Search PubMed⌕ Search

Biomedical subjects

J R Baker

Publications and source records attributed to J R Baker.

At least 91 records · Page 5Linked to original sources
PubMed1997

The interaction of plasmid DNA with polyamidoamine dendrimers: mechanism of complex formation and analysis of alterations induced in nuclease sensitivity and transcriptional activity of the complexed DNA.

The application of synthetic vectors for gene transfer has potential advantages over virus-based systems. However, little is known about the mechanisms involved in binding of synthetic materials to DNA and the nature of the DNA complexes that result from this interaction. Polyamidoamine (PAMAM) dendrimers are unique polymers with defined spherical structure. Dendrimers bind DNA to form complexes that efficiently transfect cells in vitro. We examined the formation of DNA/dendrimer complexes and found it based entirely on charge interaction. Electronmicroscopic examination of the complexes indicated that the majority of the plasmid DNA is contracted into isolated toroids, but also revealed larger, irregular aggregates of polymer and DNA. The binding of plasmid DNA to dendrimer appears to alter the secondary and tertiary structure, but does not fragment the DNA or alter its primary structure. Complexed DNA is protected against degradation by either specific nucleases or cellular extracts containing nuclease activity. While the initiation of transcription in vitro from promoters (for either T7 polymerase or eukaryotic RNA polymerase II) in dendrimer-complexed DNA is inhibited, elongation of the RNA transcript and translation do not appear to be affected. These resemble alterations of the DNA function when complexed with naturally-occurring polycations like non-acetylated histones. However, DNA complexed to dendrimer appears to maintain transcriptional activity while histone complexes at similar charge ratios do not. These results elucidate some aspects of the interaction between PAMAM dendritic polymers and DNA, and could lead to improvements in the design of polymers or formation of DNA complexes that will increase the efficiency of non-viral gene transfer.

DNA↗
PubMed1997

Entanglement in fishing gear and other causes of death in cetaceans stranded on the coasts of England and Wales.

Between August 1990 and September 1995 the carcases of 422 cetaceans of 12 species that had died around the coasts of England and Wales were examined. There were 234 harbour porpoises (Phocoena phocoena), 138 common dolphins (Delphinus delphis), and 50 individuals of 10 other species of dolphins and whales. The cause of death was diagnosed in the harbour porpoises and common dolphins was entanglement in fishing gear (bycatch). Of the cases in which the cause of death was established, 66 (38 per cent) of 176 harbour porpoises, 86 (80 percent) of 108 common dolphins, and six (19 per cent) of 31 individuals of other species had been bycaught. Neonatal starvation, pneumonia and generalised infections accounted for a further 31 per cent of the diagnosed causes of death in harbour porpoises. The proportion of stranded common dolphins that had been bycaught was consistently high except during 1995, but the proportion of stranded harbour porpoises which had been bycaught increased in each successive year.

Animals↗
PubMed1997

Dexamethasone increases expression of 5-lipoxygenase and its activating protein in human monocytes and THP-1 cells.

The aim of this study was to assess the effect of dexamethasone on 5-lipoxygenase pathway expression in human peripheral blood monocytes and the acute monocytic leukemia cell line, THP-1. Cells were conditioned over a period of days with dexamethasone, at concentrations relevant in vivo, to study the effect of the glucocorticoid on calcium-ionophore-stimulated 5-lipoxygenase product and arachidonic acid release. The effect of dexamethasone on levels of immunoreactive protein and steady-state messenger RNA encoding for 5-lipoxygenase and its activating protein (5-LAP) was also assessed. Dexamethasone increased the stimulated release of 5-lipoxygenase products from both monocytes and THP-1 cells in a dose-dependent fashion. The increase in product generation was not due to changes in the availability of arachidonic acid. However, immunoreactive protein and steady-state messenger RNA encoding for 5-lipoxygenase and 5-LAP were increased by conditioning with dexamethasone. There was no apparent effect of the glucocorticoid on LTA4-hydrolase-immunoreactive protein levels or specific activity. We conclude that dexamethasone increases 5-lipoxygenase pathway expression in both monocytes and in THP-1 cells. This effect is due, at least in part, to increases in immunoreactive protein and steady-state messenger RNA encoding for 5-lipoxygenase and 5-LAP. These results suggest a role for glucocorticoids in the regulation of 5-lipoxygenase pathway expression in mononuclear phagocytes.

5-Lipoxygenase-Activating Proteins↗
PubMed1997

O glycosylation of an Sp1-derived peptide blocks known Sp1 protein interactions.

The O-linked N-acetylglucosamine (O-GlcNAc) modification of proteins is dynamic and abundant in the nucleus and cytosol. Several transcription factors, including Sp1, have been shown to contain this modification; however, the functional role of O-GlcNAc in these proteins has not been determined. In this paper we describe the use of the previously characterized glutamine-rich transactivation domain of Sp1 (B-c) as a model to investigate the role of O-GlcNAc in Sp1's transcriptionally relevant protein-to-protein interactions with the TATA-binding-protein-associated factor (TAF110) and holo-Sp1. When the model Sp1 peptide was overexpressed in primate cells, this 97-amino-acid domain of Sp1 was found to contain a dominant O-GlcNAc residue at high stoichiometry, which allowed the mapping and mutagenesis of this glycosylation site. In vitro interaction studies between this segment of Sp1 and Drosophila TAF110 or holo-Sp1 indicate that the O-GlcNAc modification functions to inhibit the largely hydrophobic interactions between these proteins. In HeLa cells, the mutation at the mapped glycosylation site was permissive for transcriptional activation. We propose the hypothesis that the removal of O-GlcNAc from an interaction domain can be a signal for protein association. O-GlcNAc may thereby prevent untimely and ectopic interactions.

Amino Acid Sequence↗
PubMed1997

Leukotriene B4 costimulates 5-lipoxygenase activity in neutrophils via increased 5-lipoxygenase translocation.

The goal of this investigation was to assess the effect of leukotriene B4 (LTB4) on 5-lipoxygenase activity and to examine the possible mechanisms of this effect. Exogenous LTB4 significantly increased the release of endogenous LTB4 from A-23187-stimulated neutrophils. The 5-lipoxygenase product release from A-23187-stimulated neutrophils decreased in the presence of an LTB4 receptor antagonist, suggesting that LTB4 has a receptor-mediated, autocrine effect on 5-lipoxygenase activity. Neutrophil 5-lipoxygenase activity increased significantly as cell density increased. In the presence of exogenous LTB4, no significant change in [14C]arachidonic acid release from neutrophils was observed. Exogenous LTB4 increased the amount of immunoreactive 5-lipoxygenase protein detected in the nuclear fraction of disrupted cells. LTB4 receptor antagonism decreased the amount of immunoreactive 5-lipoxygenase detected in the nuclear fraction. Thus LTB4 exerts an autocrine, receptor-mediated, costimulatory effect on 5-lipoxygenase activity. This feedback appears to have biological significance and involves enhanced 5-lipoxygenase translocation to the nuclear membrane.

Arachidonate 5-Lipoxygenase↗
PubMed1997

Activated lymphocytes increase expression of 5-lipoxygenase and its activating protein in THP-1 cells.

The aim of this study was to investigate the regulation of the 5-lipoxygenase pathway of arachidonic acid metabolism by lymphocytes using the monocyte-like cell line, THP-1. When THP-1 cells were incubated over 4-7 days in 10% supernatant from lectin-activated human lymphocytes, their capacity to synthesize 5-lipoxygenase products was significantly increased. In contrast, the supernatant from nonactivated lymphocytes had no effect. The increase in capacity to synthesize 5-lipoxygenase products was mimicked by the addition of either granulocyte macrophage colony-stimulating factor (GM-CSF) or interleukin-3. These increases in synthetic capacity reflected increased enzymatic activity. Increased immunoreactive protein and mRNA for the enzymes 5-lipoxygenase and 5-lipoxygenase-activating protein were also found in cells conditioned with activated lymphocyte supernatants. Furthermore, the increase in mRNA for both enzymes was not blocked by cycloheximide, suggesting that the effect on steady-state mRNA levels does not require the synthesis of new protein. The increase in mRNA could be reproduced by GM-CSF. We conclude that lymphocytes can regulate the expression of 5-lipoxygenase in THP-1 cells over a period of days via the release of soluble factors.

5-Lipoxygenase-Activating Proteins↗
PubMed1997

Fas (APO-1, CD95)-mediated apoptosis in thyroid cells is regulated by a labile protein inhibitor.

To determine whether thyroid cell apoptosis observed in autoimmune thyroid disease could be related to activation of the Fas pathway, we examined the expression and function of Fas on thyroid follicular cells in vitro. Fas messenger RNA was found to be present using two different techniques and was expressed at equal levels in thyrocytes cultured either in the presence or absence of TSH. Fas antigen protein expression was demonstrated by Western blot of thyroid cell lysates and by immunohistochemical staining of thyrocytes, and the amount of Fas protein present did not appear to vary regardless of culture conditions. Despite expressing substantial amounts of Fas protein, thyrocytes treated with anti-Fas monoclonal antibody failed to undergo apoptosis. The addition of either interferon-gamma or interleukin-1beta to the anti-Fas-treated cell cultures also did not promote apoptotic signaling through this pathway. In contrast, the concomitant administration of cycloheximide allowed the induction of apoptosis through the activation of Fas in thyrocytes. These results suggest that Fas is constitutively expressed in thyrocytes, but that the induction of apoptosis through the Fas pathway is blocked by a labile protein inhibitor.

Antibodies, Monoclonal↗
PubMed1996

Survey of feather diseases of exhibition budgerigars in the United Kingdom.

Breeders of exhibition budgerigars have commented for some years on an increasing prevalence of feather diseases in their birds. A survey of these diseases identified 37 different conditions, but three-quarters of the cases were due to only four conditions: psittacine beak and feather disease, pulpitis, feather mites and feather cysts.

Animals↗
PubMed1996

Causes of mortality and morbidity in exhibition budgerigars in the United Kingdom.

In a survey of the causes of mortality and morbidity in exhibition budgerigars from 1984 to 1995, 1525 birds were examined post mortem. Megabacteriosis was the most common disease and trichomoniasis, enteritis, pneumonia, hepatitis and a degenerative disease of the gizzard lining were also common. A wide variety of less common diseases was also found.

Animals↗
PubMed1996

The hippocampal formation participates in novel picture encoding: evidence from functional magnetic resonance imaging.

Considerable evidence exists to support the hypothesis that the hippocampus and related medial temporal lobe structures are crucial for the encoding and storage of information in long-term memory. Few human imaging studies, however, have successfully shown signal intensity changes in these areas during encoding or retrieval. Using functional magnetic resonance imaging (fMRI), we studied normal human subjects while they performed a novel picture encoding task. High-speed echo-planar imaging techniques evaluated fMRI signal changes throughout the brain. During the encoding of novel pictures, statistically significant increases in fMRI signal were observed bilaterally in the posterior hippocampal formation and parahippocampal gyrus and in the lingual and fusiform gyri. To our knowledge, this experiment is the first fMRI study to show robust signal changes in the human hippocampal region. It also provides evidence that the encoding of novel, complex pictures depends upon an interaction between ventral cortical regions, specialized for object vision, and the hippocampal formation and parahippocampal gyrus, specialized for long-term memory.

Adult↗
PubMed1996

Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers.

Starburst polyamidoamine (PAMAM) dendrimers are a new type of synthetic polymer characterized by a branched spherical shape and a high density surface charge. We have investigated the ability of these dendrimers to function as an effective delivery system for antisense oligonucleotides and 'antisense expression plasmids' for the targeted modulation of gene expression. Dendrimers bind to various forms of nucleic acids on the basis of electrostatic interactions, and the ability of DNA-dendrimer complexes to transfer oligonucleotides and plasmid DNA to mediate antisense inhibition was assessed in an in vitro cell culture system. Cell lines that permanently express luciferase gene were developed using dendrimer mediated transfection. Transfections of antisense oligonucleotides or antisense cDNA plasmids into these cell lines using dendrimers resulted in a specific and dose dependent inhibition of luciferase expression. This inhibition caused approximately 25-50% reduction of baseline luciferase activity. Binding of the phosphodiester oligonucleotides to dendrimers also extended their intracellular survival. While dendrimers were not cytotoxic at the concentrations effective for DNA transfer, some non-specific suppression of luciferase expression was observed. Our results indicate that Starburst dendrimers can be effective carriers for the introduction of regulatory nucleic acids and facilitate the suppression of the specific gene expression.

Animals↗
PubMed1996

Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers.

Starburst polyamidoamine dendrimers are a new class of synthetic polymers with unique structural and physical characteristics. These polymers were investigated for the ability to bind DNA and enhance DNA transfer and expression in a variety of mammalian cell lines. Twenty different types of polyamidoamine dendrimers were synthesized, and the polymer structure was confirmed using well-defined analytical techniques. The efficiency of plasmid DNA transfection using dendrimers was examined using two reporter gene systems: firefly luciferase and bacterial beta-galactosidase. The transfections were performed using various dendrimers, and levels of expression of the reporter protein were determined. Highly efficient transfection of a broad range of eukaryotic cells and cell lines was achieved with minimal cytotoxicity using the DNA/dendrimer complexes. However, the ability to transfect cells was restricted to certain types of dendrimers and in some situations required the presence of additional compounds, such as DEAE-dextran, that appeared to alter the nature of the complex. A few cell lines demonstrated enhanced transfection with the addition of chloroquine, indicating endosomal localization of the complexes. The capability of a dendrimer to transfect cells appeared to depend on the size, shape, and number of primary amino groups on the surface of the polymer. However, the specific dendrimer most efficient in achieving transfection varied between different types of cells. These studies demonstrate that Starburst dendrimers can transfect a wide variety of cell types in vitro and offer an efficient method for producing permanently transfected cell lines.

Animals↗
PubMed1996

Unique autoantibody epitopes in an immunodominant region of thyroid peroxidase.

To define the autoantibody epitopes in amino acids 513-633 of thyroid peroxidase (TPO), a region frequently recognized in thyroiditis, cDNA sequences coding for peptide fragments of this region were amplified and ligated into pMalcRI and pGEX vectors for expression as recombinant fusion proteins. Western blots and enzyme-linked immunosorbent assay were then used to examine the reactivity in sera from 45 Hashimoto's and 47 Graves' disease patients. Two autoantibody epitopes within TPO amino acids 589-633 were identified; 16 of 35 patients reactive to TPO513-633 recognized the epitope of TPO592-613, while 6 patients recognized the epitope of TPO607-633. Eleven other patients with thyroiditis and two with Graves' disease recognized only the whole 589-633 fragment, and this response accounted for the Hashimoto's disease specificity. An amino acid sequence comparison of TPO592-613 with analogous regions of other peroxidase enzymes revealed significant differences in this area, and the substitution of even a single amino acid in one of the epitopes markedly decreased the binding affinity of autoantibodies. Additionally, the exclusive recognition by patients of only one of the epitopes within this region suggests a genetic restriction of the autoantibody response.

Adolescent↗
PubMed1996

Lymphocytes stimulate expression of 5-lipoxygenase and its activating protein in monocytes in vitro via granulocyte macrophage colony-stimulating factor and interleukin 3.

The aim of this study was to examine the role of lymphocytes in regulating expression of the 5-lipoxygenase pathway in monocytes. When monocytes were cultured over a period of days with lymphocytes, calcium ionophore-stimulated 5-lipoxygenase activity was enhanced. If lymphocytes alone were activated with lectins and their supernatants added to monocytes, stimulated 5-lipoxygenase activity was increased, whereas supernatants from lymphocytes cultured without lectins had no effect. Increased immunoreactive protein and mRNA for 5-lipoxygenase and 5-lipoxygenase activating protein were present in cells conditioned with lectin-activated lymphocyte supernatants. The effect of activated-lymphocyte supernatants could be mimicked by either GM-CSF or IL-3, but there was no additive effect with both cytokines. Both GM-CSF and IL-3 were present in the supernatant from lectin-activated lymphocytes at concentrations above their ED50, but were undetectable in the supernatant from nonactivated lymphocytes. The effect of lectin-activated lymphocyte supernatant could be inhibited by neutralizing antibodies to both cytokines, but not to either cytokine alone. We conclude that lymphocytes can regulate the expression of 5-lipoxygenase in monocytes, over a period of days, via the release of soluble factors, primarily GM-CSF and IL-3.

5-Lipoxygenase-Activating Proteins↗
PubMed1996

Functional magnetic resonance imaging of symptom provocation in obsessive-compulsive disorder.

BACKGROUND: The new technique of functional magnetic resonance imaging was used to investigate the mediating neuroanatomy of obsessive-compulsive disorder symptoms. METHODS: Ten patients with obsessive-compulsive disorder and 5 normal subjects were studied via functional magnetic resonance imaging during control and provoked conditions. Data analysis entailed parametric and nonparametric statistical mapping. RESULTS: Statistical maps (nonparametric; P < 10(-3)) showed activation for 70% or more of patients with obsessive-compulsive disorder in medial orbitofrontal, lateral frontal, anterior temporal, anterior cingulate, and insular cortex, as well as caudate, lenticulate, and amygdala. No normal subjects exhibited activation in any brain region. CONCLUSIONS: Results of functional magnetic resonance imaging were consistent with past studies of obsessive-compulsive disorder that used other functional neuroimaging modalities. However, paralimbic and limbic activations were more prominent in the present study.

Adult↗
PubMed1996

Presence of antigen and antibodies in serum and genital discharges of heifers after experimental intrauterine inoculation with Leptospira interrogans serovar hardjo.

The excretion of Leptospira interrogans serovar hardjo in cervico-vaginal mucus (CVM) or urine and the local and systemic immune responses to the organism were monitored in eight susceptible heifers after intrauterine inoculation while six similar heifers served as controls. All the heifers were inseminated at the subsequent oestrous periods. The overall percentage pregnancy rate (the number of pregnancies divided by the total number of inseminations) was lower in the infected heifers than in the controls though not significantly (33.3 v 50.0 per cent). Leptospires were detected, in either the urine or the CVM of six of the eight infected heifers during the study period of 15 weeks, either by direct immunofluorescence or dark ground microscopy; the bacteria did not grow in culture from any of the CVM samples. The control heifers remained free from evidence of infection. In the infected heifers, mean titres of at least 1:100 in a microscopic agglutination test were maintained for one to two weeks before declining to 1:10 to 1:30, whereas in serum IgG-ELISA tests (developed by using either protein or carbohydrate antigens), antibody titres of at least 1:100 were maintained throughout the study. During oestrous periods, IgA antibodies were detected more frequently in CVM with titres which were usually higher than the titres of IgG.

Agglutination Tests↗