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Biomedical subjects

P Webster

Publications and source records attributed to P Webster.

At least 19 recordsLinked to original sources
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↗
PubMed1994

Novel infectious particles generated by expression of the vesicular stomatitis virus glycoprotein from a self-replicating RNA.

Self-propagating infectious particles were produced in animal cells transfected with an RNA replicon encoding a single viral structural protein, the vesicular stomatitis virus glycoprotein (VSV-G). The replicon is derived from an alphavirus, Semliki Forest virus (SFV), and encodes the SFV RNA replicase, but none of the SFV structural proteins. After transfection of the replicon into tissue culture cells, expression of G protein spread from small foci throughout the culture. Supernatants from the cells contained infectious, virus-like particles that could be passaged and were neutralized by anti-VSV serum. The majority of the infectious particles were smaller and less dense than either VSV or SFV. Characterization by electron microscopy showed membrane-enveloped vesicles that contained the VSV-G protein. Infectious particles were apparently generated by budding of vesicles containing VSV-G protein and the RNA replicon. These experiments reveal that an enveloped infectious agent can be much simpler than previously thought.

Animals↗
PubMed1994

Brefeldin A sensitivity and resistance in Schizosaccharomyces pombe. Isolation of multiple genes conferring resistance.

The fungal metabolite brefeldin A (BFA) causes the inhibition of protein secretion and the disruption of the structure and function of the Golgi complex in mammalian cells. Here we show that BFA has identical effects in the fission yeast Schizosaccharomyces pombe which normally contains a Golgi complex of stacked cisternae similar to the Golgi complexes in animal cells. After treatment with BFA, secretion was inhibited, Golgi complexes disappeared, and there was an accumulation of endoplasmic reticulum. These results indicate that the effects of BFA in fungi are very similar to those in mammalian cells and provide direct evidence for an effect of BFA on Golgi morphology in fungi. Five spontaneous BFA-resistant mutants were isolated. Genetic analysis showed that the mutations conferring BFA resistance were dominant and in two separate linkage groups. One of the BFA-resistant mutations was found to be allelic to crm1, a gene affecting chromatin structure. All BFA-resistant mutants overexpressed a 20-kDa protein, and the corresponding gene obr1 was isolated and sequenced. However, obr1 overexpression was not sufficient to confer BFA resistance. Plasmids capable of conferring BFA resistance to wild type cells were isolated from libraries constructed from the two BFA-resistant mutants. These plasmids contain six different genes capable of conferring resistance when present in high copy. One of these genes encoded the transcription factor pap1, a homolog of the mammalian AP1 protein. The overexpression of pap1 probably confers BFA resistance indirectly by inducing expression of one or more other proteins. The isolation of several genes conferring BFA resistance suggests several mechanisms are involved.

Amino Acid Sequence↗
PubMed1994

Transient accumulation of new class II MHC molecules in a novel endocytic compartment in B lymphocytes.

Endocytosis of antigen by antigen-presenting cells results in the production of peptides that bind to newly synthesized class II molecules of the major histocompatibility complex. A new population of class II-enriched vesicles has been discovered in B lymphocytes that accumulate internalized antigen but are distinct from endosomes and lysosomes. These vesicles also transiently accumulate newly synthesized class II and class II-peptide complexes and appear to be a compartment specialized for the transport and loading of class II molecules.

Animals↗
PubMed1994

The invariant chain is required for intracellular transport and function of major histocompatibility complex class II molecules.

The major histocompatibility complex (MHC) class II-associated invariant chain (Ii) is thought to act as a chaperone that assists class II during folding, assembly, and transport. To define more precisely the role of Ii chain in regulating class II function, we have investigated in detail the biosynthesis, transport, and intracellular distribution of class II molecules in splenocytes from mice bearing a deletion of the Ii gene. As observed previously, the absence of Ii chain caused significant reduction in both class II-restricted antigen presentation and expression of class II molecules at the cell surface because of the intracellular accumulation of alpha and beta chains. Whereas much of the newly synthesized MHC molecules enter a high molecular weight aggregate characteristic of misfolded proteins, most of the alpha and beta chains form dimers and acquire epitopes characteristic of properly folded complexes. Although the complexes do not bind endogenously processed peptides, class II molecules that reach the surface are competent to bind peptides added to the medium, further demonstrating that at least some of the complexes fold properly. Similar to misfolded proteins, however, the alpha and beta chains are poorly terminally glycosylated, suggesting that they fail to reach the Golgi complex. As demonstrated by double label confocal and electron microscope immunocytochemistry, class II molecules were found in a subcompartment of the endoplasmic reticulum and in a population of small nonlysosomal vesicles possibly corresponding to the intermediate compartment or cis-Golgi network. Thus, although alpha and beta chains can fold and form dimers on their own, the absence of Ii chain causes them to be recognized as "misfolded" and retained in the same compartments as bona fide misfolded proteins.

Animals↗
PubMed1994

Characterization of extreme apical antigens from Toxoplasma gondii.

We have isolated 26 monoclonal antibodies which specifically recognize the extreme apex of Toxoplasma gondii, a protozoan parasite which attaches to and invades host cells via its specialized apical end. The unique apical organelles which define the phylum Apicomplexa are thought to be involved in mechanical and enzymatic aspects of invasion. Immunoblots, immunofluorescence morphology, and immunogold labeling define six classes of apically localized antigens recognized by these antibodies. Three of the classes are detergent-insoluble and localize to the conoid and the cytoplasmic face of the apical membrane, suggesting that they may be part of the parasite's membrane cytoskeleton. The remaining three classes extract with detergent and are associated with internal membrane bounded vesicles (micronemes and the upper necks of rhoptries). One class of micronemal antigens appears to be cell cycle regulated. This antigen localizes to the cytoplasm, especially the perinuclear region, in thin (recently replicated) parasites, but is apical in larger parasites.

Animals↗
PubMed1994

Subcellular localization of CFTR to endosomes in a ductal epithelium.

Plasma membrane chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) may be regulated by cellular processes that affect the cycling of CFTR with the plasma membrane. Testing this hypothesis requires cytochemical evidence for the presence of a subcellular compartment of CFTR. In this study, the subcellular distribution of CFTR in a normal epithelial cell population was characterized using immunofluorescence and immunoelectron microscopy. Two anti-CFTR antibodies, raised against different epitopes of the CFTR molecule, specifically labeled the apical pole of striated duct epithelial cells in tissue sections of rat submandibular gland. By use of electron microscopy, the CFTR immunoreactivity was associated with the apical plasma membrane and the membranes of many subapical vesicles. In this preparation, some of the CFTR-labeled vesicles were also labeled with antibodies against transferrin receptor and rab4, two markers of early endosomes and receptor-mediated endocytosis. These observations provide direct cytochemical evidence for the existence of peripherally located CFTR-expressing endosomes and support the hypothesis that membrane recycling may contribute to CFTR function.

Animals↗
PubMed1994

Prevalence of antibodies to Borrelia burgdorferi in Danish deer.

To estimate the prevalence of antibodies to Borrelia burgdorferi in the most common species of Danish deer, blood samples were collected from roe deer (Capreolus capreolus), fallow deer (Dama dama), and red deer (Cervus elaphus). A total of 156 blood samples were collected primarily from hunts in three areas of Denmark from November 1990 to December 1991. Presence of deer IgG antibodies to the Borrelia burgdorferi strain DK ECM 1 were shown by an indirect immunofluorescence assay at serum titre 1:64. Antibodies to spirochaetes were detected in 52% of roe deer, 38% of fallow deer, and 27% of red deer. There were significant differences between the number of seropositive animals in the three areas of Denmark examined, but no differences could be shown for age, sex or season. The high antibody prevalence indicates that deer are exposed to tick-borne Borrelia burgdorferi throughout Denmark.

Age Factors↗
PubMed1993

Stepwise dismantling of adenovirus 2 during entry into cells.

Adenoviruses enter their host cells by receptor-mediated endocytosis and acid-activated penetration from endosomes into the cytosol and deliver their DNA genome into the nucleus. Our results show that incoming adenovirus type 2 particles undergo a stepwise disassembly program necessary to allow progress of the virus in the entry pathway and release of the genome into the nucleus. The fibers are released, the penton base structures dissociated, the proteins connecting the DNA to the inside surface of the capsid degraded or shed, and the capsid-stabilizing minor proteins eliminated. The uncoating process starts immediately upon endocytic uptake with the loss of fibers and ends with the uptake of dissociated hexon proteins and DNA into the nucleus.

Adenoviruses, Human↗
PubMed1993

A simple modification to the LKB 7800 series Knifemaker and a balanced-break method to prepare glass knives for cryosectioning.

A novel method of using the LKB 7800 series Knifemaker to produce glass knives using a balanced break is described. The method produces knives of sufficient quality to section aldehyde-fixed, sucrose-cryoprotected, frozen biological material in a cooled cryochamber of an ultramicrotome. The modifications to the Knifemaker are minimal and, if required, the machine can be returned easily to its normal state after use.

Cryoultramicrotomy↗
PubMed1993

The transferrin receptor in African trypanosomes: identification, partial characterization and subcellular localization.

All eukaryotic cells, including African trypanosomes, require iron for growth and division, and this iron is acquired by the receptor-mediated endocytosis of iron-loaded transferrin (diFe(3+)-transferrin). In trypanosomes transferrin (Tf) has been shown to be delivered into lysosomes and may not recycle back to the cell surface as it does in mammalian cells (Grab, D. J., et al., Eur. J. Cell Biol. 59, 398-404 (1992)). Here, we describe for the first time, the characteristics of a Tf-binding protein with receptor-like properties in Trypanosoma brucei brucei. Bloodstream forms of rodent-adapted T. brucei were incubated with [35S]methionine and detergent lysates chromatographed on a Sephacryl S-300 column. Fractions were incubated with anti-Tf serum to immunoprecipitate Tf/Tf-binding protein complexes. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) the molecular mass of the major protein in the immunoprecipitate was 88 to 92 kDa. Tf-binding proteins could also be isolated using diferric Tf-Sepharose. The molecular mass of the major Tf-binding protein, as estimated from Sephacryl S-300 column chromatography, in the presence of detergent, was approximately 90 to 100 kDa and 90 kDa with SDS-PAGE. Each 90 kDa Tf-binding protein was able to bind one molecule of diferric Tf. Since monoclonal antibodies to human and bovine Tf receptors failed to react with any trypanosome proteins, antisera were raised against the T. brucei Tf-binding proteins eluted from Tf-Sepharose at low pH. These antibodies recognized a 90 kDa protein on Western blots of a T. brucei lysate and inhibited the growth of T. brucei in vitro. Immunolocalization studies, using this antiserum showed that the Tf-binding protein was localized in the flagellar pocket and within the early endosomal compartments. In the presence of protease inhibitors there was additional localization in lysosome-like organelles. The Tf-binding characteristics and localization of this 90 kDa protein suggest that this molecule is a strong candidate as a physiological receptor for Tf in these parasites.

Animals↗