Biomedical subjects

Simon C Watkins

Publications and source records attributed to Simon C Watkins.

At least 109 records · Page 6Linked to original sources

PubMed2003

Multimodal optical imaging.

The recent resurgence of interest in the use of intravital microscopy in lung research is a manifestation of extraordinary progress in visual imaging and optical microscopy. This review evaluates the tools and instrumentation available for a number of imaging modalities, with particular attention to recent technological advances, and addresses recent progress in use of optical imaging techniques in basic pulmonary research.1 Limitations of existing methods and anticipated future developments are also identified. Although there have also been major advances made in the use of magnetic resonance imaging, positron emission tomography, and X-ray and computed tomography to image intact lungs and while these technologies have been instrumental in advancing the diagnosis and treatment of patients, the purpose of this review is to outline developing optical methods that can be evaluated for use in basic research in pulmonary biology.

Animals↗

PubMed2003

Localization of the BiP molecular chaperone with respect to endoplasmic reticulum foci containing the cystic fibrosis transmembrane conductance regulator in yeast.

Almost all secreted proteins pass through the endoplasmic reticulum (ER), an organelle that is equipped to tolerate and/or degrade misfolded proteins. We report here that yeast expressing the cystic fibrosis transmembrane conductance regulator (CFTR) concentrate the protein at defined sites in the ER membrane that are not necessarily enriched for the ER molecular chaperone BiP. We propose that these sites are Russell bodies, an ER subcompartment in which misfolded proteins are stored and can be targeted for degradation.

Carrier Proteins↗

PubMed2003

Body window-enabled in vivo multicolor imaging of transplanted mouse islets expressing an insulin-Timer fusion protein.

Type 1 diabetes results from the selective destruction of insulin-producing beta cells in the islets of Langerhans, and autoimmune T cells are thought to be the mediators of this destruction. T cells are also responsible for allorejection once the islets are transplanted into a patient to reduce the negative consequences of a lack of insulin. To better understand these processes, we have developed a transgenic mouse expressing proinsulin II tagged with a live-cell fluorescent reporter protein, Timer. Timer protein is unique because it changes color from green to red in the first 24 h after synthesis. With this marker, insulin synthesis can be carefully monitored through fluorescent changes over time. To complement this new biotechnological research tool, we designed a body window to allow for in vivo imaging over time of the islets transplanted under the kidney capsule. The window device, which is sutured to replace the underlying skin and body wall over the site of islet transplantation, may be used to simultaneously observe beta cells and T cells that have been labeled with a fluorochrome distinguishable from Timer. The imaging of both insulin-producing cells and T cells may be carried out repeatedly for a week or more with no need for repeated surgery, while preserving the life of the studied animal.

Animals↗

PubMed2003

Gene transfer of manganese superoxide dismutase extends islet graft function in a mouse model of autoimmune diabetes.

Islet transplantation is a promising cure for diabetes. However, inflammation, allorejection, and recurrent autoimmune damage all may contribute to early graft loss. Pancreatic islets express lower levels of antioxidant genes than most other tissues of the body, and beta-cells in particular are sensitive to oxidative damage. Therefore, damage from oxidative stress may pose a major obstacle to islet replacement therapy in that both the islet isolation and transplantation processes generate oxygen radicals. To determine whether antioxidant gene overexpression in isolated pancreatic islets can prevent oxidative damage and prolong islet function after transplantation, we used the NOD mouse model to study oxidative stress encountered during both transplantation and autoimmune attack. We transferred an antioxidant gene, manganese superoxide dismutase (MnSOD), by adenoviral infection into isolated islets that were transplanted into streptozotocin-treated NODscid recipient mice. Functioning islet grafts were subsequently exposed to diabetogenic spleen cells and monitored until graft failure. The results show that islet grafts overexpressing MnSOD functioned approximately 50% longer than control grafts. This significant prolongation of graft function suggests that the antioxidant activity of MnSOD is beneficial to transplanted islet survival and may be used in combination with other strategies aimed at islet graft protection.

Adenoviridae↗

PubMed2002

Trafficking of the Ca2+-activated K+ channel, hIK1, is dependent upon a C-terminal leucine zipper.

We demonstrate that the C-terminal truncation of hIK1 results in a loss of functional channels. This could be caused by either (i) a failure of the channel to traffic to the plasma membrane or (ii) the expression of non-functional channels. To delineate among these possibilities, a hemagglutinin epitope was inserted into the extracellular loop between transmembrane domains S3 and S4. Surface expression and channel function were measured by immunofluorescence, cell surface immunoprecipitation, and whole-cell patch clamp techniques. Although deletion of the last 14 amino acids of hIK1 (L414STOP) had no effect on plasma membrane expression and function, deletion of the last 26 amino acids (K402STOP) resulted in a complete loss of membrane expression. Mutation of the leucine heptad repeat ending at Leu(406) (L399A/L406A) completely abrogated membrane localization. Additional mutations within the heptad repeat (L385A/L392A, L392A/L406A) or of the a positions (I396A/L403A) resulted in a near-complete loss of membrane-localized channel. In contrast, mutating individual leucines did not compromise channel trafficking or function. Both membrane localization and function of L399A/L406A could be partially restored by incubation at 27 degrees C. Co-immunoprecipitation studies demonstrated that leucine zipper mutations do not compromise multimer formation. In contrast, we demonstrated that the leucine zipper region of hIK1 is capable of co-assembly and that this is dependent upon an intact leucine zipper. Finally, this leucine zipper is conserved in another member of the gene family, SK3. However, mutation of the leucine zipper in SK3 had no effect on plasma membrane localization or function. In conclusion, we demonstrate that the C-terminal leucine zipper is critical to facilitate correct folding and plasma membrane trafficking of hIK1, whereas this function is not conserved in other gene family members.

Base Sequence↗

PubMed2002

The autosomal recessive hypercholesterolemia (ARH) protein interfaces directly with the clathrin-coat machinery.

The low density lipoprotein (LDL) receptor plays a pivotal role in cholesterol metabolism. Inherited mutations that disturb the activity of the receptor lead to elevations in plasma cholesterol levels and early-onset coronary atherosclerosis. Defects in either the LDL receptor or apolipoprotein B, the proteinaceous component of LDL particles that binds the LDL receptor, elevate circulating LDL-cholesterol levels in an autosomal-dominant fashion, with heterozygotes displaying values between homozygous and normal individuals. Rarely, similar clinical phenotypes occur with a recessive pattern of inheritance, and several genetic lesions in the autosomal recessive hypercholesterolemia (ARH) gene on chromosome 1 have been mapped in this class of patients. ARH has an N-terminal phosphotyrosine-binding (PTB) domain evolutionarily related to that found in Disabled-2 and numb, two endocytic proteins. PTB domains bind to the consensus sequence FXNPXY, corresponding to the internalization motif of the LDL receptor. We show here that in addition to the FXNPXY sequence, ARH binds directly to soluble clathrin trimers and to clathrin adaptors by a mode involving the independently folded appendage domain of the beta subunit. At steady state, ARH colocalizes with endocytic proteins in HeLa cells, and the LDL receptor fluxes through peripheral ARH-positive sites before delivery to early endosomes. Because ARH also binds directly to phosphoinositides, which regulate clathrin bud assembly at the cell surface, our data suggest that in ARH patients, defective sorting adaptor function in hepatocytes leads to faulty LDL receptor traffic and hypercholesterolemia.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Expression and localization of human lysozyme in the endosperm of transgenic rice.

In order to understand the characteristics of recombinant protein expression and sublocalization in rice ( Oryza sativa L.) endosperm, we examined the expression level of human lysozyme protein and its subcellular location in transgenic rice seeds driven by rice glutelin and globulin promoters and signal peptides. A time course of human lysozyme expression during endosperm development was analyzed. The results showed that the expression profile of recombinant protein accumulation in endosperm paralleled that of the two storage proteins. Immunofluorescence microscopy revealed that human lysozyme and storage proteins co-localized to type-II protein bodies. Both promoter-signal peptide parings targeted recombinant protein to the protein bodies. In addition, a transgenic line with a higher lysozyme expression level exhibited morphologically different protein bodies with an unbalanced composition of lysozyme and native storage proteins. The high-level expression of recombinant protein distorted the trafficking and sorting of native storage proteins in rice endosperm and affected the expression of native storage protein.

Gene Expression Regulation, Enzymologic↗

PubMed2002

Disabled-2 exhibits the properties of a cargo-selective endocytic clathrin adaptor.

Clathrin-coated pits at the cell surface select material for transportation into the cell interior. A major mode of cargo selection at the bud site is via the micro 2 subunit of the AP-2 adaptor complex, which recognizes tyrosine-based internalization signals. Other internalization motifs and signals, including phosphorylation and ubiquitylation, also tag certain proteins for incorporation into a coated vesicle, but the mechanism of selection is unclear. Disabled-2 (Dab2) recognizes the FXNPXY internalization motif in LDL-receptor family members via an N-terminal phosphotyrosine-binding (PTB) domain. Here, we show that in addition to binding AP-2, Dab2 also binds directly to phosphoinositides and to clathrin, assembling triskelia into regular polyhedral coats. The FXNPXY motif and phosphoinositides contact different regions of the PTB domain, but can stably anchor Dab2 to the membrane surface, while the distal AP-2 and clathrin-binding determinants regulate clathrin lattice assembly. We propose that Dab2 is a typical member of a growing family of cargo-specific adaptor proteins, including beta-arrestin, AP180, epsin, HIP1 and numb, which regulate clathrin-coat assembly at the plasma membrane by synchronizing cargo selection and lattice polymerization events.

Adaptor Protein Complex 2↗

PubMed2002

Chronic exposure to interleukin 1beta induces a delayed and reversible alteration in excitation-contraction coupling of cultured cardiomyocytes.

While proinflammatory cytokines can depress cardiac contractility, the mechanism by which this occurs remains unclear. To clarify the cellular effects of interleukin (IL)-1beta, we assessed contractility, calcium homeostasis, and gene expression in cardiomyocytes exposed to this proinflammatory cytokine. Neonatal rat cardiomyocytes were exposed to IL-1beta in the presence or absence of an inhibitor of nitric oxide (NO) synthase. Videomicroscopy was used to follow calcium transients (Fura-2 fluorescence) and amplitude of contraction, both unstimulated and after isoproterenol challenge. Gene expression was assessed by Northern and Western blot analyses. Both basal contractility (amplitude of contraction, maximum speed of contraction and relaxation) and amplitude of calcium transients were decreased, respectively, ca. 60% ( P< or =0.05) and ca. 40% ( P< or =0.05) after 3 days of IL-1beta exposure. Contractile function and amplitude of calcium transients returned to control values when cells where cultured an additional 3 days in the absence of IL-1beta. IL-1beta-treated cells had reduced responses to isoproterenol as evidenced by a lack of enhanced amplitude of contraction and a reduction in cAMP production. IL-1beta decreased the expression of genes important to the regulation of calcium homeostasis (phospholamban, sarcoplasmic reticulum calcium ATPase) at both the transcript and protein level. Alterations in contractile function did not occur through NO-mediated pathways. These results support the hypothesis that IL-1beta may play an important role in contractile dysfunction through alterations in calcium homeostasis.

Adrenergic beta-Agonists↗

PubMed2002

Internalization of circulating apoptotic cells by splenic marginal zone dendritic cells: dependence on complement receptors and effect on cytokine production.

Under steady-state conditions, internalization of self-antigens embodied in apoptotic cells by dendritic cells (DCs) resident in peripheral tissue followed by DC migration and presentation of self-peptides to T cells in secondary lymphoid organs are key steps for induction and maintenance of peripheral T-cell tolerance. We show here that, besides this traffic of apoptotic cells mediated by peripheral tissue-resident DCs, splenic marginal zone DCs rapidly ingest circulating apoptotic leukocytes, process apoptotic cell-derived peptides into major histocompatibility complex class II (MHC-II) molecules, and acquire CD8alpha during their mobilization to T-cell areas of splenic follicles. Because apoptotic cells activate complement and some complement factors are opsonins for phagocytosis and play roles in the maintenance of peripheral tolerance, we investigated the role of complement receptors (CRs) in relation to phagocytosis of apoptotic cells by DCs. Apoptotic cell uptake by marginal zone DCs was mediated in part via CR3 (CD11b/CD18) and, to a lesser extent, CR4 (CD11c/CD18) and was reduced significantly in vivo in hypocomplementemic animals. Following phagocytosis of apoptotic cells, DCs exhibited decreased levels of mRNA and secretion of the proinflammatory cytokines interleukin 1alpha (IL-1alpha), IL-1beta, IL-6, IL-12p70, and tumor necrosis factor alpha (TNF-alpha), without effect on the anti-inflammatory mediator transforming growth factor beta1 (TGF-beta1). This selective inhibitory effect was at least partially mediated through C3bi-CD11b/CD18 interaction. Characterization of apoptotic cell/DC interaction and its outcome provides insight into the mechanisms by which apoptotic cells affect DC function without disrupting peripheral tolerance.

Animals↗

PubMed2002

Hepatocyte Fas-associating death domain protein/mediator of receptor-induced toxicity (FADD/MORT1) levels increase in response to pro-apoptotic stimuli.

We examined the regulation of Fas-associating death domain (FADD) protein as an important adaptor molecule in apoptosis signaling and hypothesized that the regulation of FADD could contribute to hepatocyte death. FADD/mediator of receptor-induced toxicity (MORT1) is required for activation of several signaling pathways of cell death. In this study we report the interesting and unexpected result that actinomycin D increased the expression of FADD protein, and we demonstrate that other cellular stresses like ultraviolet irradiation or heat shock could also increase FADD levels in hepatocytes. In cells treated with actinomycin D, FADD levels were elevated homogeneously in the cytoplasm. The increase in cytoplasmic FADD protein by actinomycin D or FADD overexpression alone both correlated with cell death, and specific antisense inhibition of FADD expression consistently diminished approximately 30% of the cell death induced by actinomycin D. These data indicate that FADD protein expression can increase rapidly in hepatocytes exposed to broadly cytotoxic agents.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Efficiency of protein transduction is cell type-dependent and is enhanced by dextran sulfate.

Protein transduction domains (PTDs), both naturally occurring and synthetic, have been increasingly utilized to deliver biologically active agents to a variety of cell types in vitro and in vivo. We report that in addition to previously characterized arginine-rich PTDs, including TAT, lysine homopolymers were able to mediate transduction of a wide variety of cell types, as measured by flow cytometric and enzymatic assays. The efficiency of PTD-mediated transduction was influenced by the cell type tested, although polylysine homopolymers demonstrate levels of internalization that consistently exceeded those of TAT and arginine homopolymers. Transduction of arginine/lysine-rich PTDs occurred at 4 degrees C and following depletion of cellular ATP pools, albeit generally at reduced levels. Although transduction was reduced in Chinese hamster ovary mutant lines deficient in either heparan sulfate or glycosaminoglycan synthesis, uptake was restored to wild-type levels by incubating target cells with dextran sulfate. The enhancement of transduction by dextran sulfate suggests that electrostatic interactions play an important first step in the process by which PTDs and their cargo traverse the plasma membrane.

Animals↗

PubMed2002

Nerve growth factor-induced differentiation changes the cellular organization of regulated Peptide release by PC12 cells.

PC12 cells, like endocrine chromaffin cells, undergo neuronal-like differentiation in response to nerve growth factor (NGF). Here we report that this phenotype conversion produces major changes in release of a green fluorescent protein-tagged neuropeptide-hormone. First, the spatial distribution of the releasable pool is altered; peptide release from untreated cells is supported predominantly by membrane-proximal vesicles, whereas a diffuse pool at the ends of processes is used by NGF-treated cells. Second, the time course of release evoked by photolysis of caged Ca(2+) is faster after differentiation. High-resolution measurements suggest that a slow step before membrane fusion dominates the kinetics of release in untreated cells. Finally, the effect of actin microfilament depolymerization on total release is altered by NGF treatment. This implies that the mechanism that limits the size of the releasable pool is altered by phenotype conversion. Therefore, the cellular organization of peptide release is plastic and changes in response to NGF. This flexibility may be used to generate cell-specific release properties.

Actin Cytoskeleton↗

PubMed2002

Distribution of gelsolin and phosphoinositol 4,5-bisphosphate in lamellipodia during EGF-induced motility.

During induced cell motility the actin cytoskeleton at the leading edge must undergo constant reorganization. Recently, phosphoinositides have been shown to be central to cytoskeleton-membrane linkages and actin organization and turnover. Epidermal growth factor (EGF) receptor (EGFR)-mediated cell motility requires phospholipase C-gamma (PLCgamma), hydrolysis of phosphoinsotide 4,5-bisphosphate (PIP(2)) and subsequent release of gelsolin. We hypothesized this led to the mobilization of PIP(2)-binding proteins which modify the actin cytoskeleton and thus sought to determine whether the leading edge was a site of active PIP(2) hydrolysis and gelsolin redistribution to cytoskeleton. Herein, we report that during EGF-induced motility, the leading edge's submembranous region constitutes a distinct subcellular locale. The relevant phosphoinositide composition of this space was determined by probing with an antibody to PIP(2) and a green fluorescence protein (GFP)-tagged pleckstrin homology (PH) domain of PLCdelta (GFP-PH) that recognizes both PIP(2) and inositol 1,4,5-trisphosphate (IP(3)). PIP(2) was absent from leading lamellipodia despite an increase in IP(3) generation, suggesting an increase in PIP(2) hydrolysis at the leading edge. Visualized with immunofluorescence, gelsolin preferentially concentrated near the leading edge in a punctate fashion. Examining the Triton X-insoluble actin cytoskeleton fractions, we observe a PLCgamma-dependent increase of gelsolin incorporation upon EGF stimulation. At a molecular level, field emission scanning electron microscopy (FE-SEM) shows that gelsolin incorporates preferentially into the submembranous actin arcs at the leading edge of the lamellipodia. Together these data suggest a model of PIP(2) hydrolysis at the leading edge causing a localized release of PIP(2)-binding proteins-particularly gelsolin-that drives cytoskeletal rearrangement and protrusion.

Actin Depolymerizing Factors↗

PubMed2002

Intranuclear localization of apoptosis-inducing factor (AIF) and large scale DNA fragmentation after traumatic brain injury in rats and in neuronal cultures exposed to peroxynitrite.

Programmed cell death occurs after ischemic, excitotoxic, and traumatic brain injury (TBI). Recently, a caspase-independent pathway involving intranuclear translocation of mitochondrial apoptosis-inducing factor (AIF) has been reported in vitro; but whether this occurs after acute brain injury was unknown. To address this question adult rats were sacrificed at various times after TBI. Western blot analysis on subcellular protein fractions demonstrated intranuclear localization of AIF in ipsilateral cortex and hippocampus at 2-72 h. Immunocytochemical analysis showed AIF labeling in neuronal nuclei with DNA fragmentation in the ipsilateral cortex and hippocampus. Immunoelectronmicroscopy verified intranuclear localization of AIF in hippocampal neurons after TBI, primarily in regions of euchromatin. Large-scale DNA fragmentation ( approximately 50 kbp), a signature event in AIF-mediated cell death, was detected in ipsilateral cortex and hippocampi by 6 h. Neuron-enriched cultures exposed to peroxynitrite also demonstrated intranuclear AIF and large-scale DNA fragmentation concurrent with impaired mitochondrial respiration and cell death, events that are inhibited by treatment with a peroxynitrite decomposition catalyst. Intranuclear localization of AIF and large-scale DNA fragmentation occurs after TBI and in neurons under conditions of oxidative/nitrosative stress, providing the first evidence of this alternative mechanism by which programmed cell death may proceed in neurons after brain injury.

Active Transport, Cell Nucleus↗

PubMed2002

Peroxisomal localization of inducible nitric oxide synthase in hepatocytes.

Shock states induce the expression of inducible nitric oxide synthase (iNOS) in both Kupffer cells and hepatocytes in the liver, but little is known about its subcellular localization in these cells. Studies were undertaken to characterize the subcellular location of iNOS in hepatocytes in response to sepsis. By immunofluorescence analysis, intraperitoneal challenge with bacterial lipopolysaccharide induced cytosolic iNOS in Kupffer cells but punctate labeling in hepatocytes. Cultured rat hepatocytes exposed to interferon gamma, interleukin 1, and tumor necrosis factor alpha showed iNOS protein expression within peroxisomes as early as 4 hours after stimulation, as determined by colabeling for catalase or PMP70. To a lesser extent, iNOS was also observed associated with the plasma membrane and in undefined intracellular aggregates. The nitric oxide synthase (NOS) antagonist L-N-imino-ornithine (L-NIO) did not affect the expression of iNOS within peroxisomes, cytoplasmic aggregates, or cytosol but increased plasma membrane localization of iNOS. Human iNOS transduced into iNOS-null mouse hepatocytes using an adenoviral vector also localized to peroxisomes. The expression of iNOS often resulted in the disappearance of detectable catalase in many hepatocytes. In conclusion, these studies establish the peroxisome as a site of iNOS localization in hepatocytes and show a relationship between iNOS up-regulation and decreased expression of catalase.

ATP-Binding Cassette Transporters↗

PubMed2002

The modulation of hepatic injury and heat shock expression by inhibition of inducible nitric oxide synthase after hemorrhagic shock.

The role of nitric oxide (NO) in maintaining homeostasis and regulating organ function during hemorrhagic shock is complex. The inducible NO synthase (iNOS) has been hypothesized to play a critical role in the pathophysiologic consequences of severe hemorrhage. Heat shock protein (HSP) expression is increased by hemorrhage and is a marker of the magnitude of ischemic injury in the liver. HSP induction is protective against injury in animal models of inflammation and is regulated by NO in hepatocytes. To clarify the role of iNOS in hepatic injury and its relationship to HSP expression in hemorrhagic shock, NOS was inhibited with L-N-6-(1-iminoethyl) lysine (L-NIL), which is reported to be a selective inhibitor of the inducible NOS isoform. Doses of 50 microg/kg or 150 microg/kg were infused over 1 h at the end of compensated shock. Plasma ornithine carbamoyltransferase (OCT), a specific marker of liver injury, was significantly reduced after hemorrhage with low-dose L-NIL (7.1+/-1.5 IU/L) compared to saline-treated control rats (13.0+/-1.5 IU/L, P < 0.005), while high-dose L-NIL significantly increased OCT release (35.9+/-7.2 IU/L, P< 0.05 versus shock alone) despite a greater MAP after resuscitation. HSP expression (HSP-72 and HSP-32) after hemorrhage was increased by L-NIL treatment at the highest dose. We conclude that excessive NO production from iNOS contributes to shock-induced hepatic injury. Our data suggest HSP expression may reflect the degree of ischemic injury after hemorrhage.

Animals↗

PubMed2002

Platelet-endothelial cell adhesion molecule-1-directed immunotargeting to cardiopulmonary vasculature.

Therapeutic molecules conjugated with antibodies to the platelet-endothelial cell adhesion molecule-1 (PECAM-1) accumulate in the pulmonary endothelium after i.v. injection in mice. In this study, we characterized PECAM-directed targeting to the lung and heart after local versus systemic intravascular administration in a large animal model, pigs. Radiolabel tracing showed that 1 h post-i.v. injection, 35% of anti-PECAM versus 2.5% of control IgG had accumulated in the lungs. Infusion of anti-PECAM via a catheter placed in the right pulmonary artery (RPA) resulted in a 3-fold elevation of the uptake in the right lower lobe and 2-fold reduction of uptake in the left lobes in the lung. Cardiac uptake of anti-PECAM was negligible after i.v. and RPA infusion. In contrast, delivery with a catheter placed in the right coronary artery (RCA) resulted in a 4-fold elevation of cardiac uptake of anti-PECAM, but not IgG, compared with i.v. injection. To estimate the targeting of an active reporter enzyme, streptavidin-conjugated beta-galactosidase (beta-Gal) was coupled to anti-PECAM or IgG (anti-PECAM/beta-Gal and IgG/beta-Gal) and injected into the RCA. Beta-Gal activity was markedly elevated in the heart and lungs (5- and 25-fold increased, respectively) after injection of anti-PECAM/beta-Gal, but not IgG/beta-Gal. Image analysis confirmed endothelial targeting of anti-PECAM/beta-Gal in the heart and lung. In summary, anti-PECAM antibody conjugates deliver agents to the pulmonary endothelium regardless of injection route, whereas local arterial infusion permits targeting to the cardiac vasculature. This paradigm may be useful for drug targeting to endothelium in lungs, heart, and possibly other organs.

Animals↗