ULTRACAM photometry of the eclipsing cataclysmic variables GY Cnc, IR Com and HT Cas

We present high-speed, three-colour photometry of the eclipsing cataclysmic variables GY Cnc, IR Com and HT Cas. We find that the sharp eclipses in GY Cnc and IR Com are due to eclipses of the white dwarf. There is some evidence for a bright-spot on the edge of the accretion disc in GY Cnc, but not in IR Com. Eclipse mapping of HT Cas is presented which shows changes in the structure of the quiescent accretion disc. Observations in 2002 show the accretion disc to be invisible except for the presence of a bright-spot at the disc edge. 2003 observations, however, clearly show a bright inner disc and the bright-spot to be much fainter than in 2002. Although no outburst was associated with either set of quiescent observations, the system was similar to 0.6 mJy brighter in 2003, mainly due to the enhanced emission from the inner disc. We propose that these changes are due to variations in the mass-transfer rate from the secondary star and through the disc. The disc colours indicate that it is optically thin in both its inner and outer regions. We estimate the white dwarf temperature of HT Cas to be 15 000 +/- 1000 K in 2002 and 14 000 +/- 1000 K in 2003.

A rapid optical immunoassay for the screening of T-2 and HT-2 toxin in cereals and maize-based baby food

A rapid surface plasmon resonance (SPR) screening assay has been developed for the combined detection of T-2 and HT-2 toxins in naturally contaminated cereals using a sensor chip coated with an HT-2 toxin derivative and a monoclonal antibody. The antibody raised against HT-2 displayed high cross-reactivity with T-2 toxin while there was no cross-reaction observed with other commonly occurring trichothecenes. A simple extraction procedure using 40% methanol was applied to baby food, breakfast cereal, and wheat samples prior to biosensor analysis. Limits of detection (LOD) for each matrix were determined as 25 mu g kg(-1) for baby food and breakfast cereal and 26 mu g kg(-1) for wheat. Intra-assay precision (n = 6) was calculated for each matrix. The results were expressed as the relative standard deviation and determined as 2.8% (100 mu g kg(-1)) and 1.8% (200 mu g kg(-1)) in breakfast cereal, 4.6% (50 mu g kg(-1)) and 3.6% (100 mu g kg(-1)) in wheat and 0.97% (25 mu g kg(-1)) and 6.3% (50 mu g kg(-1)) in baby food. Between run precision (n = 3) performed at the same levels yielded relative standard deviations of 6.7% and 3.9% for breakfast cereals, 3.3% and 1.6% for wheat and 6.8% and 0.08% for baby food, respectively. (C) 2010 Elsevier B.V. All rights reserved.

A comparative study of qualitative immunochemical screening assays for the combined measurement of T-2/HT-2 in cereals and cereal-based products

Many different immunochemical platforms exist for the screening of naturally occurring contaminants in food from the low cost enzyme linked immunosorbent assays (ELISA) to the expensive instruments such as optical biosensors based on the phenomenon of surface plasmon resonance (SPR). The primary aim of this study was to evaluate and compare a number of these platforms to assess their accuracy and precision when applied to naturally contaminated samples containing HT-2/T-2 mycotoxins. Other important factors considered were the speed of analysis, ease of use (sample preparation techniques and use of the equipment) and ultimately the cost implications. The three screening procedures compared included an SPR biosensor assay, a commercially available ELISA and an enzyme-linked immunomagnetic electrochemical array (ELIME array). The qualitative data for all methods demonstrated very good overall agreements with each other, however on comparison with mass spectrometry confirmatory results, the ELISA and SPR assay performed slightly better than the ELIME array, exhibiting an overall agreement of 95.8% compared to 91.7%. Currently, SPR is more costly than the other two platforms and can only be used in the laboratory whereas in theory both the ELISA and ELIME array are portable and can be used in the field, but ultimately this is dependent on the sample preparation techniques employed. Sample preparative techniques varied for all methods evaluated, the ELISA was the most simple to perform followed by that of the SPR method. The ELIME array involved an additional clean-up step thereby increasing both the time and cost of analysis. Therefore in the current format, field use would not be an option for the ELIME array. In relation to speed of analysis, the ELISA outperformed the other methods.

IMMUNOCYTOCHEMICAL LOCALIZATION OF SEROTONIN (5-HT) IN THE NERVOUS-SYSTEM OF THE HYDATID ORGANISM, ECHINOCOCCUS-GRANULOSUS (CESTODA, CYCLOPHYLLIDEA)

The localization and distribution of the serotoninergic components of the nervous system in the hydatid organism, Echinococcus granulosus, were determined by immunocytochemical techniques in conjunction with confocal scanning laser microscopy (CSLM). The distribution of serotonin immunoreactivity (IR) paralleled that previously described for cholinesterase activity, although it was more widespread. Nerve cell bodies and nerve fibres immunoreactive for 5-HT were present throughout the central nervous system (CNS), occurring in the paired lateral, posterior lateral and rostellar ganglia, their connecting commissures and nerve rings in the scolex and in the ten longitudinal nerve cords that run posteriorly throughout the body of the worm. A large population of nerve cell bodies was associated with the lateral nerve cords. In the peripheral nervous system (PNS), immunoreactive nerve fibres occurred in well-developed nerve plexuses innervating the somatic musculature and the musculature of the rostellum and suckers. The genital atrium and associated reproductive ducts were richly innervated with serotoninergic nerve cell bodies and nerve fibres.

An in vitro investigation of endocrine disrupting effects of tricothecenes deoxynivalenol (DON), T-2, HT-2 toxins

Trichothecenes are a large family of chemically related mycotoxins. Deoxynivalenol (DON), T-2 and HT-2 toxins belong to this family and are produced by various species of Fusarium. The H295R steroidogenesis assay, regulation of steroidogenic gene expression and reporter gene assays (RGAs) for the detection of androgen, estrogen, progestagen and glucocorticoid (ant)agonist responses, have been used to assess the endocrine disrupting activity of DON, T-2 and HT-2 toxins.

H295R cells were used as a model for steroidogenesis and gene expression studies and exposed with either DON (0.1–1000 ng/ml), T-2 toxin (0.0005–5 ng/ml) or HT-2 toxin (0.005–50 ng/ml) for 48 h. We observed a reduction in hormone levels in media of exposed cells following radioimmunoassay. Cell viability was determined by four colorimetric assays and we observed reduced cell viability with increasing toxin concentrations partly explaining the significant reduction in hormone levels at the highest toxin concentration of all three trichothecenes.

Thirteen of the 16 steroidogenic genes analyzed by quantitative real time PCR (RT-qPCR) were significantly regulated (P < 0.05) by DON (100 ng/ml), T-2 toxin (0.5 ng/ml) and HT-2 toxin (5 ng/ml) compared to the control, with reference genes (B2M, ATP5B and ACTB). Whereas HMGR and CYP19 were down-regulated, CYP1A1 and CYP21 were up-regulated by all three trichothecenes. DON further up-regulated CYP17, HSD3B2, CYP11B2 and CYP11B1 and down-regulated NR5A1. T-2 toxin caused down-regulation of NR0B1 and NR5A1 whereas HT-2 toxin induced up-regulation of EPHX and HSD17B1 and down-regulation of CYP11A and CYP17. The expressions of MC2R, StAR and HSD17B4 genes were not significantly affected by any of the trichothecenes in the present study.

Although the results indicate that there is no evidence to suggest that DON, T-2 and HT-2 toxins directly interact with the steroid hormone receptors to cause endocrine disruption, the present findings indicate that exposure to DON, T-2 toxin and HT-2 toxin have effects on cell viability, steroidogenesis and alteration in gene expression indicating their potential as endocrine disruptors.

Role of basal extracellular Ca2+ entry during 5-HT-induced vasoconstriction of canine pulmonary arteries.

1. Measurements of artery contraction, cytosolic [Ca(2+)], and Ca(2+) permeability were made to examine contractile and cytosolic [Ca(2+)] responses of canine pulmonary arteries and isolated cells to 5-hydroxytryptamine (5-HT), and to determine the roles of intracellular Ca(2+) release and extracellular Ca(2+) entry in 5-HT responses. 2. The EC(50) for 5-HT-mediated contractions and cytosolic [Ca(2+)] increases was approximately 10(-7) M and responses were inhibited by ketanserin, a 5-HT(2A)-receptor antagonist. 3. 5-HT induced cytosolic [Ca(2+)] increases were blocked by 20 microM Xestospongin-C and by 2-APB (IC(50)=32 microM inhibitors of InsP(3) receptor activation. 4. 5-HT-mediated contractions were reliant on release of InsP(3) but not ryanodine-sensitive Ca(2+) stores. 5. 5-HT-mediated contractions and cytosolic [Ca(2+)] increases were partially inhibited by 10 microM nisoldipine, a voltage-dependent Ca(2+) channel blocker. 6. Extracellular Ca(2+) removal reduced 5-HT-mediated contractions further than nisoldipine and ablated cytosolic [Ca(2+)] increases and [Ca(2+)] oscillations. Similar to Ca(2+) removal, Ni(2+) reduced cytosolic [Ca(2+)] and [Ca(2+)] oscillations. 7. Mn(2+) quench of fura-2 and voltage-clamp experiments showed that 5-HT failed to activate any significant voltage-independent Ca(2+) entry pathways, including store-operated and receptor-activated nonselective cation channels. Ni(2+) but not nisoldipine or Gd(3+) blocked basal Mn(2+) entry. 8. Voltage-clamp experiments showed that simultaneous depletion of both InsP(3) and ryanodine-sensitive intracellular Ca(2+) stores activates a current with linear voltage dependence and a reversal potential consistent with it being a nonselective cation channel. 5-HT did not activate this current. 9. Basal Ca(2+) entry, rather than CCE, is important to maintain 5-HT-induced cytosolic [Ca(2+)] responses and contraction in canine pulmonary artery.

Anxiolytic-like effects observed in rats exposed to the elevated zero-maze following treatment with 5-HT2/5-HT3/5-HT4 ligands

The present study examined the effects of administering selective 5-HT antagonists and agonists to rats tested in the elevated zero-maze (EZM) model of anxiety. The EZM paradigm has advantages over the elevated plus-maze (EPM) paradigm with respect to measuring anxiety, yet has been utilized less frequently. Three experiments were conducted each with a diazepam control (0.25, 0.5 and 0.75 mg/kg). In the first experiment, we administered the 5-HT_2C antagonist RS 102221 (0.5, 1.0, and 2.0 mg/kg) and 5-HT_2C agonist MK-212 (0.25, 0.5 and 0.75 mg/kg); in the second experiment, we administered the 5-HT₃ antagonist Y-25130 (0.1, 1.0 and 3.0 mg/kg) and 5-HT₃ agonist SR 57227A (0.1, 1.0 and 3.0 mg/kg), and in the third experiment, we administered the 5-HT₄ antagonist RS 39604 (0.01, 0.1, 1.0 mg/kg) and 5-HT₄ agonist RS 67333 (0.01, 0.1 and 0.5 mg/kg). The administration of 5-HT_2/3/4 subtype antagonists all generated behavioral profiles indicative of anxiolytic-like effects in the EZM, which was apparent from examination of both traditional and ethological measures. While little effect was observed from 5-HT₂ and 5-HT₃ agonists, the 5-HT₄ agonist RS 67333 was found to produce a paradoxical anxiolytic-like effect similar to that produced by the 5-HT₄ antagonist RS 39604. We conclude by discussing the implications of these findings.

Specific Noncompetitive Immunoassay for HT-2 Mycotoxin Detection

Here we demonstrate a novel homogeneous one-step immunoassay, utilizing a pair of recombinant antibody antigen-binding fragments (Fab), that is specific for HT-2 toxin and has a positive readout. Advantages over the conventional competitive immunoassay formats such as enzyme-linked immunosorbent assay (ELISA) are the specificity, speed, and simplicity of the assay. Recombinant antibody HT2-10 Fab recognizing both HT-2 and T-2 toxins was developed from a phage display antibody library containing 6 × 10(7) different antibody clones. Specificity of the immunoassay was introduced by an anti-immune complex (IC) antibody binding the primary antibody-HT-2 toxin complex. When the noncompetitive immune complex assay was compared to the traditional competitive assay, an over 10-fold improvement in sensitivity was observed. Although the HT2-10 antibody has 100% cross-reactivity for HT-2 and T-2 toxins, the immune complex assay is highly specific for HT-2 alone. The assay performance with real samples was evaluated using naturally contaminated wheat reference material. The half-maximal effective concentration (EC50) value of the time-resolved fluorescence resonance energy transfer (TR-FRET) assay was 9.6 ng/mL, and the limit of detection (LOD) was 0.38 ng/mL (19 μg/kg). The labeled antibodies can be predried to the assay vials, e.g., microtiter plate wells, and readout is ready in 10 min after the sample application.

Rapid tryptophan depletion improves decision-making cognition in healthy humans without affecting reversal learning or set shifting

Rapid tryptophan (Trp) depletion (RTD) has been reported to cause deterioration in the quality of decision making and impaired reversal learning, while leaving attentional set shifting relatively unimpaired. These findings have been attributed to a more powerful neuromodulatory effect of reduced 5-HT on ventral prefrontal cortex (PFC) than on dorsolateral PFC. In view of the limited number of reports, the aim of this study was to independently replicate these findings using the same test paradigms. Healthy human subjects without a personal or family history of affective disorder were assessed using a computerized decision making/gambling task and the CANTAB ID/ED attentional set-shifting task under Trp-depleted (n=17; nine males and eight females) or control (n=15; seven males and eight females) conditions, in a double-blind, randomized, parallel-group design. There was no significant effect of RTD on set shifting, reversal learning, risk taking, impulsivity, or subjective mood. However, RTD significantly altered decision making such that depleted subjects chose the more likely of two possible outcomes significantly more often than controls. This is in direct contrast to the previous report that subjects chose the more likely outcome significantly less often following RTD. In the terminology of that report, our result may be interpreted as improvement in the quality of decision making following RTD. This contrast between studies highlights the variability in the cognitive effects of RTD between apparently similar groups of healthy subjects, and suggests the need for future RTD studies to control for a range of personality, family history, and genetic factors that may be associated with 5-HT function.

An exactly solvable four transition point model

Heavy particle collisions, in particular low-energy ion-atom collisions, are amenable to semiclassical JWKB phase integral analysis in the complex plane of the internuclear separation. Analytic continuation in this plane requires due attention to the Stokes phenomenon which parametrizes the physical mechanisms of curve crossing, non-crossing, the hybrid Nikitin model, rotational coupling and predissociation. Complex transition points represent adiabatic degeneracies. In the case of two or more such points, the Stokes constants may only be completely determined by resort to the so-called comparison- equation method involving, in particular, parabolic cylinder functions or Whittaker functions and their strong-coupling asymptotics. In particular, the Nikitin model is a two transition-point one-double-pole problem in each half-plane corresponding to either ingoing or outgoing waves. When the four transition points are closely clustered, new techniques are required to determine Stokes constants. However, such investigations remain incomplete, A model problem is therefore solved exactly for scattering along a one-dimensional z-axis. The energy eigenvalue is b(2)-a(2) and the potential comprises -z(2)/2 (parabolic) and -a(2) + b(2)/2z(2) (centrifugal/centripetal) components. The square of the wavenumber has in the complex z-plane, four zeros each a transition point at z = +/-a +/- ib and has a double pole at z = 0. In cases (a) and (b), a and b are real and unitarity obtains. In case (a) the reflection and transition coefficients are parametrized by exponentials when a(2) + b(2) > 1/2. In case (b) they are parametrized by trigonometrics when a(2) + b(2) <1/2 and total reflection is achievable. In case (c) a and b are complex and in general unitarity is not achieved due to loss of flux to a continuum (O'Rourke and Crothers, 1992 Proc. R. Sec. 438 1). Nevertheless, case (c) coefficients reduce to (a) or (b) under appropriate limiting conditions. Setting z = ht, with h a real constant, an attempt is made to model a two-state collision problem modelled by a pair of coupled first-order impact parameter equations and an appropriate (T) over tilde-tau relation, where (T) over tilde is the Stueckelberg variable and tau is the reduced or scaled time. The attempt fails because (T) over tilde is an odd function of tau, which is unphysical in a real collision problem. However, it is pointed out that by applying the Kummer exponential model to each half-plane (O'Rourke and Crothers 1994 J. Phys. B: At. Mel. Opt. Phys. 27 2497) the current model is in effect extended to a collision problem with four transition points and a double pole in each half-plane. Moreover, the attempt in itself is not a complete failure since it is shown that the result is a perfect diabatic inelastic collision for a traceless Hamiltonian matrix, or at least when both diagonal elements are odd and the off-diagonal elements equal and even.

Development in vitro of the neuromusculature of two strigeid trematodes, Apatemon cobitidis proterorhini and Cotylurus erraticus

Confocal microscopy interfaced with cytochemical procedures has been used to monitor development of the major muscle systems and associated serotoninergic (5-HT, 5-hydroxytryptamine) and peptidergic (FaRP, FMRFamide-related peptide) innervation of the strigeid trematodes, Apatemon cobitidis proterorhini and Cotylurus erraticus during cultivation in vitro. Sexually undifferentiated metacercariae were successfully grown to ovigerous adults using tissue culture medium NCTC 135, chicken serum and egg albumen. Eggs were produced after 5 days in culture but had abnormal shells and failed to embryonate. 5-HT and FaRP (the flatworm FaRP, GYIRFamide) were localised immunocytochemically in both central and peripheral nervous systems of developing worms. During cultivation, the central serotoninergic and FaRPergic neuronal pathways of the forebody became more extensive, but retained the same basic orthogonal arrangement as found in the excysted metacercaria. Longitudinal extensor and flexor muscles of the hindbody provide support for the developing reproductive complex. The male reproductive tracts were established in advance (day 3) of those of the female system (day 4); completion of the latter was marked by the appearance of the ootype/egg chamber. The inner longitudinal muscle fibres of the female tract appeared prior to the outer and more densely arranged circular muscles. Circular fibres dominate the muscle complement of both alimentary and reproductive tracts. 5-HT- and GYIRFamide-immunoreactivities were demonstrable in the central nervous system (CNS) and subtegumental parasympathetic nervous system (PNS) throughout the culture period, but innervation of the developing reproductive structures was reactive just for 5-HT. Only at the onset of egg production was FaRP-IR observed in the reproductive system and was expressed only in the innervation of the ootype, a finding consistent with the view that FaRPs may regulate egg assembly in platyhelminths.