PUFA control. The mean (+/-SEM) change in fasting triacylglycerols after the 1.7-g/d EPA+DHA intervention (-7.7 +/- 4.99%) was significantly (P < 0.05) different from the change after the 9.5-g/d ALA intervention (10.9 +/- 4.5%). The ex vivo susceptibility of LDL to oxidation was higher after the 1.7-g/d EPA+DHA intervention than after the control and ALA interventions (P < 0.05). There was no significant change in plasma a-tocopherol concentrations or in whole plasma antioxidant status in any of the groups. Conclusion: At estimated biologically equivalent intakes, dietary ALA and EPA+DHA have different physiologic effects.

Warfarin susceptibility in the lesser bandicoot rat (Bandicota bengalensis)

Warfarin is a first generation anticoagulant that relies on multiple feeding events to achieve lethality in susceptible rodents. For the Bandicoot rat, warfarin susceptibility baselines were established using the lethal feeding period (LFP) test methodology. Against a 0.003% warfarin formulation, LFP50 values of 2 and 4 days, and LFP99 values of 16 and 10 days were obtained for males and females respectively. However, consumption of rodenticide was significantly reduced after the 4th and 5th days of test, at a time when animals would be expected to experience symptoms of warfarin toxicity. This would seriously compromise the Probit analysis, particularly for estimates of higher percentiles. Possible modifications to the methodology are discussed to overcome this problem. Although the majority of animals were highly susceptible to warfarin, one female animal that survived a high dose of active ingredient (79.1 mg kg-1) may bode for future resistance

Piezotolerant small-colony variants with increased thermotolerance, antibiotic susceptibility, and low invasiveness in a clonal Staphylococcus aureus population

Following a pressure treatment of a clonal Staphylococcus aureus culture with 400 MPa for 30 min, piezotolerant variants were isolated. Among 21 randomly selected survivors, 9 were piezotolerant and all formed small colonies on several agar media. The majority of the isolates showed increased thermotolerance, impaired growth, and reduced antibiotic resistance compared to the wild type. However, several nonpiezotolerant isolates also demonstrated impaired growth and the small-colony phenotype. In agglutination tests for the detection of protein A and fibrinogen, the piezotolerant variants showed weaker agglutination reactions than the wild type and the other isolates. All variants also showed defective production of the typical S. aureus golden color, a characteristic which has previously been linked with virulence. They were also less able to invade intestinal epithelial cells than the wild type. These S. aureus variants showed phenotypic similarities to previously isolated Listeria monocytogenes piezotolerant mutants that contained mutations in ctsR. Because of these similarities, possible alterations in the ctsR hypermutable regions of the S. aureus variants were investigated through amplified fragment length polymorphism analysis. No mutations were identified, and subsequently we sequenced the ctsR and hrcA genes of three representative variants, finding no mutations. This work demonstrates that S. aureus probably possesses a strategy resulting in an abundance of multiple-stressresistant variants within clonal populations. This strategy, however, seems to involve genes and regulatory mechanisms different from those previously reported for L. monocytogenes. We are in the process of identifying these mechanisms.