Insecticide Transfer Efficiency and Lethal Load in Argentine Ants.

Trophallaxis between individual worker ants and the toxicant load in dead and live Argentine ants (Linepithema humile) in colonies exposed to fipronil and hydramethylnon experimental baits were examined using accelerator mass spectrometry (AMS). About 50% of the content of the crop containing trace levels of 14C-sucrose, 14C-hydramethylnon, and 14C-fipronil was shared between single donor and recipient ants. Dead workers and queens contained significantly more hydramethylnon (122.7 and 22.4 amol/µg ant, respectively) than did live workers and queens (96.3 and 10.4 amol/µg ant, respectively). Dead workers had significantly more fipronil (420.3 amol/µg ant) than did live workers (208.5 amol/µg ant), but dead and live queens had equal fipronil levels (59.5 and 54.3 amol/µg ant, respectively). The distribution of fipronil differed within the bodies of dead and live queens; the highest amounts of fipronil were recovered in the thorax of dead queens whereas live queens had the highest levels in the head. Resurgence of polygynous ant colonies treated with hydramethylnon baits may be explained by queen survival resulting from sublethal doses due to a slowing of trophallaxis throughout the colony. Bait strategies and dose levels for controlling insect pests need to be based on the specific toxicant properties and trophic strategies for targeting the entire colony.

A Comparison of ToxCast Test Results with In Vivo and Other In Vitro Endpoints for Neuro, Endocrine, and Developmental Toxicities: A Case Study Using Endosulfan and Methidathion.

INTRODUCTION: The U.S. Environmental Protection Agency's (EPA's) Toxicity Forecaster (ToxCast) is a potential tool for chemical prioritization, hazard identification, and risk assessment. We conducted a case study to compare ToxCast data with endpoints from other in vitro and in vivo studies for two data-rich pesticides: endosulfan and methidathion. METHODS: ToxCast assays for endocrine disruption, development (zebrafish), and neurotoxicity were qualitatively compared to traditional neurotoxicity, developmental and reproductive toxicity findings. We also used in vitro-in vivo extrapolation to convert half-maximal activity concentrations in active ToxCast assays to rat oral equivalent doses, and quantitatively compared these to the lowest observable effect level (LOEL) from in vivo studies. RESULTS: Endosulfan was inactive for GABAA R, unlike in vivo; but active with dopamine transporter assays and was neurotoxic in zebrafish as expected. Methidathion was not active for these endpoints in vivo or in vitro. Acetylcholinesterase inhibition was ToxCast-inactive, although both pesticides are inhibitors in vivo. ToxCast results were generally inactive for endosulfan estrogen receptor agonism and androgen receptor antagonism unlike in vivo. Calculated oral equivalent doses for estrogen receptor and androgen receptor pathways and for zebrafish assays for both compounds were generally consistent with in vivo LOELs. Endosulfan showed neurotoxicity and both pesticides showed developmental effects in the zebrafish assays, although methidathion is not developmentally toxic in vivo. CONCLUSIONS: ToxCast's predictions showed concordance on some endpoints and nonconcordance, consisting mainly of false inactives, in several critical endpoints, likely due to a lack of metabolic activation and limitations in assay design. Zebrafish assays were good predictors of developmental toxicity and neurotoxicity for endosulfan.

Structural studies of the final enzyme in the alpha-aminoadipate pathway-saccharopine dehydrogenase from Saccharomyces cerevisiae.

The 1.64 A structure of the apoenzyme form of saccharopine dehydrogenase (SDH) from Saccharomyces cerevisiae shows the enzyme to be composed of two domains with similar dinucleotide binding folds with a deep cleft at the interface. The structure reveals homology to alanine dehydrogenase, despite low primary sequence similarity. A model of the ternary complex of SDH, NAD, and saccharopine identifies residues Lys77 and Glu122 as potentially important for substrate binding and/or catalysis, consistent with a proton shuttle mechanism. Furthermore, the model suggests that a conformational change is required for catalysis and that residues Lys99 and Asp281 may be instrumental in mediating this change. Analysis of the crystal structure in the context of other homologous enzymes from pathogenic fungi and human sources sheds light into the suitability of SDH as a target for antimicrobial drug development.

An engineered bifunctional high affinity iron uptake protein in the yeast plasma membrane.

High affinity iron uptake in fungi is supported by a plasma membrane protein complex that includes a multicopper ferroxidase enzyme and a ferric iron permease. In Saccharomyces cerevisiae, this complex is composed of the ferroxidase Fet3p and the permease Ftr1p. Fe(II) serves as substrate for Fe-uptake by being substrate for Fet3p; the resulting Fet3p-produced Fe(III) is then transported across the membrane via Ftr1p. A model of metabolite channeling of this Fe(III) is tested here by first constructing and kinetically characterizing in Fe-uptake two Fet3p-Ftr1p chimeras in which the multicopper oxidase/ferroxidase domain of Fet3p has been fused to the Ftr1p iron permease. Although the bifunctional chimeras are as kinetically efficient in Fe-uptake as is the wild type two-component system, they lack the adaptability and fidelity in Fe-uptake of the wild type. Specifically, Fe-uptake through the Fet3p, Ftr1p complex is insensitive to a potential Fe(III) trapping agent - citrate - whereas Fe-uptake via the chimeric proteins is competitively inhibited by this Fe(III) chelator. This inhibition does not appear to be due to scavenging Fet3p-produced Fe(III) that is in equilibrium with bulk solvent but could be due to leakiness to citrate found in the bifunctional but not the two-component system. The data are consistent with a channeling model of Fe-trafficking in the Fet3p, Ftr1p complex and suggest that in this system, Fet3p serves as a redox sieve that presents Fe(III) specifically for permeation through Ftr1p.

Stromules and the dynamic nature of plastid morphology.

Investigation of plastids via green fluorescent protein (GFP) has led to the rediscovery of tubular extensions of the plastid membrane, termed stromules, for stroma-filled tubules. These unique structures are challenging our understanding of plastid structure and function. Stromules are highly dynamic, branching and elongating across the plant cell. Recent experiments indicate that cytoplasmic microtubules and microfilaments control the shape and motility of stromules. Whether stromule formation involves plastid-specific structural systems, such as the plastid division machinery, remains open to debate. Fluorescence photobleaching experiments have revealed that GFP can traffic between plastids joined by stromules. As a result, interest has grown in whether other macromolecules can also travel through these connections. Although the function of stromules is unknown, several aspects of their biology suggest they play a role in molecular exchange between plastids and other organelles.

T1 measurements in cell cultures: a new tool for characterizing contrast agents at 1.5T.

The objective of this work was to assess the feasibility and accuracy of T1 and relaxivity measurements in cell cultures using 1.5T magnetic resonance imaging (MRI) with the long-term goal to develop a tool for evaluation of novel paramagnetic agents in a realistic macromolecular environment. This initial study was carried out using MCF-7 cells treated with independently determined concentrations of Gd-DTPA. Two cell culture systems were evaluated: cell pellets and single layers of cells grown on microporous inserts. High-resolution T1 measurements of cell cultures were acquired with two dimensional Inversion Recovery Fast Spin Echo (2D-IR-FSE), three dimensional Inversion Recovery Fast Spin Echo (3D-IR-FSE), and 3D-SPGR sequences. The T1 and relaxivity accuracy of these sequences was confirmed with aqueous Gd-DTPA samples of known concentration. Relaxivities of 1.71 +/- 0.15 [mM(-1)second(-1)] and 1.55 +/- 0.50 [mM(-1)second(-1)] were measured in the cell pellets and cell monolayers, respectively, and were different from the value of 4.3 [mM(-1)second(-1)] for Gd-DTPA in water. Both cell pellets and monolayers are suitable for initial assessment of novel MR contrast agents.

Orexins/hypocretins excite rat sympathetic preganglionic neurons in vivo and in vitro.

The two recently isolated hypothalamic peptides orexin A and orexin B, also known as hypocretin 1 and 2, are reported to be important signaling molecules in feeding and sleep/wakefulness. Orexin-containing neurons in the lateral hypothalamus project to numerous areas of the rat brain and spinal cord including the intermediolateral cell column (IML) of the thoracolumbar spinal cord. An in vivo and in vitro study was undertaken to evaluate the hypothesis that orexins, acting on sympathetic preganglionic neurons (SPNs) in the rat spinal cord, increase sympathetic outflow. First, orexin A (0.3, 1, and 10 nmol) by intrathecal injection increased mean arterial pressure (MAP) and heart rate (HR) by an average of 5, 18, and 30 mmHg and 10, 42, and 85 beats/min in urethane-anesthetized rats. Intrathecal injection of saline had no significant effects. Orexin B (3 nmol) by intrathecal administration increased MAP and HR by an average of 11 mmHg and 40 beats/min. The pressor effects of orexin A were attenuated by prior intrathecal injection of orexin A antibodies (1:500 dilution) but not by normal serum albumin. Intravenous administration of the alpha(1)-adrenergic receptor antagonist prazosin (0.5 mg/kg) or the beta-adrenergic receptor antagonist propranolol (0.5 mg/kg) markedly diminished, respectively, the orexin A-induced increase of MAP and HR. Second, whole cell patch recordings were made from antidromically identified SPNs of spinal cord slices from 12- to 16-day-old rats. Superfusion of orexin A or orexin B (100 or 300 nM) excited 12 of 17 SPNs, as evidenced by a membrane depolarization and/or increase of neuronal discharges. Orexin A- or B-induced depolarizations persisted in TTX (0.5 microM)-containing Krebs solution, indicating that the peptide acted directly on SPNs. Results from our in vivo and in vitro studies together with the previous observation of the presence of orexin A-immunoreactive fibers in the IML suggest that orexins, when released within the IML, augment sympathetic outflow by acting directly on SPNs.

Functional MR imaging of vision in the deaf.

RATIONALE AND OBJECTIVES: Early loss of a sensory modality has been associated with cortical reorganization in both animal models and humans. The purpose of this study was to map visual activation with functional magnetic resonance (MR) imaging and to document possible developmental reorganization in the temporal lobe caused by early deafness. MATERIALS AND METHODS: Six prelingual, profoundly deaf subjects were compared with a similar group of six hearing subjects. Three visual tasks were performed by both groups: attention to movement in the field-of-view periphery, shape matching, and mental rotation. Echo-planar coronal MR imaging was performed at 1.5 T. RESULTS: Regions of interest encompassing the middle and posterior aspects of the superior and middle temporal gyri demonstrated a significantly (P < .05) increased activation in deaf subjects compared with hearing subjects, particularly on the right side (P < .05) and during the tasks involving motion. The most specific effect was noted during the mental-rotation task. CONCLUSION: These results support the hypothesis that portions of the temporal lobe usually involved in auditory processing are more active during certain visual tasks in deaf compared with hearing subjects. Cortical reorganization may be an important factor in the deaf population when considering the physiology of temporal lobe lesions and predicting surgical outcomes. Functional MR imaging may be helpful during preoperative assessment in individuals with deafness.

Soluble E-cadherin is a valid prognostic marker in gastric carcinoma.

BACKGROUND: Gastric cancer remains a major cause of cancer mortality globally but no good prognostic tumour marker is available. Soluble fragment of E-cadherin protein has been reported to increase in the sera of patients with cancer and recently was found to be elevated in 67% of patients with gastric cancer. AIMS: To investigate if serum soluble E-cadherin is a valid prognostic marker in gastric cancer. METHODS: Concentrations of soluble E-cadherin from 116 patients with histologically confirmed gastric adenocarcinoma and 40 healthy subjects were measured using an immunoenzymometric method with a commercially available sandwich ELISA kit based on monoclonal antibodies. RESULTS: The logarithm of the means of soluble E-cadherin concentration was significantly higher in patients with gastric cancers (mean 3.85 (SD 0.28)) than in healthy subjects (3.71 (0.18)) (p=0.001), and in palliative/conservatively treated cancers (3.91 (0.35)) than in operable cancers (3.78 (0.19)) (p=0.015). The logarithm of the concentrations correlated with tumour size (p=0.032) and carcinoembryonic antigen concentrations (p=0.001). The cut off value calculated from discriminant analysis on operability and inoperability/palliative treatment was 7025 ng/ml. Soluble E-cadherin concentrations higher than this cut off value predicted tumour (T4) depth invasion (p=0.020, confidence interval (CI) 1.008-1.668) and palliative/conservative treatment (p=0.023, CI 1.038-2.514). In contrast, the relative risks for lymph node (N2) metastasis, distant metastasis, and stage III/IV disease were 1.41, 1.33, and 1.55 respectively, despite not reaching statistical significance. CONCLUSION: Serum soluble E-cadherin is a potential valid prognostic marker for gastric cancer. A high concentration predicts palliative/conservative treatment and T4 invasion.

Enhanced sensitivity to molecular diffusion with intermolecular double-quantum coherences: implications and potential applications.

Apparent molecular self-diffusion rates for (1)H intermolecular double-quantum coherences (iDQCs) were measured in solvents covering a wide range of intrinsic diffusion coefficients at 1.5, 9.4 and 14T, and water iDQC diffusion-weighted images were obtained at 1.5T in human brains and at 9.4T in rat brains. Conventional single quantum coherence (SQC) measurements were also made in the same samples. Experimental results indicate that iDQCs are approximately twice as sensitive to diffusion as SQC. A general theoretical expression was derived, and a model was proposed to explain the phenomenon. Potential applications in DWI and brain fMRI were also discussed.

fMRI of auditory stimulation with intermolecular double-quantum coherences (iDQCs) at 1.5T.

An intermolecular double-quantum coherence (iDQC) imaging technique was used to study auditory activation in the human brain at 1.5T with a dual temporal lobe surface phased array coil and a quadrature head coil. Preliminary results demonstrate that it is feasible to obtain auditory activation maps using iDQC imaging at 1.5T, both in individual subjects using the surface coil array and with multisubject averaging of data using the head coil. The most robust activation map was obtained when a spin-echo (SE) acquisition was combined with an iDQC excitation. Since SE with conventional single quantum coherence (SQC) and similar parameters showed much reduced activation in spite of its higher signal-to-noise ratio (SNR), it was determined that activation resulting from the SE-iDQC acquisition almost entirely originates from iDQCs. In addition, the fact that the robust activation was obtained using signals at an evolution time more sensitive to changes in magnetic susceptibilities also suggests the sensitivity of iDQCs to the BOLD effect upon activation. iDQCs provide a novel MRI method which is potentially more sensitive to the BOLD effect traditionally measured with SQC. Magn Reson Med 45:356-364, 2001.

Orexins: a role in medullary sympathetic outflow.

Orexin A and B, also known as hypocretin 1 and 2, are two recently isolated hypothalamic peptides. As orexin-containing neurons are strategically located in the lateral hypothalamus, which has long been suspected to play an important role in feeding behaviors, initial studies were focused on the involvement of orexins in positive food intake and energy metabolism. Recent studies implicate a more diverse biological role of orexins, which can be manifested at different level of the neuraxis. For example, canine narcolepsy, a disorder with close phenotypic similarity to human narcolepsy, is caused by a mutation of hypocretin receptor 2 gene. Results from our immunohistochemical and functional studies, which will be summarized here, suggest that the peptide acting on neurons in the rostral ventrolateral medulla augment sympathoexcitatory outflow to the spinal cord. This finding is discussed in the context of increased sympathetic activity frequently associated with obesity.

Helicobacter pylori sero-prevalence in asthma.

Asthma is a very common respiratory disease which is characterized by the presence of cytokine-mediated airway inflammation leading to smooth muscle contraction, oedema and progressive airway damage in some cases. In light of our recent finding of an increased sero-prevalence of Helicobacter pylori in bronchiectasis, we have determined serum levels of H. pylori-specific IgG in asthmatic and control subjects. Altogether 90 consecutive asthmatic [mean age +/- SD 42.6+/-16 years and 52 female (F)] and 97 healthy control subjects (mean age +/- SD 43.2+/-13.3 years and 51 female (F); P = 0.78 and 0.39 respectively) were recruited prospectively. H. pylori sero-prevalence was not significantly different between asthmatic and control subjects (P>0.05). Serum H. pylori IgG levels did not correlate with FEV1 % predicted, FVC % predicted or duration of asthma (P>0.05). Similar to the results of previously published sero-epidemiological studies, there was a weak correlation between serum H. pylori IgG with increasing age (r = 0.43, P = 0.004). Despite the sero-epidemiological association of H. pylori infection with many inflammatory conditions, our data showed no such association for middle age asthmatic patients with mild intermittent asthma in our locality.

New image contrast mechanisms in intermolecular double-quantum coherence human MR imaging.

We have developed a novel magnetic resonance imaging (MRI) method based on the intermolecular double-quantum coherence (DQC) in humans. Combined quantum mechanical and classical formalisms were used to characterize the signal and to aid in the design of a DQC imaging sequence with conventional or echoplanar acquisitions. Imaging contrast was evaluated in volunteers using a 1.5-T clinical scanner. The results demonstrated that the DQC images have contrasts fundamentally different from the conventional images based on single-quantum coherence (SQC). Both our theoretical analysis and experiments suggest that signals from DQCs have a higher signal-to-noise ratio than those from zero-quantum coherence (ZQC) for human brain imaging. The new contrast in DQC imaging may be useful for the detection of varying microstructures, potentially improving the detection of tumors without the need for contrast agents and providing a higher sensitivity and selectivity to magnetic susceptibility distributions in functional MRI brain studies.

Endomorphins: localization, release and action on rat dorsal horn neurons.

Endomorphin (Endo) 1 and 2, two tetrapeptides isolated from the bovine and human brain, have been proposed to be the endogenous ligand for the mu-opiate receptor. A multi-disciplinary study was undertaken to address the issues of localization, release and biological action of Endo with respect to the rat dorsal horn. First, immunohistochemical studies showed that Endo-1- or Endo-2-like immunoreactivity (Endo-1- or Endo-2-LI) is selectively expressed in fiber-like elements occupying the superficial layers of the rat dorsal horn, which also exhibit a high level of mu-opiate receptor immunoreactivity. Second, release of immunoreactive Endo-2-like substances (irEndo) from the in vitro rat spinal cords upon electrical stimulation of dorsal root afferent fibers was detected by the immobilized antibody microprobe technique. The site of release corresponded to laminae I and II where the highest density of Endo-2-LI fibers was localized. Lastly, whole-cell patch clamp recordings from substantia gelatinosa (SG) neurons of rat lumbar spinal cord slices revealed two distinct actions of exogenous Endo-1 and Endo-2: (1) depression of excitatory and/or inhibitory postsynaptic potentials evoked by stimulation of dorsal root entry zone, and (2) hyperpolarization of SG neurons. These two effects were prevented by the selective mu-opiate receptor antagonist beta-funaltrexamine. The localization of endomorphin-positive fibers in superficial layers of the dorsal horn and the release of irEndo upon stimulation of dorsal root afferents together with the observation that Endo inhibits the activity of SG neurons by interacting with mu-opiate receptors provide additional support of a role of Endo as the endogenous ligand for the mu-opiate receptor in the rat dorsal horn.

Characteristics and pitfalls of contrast-enhanced, T1-weighted magnetization transfer images of the brain.

RATIONALE AND OBJECTIVES: This study was undertaken to clarify the difference in signal pattern on contrast material-enhanced T1-weighted magnetic resonance (MR) magnetization transfer (MT) images between enhancing and nonenhancing lesions in various intracranial diseases and to determine the necessity of nonenhanced MT images for evaluating lesional contrast enhancement. MATERIALS AND METHODS: MR images of 116 patients who underwent nonenhanced T1-weighted imaging, nonenhanced MT imaging, and contrast-enhanced MT imaging were reviewed. The increase in signal intensity of lesions relative to normal brain was compared between nonenhanced T1-weighted images and contrast-enhanced MT images. Signal intensity of lesions was compared with that of the striate nucleus and white matter on contrast-enhanced MT images. True enhancement was determined by comparison with nonenhanced MT images. RESULTS: In all, 143 lesions, including 86 enhancing and 57 nonenhancing lesions, were identified among 63 patients. Almost all (99%) of the enhancing lesions were hyperintense to striate nucleus on contrast-enhanced MT images, and most (>87%) showed moderate to marked signal intensity increase from nonenhanced T1-weighted images to contrast-enhanced MT images. Most (>95%) of the nonenhancing lesions showed mild or no increase in relative signal intensity, and most (75%) were iso- or hypointense to striate nucleus on contrast-enhanced MT images. A few nonenhancing lesions (4%-6%), however, showed increase in signal intensity that was indistinguishable from true enhancement without comparison to non-enhanced MT images. CONCLUSION: Nonenhanced MT images should be obtained to assess pathologic enhancement accurately.

Cocaine- and amphetamine-regulated transcript-immunoreactivity in the rat sympatho-adrenal axis.

Distribution of cocaine- and amphetamine-regulated transcript-like immunoreactivity (CART-LI) was studied in the rat spinal cord, sympathetic ganglia and adrenal glands by immunohistochemical methods, utilizing a polyclonal antiserum raised against the CART peptide fragment 55-102. CART-LI was detected in nerve fibers and in basket-like terminals surrounding many postganglionic neurons of the superior cervical ganglion (SCG), stellate, paravertebral and prevertebral ganglia. Postganglionic neurons exhibited low or non-detectable levels of CART-LI. Surgical sectioning of the cervical sympathetic trunk for 6-7 days resulted in a nearly complete loss of CART-LI fibers and terminals in the SCG. In the adrenal gland, CART-LI nerve fibers formed a plexus underneath the capsule, some of which bifurcated and made a sharp turn toward the adrenal medulla, where clusters of chromaffin cells were intensely labeled. The detection of CART-LI in sympathetic ganglia and adrenal glands extends the previous observation of the presence of CART-LI in sympathetic preganglionic neurons and further supports the notion that CART peptide(s) may function as a signaling molecule in the sympatho-adrenal axis.

In vivo intermolecular double-quantum imaging on a clinical 1.5 T MR scanner.

A novel MRI method based on the intermolecular double-quantum coherence (DQC) for soft tissues is described. DQC images of human brain were obtained for the first time on a whole-body 1.5 T scanner. The combination of quantum and classical formalisms was used to characterize multiple-quantum coherences, and to aid in the design of a DQC imaging sequence. The theoretical analysis suggests that signals from the intermolecular DQCs have higher sensitivity than those from the zero-quantum coherence (ZQC) for human brain, and the sensitivity increases with increased field strength. The DQC signal may provide a new form of contrast for MRI.

Release of endomorphin-2 like substances from the rat spinal cord.

Release of endomorphin (ENDO)-2 like substances from the dorsal horn of the isolated rat spinal cord was measured by the immobilized-antibody microprobe technique. Spinal cords were removed from anesthetized 4-6 week old rats and superfused with oxygenated Krebs solution at room temperature. Glass microprobes coated with ENDO-2 antibodies were inserted into the dorsal horn of the lumbar spinal cord 1.5 mm lateral to the midline to a depth 2.5 mm below the dorsal surface of the cord. Each probe remained in situ for 10 min periods before, during and after electrical stimulation applied to the dorsal root entry zone of the same spinal segment. There was no detectable basal release of immunoreactive endomorphin-2 like substance (irENDO) from the dorsal horns during the pre-stimulation, nor following the stimulation period. A significant release of irENDO was measured during the electrical stimulation. These results provide the first evidence of a irEndo release that is correlated spatially with the dorsal horn laminae I and II where ENDO-2-immunoreactive fibers are concentrated in the dorsal horn in response to electrical activation of primary afferent fibers.