An approach to the focal presentation of chemical stimuli

An olfactometer is described that presents temporally-discretepulses of stimuli to individual chemosensory structures. Thedevice is based on standard pressure injection techniques inwhich pulses of compressed air eject nanoliter volumes of upto six stimulants from small diameter glass micropipettes. Thedevice should be readily adaptable to different chemoreceptorpreparations.     

Stimulation of the hypothalamic-pituitary-adrenal axis and inhibition of growth hormone release via increased central noradrenaline neuronal activity by urethane anaesthesia in the rat: blockade by clonidine

Computerized gas chromatography-mass spectrometry was used to measure precisely the hypothalamic levels of noradrenaline (NA), dopamine and serotonin together with those of their major neuronal metabolites 3,4-dihydroxyphenylethyleneglycol (DHPG), 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid in normal male rats 45 min after stimulation of hypothalamic-pituitary-adrenal function by urethane (1.3 g/kg) administration. Urethane treatment resulted in a significant elevation of central noradrenergic neuronal activity (NNA) as assessed from marked rises in hypothalamic DHPG concentrations and the ratio (DHPG/NA). At the same time there was significant stimulation of ACTH and corticosterone release and inhibition of growth hormone release. These hormonal and central effects of urethane (but not anesthesia) were inhibited when the alpha 2-agonist clonidine (150 micrograms/kg) was co-administered. Urethane had no major effect on hypothalamic dopamine or serotonin status. We propose that the release of ACTH and the suppression of growth hormone release following urethane anaesthesia is a result of activation of central NNA and suggest that the hormonal responses are mediated via hypothalamic noradrenergic facilitation of corticotrophin releasing factor and somatostatin release to the anterior pituitary.     

Insulin binding in the hypothalamus of lean and genetically obese Zucker rats

Recent reports have suggested that the obesity and hyperphagia of the genetically obese Zucker rat may be related to defective insulin action or binding in the hypothalamus. We used quantitative autoradiography to determine if insulin binding is altered in specific hypothalamic nuclei associated with food intake. Insulin binding was measured in the arcuate (ARC), dorsomedial (DMN), and ventromedial (VMN) hypothalamic nuclei of 3–4-month-old lean (Fa/Fa) and genetically obese (fa/fa) Zucker rats. A consistently reproducible 15% increase in the total specific binding of 0.1 nM [¹²⁵I]-insulin was found in the ARC of the obese genotype. A slight increase in insulin binding in the DMN was also found. No difference in specific insulin binding was found between genotypes in the VMN. Nonlinear least squares analysis of competitive binding studies showed that the K_d of the ARC insulin binding site was 33% higher in the lean rats than in the obese rats, indicating an increased affinity for insulin. No difference in site number (B_max) was found in the ARC, DMN or VMN, and no evidence was found for reduced insulin binding in the hypothalamus of the obese (fa/fa) genotype. The results suggest that hyperphagia and obesity of the obese (fa/fa) Zucker rat genotype may be associated with increased insulin binding in the arcuate nucleus.     

Functional expression of dihydropyridine-insensitive calcium channels during PC12 cell differentiation by nerve growth factor (NGF), oncogenic ras,or src tyrosine kinase

1. Recombinant retroviruses were used to introduce a temperature-sensitive v-src gene and oncogenic c-Ha-ras into PC12 cells, and stable cell lines expressing these genes were established. 2. As previously reported, expression of v-src (Alema et al., 1985) or c-Ha-ras (Noda et al., 1985) in PC12 cells results in neurite outgrowth resembling that induced by NGF. We report here that v-src but not oncogenic c-Ha-ras induces a stable morphologic neuronal differentiation similar to treatment with NGF. Oncogenic c-Ha-ras-induced neurite outgrowth is not stable with long-term culture, rather the cells revert to an undifferentiated morphology with altered cell cycle kinetics. 3. The stable neuronal phenotype induced by v-src and NGF is characterized by the functional expression of dihydropyridine-insensitive calcium currents.     

Nasal trigeminal responses to toluene presented by an automated delivery system

An apparatus was developed which permits the automated deliveryof volatile chemical stimuli for use in neurophysiology experiments.A computer-controlled olfactometer, incorporating electronicmass flow controllers (EMFCs) and Teflon-lined solenoid valves,generated and delivered clean or odorized air. Neural and respiratorysignals from the animal were amplified and stored, along withtrial information (e.g. odorant concentration) on a chart recorderand video cassette recorder, both of which were controlled bythe computer. This apparatus was used to measure responses totoluene from the rat ethmoid nerve, a part of the ophthalmicdivision of the trigeminal nerve. Multi-unit responses to thiscompound were first observed at 2000 p.p.m. The magnitude ofthe response increased linearly with logarithmic increases inconcentration up to vapour saturation. Changes in respirationin response to toluene also were observed, although neural responsesoften were seen in the absence of respiratory changes.     

Vulnerability of early life intervals of Coregonus hoyi to predation by a freshwater mysid,Mysis relicta

Synopsis The bloater, Coregonus hoyi, deposits its eggs on deep sediments (70–100 m in Lake Michigan), where its eggs and embryos can be exposed to epibenthic predators. We investigated the vulnerability of early life intervals of bloaters to predation by mysids, Mysis relicta, which are mostly epibenthic by day and planktonic at night. Bloaters were raised from spawn in the laboratory and presented to field-collected mysids in laboratory predation trials. Eggs were not ingested by the mysids. Embryonic bloaters were vulnerable to predation by mysids only during the interval between hatching and swim up, usually 1–24 h under laboratory conditions. The mysids required about a day of exposure to this novel prey before they were able to kill significant numbers of the bloater embryos by making successive attacks with their thoracic legs. In experiments with experienced (2 and 3 days with bloater embryos) mysids, a functional response between embryo density and mysid predation rates was apparent. Temperature and the presence of alternative prey (zooplankton) did not alter the ‘kill rate’ (about 2.5 embryos mysid^-1d^-1) of experienced mysids at high bloater densities (>4 bloaters/mysid). However, more embryos were partially, rather than completely, ingested at 4 versus 9° C and in the presence of zooplankton.     

Experience of the use of probiotics for Salmonellae control in poultry

The Editors of Letters in Applied Microbiology will, at their discretion, publish invited and submitted 'Opinions' on subjects in the general area of Applied Microbiology. They will not be subjected to the normal refereeing procedures and reprints will not be provided. The 'Opinions' will not necessarily represent the views of the Society of Applied Bacteriology or of the Editors. The Editors may invite or readers may submit 'Responses' to published 'Opinions' , provided that they are not merely polemics, and these will also be published at the discretion of the Editors. 'Responses' will be treated precisely like 'Opinions' . They should clearly indicate, in their first sentence, the 'Opinion' to which they are a response.     

Mutations in the FAD-binding fold of alcohol oxidase from Hansenula polymorpha.

Alcohol oxidase of methylotrophic yeast is an FAD-containing enzyme. When in its active form, the enzyme is an octamer and located in the peroxisomes. To study the importance of FAD-binding on the activity, octamerization and intracellular localization of the enzyme, alcohol oxidase of Hansenula polymorpha was mutated in its presumed nucleotide-binding domain, which is formed by the N-terminal sequence. Whereas mutations of a glutamic acid residue (E42) reduced the stability of the octamer, it hardly affected enzyme activity and expression. However, replacements of three conserved glycines (G13, G15 and G18) and a conserved glutamic acid (E39) within the fold had severe effects. The mutations not only resulted in loss of enzyme activity but in reduced protein levels as well, probably due to decreased stability of the mutant alcohol oxidase. However, octamerization of the protein still occurred. The existence of inactive octameric proteins provides information about the formation pathway of this octameric flavoprotein.     

Expression of two cytochromes P450 involved in carcinogen activation in a human colon cell line

Cytochrome P450 is known to cause carcinogen activation and correspondingly increased cancer risk in animal models. In order to determine whether P450 in the colon may be involved in cancer development in the human, the human colon cell line LS174T was examined for the presence of various cytochromes P450. Two isozymes of P450 were identified in the human cell line. Expression of P450IAl or IA2 was increased by treatment of the cell line with benzanthracene; the induction was demonstrated by an increase in RNA hybridizing to a probe for P4501Al and by ethoxyresorufin deethylation activity. Western analysis of microsomes isolated from human colon tissue also demonstrated the presence of P4501A1, as well as a form which cross-reacted to an antibody to human P450IIC9. Another isozyme, P450IIE1, was identified by polymerase chain reaction amplification of RNA from LS174T cells. These results underscore the presence of cytochromes P450 in colonic tissue and provide a basis for the involvement of isozyme-specific P450 mediated reactions in carcinogenesis of the colon.Some of the data presented here were taken from a thesis submitted by D.K.H. in partial fulfillment of the requirements for the Ph.D. degree in the University of Texas Graduate School of Biomedical Sciences.     

A trace display and editing program for data from fluorescence based sequencing machines.

'Ted' (Trace editor) is a graphical editor for sequence and trace data from automated fluorescence sequencing machines. It provides facilities for viewing sequence and trace data (in top or bottom strand orientation), for editing the base sequence, for automated or manual trimming of the head (vector) and tail (uncertain data) from the sequence, for vertical and horizontal trace scaling, for keeping a history of sequence editing, and for output of the edited sequence. Ted has been used extensively in the C.elegans genome sequencing project, both as a stand-alone program and integrated into the Staden sequence assembly package, and has greatly aided in the efficiency and accuracy of sequence editing. It runs in the X windows environment on Sun workstations and is available from the authors. Ted currently supports sequence and trace data from the ABI 373A and Pharmacia A.L.F. sequencers.