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Typescript (photocopy). / Digitized by Kansas Correctional Industries

The binding of [³H]flunitrazepam (FLU) to membranes prepared from mammalian brain, retina and kidney was investigated by means of conventional filtration assay techniques. In the mouse brain a study of the ontogeny of [³H]FLU binding was conducted. Specific [³H]FLU binding was present early in development and there was a rapid increase in receptor density (Bmax) during the first 2 weeks of neonatal life. This increase could be described by the function, a·eᵏˣ, where a = 1.8 pmol/brain, k = 0.23 weeks⁻¹ and x = time in weeks (r = 0.98). By 3-4 weeks of age, adult levels of [³H]FLU binding were reached (∼115 fmol/mg tissue). Notable changes in the equilibrium dissociation constant (K(d)) during development were not observed. γ-Aminobutyric acid (GABA) has previously been shown to increase the affinity of [³H]FLU binding in the adult rat brain; in the present studies this effect was shown to be present throughout the development of the mouse brain. Kinetic analyses of the GABA enhancement of [³H]FLU binding indicated that the change in K(d) was due to a decrease in the rate constant of dissociation (k₋₁). [³H]FLU binding has been shown to occur in the mammalian retina and it has all the characteristics of cerebral [³H]FLU binding. Monosodium glutamate (MSG) is toxic to retinal neurons and it was used to ascertain the putative cellular localization of retinal BZD binding sites. Nine weeks following neonatal MSG administration, histologic evidence showed the virtual absence of ganglion cells and a marked reduction in the number of inner nuclear neurons in MSG retinae. A corresponding 73 percent decrease in GABA content and a 77 percent decrease in the Bmax of [³H]FLU were found in the retinae from MSG treated rats as compared to controls. There were no significant changes in [³H]FLU binding in the cerebella, cerebral cortices and hypothalami from MSG treated rats. The binding of [³H]FLU was characterized for the rat kidney. Binding was specific, saturable and of moderately high affinity (Bmax, ∼320 fmol/mg tissue; K(d), ∼11 nM). Drug specificity studies with renal membranes showed that inhibition of [³H]FLU binding by various BZD's did not correlate either with their pharmacologic potency as anxiolytic agents or with their potency as inhibitors of [³H]FLU binding in the brain. An intrarenal distribution of specific [³H]FLU binding was found in the bovine kidney; specific binding was greatest in the outer cortex and virtually absent in the medulla, the minor calyx and the renal artery. In rats made hypertensive by simultaneous deoxycorticosterone acetate and NaCl administration, there was a significant 35-43 percent increase in the Bmax of renal [³H]FLU binding. Binding in the cerebral cortex of these animals was unchanged. The inhibition of [³H]FLU binding by a triazolopyridazine (CL 218,872) was studied in membranes prepared from bovine retina, rat cerebral cortex, cerebellum and kidney. The affinity of CL 218,872 for the inhibition of [³H]FLU binding was greatest in the cerebellum, followed by the retina, cerebral cortex and kidney (60, 150, 200, and 1,800 nM, respectively). The slope factors (Hill coefficients) were ∼1 for the kidney, ∼0.9 for the cerebellum and ∼0.7 for the cerebral cortex and retina. A nonlinear least squares regression analysis of the data from the cerebral cortex, retina and cerebellum gave an excellent fit for a model containing 2 binding sites. In washed membrane preparations from the cerebral cortex, cerebellum and retina, Kᵢ values for CL 218,872 were significantly decreased an average of 60 percent in the presence of 100 μM GABA. (+)Bicuculline could antagonize this effect of GABA. GABA had no effect upon the Kᵢ of CL 218,872 in renal membranes.

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The centromere binding factor 1, Cbf1, of Saccharomyces cerevisiae is a bHLH/ZIP protein which has been described as a determinant of specific chromatin structures and as a tethering factor for activators of transcription at the promoters of genes of the Methionine Biosynthesis Pathway. Deletion mutants show various phenotypes, among them methionine auxotrophy, an increased rate of chromosome loss, modifications in the growth rate and modification of the chromatin structure at MET genes. Meiosis competence also becomes greatly reduced in cbf1 cells. The sequence motif (RTCACRTG) to which Cbf1p binds is found at multiple loci through the yeast genome. This thesis shows that the chromatin structure is reorganised at multiple Cbf1p binding sites in vivo, when yeast cells are starved to enter meiosis. Extensive remodelling occurs at the MET16, MET17(25), DRS2 and GDH3 loci and at the YAL060W open reading frame, as detected by in vivo digestion of chromatin with micrococcal nuclease and indirect end-labelling. The same kind of analysis showed that the remodelling of chromatin at Cbf1p binding sites is not specific for meiosis, it occurs also in similarly starved haploid cells. The lack of methionine is a key trigger of these changes. This reorganisation of chromatin is dependent on Cbf1p, since starved cbf1 cells do not display any modification in nuclease accessibility patterns at or around Cbf1p binding sites. Mutational analysis revealed that a negative charge at a putative phosphorylation site (serine residue 226) and the DNA-bindmg activity of Cbf1p are both required for the chromatin reorganisation to occur in response to starvation. CBF1 mutants which do not reorganise chromatin were also shown to be unable to enter meiosis, suggesting that the remodelling of chromatin at multiple Cbf1p binding sites may be required to enter pre-meiotic DNA replication, since such cells arrest before the initiation of this process. In summary, the results presented in this thesis are compatible with a model in which Cbf1p plays an active role as part of a mechanism sensing the nutrient availability and regulates the reorganisation of chromatin, at multiple loci through the yeast genome, in response to starvation conditions.

Thesis (Ph. D.)--University of Iowa, 2008. / Thesis supervisor: Sonya J. Franklin. Includes bibliographical references (leaves 129-134).

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