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Biomedical subjects

L J Foster

Publications and source records attributed to L J Foster.

18 recordsLinked to original sources
PubMed2005

Characterization of the elastic properties of the nuclear envelope.

Underlying the nuclear envelope (NE) of most eukaryotic cells is the nuclear lamina, a meshwork consisting largely of coiled-coil nuclear intermediate filament proteins that play a critical role in nuclear organization and gene expression, and are vital for the structural stability of the NE/nucleus. By confocal microscopy and micromanipulation of the NE in living cells and isolated nuclei, we show that the NE undergoes deformations without large-scale rupture and maintains structural stability when exposed to mechanical stress. In conjunction with image analysis, we have developed theory for a two-dimensional elastic material to quantify NE elastic behaviour. We show that the NE is elastic and exhibits characteristics of a continuous two-dimensional solid, including connections between lamins and the embedded nuclear pore complexes. Correlating models of NE lateral organization to the experimental findings indicates a heterogeneous lateral distribution of NE components on a mesoscopic scale.

Elasticity↗
PubMed2005

Physical activity patterns in nonobese and obese children assessed using minute-by-minute accelerometry.

OBJECTIVE: To determine the levels and patterns of physical activity in a sample of obese (> or =99th percentile body mass index (BMI)) and nonobese (<99th percentile BMI) children. DESIGN: Cross-sectional study. SETTING: Children were recruited from schools in Bristol and from the childhood obesity clinic, Bristol Royal Hospital for Children. Children were instructed in the use of the accelerometer either while at school or in the clinic, and wore the instrument while carrying out their normal daily activities for 7 days. PARTICIPANTS: A total of 133 children (mean age 10.5+/-0.8 y). In all 11 (16.9%) of the 65 girls and 14 (20.6%) of the 68 boys were classified as obese (above the 99th percentile for BMI and corresponding to projected adult BMI of 30). MAIN OUTCOME MEASURES: Objectively measured physical activity volume, intensity and pattern. RESULTS: Obese children were significantly less physically active overall than their nonobese counterparts (31,844+/-13,200 vs 41,844+/-10,430 counts/h; 95% confidence interval 4407 to 15592; P=0.001). Similarly the obese children spent less time in physical activity of moderate or greater intensity than the nonobese children (9.9+/-3.9 vs 12.9+/-4.2 min/h; 95% confidence interval 1.15 to 4.80; P=0.002). Hourly patterns of activity indicated a tendency in obese children to be less active than nonobese children at times when activity was more likely to be determined by free choice, particularly outside of school time. CONCLUSIONS: Obese children demonstrated patterns of physical activity that may have contributed to and are likely to sustain their obesity. Minute-by-minute accelerometry is a valuable tool to investigate physical activity patterns in obese children. It can identify periods when intervention to increase activity may be most appropriate and provide an evidence base for specific exercise prescription in primary and secondary care.

Body Mass Index↗
PubMed2004

The use of phospholipid fatty acid analysis to measure impact of acid rock drainage on microbial communities in sediments.

The impact of acid rock drainage (ARD) and eutrophication on microbial communities in stream sediments above and below an abandoned mine site in the Adelaide Hills, South Australia, was quantified by PLFA analysis. Multivariate analysis of water quality parameters, including anions, soluble heavy metals, pH, and conductivity, as well as total extractable metal concentrations in sediments, produced clustering of sample sites into three distinct groups. These groups corresponded with levels of nutrient enrichment and/or concentration of pollutants associated with ARD. Total PLFA concentration, which is indicative of microbial biomass, was reduced by >70% at sites along the stream between the mine site and as far as 18 km downstream. Further downstream, however, recovery of the microbial abundance was apparent, possibly reflecting dilution effect by downstream tributaries. Total PLFA was >40% higher at, and immediately below, the mine site (0-0.1 km), compared with sites further downstream (2.5-18 km), even after accounting for differences in specific surface area of different sediment samples. The increased microbial population in the proximity of the mine source may be associated with the presence of a thriving iron-oxidizing bacteria community as a consequence of optimal conditions for these organisms while the lower microbial population further downstream corresponded with greater sediments' metal concentrations. PCA of relative abundance revealed a number of PLFAs which were most influential in discriminating between ARD-polluted sites and the rest of the sites. These PLFA included the hydroxy fatty acids: 2OH12:0, 3OH12:0, 2OH16:0; the fungal marker: 18:2omega6; the sulfate-reducing bacteria marker 10Me16:1omega7; and the saturated fatty acids 12:0, 16:0, 18:0. Partial constrained ordination revealed that the environmental parameters with the greatest bearing on the PLFA profiles included pH, soluble aluminum, total extractable iron, and zinc. The study demonstrated the successful application of PLFA analysis to rapidly assess the toxicity of ARD-affected waters and sediments and to differentiate this response from the effects of other pollutants, such as increased nutrients and salinity.

Bacteria↗
PubMed2001

Insulin accelerates inter-endosomal GLUT4 traffic via phosphatidylinositol 3-kinase and protein kinase B.

Insulin enhances plasmalemmal-directed traffic of glucose transporter-4 (GLUT4), but it is unknown whether insulin regulates GLUT4 traffic through endosomal compartments. In L6 myoblasts expressing Myc-tagged GLUT4, insulin markedly stimulated the rate of GLUT4myc recycling. In myoblasts stimulated with insulin to maximize surface GLUT4myc levels, we followed the rates of surface-labeled GLUT4myc endocytosis and chased its intracellular distribution in space and time using confocal immunofluorescence microscopy. Surface-labeled GLUT4myc internalized rapidly (t(12) 3 min), reaching the early endosome by 2 min and the transferrin receptor-rich, perinuclear recycling endosome by 20 min. Upon re-addition of insulin, the t(12) of GLUT4 disappearance from the plasma membrane was unchanged (3 min), but strikingly, GLUT4myc reached the recycling endosome by 10 and left by 20 min. This effect of insulin was blocked by the phosphatidylinositol 3-kinase inhibitor LY294002 or by transiently transfected dominant-negative phosphatidylinositol 3-kinase and protein kinase B mutants. In contrast, insulin did not alter the rate of arrival of rhodamine-labeled transferrin at the recycling endosome. These results reveal a heretofore unknown effect of insulin to accelerate inter-endosomal travel rates of GLUT4 and identify the recycling endosome as an obligatory stage in insulin-dependent GLUT4 recycling.

Animals↗
PubMed2001

Centrifugally-spun polyhydroxybutyrate fibres: effect of process solvent on structure, morphology and cell response.

The structure and morphology of a novel form of poly(beta-hydroxybutyrate) produced by gel-spinning is described. The entangled fibrous nature of the material, which resembles 'cotton wool' suggests possible functions in wound scaffolding devices. The surface structure and fibre diameter distribution of the fibres have been investigated using phase contrast and scanning electron microscopy. Fibres were found to possess a variety of surface irregularities, such as pores and indentations. with diameters mainly in the range 1-15 microm. Additionally, individual fibres were occasionally found to be fused or forked together with neighbours. The effects of blending with various polysaccharides and of altering the process solvent on fibre morphology were also investigated. Under hydrolytic degradation conditions (pH 10.6, 70 degrees C) the fibres degraded by gradual fragmentation and erosion to fibre fragments, particulate matter and eventually to monomer. Altering the production process influenced both the fibre diameter distributions and surface morphology of the constituent fibres. Mammalian and human epithelial cells were used to study the cellular interaction with the spun fibres. SEM studies show that there is little or no cell adhesion to the unmodified fibres, but surface treatment by means of acid and alkali washes promoted cell proliferation on the materials, probably as a result of the introduction of hydroxyl and carboxyl at the surface. Fabrication of non-woven mats, which were subsequently acid or alkali treated, provided a conventional way of forming a cell-adhesive matrix which may have potential value as a wound scaffold. Neither cell line exhibited any cytotoxic response to these polymers.

Animals↗
PubMed2000

A functional role for VAP-33 in insulin-stimulated GLUT4 traffic.

Soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are critical proteins in membrane fusion, in both regulated and constitutive vesicular traffic. In addition, proteins that interact with the SNAREs are thought to regulate fusion. Vesicle-associated membrane protein-2 (VAMP-2) is a SNARE protein involved in insulin-dependent glucose transporter 4 (GLUT4) traffic. VAMP-2 is required for productive GLUT4 incorporation into the plasma membrane. VAMP-associated protein of 33 kDa (VAP-33) is an integral membrane protein that binds VAMPs in vitro, and is hypothesized to be a regulator of VAMPs. In L6 skeletal myoblasts, which display insulin-dependent traffic of GLUT4, we show that VAP-33 colocalized significantly with VAMP-2 using indirect confocal immunofluorescence and biochemical cosegregation. Overexpression of wild-type VAP-33 in L6 myoblasts attenuated the insulin-dependent incorporation of myc-tagged GLUT4 into the plasma membrane, and this response was restored by co-overexpression of VAMP-2 linked to green fluorescent protein. Antibodies to VAP-33 microinjected into 3T3-L1 adipocytes abrogated the insulin-stimulated translocation of GLUT4 to the plasma membrane, as measured in adhered plasma membrane lawns. Immunopurified VAMP-2-containing compartments from L6 myotubes and 3T3-L1 adipocytes showed significant levels of VAP-33. We propose that VAP-33 may be a regulator of VAMP-2 availability for GLUT4 traffic and other vesicle fusion events.

Animals↗
PubMed2000

Mechanism and regulation of GLUT-4 vesicle fusion in muscle and fat cells.

Twenty years ago it was shown that recruitment of glucose transporters from an internal membrane compartment to the plasma membrane led to increased glucose uptake into fat and muscle cells stimulated by insulin. The final step of this process is the fusion of glucose transporter 4 (GLUT-4)-containing vesicles with the plasma membrane. The identification of a neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex as a requirement for synaptic vesicle-plasma membrane fusion led to the search for homologous complexes outside the nervous system. Indeed, isoforms of the neuronal SNAREs were identified in muscle and fat cells and were shown to be required for GLUT-4 incorporation into the cell membrane. In addition, proteins that bind to nonneuronal SNAREs were cloned and proposed to regulate vesicle fusion. We have summarized the molecular mechanisms leading to membrane fusion in nonneuronal systems, focusing on the role of SNAREs and accessory proteins (Munc18c, synip, Rab4, and VAP-33) in incorporation of GLUT-4 into the plasma membrane. Potential modes of regulation of this process are discussed, including SNARE phosphorylation and interaction with the cytoskeleton.

Adipocytes↗
PubMed1999

Intracellular depolymerase activity in isolated inclusion bodies containing polyhydroxyalkanoates with long alkyl and functional substituents in the side chain.

The in vitro degradation of isolated Pseudomonas oleovorans inclusion bodies containing either poly-3-hydroxynonanoate (PHN), or poly(-3-hydroxy-5-phenylvalerate) (PHPV), or a mixture of these two polymers was investigated. When incubated at 30 degrees C and pH 9, inclusion bodies containing either polyhydroxyoctanoate (PHO), PHN or PHPV exhibited similar degradation rates of approximately 0.94 (+/- 3%) mg/h. The PHN and PHPV components for inclusion bodies containing a mixture of PHN and PHPV showed similar degradation rates; that is the ratios showed little change and remained at approximately 50 wt.% (+/- 3%) for each component. These results contrast markedly with in vivo studies for similar inclusion bodies in whole cells. The results suggest that the synthesis and degradation of these novel polyhydroxyalkanoates by P. oleovorans proceeds by the same enzymatic pathway. In addition, comparisons between the in vivo and in vitro polymer degradation suggest that the activity of the intracellular depolymerase does not control the rate limiting step of PHPV degradation in vivo. Instead, the presence of an aromatic group in the repeating units of this polymer may inhibit the utilization of the monomeric units of PHPV as a reserve carbon source by the cells.

Bacterial Proteins↗
PubMed1999

SNAP23 promotes insulin-dependent glucose uptake in 3T3-L1 adipocytes: possible interaction with cytoskeleton.

The acute stimulation of glucose uptake by insulin in fat and muscle cells is primarily the result of translocation of facilitative glucose transporter 4 (GLUT-4) from an internal compartment to the plasma membrane. Here, we investigate the role of SNAP23 (a 23-kDa molecule resembling the 25-kDa synaptosome associated protein) in GLUT-4 translocation and glucose uptake in 3T3-L1 adipocytes. Microinjection of a polyclonal antibody directed to the carboxy terminus of SNAP23 inhibited GLUT-4 incorporation into the membrane in response to insulin, whereas microinjection of full-length recombinant SNAP23 enhanced the insulin effect. Introduction of recombinant SNAP23 into chemically permeabilized cells also enhanced insulin-stimulated glucose transport. These results indicate that SNAP23 is required for insulin-dependent, functional incorporation of GLUT-4 into the plasma membrane and that the carboxy terminus of the protein is essential for this process. SNAP23 is therefore likely to be a fusion catalyst along with syntaxin-4 and vesicle-associated membrane protein (VAMP)-2. Furthermore, the endogenous content of SNAP23 appears to be limiting for insulin-dependent GLUT-4 exposure at the cell surface. A measurable fraction of SNAP23 was sedimented with cytoskeletal elements when extracted with Triton X-100, unlike VAMP-2 and syntaxin-4, which were exclusively soluble in detergent. We hypothesize that SNAP23 and its interaction with the cytoskeleton may be targets for regulation of GLUT-4 traffic.

3T3 Cells↗
PubMed1998

Binary interactions of the SNARE proteins syntaxin-4, SNAP23, and VAMP-2 and their regulation by phosphorylation.

The SNARE hypothesis proposes that synaptic vesicles dock at presynaptic membranes via interactions among the vesicular, integral membrane proteins VAMP (vesicle-associated membrane protein) and synaptotagmin and the target membrane proteins SNAP25 (synaptosome-associated protein with an Mr of 25 kDa) and syntaxin-1. Non-neuronal cells express isoforms of these proteins, believed to mediate secretory vesicle docking and/or fusion. Secretion in neuronal and non-neuronal systems differs in time course, Ca2+ dependence, and regulatory input. It is not known whether the non-neuronal protein isoforms form complexes akin to those of their neuronal counterparts. In this study, we defined the binding characteristics of three SNARE proteins: SNAP23, VAMP-2, and syntaxin-4. Binary, saturable interactions among all three partners (VAMP-2-syntaxin-4, VAMP-2-SNAP23, and SNAP23-syntaxin-4) were measured in vitro. Unlike its neuronal counterpart, SNAP23 did not potentiate VAMP-2 binding to its putative t-SNARE partner, syntaxin-4. The susceptibility of SNARE proteins to phosphorylation by exogenous kinases and their impact on binary interactions were explored. Syntaxin-4 was efficiently phosphorylated by casein kinase II (CKII) and cAMP-dependent protein kinase (PKA) (incorporating 0.8 and 3.9 mol of phosphate/mol of syntaxin-4, respectively), while syntaxin-1 was only strongly phosphorylated by CKII. Each of the syntaxin isoforms was weakly phosphorylated by protein kinase C (PKC) (<0.05 mol of phosphate/mol of syntaxin-4). Importantly, PKA but not casein kinase II phosphorylation of syntaxin-4 disrupted its binding to SNAP23. We hypothesize that PKA may modulate syntaxin-4-dependent SNARE complex formation to regulate exocytosis in non-neuronal cells.

Animals↗
PubMed1998

Perturbation of dynamin II with an amphiphysin SH3 domain increases GLUT4 glucose transporters at the plasma membrane in 3T3-L1 adipocytes. Dynamin II participates in GLUT4 endocytosis.

The GLUT4 glucose transporter continuously recycles between the cell surface and an endosomal compartment in adipocytes. Insulin decreases the rate of GLUT4 endocytosis in addition to increasing its exocytosis. Endocytosis of the transporter is thought to occur at least in part via the clathrin-mediated endocytic system. The protein dynamin is involved in the final stages of clathrin-coated vesicle formation. Here we show that the dynamin II isoform is expressed in 3T3-L1 adipocytes and is present in isolated plasma membrane and low density microsomal fractions. Insulin reduced the levels of dynamin II associated with the plasma membrane by about half, raising the possibility that the hormone may reduce GLUT4 endocytosis by removing dynamin from the cell surface. A fusion protein containing the amphiphysin SH3 domain selectively bound dynamin II from 3T3-L1 adipocyte cell lysates. Microinjection of the fusion protein into these cells inhibited transferrin endocytosis and increased the levels of GLUT4 at the cell surface. Glutathione S-transferase alone, the SH3 domains of spectrin and Crk, and a mutated amphiphysin SH3 domain unable to bind dynamin II did not affect GLUT4 distribution. However, a peptide containing the dynamin II sequence that binds amphiphysin increased the surface presence of GLUT4. Moreover, in cells first treated with insulin to externalize GLUT4, the dynamin peptide, but not an unrelated control peptide, inhibited GLUT4 internalization upon insulin removal. These results suggest that interactions of dynamin II with amphiphysin may play an important role in GLUT4 endocytosis. We hypothesize that insulin may reduce GLUT4 endocytosis by regulating the function of dynamin II at the cell surface, as part of the mechanism to increase glucose uptake.

3T3 Cells↗
PubMed1996

Intracellular depolymerase functionality and location in Pseudomonas oleovorans inclusions containing polyhydroxyoctanoate.

Microbial poly-3-hydroxyoctanoate inclusion bodies produced by Pseudomonas oleovorans when grown on n-octanoic acid, are complex macromolecular structures consisting of polyester, organized paracrystalline lattice arrays and lipids. While it is known that the polymer in the granules maintains its native, amorphous state while it is surrounded by the components of this complex, the precise functions of the various components during polymer production and utilization have yet to be established. By utilizing electron microscopy, SDS-PAGE, and gel filtration chromatography along with in vitro assays for depolymerase activity, the present study demonstrates that a protein species with molecular weight of approximately 32 kDa is the depolymerase protein of the polymer inclusion. When exogenous carbon was exhausted, cell viability required utilization of the stored polyester. Under these conditions, the concentration of the depolymerase increased while the concentrations of the polymerase decreased. Thus, the association of the depolymerase with the granules was shown to be under metabolic regulation relative to the polymerase. The results from the present studies show that careful manipulation of the substrate concentration can selectively, and differentially, alter the level of inclusion associated proteins as well as the quantity and quality of the polyester which is accumulated.

Bacterial Proteins↗
PubMed1996

Intracellular depolymerase and polyhydroxyoctanoate granule integrity in Pseudomonas oleovorans.

When polyhydroxyoctanoate (PHO) was produced by Pseudomonas oleovorans during a regimen of intermittent feeding on octanoic acid, there was a significant change in both the polymer associated proteins and the composition of the enclosed polymer. The polymer granules were isolated with their protein coat intact and the enzymatic hydrolysis of the polymer within this cell free system was determined. The degradation rate for the PHO in these native granules reached a maximum of 1.17 mg/h at an optimum pH of 9 when incubated at 30 degrees C. A study of the effect of various inhibitors on depolymerase activity suggested that the enzyme most likely has disulfide linkages and serine residues at its active site. Ultrastructure studies suggested this loss of enzyme activity was correlated with significant organizational degeneration in the proteins associated with the PHO inclusion body. Once solubilized from the granule, the depolymerase itself remained enzymatically active, and addition of this released material to other granule preparations increased the rate of polymer granule degradation. Similarly, when colloidal suspensions of purified, amorphous PHO were placed in contact with that depolymerase, they also underwent rapid degradation. In contrast, when crystalline solvent-cast PHO films were placed in contact with this enzyme, no degradative activity was observed.

Bacterial Proteins↗
PubMed1995

Enzymatic assay of hydroxybutyric acid monomer formation in poly(beta-hydroxybutyrate) degradation studies.

A novel method for monitoring the degradation of poly(beta-hydroxybutyrate) based on the enzymatic assay of beta-hydroxybutyrate (HBA) monomers has been developed. The method is particularly applicable to forms of the polymer, such as fibre and microcapsules, for which conventional surface and gravimetric monitoring techniques are not readily applicable. The method involves the use of enzyme HBA dehydrogenase in a reaction that converts nicotinamide adenine dinucleotide (NAD) to its reduced form (NADH). The conversion is associated with an increase in light absorption at 340 nm which thus serves to indicate the concentration of HBA monomer in the sample. The application of the methodology to the degradation of poly(beta-hydroxybutyrate) gel spun fibres has been used to demonstrate its potential use as a quantitative monitoring technique in the study of the hydrolysis of this polymer.

Hydrogen-Ion Concentration↗
PubMed1994

Quantitative determination of intracellular depolymerase activity in Pseudomonas oleovorans inclusions containing poly-3-hydroxyalkanoates with long alkyl substituents.

Research regarding the accurate, quantitative degradation of novel poly-3-hydroxyalkanoates has been restricted by the absence of an appropriate monitoring technique. The calibration of a gas chromatograph to poly-3-hydroxyoctanoate reveals a linear relationship between the area under gas chromatograph tracings and polymer weight. With this new method, poly-3-hydroxy-octanoate granules isolated from Pseudomonas oleovorans, which were incubated at 30 degrees C in an alkaline buffer, exhibited a linear degradation rate. Degradation was inhibited by the presence of Triton X-100 and phenylmethylsulfonyl fluoride. The depolymerase was demonstrated to be associated with the polymer granule complex and most likely possessed serine residues at its active site.

Chromatography, Gas↗
PubMed1979

Bronchodilator effects on gastric acid secretion.

Nine patients with chronic obstructive pulmonary disease were given oral aminophylline, intravenous aminophylline, and various inhaled and oral adrenergic bronchodilators to determine the effect of these agents on gastric acid secretion and gastrin release. Inhaled epinephrine hydrochloride resulted in an increase in basal acid output of borderline significance (.05 less than P less than .10). Oral aminophylline caused a significant increase in basal acid output from 2.43 to 4.06 mEq (P less than .05). Intravenous aminophylline also caused a significant increase in basal acid output from 0.66 to 2.19 mEq (P less than .01). There were no statistically significant changes in serum gastrin levels after administration of any of the bronchodilators. Aminophylline should be used with caution, if at all, in patients with peptic ulcer disease. In these patients, a beta agonist should be used for initial therapy. If the addition of aminophylline is necessary, antacids should be given.

Administration, Oral↗
PubMed1978

Effectiveness of oxygen therapy in hypoxic polycythemic smokers.

Eleven hypoxic (arterial oxygen pressure [PaO2] less than or equal to 61 mm Hg), polycythemic (hematocrit reading greater than or equal to 54 percent) patients were studied to determine the effect of the carboxyhemoglobin level on their response to therapy with supplemental oxygen. Five nonsmokers with a mean carboxyhemoglobin level of 2.8 percent showed an excellent response to therapy with supplemental oxygen, with a decrease in hematocrit reading from 57 percent to 48 percent (P less than 0.0025) as the PaO2 increased from 53 mm Hg to 69 mm Hg (P less than 0.01) and the arterial oxygen saturation (SaO2) increased from 86 percent to 94 percent (P less than 0.0025). Six smokers with a mean carboxyhemoglobin level of 13 percent showed an incomplete response to therapy with supplemental oxygen, with a decrease in hematocrit reading from 62 percent to 57 percent (P less than 0.0025) as the PaO2 increased from 53 mm Hg to 69 mm Hg (P less than 0.0025) and the SaO2 increased from 82 percent to 88 percent (P less than 0.001). In spite of similar values for the PaO2, the smokers had higher hematocrit readings before and during therapy with oxygen (P less than 0.01), presumably due to superimposed desaturation by carboxyhemoglobin. We concluded that polycythemia in hypoxic smokers is due to additive effects of hypoxia and a high carboxyhemoglobin level. The former is responsive to therapy with oxygen, while the latter is not. To achieve a "complete" response to therapy with supplemental oxygen, hypoxic polycythemic smokers should quit smoking.

Carboxyhemoglobin↗