The Interplay Between KSHV-encoded Viral Cyclin and CDK-inhibitors p27Kip1 and p21Cip1 -implications for Viral Lymphomagenesis

Cell division, which leads to the birth of two daughter cells, is essential for the growth and development of all organisms. The reproduction occurs in a series of events separated in time, designated as the cell cycle. The cell cycle progression is controlled by the activity of cyclin-dependent kinases (CDK). CDKs pair with cyclins to become catalytically active and phosphorylate a broad range of substrates required for cell cycle progression. In addition to cyclins, CDKs are regulated by inhibitory and activating phosphorylation events, binding to CDK-inhibitory proteins (CKI), and also by subcellular localization. The control of the CDK activity is crucial in preventing unscheduled progression of the cell cycle with mistakes having potentially hazardous consequences, such as uncontrolled proliferation of the cells, a hallmark of cancer. The mammalian cell cycle is a target of several DNA tumor viruses that can deregulate the host s cell cycle with their viral oncoproteins. A human herpesvirus called Kaposi s sarcoma herpesvirus (KSHV) is implicated in the cause of Kaposi s sarcoma (KS) and lymphoproliferative diseases such as primary effusion lymphomas (PEL). KSHV has pirated several cell cycle regulatory genes that it uses to manipulate its host cell and to induce proliferation. Among these gene products is a cellular cyclin D homologue, called viral cyclin (v-cyclin) that can activate cellular CDKs leading to the phosphorylation of multiple target proteins. Intriguingly, PELs that are naturally infected with KSHV consistently express high levels of CDK inhibitor protein p27Kip1 and still proliferate actively. The aim of this study was to investigate v-cyclin complexes and their activity in PELs, and search for an explanation why CKIs, such as p27Kip1 and p21Cip1 are unable to inhibit cell proliferation in this type of lymphoma. In this study, we found that v-cyclin binds to p27Kip1 in PELs, and confirmed this novel interaction also in the overexpression models. We observed that p27Kip1 associated with v-cyclin was also phosphorylated by a v-cyclin-associated kinase and identified cellular CDK6 as the major kinase partner of v-cyclin responsible for this phosphorylation. Analysis of the p27Kip1 residues targeted by v-cyclin-CDK6 revealed that serine 10 (S10) is the major phosphorylation site during the latent phase of the KSHV replication cycle. This phosphorylation led to the relocalization of p27Kip1 to the cytoplasm, where it is unable to inhibit nuclear cyclin-CDK complexes. In the lytic phase of the viral replication cycle, the preferred phosphorylation site on p27Kip1 by v-cyclin-CDK6 changed to threonine 187 (T187). T187 phosphorylation has been shown to lead to ubiquitin-mediated degradation of p27Kip1 and downregulation of p27Kip1 was also observed here. v-cyclin was detected also in complex with p21Cip1, both in overexpression models and in PELs. Phosphorylation of p21Cip1 on serine 130 (S130) site by v-cyclin-CDK6 functionally inactivated p21Cip1 and led to the circumvention of G1 arrest induced by p21Cip1. Moreover, p21Cip1 phosphorylated by v-cyclin-associated kinase showed reduced binding to CDK2, which provides a plausible explanation why p21Cip1 is unable to inhibit cell cycle progression upon v-cyclin expression. Our findings clarify the mechanisms on how v-cyclin evades the inhibition of cell cycle inhibitors and suggests an explanation to the uncontrolled proliferation of KSHV-infected cells.

Transcriptional regulation by the orphan nuclear receptor ERRgamma

The nuclear receptor (NR) superfamily is comprised of receptors for small lipopfilic ligands such as steroid hormones, thyroid hormone, retinoids, and vitamin D. NRs are ligand-inducible transcription factors capable of both activating and repressing their target gene expression. They control a wide range of biological functions connected to growth, development, and homeostasis. In addition to the ligand-regulated receptors, the family includes a large group of receptors whose physiological ligands are unknown. These receptors are referred to as orphan NRs. Estrogen-related receptor gamma (ERRgamma) belongs to the ERR subfamily of orphan NRs together with the related ERRalpha and ERRbeta. ERRs share amino acid sequence homology with the classical estrogen receptors (ERs) but they are unable to bind natural estrogenic ligands. ERRgamma is expressed in several embryonic and adult tissues but its biological role is still largely unknown. ERRgamma activates reporter gene expression in transfected cells independently of added hormones implying that ERRgamma harbors constitutive activity. However, the intrinsic activity of ERRgamma can be inhibited by synthetic compounds such as the selective estrogen receptor modulator 4-hydroxytamoxifen (4-OHT). Ligands of NRs can act as agonists that activate transcription, as antagonists that prevent activation of transcription, or as inverse agonists that antagonize the constitutive transcriptional activity of receptor. Most of the synthetic ERRgamma ligands act as inverse agonists but recently, a synthetic ERRgamma agonist GSK4716 was identified. This demonstrates that it is possible to design and identify agonists for ERRgamma. Prior to this thesis work, the structural and functional characteristics of ERRgamma were largely unknown. The aim of this study was to define the functional requirements for ERRgamma-mediated transcriptional regulation and to examine the cross-talk between ERRgamma and other NRs. Due to the fact that natural physiological ligands of ERRgamma are unknown, another aim of this study was to seek new natural compounds that may affect transcriptional activity of ERRgamma. Plant-derived phytoestrogens have previously been shown to act as ligands for ERs and ERRalpha, and therefore the effects of these compounds were also studied on ERRgamma-mediated transcriptional regulation. This work demonstrated that ERRgamma-mediated transcriptional regulation was dependent on DNA-binding, dimerization and activation function-2. Heterodimerization with ERRalpha inhibited the transcriptional activity of ERRgamma. In addition to 4-OHT, another anti-estrogen, 4-hydroxytoremifene (4-OHtor), was identified as an inverse agonist of ERRgamma. Interestingly, ERRgamma activated transcription in the presence of 4-OHT and 4-OHtor on activator protein-1 binding sites. ERRgamma was found to interact with another orphan NR Nurr1 by repressing the ability of Nurr1 to activate transcription of the osteopontin gene. Transcriptional activity of ERRgamma was shown to be stimulated by the phytoestrogen equol. Structural model analysis and mutational experiments indicated that equol was able to bind to the ligand binding domain of ERRgamma. The growth inhibitory effect of ERRgamma on prostate cancer cells was found to be enhanced by equol. In summary, this study demonstrates that despite the absence of an endogenous physiological ligand, the activity of ERRgamma can be modulated in other ways such as dimerization with related receptors or by cross-talk with other transcription factors as well as by binding some synthetic or natural compounds.

Nuclear magnetic resonance spectra of oriented bicyclic systems containing heteroatom(s):the spectrum of 2-thiocoumarin

From the proton nmr studies of 2-thiocoumarin and coumarin, it is concluded that the relative interproton distances in the two oxygen heteroatom bicyclic systems are similar. The values for the phenyl ring protons do not deviate significantly from the regular hexagonal geometry, unlike bicyclic systems with nitrogens as the heteroatoms, such as diazanaphthalenes. Larger values of the indirect spin-spin couplings within the protons of the ring containing the oxygen heteroatom, compared to the values between the ortho protons in the phenyl rings in coumarin and 2-thiocoumarin, correspond to the olefinic nature of these protons. This is in contrast to results for the nitrogen heterocycles where both the rings are aromatic.

Photoacoustic investigation of phase transitions in solids

The use of the photoacoustic effect in the investigation of first- and second-order phase transitions has been examined. Changes in the amplitude of the photoacoustic signal across the phase transition are compared with changes in thermal properties such as specific heat and thermal diffusivity. The systemsstudied include NaN02, TlN03, CsN03, NH4N03, BaTiO,, COO, Cu,HgI,, V02 andV305. The current photoacoustic studies are discussed in the light of the theoretical models available.

Electrochemical phase formation (ECPF) and macrogrowth Part I. Hemispherical models

The phenomenological theory of hemispherical growth is generalised to time-dependent nucleation and growth-rates. Special cases, which include models with diffusion-controlled rates, are analysed. Expressions are obtained for small and large time behaviour and peak characteristics of potentiostatic transients, and their use in model parameter estimation is discussed. Two earlier equations are corrected. Numerically calculated transients which are presented exhibit some interesting features such as a maximum preceding the steady state, oscillations and shoulder.

Electrochemical phase formation (ECPF) and macrogrowth Part II. Two-rate models

A general theory is evolved for a class of macrogrowth models which possess two independent growth-rates. Relations connecting growth-rates to growth geometry are established and some new growth forms are shown to result for models with passivation or diffusion-controlled rates. The corresponding potentiostatic responses, their small and large time behaviours and peak characteristics are obtained. Numerical transients are also presented. An empirical equation is derived as a special case and an earlier equation is corrected. An interesting stochastic result pertaining to nucleation events in the successive layers is proved.

Enhancement of organic phase controlled extraction of copper by activated carbon

The addition of activated carbon particles (Darco-G, average size 4.3,μm) is shown to enhance the initial rate of extraction of copper in a Lewis cell by a mixture of α- and β-hydroxyoximes, when the rate of extraction is controlled by resistances in the organic phase. It is likely that the copper complex is adsorbed by carbon near the interace and partially released in the bulk. The enhancing effect of carbon vanishes when toluene is used as a diluent instead of heptane, presumably because toluene preferentially adsorbs on its surface.

Orphan nuclear receptor subfamily NR4A their interplay with other nuclear receptors and functions in osteoblasts

Nurr1, NGFI-B and Nor1 (NR4A2, NR4A1 and NR4A3, respectively) belong to the NR4A subfamily of nuclear receptors. The NR4A receptors are orphan nuclear receptors which means that activating or repressing ligands for these receptors have not been found. NR4A expression is rapidly induced in response to various stimuli including growth factors and the parathyroid hormone (PTH). The studies concerning the NR4A receptors in the central nervous system have demonstrated that they have a major role in the development and function of the dopaminergic neurons of the midbrain and in regulating hypothalamus-pituitary-adrenal-axis. However, the peripheral functions of the NR4A family are largely unknown. Cultured mouse primary osteoblasts, a preosteoblastic cell line and several osteoblastic cell lines were used to investigate the role of NR4A receptors in osteoblasts. NR4A receptors were shown to directly bind to and activate the promoter of the osteopontin gene (OPN) in osteoblastic cells, thus regulating its expression. OPN is a major bone matrix protein expressed throughout the differentiation of preosteoblastic cells into osteoblasts. The activation of the OPN promoter was shown to be dependent on the activation function-1 located in the N-terminal part of Nurr1 and to occur in both monomeric and RXR heterodimeric forms of NR4A receptors. Furthermore, PTH was shown to upregulate OPN expression through the NR4A family. It was also demonstrated that the fibroblast growth factor-8b (FGF-8b) induces the expression of NR4A receptors in osteoblasts as immediate early genes. This induction involved phosphatidylinositol-3 kinase, protein kinase C, and mitogen activated protein kinase, which are all major pathways of FGF signalling. Nurr1 and NGFI-B were shown to induce the proliferation of preosteoblastic cells and to reduce their apoptosis. FGF-8b was shown to stimulate the proliferation of osteoblastic cells through the NR4A receptors. These results suggest that NR4A receptors have a role both in the differentiation of osteoblasts and in the proliferation and apoptosis of preosteoblast. The NR4A receptors were found to bind to the same response element on OPN as the members of the NR3B family of orphan receptors do. Mutual repression was observed between the NR4A receptors and the NR3B receptors. This repression was shown to be dependent on the DNA-binding domains of both receptor families, but to result neither from the competition of DNA binding nor from the competition for coactivators. As the repression was dependent on the relative expression levels of the NR4As and NR3Bs, it seems likely that the ratio of the receptors mediates their activity on their response elements. Rapid induction of the NR4As in response to various stimuli and differential expression of the NR3Bs can effectively control the gene activation by the NR4A receptors. NR4A receptors can bind DNA as monomers, and Nurr1 and NGFI-B can form permissive heterodimers with the retinoid X receptor (RXR). Permissive heterodimers can be activated with RXR agonists, unlike non-permissive heterodimers, which are formed by RXR and retinoic acid receptor or thyroid hormone receptor (RAR and TR, respectively). Non-permissive heterodimers can only be activated by the agonists of the heterodimerizing partner. The mechanisms behind differential response to RXR agonists have remained unresolved. As there are no activating or repressing ligands for the NR4A receptors, it would be important to find out, how they are regulated. Permissiviness of Nurr1/RXR heterodimers was linked to the N-terminal part of Nurr1 ligand-binding domain. This region has previously been shown to mediate the interaction between NRs and corepressors. Non-permissive RAR and TR, permissive Nurr1 and NGFI-B, and RXR were overexpressed with corepressors silencing mediator for retinoic acid and thyroid hormone receptors (SMRT), and with nuclear receptor corepressor in several cell lines. Nurr1 and NGFI-B were found to be repressed by SMRT. The interaction of RXR heterodimers with corepressors was weak in permissive heterodimers and much stronger in non-permissive heterodimers. Non-permissive heterodimers also released corepressors only in response to the agonist of the heterodimeric partner of RXR. In the permissive Nurr1/RXR heterodimer, however, SMRT was released following the treatment with RXR agonists. Corepressor release in response to ligands was found to differentiate permissive heterodimers from non-permissive ones. Corepressors were thus connected to the regulation of NR4A functions. In summary, the studies presented here linked the NR4A family of orphan nuclear receptors to the regulation of osteoblasts. Nurr1 and NGFI-B were found to control the proliferation and apoptosis of preosteoblasts. The studies also demonstrated that cross-talk with the NR3B receptors controls the activity of these orphan receptors. The results clarified the mechanism of permissiviness of RXR-heterodimers. New information was obtained on the regulation and functions of NR4A receptors, for which the ligands are unknown.

A novel investigation of vapor-phase charge-transfer complexes of halogens with n-donors by electron energy loss spectroscopy

Complexes of I2 with diethyl ether and triethylamine and of Br, with diethyl ether have been investigated in the vapor phase for the first time by employing electron energy loss spectroscopy. Besides the CT bands, blue-shifted vacuum-UV bands of the halogens have been assigned; the amine-I, system appears to exhibit two CT bands,associated with two different excited states of the complex.

Electron-Energy Loss Spectroscopy (Eels) Of Organic-Molecules In Vapor-Phase - Design And Fabrication Of An Indigenous Eel Spectrometer

An indigenous electron energy loss spectrometer has been designed and fabricated for the study of free molecules. The spectrometer enables the recording of low-resolution electronic spectra of molecules inthe vapour phase with ready access to the vacuum ultraviolet region. Electron energy loss spectra of aliphatic alcohols and carbonyl compounds as wellas of benzene derivatives have been recorded with the indigenous spectrometer and the electronic transitions in these molecules discussed.

VG10024 Consumer insights into vegetable consumption phase two

To develop the psychological concepts identified in Horticulture Australia Limited (HAL) study VG08000 into a survey instrument. To conduct a quantitative study that provides a psychological understanding of consumer segments.

In Situ Phase Separation Following Dehydration in Bimetallic Sulfates: A Variable-Temperature X-Ray Diffraction Study

Phase separation resulting in a single-crystal-single-crystal transition accompanied by a polycrystalline phase following the dehydration of hydrated bimetallic sulfates [Na2Mn1.167(SO4)(2)S0.33O1.167 center dot 2H(2)O and K4Cd3-(SO4)(5)center dot 3H(2)O] has been investigated by in situ variable-temperature single-crystal X-ray diffraction. With two examples, we illustrate the possibility of generating structural frameworks following dehydration in bimetallic sulfates, which refer to the possible precursor phases at that temperature leading to the mineral formation. The room-temperature structure of Na2Mn1.167(SO4)(2)S0.33O1.167 center dot 2H(2)O is trigonal, space group R (3) over bar. On heating the crystal in situ on the diffractometer, the diffraction images display spherical spots and concentric rings suggesting phase separation, with the spherical spots getting indexed in a monoclinic space group, C2/c. The structure determination based on this data suggests the formation of Na2Mn(SO4)(2). However, the diffraction images from concentric rings could not be indexed. In the second example, the room-temperature structure is determined to be K4Cd3(SO4)(5)center dot 3H(2)O, crystallizing in a monoclinic space group, P2(1)/n. On heating the crystal in situ, the diffraction images collected also have both spherical spots and diffuse rings. The spherical spots could be indexed to a cubic crystal system, space group P2(1)3, and the structure is K4Cd3(SO4)(3). The possible mechanism for the phase transition in the dehydration regime resulting in this remarkable single-crystal to single-crystal transition with the appearance of a surrogate polycrystalline phase is proposed.

Sustainable Grazing in the Channel Country Floodplains (phase 2) : A technical report on findings between March 2003 and June 2006

‘Sustainable Grazing in the Channel Country Floodplains’ was initiated by industry to redress the lack of objective information for sustainable management in the floodplains of Cooper Creek and the Diamantina and Georgina Rivers. The project has maintained links with the grazing community and has extensively drawn upon expert local experience and knowledge. The project has provided tools for managers to better anticipate the size of beneficial flooding arising from rains in the upper catchment and to more objectively assess the value of the pasture resulting from flooding. The latest information from the project has enabled customisation of the EDGENetwork™ Grazing Land Management training package for the Channel Country. In combination, these tools will assist in making earlier cattle stocking decisions, including when cattle may need to be mustered out of floodplain paddocks, how many additional cattle will be required to take advantage of the flood–grown pasture, and the timing of cattle turnoff. These will reduce costs by providing a greater lead time to plan cattle movements and purchases, and may enhance the sustainability of the resource base by better matching cattle numbers with the feed on offer.