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Biomedical subjects

B Fabry

Publications and source records attributed to B Fabry.

At least 19 recordsLinked to original sources
PubMed2004

Linearity and time-scale invariance of the creep function in living cells.

We report here the creep function measured in three cell types, after a variety of interventions, and over three time decades (from 3 ms to 3.2 s). In each case the response conformed to a power law, implying that no distinct molecular relaxation times or time constants could characterize the response. These results add to a growing body of evidence that stands in contrast to widely used viscoelastic models featuring at most a few time constants. We show instead that the ability of the matrix to deform is time-scale invariant and characterized by only one parameter: the power law exponent that controls the transition between solid-like and liquid-like behaviour. Moreover, we validate linearity by comparison of measurements in the time and frequency domains.

Cell Movement↗
PubMed2001

Scaling the microrheology of living cells.

We report a scaling law that governs both the elastic and frictional properties of a wide variety of living cell types, over a wide range of time scales and under a variety of biological interventions. This scaling identifies these cells as soft glassy materials existing close to a glass transition, and implies that cytoskeletal proteins may regulate cell mechanical properties mainly by modulating the effective noise temperature of the matrix. The practical implications are that the effective noise temperature is an easily quantified measure of the ability of the cytoskeleton to deform, flow, and reorganize.

Cytoskeletal Proteins↗
PubMed2001

Twisting integrin receptors increases endothelin-1 gene expression in endothelial cells.

A magnetic twisting stimulator was developed based on the previously published technique of magnetic twisting cytometry. Using ligand-coated ferromagnetic microbeads, this device can apply mechanical stresses with varying amplitudes, duration, frequencies, and waveforms to specific cell surface receptors. Biochemical and biological responses of the cells to the mechanical stimulation can be assayed. Twisting integrin receptors with RGD (Arg-Gly-Asp)-containing peptide-coated beads increased endothelin-1 (ET-1) gene expression by >100%. In contrast, twisting scavenger receptors with acetylated low-density lipoprotein-coated beads or twisting HLA antigen with anti-HLA antibody-coated beads did not lead to alterations in ET-1 gene expression. In situ hybridization showed that the increase in ET-1 mRNA was localized in the cells that were stressed with the RGD-coated beads. Blocking stretch-activated ion channels with gadolinium, chelating Ca2+ with EGTA, or inhibiting tyrosine phosphorylation with genistein abolished twist-induced ET-1 mRNA elevation. Abolishing cytoskeletal tension with an inhibitor of the myosin ATPase, with an inhibitor of myosin light chain kinase, or with an actin microfilament disrupter blocked twisted-induced increases in ET-1 expression. Our results are consistent with the hypothesis that the molecular structural linkage of integrin-cytoskeleton is an important pathway for stress-induced ET-1 gene expression.

Actins↗
PubMed2001

Selected contribution: time course and heterogeneity of contractile responses in cultured human airway smooth muscle cells.

We measured the time course and heterogeneity of responses to contractile and relaxing agonists in individual human airway smooth muscle (HASM) cells in culture. To this end, we developed a microrheometer based on magnetic twisting cytometry adapted with a novel optical detection system. Ferromagnetic beads (4.5 microm) coated with Arg-Gly-Asp peptide were bound to integrins on the cell surface. The beads were twisted in a sinusoidally varying magnetic field at 0.75 Hz. Oscillatory bead displacements were recorded using a phase-synchronized video camera. The storage modulus (cell stiffness; G'), loss modulus (friction; G"), and hysteresivity (eta; ratio of G" to G') could be determined with a time resolution of 1.3 s. Within 5 s after addition of histamine (100 microM), G' increased by 2.2-fold, G" increased by 3.0-fold, and eta increased transiently from 0.27 to 0.34. By 20 s, eta decreased to 0.25, whereas G' and G" remained above baseline. Comparable results were obtained with bradykinin (1 microM). These changes in G', G", and eta measured in cells were similar to but smaller than those reported for intact muscle strips. When we ablated baseline tone by adding the relaxing agonist dibutyryl cAMP (1 mM), G' decreased within 5 min by 3.3-fold. With relaxing and contracting agonists, G' could be manipulated through a contractile range of 7.3-fold. Cell populations exhibited a log-normal distribution of baseline stiffness (geometric SD = 2.8) and a heterogeneous response to both contractile and relaxing agonists, partly attributable to variability of baseline tone between cells. The total contractile range of the cells (from maximally relaxed to maximally stimulated), however, was independent of baseline stiffness. We conclude that HASM cells in culture exhibit a clear, although heterogeneous, response to contractile and relaxing agonists and express the essential mechanical features characteristic of the contractile response observed at the tissue level.

Cells, Cultured↗
PubMed2000

Analysis of cell mechanics in single vinculin-deficient cells using a magnetic tweezer.

A magnetic tweezer was constructed to apply controlled tensional forces (10 pN to greater than 1 nN) to transmembrane receptors via bound ligand-coated microbeadswhile optically measuring lateral bead displacements within individual cells. Use of this system with wild-type F9 embryonic carcinoma cells and cells from a vinculin knockout mouse F9 Vin (-/-) revealed much larger differences in the stiffness of the transmembrane integrin linkages to the cytoskeleton than previously reported using related techniques that measured average mechanical properties of large cell populations. The mechanical properties measured varied widely among cells, exhibiting an approximately log-normal distribution. The median lateral bead displacement was 2-fold larger in F9 Vin (-/-) cells compared to wild-type cells whereas the arithmetic mean displacement only increased by 37%. We conclude that vinculin serves a greater mechanical role in cells than previously reported and that this magnetic tweezer device may be useful for probing the molecular basis of cell mechanics within single cells.

Animals↗
PubMed2000

Mechanical control of cyclic AMP signalling and gene transcription through integrins.

This study was carried out to discriminate between two alternative hypotheses as to how cells sense mechanical forces and transduce them into changes in gene transcription. Do cells sense mechanical signals through generalized membrane distortion or through specific transmembrane receptors, such as integrins? Here we show that mechanical stresses applied to the cell surface alter the cyclic AMP signalling cascade and downstream gene transcription by modulating local release of signals generated by activated integrin receptors in a G-protein-dependent manner, whereas distortion of integrins in the absence of receptor occupancy has no effect.

3T3 Cells↗
PubMed2000

Mechanical properties of cultured human airway smooth muscle cells from 0.05 to 0.4 Hz.

We investigated the rheological properties of living human airway smooth muscle cells in culture and monitored the changes in rheological properties induced by exogenous stimuli. We oscillated small magnetic microbeads bound specifically to integrin receptors and computed the storage modulus (G') and loss modulus (G") from the applied torque and the resulting rotational motion of the beads as determined from their remanent magnetic field. Under baseline conditions, G' increased weakly with frequency, whereas G" was independent of the frequency. The cell was predominantly elastic, with the ratio of G" to G' (defined as eta) being approximately 0. 35 at all frequencies. G' and G" increased together after contractile activation and decreased together after deactivation, whereas eta remained unaltered in each case. Thus elastic and dissipative stresses were coupled during changes in contractile activation. G' and G" decreased with disruption of the actin fibers by cytochalasin D, but eta increased. These results imply that the mechanisms for frictional energy loss and elastic energy storage in the living cell are coupled and reside within the cytoskeleton.

Cell Movement↗
PubMed1999

Conformational changes induced by phosphorylation of the FixJ receiver domain.

BACKGROUND: A variety of bacterial adaptative cellular responses to environmental stimuli are mediated by two-component signal transduction pathways. In these phosphorelay cascades, histidine kinases transphosphorylate a conserved aspartate in the receiver domain, a conserved module in the response regulator superfamily. The main effect of this phosphorylation is to alter the conformation of the response regulator in order to modulate its biological function. The response regulator FixJ displays a typical modular arrangement, with a phosphorylatable N-terminal receiver domain and a C-terminal DNA-binding domain. In the symbiotic bacterium Sinorhizobium meliloti, phosphorylation of this response regulator activates transcription of nitrogen-fixation genes. RESULTS: The crystal structures of the phosphorylated and of the unphosphorylated N-terminal receiver domain of FixJ (FixJN) were solved at 2.3 A and 2.4 A resolution, respectively. They reveal the environment of the phosphoaspartate in the active site and the specific conformational changes leading to activation of the response regulator. Phosphorylation of the conserved aspartate induces major structural changes in the beta 4-alpha 4 loop, and in the signaling surface alpha 4-beta 5 that mediates dimerization of the phosphorylated full-length response regulator. A site-directed mutant at this protein-protein interface decreases the affinity of the phosphorylated response regulator for the fixK promoter tenfold. CONCLUSIONS: The cascade of phosphorylation-induced conformational changes in FixJN illustrates the role of conserved residues in stabilizing the phosphoryl group in the active site, triggering the structural transition and achieving the post-phosphorylation signaling events. We propose that these phosphorylation-induced conformational changes underly the activation of response regulators in general.

Amino Acid Sequence↗
PubMed1999

Structural transitions in the FixJ receiver domain.

BACKGROUND: Two-component signal transduction pathways are sophisticated phosphorelay cascades widespread in prokaryotes and also found in fungi, molds and plants. FixL/FixJ is a prototypical system responsible for the regulation of nitrogen fixation in the symbiotic bacterium Sinorhizobium meliloti. In microaerobic conditions the membrane-bound kinase FixL uses ATP to transphosphorylate a histidine residue, and the response regulator FixJ transfers the phosphoryl group from the phosphohistidine to one of its own aspartate residues in a Mg(2+)-dependent mechanism. RESULTS: Seven X-ray structures of the unphosphorylated N-terminal receiver domain of FixJ (FixJN) have been solved from two crystal forms soaked in different conditions. Three conformations of the protein were found. In the first case, the protein fold impairs metal binding in the active site and the structure reveals a receiver domain that is self-inhibited for catalysis. In the second conformation, the canonical geometry of the active site is attained, and subsequent metal binding to the protein induces minimal conformational changes. The third conformation illustrates a non-catalytic form of the protein where unwinding of the N terminus of helix alpha 1 has occurred. Interconversion of the canonical and self-inhibited conformations requires a large conformational change of the beta 3-alpha 3 loop region. CONCLUSIONS: These unphosphorylated structures of FixJN stress the importance of flexible peptide segments that delineate the active site. Their movements may act as molecular switches that define the functional status of the protein. Such observations are in line with structural and biochemical results obtained on other response regulator proteins and may illustrate general features that account for the specificity of protein-protein interactions.

Amino Acid Sequence↗
PubMed1999

Additional inspiratory work of breathing imposed by tracheostomy tubes and non-ideal ventilator properties in critically ill patients.

OBJECTIVE: To determine the tracheostomy tube-related additional work of breathing (WOBadd) in critically ill patients and to show its reduction by different ventilatory modes. DESIGN: Prospective, clinical study. SETTING: Medical ICU of a university teaching hospital. INTERVENTION: Standard tracheostomy due to prolonged respiratory failure. MEASUREMENTS AND RESULTS: Ten tracheostomized, spontaneously breathing patients were investigated. As the tube resistance depends on gas flow, patients were subdivided according to minute ventilation into a low ventilation group (= 10 l/min; n = 5) and a high ventilation group (> 10 l/min; n = 5). The WOBadd due to tube resistance and non-ideal ventilator properties was calculated on the basis of the tracheal pressure measured. Ventilatory modes investigated were: continuous positive airway pressure (CPAP), inspiratory pressure support (IPS) of 5, 10, and 15 cm H2O above PEEP, and automatic tube compensation (ATC). In the low ventilation group, WOBadd during CPAP was 0.382+/-0.106 J/l. It was reduced to below 15% of that value by ATC or IPS more than 5 cm H2O. In the high ventilation group WOBadd during CPAP increased to 0.908+/-0.142 J/l. In this group, however, only ATC was able to reduce WOBadd below 15% of the value observed in the CPAP mode. CONCLUSIONS: The results indicate that, depending on respiratory flow rate, (1) tracheostomy tubes can cause a considerable amount of WOBadd, and (2) ATC, in contrast to IPS, is a suitable mode to compensate for WOBadd at any ventilatory effort of the patient.

Aged↗
PubMed1999

Role of ERK MAP kinases in responses of cultured human airway smooth muscle cells to IL-1beta.

We have previously reported that interleukin (IL)-1beta causes beta-adrenergic hyporesponsiveness in cultured human airway smooth muscle cells by increasing cyclooxygenase-2 (COX-2) expression and prostanoid formation. The purpose of this study was to determine whether extracellular signal-regulated kinases (ERKs) are involved in these events. Levels of phosphorylated ERK (p42 and p44) increased 8.3- and 13-fold, respectively, 15 min after treatment with IL-1beta (20 ng/ml) alone. Pretreating cells with the mitogen-activated protein kinase kinase inhibitor PD-98059 or U-126 (2 h before IL-1beta treatment) decreased ERK phosphorylation. IL-1beta (20 ng/ml for 22 h) alone caused a marked induction of COX-2 and increased basal PGE(2) release 28-fold (P < 0.001). PD-98059 (100 microM) and U-126 (10 microM) each decreased COX-2 expression when administered before IL-1beta treatment. In control cells, PD-98059 and U-126 had no effect on basal or arachidonic acid (AA; 10 microM)-stimulated PGE(2) release, but both inhibitors caused a significant decrease in bradykinin (BK; 1 microM)-stimulated PGE(2) release, consistent with a role for ERK in the activation of phospholipase A(2) by BK. In IL-1beta-treated cells, prior administration of PD-98059 caused 81, 92 and 40% decreases in basal and BK- and AA-stimulated PGE(2) release, respectively (P < 0.01), whereas administration of PD-98059 20 h after IL-1beta resulted in only 38 and 43% decreases in basal and BK-stimulated PGE(2) release, respectively (P < 0.02) and had no effect on AA-stimulated PGE(2) release. IL-1beta attenuated isoproterenol-induced decreases in human airway smooth muscle stiffness as measured by magnetic twisting cytometry, and PD-98059 or U-126 abolished this effect in a concentration-dependent manner. These results are consistent with the hypothesis that ERKs are involved early in the signal transduction pathway through which IL-1beta induces PGE(2) synthesis and beta-adrenergic hyporesponsiveness and that ERKs act by inducing COX-2 and activating phospholipase A(2).

Blotting, Western↗
PubMed1999

Dynamic delay time compensation for sampling capillaries used in respiratory mass spectrometry.

In intensive care patients who receive ventilatory support or full mechanical ventilation, valuable information can be drawn from gas exchange measurements. In this setting, the most favorable method for gas exchange measurement is by simultaneous recording of gas concentrations and gas flow, and by time resolved multiplication and accumulation. This paper presents a new method to compensate for the signal delay time which occurs when a sampling capillary is used for measuring gas concentrations with a respiratory mass spectrometer or some equivalent sidestream gas analyzer. The signal delay of gas concentrations must be accurately compensated to avoid error accumulation in gas exchange calculation. A delay time can be easily measured with a test gas in a laboratory setup and be readily compensated for during the measurements in a ventilated patient. This is a standard procedure which gives reasonable results under normal conditions. Special attention is however required in cases where the gas viscosity changes due to large changes in gas composition, e.g., those used for diagnostic breathing or ventilatory maneuvers. Such changes of viscosity will influence the delay time of the capillary, because they affect its flow resistance. As a consequence they will degrade the quality of measurements when done with a simple fixed delay compensation. The method described here consists of an algorithm which enables compensation for such a temporally changing delay time due to changes in gas composition.

Algorithms↗
PubMed1998

Effects of mechanical unloading/loading on respiratory loop gain and periodic breathing in man.

We investigated the effect of mechanical unloading and loading on Cheyne-Stokes respiration (CSR) in seven intubated patients with preexisting CSR. For mechanical loading patients had to breathe against the resistance of the endotracheal tube. For mechanical unloading patients were supported with a volume-proportional pressure support in the proportional assist ventilation (PAV) mode whilst the flow-dependent (nonlinear) endotracheal tube resistance was continuously compensated for by means of the automatic tube compensation (ATC) mode. Mechanical unloading aggravated CSR as revealed by a prolongation of apnea time and by an increase in the so-called strength index whereas mechanical loading shortened apnea time and decreased strength index. To test whether the observed changes are caused by the effect of mechanical unloading/loading on respiratory loop gain (relationship between minute ventilation and arterial CO2 tension), the response of respiratory loop gain on mechanical unloading/loading was determined in five healthy subjects (without CSR). In each subject, mechanical unloading increased respiratory loop gain whereas mechanical loading decreased it.

Adult↗
PubMed1997

Characterization of the promoter of human adipocyte hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The human HSL gene is composed of nine exons encoding the adipocyte form and a testis-specific coding exon. Northern blot analyses showed that human adipocytes express a 2.8 kb HSL mRNA, suggesting the presence of a short (20-150 bp) 5' untranslated region (5'-UTR). A single 5'-UTR of approx. 70 nt was detected in RNase H mapping experiments. Two 5'-UTRs of 70 and 170 nt respectively were obtained by rapid amplification of cDNA ends and cDNA library screenings. RNase protection experiments, with probes derived from the two products, showed that human adipocyte HSL mRNA contains only the 70 nt product. Primer extension analysis mapped the transcriptional start site 74 nt upstream of the start codon. In HT29, a human cell line expressing HSL, the presence of the short or the long 5'-UTR is mutually exclusive. The short and long 5'-UTR exons were located 1.5 and approx. 13 kb respectively upstream of the first coding exon. Various portions of the 5'-flanking region upstream of the short product exon were linked to the luciferase gene and transfected into cells that express HSL (HT29 cells and rat adipocytes) and do not express HSL (HeLa cells). High luciferase activity was found for constructs containing the sequence between nt -2400 and -86, but not for shorter constructs. An analysis of 14 kb of genomic sequence revealed the presence of five DNase I hypersensitive sites associated with active gene transcription. Three of the sites are located in the vicinity of the transcriptional start site and could be linked to the minimal promoter activity. Two of the sites are located downstream of the exon containing the start codon, suggesting the presence of intronic regulatory elements.

Adipocytes↗
PubMed1997

Breathing pattern and additional work of breathing in spontaneously breathing patients with different ventilatory demands during inspiratory pressure support and automatic tube compensation.

OBJECTIVE: We designed a new ventilatory mode to support spontaneously breathing, intubated patients and to improve weaning from mechanical ventilation. This mode, named Automatic Tube Compensation (ATC), compensates for the flow-dependent pressure drop across the endotracheal tube (ETT) and controls tracheal pressure to a constant value. In this study, we compared ATC with conventional patient-triggered inspiratory pressure support (IPS). DESIGN: A prospective, interventional study. SETTING: A medical intensive care unit (ICU) and an ICU for heart and thoracic surgery in a university hospital. PATIENTS: We investigated two groups of intubated, spontaneously breathing patients: ten postoperative patients without lung injury, who had a normal minute ventilation (VE) of 7.6 +/- 1.7 l/min, and six critically ill patients who showed increased ventilatory demand (VE = 16.8 +/- 3.0 l/ min). INTERVENTIONS: We measured the breathing pattern [VE, tidal volume (VT), and respiratory rate (RR)] and additional work of breathing (WOBadd) due to ETT resistance and demand valve resistance. Measurements were performed under IPS of 5, 10, and 15 mbar and under ATC. RESULTS: The response of VT, RR, and WOBadd to different ventilatory modes was different in both patient groups, whereas VE remained unchanged. In postoperative patients, ATC, IPS of 10 mbar, and IPS of 15 mbar were sufficient to compensate for WOBadd. In contrast, WOBadd under IPS was greatly increased in patients with increased ventilatory demand, and only ATC was able to compensate for WOBadd. CONCLUSIONS: The breathing pattern response to IPS and ATC is different in patients with differing ventilatory demand. ATC, in contrast to IPS, is a suitable mode to compensate for WOBadd in patients with increased ventilatory demand. When WOBadd was avoided using ATC, the patients did not need additional pressure support.

Adult↗
PubMed1997

Respiratory comfort of automatic tube compensation and inspiratory pressure support in conscious humans.

OBJECTIVE: To compare the new mode of ventilatory support, which we call automatic tube compensation (ATC), with inspiratory pressure support (IPS) with respect to perception of respiratory comfort. ATC unloads the resistance of the endotracheal tube (ETT) in inspiration by increasing the airway pressure, and in expiration by decreasing the airway pressure according to the non-linear pressure-flow relationship of the ETT. DESIGN: Prospective randomized single blind cross-over study. SETTING: Laboratory of the Section of Experimental Anaesthesiology (Clinic of Anaesthesiology; University of Freiburg). SUBJECTS: Ten healthy volunteers. INTERVENTIONS: The subjects breathed spontaneously through an ETT of 7.5 mm i.d. Three different ventilatory modes, each with a PEEP of 5 cmH2O, were presented in random order using the Dräger Evita 2 ventilator with prototype software: (1) IPS (10 cmH2O, 1 s ramp), (2) inspiratory ATC (ATC-in), (3) inspiratory and expiratory ATC (ATC-in-ex). MEASUREMENTS AND MAIN RESULTS: Immediately following a mode transition, the volunteers answered with a hand sign to show how they perceived the new mode compared with the preceding mode in terms of gain or loss in subjective respiratory comfort: "better", "unchanged" or "worse". Inspiration and expiration were investigated separately analyzing 60 mode transitions each. Flow rates were continuously measured. The transition from IPS to either type of ATC was perceived positively, i.e. as increased comfort, whereas the opposite transition from ATC to IPS was perceived negatively, i.e. as decreased comfort. The transition from ATC-in to ATC-in-ex was perceived positively whereas the opposite mode transition was perceived negatively in expiration only. Tidal volume was 1220 +/- 404 ml during IPS and 1017 +/- 362 ml during ATC. The inspiratory peak flow rate was 959 +/- 78 ml/s during IPS and 1048 +/- 197 ml/s during ATC. CONCLUSIONS: ATC provides an increase in respiratory comfort compared with IPS. The predominant cause for respiratory discomfort in the IPS mode seems to be lung over-inflation.

Adult↗
PubMed1997

Delayed derecruitment after removal of PEEP in patients with acute lung injury.

BACKGROUND: A step decrease in positive end-expiratory airway pressure (PEEP) is not followed by an instantaneous loss of the PEEP-induced increase in end-expiratory lung volume (EELV). Rather, the reduction of EELV is delayed, while adverse PEEP effects on hemodynamics are immediately attenuated upon the drop in airway pressure. Step PEEP increments were applied to the lungs of patients with acute lung injury. It was investigated retrospectively whether enlargement of end-expiratory lung volume and changes in lung mechanics persist 45 min after removal of the PEEP increment. METHODS: In 14 patients with acute lung injury (LIS score 2.7) EELV and volume-dependent dynamic compliance of the respiratory system (Cdyn,rs) were determined 45 min after removal of an additional PEEP increment (0.64 kPa added to baseline PEEP of 1.0 kPa). RESULTS: Nine patients kept an EELV gain of 13% (SD 7) and showed improved Cdyn,rs. In 5 patients, EELV was reduced (by 9% (SD 6)) and Cdyn,rs unchanged after removal of the PEEP increment compared to baseline. CONCLUSION: A subgroup of patients with acute lung injury, the characteristics of which remain to be defined, benefit from prolonged recruitment effects up to 45 min after removal of a PEEP increment, while sequelae of continuously increased airway pressures are minimised.

Adult↗