Brain histamine H 3 receptors are predominantly presynaptic and serve an important autoregulatory function for the release of histamine and other neurotransmitters. They have been implicated in a variety of brain functions, including arousal, locomotor activity, thermoregulation, food intake, and memory. The recent cloning of the H 3 receptor in our laboratory has made it possible to create a transgenic line of mice devoid of H 3 receptors. This paper provides the first description of the H 3 receptor-deficient mouse (H 3 Ϫ/Ϫ ), including molecular and pharmacologic verification of the receptor deletion as well as phenotypic screens. The H 3 Ϫ/Ϫ mice showed a decrease in overall locomotion, wheel-running behavior, and body temperature during the dark phase but maintained normal circadian rhythmicity. H 3 Ϫ/Ϫ mice were insensitive to the wake-promoting effects of the H 3 receptor antagonist thioperamide. We also observed a slightly decreased stereotypic response to the dopamine releaser, methamphetamine, and an insensitivity to the amnesic effects of the cholinergic receptor antagonist, scopolamine. These data indicate that the H 3 receptor-deficient mouse represents a valuable model for studying histaminergic regulation of a variety of behaviors and neurotransmitter systems, including dopamine and acetylcholine.The neurotransmitter histamine, which originates from tuberomamillary nuclei in the posterior hypothalamus, projects diffusely throughout the central nervous system (CNS) and has been implicated in the regulation of many functions, including sleep/wake, food and water intake, thermoregulation, memory, and other homeostatic processes (Wada et al., 1991;Brown et al., 2001). Four subtypes (H 1 , H 2 , H 3 , and H 4 ) of histamine receptors are currently recognized (Hill et al., 1997;Hough, 2001). The H 3 subtype is predominantly located presynaptically and serves as an autoreceptor to regulate the synthesis and release of histamine (Hill et al., 1997). The H 3 subtype also has heteroreceptor functions and influences CNS dopamine, ␥-aminobutyric acid, noradrenaline, acetylcholine, and serotonin levels (Arrang et al., 1983(Arrang et al., , 1987bSchlicker et al., 1988;Clapham and Kilpatrick, 1992;Hill et al., 1997). Behavioral correlates of H 3 receptor function have primarily been studied in the context of pharmacologically blocking the receptor using the specific H 3 receptor antagonist, thioperamide. For instance, thioperamide has been used to increase the amount of wakefulness (Monti et al., 1991), to prevent scopolamine-induced amnesia (Giovannini et al., 1999), and to decrease food intake (Itoh et al., 1999;Attoub et al., 2001) in rats. The recent cloning of the H 3 receptor in our laboratory (Lovenberg et al., 1999) has made it possible to create a transgenic line of mice devoid of H 3 receptors and to explore at a molecular level the importance of this receptor in a variety of behaviors. This paper provides the first description of 1) generating the H 3 receptor knockout mice, 2) verifying the d...

Undeclared food allergens account for 30-40% of food recalls in the United States. Compliance with ingredient labeling regulations and the implementation of effective manufacturing allergen control plans require the use of reliable methods for allergen detection and quantitation in complex food products. The objectives of this work were to (1) produce industry-processed model foods incurred with egg, milk, and peanut allergens, (2) compare analytical method performance for allergen quantitation in thermally processed bakery products, and (3) determine the effects of thermal treatment on allergen detection. Control and allergen-incurred cereal bars and muffins were formulated in a pilot-scale industry processing facility. Quantitation of egg, milk, and peanut in incurred baked goods was compared at various processing stages using commercial enzyme-linked immunosorbent assay (ELISA) kits and a novel multi-allergen liquid chromatography (LC)-tandem mass spectrometry (MS/MS) multiple-reaction monitoring (MRM) method. Thermal processing was determined to negatively affect the recovery and quantitation of egg, milk, and peanut to different extents depending on the allergen, matrix, and analytical test method. The Morinaga ELISA and LC-MS/MS quantitative methods reported the highest recovery across all monitored allergens, whereas the ELISA Systems, Neogen BioKits, Neogen Veratox, and R-Biopharm ELISA Kits underperformed in the determination of allergen content of industry-processed bakery products.

Ultra performance liquid chromatography (UPLC) employs particles smaller than 2 m in diameter to achieve superior resolution, speed, and sensitivity compared with high-performance liquid chromatography (HPLC). We have tested the suitability of UPLC for the analysis of deuterated peptides in hydrogen exchange mass spectrometry experiments. Superior resolution and sample throughput were obtained with UPLC versus HPLC. For highly deuterated model peptides, deuterium loss using UPLC was greater than the deuterium loss observed using a conventional HPLC system, primarily as a result of the injection requirements of the UPLC system. Partially deuterated cytochrome c peptides also lost more deuterium in UPLC versus HPLC, although the effect was not as pronounced as it was for the highly deuterated model peptides. The exceptional chromatographic aspects of UPLC make it a very attractive alternative to HPLC for hydrogen exchange mass spectrometry

Pepsin digestion prior to mass analysis increases the spatial resolution of hydrogen exchange mass spectrometry experiments. Online digestion with immobilized pepsin is advantageous for several reasons including better digestion efficiency. We have found that certain immobilized pepsin columns cause substantial deuterium back-exchange, rendering the data unusable. When pepsin immobilized on a POROS support was used for online digestion, back-exchange was within the expected range and was similar to the back-exchange of deuterated peptides produced by in-solution pepsin digestion. However, when pepsin immobilized onto selected polystyrene-divinylbenzene supports was used for online digestion with the same system, deuterium loss was extremely high. The effect seems linked to the properties of the solid support used to conjugate the pepsin.

Voltage-gated sodium channels (Navs) play critical roles in action potential generation and propagation. Nav channelopathy as well as pathological sensitization contribute to allodynia and hyperalgesia. Recent evidence has demonstrated the significant roles of Nav subtypes (Nav1.3, 1.7, 1.8, and 1.9) in nociceptive transduction, and therefore these Navs may represent attractive targets for analgesic drug discovery. Animal toxins are structurally diverse peptides that are highly potent yet selective on ion channel subtypes and therefore represent valuable probes to elucidate the structures, gating properties, and cellular functions of ion channels. In this review, we summarize recent advances on peptide toxins from animal venom that selectively target Nav1.3, 1.7, 1.8, and 1.9, along with their potential in analgesic drug discovery.

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Neurosteroids are modulators of neuronal function that may play important roles in brain maturation. We determined whether chronic prenatal ethanol exposure altered neurosteroid levels in the developing brain. Rat dams were exposed to: (i) a 5% ethanol-containing liquid diet that produces peak maternal blood alcohol levels near the legal intoxication limit (approximately 0.08 g/dL); (ii) an isocaloric liquid diet containing maltose-dextrin instead of ethanol with pair-feeding; (iii) rat chow ad libitum. Neurosteroid levels were assessed in offspring brains using radioimmunoassay or gas chromatography-mass spectrometry techniques. A prenatal ethanol exposure-induced increase in pregnenolone sulfate levels, but not dehydroepiandrosterone sulfate levels, was evident at the earliest time point studied (embryonic day 14). This effect lasted until post-natal day 5. Levels of other neurosteroids were assessed at embryonic day 20; pregnenolone levels, but not allopregnanolone levels, were elevated. Pregnenolone sulfate levels were not altered in the maternal brain. Neither pregnenolone nor pregnenolone sulfate levels were significantly altered in the fetal liver, placenta and maternal blood, indicating that the effect of ethanol is not secondary to accumulation of peripherally-produced steroids. Fetal ethanol exposure has been shown to decrease both cellular and behavioral responsiveness to neurosteroids, and our findings provide a plausible explanation for this effect.

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