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P Webster

Publications and source records attributed to P Webster.

At least 55 records · Page 3Linked to original sources
PubMed1997

Gi regulation of secretory vesicle swelling examined by atomic force microscopy.

In the last decade, several monomeric and heterotrimeric guanine nucleotide binding proteins have been identified to associate with secretory vesicles and to be implicated in exocytosis. Vesicle volume also has been proposed to play a regulatory role in secretory vesicle fusion at the plasma membrane. However, the molecular mechanism of function of the guanine nucleotide binding proteins and of the regulation of secretory vesicle volume in the exocytotic process remains unclear. In this study, we report association of the secretory vesicle membrane with the alpha subunit of a heterotrimeric GTP binding protein G(alpha i3) and implicate its involvement in vesicle swelling. Using an atomic force microscope in combination with confocal microscopy, we were able to study the dynamics of isolated zymogen granules, the secretory vesicles in exocrine pancreas. Exposure of zymogen granules to GTP resulted in a 15-25% increase in vesicle height as measured by the atomic force microscope and a similar increase in vesicle diameter as determined by confocal microscopy. Mas7, an active mastoparan analog known to stimulate Gi proteins, was found to stimulate the GTPase activity of isolated zymogen granules and cause swelling. Increase in vesicle size in the presence of GTP, NaF, and Mas7 were irreversible and KCl-sensitive. Ca2+ had no effect on zymogen granule size. Taken together, the results indicate that G(alpha i3) protein localized in the secretory vesicle membrane mediates vesicle swelling, a potentially important prerequisite for vesicle fusion at the cell plasma membrane.

Animals↗
PubMed1997

Association of tissue factor pathway inhibitor with human umbilical vein endothelial cells.

Tissue factor pathway inhibitor (TFPI) is a serine protease inhibitor of the extrinsic coagulation system, synthesized in endothelial cells, which has recently been shown to play an important role in the regulation of activated coagulation factors at the endothelial cell surface. In the present study we investigated the subcellular localization and metabolism of TFPI in human umbilical vein endothelial cells (HUVEC). Immunocytochemical labeling of HUVEC with anti-TFPI showed specific labeling associated with the cell surface and with many intracellular organelles including the Golgi complex. Further characterization of these organelles was performed by colocalizing the anti-TFPI with 3-(2, 4-dinitroanilino)'-amino-N-methyldipropylamine (DAMP; to demonstrate low pH), mannose phosphate receptor (endosomes), and LAMP 1 (late endocytic compartments). TFPI also colocalized with antibodies to the human transferrin receptor, a marker for early endocytic, recycling compartment. Endogenous TFPI colocalized with biotin in intracellular vesicles during endocytosis after biotinylation of the cell surface, which indicated that TFPI was being co-internalized with the surface biotin. The binding of exogenously added 125I-TFPI increased linearly to HUVEC over the concentration range of 0 to 32 nmol/L without saturation, the binding was not affected by up to a thousand-fold molar excess of unlabeled TFPI, and heparin inhibited the binding dose dependently. An intact C-terminal domain was important for the interaction between TFPI and the cell surface of HUVEC, because less than 10% of a C-terminal truncated form of TFPI (TFPI1-161) was bound after addition of equimolar concentrations of full-length TFPI. Exogenously added 125I-TFPI was not degraded in HUVEC during 4 hours at 37 degrees C. The presence of TFPI in endocytic and recycling compartments support the hypothesis that endogenous, membrane-anchored TFPI could be internalized for subsequent recycling back to the cell surface.

Endothelium, Vascular↗
PubMed1997

Delivery of B cell receptor-internalized antigen to endosomes and class II vesicles.

B cell receptor (BCR)-mediated antigen processing is a mechanism that allows class II-restricted presentation of specific antigen by B cells at relatively low antigen concentrations. Although BCR-mediated antigen processing and class II peptide loading may occur within one or more endocytic compartments, the functions of these compartments and their relationships to endosomes and lysosomes remain uncertain. In murine B cells, at least one population of class II- containing endocytic vesicles (i.e., CIIV) has been identified and demonstrated to be distinct both physically and functionally from endosomes and lysosomes. We now demonstrate the delivery of BCR-internalized antigen to CIIV within the time frame during which BCR-mediated antigen processing and formation of peptide-class II complexes occurs. Only a fraction of the BCR-internalized antigen was delivered to CIIV, with the majority of internalized antigen being delivered to lysosomes that are largely class II negative. The extensive colocalization of BCR-internalized antigen and newly synthesized class II molecules in CIIV suggests that CIIV may represent a specialized subcellular compartment for BCR-mediated antigen processing. Additionally, we have identified a putative CIIV-marker protein, immunologically related to the Igalpha subunit of the BCR, which further illustrates the unique nature of these endocytic vesicles.

Animals↗
PubMed1997

Lysosomes behave as Ca2+-regulated exocytic vesicles in fibroblasts and epithelial cells.

Lysosomes are considered to be a terminal degradative compartment of the endocytic pathway, into which transport is mostly unidirectional. However, specialized secretory vesicles regulated by Ca2+, such as neutrophil azurophil granules, mast cell-specific granules, and cytotoxic lymphocyte lytic granules, share characteristics with lysosomes that may reflect a common biogenesis. In addition, the involvement of Ca2+ transients in the invasion mechanism of the parasite Trypanosoma cruzi, which occurs by fusion of lysosomes with the plasma membrane, suggested that lysosome exocytosis might be a generalized process present in most cell types. Here we demonstrate that elevation in the intracellular free Ca2+ concentration of normal rat kidney (NRK) fibroblasts induces fusion of lysosomes with the plasma membrane. This was verified by measuring the release of the lysosomal enzyme beta-hexosaminidase, the appearance on the plasma membrane of the lysosomal glycoprotein lgp120, the release of fluid-phase tracers previously loaded into lysosomes, and the release of the lysosomally processed form of cathepsin D. Exposure to the Ca2+ ionophore ionomycin or addition of Ca2+-containing buffers to streptolysin O-permeabilized cells induced exocytosis of approximately 10% of the total lysosomes of NRK cells. The process was also detected in other cell types such as epithelial cells and myoblasts. Lysosomal exocytosis was found to require micromolar levels of Ca2+ and to be temperature and ATP dependent, similar to Ca2+-regulated secretory mechanisms in specialized cells. These findings highlight a novel role for lysosomes in cellular membrane traffic and suggest that fusion of lysosomes with the plasma membrane may be an ubiquitous form of Ca2+-regulated exocytosis.

Adenosine Triphosphate↗
PubMed1997

A plastid of probable green algal origin in Apicomplexan parasites.

Protozoan parasites of the phylum Apicomplexa contain three genetic elements: the nuclear and mitochondrial genomes characteristic of virtually all eukaryotic cells and a 35-kilobase circular extrachromosomal DNA. In situ hybridization techniques were used to localize the 35-kilobase DNA of Toxoplasma gondii to a discrete organelle surrounded by four membranes. Phylogenetic analysis of the tufA gene encoded by the 35-kilobase genomes of coccidians T. gondii and Eimeria tenella and the malaria parasite Plasmodium falciparum grouped this organellar genome with cyanobacteria and plastids, showing consistent clustering with green algal plastids. Taken together, these observations indicate that the Apicomplexa acquired a plastid by secondary endosymbiosis, probably from a green alga.

Animals↗
PubMed1997

A cytosolic serine endopeptidase from Trypanosoma cruzi is required for the generation of Ca2+ signaling in mammalian cells.

An early event in the Trypanosoma cruzi cell invasion process, the recruitment of host lysosomes, led us to investigate the involvement of signal transduction. Infective trypomastigotes were found to contain a soluble Ca2+-signaling activity for mammalian cells that is sensitive to protease inhibitors. Inhibitor and substrate utilization profiles were used to purify a candidate peptidase for involvement in this process, from which we isolated a full-length cDNA clone. The sequence revealed a novel enzyme, denominated T. cruzi oligopeptidase B, which is homologous to members of the prolyl oligopeptidase family of serine hydrolases, known to participate in the maturation of biologically active peptides. The T. cruzi oligopeptidase B was expressed as a fully active product in Escherichia coli, and antibodies to the recombinant enzyme inhibited both peptidase activity and Ca2+ signaling induced in normal rat kidney cells by trypomastigote extracts. Our data suggest that the T. cruzi oligopeptidase B participates in processing events in the cytoplasm of the parasites, generating a factor with Ca2+-signaling activity for mammalian cells.

Amino Acid Sequence↗
PubMed1997

Localization of SH-PTP1 to synaptic vesicles: a possible role in neurotransmission.

The tyrosine kinase pp60src is known to phosphorylate synaptophysin and in doing so may regulate neurotransmitter release. The tyrosyl phosphorylated state of synaptophysin is dependent on pp60src kinase and the unknown protein tyrosine phosphate phosphohydrolase (PTPase, EC 3.1.3.48). Here we report the protein tyrosine phosphate phosphohydrolase SH-PTP1, to associate with synaptic vesicles and interact with synaptophysin. These studies identify SH-PTP1 as a new member of the secretory machinery at the nerve terminal and suggest its involvement in neurotransmission.

Animals↗
PubMed1997

Association of host cell endoplasmic reticulum and mitochondria with the Toxoplasma gondii parasitophorous vacuole membrane: a high affinity interaction.

The parasitophorous vacuole membrane (PVM) of the obligate intracellular protozoan parasite Toxoplasma gondii forms tight associations with host mitochondria and the endoplasmic reticulum (ER). We have used a combination of morphometric and biochemical approaches to characterize this unique phenomenon, which we term PVM-organelle association. The PVM is separated from associated mitochondria and ER by a mean distance of 12 and 18 nm, respectively. The establishment of PVM-organelle association is dependent on active parasite entry, but does not require parasite viability for its maintenance. Association is not a consequence of spatial constraints imposed on the growing vacuole. Morphometric analysis indicates that the extent of mitochondrial association with the PVM stays constant as the vacuole enlarges, whereas the extent of ER association decreases. Disruption of host cell microtubules partially blocks the establishment but not the maintenance of PVM-mitochondrial association, and has no significant effect on PVM-ER association. PVM-organelle association is maintained following disruption of infected host cells, as assessed by electron microscopy and by sub-cellular fractionation showing co-migration of fixed PVM and organelle markers. Taken together, the data suggest that a high affinity, potentially protein-protein interaction between parasite and organelle components is responsible for PVM-organelle association.

Animals↗
PubMed1997

Homotypic fusion between aggregated lysosomes triggered by elevated [Ca2+]i in fibroblasts.

Previous studies demonstrated that microinjection of antibodies to the cytoplasmic domain of the lysosomal glycoprotein lgp120 induces aggregation of lysosomes in NRK cells. Here we show that the antibody-clustered vesicles do not co-localize with MPR and ss-COP-containing organelles, confirming their lysosomal nature. Observations by transmission and high voltage electron microscopy indicated that, although tightly apposed to each other, aggregated lysosomes remained as separate vesicles, with an average diameter of 0.3-0.4 micron. However, when cells microinjected with antibody were exposed to the Ca2+ ionophore ionomycin, large vesicles were formed within the lysosome clusters, suggesting the occurrence of lysosome-lysosome fusion. Stereological measurements of lysosome diameters on confocal and transmission electron microscopy indicated that the large lgp120-positive vesicles could have originated from the fusion of 3 up to 15 individual lysosomes. To verify if agents that mobilize Ca2+ from intracellular stores had the same effect, anti-lgp120-microinjected cells were treated with thapsigargin, and with the receptor-mediated agonists bombesin and thrombin. Thapsigargin also induced the formation of large lgp120-containing vesicles, detected by both confocal and transmission electron microscopy. Analysis of antibody-clustered lysosomes in streptolysin O-permeabilized cells indicated that an intracellular free Ca2+ concentration of 1 microM was sufficient to trigger formation of large lysosomes.

Animals↗
PubMed1996

Identification and localization of rab6, separation of rab6 from ERD2 and implications for an 'unstacked' Golgi, in Plasmodium falciparum.

The rab6 gene product in mammalian cells and yeast is localized to and regulates protein transport in the medial and trans Golgi cisternae, as well as the trans Golgi network. We have identified a homologue in the malaria parasite Plasmodium falciparum which displays a rab-like sequence that is 62.4% identical to mammalian rab6. In addition the parasite gene (Pfrab6 gene) contains an N-terminal hydrophobic domain, unique to P. falciparum. Antibodies developed to Pfrab6 localize protein in 4-7 well-resolved sites in a ring-stage parasite, as detected by high resolution fluorescence microscopy. This suggests that there are multiple, distinct foci of medial/trans Golgi membranes in a ring. ERD2 is a cis Golgi marker in mammalian cells. The plasmodial homologue of ERD2 (PfERD2) is concentrated in a single perinuclear region in a ring-stage parasite. This site is distinct from the Pfrab6 membranes, indicating that early and late Golgi markers can be segregated in P. falciparum. Mammalian cells contain a single Golgi complex where cis medial and trans markers are tightly stacked in closely apposed cisternae. In P. falciparum-rings however, rab6-associated membranes are not invariably 'stacked' with an ERD2 structure. In immunoelectron microscopy studies, both the PfERD2- and Pfrab6-associated membranes appear tubulovesicular in nature, devoid of cisternal morphology. Hence the Golgi of ring stage parasites may comprise of multiple, 'unstacked' tubulovesicular clusters, suggesting a primitive organization of the organelle in Plasmodia.

Amino Acid Sequence↗
PubMed1996

The role of the adenovirus protease on virus entry into cells.

Adenovirus uncoating is a stepwise process which culminates in the release of the viral DNA into the nucleus through the nuclear pore complexes and dissociation of the capsid. Using quantitative biochemical, immunochemical and morphological methods, we demonstrate that inhibitors of the cystine protease, L3/p23, located inside the capsid block the degradation of the capsid-stabilizing protein VI, and prevent virus uncoating at the nuclear membrane. There was no effect on virus internalization, fiber shedding and virus binding to the nuclear envelope. The viral enzyme (dormant in the extracellular virus) was activated by two separate signals, neither of which was sufficient alone; virus interaction with the integrin receptor (inhibited with RGD peptides) and re-entry of the virus particle into a reducing environment in the endosome or the cytosol. Incorrectly assembled mutant viruses that lack the functional protease (ts1) failed at releasing fibers and penetrating into the cytosol. The results indicated that L3/p23 is needed not only to assemble an entry-competent virus but also to disassemble the incoming virus.

Adenoviruses, Human↗
PubMed1996

A shared care approach in obesity management: the general practitioner and a hospital based service.

OBJECTIVE: To describe the process of establishing a Shared Care obesity management programme between general practitioners and a hospital based specialist obesity service and to compare outcomes of the Shared Care programme (SC) to an established hospital based programme (MOS). DESIGN: A comparative study of two obesity management programmes. Patients were matched on gender, age and BMI (kg/m2). SUBJECTS: 29 female and eight male (age: 47.0 +/- 2 years, BMI 35.9 +/- 0.8 kg/m2) patients enrolled in the Shared Care programme (SC) were matched to 81 female and 20 male (age: 45.8 +/- 1.1 years, BMI 35.7 +/- 0.4 kg/m2) patients enrolled in a hospital based programme (MOS). MAIN OUTCOME MEASURES: Relative and absolute weight loss and retention rate were compared between the programmes at 10 and 26 weeks. Food habits were assessed at enrolment and week 10 of the programme in the SC group by a Food Habits Questionnaire and cognitive restraint, disinhibition and hunger were assessed by the Eating Inventory Questionnaire. RESULTS: Shared Care patients (n = 28) lost significantly more weight than the MOS patients (n = 60) (SC 4.8 +/- 0.6 kg and MOS 2.6 +/- 0.4 kg; p = 0.0016) over the 10 weeks of the programme. At 26 weeks both groups demonstrated a 5 kg weight loss. There was a significant improvement in food habits, and cognitive restraint, disinhibition and hunger over the 10 weeks of the programme in the SC group. Patient satisfaction was reflected in a better retention rate at 26 weeks by the Shared Care group. CONCLUSION: The study demonstrated that the obese patient managed in a shared care setting achieved better weight loss in the short term and attrition was lower in the longer term than a similar patient attending a specialist service based in a hospital.

Adult↗
PubMed1995

Steroid-responsive interstitial lung disease in patients receiving 2'-deoxy-5-fluorouridine-infusion chemotherapy. A report of three cases.

BACKGROUND: Continuous infusion of 2'-deoxy-5-fluorouridine (FUdR) has shown promise in its activity against metastatic renal cell carcinoma. Its side-effect profile is dominated by gastrointestinal toxicity; there are no known adverse pulmonary reactions. To the authors' knowledge, this is the first report on the development of lung toxicity in three patients receiving FUdR-infusion chemotherapy for metastatic renal cell carcinoma. METHODS: The hospital charts of three patients presenting with pulmonary symptoms during FUdR chemotherapy were reviewed. A literature search was performed regarding FUdR-related pulmonary toxicity. RESULTS: Nonproductive cough, dyspnea, and fever appeared within the 10th chemotherapy cycle. Chest radiographs showed interstitial disease in each case, accompanied by a restrictive pattern by pulmonary-function testing. Lung biopsies were performed on two patients showing a pattern of interstitial inflammation. Discontinuing FUdR and instituting steroidal therapy invariably improved symptoms, as was evident by chest radiographs and pulmonary function tests. In one patient, resuming FUdR treatment resulted in a recurrence of the respiratory symptoms, which was controlled with an increased steroidal dose. All three patients required low dose steroids to maintain their baseline respiratory functions. CONCLUSIONS: 2'-deoxy-5-fluorouridine-related lung toxicity is an uncommon event and occurs late in the treatment course. It is rapidly symptomatic and responds readily to steroidal therapy.

Aged↗
PubMed1995

Invariant chain cleavage and peptide loading in major histocompatibility complex class II vesicles.

B lymphocytes contain a novel population of endocytic vesicles involved in the transport of newly synthesized major histocompatibility complex (MHC) class II alpha beta chains and alpha beta peptide complexes to the cell surface. We now present evidence that these class II-enriched vesicles (CIIV) are also likely to be a site for the loading of immunogenic peptides onto MHC molecules. We used the serine protease inhibitor leupeptin to accumulate naturally occurring intermediates in the degradation of alpha beta-invariant chain complexes and to slow the intracellular transport of class II molecules. As expected, leupeptin caused an accumulation of Ii chain and class II molecules (I-A(d)) in endosomes and lysosomes. More importantly, however, it enhanced the selective accumulation of a 10-kD invariant chain fragment associated with sodium dodecyl sulfate (SDS)-labile (empty) alpha beta dimers in CIIV. This was followed by the dissociation of the 10-kD fragment, formation of SDS-stable (peptide-loaded) alpha beta dimers, and their subsequent appearance at the cell surface. Thus, CIIV are likely to serve as a specialized site, distinct from endosomes and lysosomes, that hosts the final steps in the dissociation of invariant chain from class II molecules and the loading of antigen-derived peptides onto newly synthesized alpha beta dimers.

Animals↗
PubMed1995

The Pisum sativum TubA1 gene, a member of a small family of alpha-tubulin sequences.

alpha- and beta-tubulin proteins are subunits of microtubules, which as primary elements of the plant cytoskeleton play major roles in plant cell division and cell morphogenesis. Several higher-plant alpha- and beta-tubulin gene families have been reported to have at least six to nine members each. Using genomic Southern hybridizations and polymerase chain reaction (PCR) experiments, we have found that the Pisum sativum (garden pea) genome has only four copies of alpha-tubulin sequences and a similar number of beta-tubulin sequences. We have characterized the pea alpha-tubulin gene TubA1. Its nucleotide sequence predicts a 452 amino acid product which is 89-98% identical to those predicted for other plant alpha-tubulins. By S1 nuclease analysis we have located the transcript start site at 102 bases upstream of the ATG. We have also shown that the TubA1 gene is expressed by northern hybridization with a gene-specific probe.

Base Sequence↗