Tricyclic quinoxalines as ligands for the strychnine-insensitive glycine site

The synthesis of a series of tricyclic quinoxalines is described. These compounds exhibit good affinity for both the strychnine-insensitive glycine site of the NMDA receptor and the AMPA receptor.     

Analysis of the embryonic lineage of vertebrate taste buds

In all vertebrates, taste buds are the last sensory receptorsto appear late in embryonic development. They are thought toarise locally from the oropharyngeal epithelium, although thishypothesis has not been tested experimentally. Alternatively,taste buds have been proposed to arise from neurocctodermalcells that migrate from peripheral neurogenic sources to theoropharyngeal epithelium and give rise to taste bud precursorcells. In order to determine the exact embryonic lineage ofthe cells of vertebrate taste buds, we have employed a combinationof endogenous and exogenous cell marking techniques to followneuroectodermal and endodermal cells through development. Wefind, in the ambystomatid salamander used in our studies, tastebuds arise locally within the endodermally-derived epitheliumlining the oropharyngeal cavity, and do not receive a contributionfrom neuroectodermal sources, i.e. ectodermal placodes or cephalicneural crest.     

Purification,properties, and kinetic mechanism of coenzyme A-linked aldehyde dehydrogenase from Clostridium kluyveri

Coenzyme A-linked aldehyde dehydrogenase from Clostridium kluyveri was purified from the soluble fraction of crude extracts and its physical and kinetic properties were studied. The enzyme was purified approximately 90-fold over crude extracts to a specific activity of 50 units/mg protein and was estimated to be 40% pure by polyacrylamide gel electrophoresis. From active enzyme centrifugation studies, aldehyde dehydrogenase was found to have a sedimentation coefficient of s_{20, w} = 7.4. The Stokes radius of the enzyme was determined by gel filtration and found to be 9.5 nm in the presence of substrates and 11.0 nm in the absence of substrates. Using the values found for the sedimentation coefficient and the Stokes radius, the molecular weight of the enzyme in the presence of substrates was calculated to be 290,000 and the frictional ratio, 2.2. Aldehyde dehydrogenase can utilize thiols other than CoA as acetyl acceptors. A number of methods were employed in order to exclude the possibility that these thiols act merely by recycling nonenzymatically trace amounts of CoA that might be in the enzyme preparation. From steady-state kinetic measurements, a ping pong mechanism was proposed in which NAD⁺ binds to free enzyme, acetaldehyde binds next, and NADH is released before CoA binds and acetyl-CoA released. At K_m levels of other substrates, substrate inhibition by CoA was observed. The nature of the substrate inhibition is discussed.     

Differential Allocation by Female Zebrafish (Danio rerio) to Different-Sized Males – An Example in a Fish Species Lacking Parental Care

Organisms allocate resources to reproduction in response to the costs and benefits of current and future reproductive opportunities. According to the differential allocation hypothesis, females allocate more resources to high-quality males. We tested whether a fish species lacking parental care (zebrafish, Danio rerio) expresses male size-dependent differential allocation in monogamous spawning trials. In addition, we tested whether reproductive allocation by females is affected by previous experience of different-quality males, potentially indicating plasticity in mate choice. To that end, females were conditioned to large, small or random-sized males (controls) for 14 days to manipulate females'' expectations of the future mate quality. Females showed a clear preference for large males in terms of spawning probability and clutch size independent of the conditioning treatment. However, when females experienced variation in male size (random-sized conditioning treatment) they discriminated less against small males compared to females conditioned to large and small males. This might suggest that differential allocation and size-dependent sexual selection is of less relevance in nature than revealed in the present laboratory study.     

Vasopressin analog delays extinction of classically conditioned bradycardia

Albino rabbits were subjected to Pavlovian (classical) conditioning and extinction of concomitant heart rate and eyeblink responses. Sixty minutes before each of three extinction sessions animals were treated with 5 or 20 μg/kg of deamino-dicarba-arginine-8-vasopressin or saline. Vasopressin treatment delayed extinction of bradycardiac conditioned responses but did not affect concomitant eyeblink conditioned responses. It was concluded that classically conditioned autonomic responses may be useful tools for studying the effects of peptides on learning.     

Chronic intracerebroventricular infusion of the antiopioid peptide, Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (NPFF), downregulates mu opioid binding sites in rat brain

Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (NPFF), an endogenous mammalian antiopioid peptide, has been shown by other laboratories to attenuate the acute antinociceptive effects of morphine, the development of morphine tolerance, and naloxone-induced withdrawal in morphine-dependent rats. The present study determined the effect of chronic NPFF on mu opioid receptors and mRNA for the endogenous opioids dynorphin and enkephalin. Rats received ICV infusions of either saline or NPFF (5 μg/h) for 13 days via Alzet 2002 osmotic minipumps. Homogenate binding studies, which used whole brain membranes, demonstrated that NPFF decreased the B_max of mu binding sites (labeled by [³H][ -Ala²-MePhe⁴,Gly-ol⁵]enkephalin) from 262 ± 12 to 192 ± 12 fmolmg protein, and increased the K_d from 1.1 to 2.3 nM. Quantitative receptor autoradiography and in situ hybridization experiments were conducted with sections collected at the level of the striatum. The density of mu opioid binding sites labeled by [³H][ -Ala²-MePhe⁴,Gly-ol⁵]enkephalin was decreased in all brain areas measured except the corpus callosum, and there was no change in dynorphin mRNA or enkephalin mRNA in the caudate, the nucleus accumbens, or the ventral pallidum. Rats chronically administered ICV morphine sulfate (20 μg/h) for 14 days developed tolerance to morphine and a low degree of dependence, as measured by naloxone-precipitated withdrawal. Chronic administration of NPFF concurrently with morphine sulfate did not significantly alter naloxone-induced withdrawal signs or the development of morphine tolerance. Viewed collectively with previous findings that chronic ICV infusion of anti-NPFF IgG upregulates mu receptors, these data provide additional evidence that the density of CNS mu receptors is tonically regulated by NPFF in the extracellular fluid. The action of NPFF to decrease mu receptors is consistent with an antiopioid role for this peptide; however, the fact that NPFF (administered into the lateral ventricle) did not appreciably alter expression of morphine tolerance and dependence contrasts with previous findings and reinforces the view that this effect is most reliably seen after third ventricle administration.     

IDENTIFICATION OF THE CLASS PRYMNESIOPHYCEAE AND THE GENUS PHAEOCYSTIS WITH RIBOSOMAL RNA–TARGETED NUCLEIC ACID PROBES DETECTED BY FLOW CYTOMETRY1

Target regions specific for the class Prymnesiophyceae and the genus Phaeocystis (Har.) Lag. were identified from 18S ribosomal RNA coding regions, and two complementary probes were designed (PRYMN01 and PHAEO01). Detection of whole cells hybridized with these probes labeled with fluorescein isothiocyanate was difficult using epifluorescence microscopy because autofluorescence of the chlorophylls seriously interfered with the fluorescence of the probes. In contrast, flow cytometry proved very useful to detect and quantify the fluorescence of the hybridized cells. Hybridization conditions were optimized, especially with respect to formamide concentration. Both probes were tested on a large array of both target and nontarget strains. Positive and negative controls were also analyzed. Specificity was tested by adding a competing nonlabeled probe. Whereas probe PHAEO01 seems to have good specificity, probe PRYMN01 appeared less specific and must be used with stringent positive and negative controls.