Biomedical subjects

S Naylor

Publications and source records attributed to S Naylor.

At least 19 recordsLinked to original sources

PubMed2006

EIAV vector-mediated delivery of endostatin or angiostatin inhibits angiogenesis and vascular hyperpermeability in experimental CNV.

We evaluated the efficacy of equine infectious anaemia virus (EIAV)-based lentiviral vectors encoding endostatin (EIAV.endostatin) or angiostatin (EIAV.angiostatin) in inhibiting angiogenesis and vascular hyperpermeability in the laser-induced model of choroidal neovascularisation (CNV). Equine infectious anaemia virus.endostatin, EIAV.angiostatin or control (EIAV.null) vectors were administered into the subretinal space of C57Bl/6J mice. Two weeks after laser injury CNV areas and the degree of vascular hyperpermeability were measured by image analysis of in vivo fluorescein angiograms. Compared with EIAV.null-injected eyes, EIAV.endostatin resulted in a 59.5% (P<0.001) reduction in CNV area and a reduction in hyperpermeability of 25.6% (P<0.05). Equine infectious anaemia virus.angiostatin resulted in a 50.0% (P<0.05) reduction in CNV area and a 23.9% (P<0.05) reduction in hyperpermeability. Equine infectious anaemia virus.endostatin, but not EIAV.angiostatin significantly augmented the frequency of apoptosis within the induced CNV as compared with injected controls. TdT-dUTP terminal nick end labeling analysis 5 weeks post-injection, and histological and retinal flatmount analysis 12 months post-injection revealed no evidence of vector- or transgene expression-related deleterious effects on neurosensory retinal cells, or mature retinal vasculature in non-lasered eyes. Highly expressing EIAV-based vectors encoding endostatin or angiostatin effectively control angiogenesis and hyperpermeability in experimental CNV without long-term deleterious effects, supporting the use of such a strategy in the management of patients with exudative age-related macular degeneration.

Angiogenesis Inhibitors↗

PubMed2006

Stable and efficient intraocular gene transfer using pseudotyped EIAV lentiviral vectors.

BACKGROUND: We have developed minimal non-primate lentiviral vectors based on the equine infectious anaemia virus (EIAV). We evaluated the in vivo expression profiles of these vectors delivered regionally to ocular tissues to define their potential utility in ocular gene therapy. METHODS: EIAV vectors pseudotyped with VSV-G or rabies-G envelope proteins were delivered subretinally, intravitreally or into the anterior chambers (intracameral administration) in mice. Reporter gene (eGFP) expression was analysed using in vivo retinal imaging or histological examination of eyes and brains at intervals between 3 days and 16 months. We investigated the effects of vector titre, pseudotype, genome configuration, site of intraocular administration, intentional retinal trauma and the degree of retinal maturation on the spatial and temporal expression profiles of these vectors. RESULTS: Subretinal vector delivery resulted in efficient and stable transduction of retinal pigment epithelial (RPE) cells and variable transduction of photoreceptors up to 16 months post-injection. Retinal trauma facilitated the local transduction of neurosensory retinal cells. Intracameral administration of VSV-G- but not rabies-G-pseudotyped vectors produced stable eGFP expression in corneal endothelial cells and trabecular meshwork. CONCLUSIONS: The cellular tropism and expression kinetics of optimised EIAV vectors after intraocular administration make them attractive vehicles for delivering therapeutic genes in the management of inherited and acquired retinal and anterior segment disorders.

Animals↗

PubMed2005

Phenotypic and functional characterisation of follicle-associated epithelium of rectal lymphoid tissue.

Lymphoid follicles cluster in the terminal rectum of various animal species and of man and hence this site may be important in the development of immune responses to pathogens. For the induction of immune responses at mucosal sites, interplay is required between various cell types performing functions ranging from antigen-sampling cells via antigen-presenting cells to antigen-specific lymphocytes. Therefore, we have characterised the cell populations and relevant functioning of follicle-associated epithelium (FAE) and associated follicles in the terminal portion of rectum in cattle as a representative mammal. Immunohistochemical studies of this region identified immune cell subsets (CD4+, CD8+, WC 1+gammadelta, CD2+, CD 21+ and CD 40+ cells) characteristic of an immune-inductive site. Examination of FAE identified a subset of cells with structural and functional features of antigen-sampling M-cells. Cells of the FAE and adjacent follicle-associated crypts expressed vimentin and a subset of these cells internalised microparticles, a further attribute of M-cells. The FAE cells were phenotypically heterogeneous and therefore the function and phenotype of these cell subsets requires further characterisation, particularly with respect to their potentially important role in the interaction of hosts with pathogens and the development of immune responses.

Animals↗

PubMed2004

Genetic amplification of the transcriptional response to hypoxia as a novel means of identifying regulators of angiogenesis.

The cellular response to hypoxia involves the promotion of angiogenesis, leading to increased blood flow and oxygenation. The macrophage has been identified as an orchestrator of this response in several pathologies, through the release of angiogenic factors in response to hypoxia. We have produced the first comprehensive transcriptome analysis of hypoxic primary human macrophages with respect to the regulation of angiogenesis. There is a marked induction of genes encoding factors known to stimulate angiogenesis, rather than factors that inhibit this process. We show that overexpression of the transcription factor EPAS1 using a recombinant adenoviral vector amplifies the induction of genes encoding angiogenic proteins in response to hypoxia. This defines a new strategy for enhancing transcriptome and proteome analyses by overexpressing disease-implicated genes using viral gene transfer methodologies.

Adenoviridae↗

PubMed2003

Hypoxia-mediated tumour targeting.

Hypoxia is a common physiological feature of tumours. It activates a signalling cascade that culminates in the stabilization of the HIF-1 transcription factor and activation of genes that possess a hypoxia response element (HRE). We have used an optimized hypoxia responsive promoter (OBHRE) to investigate hypoxia-targeted gene expression in vivo in the context of an adenovirus vector. The OBHRE promoter showed limited activity in the liver or spleen such that expression was 1000-fold lower than that driven by the strong CMV/IE promoter. However, in the context of the tumour microenvironment, the OBHRE promoter achieved expression levels comparable to that of the CMV/IE promoter. Next, we showed that an adenovirus expressing the human cytochrome P450 (CYP2B6) regulated by the OBHRE promoter delays tumour growth in response to the prodrug cyclophosphamide (CPA). Finally, we exploited the hepatotropism of adenovirus to investigate whether the OBHRE promoter could mitigate the hepatotoxicity of a recombinant adenovirus expressing thymidine kinase (TK) in the context of the prodrug ganciclovir (GCV). High-dose Ad.CMVTK/GCV treatment caused significant liver necrosis whereas the same dose of Ad.HRETK was well tolerated. These in vivo data demonstrate that hypoxia-targeted gene expression via the OBHRE promoter can be used to increase the therapeutic window of cytotoxic cancer gene therapy.

Adenoviridae↗

PubMed2003

Hypoxia-regulated transgene expression in experimental retinal and choroidal neovascularization.

Recombinant AAV vectors mediate efficient and sustained transgene expression in retinal tissues and offer a powerful approach to the local, sustained delivery of angiostatic proteins for the treatment of ocular neovascular disorders. The application of such strategies may also require regulated gene expression to minimize the potential for unwanted adverse effects. In this study, we have evaluated the effect of a hypoxia-responsive element (HRE) on the kinetics of recombinant adeno-associated (rAAV)-mediated reporter gene expression in murine models of retinal and choroidal neovascularization. In murine ischaemia-induced retinal neovascularization, intravitreal delivery of rAAV.HRE.GFP results in reporter gene expression specifically at sites of vascular closure during the period of active neovascularization and not after vector delivery in normal controls. In murine laser-induced choroidal neovascularization, subretinal delivery of rAAV.HRE.GFP results in reporter gene expression at sites of active neovascularization but not elsewhere or after vector delivery in normal controls. HRE-driven gene expression offers an attractive strategy for the targeted and regulated delivery of angiostatic proteins to the retina in the management of neovascular disorders.

Animals↗

PubMed2003

Treatment of freshwater fish farm effluent using constructed wetlands: the role of plants and substrate.

Freshwater fish farm effluents have low nutrient concentrations but high flow rates, resulting in pollutant load, especially phosphorus (P), causing eutrophication. The feasibility was tested of a treatment combining, within a single constructed wetland, the contribution of macrophytes for reducing organic matter and nitrogen (N), with the high efficiency of steel slag and limestone for P removal. Twenty subsurface flow (SSF) basins of 280 L with different combinations of plants (Phragmites communis or Typha latifolia) and substrates (steel slag, limestone, gravel, peat) were fed with a reconstituted fish farm effluent in a greenhouse experiment. Pollutant removal was generally very good under all treatments. N and organic matter removal were correlated with plant biomass while P removal was better in substrates with steel slag and limestone. However, the high pH of the P-adsorbing substrate was detrimental to plant growth so that no combination of plants and substrates could maximise in one step the simultaneous removal of all evaluated pollutants. Therefore, the use of two sequential units is recommended, a first one consisting of a macrophyte planted basin using a neutral substrate to remove organic matter and N, followed by a second unplanted basin containing only a P-adsorbing substrate.

Animals↗

PubMed2002

A double-blind, randomized, placebo-controlled trial of essential fatty acid supplementation in the maintenance of remission of ulcerative colitis.

BACKGROUND: Essential fatty acid supplementation has been found to ameliorate certain chronic inflammatory diseases. This effect is thought to be mediated through the modulation of eicosanoid synthesis. Pro-inflammatory eicosanoids have been implicated in ulcerative colitis. AIM: To investigate the possible therapeutic benefit of essential fatty acids in quiescent ulcerative colitis to reduce the frequency of disease relapse. METHODS: A randomized, double-blind, placebo-controlled study was performed with a treatment duration of 12 months. Patients with quiescent disease received either trial medication (gamma-linolenic acid, 1.6 g, eicosapentaenoic acid, 270 mg, and docosahexaenoic acid, 45 mg, per day) or placebo (sunflower oil, 500 mg/day). The primary end-point was disease activity, assessed by a previously validated clinical index, sigmoidoscopic appearance and histology. RESULTS: Sixty-three patients were randomized, 31 to receive essential fatty acid treatment and 32 to receive placebo. Disease relapse rates were similar at 12 months (placebo, 38%; essential fatty acids, 55%), as were changes in sigmoidoscopic grade from baseline. CONCLUSIONS: The supplementation of the diet with this combination of essential fatty acids does not prolong the period of disease remission of ulcerative colitis.

Administration, Oral↗

PubMed2001

Analysis of in vivo-derived amyloid-beta polypeptides by on-line two-dimensional chromatography-mass spectrometry.

The presence of senile plaques composed of amyloid-beta (Abeta) polypeptides within brain tissue is normally used as a definitive postmortem diagnosis for Alzheimer's Disease (AD). Therefore, these polypeptides have been investigated as potential biomarkers of the disease state. However, at present, there is a lack of a robust assay for the detection of such polypeptides derived from in vivo sources. Such an assay is essential for analysis of biological samples from model AD systems. To overcome this problem we have developed a new single-step assay utilizing two dimensional-chromatography in conjunction with mass spectrometry. The method consists of on-line size-exclusion chromatography (SEC) to provide initial separation of analytes from the sample (based on their molecular weight) coupled with sample preconcentration prior to analysis by microbore high-performance liquid chromatography-mass spectrometry (HPLC-MS). This provides an extremely versatile and powerful assay which can separate specific analytes from cell lysate in a single step without further sample handling. The use of mass spectrometry as the detection system yields much more structural information than can be obtained from traditional ELISA and sandwich ELISA antibody assays. Furthermore, the on-line sample cleanup protocol minimizes sample handling and facilitates assay automation. Utilizing this new assay we have been able to detect Abeta 1-40 and Abeta 1-42 at cellular concentration levels directly from cell lysates. Moreover, we have detected multiple peptide responses within the same analysis, some of which have been tentatively identified as other ragged C-termini Abeta polypeptides derived from Abeta 1-42, based on their molecular weight, as well as oxidized Abeta polypeptides.

Amyloid beta-Peptides↗

PubMed2001

Online single-step analysis of blood proteins: the transferrin story.

The serum iron transport protein human transferrin (hTf) is a glycoprotein (MW approximately 79.6 kDa) containing two Asn-linked sites of glycosylation. The presence of specific glycoforms of hTf has been used as an indicator of carbohydrate-deficient glycoprotein syndrome (CDGS) or an indicator of alcohol abuse. The exact nature of the glycoforms described in the literature is controversial. In this work we demonstrate that the altered hTf glycoforms have lost one or both complete glycan side chains. Furthermore, we demonstrate using a combination of online immunoaffinity-postconcentration-mass spectrometry in conjunction with a blood spot cartridge that we can determine the relative quantities of the hTf glycoforms using <5 microL blood in under 30 min. This is in contrast to previous methods that used 1 mL and took 4 days. We show that this method can be useful to analyze hTf from CDGS and alcoholic patients.

Glycoproteins↗

PubMed2001

Microelectrospray ionization analysis of noncovalent interactions within the electron transferring flavoprotein.

Cofactor associations within the electron transferring flavoprotein (ETF) were studied in real time using microelectrospray ionization-mass spectrometry (muESI-MS). Initial analysis of porcine (pETF) and human ETF (hETF) revealed only the holoprotein. When muESI-MS source energies were increased, both pETF and hETF readily lost AMP. Analysis of hETF and pETF in methanol revealed intact alpha- and beta-subunits, and beta-subunit with AMP. The pETF also contained beta-subunit with FAD and beta-subunit with both cofactors. In contrast to crystal structure predictions, AMP dissociates more readily than FAD, and the pETF beta-subunit has an intimate association with FAD. This work demonstrates the complementarity of muESI-MS with NMR X-ray and optical spectroscopy in the analysis of noncovalent complexes.

Animals↗

PubMed2001

The role of mass spectrometry in vaccine development.

For the most part, vaccine development to date has been empiric. While sometimes successful, such a strategy is 'hit or miss', and fails to advance the basic science of vaccine development. Preferable would be tools that allow for a more directed development of vaccines at either the population or sub-population level. Characteristics of useful tools in vaccine development should include the ability to identify and characterize the spectrum of antigenic peptides presented by MHC molecules to which the immune system responds by the development of protective immune responses. In addition, because the explosion in human genomics allows the ability to understand MHC haplotypes at the population level, as well as an enhanced understanding of MHC binding motifs, new tools might further allow for an understanding of which vaccine antigens are capable of being bound and presented to the immune system by MHC molecules. New mass spectrometry technology fulfils these criteria, and may well lead to a revolution in the design of new vaccines. This paper will review the basics of mass spectrometry techniques as applied to the identification and characterization of vaccine peptide antigens, and discusses how these tools can be applied to vaccine development.

Chromatography, High Pressure Liquid↗

PubMed2001

Determination of in vivo adducts of disulfiram with mitochondrial aldehyde dehydrogenase.

Extensive use for disulfiram (DSF) has been found in the aversion therapy treatment of recovering alcoholics. Although it is known to irreversibly inhibit hepatic aldehyde dehydrogenase (ALDH), the specific mechanism of in vivo inhibition of the enzyme by the drug has not been determined yet. We have demonstrated in this report a novel, but simple and rapid method for structurally characterizing in vivo derived protein-drug adducts by linking on-line sample processing to HPLC-electrospray ionization mass spectrometry (HPLC-MS) and HPLC-tandem mass spectrometry (HPLC-MS/MS). Employing this approach, rats were administered DSF, and their liver mitochondria were isolated and solubilized. Both native and in vivo DSF-treated mitochondrial ALDH (mALDH) were purified in one step with an affinity cartridge. The in vivo DSF-treated mALDH showed 77% inhibition in enzyme activity as compared with that of the control. Subsequently, the control and DSF-inhibited mALDH were both subjected to HPLC-MS analyses. We were able to detect two adducts on DSF-inhibited mALDH, as indicated by the mass increases of approximately 71 and approximately 100 Da. To unequivocally determine the site and structure of these adducts, on-line pepsin digestion-HPLC-MS and HPLC-MS/MS were performed. We observed two new peptides at MH(+) = 973.7 and MH(+) = 1001.8 in the pepsin digestion of DSF-inhibited enzyme. These two peptides were subsequently subjected to HPLC-MS/MS for sequence determination. Both peptides possessed the sequence FNQGQC(301)C(302)C(303), derived from the enzyme active site region, and were modified at Cys(302) by N-ethylcarbamoyl (+71 Da) and N-diethylcarbamoyl (+99 Da) adducts. These findings indicated that N-dealkylation may be an important step in DSF metabolism, and that the inhibition of ALDH occurred by carbamoylation caused by one of the DSF metabolites, most likely S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO). Finally, there was no evidence of the presence of an intramolecule disulfide bridge modification on the peptide FNQGQCCC.

Alcohol Deterrents↗

PubMed2001

A physical map of the human genome.

The human genome is by far the largest genome to be sequenced, and its size and complexity present many challenges for sequence assembly. The International Human Genome Sequencing Consortium constructed a map of the whole genome to enable the selection of clones for sequencing and for the accurate assembly of the genome sequence. Here we report the construction of the whole-genome bacterial artificial chromosome (BAC) map and its integration with previous landmark maps and information from mapping efforts focused on specific chromosomal regions. We also describe the integration of sequence data with the map.

Chromosomes, Artificial, Bacterial↗

PubMed2001

Overview--in vitro inhibition of aldehyde dehydrogenase by disulfiram and metabolites.

Disulfiram (DSF) has found extensive use in the aversion therapy treatment of recovering alcoholics. It is known that DSF or a metabolite irreversibly inhibits aldehyde dehydrogenase (ALDH). However, the actual mechanism of inhibition is still not known. In this work we describe the in vitro interactions of DSF, as well as a principal metabolite S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO), with both recombinant rat liver mitochondrial monomeric ALDH (rmALDH) and homotetrameric rmALDH. We show that DSF directly inhibits rmALDH (IC(50)=36.4 microM) by inducing the formation of an intramolecular disulfide bond. We also demonstrate by HPLC-MS analysis of a Glu-C digest of DSF-treated rmALDH that the intramolecular disulfide bridge formed involves two of the three cysteines located at the active site of the enzyme. Using a combination of HPLC-MS and HPLC-MS/MS, we further show that the electrophilic metabolite MeDTC-SO also inhibits rmALDH (IC(50)=4.62 microM). We isolate and identify a carbamoylated peptide at Cys(302) with the sequence FNQGQC(301)C(302)C(303). Hence we show that MeDTC-SO exhibits its inhibitory effect by covalently modifying the -SH side-chain of Cys(302), present at the active site rmALDH. Finally we show using SEC-MS that both DSF and MeDTC-SO do not prevent formation of the homotetramer of rmALDH, but inhibit the enzyme by acting directly at the active site of specific monomers of rmALDH.

Aldehyde Dehydrogenase↗

PubMed2001

In vivo inhibition of aldehyde dehydrogenase by disulfiram.

Disulfiram (DSF) has found extensive use in the aversion therapy treatment of recovering alcoholics. Although it is known to irreversibly inhibit hepatic aldehyde dehydrogenase (ALDH), the specific mechanism of in vivo inhibition of the enzyme by the drug has not yet been determined. In this report, we demonstrate a novel, but simple and rapid method for structurally characterizing in vivo derived protein-drug adducts by linking on-line sample processing to HPLC-electrospray ionization mass spectrometry (HPLC-MS) and HPLC-tandem mass spectrometry (HPLC-MS/MS). Employing this approach, rats were administered DSF, and their liver mitochondria were isolated and solubilized. Both native and in vivo DSF-treated mitochondrial ALDH (rmALDH) were purified in one-step with an affinity cartridge. The in vivo DSF-treated rmALDH showed 77% inhibition in enzyme activity as compared to that of the control. Subsequently, the control and DSF-inhibited rmALDH were both subjected to HPLC-MS analyses. We were able to detect two adducts on DSF-inhibited rmALDH as indicated by the mass increases of approximately 71 and approximately 100 Da. To unequivocally determine the site and structure of these adducts, on-line pepsin digestion-HPLC-MS and HPLC-MS/MS were performed. We observed two new peptides at MH(+)=973.7 and 1001.8 in the pepsin digestion of DSF-inhibited enzyme. These two peptides were subsequently subjected to HPLC-MS/MS for sequence determination. Both peptides possessed the sequence FNQGQC(301)C(302)C(303), derived from the enzyme active site region, and were modified at Cys(302) by N-ethylcarbamoyl (+71 Da) and N-diethylcarbamoyl (+99 Da) adducts. These findings indicated that N-dealkylation may be an important step in DSF metabolism, and that the inhibition of ALDH occurred by carbamoylation caused by one of the DSF metabolites, most likely S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO).

Aldehyde Dehydrogenase↗

PubMed2001

Modulation effects of zinc on the formation of vitamin D receptor and retinoid X receptor alpha-DNA transcription complexes: analysis by microelectrospray mass spectrometry.

The vitamin D receptor (VDR) binds zinc, and the activity of vitamin D dependent genes in cells is influenced by intracellular zinc concentrations. To determine whether zinc influences vitamin D action in cells by modulating the formation of VDR and retinoid x receptor alpha (RXR alpha) heterodimer-DNA complexes, we used microelectrospray ionization mass spectrometry (microESI-MS) to assess receptor-DNA interactions in the presence of varying amounts of zinc. In the absence of DNA, VDR and RXR alpha proteins were primarily monomeric with small amounts of protein homodimers also observed. Zn(2+) (up to 300 microM) did not change VDR or RXR alpha monomer/homodimer ratios. Mass spectra of VDR combined with RXR alpha were a sum of individual protein spectral data. Zn(2+) had no effect on the interactions of receptors. With increasing amounts of Zn(2+), additional Zn(2+) ions were detected bound to VDR and RXR alpha. microESI-MS analyses of RXR alpha in the presence of an osteopontin vitamin D DNA response element (OP-VDRE) showed RXR alpha homodimer/OP-VDRE complexes. DNA-protein complex formation increased on addition of Zn(2+) up to 200 microM; at 300 microM, Zn(2+) dissociation of the RXR alpha homodimer/OP-VDRE complexes occurred, coincident with the appearance of RXR alpha monomeric protein. When microESI-MS analyses were carried out with VDR and OP-VDRE, VDR homodimer/OP-VDRE complexes were not detected. Addition of Zn(2+) did not result in VDR/OP-VDRE complex formation. Heterodimeric VDR/RXR alpha complexes with OP-VDRE were detected by microESI-MS. Addition of 300 microM Zn(2+) resulted in dissociation of the heterodimeric VDR/RXR alpha/OP-VDRE complex. Addition of Mg(2+) in place of Zn(2+) did not alter protein/OP-VDRE complexes. Our results show that zinc modulates steroid hormone receptor-DNA interactions.

Animals↗

PubMed2001

RNA-RNA noncovalent interactions investigated by microspray ionization mass spectrometry.

Electrospray ionization mass spectrometry is playing an increasing role in the study of noncovalent interactions involving biomolecules. RNA-RNA complexes are important in many areas of biology, including RNA catalysis, RNA splicing, ribosome function, and gene regulation. Here, microelectrospray mass spectrometry (microESI-MS) is used to study noncovalent base-pairing interactions between RNA oligonucleotides, an area not previously explored by this technique. Using a set of complementary RNA oligonucleotides, we demonstrate the formation of the expected double-helical RNA complexes composed of three distinct oligonucleotides. The ability to study specific RNA noncovalent interactions by microESI-MS has the potential to provide a unique method by which to analyze and assign precise molecular masses to RNA-RNA complexes.

Base Sequence↗