Nicholas Bright | University of Cambridge (original) (raw)

Papers by Nicholas Bright

Research paper thumbnail of Reversible assembly and disassembly of V-ATPase during the lysosome regeneration cycle

bioRxiv (Cold Spring Harbor Laboratory), Dec 21, 2023

Research paper thumbnail of Targeting of a phogrin-green fluorescent protein chimaera to insulin secretory granules of pancreatic beta-cells in transgenic mice

PubMed, Dec 1, 2002

We have generated a transgenic mouse expressing a chimaeric phogrin-enhanced green fluorescent pr... more We have generated a transgenic mouse expressing a chimaeric phogrin-enhanced green fluorescent protein (EGFP) targeted to pancreatic beta-cells by the rat insulin II promoter. The transgenic animals appear healthy, have normal weight gain and normal glucose tolerance. Morphological analyses of adult transgenic mice revealed that the fluorescent reporter molecule was specifically expressed in beta-cells of the pancreatic islet and not in extra-pancreatic tissues. The distribution of phogrin-EGFP in beta-cells, however, was heterogeneous with three distinct populations distinguishable by FACS analysis and immunofluorescence microscopy. Superficially-localized islets in the whole pancreas were readily visualized in the animals as was the developing endocrine pancreas in undissected 15.5 day old mouse embryos. We envisage that the animal will be an important resource for future investigations of islet development, regeneration and the molecular cell biology of insulin secretion.

Research paper thumbnail of Tumor Necrosis Factor-α Induces Apoptosis of Human Adipose Cells

Diabetes, Dec 1, 1997

Tumor necrosis factor-a (TNF-a) production by adipocytes is elevated in obesity, as shown by incr... more Tumor necrosis factor-a (TNF-a) production by adipocytes is elevated in obesity, as shown by increased adipose tissue TNF-a mRNA and protein levels and by increased circulating concentrations of the cytokine. Furthermore, TNF-a has distinct effects on adipose tissue including induction of insulin resistance, induction of leptin production, stimulation of lipolysis, suppression of lipogenesis, induction of adipocyte dedifferentiation, and impairment of preadipocyte differentiation in vitro. Taken together, these effects all tend to decrease adipocyte volume and number and suggest a role for TNF-a in limiting increase in fat mass. The aim of the present study was to determine if TNF-a could induce apoptosis in human adipose cells, hence delineating another mechanism by which the cytokine could act to limit the development of, or extent of, obesity. Cultured human preadipocytes and mature adipocytes in explant cultures were exposed in vitro to human TNF-a at varying concentrations for up to 24 h. Apoptosis was assessed using morphological (histology, nuclear morphology following acridine orange staining, electron microscopy) and biochemical (demonstration of internucleosomal DNA cleavage by gel electrophoresis and of annexin V staining using immunocytochemistry) criteria. In control cultures, apoptotic indexes were between 0 and 2.3% in all experiments. In the experimental systems, TNF-a induced apoptosis in both preadipocytes and adipocytes, with indexes between 5 and 25%. Therefore, TNF-a induces apoptosis of human preadipocytes and adipocytes in vitro. In view of the major metabolic role of TNF-a in human adipose tissue, and the knowledge that adipose tissue is dynamic (with cell acquisition via preadipocyte replication/differentiation and cell loss via apoptosis), these findings describe a further mechanism whereby adipose tissue mass may be modified by TNF-a. Dia

Research paper thumbnail of The ontogeny and heterogeneity of Fc-gamma receptor bearing cells in the human placenta and fetal membranes

ProQuest eBooks, 1994

The human fetus is donated passive immunity, via the placenta, in the form of maternal IgG class ... more The human fetus is donated passive immunity, via the placenta, in the form of maternal IgG class antibodies. This provides protection against pathogens while the fetus is immunologically immature. Immunocytochemistry has been performed on placental tissues to localise endogenous IgG and three subtypes of IgG receptor designated FcgammaRI (CD64), FcgammaRII (CDw32) and FcgammaRIII (CD16). Ultrastructural immunocytochemistry has been used to investigate the subcellular distribution of IgG. FcgammaRI is expressed by mesenchymal cells of term and first trimester placentae. FcgammaRII is expressed by endothelial cells and FcgammaRIII is expressed by syncytiotrophoblast. Endogenous IgG is restricted to the syncytiotrophoblast in first trimester villi indicating that transport is inhibited in early stages of gestation. At term IgG is associated with extracellular matrix, serum, endothelial cells, mesenchymal cells and syncytiotrophoblast suggesting that it is efficiently transported across the trophoblast. Ultrastructural observations of IgG distribution indicate that it is taken up by receptor-mediated endocytosis into the syncytiotrophoblast and reaches an endosome-like compartment. In term amniochorion FcgammaRI, FcgammaRII and FcgammaRIII are expressed by leucocytes in the maternal decidua and heterogeneous populations of mesenchymal cells in the fetal connective tissues suggesting that they possess an immunoregulatory role. Endogenous IgG is restricted to the extracellular matrix and cells possessing Fcgamma receptors. Cytotrophoblast and amniotic epithelial cells, which do not possess Fcgamma receptors, contain no endogenous antibodies. The hypothesis is proposed that cytotrophoblast cells are a barrier to IgG transmission across the placenta. This accounts for the paradox that there are low levels of transport in the first trimester when the syncytiotrophoblast expresses Fcgamma receptors. Cytotrophoblast cells form an almost continuous layer underlying the syncytiotrophoblast in the first trimester which becomes attenuated as the placenta matures. Furthermore, IgG may diffuse non-specifically between cytotrophoblast cells of the amniochorion and thus contribute to the acquisition of passive immunity. These data suggest that IgG is transmitted across the vascular system of the placenta and is not acquired via amniotic fluid

Research paper thumbnail of Localisation of Nramp1 in macrophages: modulation with activation and infection

Journal of Cell Science, Oct 1, 1998

The Lsh/Ity/Bcg gene was identified in mice for its role in regulating resistance and susceptibil... more The Lsh/Ity/Bcg gene was identified in mice for its role in regulating resistance and susceptibility to intracellular pathogens: Leishmania donovani, Salmonella typhimurium, Mycobacterium bovis and M. intracellulare (reviewed by Blackwell, 1989; Schurr et al., 1991). It is also a major candidate gene for autoimmune disease susceptibility in man (reviewed by Blackwell and Searle, 1998). A candidate for Lsh/Ity/Bcg, Nramp1 (natural resistance-associated macrophage protein), was identified by positional cloning (Vidal et al., 1993) and the full-length sequence obtained (Barton et al., 1994), and its candidacy was confirmed using transfected macrophages in vitro (Barton et al., 1995) and gene disruption in vivo (Vidal et al., 1995). Murine Nramp1 expression is macrophage-restricted and affects the capacity of the host to control intracellular replication of microorganisms , all of which ultimately reside in the phagolysosome. Susceptibility is associated with a nonconservative amino acid substitution at position 169 (glycine resistant to aspartic acid susceptible), caused by a point mutation in the region encoding the fourth putative transmembrane spanning domain (TMD; Vidal et al., 1995). The polypeptide Nramp1 is a hydrophobic integral membrane protein, with the following predicted features: 10-12 TMDs, a

Research paper thumbnail of ATP Regulation in Adult Rat Cardiomyocytes

Journal of Biological Chemistry, Sep 1, 2006

The mechanisms that enable the heart to rapidly increase ATP supply in line with increased demand... more The mechanisms that enable the heart to rapidly increase ATP supply in line with increased demand have not been fully elucidated. Here we used an adenoviral system to express the photoproteins luciferase and aequorin, targeted to the mitochondria or cytosol of adult cardiomyocytes, to investigate the interrelationship between ATP and Ca 2؉ in these compartments. In neither compartment were changes in free [ATP] observed upon increased workload (addition of isoproterenol) in myocytes that were already beating. However, when myocytes were stimulated to beat rapidly from rest, in the presence of isoproterenol, a significant but transient drop in mitochondrial [ATP] ([ATP] m) occurred (on average to 10% of the initial signal). Corresponding changes in cytosolic [ATP] ([ATP] c) were much smaller (<5%), indicating that [ATP] c was effectively buffered in this compartment. Although mitochondrial [Ca 2؉ ] ([Ca 2؉ ] m) is an important regulator of respiratory chain activity and ATP production in other cells, the kinetics of mitochondrial Ca 2؉ transport are controversial. Parallel experiments in cells expressing mitochondrial aequorin showed that the drop in [ATP] m occurred over the same time scale as average [Ca 2؉ ] m was increasing. Conversely, in the absence or presence of isoproterenol, clear beat-to-beat peaks in [Ca 2؉ ] m were observed at 0.9 or 1.3 M, respectively, concentrations similar to those observed in the cytosol. These results suggest that mitochondrial Ca 2؉ transients occur during the contractile cycle and are translated into a time-averaged increase in mitochondrial ATP production that keeps pace with increased cytosolic demand.

Research paper thumbnail of Lysosome-endosome fusion and lysosome biogenesis

Journal of Cell Science, May 1, 2000

Recent data both from cell-free experiments and from cultured cells have shown that lysosomes can... more Recent data both from cell-free experiments and from cultured cells have shown that lysosomes can fuse directly with late endosomes to form a hybrid organelle. This has a led to a hypothesis that dense core lysosomes are in essence storage granules for acid hydrolases and that, when the former fuse with late endosomes, a hybrid organelle for digestion of endocytosed macromolecules is created. Lysosomes are then reformed from hybrid organelles by a process involving condensation of contents. In this Commentary we review the evidence for formation of the hybrid organelles and discuss the current status of our understanding of the mechanisms of fusion and lysosome reformation. We also review lysosome biosynthesis, showing how recent studies of lysosome-like organelles including the yeast vacuole, Drosophila eye pigment granules and mammalian secretory lysosomes have identified novel proteins involved in this process.

Research paper thumbnail of COMMENTARY Lysosome-endosome fusion and lysosome biogenesis

Recent data both from cell-free experiments and from cultured cells have shown that lysosomes can... more Recent data both from cell-free experiments and from cultured cells have shown that lysosomes can fuse directly with late endosomes to form a hybrid organelle. This has a led to a hypothesis that dense core lysosomes are in essence storage granules for acid hydrolases and that, when the former fuse with late endosomes, a hybrid organelle for digestion of endocytosed macromolecules is created. Lysosomes are then re-formed from hybrid organelles by a process involving condensation of contents. In this Commentary we review the evidence for formation of the hybrid organelles and discuss the current status of our understanding of the mechanisms of fusion and lysosome re-formation. We also review lysosome biosynthesis, showing how recent studies of lysosome-like organelles including the yeast vacuole, Drosophila eye pigment granules and mammalian secretory lysosomes have identified novel proteins involved in this process. SUMMARY

Research paper thumbnail of Fusion with

Autophagy-receptors link myosin VI to autophagosomes to mediate Tom1-dependent autophagosome matu... more Autophagy-receptors link myosin VI to autophagosomes to mediate Tom1-dependent autophagosome maturation and

Research paper thumbnail of Supplemental Experimental Procedures Materials

Antibodies were obtained from the following: a-flotillin1 and a-flotil-lin2 mAbs and a-caveolin1 ... more Antibodies were obtained from the following: a-flotillin1 and a-flotil-lin2 mAbs and a-caveolin1 pAb (BD Biosciences, Oxford); anti-flotil-lin1 and anti-flotillin2 rabbit pAbs (Santa Cruz Biotechnology, Hei-delberg, Germany); and anti-CD59 mAB and anti-CD59 goat pAB (R&D Systems, Abingdon, UK). Fluorescently labeled secondary antibodies were obtained from Molecular Probes (Cambridge Bio-science, Cambridge). A polyclonal antibody was raised against flotillin1 in rabbits, with full-length flotillin1 used as an antigen. The antibody was affinity purified with full-length flotillin1 coupled to CnBr-activated sepharose. Cell Culture, Transfections, and Immunofluorescence HeLa and MEF cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), 10 % fetal calf serum (FCS), and 1 % Pen and Strep at 10 % CO2. MEFs were freshly isolated from wild-type (WT) and caveolin null mice and discarded after passage six. All experiments

Research paper thumbnail of The ontogeny and heterogeneity of Fc-gamma receptor bearing cells in the human placenta and fetal membranes

The human fetus is donated passive immunity, via the placenta, in the form of maternal IgG class ... more The human fetus is donated passive immunity, via the placenta, in the form of maternal IgG class antibodies. This provides protection against pathogens while the fetus is immunologically immature. Immunocytochemistry has been performed on placental tissues to localise endogenous IgG and three subtypes of IgG receptor designated FcgammaRI (CD64), FcgammaRII (CDw32) and FcgammaRIII (CD16). Ultrastructural immunocytochemistry has been used to investigate the subcellular distribution of IgG. FcgammaRI is expressed by mesenchymal cells of term and first trimester placentae. FcgammaRII is expressed by endothelial cells and FcgammaRIII is expressed by syncytiotrophoblast. Endogenous IgG is restricted to the syncytiotrophoblast in first trimester villi indicating that transport is inhibited in early stages of gestation. At term IgG is associated with extracellular matrix, serum, endothelial cells, mesenchymal cells and syncytiotrophoblast suggesting that it is efficiently transported across the trophoblast. Ultrastructural observations of IgG distribution indicate that it is taken up by receptor-mediated endocytosis into the syncytiotrophoblast and reaches an endosome-like compartment. In term amniochorion FcgammaRI, FcgammaRII and FcgammaRIII are expressed by leucocytes in the maternal decidua and heterogeneous populations of mesenchymal cells in the fetal connective tissues suggesting that they possess an immunoregulatory role. Endogenous IgG is restricted to the extracellular matrix and cells possessing Fcgamma receptors. Cytotrophoblast and amniotic epithelial cells, which do not possess Fcgamma receptors, contain no endogenous antibodies. The hypothesis is proposed that cytotrophoblast cells are a barrier to IgG transmission across the placenta. This accounts for the paradox that there are low levels of transport in the first trimester when the syncytiotrophoblast expresses Fcgamma receptors. Cytotrophoblast cells form an almost continuous layer underlying the syncytiotrophoblast in the first trimester which becomes attenuated as the placenta matures. Furthermore, IgG may diffuse non-specifically between cytotrophoblast cells of the amniochorion and thus contribute to the acquisition of passive immunity. These data suggest that IgG is transmitted across the vascular system of the placenta and is not acquired via amniotic fluid

Research paper thumbnail of The effects of cytochalasin D and phorbol myristate acetate on the apical endocytosis of ricin in polarised Caco-2 cells

Journal of Cell Science, 1996

Apical endocytosis of 125I-ricin in Caco-2 cells was inhibited >95% by hypertonic and/or acid ... more Apical endocytosis of 125I-ricin in Caco-2 cells was inhibited >95% by hypertonic and/or acid media, consistent with the major uptake route being clathrin-mediated. The presence of apical cell surface bound ricin-gold in clathrin coated pits and vesicles was observed by electron microscopy. An electron microscopic investigation in which ricin-gold bound to the apical surface was quantitated, showed that cytochalasin D, which inhibits apical but not basolateral endocytosis, prevented movement of ricin-gold along the microvillar surface. This was consistent with an actin bound mechanochemical motor within microvilli driving the movement of membranous components towards the cell body. Cytochalasin D also caused an increase in the number of coated pits observed at the apical cell surface relative to the number observed in untreated cells. Stimulation of apical endocytosis of ricin by phorbol 12-myristate 13-acetate showed the characteristics of being mediated by protein kinase C, was...

Research paper thumbnail of Inhibition of SNARE-mediated membrane fusion by VARP

Acta Crystallographica Section A Foundations of Crystallography, 2011

Microsymposia C185 MS crystals and to outline the frontier comes through between crystalline and ... more Microsymposia C185 MS crystals and to outline the frontier comes through between crystalline and quasi-crystalline local rules.

Research paper thumbnail of Targeting of a phogrin-green fluorescent protein chimaera to insulin secretory granules of pancreatic beta-cells in transgenic mice

Diabetes & metabolism, 2002

We have generated a transgenic mouse expressing a chimaeric phogrin-enhanced green fluorescent pr... more We have generated a transgenic mouse expressing a chimaeric phogrin-enhanced green fluorescent protein (EGFP) targeted to pancreatic beta-cells by the rat insulin II promoter. The transgenic animals appear healthy, have normal weight gain and normal glucose tolerance. Morphological analyses of adult transgenic mice revealed that the fluorescent reporter molecule was specifically expressed in beta-cells of the pancreatic islet and not in extra-pancreatic tissues. The distribution of phogrin-EGFP in beta-cells, however, was heterogeneous with three distinct populations distinguishable by FACS analysis and immunofluorescence microscopy. Superficially-localized islets in the whole pancreas were readily visualized in the animals as was the developing endocrine pancreas in undissected 15.5 day old mouse embryos. We envisage that the animal will be an important resource for future investigations of islet development, regeneration and the molecular cell biology of insulin secretion.

Research paper thumbnail of Tumor necrosis factor is delivered to mitochondria where a tumor necrosis factor-binding protein is localized

Laboratory investigation; a journal of technical methods and pathology, 1998

The roles of the known tumor necrosis factor (TNF) receptors (TNFR-I and TNFR-II) and their assoc... more The roles of the known tumor necrosis factor (TNF) receptors (TNFR-I and TNFR-II) and their associated signaling pathways in mediating the diverse actions of TNF remain incompletely defined. We have found that a proportion of exogenous TNF is delivered to mitochondria as well as to lysosomes. Using confocal and immunoelectron microscopy and Western blotting of subcellular fractions, we have identified a 60-kd protein in the inner mitochondrial membrane that is recognized by a monoclonal antibody to TNFR-II. In isolated mitochondria, this protein binds [125I]-TNF. This provides evidence of a mitochondrial binding protein for an extracellular ligand and demonstrates the presence of a pathway capable of delivering TNF from the cell surface to mitochondria. These findings suggest that TNF effects on cells may be due in part to a direct effect on mitochondria.

Research paper thumbnail of Necrosis factor is trafficked to mitochondria where immunoreactive tumor necrosis factor receptor 2 is localized

Research paper thumbnail of Tumour necrosis factor is delivered to a mitochondrial receptor

Research paper thumbnail of Lysosome-endosome fusion and lysosome biogenesis

Journal of Cell Science, 2000

Recent data both from cell-free experiments and from cultured cells have shown that lysosomes can... more Recent data both from cell-free experiments and from cultured cells have shown that lysosomes can fuse directly with late endosomes to form a hybrid organelle. This has a led to a hypothesis that dense core lysosomes are in essence storage granules for acid hydrolases and that, when the former fuse with late endosomes, a hybrid organelle for digestion of endocytosed macromolecules is created. Lysosomes are then re-formed from hybrid organelles by a process involving condensation of contents. In this Commentary we review the evidence for formation of the hybrid organelles and discuss the current status of our understanding of the mechanisms of fusion and lysosome re-formation. We also review lysosome biosynthesis, showing how recent studies of lysosome-like organelles including the yeast vacuole, Drosophila eye pigment granules and mammalian secretory lysosomes have identified novel proteins involved in this process.

Research paper thumbnail of Ultrastructural distribution of endogenous immunoglobulin-G in human term amniochorion

Cell and Tissue Research, 1995

Maternal immunoglobulin-G (IgG) is known to be transported across the placental syncytiotrophobla... more Maternal immunoglobulin-G (IgG) is known to be transported across the placental syncytiotrophoblast during the period when the human fetus is incapable of manufacturing these defensive molecules. In this study we investigated the possible role of the amniochorion, that surrounds the amniotic cavity in which the fetus lies, in the transfer of immunoglobulin. Endogenous IgG was localised in the amniochorion by confocal immunofluorescence microscopy and by ultrastructural labelling of ultrathin frozen tissue sections using the prorein A-gold technique. Immunoreactivity was identified in the extracellular matrix tissues and necrotic amniotic epithelial cells. Healthy amniotic epithelial cells and cytotrophoblast ceils of the chorion laeve were devoid of endogenous IgG. These results suggest a possible nonspecific paracellular transport pathway between cytotrophoblast cells, which may conceivably contribute to the acquisition of passive immunity by the fetus, and offer a rational explanation for the presence of small quantities of maternal IgG in the amniotic fluid.

Research paper thumbnail of Confocal and conventional immunofluorescence and ultrastructural localisation of intracellular strength-giving components of human amniochorion

Journal of anatomy, 1993

Key cytoskeletal polypeptides of human fetal membranes have been localised at subcellular level u... more Key cytoskeletal polypeptides of human fetal membranes have been localised at subcellular level using confocal and conventional indirect immunofluorescence microscopy. Correlation with electron microscope data has allowed us to examine how cellular compartments of this multilaminar tissue maintain their mechanical integrity until the time of membrane rupture at parturition. Evidence is presented for myofibroblastic characteristics of cells in both the fibroblast and reticular layers which may therefore have tension-generating, position-adjustment and wound-healing roles in the amniochorion. Desmin and vimentin are coexpressed in these cells, but a small localised population of cells in the fibroblast layer contains vimentin alone. An interaction of cytokeratin filaments with nuclei and desmosomes of amniotic epithelium in vivo is demonstrated, indicating that nuclei of cells of ectodermal origin are integrated into a mechanical structure extending throughout the tissue as a whole. C...

Research paper thumbnail of Reversible assembly and disassembly of V-ATPase during the lysosome regeneration cycle

bioRxiv (Cold Spring Harbor Laboratory), Dec 21, 2023

Research paper thumbnail of Targeting of a phogrin-green fluorescent protein chimaera to insulin secretory granules of pancreatic beta-cells in transgenic mice

PubMed, Dec 1, 2002

We have generated a transgenic mouse expressing a chimaeric phogrin-enhanced green fluorescent pr... more We have generated a transgenic mouse expressing a chimaeric phogrin-enhanced green fluorescent protein (EGFP) targeted to pancreatic beta-cells by the rat insulin II promoter. The transgenic animals appear healthy, have normal weight gain and normal glucose tolerance. Morphological analyses of adult transgenic mice revealed that the fluorescent reporter molecule was specifically expressed in beta-cells of the pancreatic islet and not in extra-pancreatic tissues. The distribution of phogrin-EGFP in beta-cells, however, was heterogeneous with three distinct populations distinguishable by FACS analysis and immunofluorescence microscopy. Superficially-localized islets in the whole pancreas were readily visualized in the animals as was the developing endocrine pancreas in undissected 15.5 day old mouse embryos. We envisage that the animal will be an important resource for future investigations of islet development, regeneration and the molecular cell biology of insulin secretion.

Research paper thumbnail of Tumor Necrosis Factor-α Induces Apoptosis of Human Adipose Cells

Diabetes, Dec 1, 1997

Tumor necrosis factor-a (TNF-a) production by adipocytes is elevated in obesity, as shown by incr... more Tumor necrosis factor-a (TNF-a) production by adipocytes is elevated in obesity, as shown by increased adipose tissue TNF-a mRNA and protein levels and by increased circulating concentrations of the cytokine. Furthermore, TNF-a has distinct effects on adipose tissue including induction of insulin resistance, induction of leptin production, stimulation of lipolysis, suppression of lipogenesis, induction of adipocyte dedifferentiation, and impairment of preadipocyte differentiation in vitro. Taken together, these effects all tend to decrease adipocyte volume and number and suggest a role for TNF-a in limiting increase in fat mass. The aim of the present study was to determine if TNF-a could induce apoptosis in human adipose cells, hence delineating another mechanism by which the cytokine could act to limit the development of, or extent of, obesity. Cultured human preadipocytes and mature adipocytes in explant cultures were exposed in vitro to human TNF-a at varying concentrations for up to 24 h. Apoptosis was assessed using morphological (histology, nuclear morphology following acridine orange staining, electron microscopy) and biochemical (demonstration of internucleosomal DNA cleavage by gel electrophoresis and of annexin V staining using immunocytochemistry) criteria. In control cultures, apoptotic indexes were between 0 and 2.3% in all experiments. In the experimental systems, TNF-a induced apoptosis in both preadipocytes and adipocytes, with indexes between 5 and 25%. Therefore, TNF-a induces apoptosis of human preadipocytes and adipocytes in vitro. In view of the major metabolic role of TNF-a in human adipose tissue, and the knowledge that adipose tissue is dynamic (with cell acquisition via preadipocyte replication/differentiation and cell loss via apoptosis), these findings describe a further mechanism whereby adipose tissue mass may be modified by TNF-a. Dia

Research paper thumbnail of The ontogeny and heterogeneity of Fc-gamma receptor bearing cells in the human placenta and fetal membranes

ProQuest eBooks, 1994

The human fetus is donated passive immunity, via the placenta, in the form of maternal IgG class ... more The human fetus is donated passive immunity, via the placenta, in the form of maternal IgG class antibodies. This provides protection against pathogens while the fetus is immunologically immature. Immunocytochemistry has been performed on placental tissues to localise endogenous IgG and three subtypes of IgG receptor designated FcgammaRI (CD64), FcgammaRII (CDw32) and FcgammaRIII (CD16). Ultrastructural immunocytochemistry has been used to investigate the subcellular distribution of IgG. FcgammaRI is expressed by mesenchymal cells of term and first trimester placentae. FcgammaRII is expressed by endothelial cells and FcgammaRIII is expressed by syncytiotrophoblast. Endogenous IgG is restricted to the syncytiotrophoblast in first trimester villi indicating that transport is inhibited in early stages of gestation. At term IgG is associated with extracellular matrix, serum, endothelial cells, mesenchymal cells and syncytiotrophoblast suggesting that it is efficiently transported across the trophoblast. Ultrastructural observations of IgG distribution indicate that it is taken up by receptor-mediated endocytosis into the syncytiotrophoblast and reaches an endosome-like compartment. In term amniochorion FcgammaRI, FcgammaRII and FcgammaRIII are expressed by leucocytes in the maternal decidua and heterogeneous populations of mesenchymal cells in the fetal connective tissues suggesting that they possess an immunoregulatory role. Endogenous IgG is restricted to the extracellular matrix and cells possessing Fcgamma receptors. Cytotrophoblast and amniotic epithelial cells, which do not possess Fcgamma receptors, contain no endogenous antibodies. The hypothesis is proposed that cytotrophoblast cells are a barrier to IgG transmission across the placenta. This accounts for the paradox that there are low levels of transport in the first trimester when the syncytiotrophoblast expresses Fcgamma receptors. Cytotrophoblast cells form an almost continuous layer underlying the syncytiotrophoblast in the first trimester which becomes attenuated as the placenta matures. Furthermore, IgG may diffuse non-specifically between cytotrophoblast cells of the amniochorion and thus contribute to the acquisition of passive immunity. These data suggest that IgG is transmitted across the vascular system of the placenta and is not acquired via amniotic fluid

Research paper thumbnail of Localisation of Nramp1 in macrophages: modulation with activation and infection

Journal of Cell Science, Oct 1, 1998

The Lsh/Ity/Bcg gene was identified in mice for its role in regulating resistance and susceptibil... more The Lsh/Ity/Bcg gene was identified in mice for its role in regulating resistance and susceptibility to intracellular pathogens: Leishmania donovani, Salmonella typhimurium, Mycobacterium bovis and M. intracellulare (reviewed by Blackwell, 1989; Schurr et al., 1991). It is also a major candidate gene for autoimmune disease susceptibility in man (reviewed by Blackwell and Searle, 1998). A candidate for Lsh/Ity/Bcg, Nramp1 (natural resistance-associated macrophage protein), was identified by positional cloning (Vidal et al., 1993) and the full-length sequence obtained (Barton et al., 1994), and its candidacy was confirmed using transfected macrophages in vitro (Barton et al., 1995) and gene disruption in vivo (Vidal et al., 1995). Murine Nramp1 expression is macrophage-restricted and affects the capacity of the host to control intracellular replication of microorganisms , all of which ultimately reside in the phagolysosome. Susceptibility is associated with a nonconservative amino acid substitution at position 169 (glycine resistant to aspartic acid susceptible), caused by a point mutation in the region encoding the fourth putative transmembrane spanning domain (TMD; Vidal et al., 1995). The polypeptide Nramp1 is a hydrophobic integral membrane protein, with the following predicted features: 10-12 TMDs, a

Research paper thumbnail of ATP Regulation in Adult Rat Cardiomyocytes

Journal of Biological Chemistry, Sep 1, 2006

The mechanisms that enable the heart to rapidly increase ATP supply in line with increased demand... more The mechanisms that enable the heart to rapidly increase ATP supply in line with increased demand have not been fully elucidated. Here we used an adenoviral system to express the photoproteins luciferase and aequorin, targeted to the mitochondria or cytosol of adult cardiomyocytes, to investigate the interrelationship between ATP and Ca 2؉ in these compartments. In neither compartment were changes in free [ATP] observed upon increased workload (addition of isoproterenol) in myocytes that were already beating. However, when myocytes were stimulated to beat rapidly from rest, in the presence of isoproterenol, a significant but transient drop in mitochondrial [ATP] ([ATP] m) occurred (on average to 10% of the initial signal). Corresponding changes in cytosolic [ATP] ([ATP] c) were much smaller (<5%), indicating that [ATP] c was effectively buffered in this compartment. Although mitochondrial [Ca 2؉ ] ([Ca 2؉ ] m) is an important regulator of respiratory chain activity and ATP production in other cells, the kinetics of mitochondrial Ca 2؉ transport are controversial. Parallel experiments in cells expressing mitochondrial aequorin showed that the drop in [ATP] m occurred over the same time scale as average [Ca 2؉ ] m was increasing. Conversely, in the absence or presence of isoproterenol, clear beat-to-beat peaks in [Ca 2؉ ] m were observed at 0.9 or 1.3 M, respectively, concentrations similar to those observed in the cytosol. These results suggest that mitochondrial Ca 2؉ transients occur during the contractile cycle and are translated into a time-averaged increase in mitochondrial ATP production that keeps pace with increased cytosolic demand.

Research paper thumbnail of Lysosome-endosome fusion and lysosome biogenesis

Journal of Cell Science, May 1, 2000

Recent data both from cell-free experiments and from cultured cells have shown that lysosomes can... more Recent data both from cell-free experiments and from cultured cells have shown that lysosomes can fuse directly with late endosomes to form a hybrid organelle. This has a led to a hypothesis that dense core lysosomes are in essence storage granules for acid hydrolases and that, when the former fuse with late endosomes, a hybrid organelle for digestion of endocytosed macromolecules is created. Lysosomes are then reformed from hybrid organelles by a process involving condensation of contents. In this Commentary we review the evidence for formation of the hybrid organelles and discuss the current status of our understanding of the mechanisms of fusion and lysosome reformation. We also review lysosome biosynthesis, showing how recent studies of lysosome-like organelles including the yeast vacuole, Drosophila eye pigment granules and mammalian secretory lysosomes have identified novel proteins involved in this process.

Research paper thumbnail of COMMENTARY Lysosome-endosome fusion and lysosome biogenesis

Recent data both from cell-free experiments and from cultured cells have shown that lysosomes can... more Recent data both from cell-free experiments and from cultured cells have shown that lysosomes can fuse directly with late endosomes to form a hybrid organelle. This has a led to a hypothesis that dense core lysosomes are in essence storage granules for acid hydrolases and that, when the former fuse with late endosomes, a hybrid organelle for digestion of endocytosed macromolecules is created. Lysosomes are then re-formed from hybrid organelles by a process involving condensation of contents. In this Commentary we review the evidence for formation of the hybrid organelles and discuss the current status of our understanding of the mechanisms of fusion and lysosome re-formation. We also review lysosome biosynthesis, showing how recent studies of lysosome-like organelles including the yeast vacuole, Drosophila eye pigment granules and mammalian secretory lysosomes have identified novel proteins involved in this process. SUMMARY

Research paper thumbnail of Fusion with

Autophagy-receptors link myosin VI to autophagosomes to mediate Tom1-dependent autophagosome matu... more Autophagy-receptors link myosin VI to autophagosomes to mediate Tom1-dependent autophagosome maturation and

Research paper thumbnail of Supplemental Experimental Procedures Materials

Antibodies were obtained from the following: a-flotillin1 and a-flotil-lin2 mAbs and a-caveolin1 ... more Antibodies were obtained from the following: a-flotillin1 and a-flotil-lin2 mAbs and a-caveolin1 pAb (BD Biosciences, Oxford); anti-flotil-lin1 and anti-flotillin2 rabbit pAbs (Santa Cruz Biotechnology, Hei-delberg, Germany); and anti-CD59 mAB and anti-CD59 goat pAB (R&D Systems, Abingdon, UK). Fluorescently labeled secondary antibodies were obtained from Molecular Probes (Cambridge Bio-science, Cambridge). A polyclonal antibody was raised against flotillin1 in rabbits, with full-length flotillin1 used as an antigen. The antibody was affinity purified with full-length flotillin1 coupled to CnBr-activated sepharose. Cell Culture, Transfections, and Immunofluorescence HeLa and MEF cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), 10 % fetal calf serum (FCS), and 1 % Pen and Strep at 10 % CO2. MEFs were freshly isolated from wild-type (WT) and caveolin null mice and discarded after passage six. All experiments

Research paper thumbnail of The ontogeny and heterogeneity of Fc-gamma receptor bearing cells in the human placenta and fetal membranes

The human fetus is donated passive immunity, via the placenta, in the form of maternal IgG class ... more The human fetus is donated passive immunity, via the placenta, in the form of maternal IgG class antibodies. This provides protection against pathogens while the fetus is immunologically immature. Immunocytochemistry has been performed on placental tissues to localise endogenous IgG and three subtypes of IgG receptor designated FcgammaRI (CD64), FcgammaRII (CDw32) and FcgammaRIII (CD16). Ultrastructural immunocytochemistry has been used to investigate the subcellular distribution of IgG. FcgammaRI is expressed by mesenchymal cells of term and first trimester placentae. FcgammaRII is expressed by endothelial cells and FcgammaRIII is expressed by syncytiotrophoblast. Endogenous IgG is restricted to the syncytiotrophoblast in first trimester villi indicating that transport is inhibited in early stages of gestation. At term IgG is associated with extracellular matrix, serum, endothelial cells, mesenchymal cells and syncytiotrophoblast suggesting that it is efficiently transported across the trophoblast. Ultrastructural observations of IgG distribution indicate that it is taken up by receptor-mediated endocytosis into the syncytiotrophoblast and reaches an endosome-like compartment. In term amniochorion FcgammaRI, FcgammaRII and FcgammaRIII are expressed by leucocytes in the maternal decidua and heterogeneous populations of mesenchymal cells in the fetal connective tissues suggesting that they possess an immunoregulatory role. Endogenous IgG is restricted to the extracellular matrix and cells possessing Fcgamma receptors. Cytotrophoblast and amniotic epithelial cells, which do not possess Fcgamma receptors, contain no endogenous antibodies. The hypothesis is proposed that cytotrophoblast cells are a barrier to IgG transmission across the placenta. This accounts for the paradox that there are low levels of transport in the first trimester when the syncytiotrophoblast expresses Fcgamma receptors. Cytotrophoblast cells form an almost continuous layer underlying the syncytiotrophoblast in the first trimester which becomes attenuated as the placenta matures. Furthermore, IgG may diffuse non-specifically between cytotrophoblast cells of the amniochorion and thus contribute to the acquisition of passive immunity. These data suggest that IgG is transmitted across the vascular system of the placenta and is not acquired via amniotic fluid

Research paper thumbnail of The effects of cytochalasin D and phorbol myristate acetate on the apical endocytosis of ricin in polarised Caco-2 cells

Journal of Cell Science, 1996

Apical endocytosis of 125I-ricin in Caco-2 cells was inhibited >95% by hypertonic and/or acid ... more Apical endocytosis of 125I-ricin in Caco-2 cells was inhibited >95% by hypertonic and/or acid media, consistent with the major uptake route being clathrin-mediated. The presence of apical cell surface bound ricin-gold in clathrin coated pits and vesicles was observed by electron microscopy. An electron microscopic investigation in which ricin-gold bound to the apical surface was quantitated, showed that cytochalasin D, which inhibits apical but not basolateral endocytosis, prevented movement of ricin-gold along the microvillar surface. This was consistent with an actin bound mechanochemical motor within microvilli driving the movement of membranous components towards the cell body. Cytochalasin D also caused an increase in the number of coated pits observed at the apical cell surface relative to the number observed in untreated cells. Stimulation of apical endocytosis of ricin by phorbol 12-myristate 13-acetate showed the characteristics of being mediated by protein kinase C, was...

Research paper thumbnail of Inhibition of SNARE-mediated membrane fusion by VARP

Acta Crystallographica Section A Foundations of Crystallography, 2011

Microsymposia C185 MS crystals and to outline the frontier comes through between crystalline and ... more Microsymposia C185 MS crystals and to outline the frontier comes through between crystalline and quasi-crystalline local rules.

Research paper thumbnail of Targeting of a phogrin-green fluorescent protein chimaera to insulin secretory granules of pancreatic beta-cells in transgenic mice

Diabetes & metabolism, 2002

We have generated a transgenic mouse expressing a chimaeric phogrin-enhanced green fluorescent pr... more We have generated a transgenic mouse expressing a chimaeric phogrin-enhanced green fluorescent protein (EGFP) targeted to pancreatic beta-cells by the rat insulin II promoter. The transgenic animals appear healthy, have normal weight gain and normal glucose tolerance. Morphological analyses of adult transgenic mice revealed that the fluorescent reporter molecule was specifically expressed in beta-cells of the pancreatic islet and not in extra-pancreatic tissues. The distribution of phogrin-EGFP in beta-cells, however, was heterogeneous with three distinct populations distinguishable by FACS analysis and immunofluorescence microscopy. Superficially-localized islets in the whole pancreas were readily visualized in the animals as was the developing endocrine pancreas in undissected 15.5 day old mouse embryos. We envisage that the animal will be an important resource for future investigations of islet development, regeneration and the molecular cell biology of insulin secretion.

Research paper thumbnail of Tumor necrosis factor is delivered to mitochondria where a tumor necrosis factor-binding protein is localized

Laboratory investigation; a journal of technical methods and pathology, 1998

The roles of the known tumor necrosis factor (TNF) receptors (TNFR-I and TNFR-II) and their assoc... more The roles of the known tumor necrosis factor (TNF) receptors (TNFR-I and TNFR-II) and their associated signaling pathways in mediating the diverse actions of TNF remain incompletely defined. We have found that a proportion of exogenous TNF is delivered to mitochondria as well as to lysosomes. Using confocal and immunoelectron microscopy and Western blotting of subcellular fractions, we have identified a 60-kd protein in the inner mitochondrial membrane that is recognized by a monoclonal antibody to TNFR-II. In isolated mitochondria, this protein binds [125I]-TNF. This provides evidence of a mitochondrial binding protein for an extracellular ligand and demonstrates the presence of a pathway capable of delivering TNF from the cell surface to mitochondria. These findings suggest that TNF effects on cells may be due in part to a direct effect on mitochondria.

Research paper thumbnail of Necrosis factor is trafficked to mitochondria where immunoreactive tumor necrosis factor receptor 2 is localized

Research paper thumbnail of Tumour necrosis factor is delivered to a mitochondrial receptor

Research paper thumbnail of Lysosome-endosome fusion and lysosome biogenesis

Journal of Cell Science, 2000

Recent data both from cell-free experiments and from cultured cells have shown that lysosomes can... more Recent data both from cell-free experiments and from cultured cells have shown that lysosomes can fuse directly with late endosomes to form a hybrid organelle. This has a led to a hypothesis that dense core lysosomes are in essence storage granules for acid hydrolases and that, when the former fuse with late endosomes, a hybrid organelle for digestion of endocytosed macromolecules is created. Lysosomes are then re-formed from hybrid organelles by a process involving condensation of contents. In this Commentary we review the evidence for formation of the hybrid organelles and discuss the current status of our understanding of the mechanisms of fusion and lysosome re-formation. We also review lysosome biosynthesis, showing how recent studies of lysosome-like organelles including the yeast vacuole, Drosophila eye pigment granules and mammalian secretory lysosomes have identified novel proteins involved in this process.

Research paper thumbnail of Ultrastructural distribution of endogenous immunoglobulin-G in human term amniochorion

Cell and Tissue Research, 1995

Maternal immunoglobulin-G (IgG) is known to be transported across the placental syncytiotrophobla... more Maternal immunoglobulin-G (IgG) is known to be transported across the placental syncytiotrophoblast during the period when the human fetus is incapable of manufacturing these defensive molecules. In this study we investigated the possible role of the amniochorion, that surrounds the amniotic cavity in which the fetus lies, in the transfer of immunoglobulin. Endogenous IgG was localised in the amniochorion by confocal immunofluorescence microscopy and by ultrastructural labelling of ultrathin frozen tissue sections using the prorein A-gold technique. Immunoreactivity was identified in the extracellular matrix tissues and necrotic amniotic epithelial cells. Healthy amniotic epithelial cells and cytotrophoblast ceils of the chorion laeve were devoid of endogenous IgG. These results suggest a possible nonspecific paracellular transport pathway between cytotrophoblast cells, which may conceivably contribute to the acquisition of passive immunity by the fetus, and offer a rational explanation for the presence of small quantities of maternal IgG in the amniotic fluid.

Research paper thumbnail of Confocal and conventional immunofluorescence and ultrastructural localisation of intracellular strength-giving components of human amniochorion

Journal of anatomy, 1993

Key cytoskeletal polypeptides of human fetal membranes have been localised at subcellular level u... more Key cytoskeletal polypeptides of human fetal membranes have been localised at subcellular level using confocal and conventional indirect immunofluorescence microscopy. Correlation with electron microscope data has allowed us to examine how cellular compartments of this multilaminar tissue maintain their mechanical integrity until the time of membrane rupture at parturition. Evidence is presented for myofibroblastic characteristics of cells in both the fibroblast and reticular layers which may therefore have tension-generating, position-adjustment and wound-healing roles in the amniochorion. Desmin and vimentin are coexpressed in these cells, but a small localised population of cells in the fibroblast layer contains vimentin alone. An interaction of cytokeratin filaments with nuclei and desmosomes of amniotic epithelium in vivo is demonstrated, indicating that nuclei of cells of ectodermal origin are integrated into a mechanical structure extending throughout the tissue as a whole. C...