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S100B is a member of the S100 calcium binding protein family and is highly expressed within astrocytes in the brain. Elevated levels of S100B are associated with brain and central nervous system disorders, due to the breakdown of the blood brain barrier. Therefore S100B is routinely used as a marker of disease. Traditionally S100B was thought only as a cell breakdown product but increasing evidence suggests that it may play a role in exacerbating inflammation, however this role is not clear. S100B is known to be present within the eye but its role in retinal inflammation has not been investigated. The aim of this project was therefore to examine the role of S100B using the animal model experimental autoimmune uveoretinitis (EAU). This is a well-established model for the sight-threatening human condition posterior endogenous uveoretinitis. In this disease model an autoimmune response is induced leading to retinal inflammation. Using S100B knockout mice, I have shown a significantly reduced level of disease, as determined by clinical and histological grading. Real-time PCR array analysis of diseased matched retinas indicated down regulation of cytokines and chemokines in S100B knockout mice. In vitro experiments on a macrophage cell line confirmed S100B to have a pro-inflammatory effect on macrophages, the main effector cell in EAU, with up-regulation of cytokine and chemokine expression. In particular IL-1β, CCR1 and CCL22 showed a marked increase in gene expression in response to S100B which was confirmed by real-time PCR. Increased protein production of IL-1β (pro-form), CCR1 and CCL22 was also confirmed. S100B inhibited activation of T cells separated from spleens, as shown by reduced CD25+ expression and IL-2 production. IFN-γ and IL-17 production however was not affected. CCL2 and IL-6 are main inflammatory mediators produced by retinal pigment epithelial cells which are known to be elevated during retinal inflammation. S100B promoted CCL2 and IL-6 production in retinal pigment epithelial cells at different concentrations. The work carried out in this thesis provides additional understanding of the actions of extracellular S100B on immune system cells and its potential role in posterior uveitis.

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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Every aerobic organism expresses cytochrome c oxidase to catalyze reduction of molecular oxygen to water, and takes advantage of this energy releasing reaction to produce an electrochemical gradient used in cellular energy production. The protein SCO (Synthesis of cytochrome c oxidase) is a required assembly factor for the oxidase, conserved across many species. SCO is implicated in the assembly of one of two copper centres (ie., CuA) of cytochrome oxidase. The exact mechanism of SCO’s participation in CuA assembly is not known. SCO has been proposed to bind and deliver copper, or alternatively to act in reductive preparation of the CuA site within the oxidase. In this body of work, the strength and stability of Cu(II) binding to Bacillus subtilis SCO is explored via electronic absorption and fluorescence spectroscopies and by calorimetric methods. An equilibrium dissociation constant (Kd) of 3.5x10-12 M was determined as an upper limit for the BsSCO-Cu(II) interaction, via differential scanning calorimetry. In the first reported case for a SCO homolog, dissociation kinetics of Cu(II) from BsSCO were characterized, and found to be dependent on both ionic strength and the presence of free Cu(II) in solution. Further differential scanning calorimetry experiments performed at high ionic strength support a two-step model of BsSCO and Cu(II) binding. The implications of this model for the BsSCO-Cu(II) interaction are presented in relation to the mechanism of interaction between SCO and the CuA site of cytochrome c oxidase. / Thesis (Master, Biochemistry) -- Queen's University, 2007-09-21 16:00:23.621

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