Biomedical subjects

D P Lew

Publications and source records attributed to D P Lew.

At least 73 records · Page 4Linked to original sources

PubMed1993

Inositol 1,4,5-trisphosphate binding sites copurify with the putative Ca-storage protein calreticulin in rat liver.

Rat liver was homogenized and subjected to differential centrifugation. When the low speed nuclear pellet was processed on a Percoll gradient, plasma membrane markers and Ins(1,4,5)P3 binding activity purified together. The high speed (microsomal) fraction was subfractionated by sucrose density gradient centrifugation, resulting in 10-fold enrichment of [32P]-Ins(1,4,5)P3 binding. In the sucrose density gradient fractions there was an inverse relationship between the enrichment of plasma membrane markers and Ins(1,4,5)P3 binding sites. Endoplasmic reticulum markers showed a moderate enrichment in the fractions displaying high Ins(1,4,5)P3 binding activity. Calcium binding proteins in the homogenate and in the microsomal subfractions were separated by SDS/PAGE. A 60 kD protein, stained metachromatically with Stains-All was identified as calreticulin with immunoblotting. Its enrichment pattern was similar to that of Ins(1,4,5)P3 binding sites, indicating the co-existence of these two elements of Ca(2+)-metabolism in the same intracellular compartment in the liver.

Animals↗

PubMed1993

Fibronectin is more active than fibrin or fibrinogen in promoting Staphylococcus aureus adherence to inserted intravascular catheters.

To further define the role of fibrin(ogen) and fibronectin in Staphylococcus aureus adherence to central venous catheters, the amount, chemical integrity, and biologic activity of these proteins adsorbed on lines inserted in hospitalized patients were prospectively studied. Polyurethane cannulas promoted a significantly lower adherence of S. aureus than polyvinyl chloride (P < .01) or Hickman (P < .001) cannulas and contained the lowest amount of immunologically assayed fibronectin but not of fibrin(ogen). Fibrinogen showed an extensive loss of adherence-promoting activity on inserted cannulas, which was related to its proteolytic breakdown, as detected by SDS-PAGE and immunoblots with antifibrinogen antibodies and confirmed by in vitro studies with purified protein fragments. In contrast, either intact or fragmented fibronectin, although present in much lower amounts than fibrin(ogen), could actively promote S. aureus adherence onto intravenous catheters.

Anticoagulants↗

PubMed1993

Proton channels, plasma membrane potential, and respiratory burst in human neutrophils.

When confronted with invading microorganisms, neutrophils undergo a number of nearly synchronous reactions including the generation of microbicidal reactive oxygen intermediates by the NADPH oxidase. These reactions are accompanied by a slow depolarization, from resting values of-60 mV to levels probably exceeding 0 mV. The depolarization is transient, indicating that a compensatory charge transport mechanism is activated. Activation of the oxidase system causes a massive burst of metabolic acid generation that would, if uncompensated, lower the intracellular pH of neutrophils by over 5 units, to lethal levels (pH = 2). Neutrophils must therefore possess particularly effective regulatory systems to avoid excessive cytosolic acidification. The recently described H+ conductance of neutrophils may counteract both the acidification and the depolarization. Activation of the H+ conductance occurs at depolarizing voltages and is promoted by cytosolic acidification, a combination that takes place during the respiratory burst. The NADPH oxidase of neutrophils is thus associated to an unusual, particularly efficient mechanism of H+ export and charge compensation. The sequential activation of these two systems causes neutrophils to depolarize through the activation of an electron transport chain, and to repolarize through the activation of a H+ conductance.

Cell Membrane↗

PubMed1993

Highly co-operative Ca2+ activation of intermediate-conductance K+ channels in granulocytes from a human cell line.

1. To study Ca(2+)-activated K+ currents in dimethyl sulphoxide (DMSO)-differentiated HL-60 cells (HL-60 granulocytes), we have combined the patch clamp technique with microfluorimetric measurements of the cytosolic free Ca2+ concentration ([Ca2+]i). 2. Elevations of [Ca2+]i induced by the receptor agonist N-formyl-L-methionyl-L-phenylalanine (f-MLP), by cellular spreading or by the Ca2+ ionophore ionomycin, activated whole-cell currents. The kinetics of the current elevations closely paralleled the kinetics of the elevations in [Ca2+]i. Cellular spreading induced oscillations in [Ca2+]i and parallel oscillatory changes in the amplitude of the recorded currents. 3. The reversal potential of the Ca(2+)-activated current was a function of the extracellular K+ concentration (56.1 mV per log [K+]), demonstrating that the underlying conductance was selective for K+. 4. The current was blocked by charybdotoxin, but insensitive to apamin. 5. The whole-cell current was inwardly rectifying. No time-dependent activation or inactivation of the current could be observed within the range of voltages tested (-100 to +100 mV). 6. The dependence of the current amplitude on the measured [Ca2+]i revealed a half-maximal activation at approximately 350 nM [Ca2+]i, and a highly co-operative activation by [Ca2+]i with an apparent Hill coefficient of approximately 8. Neither the half-maximal activation by [Ca2+]i nor the apparent Hill coefficient depended on the voltage, and they were identical for Ca2+ elevations caused by the ionophore and the receptor agonist. 7. Analysis of Ca(2+)-activated single-channel events in cell-attached recordings revealed an inwardly rectifying K+ channel with a slope conductance of 35 pS. Fluctuation analysis of the Ca(2+)-activated whole-cell current suggested an underlying single-channel conductance of a similar size (28 pS). 8. In summary, we describe a charybdotoxin-sensitive, intermediate-conductance Ca(2+)-activated K+ channel in HL-60 granulocytes. The characteristics of the Ca2+ activation of this current (i.e. sensitivity to submicromolar [Ca2+]i, high co-operativity and voltage independence) are similar to the Ca2+ activation of the apamin-sensitive small-conductance K+ channel. Our results also suggest that [Ca2+]i elevations are the predominant, if not the only, activators of this channel during physiological stimulation of HL-60 granulocytes.

Apamin↗

PubMed1993

Influence of rifampin on fleroxacin pharmacokinetics.

Staphylococcus aureus infections have been successfully treated in animal models with the combination of fleroxacin and rifampin. We studied the influence of rifampin, a potent cytochrome P-450 inducer, on the pharmacokinetics and biotransformation of fleroxacin in 14 healthy young male volunteers. Subjects were given 400 mg of fleroxacin orally once a day for 3 days to reach steady state. After a wash-out period of 2 days, the same subjects received 600 mg of rifampin orally once daily for 7 days. On days 5 to 7 of rifampin treatment, 400 mg of fleroxacin was again administered once daily. Concentrations of fleroxacin as well as its two major urinary metabolites, N-demethyl- and N-oxide-fleroxacin, in plasma and urine were determined by reverse-phase high-performance liquid chromatography. The extent of hepatic enzyme induction by rifampin was confirmed by a significant increase of 6-beta-hydroxycortisol urinary output from 160.8 +/- 41.4 to 544.8 +/- 120.7 micrograms/4 h. There were no significant changes in the peak fleroxacin concentration in plasma (6.3 +/- 1.2 versus 6.2 +/- 1.9 mg/liter), time to maximum concentration of fleroxacin in plasma (1.1 +/- 0.9 versus 1.3 +/- 1.1 h), or renal clearance (58.3 +/- 16.4 versus 61.9 +/- 19.2 ml/min). The area under the curve AUC (71.4 +/- 15.8 versus 62.2 +/- 13.7 mg.h/liter) and the terminal half-life of fleroxacin (11.4 +/- 2.2 versus 9.2 +/- 1.1 h) decreased (P < 0.05), while the total plasma clearance increased from 97.7 +/- 21.6 to 112.3 +/- 25.8 ml/min (P < 0.01). Despite being statistically significant, this 15% increase in total plasma clearance does not appear to be clinically relevant. Metabolic clearance by N demethylation was increased ( 6.9 +/- 2.4 versus 12.5 +/- 3.2 ml/min; P < 0.01), whereas clearance by N oxidation did not change (5.8 +/- 1.1 versus 5.8 +/- 1.5 ml/min). Fleroxacin elimination was slightly increased (about 15%) through induction of metabolic clearance to N-demethyl-fleroxacin. Since fleroxacin levels remained above the MIC for 90% of the tested isolates of methicillin-susceptible S. aureus for at least 24 h, dose adjustment does not appear necessary, at least for short-term treatments.

Adult↗

PubMed1993

Susceptibility of Staphylococcus aureus growing on fibronectin-coated surfaces to bactericidal antibiotics.

Several recent studies have shown that bacteria either grown in vitro as adherent biofilms or recovered from infected prosthetic devices have decreased susceptibilities to antimicrobial killing. To further study the microbial and environmental factors responsible for this decreased antibiotic susceptibility, we developed an in vitro model of surface-adherent Staphylococcus aureus growing on polymethylmethacrylate coverslips coated with pure fibronectin. After exponential growth for 4 h, the population of fibronectin-attached S. aureus remained constant for a further 48-h period, as evaluated by CFU counts of organisms quantitatively removed from the coverslips. At selected time points, surface-bound organisms were exposed to bactericidal concentrations of either oxacillin, vancomycin, fleroxacin, or gentamicin in short-term (0.5 to 2 h) or long-term (24 h) killing assays. Whereas at 2 h surface-growing organisms were still optimally killed by all antimicrobial agents, at 4 and 24 h attached bacteria expressed markedly altered susceptibilities to these agents. The decrease in susceptibility was moderate for fleroxacin, more important for oxacillin and vancomycin, and extensive for gentamicin. When surface-attached S. aureus was compared with bacteria grown in a fluid phase, both populations showed a parallel time-dependent decrease in their susceptibilities to either oxacillin, vancomycin, or fleroxacin. In contrast, attached organisms became considerably more resistance to gentamicin than suspended bacteria did. Subpopulations of organisms spontaneously released from coverslips during antibiotic exposure also showed markedly reduced susceptibilities to antimicrobial killing. This simple model of S. aureus colonization of in vitro fibronectin-coated surfaces might represent a useful approach to the study of the physiological and biochemical changes that underlie the decreased antibiotic susceptibilities of biomaterial-attached organisms.

Anti-Bacterial Agents↗

PubMed1993

Ion channels and receptor-mediated Ca2+ influx in neutrophil granulocytes.

Although generally classified as non-excitable cells, human neutrophils possess a variety of ion channels that play a crucial role in the regulation of cellular activity. The mechanism of receptor-mediated Ca2+ influx in neutrophils is complex. Receptor agonists empty intracellular Ca2+ stores via generation of Ins(1,4,5)P3. The emptying of intracellular Ca2+ stores leads by an hitherto not understood mechanism to the activation of Ca2+ influx across the plasma membrane. Neutrophils possess at least 2 types of K+ channels. Voltage-activated K+ channels, important for the maintenance of the resting potential, and Ca2+ activated K+ channels, important for the repolarization after cellular activation. Neutrophils also possess voltage- and pH activated H+ channels that serve to extrude protons, generated by the neutrophil respiratory burst. Neutrophils depolarize in response to activation by agonists. The mechanism of neutrophil depolarization involves electron transport by the respiratory burst oxidase. Neutrophil depolarization serves as a negative feed-back mechanism, it also activates the H+ channels and thereby stimulates extrusion of protons.

Calcium↗

PubMed1993

Proton currents in human granulocytes: regulation by membrane potential and intracellular pH.

1. To determine whether conductive pathways contribute to the H+ efflux from granulocytes, we used the whole-cell patch-clamp technique combined with microfluorimetric determinations of cytosolic pH (pHi) in single, dimethylsulphoxide-differentiated HL-60 cells. 2. In voltage-clamp mode, depolarization of the cell from the resting potential (around -60 mV) to +60 mV caused an increase in pHi that was accompanied by a sizeable outward current. 3. Ion substitution experiments and analysis of the reversal potential of tail currents indicated that the outward current is carried largely by H+ ions. 4. Full activation of the H+ current occurred within 1-2s after depolarization and deactivation within 100-200 ms upon repolarization. 5. This H+ conductance was strongly dependent on pHi, being larger at acidic pH. In addition, at low pHi the threshold for voltage activation of the H+ conductance was shifted to more negative values. 6. Addition of millimolar concentrations of Cd2+ and Zn2+ to the bath solution reduced the maximum H+ conductance and shifted the voltage dependence of the H+ conductance to more positive potentials. The effects were reversible. 7. In conclusion, our results demonstrate that granulocytic HL-60 cells possess a voltage-gated and pHi-sensitive H+ conductance. Because both a depolarization and a cytosolic acidification occur during the activation of granulocytes, this conductance may play a role in pHi homeostasis of granulocytes during microbial killing.

Cations, Divalent↗

PubMed1993

Actin dynamics in human neutrophils during adhesion and phagocytosis is controlled by changes in intracellular free calcium.

The role of changes in cytosolic free calcium concentration ([Ca2+]i) in the assembly and disassembly of actin during adhesion and phagocytosis was evaluated. Rhodamine-phalloidin staining combined with quantitative fluorescence and confocal laser scanning microscopy was used to measure local F-actin changes in single adherent human neutrophils phagocytosing yeast particles on different surfaces and under different calcium conditions. Cells were suspended in a) calcium-containing medium (CCM) or b) calcium-free medium (CFM) or c) were first depleted of calcium (i.e., MAPT/AM-loaded in CFM) and then suspended in CFM (MAPT). In parallel, local [Ca2+]i changes were monitored using a fura-2 ratio imaging system. In CCM or CFM, attachment to the substrate and formation of pseudopods around a yeast particle generated, within a few seconds, rises in [Ca2+]i, both around the phagosome and in the cell body. During continued phagocytosis, [Ca2+]i was more elevated around the phagosome compared to the rest of the cell. No [Ca2+]i fluctuations were observed in MAPT cells. Adhesion and phagocytosis led to a several-fold increase in F-actin. The increase was transient in cells in CCM and CFM, but remained high in Ca-depleted neutrophils. A distinct ring of F-actin was formed around a phagosome with a yeast particle. Twenty min after ingestion the amount of this actin decreased more than 50% in CCM and CFM cells but increased by 40 to 100% in MAPT cells. The accumulation of F-actin in MAPT cells was reduced to resting levels by adding Ca2+ and ionomycin after ingestion. This treatment reestablished the periphagosomal [Ca2+]i rises, as observed in CCM cells. In conclusion, the present study shows that the actin polymerization, occurring in human neutrophils during adhesion and phagocytosis, is not influenced by changes in [Ca2+]i, whereas the subsequent depolymerization is. The accumulation of actin filaments around the phagosome in calcium-depleted cells could be involved in the inhibition of phagolysosome fusion seen in the absence of [Ca2+]i changes (Jaconi et al., J. Cell Biol. 110, 1555-1564 (1990)). This suggests that the actin network, controlled by [Ca2+]i, regulates the movement of granules during phagocytosis.

Actins↗

PubMed1992

Differential effects on neutrophil activation of staurosporin and its protein kinase C-selective derivative cgp 41231.

The relative insensitivity of the chemoattractant-induced respiratory burst to non-specific kinase inhibitors, such as staurosporin, is widely considered as evidence against the involvement of protein kinase C in signal transduction by chemoattractants. In this study we compared the effect on neutrophil activation of the non-specific kinase inhibitor staurosporin with the effect of its protein kinase C-selective derivative cgp 41251. Staurosporin activates secondary granule release by itself and enhances chemoattractant-induced primary granule release; it inhibits superoxide production in response to phorbol esters at low concentrations, but superoxide production in response to chemoattractants only at considerably higher concentrations. In contrast, cgp 41251 did not interfere with granule release, but inhibited phorbol ester- and chemoattractant-induced superoxide production with similar potency. These results suggest that many of the staurosporin effects, including its low potency to inhibit chemoattractant-induced superoxide production, are due to protein kinase C-independent effects. The results obtained with cgp 41251 are compatible with a role of protein kinase C in the mediation of the chemoattractant-induced respiratory burst of human neutrophils.

Alkaloids↗

PubMed1992

Cyclopiazonic acid depletes intracellular Ca2+ stores and activates an influx pathway for divalent cations in HL-60 cells.

The filling state of intracellular Ca2+ stores has been proposed to regulate Ca2+ influx across the plasma membrane in a variety of tissues. To test this hypothesis, we have used three structurally unrelated inhibitors of the Ca(2+)-ATPase of intracellular Ca2+ stores and investigated their effect on Ca2+ homeostasis in HL-60 cells. Without increasing cellular inositol (1,4,5)trisphosphate levels, all three inhibitors (cyclopiazonic acid, thapsigargin, and 2,5-Di-tert-butylhydroquinone) released Ca2+ from intracellular stores, resulting in total depletion of agonist-sensitive Ca2+ stores. The Ca2+ release was relatively slow with a lag time of 5 s and a time to peak of 60 s. After a lag time of approximately 15 s, all three Ca(2+)-ATPase inhibitors activated a pathway for divalent cation influx across the plasma membrane. At a given concentration of an inhibitor, the plasma membrane permeability for divalent cations closely correlated with the extent of depletion of Ca2+ stores. The influx pathway activated by Ca(2+)-ATPase inhibitors conducted Ca2+, Mn2+, Co2+, Zn2+, and Ba2+ and was blocked, at similar concentrations, by La3+, Ni2+, Cd2+, as well as by the imidazole derivate SK&F 96365. The divalent cation influx in response to the chemotactic peptide fMLP had the same characteristics, suggesting a common pathway for Ca2+ entry. Our results support the idea that the filling state of intracellular Ca2+ stores regulates Ca2+ influx in HL-60 cells.

Biological Transport↗

PubMed1992

Purification of an inositol 1,4,5-trisphosphate-binding calreticulin-containing intracellular compartment of HL-60 cells.

To investigate the identity of Ins(1,4,5)P3-sensitive intracellular Ca2+ stores in myeloid cells, we have developed a method that yields subcellular fractions highly enriched in Ins(1,4,5)P3 binding. HL-60 cells were disrupted by nitrogen cavitation, and subcellular fractions were obtained by differential centrifugation, followed by Percoll- and sucrose-density-gradient separations. A subcellular fraction enriched 26-fold in Ins(1,4,5)P3-binding sites was obtained. This fraction showed no enrichment in plasma-membrane markers and only a comparatively moderate enrichment (7-fold) in endoplasmic-reticulum markers. The ratio between specific enrichment of Ins(1,4,5)P3 binding and endoplasmic-reticulum markers in the different fractions varied over 50-fold, from less than 0.1 to greater than 5. The purified Ins(1,4,5)P3-binding fraction was enriched to a similar extent (27-fold) in the putative intravesicular Ca(2+)-storage protein calreticulin. Our results favour the concept of a distinct Ins(1,4,5)P3-binding, calreticulin-containing compartment (i.e. the calciosome) in HL-60 cells.

Calcium-Binding Proteins↗

PubMed1992

Calcium ions are required for the intracellular routing of insulin and its receptor.

We have studied the role of the cytosolic-free calcium concentration ([Ca2+]i) on the early and later internalization steps of insulin and its receptor. As before, we find that the rate of 125I-insulin internalization in HL60 cells remains normal when [Ca2+]i is lowered 10 times below normal resting level by the use of an intracellular Ca2+ chelator. By contrast, the subsequent intracellular steps, i.e. insulin receptor recycling and insulin degradation, are inhibited in calcium-depleted cells. Under low [Ca2+]i conditions, the association of 125I-insulin with late endosomes and lysosomes is also reduced. This suggests that calcium ions are required for fusion processes occurring at the endosomal or postendosomal stage of internalization. Thus, by regulating insulin receptor recycling and by controlling insulin degradation, Ca2+ ions play a key role in the regulation of insulin action.

Ammonium Chloride↗

PubMed1992

Contribution of tumor necrosis factor to host defense against staphylococci in a guinea pig model of foreign body infections.

The contribution of the cytokine tumor necrosis factor (cachectin; TNF) to host defenses against staphylococcal foreign body infections was studied in vivo. In tissue cages subcutaneously implanted into guinea pigs, progressive infection was initiated by a very low inoculum (100 cfu) of Staphylococcus aureus with a success rate of 100%, as is frequently encountered in related clinical situations. Locally injected autologous bacterial components derived from the cell wall of S. aureus, in particular peptidoglycan, were very active in raising TNF levels in tissue cage fluid and in preventing the development of infection by the 100% infective dose of the test strain. Furthermore, injection of murine recombinant TNF into tissue cages could substitute for the bacterial components in preventing experimental infection by S. aureus. The protective effect of TNF-eliciting bacterial components could be neutralized by anti-TNF antibodies. A local increase in TNF levels might improve host defenses against staphylococcal foreign body infections.

Animals↗

PubMed1992

Comparative in-vitro activity of new quinolones against clinical isolates and resistant mutants.

The in-vitro activity of five new fluoroquinolones, WIN 57273, sparfloxacin, fleroxacin, temafloxacin and ciprofloxacin was determined against 543 recent clinical isolates and eight quinolone resistant strains derived by mutation and their five parent strains. WIN 57273 was the most active compound against Gram-positive bacteria, sparfloxacin had a broad spectrum which was similar to that of ciprofloxacin. Ciprofloxacin showed the greatest activity against Gram-negative bacteria. Temafloxacin showing some activity against Gram-positive organisms and Acinetobacter spp. Fleroxacin was the least active compound studied. Compared to wild type parent strains, the mutated strains produced the following results. In Enterobacter cloacae OmpF deficiency increased the MICs of all quinolones by 8-32-fold. In Pseudomonas aeruginosa OmpF deficiency had a limited effect, Omp D2 deficiency combined with an increased lipopolysaccharide content produced greater resistance, i.e. 4-16-fold; mutations in gyrase were associated with variously increased MICs, depending on the strain and compound tested.

4-Quinolones↗

PubMed1992

In-vitro and in-vivo evaluation of the antistaphylococcal activity of S-5556, a new 16-membered macrolide.

During recent years, a resurgence of interest in the macrolides had led to the discovery of new derivatives of erythromycin with improved antibacterial activity and pharmacokinetic properties. In this study the in-vitro and in-vivo antistaphylococcal activity of S-5556, a 16-membered macrolide, was evaluated. In vitro, S-5556 was slightly less active than erythromycin against methicillin-susceptible Staphylococcus aureus. In contrast, it had superior activity for methicillin-resistant S. aureus (MRSA); several of these strains with inducible resistance to the macrolides-lincosamides-streptogramins group were susceptible to S-5556 whereas erythromycin was inactive. The combination of S-5556 with oxacillin was synergic for most MRSA strains tested. In vivo, a single prophylactic dose of S-5556 prevented 75%-100% of the cases of acute staphylococcal subcutaneous foreign body infection in a guinea pig-model. In a rat-model of chronic implant infection due to a methicillin- and erythromycin-resistant S. aureus strain, S-5556 significantly decreased the bacterial concentration around the foreign material, however resistant mutants emerged.

Animals↗

PubMed1992

Regulation of Ca2+ influx in myeloid cells. Role of plasma membrane potential, inositol phosphates, cytosolic free [Ca2+], and filling state of intracellular Ca2+ stores.

To study the mediation of Ca2+ influx by second messengers in myeloid cells, we have combined the whole-cell patch clamp technique with microfluorimetric measurements of [Ca2+]i. Me2SO-differentiated HL-60 cells were loaded with the fluorescent Ca2+ indicator Indo-1, allowed to adhere to glass slides, and patch-clamped. Receptor agonists and Ca(2+)-ATPase inhibitors were applied by superfusion and inositol phosphates by microperfusion through the patch pipette. In voltage-clamped cells, [Ca2+]i elevations with a sustained phase could be induced by (a) the chemoattractant receptor agonist FMLP, (b) the Ca(2+)-releasing second messenger myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P3], as well as its nonmetabolizable analogues, and (c) the Ca(2+)-ATPase inhibitor cyclopiazonic acid, which depletes intracellular Ca2+ stores. In the absence of extracellular Ca2+, responses to all stimuli were short-lasting, monophasic transients; however, subsequent addition of Ca2+ to the extracellular medium led to an immediate [Ca2+]i increase. In all cases, the sustained phase of the [Ca2+]i elevations could be inhibited by millimolar concentrations of extracellular Ni2+, and its amplitude could be decreased by depolarization of the plasma membrane. Thus, the sustained phase of the Ca2+ elevations was due to Ca2+ influx through a pathway sensitive to the electrical driving force and to Ni2+. No Ca2+ influx could be observed after (a) plasma membrane depolarization in resting cells, (b) an imposed [Ca2+]i transient independent of receptor activation, or (c) microperfusion of myo-inositol(1,3,4,5)tetrahisphosphate (Ins(1,3,4,5)P4). Also, Ins(1,3,4,5)P4 did not have additive effects when co-perfused with a submaximal concentration of Ins(1,4,5)P3. Our results suggest that, in myeloid cells, activation of chemoattractant receptors induces an electrogenic, Ni(2+)-sensitive Ca2+ influx via generation of Ins(1,4,5)P3. Ins(1,4,5)P3 might activate Ca2+ influx directly, or by depletion of intracellular Ca2+ stores, but not via [Ca2+]i increase or Ins(1,3,4,5)P4 generation.

Calcium↗

PubMed1992

Inhibition by heparin and derivatized dextrans of Staphylococcus aureus adhesion to fibronectin-coated biomaterials.

Recent data on cardiovascular device-centered infections suggest that some plasma and extracellular matrix proteins contribute to bacterial adhesion and colonization on biomaterials. We previously developed an in vitro assay to study the Staphylococcus aureus adhesion-promoting effect of surface-adsorbed fibronectin on flat PMMA coverslips coated with a monolayer amount of fibronectin. We screened the potential anti-adhesive properties of a group of substituted dextrans, previously shown to exhibit potent anticoagulant and anticomplementary activities. In comparison to unsubstituted dextran which showed no significant (< 20%) adhesion inhibition at 1 mg/ml, dextrans increasingly substituted with carboxylic and benzylamide groups (CMBD) exhibited increasing anti-adhesive activities. Three CMBD derivatives showing an increasing proportion (5-14%) of benzylamide groups showed inhibition of bacterial adhesion increasing from 33 to 51% at 1 mg/ml. Another category of substituted dextrans having a variable proportion (2-26%) of sulfonated benzylamide groups (CMBDS) produced active inhibition of S. aureus adhesion. In comparison to these heparin-like dextran derivatives, native heparin produced inhibition values of S. aureus adhesion which were intermediate between those of CMBD and CMBDS compounds. Furthermore, the anti-adhesive activity was still expressed when substituted dextrans were preincubated with fibronectin-coated PMMA but washed away at the time when radiolabeled bacteria were added to the adhesion assay. This indicates that the anti-adhesive effects of CMBDS could be exerted at the level of the S. aureus binding site of fibronectin. In conclusion, S. aureus adhesion on fibronectin-coated biomaterials can be efficiently blocked in vitro by soluble compounds such as dextran derivatives.

Adsorption↗