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Biomedical subjects

K A Foster

Publications and source records attributed to K A Foster.

At least 19 recordsLinked to original sources
PubMed2001

An improved survival model of hypoxia/ischaemia in the piglet suitable for neuroprotection studies.

The purpose of this study was to develop a newborn piglet model of hypoxia/ischaemia which would better emulate the clinical situation in the asphyxiated human neonate and produce a consistent degree of histopathological injury following the insult. One-day-old piglets (n=18) were anaesthetised with a mixture of propofol (10 mg/kg/h) and alfentinal (55.5 microg/kg/h) i.v. The piglets were intubated and ventilated. Physiological variables were monitored continuously. Hypoxia was induced by decreasing the inspired oxygen (FiO(2)) to 3-4% and adjusting FiO(2) to maintain the cerebral function monitor peak amplitude at < or =5 microV. The duration of the mild insult was 20 min while the severe insult was 30 min which included 10 min where the blood pressure was allowed to fall below 70% of baseline. Control piglets (n=4 of 18) were subjected to the same protocol except for the hypoxic/ischaemic insult. The piglets were allowed to recover from anaesthesia then euthanased 72 h after the insult. The brains were perfusion-fixed, removed and embedded in paraffin. Coronal sections were stained by haematoxylin/eosin. A blinded observer examined the frontal and parietal cortex, hippocampus, basal ganglia, thalamus and cerebellum for the degree of damage. The total mean histology score for the five areas of the brain for the severe insult was 15.6+/-4.4 (mean +/-S.D., n=7), whereas no damage was seen in either the mild insult (n=4) or control groups. This 'severe damage' model produces a consistent level of damage and will prove useful for examining potential neuroprotective therapies in the neonatal brain.

Animals↗
PubMed2001

A mechanistic model for Ncd directionality.

Ncd is a kinesin-related protein that drives movement to the minus-end of microtubules. Pre-steady-state kinetic experiments have been employed to investigate the cooperative interactions between the motor domains of the MC1 dimer and to establish the ATPase mechanism. Our results indicate that the active sites of dimeric Ncd free in solution are not equivalent; ADP is held more tightly at one site than at the other. Upon microtubule binding, fast release of ADP from the first motor domain is stimulated at 18 s(-1), yet rate-limiting ADP release from the second motor domain occurs at 1.4 s(-1). We propose that the head with the low affinity for ADP binds the microtubule first to establish the directional bias of the microtubule.Ncd intermediate where one motor domain is bound to the microtubule with the second head detached and directed toward the minus-end of the microtubule. The force generating cycle is initiated as ATP binds to the empty site of the microtubule-bound head. ATP hydrolysis at head 1 is required for head 2 to bind to the microtubule. The kinetics indicate that two ATP molecules are required for a single step and force generation for minus-end directed movement generated by this non-processive dimeric motor.

Adenosine Diphosphate↗
PubMed2001

Microparticulate uptake mechanisms of in-vitro cell culture models of the respiratory epithelium.

The objective of this study was to examine the uptake mechanisms of fluorescent polystyrene microspheres of various diameters and surface chemistry by two human cell lines derived from the respiratory epithelium, A549 and Calu-3. Briefly, A549 and Calu-3 cells were grown to confluence in 12-well cluster plates and the uptake of fluorescent microspheres by the cells was determined at various time points. The amount of microspheres internalized by the cells was determined by correcting for non-specific binding to the cell surface. The data showed that A549 cells appeared to have more phagocytic activity than Calu-3 cells. Albumin-coated microspheres as large as 3 microm diameter can be internalized by A549 cells. The amount of internalization by A549 cells observed for 0.5-microm diameter albumin-coated microspheres was approximately 10-times greater than that observed for 1-microm diameter spheres and approximately 100-times greater than values observed for 2- and 3-microm diameter beads. Transmission electron micrographs confirmed that the microspheres were internalized by the cells. Uptake experiments conducted with Calu-3 cells indicated that albumin-coated microspheres were neither bound nor internalized by the cells. The effect of microsphere surface chemistry on the uptake mechanism indicated that amidine microspheres were internalized more rapidly and to a greater extent by both A549 and Calu-3 cells than carboxylate microspheres and non-coated microspheres. This phenomenon is thought to be attributed to masking of the negative polystyrene core by the positive amidine functional group; this effect was less marked for the carboxylate microspheres. These results suggest that A549 and Calu-3 cells can internalize microspheres and that size and effective charge played an important role in the uptake process.

Albumins↗
PubMed2000

Characterization of the Calu-3 cell line as a tool to screen pulmonary drug delivery.

The objective of this research was to examine the human sub-bronchial gland cell line, Calu-3, and assess its potential as a metabolic and transport model to study drug delivery to the respiratory epithelium. The present studies were conducted using Calu-3 cells grown in Transwells(R) or in multiwell cluster plates. TEER values for Calu-3 monolayers were determined using the World Precision Instrument Voltohmmeter and STX-2 electrode. The results confirmed that Calu-3 cells form tight monolayers and give appreciable TEER values in culture when grown under air-interface conditions. Permeability data for small lipophilic molecules across Calu-3 monolayers suggested that the cell line is a suitable model to examine the transport of low molecular weight substances and xenobiotics. Calu-3 cells were also found to efflux FITC-transferrin (MW 80000) in a polarized manner. The metabolic capacity of Calu-3 cells was also examined. The P4501A1 and P4502B isozymes were determined to be functional, but not inducible, with fluorescent resorufin assays. The data indicated that the Calu-3 cell line may be useful for studying the contributions of bronchial epithelial cells to mechanisms of drug delivery at the respiratory epithelium.

Administration, Inhalation↗
PubMed2000

Kinetic studies of dimeric Ncd: evidence that Ncd is not processive.

Ncd is a kinesin-related motor protein which drives movement to the minus-end of microtubules. The kinetics of Ncd were investigated using the dimeric construct MC1 (Leu(209)-Lys(700)) expressed in Escherichia coli strain BL21(DE) as a nonfusion protein [Chandra, R., Salmon, E. D., Erickson, H. P., Lockhart, A., and Endow, S. A. (1993) J. Biol. Chem. 268, 9005-9013]. Acid chemical quench flow methods were used to measure directly the rate of ATP hydrolysis, and stopped-flow kinetic methods were used to determine the kinetics of mantATP binding, mantADP release, dissociation of MC1 from the microtubule, and binding of MC1 to the microtubule. The results define a minimal kinetic mechanism, M.N + ATP M.N.ATP M.N.ADP.P N. ADP.P N.ADP + P M.N.ADP M.N + ADP, where N, M, and P represent Ncd, microtubules, and inorganic phosphate respectively, with k(+1) = 2.3 microM(-1) s(-1), k(+2) =23 s(-1), k(+3) =13 s(-1), k(+5)= 0.7 microM(-)(1) s(-)(1), and k(+6) = 3.7 s(-)(1). Phosphate release (k(+4)) was not measured directly although it is assumed to be fast relative to ADP release because Ncd is purified with ADP tightly bound at the active site. ATP hydrolysis occurs at 23 s(-)(1) prior to Ncd dissociation at 13 s(-)(1). The pathway for ATP-promoted detachment (steps 1-3) of Ncd from the microtubule is comparable to kinesin's. However, there are two major differences between the mechanisms of Ncd and kinesin. In contrast to kinesin, mantADP release for Ncd at 3.7 s(-)(1) is the slowest step in the pathway and is believed to limit steady-state turnover. Additionally, the burst amplitude observed in the pre-steady-state acid quench experiments is stoichiometric, indicating that Ncd, in contrast to kinesin, is not processive for ATP hydrolysis.

Adenosine Triphosphatases↗
PubMed2000

Cloning and characterization of the Dictyostelium discoideum cycloartenol synthase cDNA.

Cycloartenol synthase converts oxidosqualene to cycloartenol, the first carbocyclic intermediate en route to sterols in plants and many protists. Presented here is the first cycloartenol synthase gene identified from a protist, the cellular slime mold Dictyostelium discoideum. The cDNA encodes an 81-kDa predicted protein 50-52% identical to known higher plant cycloartenol synthases and 40-49% identical to known lanosterol synthases from fungi and mammals. The encoded protein expressed in transgenic Saccharomyces cerevisiae converted synthetic oxidosqualene to cycloartenol in vitro. This product was characterized by 1H and 13C nuclear magnetic resonance and gas chromatography-mass spectrometry. The predicted protein sequence diverges sufficiently from the known cycloartenol synthase sequences to dramatically reduce the number of residues that are candidates for the catalytic difference between cycloartenol and lanosterol formation.

Amino Acid Sequence↗
PubMed2000

Sensitivity of embryonic rat dorsal root ganglia neurons to Clostridium botulinum neurotoxins.

Clostridium botulinum neurotoxins (BoNT) are zinc dependent endopeptidases which, once internalised into the neuronal cytosol, block neurotransmission by proteolysis of membrane-associated proteins putatively involved in synaptic vesicle docking and fusion with the plasma membrane. Although many studies have used a variety of cellular systems to study the neurotoxins, most require relatively large amounts of toxin or permeabilisation to internalise the neurotoxin. We present here a primary culture of embryonic rat dorsal root ganglia (DRG) neurons that exhibits calcium-dependent substance P secretion when depolarised with elevated extracellular potassium and is naturally BoNT sensitive. The DRG neurons showed a different IC50 for each of the toxins tested with a 1000 fold difference between the most and least potent neurotoxins (0.05, 0.3, 30 and approximately 60 nM for A, C, F and B, respectively). BoNT/A cleavage of SNAP-25 was seen as early as 2 h, but substance P secretion was not significantly inhibited until 4 h intoxication and the effects of BoNT/A were observed for as long as 15 days. This primary neuronal culture system represents a new and sensitive cellular model for the in vitro study of the botulinum neurotoxins.

Animals↗
PubMed2000

An isolated in-situ rat head perfusion model for pharmacokinetic studies.

PURPOSE: To develop a viable, single pass rat head perfusion model useful for pharmacokinetic studies. METHODS: A viable rat head preparation, perfused with MOPS-buffered Ringer's solution, was developed. Radiolabelled markers (red blood cells, water and sucrose) were injected in a bolus into the internal carotid artery and collected from the posterior facial vein over 28 minutes. The double inverse Gaussian function was used to estimate the statistical moments of the markers. RESULTS: The viability of the perfusion was up to one hour, with optimal perfusate being 2% bovine serum albumin at 37 degrees C, pH 7.4. The distribution volumes for red blood cells, sucrose and water (from all studies, n = 18) were 1.0 +/- 0.3 ml, 6.4 +/- 4.2 ml and 18.3 +/- 11.9 ml, respectively. A high normalised variance for red blood cells (3.1 +/- 2.0) suggests a marked vascular heterogeneity. A higher normalised variance for water (6.4 +/- 3.3) is consistent with additional diffusive/permeability limitations. CONCLUSIONS: Analysis of the physiological parameters derived from the moments suggested that the kinetics of the markers were consistent with distribution throughout the head (weight 25 g) rather than just the brain (weight 2 g). This model should assist in studying solute pharmacokinetics in the head.

Animals↗
PubMed2000

Inhibition of vesicular secretion in both neuronal and nonneuronal cells by a retargeted endopeptidase derivative of Clostridium botulinum neurotoxin type A.

Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types by a mechanism that involves cleavage of specific components of the vesicle docking/fusion complex, the SNARE complex. A derivative of the type A neurotoxin from Clostridium botulinum (termed LH(N)/A) that retains catalytic activity can be prepared by proteolysis. The LH(N)/A, however, lacks the putative native binding domain (H(C)) of the neurotoxin and is thus unable to bind to neurons and effect inhibition of neurotransmitter release. Here we report the chemical conjugation of LH(N)/A to an alternative cell-binding ligand, wheat germ agglutinin (WGA). When applied to a variety of cell lines, including those that are ordinarily resistant to the effects of neurotoxin, WGA-LH(N)/A conjugate potently inhibits secretory responses in those cells. Inhibition of release is demonstrated to be ligand mediated and dose dependent and to occur via a mechanism involving endopeptidase-dependent cleavage of the natural botulinum neurotoxin type A substrate. These data confirm that the function of the H(C) domain of C. botulinum neurotoxin type A is limited to binding to cell surface moieties. The data also demonstrate that the endopeptidase and translocation functions of the neurotoxin are effective in a range of cell types, including those of nonneuronal origin. These observations lead to the conclusion that a clostridial endopeptidase conjugate that can be used to investigate SNARE-mediated processes in a variety of cells has been successfully generated.

Animals↗
PubMed2000

Experimental methods for studying drug uptake in the head and brain.

A number of techniques have been developed to study the disposition of drugs in the head and, in particular, the role of the blood-brain barrier (BBB) in drug uptake. The techniques can be divided into three groups: in-vitro, in-vivo and in-situ. The most suitable method depends on the purpose(s) and requirements of the particular study being conducted. In-vitro techniques involve the isolation of cerebral endothelial cells so that direct investigations of these cells can be carried out. The most recent preparations are able to maintain structural and functional characteristics of the BBB by simultaneously culturing endothelial cells with astrocytic cells. The main advantages of the in-vitro methods are the elimination of anaesthetics and surgery. In-vivo methods consist of a diverse range of techniques and include the traditional Brain Uptake Index and indicator diffusion methods, as well as microdialysis and positron emission tomography. In-vivo methods maintain the cells and vasculature of an organ in their normal physiological states and anatomical position within the animal. However, the shortcomings include renal and hepatic elimination of solutes as well as the inability to control blood flow. In-situ techniques, including the perfused head, are more technically demanding. However, these models have the ability to vary the composition and flow rate of the artificial perfusate. This review is intended as a guide for selecting the most appropriate method for studying drug uptake in the brain.

Animals↗
PubMed2000

A conjugate composed of nerve growth factor coupled to a non-toxic derivative of Clostridium botulinum neurotoxin type A can inhibit neurotransmitter release in vitro.

Nerve growth factor (NGF) receptor binding, internalisation and transportation of NGF has been identified as a potential route of delivery for other molecules. A derivative of Clostridium botulinum neurotoxin type A (LHN) that retains catalytic activity but has significantly reduced cell-binding capability has been prepared and chemically coupled to NGF. Intact clostridial neurotoxins potently inhibit neurotransmitter release at the neuromuscular junction by proteolysis of specific components of the vesicle docking/fusion complex. Here we report that the NGF-LHN/A conjugate, when applied to PC12 cells, significantly inhibited neurotransmitter release and cleaved the type A toxin substrate. This work represents the successful use of NGF as a targeting moiety for the delivery of a neurotoxin fragment.

Animals↗
PubMed1999

Development of in vitro assays for the detection of botulinum toxins in foods.

Currently the only accepted method for the detection of botulinum neurotoxin in contaminated samples is the mouse bioassay. Although highly sensitive this test has a number of drawbacks: it is expensive to perform, lacks specificity and involves the use of animals. With increasing resistance to such animal tests there is a need to replace the bioassay with a reliable in vitro test. Over the past six years it has been demonstrated that all the botulinum neurotoxins act intracellularly as highly specific zinc endoproteases, cleaving proteins involved in the control of secretion of neurotransmitters. In the work described, this enzymatic activity has been utilised in assay formats for the detection in foods of neurotoxin of the serotypes involved in food-borne outbreaks in man. These assays have been shown to have a greater sensitivity, speed and specificity than the mouse bioassay. It is envisaged that such assays will prove realistic alternatives to animal-based tests.

Animals↗
PubMed1999

Novel assays for the detection of botulinum toxins in foods.

Currently the only accepted method for the detection of botulinum neurotoxin in contaminated samples is the mouse bio-assay. Although highly sensitive this test has a number of drawbacks: it is expensive to perform, lacks specificity and involves the use of animals. With increasing resistance to such animal tests there is a need to replace the bio-assay with a reliable in vitro test. Over the past six years it has been demonstrated that all the botulinum neurotoxins act intracellularly as highly specific zinc endoproteases, cleaving proteins involved in the control of secretion of neurotransmitters. In the work described, this enzymatic activity has been utilised in assay formats for the detection in foods of neurotoxin from the serotypes involved in food-borne outbreaks in man. These assays have been shown to have a greater sensitivity, speed and specificity than the mouse bio-assay. It is envisaged that such assays will prove realistic alternatives to animal based tests.

Animals↗
PubMed1998

Equilibrium binding studies of non-claret disjunctional protein (Ncd) reveal cooperative interactions between the motor domains.

Non-claret disjunctional protein (Ncd) is a minus end-directed microtubule motor required for normal spindle assembly and integrity during Drosophila oogenesis. We have pursued equilibrium binding experiments to examine the affinity of Ncd for microtubules in the presence of the ATP nonhydrolyzable analog 5'-adenylyl-beta, gamma-imidodiphosphate (AMP-PNP), ADP, or ADP + Pi using both dimeric (MC1) and monomeric (MC6) Ncd constructs expressed in Escherichia coli. Both MC1 and MC6 sediment with microtubules in the absence of added nucleotide as well as in the presence of either ADP or AMP-PNP. Yet, in the presence of ADP + Pi, there is a decrease in the affinity of both MC1 and MC6 for microtubules. The data for dimeric MC1 show that release of the dimer to the supernatant is sigmoidal with the apparent Kd(Pi) for the two phosphate sites at 23.3 and 1.9 mM, respectively. The results indicate that binding at the first phosphate site enhances binding at the second site, thus cooperatively stimulating release. Stopped-flow kinetics indicate that MgATP promotes dissociation of the Mt.MC1 complex at 14 s-1, yet AMP-PNP has no effect on the Mt.MC1 complex. These results are consistent with a model for the ATPase cycle in which ATP hydrolysis occurs on the microtubule followed by detachment as the Ncd.ADP.Pi intermediate.

Adenosine Diphosphate↗
PubMed1998

Characterization of the A549 cell line as a type II pulmonary epithelial cell model for drug metabolism.

Multiple cell types contribute to the pulmonary barrier including Type I and Type II alveolar epithelium. The objective of this research was to establish and characterize an in vitro model of Type II alveolar epithelium using the A549 human lung adenocarcinoma cell line. A549 cells form confluent monolayers with Type II characteristic morphology and tannic acid staining for typical lamellar bodies. A549 cells possess P450 IA1 and P450 IIB6 as determined by Western blots. Both CYPIA1 and CYPIIB6 P450 isozymes were determined to be functional with the fluorescent resorufin assay. Only the IA1 isozyme was observed to be inducible with selected polycyclic hydrocarbons. Uptake and transport experiments were carried out in cluster plates and in Snapwells. Cationized ferritin, a nonspecific absorbtive marker, was found to be taken up by the cells in a concentration-, time-, and temperature-dependent fashion. Lucifer yellow, a fluid-phase marker, was not internalized by the A549 cells. Transferrin, a representative receptor-mediated endocytic marker, was found to be taken up by the cells in a concentration-dependent and competitive fashion. Transport experiments involving fluorescein-transferrin also showed that A549 monolayers were polarized, with a greater amount of intracellular transferrin being transported out of the basolateral side of the cells. The experimental data agree favorably with literature for primary cultures of Type II pulmonary epithelial cells. These results indicated that the A549 cell line may be useful for the studying the metabolic and macromolecule processing contributions of alveolar Type II cells to mechanisms of drug delivery at the pulmonary epithelium.

Biological Transport↗
PubMed1998

Comparison of the effects of potential parenteral vehicles for poorly water soluble anticancer drugs (organic cosolvents and cyclodextrin solutions) on cultured endothelial cells (HUV-EC).

The effect of dilution of parenteral vehicles (organic cosolvent and 0.1 M cyclodextrin solutions) on cultured endothelial cells (HUV-EC) were compared in vitro. Cell morphology was observed by phase contrast light microscopy and cell viability by measuring 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction or intracellular lactate dehydrogenase (LDH) activity and total protein. Disruption of the HUV-EC monolayer was observed at dilutions of 1 in 20 for the melphalan and PEP cosolvents, 1 in 100 for an investigational drug cosolvent, and 1 in 10 for 0.1 M dimethyl-beta-cyclodextrin. In comparison, 0.1 M SBE7M- and HP-beta-cyclodextrin caused only minor disruption at a 1 in 5 dilution. MTT reduction, intracellular LDH, and total protein were decreased following exposure to 1 in 10 dilution of the melphalan cosolvent. For other test solutions, intracellular LDH activity and total protein were measured, and reductions were observed following exposure to 1 in 10, 20, and 50 dilutions of the investigational drug cosolvent and 1 in 5 dilution of DM-beta-cyclodextrin (0.1 M). At a dilution of 1 in 10, no delayed toxicity was observed for cosolvents or cyclodextrin solutions. Hence, 0.1 M SBE7M- or HP-beta-cyclodextrin formulations may be less damaging to the venous endothelium at the site of injection than organic cosolvent formulations.

Antineoplastic Agents↗
PubMed1997

Botulinum neurotoxin B inhibits insulin-stimulated glucose uptake into 3T3-L1 adipocytes and cleaves cellubrevin unlike type A toxin which failed to proteolyze the SNAP-23 present.

Types A, B, and C1 botulinum neurotoxin (BoNT), a group of selective Zn2+-dependent endoproteases, have been instrumental in demonstrating that their respective substrates [synaptosomal-associated protein with Mr = 25 kDa (SNAP-25), synaptobrevin (Sbr), and syntaxin] are essential for regulated exocytosis from nerve terminals and neuroendocrine cells. The colocalization of Sbr, or its homologue cellubrevin (Cbr), in the majority of the glucose transporter-isotype 4 (GLUT4)-containing vesicles from adipocytes implicates their involvement in insulin-stimulated glucose uptake, which results in part from enhanced fusion of these vesicles with the plasmalemma. In this study, exposure of cultured 3T3-L1 adipocytes to BoNT/B in a low-ionic strength medium was found to block insulin-evoked glucose uptake by up to 64%. BoNT/B was shown by immunoblotting to cause extensive proteolysis of Cbr and Sbr resulting in a significant blockade of the insulin-stimulated translocation of GLUT4 to the plasmalemma. This establishes that these two toxin substrates contribute to the insulin-regulated fusion of GLUT4-containing vesicles with the plasmalemma, at least in this differentiated 3T3-L1 clone. Although SNAP-25 was not detectable in the differentiated adipocytes, its functional homologue SNAP-23 is abundant and largely confined to the plasmalemma. SNAP-23 proved to be resistant to cleavage by BoNT/A. Consistent with these results, type A did not block insulin-induced glucose uptake, precluding a demonstration of its likely importance in this process.

3T3 Cells↗