pHi controls cytoplasmic calcium in rat parotid cells.

The goal of this investigation was to determine if cytoplasmic pH (pHi) modulated the basal level of the concentration of calcium ions in the cytoplasm (Cai) in rat parotid cells. We investigated the effects of various experimental manipulations on both pHi and Cai as measured with BCECF and the calcium photoprotein aequorin, respectively. We found that various experimental manipulations that increased pHi, such as exposure of the cells to NH4Cl, a decrease of the partial pressure of CO2 or an increase in extracellular pH in the presence of nigericin invariably increased Cai. Moreover, experimental manipulations which lowered Cai, such as a reduction of extracellular [NaHCO3] or the removal of loaded NH4 invariably decreased Cai. Thus pHi and Cai are directly related in parotid cells. Since recent studies have shown that Cai directly influences pHi, we suggest that Cai-handling and pHi-handling are tightly linked in parotid cells.     

A reverse-phase liquid chromatographic assay for flavin-containing monooxygenase activity

A rapid, convenient assay for flavin-containing monooxygenase activity is described. The method is based on direct analysis of quenched incubation mixtures by reverse-phase liquid chromatography, and utilizes p-nitrophenyl-1,3-oxathiolane as the substrate. The synthesis of the substrate and the product are described. The usefulness of p-nitrophenyl-1,3-oxathiolane S-oxide formation as a measure of flavin-containing monooxygenase activity was demonstrated using highly purified and microsomal hog and rat liver flavin-containing monooxygenase. The assay is especially useful for determining stereoselectivity of flavin-containing monooxygenase activity in small amounts of crude tissue preparations.     

Mitochondrial proteomic analysis of a cell line model of familial amyotrophic lateral sclerosis

Mutations in copper-zinc superoxide dismutase (SOD1) have been linked to a subset of familial amytrophic lateral sclerosis (fALS), a fatal neurodegenerative disease characterized by progressive motor neuron death. An increasing amount of evidence supports that mitochondrial dysfunction and apoptosis activation play a critical role in the fALS etiology, but little is known about the mechanisms by which SOD1 mutants cause the mitochondrial dysfunction and apoptosis. In this study, we use proteomic approaches to identify the mitochondrial proteins that are altered in the presence of a fALS-causing mutant G93A-SOD1. A comprehensive characterization of mitochondrial proteins from NSC34 cells, a motor neuron-like cell line, was achieved by two independent proteomic approaches. Four hundred seventy unique proteins were identified in the mitochondrial fraction collectively, 75 of which are newly discovered proteins that previously had only been reported at the cDNA level. Two-dimensional gel electrophoresis was subsequently used to analyze the differences between the mitochondrial proteomes of NSC34 cells expressing wild-type and G93A-SOD1. Nine and 36 protein spots displayed elevated and suppressed abundance respectively in G93A-SOD1-expressing cells. The 45 spots were identified by MS, and they include proteins involved in mitochondrial membrane transport, apoptosis, the respiratory chain, and molecular chaperones. In particular, alterations in the post-translational modifications of voltage-dependent anion channel 2 (VDAC2) were found, and its relevance to regulating mitochondrial membrane permeability and activation of apoptotic pathways is discussed. The potential role of other proteins in the mutant SOD1-mediated fALS is also discussed. This study has produced a short list of mitochondrial proteins that may hold the key to the mechanisms by which SOD1 mutants cause mitochondrial dysfunction and neuronal death. It has laid the foundation for further detailed functional studies to elucidate the role of particular mitochondrial proteins, such as VDAC2, in the pathogenesis of familial ALS.     

Cellular isoform of the scrapie agent protein participates in lymphocyte activation

The scrapie agent protein (Sp33-37 or PrPSc) is the disease-associated isoform of a normal cellular membrane protein (Cp33-37 or PrPC) of unknown function. We report that normal human lymphocytes and lymphoid cell lines, but not erythrocytes or granulocytes, express PrPC mRNA and protein. PrPC is detectable on the surface of lymphocytes; the surface immunoreactivity is sensitive to phosphatidylinositol-specific phospholipase C, indicating glycosyl-phosphatidylinositol membrane anchorage. Lymphocyte PrPC surface abundance is increased by cell activation, and polyclonal antibodies to PrPC suppress mitogen-induced activation. We conclude that PrPC is a lymphocyte surface molecule that may participate in cell activation.     

A poly(ethylene) glycolylated peptide for ocular delivery compacts DNA into nanoparticles for gene delivery to post‐mitotic tissues in vivo

Background

We have previously shown that a novel synthetic peptide for ocular delivery (POD) can efficiently compact DNA and deliver it to cells in vitro. This observation prompted us to develop use of POD as a nonviral vector in vivo.

Methods

POD peptide was modified using poly(ethylene) glycol (PEG‐POD) and used to compact DNA into nanoparticles that were then analysed using electron microscopy, dynamic light scattering, and fluorescent labeling. Transfection efficiency and localization were determined 48 h post‐injection into the subretinal space of the mouse eye using luciferase and LacZ, respectively. Efficiency of ocular transfection was compared to two other PEGylated peptides: PEG‐TAT and PEG‐CK30.

Results

PEG‐POD can compact DNA and form discrete nanoparticles of approximately 136 nm that can penetrate and transduce the retinal pigment epithelium (RPE) in vivo. PEG‐POD significantly increased expression of plasmid DNA by 215‐fold, PEG‐TAT by 56.52‐fold, and PEG‐CK30 by 24.73‐fold relative to DNA injected alone. In all cases β‐galactosidase was observed primarily in the RPE layer after subretinal injection. Electrophysiological analyses of PEG‐POD transduced retina indicates an absence of PEG‐POD‐mediated toxicity. PEG‐POD can protect plasmid DNA from DNaseI digestion, resulting in significant transfection of the lung after intravenous injection in mice.

Conclusions

PEG‐POD was found to significantly increase gene delivery relative to both DNA alone and other pegylated peptides. These findings highlight the use of pegylated peptides, and specifically PEG‐POD, as novel gene delivery vectors. Copyright © 2009 John Wiley & Sons, Ltd.     

Non-viral in vivo immune gene therapy of cancer: combined strategies for treatment of systemic disease

Many patients with various types of cancers have already by the time of presentation, micrometastases in their tissues and are left after treatment in a minimal residual disease state [Am J Gastroenterol 95(12), 2000]. To prevent tumour recurrence these patients require a systemic based therapy, but current modalities are limited by toxicity or lack of efficacy. We have previously reported that immune reactivity to the primary tumour is an important regulator of micrometastases and determinant of prognosis. This suggests that recruitment of specific anti-tumour mechanisms within the primary tumour could be used advantageously for tumour control as either primary or neo-adjuvant treatments. Recently, we have focused on methods of stimulating immune eradication of solid tumours and minimal residual disease using gene therapy approaches. Gene therapy is now a realistic prospect and a number of delivery approaches have been explored, including the use of viral and non-viral vectors. Non-viral vectors have received significant attention since, in spite of their relative delivery inefficiency, they may be safer and have greater potential for delivery of larger genetic units. By in vivo electroporation of the primary tumour with plasmid expressing GM-CSF and B7-1, we aim to stimulate immune eradication of the treated tumour and associated metastases. In this symposium report, we describe an effective gene based approach for cancer immunotherapy by inducing cytokine and immune co-stimulatory molecule expression by the growing cells of the primary tumour using a plasmid electroporation gene delivery strategy. We discuss the potential for enhancement of this therapy by its application as a neoadjuvant to surgical excision and by its use in combination with suppressor T cell depletion.This article is a symposium paper from the Annual Meeting of the “International Society for Cell and Gene Therapy of Cancer”, held in Shenzhen, China, on 9–11 December 2005.     

Comparative LCA of treatment options for US scrap tires: material recycling and tire-derived fuel combustion

Purpose

This life cycle assessment (LCA) study compares two prevalent end-of-life (EOL) treatment methods for scrap tires: material recycling and energy recovery. The primary intended use of the study results is to inform stakeholders of the relative environmental burdens and trade-offs associated with these two EOL vehicle tire treatment methods. The study supports prioritization of the waste treatment hierarchy for this material stream in the US.

Methods

This LCA compares (1) material recycling through ambient-temperature mechanical processing and (2) energy recovery through co-incineration of both whole and preprocessed scrap tires at a cement kiln. The avoided burden recycling methodology reflects the substitution of virgin synthetic rubber used in asphalt modification with the ground tire rubber from material recycling and the substitution of conventional kiln fuels with the tire-derived fuel (TDF). Both attributional (ALCA) and consequential (CLCA) methodologies are used: the ALCA assesses the environmental profiles of the treatment methods and the CLCA examines the potential effects of shifting more scrap tires to material recycling. The attributional portion of the LCA study was conducted in accordance with ISO standards 14044 series.

Results

The results in both methodological approaches indicate that the material recycling scenario provides greater impact reductions than the energy recovery scenario in terms of the examined environmental impact potentials: energy demand, iron ore consumption, global warming potential, acidification, eutrophication, smog formation, and respiratory effects. The additional impact reductions from material recycling are significant, and the establishment of new infrastructure required for a shift to material recycling incurs relatively insignificant burdens. Sensitivity analyses indicate that this conclusion does not change for (1) a range of TDF heating values, (2) a decrease in the mixed scrap tire rubber-to-steel composition ratio, (3) two alternative electricity grid fuel mixes with higher and lower carbon dioxide emission rankings than that of the baseline scenario, or (4) a comparison of material recycling to energy recovery when TDF is used in pulp and paper mills instead of cement kilns.

Conclusions

These results provide a basis for more informed decision-making when prioritizing scrap tire waste treatment hierarchy.     

Binding of bovine T194A PrPC by PrPSc-specific antibodies

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that are based on the misfolding of a cellular prion protein (PrP^C) into an infectious, pathological conformation (PrP^Sc). There is proof-of-principle evidence that a prion vaccine is possible but this is tempered with concerns of the potential dangers associated with induction of immune responses to a widely-expressed self-protein. By targeting epitopes that are specifically exposed upon protein misfolding, our group developed a vaccine that induces PrP^Sc-specific antibody responses. Here we consider the ability of this polyclonal antibody (SN6b) to bind to a mutant of PrP^C associated with spontaneous prion disease. Polyclonal antibodies were selected to mimic the vaccination outcome and also explore all possible protein conformations of the recombinant bovine prion protein with mutation T194A [bPrP(T194A)]. This mutant is a homolog of the human T183A mutation of PrP^C that is associated with early onset of familial dementia. With nanopore analysis, under non-denaturing conditions, we observed binding of the SN6b antibody to bPrP(T194A). This interaction was confirmed through ELISAs as well as immunoprecipitation of the recombinant and cellularly expressed forms of bPrP(T194A). This interaction did not promote formation of a protease resistant conformation of PrP in vitro. Collectively, these findings support the disease-specific approach for immunotherapy of prion diseases but also suggest that the concept of conformation-specific immunotherapy may be complicated in individuals who are genetically predisposed to PrP^C misfolding.