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Biomedical subjects

P Webster

Publications and source records attributed to P Webster.

At least 37 records · Page 2Linked to original sources
PubMed1999

Involvement of MIIC-like late endosomes in B cell receptor-mediated antigen processing in murine B cells.

Currently, the involvement of classical vs novel endocytic compartments in the phenomenon of B cell receptor (BCR)-mediated Ag processing is a matter of considerable debate. In murine B cells, class II vesicles (CIIV) represent a novel endocytic compartment involved in BCR-mediated Ag processing and class II peptide loading. Alternatively, in human B cells, the MHC class II-enriched compartment (MIIC) represents a lysosome (L)-like endocytic compartment that appears to be involved in this process. Presently, the relationship between CIIV, MIIC, and classical endosomes and L remains to be determined. Using density gradient centrifugation, a subcellular compartment morphologically and immunologically similar to human MIIC has been identified, isolated, and characterized in murine B cells. These MIIC-like vesicles represent a population of class II-positive late endosomes (LE) and are distinct from CIIV. MIIC-like LE are uniquely marked by the thiol protease cathepsin B, and along with mature L, appear to be the major repository of DM molecules in these cells. Importantly, both MIIC-like LE and CIIV isolated from Ag-pulsed B cells contain BCR-internalized Ag as well as antigenic peptide-class II complexes.

Animals↗
PubMed1999

Acceleration of the folding of acylphosphatase by stabilization of local secondary structure.

The addition of trifluoroethanol or hexafluoroisopropanol converts the apparent two-state folding of acylphosphatase, a small alpha/beta protein, into a multistate mechanism where secondary structure accumulates significantly in the denatured state before folding to the native state. This results in a marked acceleration of folding as revealed by following the intrinsic fluorescence and circular dichroism changes upon folding. The folding rate is at a maximum when the secondary-structure content of the denatured state corresponds to that of the native state, while further stabilization of secondary structure decreases the folding rate. These findings indicate that stabilization of intermediate structure can either enhance or retard folding depending on its nature and content of native-like interactions.

Acid Anhydride Hydrolases↗
PubMed1999

Congenital chylothorax in neonatal thyrotoxicosis.

We report a patient with congenital chylothorax who also had neonatal thyrotoxicosis secondary to maternal Graves' disease. Fetal tachycardia with hydrops was detected at 28 weeks' gestational age. The fetus responded to antithyroid medication in utero but had persistent bilateral pleural effusion. At birth, he had respiratory distress due to massive pleural effusion. Cytologic studies of pleural fluid were consistent with chylothorax. To the best of our knowledge, the association of congenital chylothorax with fetal (neonatal) thyrotoxicosis, has not been reported previously.

Adult↗
PubMed1999

Efficiency analysis of a multi-specialty telemedicine service.

OBJECTIVE: The goal of this project was to assess case turn-around times for store-and-forward and real-time video consultations in the Arizona Telemedicine Program. MATERIALS AND METHODS: Five components contributing to total case turn-around time were analyzed. Each parameter was submitted to statistical analysis and compared for store-and-forward and real-time sessions. RESULTS: Turn-around for real-time are longer than for store-and-forward sessions. Real-time sessions take longer, from when the consulting clinician is contacted to the time the case is reviewed. This is compounded by the fact that real-time sessions sometimes need to be rescheduled. For both types of consults, the time to deliver the final report is the longest segment of the total turn-around time. CONCLUSION: Several factors contribute to case turn-around times. By identifying and analyzing each contributing factor, it is possible to revise consult protocols to improve efficiency.

Arizona↗
PubMed1998

Cloning, expression, and localization of a novel gamma-adaptin-like molecule.

We describe the cloning, expression, and localization of gamma2-adaptin, a novel isoform of gamma-adaptin. The predicted human and mouse gamma2-adaptin proteins are approximately 90 kDa and 64.4% and 61.7%) identical to gamma-adaptin, respectively. gamma2-Adaptin was localized to the Golgi, its localization distinct from gamma-adaptin. The membrane association of gamma- and gamma2-adaptin could further be distinguished by differential sensitivity to the fungal metabolite brefeldin A, gamma2-adaptin binding being insensitive to drug treatment. Together, these results suggest that gamma2-adaptin plays a role in membrane transport distinct from that played by gamma-adaptin.

Adaptor Protein Complex gamma Subunits↗
PubMed1998

Effect of microwave irradiation on antibody labeling efficiency when applied to ultrathin cryosections through fixed biological material.

To study the effect of microwaves on immunolabeling, ultrathin cryosections or diluted antibodies were irradiated prior to antibody application, and gold labeling was quantified. In addition, affinity purified, polyclonal antibodies and protein A-gold were applied to ultrathin cryosections of aldehyde-fixed material in the presence and absence of microwaves. Amylase, a soluble protein secreted by pancreatic acinar cells, MHC class II, an integral membrane protein, and 3-(2,4-dinitroanilino)-3-amino-N-methyldipropylamine (DAMP), an exogenously added antigen, were localized with monospecific antibodies. Each was chosen for their contrasting subcellular characteristics. Results demonstrated that for some antigens, antibody labeling efficiency was quantitatively improved by microwave irradiation of sections prior to antibody application. Irradiation of diluted antibodies prior to their application also resulted in improved labeling. In contrast, the results obtained using rapid immunolabeling protocols in the presence of microwaves resulted in labeling levels similar to those obtained in the absence of microwaves. We conclude that microwave irradiation can improve the labeling efficiency of some antibodies. However, improvements in labeling density are dependent on the antigen under study and on variable irradiation times, unique to each antibody. This suggests that the routine use of microwaves to reduce incubation times may not be a viable alternative to bench protocols.

Amylases↗
PubMed1998

The protozoan parasite Toxoplasma gondii targets proteins to dense granules and the vacuolar space using both conserved and unusual mechanisms.

All known proteins that accumulate in the vacuolar space surrounding the obligate intracellular protozoan parasite Toxoplasma gondii are derived from parasite dense granules. To determine if constitutive secretory vesicles could also mediate delivery to the vacuolar space, T. gondii was stably transfected with soluble Escherichia coli alkaline phosphatase and E. coli beta-lactamase. Surprisingly, both foreign secretory reporters were delivered quantitatively into parasite dense granules and efficiently secreted into the vacuolar space. Addition of a glycosylphosphatidylinositol membrane anchor rerouted alkaline phosphatase to the parasite surface. Alkaline phosphatase fused to the transmembrane domain and cytoplasmic tail from the endogenous dense granule protein GRA4 localized to dense granules. The protein was secreted into a tuboreticular network in the vacuolar space, in a fashion dependent upon the cytoplasmic tail, but not upon a tyrosine-based motif within the tail. Alkaline phosphatase fused to the vesicular stomatitis virus G protein transmembrane domain and cytoplasmic tail localized primarily to the Golgi, although staining of dense granules and the intravacuolar network was also detected; truncating the cytoplasmic tail decreased Golgi staining and increased delivery to dense granules but blocked delivery to the intravacuolar network. Targeting of secreted proteins to T. gondii dense granules and the plasma membrane uses general mechanisms identified in higher eukaryotic cells but is simplified and exaggerated in scope, while targeting of secreted proteins beyond the boundaries of the parasite involves unusual sorting events.

Alkaline Phosphatase↗
PubMed1998

The agent of Human Granulocytic Ehrlichiosis resides in an endosomal compartment.

The composition of cytoplasmic vacuoles containing the agent of Human Granulocytic Ehrlichiosis (HGE) was studied to investigate how this pathogen exists within infected host cells. Electron microscopy demonstrated that the HGE organism resides in a membrane-bound compartment within HL-60 cells: early forms of the HGE agent have a round reticular appearance while later structures are small and dense. Vacuoles containing HGE bacteria incorporated endocytosed colloidal gold particles, suggesting that they are part of the endocytic pathway. Antibodies directed to the mannose-6-phosphate receptor labeled vacuole membranes. Antibodies to the transferrin receptor and to the lysosomal membrane glycoprotein LAMP 1 did not. Moreover, 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine, which normally accumulates in compartments with low pH, was not present inside these vacuoles. These results suggest that vacuoles containing the agent of HGE fail to mature into phagolysosomes. We conclude that the agent of HGE appears to enter and modify part of the endocytic pathway.

Cell Compartmentation↗
PubMed1998

Vasoactive intestinal peptide, forskolin, and genistein increase apical CFTR trafficking in the rectal gland of the spiny dogfish, Squalus acanthias. Acute regulation of CFTR trafficking in an intact epithelium.

Defective trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common cause of cystic fibrosis. In chloride-secreting epithelia, it is well established that CFTR localizes to intracellular organelles and to apical membranes. However, it is controversial whether secretagogues regulate the trafficking of CFTR. To investigate whether acute hormonal stimulation of chloride secretion is coupled to the trafficking of CFTR, we used the intact shark rectal gland, a model tissue in which salt secretion is dynamically regulated and both chloride secretion and cellular CFTR immunofluorescence can be quantified in parallel. In rectal glands perfused under basal conditions without secretagogues, Cl- secretion was 151+/-65 microeq/h/g. Vasoactive intestinal peptide (VIP), forskolin, and genistein led to 10-, 6-, and 4-fold increases in Cl- secretion. In basal glands, quantitative confocal microscopy revealed CFTR immunofluorescence extending from the apical membrane deeply into the cell (7.28+/-0.35 micron). During stimulation with secretagogues, apical extension of CFTR immunofluorescence into the cell was reduced significantly to 3.24+/-0.08 micron by VIP, 4.08+/-0.13 by forskolin, and 3.19+/-0.1 by genistein (P < 0.001). Moreover, the peak intensity of CFTR fluorescence shifted towards the apical membrane (peak fluorescence 2.5+/-0.13 micron basal vs. 1.51+/-0.06, 1.77+/-0.1, and 1.38+/-0.05 for VIP, forskolin, and genistein; all P < 0.001). The increase in both Cl- secretion and apical CFTR trafficking reversed to basal values after removal of VIP. These data provide the first quantitative morphological evidence for acute hormonal regulation of CFTR trafficking in an intact epithelial tissue.

Animals↗
PubMed1998

Analysis of trypanosomal endocytic organelles using preparative free-flow electrophoresis.

In this paper we demonstrate the power of preparative free-flow electrophoresis (FFE) for the study of endocytosis by African trypanosomes. Endocytosis of extracellular macromolecules by these parasites occurs through a specialized region of the parasite called the flagella pocket. The uptake of fluid phase markers such as horseradish peroxidase (HRP) into the various compartments of the endocytic pathway of bloodstream forms of Trypanosoma brucei brucei was manipulated by regulating the external environment (e.g., by altering the temperature of incubation). The various subcellular compartments were then separated by free-flow electrophoresis (FFE) or isopycnic density gradient centrifugation and analyzed for marker uptake. At low temperatures, HRP was found predominantly in the flagellar pocket. Increasing the temperature resulted in a time-dependent uptake of HRP into more positively charged endosomal fractions. However, little HRP activity was detected in lysosomal compartments, suggesting that either HRP had not yet entered the lysosome or was degraded immediately upon entry. Through the use of FFE we were able to identify and analyze compartments of the endosomal pathway that were not possible to identify by density gradient centrifugation alone. Although the differences in FFE separation of the endocytic compartments as seen in HRP uptake were striking, the minor changes seen within the lysosomal system were more subtle, as depicted in the protease profiles. In conclusion, we show that preparative FFE is a powerful technique for the analysis and separation of flagellar pocket-derived membranes from other endosomal and lysosomal compartments of African trypanosomes.

Animals↗
PubMed1998

Transport of residual endocytosed products into terminal lysosomes occurs slowly in rat liver endothelial cells.

Receptor-mediated endocytosis of circulating collagen is a major physiological scavenger function of the liver endothelial cell and an important catabolic event in the complete turnover of this abundant connective tissue protein. In the present study, transport of collagen through the endocytic pathway was investigated in cultured liver endothelial cells. Collagen conjugated to fluorescein isothiocyanate, to allow detection of the ligand by fluorescence and immunoelectron microscopy, was found sequentially in three different organelles that compose the basic degradative endocytic pathway of eukaryotic cells: early endosomes, late endosomes, and terminal lysosomes. Early endosomes were identified as vesicles positive for early endosome antigen 1 (EEA1). Late endosomes were distinguished as structures positive for the late endosomal/lysosomal marker rat lysosomal membrane glycoprotein 120, but negative for EEA1 and lysosomally targeted BSA-gold. Lysosomes were defined by their content of BSA-gold, injected 24 hours before isolation of cells. Coated pits and coated vesicles mediated an extremely rapid internalization. Shortly after internalization and during the first 20 minutes, ligand was found in early endosomes. From 20 minutes on, ligand started to appear in late endosomes (23%), and by 2 hours the transfer was largely complete (82.5%). Only 2.5% of ligand was transferred to the lysosomes after 2 hours, and this number slowly increased to 21% and 53% after 6 and 16 hours, respectively. We conclude that 1) EEA1 is a useful marker for tracing early events of endocytosis in liver endothelial cells; 2) in contrast to the rapid internalization, transit of internalized ligand through early sorting endosomes generally takes from 20 minutes to 2 hours; and 3) exit from the late endosomes is very slow, requiring several hours.

Animals↗
PubMed1998

The native membrane fusion machinery in cells.

Recombinant SNAREs have been demonstrated as the minimal membrane fusion machinery. The participation of additional proteins in the regulation of membrane fusion has been suggested. In this study we provide nanometer-resolution images of native NSF oligomers and SNARE complexes isolated from neurons and the pancreas. Our study reveals the presence of new coiled rod-like structures in association with the SNARE complex only in neuronal tissue. Neuronal SNAREs were found coiled and super-coiled with these structures. The existence of NSF as pentamers in its native state is also demonstrated. The extent of coiling and super-coiling of SNAREs may regulate the potency and efficacy of membrane fusion in cells.

Animals↗
PubMed1998

Trichinella spiralis, T. britovi, and T. nativa: infectivity, larval distribution in muscle, and antibody response after experimental infection of pigs.

The infectivity of Trichinella spiralis, T. nativa, and T. britovi was experimentally compared in pigs. Blood sampling was performed weekly, and muscle juices were obtained at slaughter 10 weeks after inoculation. Muscle larvae were found in all of four pigs inoculated with T. spiralis [mean 190 larvae per gram (lpg)] and in three of four pigs inoculated with T. britovi (mean 7 lpg). No larvae were found in pigs inoculated with T. nativa. For T. spiralis and T. britovi, the neck muscle (m. splenius) appears to be a predilection site in addition to the tongue, the diaphragm, and the jaw. High antibody responses were found in all experimental groups, independent of the antigen used, and even in pigs in which no muscle larvae were recovered. The strong and consistent antibody response found with meat juice indicates the usefulness of this material where a blood sample is not obtainable, e.g. meat samples from wild animals. Immunoblotting (Western blots) on slaughter sera revealed no species specificity when comparing homologous versus heterologous staining.

Animals↗
PubMed1998

Evaluation of the infectivity of Trichinella spp. for reptiles (Caiman sclerops).

Experimental inoculation with nine well-characterised Trichinella isolates was performed on caimans (Caiman sclerops) to determine their infectivity for reptiles belonging to the family Crocodilidae. As controls, the same larval batches of Trichinella isolates were inoculated into mice and guinea pigs. It was suggested that Trichinella pseudospiralis was more likely to infect reptiles than encapsulating species, but whereas all Trichinella species established in mice and guinea pigs, the caimans remained negative. The finding that caimans could not be experimentally infected contrasts with a recent report on infections in farmed crocodiles (Crocodylus niloticus).

Alligators and Crocodiles↗