The molecular biology of macrophage activating and deactivating cytokines and their receptors was discussed. Comparison of IFN-γ amino acid sequences of several mammalian species reveals a low conservation of amino acids. The interaction of IFN-γ with its receptor system is complicated and coherent with the species specificity of IFN-γ. Identification by PCR of an IFN-γ-like gene in the trout genome was not possible. In contrast with IFN-γ, TGF-β is very conserved in its amino acid sequence. The PCR-amplification of a TGF-β fragment from amphibian, <I>Xenopus</I>, and rainbow trout cDNA libraries was possible. Two oligonucleotide primers were used in PCRs to amplify a 360 bp fragment of trout Mhc class II β chain. Using these two oligos, this 360 bp fragment could be amplified from trout spleen cDNA library and HK leucocytes. cDNA synthesized from RNA extracted from ConA/PMA stimulated HK leucocytes was used as template DNA in PCR, and a class II specific fragment was amplified. This class II fragment could not be amplified from HK macrophages treated with a MAF containing supernatant, although HK macrophages treated with a control supernatant did express class II molecules. This could suggest that priming or activation of trout macrophages results in a decreased expression of Mhc class II antigens. A novel method for analysing 5'nucleotidase activity of head kidney macrophages was optimised for use with cell monolayers, with respect to the effect of cell numbers, temperature and substrate concentration. Both lysed and whole cells could be used for determination of 5'nucleotidase activity. Maximal 5'nucleotidase activity was found in the range of 27°C to 33°C and using a substrate concentration of ≥ 1 μmol AMP ml^-1 for whole cells and ≥ 1.5 μmol AMP ml^-1 for lysed cells. 5'nucleotidase activity was also correlated with respiratory burst activity in cells treated with a variety of supernatants containing MAF activity. A significant inverse relationship between these two activities was found. MAF-treated cells were also found to lose 5'nucleotidase activity faster than control cells in the presence of cycloheximide, suggesting such cells may have a higher membrane turnover of this enzyme.

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: John J.G. Tesmer. Vita. Includes bibliographical references. Also available from UMI.

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 197-201). Also available on the World Wide Web.

Universiẗat, Diss., 2005--Giessen.

Universiẗat, Diss., 2005--Giessen.

The purpose of this study was to determine the effects of PAF treatment of rabbit spermatozoa on in vitro fertilization and subsequent blastocyst formation. Rabbit spermatozoa were exposed to PAF (10-7 M ), lyso-PAF (10-7 M ), or HIS (385 mOsm/kg) for 15 min prior to insemination of ovulated oocytes. Fertilized oocytes were cultured to the hatched blastocyst stage.

The pedunculopontine nucleus (PPN), the cholinergic arm of the reticular activating system (RAS), is known to modulate waking and rapid eye movement (REM) sleep. REM sleep decreases between 10 and 30 days postnatally in the rat, with the majority occurring between 12 and 21 days. We investigated the possibility that changes in the cholinergic, muscarinic and/or nicotinic, input to PPN neurons could explain at least part of the developmental decrease in REM sleep. We recorded intracellularly from PPN neurons in 12-21 day rat brainstem slices maintained in artificial cerebrospinal fluid (aCSF) and found that application of the nicotinic agonist 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) depolarized PPN neurons early in development, then hyperpolarized PPN neurons by day 21. Most of the effects of DMPP persisted following application of the sodium channel blocker tetrodotoxin (TTX), and in the presence of glutamatergic, serotonergic, noradrenergic and GABAergic antagonists, but were blocked by the nicotinic antagonist mecamylamine (MEC). The mixed muscarinic agonist carbachol (CAR) hyperpolarized all type II (A current) PPN cells and depolarized all type I (low threshold spike-LTS current) and type III (A + LTS current) PPN cells, but did not change effects during the period known for the developmental decrease in REM sleep. The effects of CAR persisted in the presence of TTX but were mostly blocked by the muscarinic antagonist atropine (ATR), and the remainder by MEC. We conclude that, while the nicotinic inputs to the PPN may help modulate the developmental decrease in REM sleep, the muscarinic inputs appear to modulate different types of cells differentially.

Rapid eye movement sleep decreases between 10 and 30 days postnatally in the rat. The pedunculopontine nucleus is known to modulate waking and rapid eye movement sleep, and pedunculopontine nucleus neurons are thought to be hyperpolarized by noradrenergic input from the locus coeruleus. The goal of the study was to investigate the possibility that a change in α-2 adrenergic inhibition of pedunculopontine nucleus cells during this period could explain at least part of the developmental decrease in rapid eye movement sleep. We, therefore, recorded intracellularly in 12-21 day rat brainstem slices maintained in oxygenated artificial cerebrospinal fluid. Putative cholinergic vs. non-cholinergic pedunculopontine nucleus neurons were identified using nicotinamide adenine dinucleotide phosphate diaphorase histochemistry and intracellular injection of neurobiotin (Texas Red immunocytochemistry). Pedunculopontine nucleus neurons also were identified by intrinsic membrane properties, type I (low threshold spike), type II (A) and type III (A+low threshold spike), as previously described. Clonidine (20 μM) hyperpolarized most cholinergic and non-cholinergic pedunculopontine nucleus cells. This hyperpolarization decreased significantly in amplitude (mean±S.E.) from -6.8±1.0 mV at 12-13 days, to -3.0±0.7 mV at 20-21 days. However, much of these early effects (12-15 days) were indirect such that direct effects (tested following sodium channel blockade with tetrodotoxin (0.3 μM)) resulted in hyperpolarization averaging -3.4±0.5 mV, similar to that evident at 16-21 days. Non-cholinergic cells were less hyperpolarized than cholinergic cells at 12-13 days (-1.6±0.3 mV), but equally hyperpolarized at 20-21 days (-3.3±1.3 mV). In those cells tested, hyperpolarization was blocked by yohimbine, an α-2 adrenergic receptor antagonist (1.5 μM). These results suggest that the α-2 adrenergic receptor on cholinergic pedunculopontine nucleus neurons activated by clonidine may play only a modest role, if any, in the developmental decrease in rapid eye movement sleep. Clonidine blocked or reduced the hyperpolarization-activated inward cation conductance, so that its effects on the firing rate of a specific population of pedunculopontine nucleus neurons could be significant. In conclusion, the α-2 adrenergic input to pedunculopontine nucleus neurons appears to consistently modulate the firing rate of cholinergic and non-cholinergic pedunculopontine nucleus neurons, with important effects on the regulation of sleep-wake states.

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