No description available.

Calcium/phospholipid-dependent protein kinase (PKC) was partially purified from P1798 lymphosarcoma. Phospholipid-dependence was specific for phosphatidylserine. PKC phosphorylated Histone 1, with an apparent K_m of 14.1 μM. Chlorpromazine, a lipid-binding drug, inhibited PKC activity by 100%. Further studies were undertaken to establish analytical conditions which could be applied to the study of PKC in intact cells. The conditions included (1) determining optimum cell concentration for measuring PKC activity, (2) recovering PKC into the soluble fraction of cell extracts, (3) evaluating calcium and phospholipid requirements of PKC in this fraction, and (4) inhibiting PKC in this fraction. Final studies involved treatment of intact cells with potential activators. Both phytohaemagglutinin and a phorbol ester increased PKC activation.

No description available.

No description available.

Viral RNA dependent RNA polymerases (RdRps) are enzymes which enable RNA viruses to replicate their genome and to prepare mRNA for translation of viral proteins. Due to its relative evolutionary conservation RdRps are good targets for drug design. In this work we present a structure of the RdRp (3Dpol ) of Aichi virus, which has not been solved yet. Aichi virus is a human pathogen that causes gastroenteritis. Aichi virus is also used as a model organism for studying cognate viruses which virulence is more dangerous, for example: Rhinovirus, Hepatitis A virus, SARS virus, hepatitis C virus, yellow fever, and West-Nile virus. In addition to structural studies of Aichi virus 3Dpol we also tested a previously published hypothesis that, 3Dpol is recruited to the membrane through phosphatidylinositol 4 phosphate (PI4P) - an important regulatory lipid. Membranes highly enriched in PI4P are formed in cells infected by single stranded positive sense RNA (plus ssRNA) viruses. Finally we tested the influence of ribonucleotides on the 3Dpol protein stability. (In Czech)

A dissertation submitted in fulfilment of the requirements for the degree of Master of Science at the University of the Witwatersrand. Johannesburg February 1994 / Glutathione S-transferases are multifunctional intracellular proteins. They catalyse the conjugation of glutathione to endogenous'or foreign electrophiles, and also bind non-substrate ligands. Class Pi glutathione S-transferase (pGSTPl~l) was purified from porcine lung to a specific. activity of 6.63p.ffiol/min/mg. The homodimeric protein has a molecular weight of about 4~.7kD and an isoelectric point of 8.6. Anionic ligand-binding properties of this isoenzyme were investigated. Steady-state fluorescence methods were used to determine ~ values for 8-anilino··l~naphtha1enesulphonic acid (K, == 17.1p.M and 11.1J.tM using fluorescence enhancement techniques and quenching techniques respectively), bromosulphophtbalein (Kcl=1.1p.M at pH 6.5 and 2.4/jM at pH 7.5) and glutathione {~=1201I.M). The affinity of bromosulphophthalein for the enzyme, in the presence of 10mM glutathione was slightly enhanced (~=O.7.uM at pH 6.5). The energy transfer betwecz the protein's tryptophan residues and 8-anUino-l-naphthalene sulphonic acid was observed and found to be about 56% efficient. The impact of ligand binding on both protein structure and catalytic activity were assessed. Kinetic studies show that the active site of the enzyme is not the primary binding site for the non-substrate ligands, but that the binding of bromosulphophthalein and to a lesser extent 8~ani1ino-l-!.~phtha1ene sulphonic acid, does affect the active site of the enzyme, especially aner saturating concentrations of the ligand. This may be the result of a small ligand-induced conformational change. Fluorescence studies also indicate that the primary site for anionic ligand binding is not in close proximity to either Trp28 or Trp38 in domain I, Competition studies indicated that the two anionic ligands bind the Same site, < Prorein fluorescence, chemical modification « and size-exclusion HPLC data indicate that ligand binding does 110t induce gross conformational changes in the protein.

Thesis (M.Sc.)--University of the Witwatersrand, Faculty of Science, School of Molecular and Cell Biology, 2018. / ABSTRACT The Retinoblastoma binding protein 6 is dysregulated in most cancers, indicating it may play a role in metabolic reprograming- a hallmark of carcinogenesis. Its human isoforms have been shown to play diverse roles in apoptosis. This study aimed to elucidate biochemical roles of RBBP6 isoforms in metabolic reprogramming during carcinogenesis. Drosophila melanogaster wild type and p53 null mutants were treated with drug permutations of irinotecan (DNA damaging agent) and exogenous pyruvate to perturb metabolism. Moreover, using RT-PCR and Western blot expression profiles of SNAMA (Drosophila Orthologue of RBBP6) isoforms were shown followed by survival studies to investigate the effects of these drugs. Furthermore, using bioinformatics the domains of RBBP6 isoforms in various species were shown. Results indicate that RBBP6 isoforms show contrasting expression patterns. Furthermore, exogenous pyruvate protects the wild type flies from irinotecan toxicity while killing p53 null mutants. RBBP6 proves to be a potential druggable target for chemotherapy. / EM2018

Typescript (photocopy). / Digitized by Kansas Correctional Industries

It has become increasingly apparent that the future of electronic devices can and will rely on the functionality provided by single or few dopant atoms. The most scalable physical system for quantum technologies, i.e. sensing, communication and computation, are spins in crystal lattices. Diamond is an excellent host crystal offering long room temperature spin coherence times and there has been exceptional experimental work done with the nitrogen vacancy center in diamond demonstrating many forms of spin control. Transition metal dopants have additional advantages, large spin-orbit interaction and internal core levels, that are not present in the nitrogen vacancy center. This work explores the implications of the internal degrees of freedom associated with the core d levels using a tight-binding model and the Koster-Slater technique. The core d levels split into two separate symmetry states in tetrahedral bonding environments and result in two levels with different wavefunction spatial extents. For 4d semiconductors, e.g. GaAs, this is reproduced in the tight-binding model by adding a set of d orbitals on the location of the transition metal impurity and modifying the hopping parameters from impurity to its nearest neighbors. This model does not work in the case of 3d semiconductors, e.g. diamond, where there is no physical reason to drastically alter the hopping from 3d dopant to host and the difference in wavefunction extent is not as pronounced. In the case of iron dopants in gallium arsenide the split symmetry levels in the band gap are responsible for a decrease in tunneling current when measured with a scanning tunneling microscope due to interference between two elastic tunneling paths and comparison between wavefunction measurements and tight-binding calculations provides information regarding material parameters. In the case of transition metal dopants in diamond there is less distinction between the symmetry split d levels. When considering pairs of transition metal dopants an important quantity is the exchange interaction between the two, which is a measure of how fast a gate can be operated between the pair and how well entanglement can be created. The exchange interaction between pairs of transition metal dopants has been calculated in diamond for several directions in the (110) plane, and for select transition metal dopants in gallium arsenide. In tetrahedral semiconductors transition metal dopants provide an internal degree of freedom due to the symmetry split d levels and this included functionality makes them special candidates for single spin based quantum technologies as well as physical systems to learn about fundamental physics.

The multigene family of Ca2+-binding S100 proteins comprises 22 members that have various important intra- and extracellular roles. The three inflammation-associated members of this family???S100A8, S100A9 and S100A12 (collectively termed &quotcalgranulins&quot)???are constitutive neutrophil and monocyte proteins also expressed by macrophages within acute and chronic inflammatory lesions, but not in tissue macrophages. They are expressed in human/murine wounds and by appropriately activated macrophages, microvascular endothelial cells and keratinocytes in vitro. The &quot calgranulins&quot are implicated in leukocyte activation/deactivation, fatty acid transport, leukocyte/fibroblast chemotaxis, transmigration and adhesion, embryogenesis, wound healing, protection against oxidants and antibacterial defence. Chapter 3 of this thesis explores growth-factor- and cytokine-mediated regulation and expression of S100A8 and S100A9 in fibroblasts, and demonstrates spatio-temporal expression of S100A8 in rat dermal wounds. Fibroblasts are stromal resident cells with important regulatory immune-inflammatory functions in wound healing, tissue remodelling and fibrosis. Fibroblast migration, proliferation, differentiation and their synthetic repertoire are modulated by various factors including extracellular matrix components, growth factors, prostaglandins, reactive oxygen species and cytokines. Fibroblast growth factor-2 (FGF-2), interleukin-1?(IL-1? and platelet-derived growth factor (PDGF) are potent fibroblast mitogens; PDGF and transforming growth factor-? (TGF-? are fibroblast chemoattractants. FGF-2 and IL-1?promote fibroblast proliferation, whereas TGF-?promotes myofibroblast differentiation and collagen production. Lipopolysaccharide (LPS), interferon ?(IFN?, tumour-necrosis factor ? (TNF?, TGF-?and PDGF did not induce the S100A8 gene in fibroblasts whereas FGF-2 (25 ng/ml) maximally induced mRNA 12 hr. after stimulation and this declined over 36 hr. The FGF-2 response was strongly enhanced and prolonged by optimal levels of heparin (1-10 IU/ml), maximally at 18 hr. post-stimulation. FGF-2/heparin-induced responses depended on cell-cell contact in vitro. IL-1?(10 U/ml) alone, or in synergy with FGF-2/heparin strongly induced the gene in 3T3 and primary fibroblasts. Dexamethasone (10???6 M) enhanced LPS- and FGF-2/-IL-1?induced responses. S100A9 mRNA was not induced by any of these mediators. Induction of S100A8 in the absence of S100A9 was confirmed in primary fibroblast-like cells by real-time reverse-transcriptase polymerase chain-reaction. FGF-2-heparin- and IL-1?induced mRNA expression depended on de-novo protein synthesis and was partially mediated by the mitogenactivated protein kinase pathway of activation. Preliminary promoter deletion analyses indicated that FGF-2-responsive elements in the gene promoter were distinct from those responsive to IL-1? TGF-?(2 ng/ml) significantly suppressed gene induction mediated by FGF-2 ?heparin/LPS/dexamethasone, but not by IL-1? TGF-?may compromise mRNA stability. Protein levels in FGF-2-heparin-IL-1?stimulated fibroblasts correlated well with mRNA levels and expression was mainly cytoplasmic. Immunohistochemistry indicated S100A8 associated with keratinocytes, neutrophils, macrophage-like cells and some hair follicles in wounded rat skin. Rat wounds also contained numerous S100A8- positive fibroblast-like cells 2 and 4 days post-injury; numbers declined by 7 days. Upregulation of S100A8 by FGF-2/IL-1? down-regulation by TGF-? and time-dependent expression of S100A8 in wound fibroblasts suggest a role in fibroblast differentiation at sites of inflammation and repair. Intracellular fibroblast-derived S100A8 may also regulate intracellular redox equilibrium and antioxidant defence. Atherosclerosis is a progressive chronic disease with complex aetiology and pathogenesis. S100A1 and S100B are associated with dendritic cells and lymphocytes in experimental rodent and human atherosclerotic lesions. Monocytes and macrophages in plaques of ApoE???/??? mice express S100A9 but not S100A8. Myeloperoxidase and HOClmediated oxidative mechanisms are fundamental in the pathogenesis of atherosclerosis and S100A8 is exquisitely sensitive to HOCl oxidation which generates sulphinamide bonds, novel non-reducible cysteine-lysine covalent bonds. Chapter 4 of this thesis presents novel evidence that, in contrast to the murine ApoE???/??? model, the three human &quot calgranulins&quot were expressed in human atherosclerotic plaques, but not in normal arteries. High levels of S100A8, S100A9 and S100A12 were evident in macrophages and foam cells. Some neovessels were anti-S100A8-/anti-S100A9-immunoreactive; S100A9 staining was also evident on the extracellular matrix. Patterns of expression of S100A8, S100A9 and S100A12 were overlapping in serial sections, except that only smooth muscle cells were S100A12-positive. S100A8 and S100A9 mRNA were also expressed by macrophages, foam cells and endothelial cells, indicating gene up-regulation rather than passive protein uptake. Western blotting of plaque extracts revealed monomeric S100A8, S100A9 and S100A12 and larger complexes. Some were resistant to reduction, suggesting non-disulfide covalent cross-linking, possibly via sulphinamide bonds. Stable S100A8-S100A9 complexes were also detected after immunoaffinity purification. In an in-vitro system, molar ratios of HOCl of &gt1 generated stable complexes of S100A8 and S100A9 whereas ~800 and ~100-fold excess HOCl oxidises apolipoprotein B-100 and BSA, respectively. S100A8 and S100A9 protected low-density lipoprotein (LDL) against HOCl oxidation in a thiol-independent manner. Because HOCl-oxidised S100s did not contain epitopes recognised by an antibody used to detect HOCl-oxidised proteins in plaque, levels of oxidised proteins in plaque are likely to be significantly greater than described. S100A8 and S100A9 may protect LDL by functioning as HOCl-scavengers. However, chronic oxidative cross-linking of S100A8 and S100A9 with other proteins and extracellular matrix components may contribute to plaque pathogenesis. These studies support key roles for the &quot calgranulins&quot in chronic inflammation, wound healing and atherogenesis possibly by regulating cellular differentiation, activation and modulation of redox-dependent mechanisms.