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Biomedical subjects

F M Harris

Publications and source records attributed to F M Harris.

18 recordsLinked to original sources
PubMed2001

Physical properties of erythrocyte ghosts that determine susceptibility to secretory phospholipase A2.

Artificial membranes may be resistant or susceptible to catalytic attack by secretory phospholipase A(2) (sPLA(2)) depending on the physical properties of the membrane. Living cells are normally resistant but become susceptible during trauma, apoptosis, and/or a significant elevation of intracellular calcium. Intact erythrocytes and ghosts were studied to determine whether the principles learned from artificial systems apply to biological membranes. Membrane properties such as phospholipid and/or protein composition, morphology, and microscopic characteristics (e.g. fluidity) were manipulated by preparing ghosts under different experimental conditions such as in the presence or absence of divalent cations with or without ATP. The properties of each membrane preparation were assessed by biochemical and physical means (fluorescence spectroscopy and electron and two-photon microscopy using the membrane probes bis-pyrene and laurdan) and compared with sPLA(2) activity. The properties that appeared most relevant were the degree of phosphatidylserine exposure on the outer face of the membrane and changes to the membrane physical state detected by bis-pyrene and laurdan. Specifically, vulnerability to hydrolysis by sPLA(2) was associated with an increase in bilayer order apparently reflective of expansion of membrane regions of diminished fluidity. These results argue that the general principles identified from studies with artificial membranes apply to biological systems.

Erythrocyte Membrane↗
PubMed2001

Mechanisms by which intracellular calcium induces susceptibility to secretory phospholipase A2 in human erythrocytes.

Exposure of human erythrocytes to the calcium ionophore ionomycin rendered them susceptible to the action of secretory phospholipase A(2) (sPLA(2)). Analysis of erythrocyte phospholipid metabolism by thin-layer chromatography revealed significant hydrolysis of both phosphatidylcholine and phosphatidylethanolamine during incubation with ionomycin and sPLA(2). Several possible mechanisms for the effect of ionomycin were considered. Involvement of intracellular phospholipases A(2) was excluded since inhibitors of these enzymes had no effect. Assessment of membrane oxidation by cis-parinaric acid fluorescence and comparison to the oxidants diamide and phenylhydrazine revealed that oxidation does not participate in the effect of ionomycin. Incubation with ionomycin caused classical physical changes to the erythrocyte membrane such as morphological alterations (spherocytosis), translocation of aminophospholipids to the outer leaflet of the membrane, and release of microvesicles. Experiments with phenylhydrazine, KCl, quinine, merocyanine 540, the calpain inhibitor E-64d, and the scramblase inhibitor R5421 revealed that neither phospholipid translocation nor vesicle release was required to induce susceptibility. Results from fluorescence spectroscopy and two-photon excitation scanning microscopy using the membrane probe laurdan argued that susceptibility to sPLA(2) is a consequence of increased order of membrane lipids.

Biological Transport↗
PubMed2001

Binding of steroidogenic acute regulatory protein to synthetic membranes suggests an active molten globule.

Steroidogenic acute regulatory protein (StAR) mediates cholesterol transport from the outer to the inner mitochondrial membrane during steroid biosynthesis. The mechanism of StAR's action is not established. To address mechanistic issues, we assessed the binding of StAR to artificial membranes by fluorescence resonance energy transfer using endogenous StAR tryptophan residues as the donor and dansyl-phosphatidylethanolamine in the bilayer as the acceptor. Mixing StAR with dansyl-labeled vesicles composed of phosphatidylcholine increased the fluorescence intensity of dansyl emission excited at 280 nm by 10-40%. This interaction was dependent on pH, with a maximum at pH 3.0-3.5 and essentially no change above pH 5. Binding experiments at different temperatures and various combinations of phosphatidylcholine, phosphatidylglycerol, cardiolipin, and cholesterol showed that binding involves an electrostatic step and one or more other steps. Although binding prefers a thermodynamically ordered bilayer, the rate-limiting step occurs either when the bilayer is in a fluid state or when there is cholesterol-induced membrane heterogeneity. Experiments with fluorescence and light scattering indicate that StAR binding promotes ordering and aggregation of anionic membranes. The inactive StAR mutant R182L had lower affinity for the membrane, and the partially active mutant L275P had intermediate affinity. Far-UV CD spectroscopy of StAR in PC membranes show more beta-structure than in aqueous buffers, and the presence of cardiolipin or cholesterol in the membrane fosters a molten globule state. Our data suggest that StAR binds to membranes in a partially unfolded molten globule state that is relevant to the activity of the protein.

Amino Acid Substitution↗
PubMed1999

Kinetic analysis of cyclic CMP-specific and multifunctional phosphodiesterases by quantitative positive-ion fast-atom bombardment mass spectrometry.

Two enzymes, cyclic CMP-specific phosphodiesterase and multifunctional phosphodiesterase, are responsible for the hydrolysis of cytidine 3',5'-cyclic monophosphate in living cells. Quantitation of both enzymes has been carried out by positive-ion fast-atom bombardment mass spectrometric analysis of the enzyme incubates after termination of the reaction. The kinetic data obtained are in close agreement with parallel data obtained by the conventional radiometric assay. The extra facility of the mass spectrometry based assay to monitor several incubation components simultaneously has been exploited to study the concurrent hydrolysis of alternate cyclic nucleotide substrates and provides kinetic parameters of significance in interpreting substrate-enzyme interactions. This is extended by the use of collisionally-induced dissociation of the protonated molecules of the liberated products to identify the mononucleotide isomers resulting from the cyclic nucleotide hydrolysis.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗
PubMed1998

Fast-atom bombardment tandem mass spectrometry of cyclic nucleotide analogues used as site-selective activators of cyclic nucleotide-dependent protein kinases.

The mass spectrometric behaviour of six cyclic nucleotide analogues which activate cyclic AMP-dependent protein kinase was studied by positive-ion fast-atom bombardment (FAB) and collision-induced dissociation (CID) mass-analysed ion kinetic energy (MIKE) spectrometry. The compounds studied were 1,N6-ethenoadenosine-3',5'-cyclic monophosphate, (epsilon-cyclic AMP) and 2'-aza-1,N6-ethenoadenosine-3',5'-cyclic monophosphate, which each activate both isoforms of cyclic AMP-dependent protein kinase and have similar affinity for both the 'fast' and the 'slow' regulatory site of each isoform, N6-phenyl-cyclic AMP, which is selective for the 'fast' regulatory site of each isoform, and 6-chloropurine riboside-3',5'-cyclic monophosphate, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphate and 8-(4-chlorophenylthio)-adenosine-3',5'-cyclic monophosphate, which are each selective for the 'slow' regulatory site and preferentially activate isoform II. The FAB- and CID/MIKE spectra of the analogues are discussed in relation to their use in studies of the regulation of protein kinase activity by quantitative FAB mass spectrometry.

Cyclic AMP↗
PubMed1997

Estimation of cytidylyl cyclase activity and monitoring of side-product formation by fast-atom bombardment mass spectrometry.

The enzyme cytidylyl cyclase catalyses the conversion of cytidine 5'-triphosphate into cytidine 3',5'-cyclic monophosphate, a third naturally occurring cyclic nucleotide currently under investigation to assign a biochemical function. Quantitation of the activity of this enzyme has been carried out by the positive-ion fast-atom bombardment mass spectrometric analysis of the enzyme incubation mixture after the reaction has been terminated. The data obtained are in good agreement with those obtained from the conventional radiometric and radioimmunoassays of the same enzyme preparations. The advantage of the mass spectrometer-based assay is the facility for multiple component monitoring. Thus, the production of the cytidine diphosphates and monophosphates, and the production of four cytidine 3',5'-cyclic monophosphate analogues as side-products, were simultaneously estimated. The identities of two of the side-products, 2'-O-glutamyl- and 2'-O-aspartyl-cytidine-3',5'-cyclic monophosphate, and of the cytidine 3',5'-cyclic monophosphate product, were confirmed by mass-analysed ion kinetic energy spectra from the collision-induced dissociation of the protonated molecules.

Animals↗
PubMed1997

Kinetic analysis and multiple component monitoring of effectors of adenylyl cyclase activity by quantitative fast-atom bombardment mass spectrometry.

The enzyme adenylyl cyclase catalyses the conversion of adenosine 5'-triphosphate (ATP) to adenosine-3',5'-cyclic monophosphate (cyclic AMP), and is an important pharmaceutical target. Quantitation of this enzyme's activity has been carried out by positive-ion fast-atom bombardment mass spectrometric analysis of the enzyme incubation mixture after the reaction has been terminated. The kinetic data obtained are in good agreement with those obtained by the conventional radiometric assay, and this mass spectrometry-based assay offers the facility to monitor the turnover of several components of the incubation simultaneously. This is utilized to study the relative efficiencies of two ATP-regenerating systems, three phosphodiesterase inhibitors and two modified substrates, and to monitor the uptake and conversion of two competing substrates, adenosine 5' triphosphate and 2'-deoxyadenosine-5-triphosphate, to cyclic AMP and to cyclic deoxyAMP, respectively.

Adenosine Triphosphate↗
PubMed1997

Clinical utility of hygroscopic heat and moisture exchangers in intensive care patients.

OBJECTIVE: To compare the degree of bacterial circuit colonization, frequency of ventilator-associated pneumonia (VAP), character of respiratory secretions, rewarming of hypothermic patients, disposable costs, and air flow resistance in intensive care patients ventilated using either a heat and moisture exchanger (HME) or hot water (HW) humidifier circuit. DESIGN: A prospective, randomized blinded trial of patients in the intensive care unit undergoing mechanical ventilation. SETTING: A metropolitan teaching hospital. PATIENTS: One hundred sixteen patients undergoing mechanical ventilation for a minimum period of 48 hrs were enrolled. INTERVENTIONS: Patients were randomized to three ventilation groups using a) an HW circuit with a 2-day circuit change (n = 41); or b) a bacterial-viral filtering HME in the circuit, with either a 2-day (n = 42); or c) a 4-day circuit change (n = 33). MEASUREMENTS AND MAIN RESULTS: Circuit colonization was assessed using quantitative culture of washings taken from the circuit tubing and semiquantitative culture of swabs from the Y connectors. Sixty-seven percent of HW circuits became contaminated compared with 12% in the two HME groups (p < .0001). Median colony counts were lower in the HME groups (p < .0001). If circuits at first circuit change were contaminated in the HW group, 89% of subsequent circuit changes became contaminated compared with 0% and 25% for the 2- and 4-day HME groups, respectively. The frequency of VAP, the time to resolution of admission hypothermia, and the volume and fluidity of secretions were similar for all groups. The resistance of the HME after 24 hrs of use was < 0.025 cm H2O/L at gas flows of 40 L/min. HME use resulted in a cost reduction of $1.48 (Australian)/day. CONCLUSIONS: Circuits with a bacterial-viral filtering HME are less readily colonized by bacteria. Contamination is a random event. Humidification technique has no influence on the frequency rate of VAP, the effectiveness of rewarming, nor the character of the respiratory secretions. Breathing resistance is generally low and disposable costs are reduced when an HME is used.

APACHE↗
PubMed1995

Assay of adenosine 3',5'-cyclic monophosphate-dependent protein kinase activity by quantitative fast atom bombardment mass spectrometry.

Cyclic AMP-dependent protein kinase is conventionally assayed by measuring the incorporation of radiolabeled phosphate into a histone substrate. Here the assay of the protein kinase is carried out by the positive-ion fast atom bombardment mass spectrometric analysis of the enzyme incubation mixture after the reaction has been terminated. The data obtained are in good agreement with those obtained from the conventional radiometric assay of the same kinase preparation. The inherent advantage of this mass spectrometric assay is the capacity for multiple component monitoring; in addition to the kinase activity, the ability of the enzyme to bind cyclic nucleotides, together with integral ATPase and phosphodiesterase activity, can also be estimated from the same spectra.

Cyclic AMP↗
PubMed1994

Proton-transfer reactions of mass-selected multiply charged ions.

A triple-quadrupole spectrometer has been used to study proton-transfer reactions of multiply charged ions generated by electrospray ionization. Doubly and triply charged ions generated from the peptides Arg-Lys-Glu-Val-Tyr and Met-Lys-bradykinin, respectively, were found to undergo proton-transfer reactions with ammonia molecules contained in the RF-only quadrupole collision-gas cell of the spectrometer. With horse-heart myoglobin in the source, ions having charges of 20+, 19+, 16+ and 14+ were selected in turn by the first quadrupole and their proton-transfer reactions with ammonia investigated. For each ion, numerous product ions were detected having charges (n-1)+, (n-2)+, (n-3)+ ... where n was the charge on the reacting parent ion. The possibility of using the experimental technique to measure approximately the proton affinities of multiply charged ions is discussed. Also, a procedure is outlined for identifying the charge states of product ions resulting from collision-induced dissociation of multiply charged ions.

Amino Acid Sequence↗
PubMed1993

Analysis of cyclic nucleotide-related enzymes by continuous-flow fast-atom bombardment mass spectrometry.

Continuous-flow fast-atom bombardment mass spectrometry has been developed to directly monitor cyclic nucleotide (substrate) and its product levels from an on-going phosphodiesterase reaction. Analysis of cAMP and cCMP phosphodiesterase incubates have been performed where the temporal evolution of the enzymic reaction is monitored and the effect of enzyme concentration upon the rate of reaction determined. Quantitative data on the enzyme kinetics have been obtained, in the form of Lineweaver-Burke plots, that are shown to correlate well with well-established radiometric methods.

Animals↗
PubMed1992

Quantitation by fast-atom bombardment mass spectrometry: assay of cytidine 3',5'-cyclic monophosphate-responsive protein kinase.

A protein kinase, stimulated by cytidine 3',5'-cyclic monophosphate, is conventionally assayed by monitoring the incorporation of radiolabelled phosphate from adenosine triphosphate into a histone substrate. Here the assay of the protein kinase is carried out by positive-ion fast-atom bombardment mass spectrometric analysis of the enzyme incubation mixture after the reaction has been terminated. The data so obtained show good agreement with data obtained by the conventional radiometric assay: the intrinsic advantage of the mass spectrometric assay is the capacity for multiple component monitoring; the ability of the kinase to bind competing cyclic nucleotides together with integral adenosine triphosphatase (ATPase) and phosphodiesterase activity can also be assessed.

Adenosine Triphosphate↗
PubMed1989

Quantitation by fast-atom bombardment/mass-analysed ion kinetic energy spectrometry: kinetic analysis of cyclic nucleotide phosphodiesterase activity.

Quantitation of cyclic nucleotide phosphodiesterase activity by means of fast-atom bombardment (FAB) mass spectrometry with mass-analysed ion kinetic energy (MIKE) spectrum scanning is described. Characteristic peaks of the substrate, cyclic AMP, and product, AMP, were identified in positive-ion FAB mass spectra and MIKE scans of the protonated molecules. By spiking enzyme incubates with known quantities of cyclic AMP and AMP and measuring peak heights in the MIKE spectra of both spiked and unspiked samples, the concentrations of cyclic AMP and AMP in solution at the end of a series of enzyme incubations have been estimated. From the data obtained the Km and Vmax of the enzymes were calculated as 181 microM and 28.6 nmol/min respectively, showing excellent agreement with values of the Michaelis constant, Km = 205 microM and the maximum velocity Vmax = 33.2 nmol/min obtained by radioactive assay.

3',5'-Cyclic-AMP Phosphodiesterases↗