Induction of neurites by the regulatory domains of PKCdelta and epsilon is counteracted by PKC catalytic activity and by the RhoA pathway

We have shown that protein kinase C (PKC) epsilon, independently of its kinase activity, via its regulatory domain (RD), induces neurites in neuroblastoma cells. This study was designed to evaluate whether the same effect is obtained in nonmalignant neural cells and to dissect mechanisms mediating the effect. Overexpression of PKCepsilon resulted in neurite induction in two immortalised neural cell lines (HiB5 and RN33B). Phorbol ester potentiated neurite outgrowth from PKCepsilon-overexpressing cells and led to neurite induction in cells overexpressing PKCdelta. The effects were potentiated by blocking the PKC catalytic activity with GF109203X. Furthermore, kinase-inactive PKCdelta induced more neurites than the wild-type isoform. The isolated regulatory domains of novel PKC isoforms also induced neurites. Experiments with PKCdelta-overexpressing HiB5 cells demonstrated that phorbol ester, even in the presence of a PKC inhibitor, led to a decrease in stress fibres, indicating an inactivation of RhoA. Active RhoA blocked PKC-induced neurite outgrowth, and inhibition of the RhoA effector ROCK led to neurite outgrowth. This demonstrates that neurite induction by the regulatory domain of PKCdelta can be counteracted by PKCdelta kinase activity, that PKC-induced neurite outgrowth is accompanied by stress fibre dismantling indicating an inactivation of RhoA, and that the RhoA pathway suppresses PKC-mediated neurite outgrowth.     

Dynamics of transgene expression in a neural stem cell line transduced with lentiviral vectors incorporating the cHS4 insulator

Transplantation of genetically manipulated cells to the central nervous system holds great promise for the treatment of several severe neurological disorders. The success of this strategy relies on sufficient levels of transgene expression after transplantation. This has been difficult to achieve, however, due to transgene silencing. In this study, we transduced the neural stem cell line RN33B with self-inactivating lentiviral vectors and analyzed transgenic expression of green fluorescent protein (GFP) in several different settings both in vitro and after transplantation to the brain. We found that the transgene was affected of silencing both when transduced cells were proliferating and after differentiation. To prevent silencing, the cHS4 insulator was incorporated into the lentiviral vector. We found that a vector carrying the cHS4 insulator was partially protected against differentiation-dependent downregulation in vitro and in vivo. However, in proliferating cells, we found evidence for variegation and positional effects that were not prevented by the cHS4 insulator, suggesting that the mechanism behind silencing in proliferating cells is not the same mechanism influencing differentiation-dependent silencing. Taken together, these findings favor vector optimization as a strategy for achieving efficient ex vivo gene transfer in the central nervous system.     

Chromatin from two classes of platyhelminthes display both protist H1 and higher eukaryote core histones

Histones from the parasitic platyhelminthes, Echinococcus granulosus and Fasciola hepatica, were systematically characterized. Core histones H2A, H2B, H3 and H4, which were identified on the basis of amino acid sequencing and mass spectrometry data, showed conserved electrophoretic patterns. Histones H1, identified on the basis of physicochemical properties, amino acid composition and amino acid sequencing, showed divergence, both in their number and electrophoretic mobilities, between the two species and among other organisms. According to these data, core histones but not H1 histones, would be stabilized during evolution at the level of platyhelminthes.     

Alpha-synuclein up-regulation and aggregation during MPP+-induced apoptosis in neuroblastoma cells: intermediacy of transferrin receptor iron and hydrogen peroxide

1-Methyl-4-phenylpyridinium (MPP(+)) is a neurotoxin that causes Parkinson's disease in experimental animals and humans. Despite the fact that intracellular iron was shown to be crucial for MPP(+)-induced apoptotic cell death, the molecular mechanisms for the iron requirement remain unclear. We investigated the role of transferrin receptor (TfR) and iron in modulating the expression of alpha-synuclein (alpha-syn) in MPP(+)-induced oxidative stress and apoptosis. Results show that MPP(+) inhibits mitochondrial complex-1 and aconitase activities leading to enhanced H(2)O(2) generation, TfR expression and alpha-syn expression/aggregation. Pretreatment with cell-permeable iron chelators, TfR antibody (that inhibits TfR-mediated iron uptake), or transfection with glutathione peroxidase (GPx1) enzyme inhibits intracellular oxidant generation, alpha-syn expression/aggregation, and apoptotic signaling as measured by caspase-3 activation. Cells overexpressing alpha-syn exacerbated MPP(+) toxicity, whereas antisense alpha-syn treatment totally abrogated MPP(+)-induced apoptosis in neuroblastoma cells without affecting oxidant generation. The increased cytotoxic effects of alpha-syn in MPP(+)-treated cells were attributed to inhibition of mitogen-activated protein kinase and proteasomal function. We conclude that MPP(+)-induced iron signaling is responsible for intracellular oxidant generation, alpha-syn expression, proteasomal dysfunction, and apoptosis. Relevance to Parkinson's disease is discussed.     

Expression of L-ornithine Ndelta-oxygenase (PvdA) in fluorescent Pseudomonas species: an immunochemical and in silico study

Omega-amino acid monooxygenases (EC 1.14.13.-), catalysing the formation of hydroxamate precursors of microbial siderophores (e.g., pyoverdine), have so far eluded structural and biochemical characterisation. Here, the expression of recombinant L-ornithine-Ndelta-oxygenase (PvdA) from Pseudomonas aeruginosa PAO1 is reported. A library of eight monoclonal antibodies (MAbs) directed against PvdA has been generated. Two MAb families recognising the N- and C-terminal regions of PvdA were identified. The MAbs made it possible to demonstrate that 45-48 kDa PvdA homologues are expressed in response to iron limitation by different species and strains of fluorescent pseudomonads. Despite the different degrees in sequence similarity between P. aeruginosa PvdA and putative homologues from Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, Burkholderia cepacia, and Ralstonia solanacearum, in silico domain scanning predicts an impressive conservation of putative cofactor and substrate binding domains. The MAb library was also used to monitor PvdA expression during the transition of P. aeruginosa from iron-sufficient to iron-deficient growth.     

Defective phosphatidic acid-phospholipase C signaling in diabetic cardiomyopathy

The effects of exogenous phosphatidic acid (PA) on Ca2+ transients and contractile activity were studied in cardiomyocytes isolated from chronic streptozotocin-induced diabetic rats. In control cells, 25 microM PA induced a significant increase in active cell shortening and Ca2+ transients. PA increased IP3 generation in the control cardiomyocytes and its inotropic effects were blocked by a phospholipase C inhibitor. In cardiomyocytes from diabetic rats, PA induced a 25% decrease in active cell shortening and no significant effect on Ca2+ transients. Basal and PA-induced IP3 generation in diabetic rat cardiomyocytes was 3-fold lower as compared to control cells. Sarcolemmal membrane PLC activity was impaired. Insulin treatment of the diabetic animals resulted in a partial recovery of PA responses. Our results, therefore, identify an important defect in the PA-PLC signaling pathway in diabetic rat cardiomyocytes, which may have significant implications for heart dysfunction during diabetes.     

The function and characteristics of tyrosyl radical cofactors

Amino-acid radicals are involved in the catalytic cycles of a number of enzymes. The main focus of this mini-review is to discuss the function and properties of tyrosyl radical cofactors. We start by briefly summarizing the experimental studies that led to the detection and identification of the two redox-active tyrosines, denoted Y(Z) and Y(D), found in the water-oxidizing photosystem II (PSII) enzyme. More recent work that shows that the histidine-cross-linked tyrosine located in the active site of cytochrome c oxidase forms a radical during the catalytic oxygen-oxygen bond-cleavage process is also described. Advanced spectroscopic and structural studies have been performed to investigate the spin-density distribution, the protonation state and the hydrogen bonding of redox-active tyrosines. These studies have shown that the radical spin-density distribution is highly insensitive to the environment and that it is typical of a deprotonated species. In contrast, the hydrogen bonding and the nature of the proton acceptor or network of acceptors vary substantially in different systems. This is important for the function of the tyrosyl radical, as will be emphasized in a detailed discussion on the proposed function of Y(Z) as a proton coupled electron-transfer cofactor in photosynthetic water oxidation. Amino-acid radical enzymes are typically large complexes containing multiple subunits, chromophores and redox cofactors. The structural and mechanistic complexity of these systems has hampered the detailed characterization of their radical cofactors. In the final section of this mini-review, we will describe a project aimed at investigating how the protein controls the thermodynamic and kinetic redox properties of aromatic residues by using de novo protein design. Two model proteins of different size have been constructed. The smaller protein is a 67-residue three-helix bundle containing either a single buried tryptophan or tyrosine residue. The high-resolution NMR structure of the tryptophan-containing protein, denoted alpha(3)W, shows that the aromatic side chain is involved in a pi-cation interaction with a nearby lysine. The effects of this interaction on the tryptophan reduction potential were investigated by electrochemical and quantum mechanical methods. The calculations predict that the pi-cation interaction increases the potential, which is consistent with the electrochemical characterization of alpha(3)W. A larger 117-residue four-helix bundle, alpha(4)W, has more recently been constructed to complement the work on the three-helix-bundles and expand the family of model radical proteins.     

Accelerated maturation of primate testis by xenografting into mice

Testicular maturation and sperm production throughout the life of the male form the basis of male fertility. It is difficult to elucidate the intricate processes controlling testicular maturation and spermatogenesis in primates in vivo due to the long time span required for sexual maturation and also to the lack of accessible in vitro or in vivo models of primate spermatogenesis. Ectopic xenografting of neonatal testis tissue into mice provides an accessible model to study and manipulate the propagation and differentiation of male germ cells from immature donor animals. However, it was not clear whether this approach would be applicable to slowly maturing primates. Here we report that grafting of testis tissue from immature rhesus monkeys (Macaca mulatta) into host mice resulted in the acceleration of testicular maturation and production of fertilization-competent sperm in testis xenografts. The system reported here provides a powerful, practical approach to study timing and control of testicular maturation and regulation of primate spermatogenesis without the necessity for experimentation in primates. This approach could potentially be applied to produce fertile sperm from sexually immature individuals of rare or valuable primate species or from prepubertal boys undergoing sterilizing therapy for cancer.