Mitochondrial function is dependent upon mitochondrial protein import (MPI), a complex process that transports nuclear-encoded proteins into mitochondria. Little is known about MPI in neurons. We examined the effects of Resveratrol (RSV), a polyphenolic antioxidant compound from grapes, on MPI in a neuronal cell model, differentiated PC12 cells. RSV (50µM, 24h) increased levels of mtGFP, a nuclear encoded mitochondrially targeted green fluorescent protein, and mtHsp70, a physiological mitochondrial heat shock protein, in mitochondria. In addition RSV also increased levels of Tom20, a key translocase of the outer mitochondrial membrane. The RSV mediated increases in mitochondrial proteins were independent of increases in mitochondrial mass or changes in intramitochondrial degradation. RSV also reduced mitochondria membrane potential and decreased basal levels of reactive oxygen species. Taken together, these findings show that RSV increases MPI and that this effect may be an important mechanism in the reported neuroprotective effects of RSV.

Mitochondrial function is dependent upon mitochondrial protein import (MPI), a complex process that transports nuclear-encoded proteins into mitochondria. Little is known about MPI in neurons. We examined the effects of Resveratrol (RSV), a polyphenolic antioxidant compound from grapes, on MPI in a neuronal cell model, differentiated PC12 cells. RSV (50µM, 24h) increased levels of mtGFP, a nuclear encoded mitochondrially targeted green fluorescent protein, and mtHsp70, a physiological mitochondrial heat shock protein, in mitochondria. In addition RSV also increased levels of Tom20, a key translocase of the outer mitochondrial membrane. The RSV mediated increases in mitochondrial proteins were independent of increases in mitochondrial mass or changes in intramitochondrial degradation. RSV also reduced mitochondria membrane potential and decreased basal levels of reactive oxygen species. Taken together, these findings show that RSV increases MPI and that this effect may be an important mechanism in the reported neuroprotective effects of RSV.

Recent studies suggest that carbon monoxide (CO) is another gas molecule that has similar biological actions as nitric oxide (NO). The purpose of this study is to investigate the relationship between NO and CO in the survival of naïve rat pheochromocytoma PC12 cells. Western blot analysis revealed that all three isoforms of nitric oxide synthase (NOS) exhibited low expression and two isoforms of heme oxygenase (HO), especially HO-1, exhibited higher expression in PC12 cells under basal condition. Exposure of PC12 cells for 24 h to the NO scavenger, carboxy-2-phenyl-4,4,5,5,- tetramethylimidazoline-1-oxy-1-3-oxide (carboxy-PTIO, 2 £gmol) or HO inhibitor, zinc protoporphyrinIX (ZnPP, 25 nmol) resulted in a progressive reduction in mitochondria dehydrogenase activity reflected cell viability as determined by the WST-1 (4-[3-(4-lodophenyl)- 2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay. On the other hand, incubation with NO donors, amino-3-morpholinyl- 1,2,3-oxadiazolium chloride (SIN-1, 1 £gmol) or S-Nitroso-N-acetyl- penicillamine (SNAP, 1 £gmol), or the CO precursor, hematin (500 nmol), resulted in an elevation in cell viability. The progressive reduction in cell viability induced by carboxy-PTIO (2 £gmol) or ZnPP (25 nmol) was significantly blunted by co-treatment with SIN-1 (1 £gmol). However, incubation with the NO precursor, L-arginine (L-Arg, 2 £gmol), or the selective inhibitors for nNOS, iNOS or eNOS, N£s-propyl-L-arginine (NPLA, 100 pmol), S-methylisothiourea (SMT, 10 nmol) or N5-1-Iminoethyl-L-ornithine dihydrochloride (L-NIO, 4 nmol) did not significantly alter cell viability. Co-treatment with carboxy-PTIO (2 £gmol) and L-Arg (2 £gmol) was also ineffective. These results suggest that NO or CO contributes to the survival of naïve PC12 cells.

No description available.

Injuries to the central nervous system (CNS) have traumatic consequences such as irreparable disability due to the inability of the CNS to regenerate injured nerve fibres. The aim of the work presented here was to develop a scaffold which potentially provides guidance to axons in the injured spinal cord thus facilitating signal transduction. A poly-(lactic-co-glycolic acid) (PLGA, PLA:PGA ratio of 75:25) flat sheet membrane scaffold was created using phase inversion with N-methyl pyrrolidinone (NMP) as the solvent and water as the non-solvent for immersion precipitation. PLGA flat sheet membranes were exposed to surface treatments including aminolysis, peptide immobilisation and ozonation in order to achieve higher cell attachment of PC12 cells, a cell line which was cloned from a solid pheochromocytoma tumour of white rats, and used as a tool for measurement of regeneration. Cell attachment studies revealed no significant difference in cell attachment between modified and not-modified PLGA flat sheet membranes. However, the absence of foetal calf serum (FCS) resulted in fivefold higher cell attachment compared to medium supplemented with 10% FCS. A second scaffold was produced by electrospinning 10% (w/w) PLGA in a chloroform:methanol (CHCl3:MeOH) mixture in ratio of 3:1 resulting in a nanofibrous scaffold. Optimum settings for electrospinning were found to be 3 ml/h feeding rate, 15kV applied voltage and 11cm collector-to-needle distance. Random and aligned PLGA nanofibres were produced, with a fibre diameter of 530±140nm. PC12 cells attached and differentiated to the nanofibrous scaffold. When exposed to NGF these cells stopped dividing and extended neurites. On random fibres, neurite orientation was random, whereas on aligned fibres 63% of neurites grew with the fibre orientation ±15��ᵒ. After 7 days of exposure to NGF, cells had 1-4 neurites on random fibres, reaching a maximum length of 188μm, whereas on aligned fibres, cells had 1-2 neurites, reaching a maximum length of 400μm. PLGA nanofibres were also investigated as a delivery vehicle for bioactive molecules. For this, poly-L-lysine (PLL) was incorporated into electrospun PLGA nanofibres via emulsion electrospinning. PLGA-PLL nanofibres were significantly larger than PLGA nanofibres having a diameter of 830±190nm. In order to visualise the incorporation of PLL, FITC-PLL was electrospun und the resulting nanofibres fluoresced greed. Attachment of PC12s to PLGA-PLL nanofibres was not significantly different compared to PLGA nanofibres. Aligned PLGA-PLL nanofibres were shown to promote neurite outgrowth of PC12s with resulting neurites of up to twice the length compared to aligned PLGA nanofibres. The results suggest that PLGA nanofibres strongly influences neurite organisation, which is potentially useful for future therapeutic approaches. The work in this thesis has shown that electrospun PLGA nanofibre mats have the potential to be used as scaffolds for spinal cord repair addressing topographical guidance and delivery of bioactive molecules to the site of injury.

The Bcl-2 family of proteins contains both anti-apoptotic (e.g.Bcl-2, Bcl-xL) and pro-apoptotic (e.g.Bad, Bcl-xS) proteins. The Bcl-xL and Bcl-xS are splice variants, but have different functions during apoptosis. The pro-survival kinase Akt can phosphorylate certain Bcl-2-related proteins, specifically on serine residues, to regulate their function and localization. This is an extension of the work from our laboratorys finding that haloperidol induces PC12 cell death by inducing Bcl-xS which then translocates from cytosol to mitochondria where it facilitates the release of cytochrome c. The toxicity induced by Bcl-xS is reversed by expression of constitutively active Akt. I hypothesized that Akt-mediated post-translational modification may be important for regulating the function of Bcl-xS and Bcl-xL.<p> Three specific serine residues were ultimately chosen for the characterization of Bcl-xS/xL function: Ser62 (inactivation mutant), Ser106 (putative Akt phosphorylation motif), and Ser165 in Bcl-xS (and the corresponding Ser228 in Bcl-xL) (immediately upstream of hydrophobic tail). The individual substitution of all three Serines with Alanines (which precludes phosphorylation at that site) in Bcl-xS did not affect the expression of the protein, but they did induce varying degrees of cytotoxicity in both PC12 and HEK cultures. I focused on the Ser106 substitution mutant given my hypothesis that Akt targeted this site. Overexpression of Bcl-xS(S106A) was toxic in both PC12 and HEK cultures, as expected, and this coincided with the appearance of the Bcl-xS(S106A) protein in the mitochondrial fraction. The release of cytochrome c from PC12 cell mitochondria coincided with the co-immunoprecipitation of the Bcl-xS protein with VDAC (voltage-dependent anion channel), a channel-forming protein that is known to mediate cytochrome c release, and with the initiation of caspase-dependent events. This was not the case in HEK cells, where the mitochondrial VDAC seemed to be diminished and the toxicity was cytochrome c-independent as well as caspase-independent. In addition, I was able to demonstrate that the S106A substituted protein was not able to co-immunoprecipitate with Akt, supporting Ser106 as a potential target for the Akt protein. I then studied the effects of the homologous substitutions in Bcl-xL on cell function. I chose to use treatment with the potent inducer of apoptosis, staurosporine, as a model of cytotoxicity. Again, substituted proteins exerted toxicity, but they did not potentiate the effects of staurosporine, at least not on MTT conversion. I did notice, however, that there was a clear morphological change with certain concentrations of staurosporine, and subsequently demonstrated that the Bcl-xL(S106A) protein potentiated PC12 cell differentiation induced by staurosporine. This protein also co-immunoprecipitated better with Akt, which was unexpected given my results with the Bcl-xS(S106A) protein described above. Perhaps the extra amino acids in Bcl-xL account for this.<p> It is clear that the phosphorylation of Bcl-xS and Bcl-xL proteins is an important means of regulating their function and localization within the cell. These data support the S106 residues in both Bcl-xS and Bcl-xL as novel targets for the pro-survival Akt kinase, and indicate a role for this/these residue(s) in cellular functions as diverse as apoptosis and differentiation.

The Bcl-2 family of proteins contains both anti-apoptotic (e.g.Bcl-2, Bcl-xL) and pro-apoptotic (e.g.Bad, Bcl-xS) proteins. The Bcl-xL and Bcl-xS are splice variants, but have different functions during apoptosis. The pro-survival kinase Akt can phosphorylate certain Bcl-2-related proteins, specifically on serine residues, to regulate their function and localization. This is an extension of the work from our laboratorys finding that haloperidol induces PC12 cell death by inducing Bcl-xS which then translocates from cytosol to mitochondria where it facilitates the release of cytochrome c. The toxicity induced by Bcl-xS is reversed by expression of constitutively active Akt. I hypothesized that Akt-mediated post-translational modification may be important for regulating the function of Bcl-xS and Bcl-xL.<p> Three specific serine residues were ultimately chosen for the characterization of Bcl-xS/xL function: Ser62 (inactivation mutant), Ser106 (putative Akt phosphorylation motif), and Ser165 in Bcl-xS (and the corresponding Ser228 in Bcl-xL) (immediately upstream of hydrophobic tail). The individual substitution of all three Serines with Alanines (which precludes phosphorylation at that site) in Bcl-xS did not affect the expression of the protein, but they did induce varying degrees of cytotoxicity in both PC12 and HEK cultures. I focused on the Ser106 substitution mutant given my hypothesis that Akt targeted this site. Overexpression of Bcl-xS(S106A) was toxic in both PC12 and HEK cultures, as expected, and this coincided with the appearance of the Bcl-xS(S106A) protein in the mitochondrial fraction. The release of cytochrome c from PC12 cell mitochondria coincided with the co-immunoprecipitation of the Bcl-xS protein with VDAC (voltage-dependent anion channel), a channel-forming protein that is known to mediate cytochrome c release, and with the initiation of caspase-dependent events. This was not the case in HEK cells, where the mitochondrial VDAC seemed to be diminished and the toxicity was cytochrome c-independent as well as caspase-independent. In addition, I was able to demonstrate that the S106A substituted protein was not able to co-immunoprecipitate with Akt, supporting Ser106 as a potential target for the Akt protein. I then studied the effects of the homologous substitutions in Bcl-xL on cell function. I chose to use treatment with the potent inducer of apoptosis, staurosporine, as a model of cytotoxicity. Again, substituted proteins exerted toxicity, but they did not potentiate the effects of staurosporine, at least not on MTT conversion. I did notice, however, that there was a clear morphological change with certain concentrations of staurosporine, and subsequently demonstrated that the Bcl-xL(S106A) protein potentiated PC12 cell differentiation induced by staurosporine. This protein also co-immunoprecipitated better with Akt, which was unexpected given my results with the Bcl-xS(S106A) protein described above. Perhaps the extra amino acids in Bcl-xL account for this.<p> It is clear that the phosphorylation of Bcl-xS and Bcl-xL proteins is an important means of regulating their function and localization within the cell. These data support the S106 residues in both Bcl-xS and Bcl-xL as novel targets for the pro-survival Akt kinase, and indicate a role for this/these residue(s) in cellular functions as diverse as apoptosis and differentiation.

<p>Background: Silver nanoparticles (AgNPs) act as antimicrobials by releasing monovalent silver (Ag+) and are increasingly used in consumer products, thus elevating exposures in human and environmental populations. Materials and Methods: We evaluated Ag+ in a standard model of neuronal cell replication and differentiation, and then determined whether there were similar effects of the ion in vivo using zebrafish. Next, we compared Ag+ and AgNP exposures in the same two models and incorporated the effects of particle coating, size and composition. Conclusions: This work is the first to show that both Ag+ and AgNPs are developmental neurotoxicants in vitro and in vivo. Moreover, although both the soluble ion and the particles impair measures of neurodevelopment, the outcomes and underlying mechanisms of each toxicant are often wholly distinct. Superimposed on the dichotomies between Ag+ and AgNP exposures are clear effects of particle coating, size and composition that will necessitate evaluation of individual AgNP types when considering potential environmental and human health effects. The results presented here provide hazard identification that can help isolate the models and endpoints necessary for developing a risk assessment framework for the growing use of AgNPs.</p> / Dissertation

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / "July 1998." Typescript. Vita. Includes bibliographical references (leaves 138-170). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [97]-122).