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Biomedical subjects

M D Schaller

Publications and source records attributed to M D Schaller.

At least 19 recordsLinked to original sources
PubMed2003

Pharmacokinetics and dosage adaptation of meropenem during continuous venovenous hemodiafiltration in critically ill patients.

Meropenem, a carbapenem broad-spectrum antibiotic, is regularly used in patients undergoing continuous venovenous hemodiafiltration (CVVHDF). Its disposition was studied over one dosage interval in 15 patients under CVVHDF on a steady regimen of 500 or 1000 mg every 8 to 12 hours. Meropenem levels were measured in plasma and filtrate-dialysate by high-performance liquid chromatography (HPLC) with UV detection. The mean CVVHDF flow rates were 7.1 +/- 0.9 L/h for blood (mean +/- SD), 0.5 +/- 0.3 L/h for predilution solution, 1.2 +/- 0.3 L/h for countercurrent dialysate, and 1.8 +/- 0.5 L/h for the total filtrate-dialysate. The pharmacokinetic analysis was based both on a noncompartmental approach and on a four-compartment modeling. The mean (coefficient of variation [CV]) total body clearance, volume of distribution at steady state, and mean residence time were, respectively, 5.0 L/h (46%), 14.3 L (29%), and 4.8 h (36%). The hemodiafiltration clearances calculated from plasma data alone and plasma with filtrate-dialysate data were 1.2 L/h (26%) and 1.6 L/h (39%), respectively. The compartmental model was used to optimize the therapeutic schedule of meropenem, considering reference minimal inhibitory concentration (MIC) of sensitive strains (4 mg/L). The results indicate that two different therapeutic schedules of meropenem are equally applicable to patients receiving CVVHD: either 750 mg tid or 1500 bid.

Acute Kidney Injury↗
PubMed2002

Determination of meropenem in plasma and filtrate-dialysate from patients under continuous veno-venous haemodiafiltration by SPE-LC.

Meropenem, a carbapenem antibiotic displaying a broad spectrum of antibacterial activity, is administered in Medical Intensive Care Unit to critically ill patients undergoing continuous veno-venous haemodiafiltration (CVVHDF). However, there are limited data available to substantial rational dosing decisions in this condition. In an attempt to refine our knowledge and propose a rationally designed dosage regimen, we have developed a HPLC method to determine meropenem after solid-phase extraction (SPE) of plasma and dialysate fluids obtained from patients under CVVHDF. The assay comprises the simultaneous measurement of meropenem's open-ring metabolite UK-1a, whose fate has never been studied in CVVHDF patients. The clean-up procedure involved a SPE on C18 cartridge. Matrix components were eliminated with phosphate buffer pH 7.4 followed by 15:85 MeOH-phosphate buffer pH 7.4. Meropenem and UK-1a were subsequently desorbed with MeOH. The eluates were evaporated under nitrogen at room temperature (RT) and reconstituted in phosphate buffer pH 7.4. Separation was performed at RT on a Nucleosil 100-5 microm C18 AB cartridge column (125 x 4 mm I.D.) equipped with a guard column (8 x 4 mm I.D.) with UV-DAD detection set at 208 nm. The mobile phase was 1 ml min(-1), using a step-wise gradient elution program: %MeOH/0.005 M tetrabutylammonium chloride pH 7.4; 10/90-50/50 in 27 min. Over the range of 5-100 microg ml(-1), the regression coefficient of the calibration curves (plasma and dialysate) were >0.998. The absolute extraction recoveries of meropenem and UK-1a in plasma and filtrate-dialysate were stable and ranged from 88-93 to 72-77% for meropenem, and from 95-104 to 75-82% for UK-1a. In plasma and filtrate-dialysate, respectively, the mean intra-assay precision was 4.1 and 2.6% for meropenem and 4.2 and 3.7% for UK-1a. The inter-assay variability was 2.8 and 3.6% for meropenem and 2.3 and 2.8% for UK-1a. The accuracy was satisfactory for both meropenem and UK-1a with deviation never exceeding 9.0% of the nominal concentrations. The stability of meropenem, studied in biological samples left at RT and at +4 degrees C, was satisfactory with < 5% degradation after 1.5 h in blood but reached 22% in filtrate-dialysate samples stored at RT for 8 h, precluding accurate measurements of meropenem excreted unchanged in the filtrate-dialysate left at RT during the CVVHDF procedure. The method reported here enables accurate measurements of meropenem in critically ill patients under CVVHDF, making dosage individualisation possible in such patients. The levels of the metabolite UK-1a encountered in this population of patients were higher than those observed in healthy volunteers but was similar to those observed in patients with renal impairment under hemodialysis.

Anti-Bacterial Agents↗
PubMed2001

Multiple stimuli induce tyrosine phosphorylation of the Crk-binding sites of paxillin.

Paxillin is a focal-adhesion-associated, tyrosine-phosphorylated protein. In cells transformed by the src, crk or BCR-Abl oncogenes, the phosphotyrosine content of paxillin is elevated. In normal cells paxillin functions in signalling following integrin-dependent cell adhesion or exposure to a number of stimuli, including growth factors and neuropeptides. These stimuli induce tyrosine phosphorylation of paxillin, regulating the association of Src homology 2 domain-containing signalling molecules with paxillin. There are multiple sites of tyrosine phosphorylation on paxillin. To elucidate the role of paxillin in transducing signals in response to various stimuli, it is essential to identify all of the sites of phosphorylation on paxillin and to define which residues are phosphorylated in response to distinct stimuli. We describe two new sites of tyrosine phosphorylation on paxillin, residues at positions 40 and 88. Using paxillin variants with phenylalanine substitutions at phosphorylation sites and phospho-specific paxillin antibodies, tyrosine phosphorylation of paxillin in response to distinct stimuli was examined. The results demonstrate that Tyr(31) and Tyr(118), which are binding sites for Crk, are major sites of tyrosine phosphorylation following cell adhesion or stimulation with platelet-derived growth factor or angiotensin II. Thus multiple stimuli may elicit similar signalling events downstream of paxillin.

Animals↗
PubMed2001

Paxillin: a focal adhesion-associated adaptor protein.

Paxillin is a focal adhesion-associated, phosphotyrosine-containing protein that may play a role in several signaling pathways. Paxillin contains a number of motifs that mediate protein-protein interactions, including LD motifs, LIM domains, an SH3 domain-binding site and SH2 domain-binding sites. These motifs serve as docking sites for cytoskeletal proteins, tyrosine kinases, serine/threonine kinases, GTPase activating proteins and other adaptor proteins that recruit additional enzymes into complex with paxillin. Thus paxillin itself serves as a docking protein to recruit signaling molecules to a specific cellular compartment, the focal adhesions, and/or to recruit specific combinations of signaling molecules into a complex to coordinate downstream signaling. The biological function of paxillin coordinated signaling is likely to regulate cell spreading and motility.

Amino Acid Motifs↗
PubMed2001

Protective effects of hypercapnic acidosis on ventilator-induced lung injury.

To investigate whether respiratory acidosis modulates ventilator-induced lung injury (VILI), we perfused (constant flow) 21 isolated sets of normal rabbit lungs, ventilated them for 20 min (pressure controlled ventilation [PCV] = 15 cm H(2)O) (Baseline) with an inspired CO(2) fraction adjusted for the partial pressure of CO(2) in the perfusate (PCO(2) approximately equal to 40 mm Hg), and then randomized them into three groups. Group A (control: n = 7) was ventilated with PCV = 15 cm H(2)O for three consecutive 20-min periods (T1, T2, T3). In Group B (high PCV/normocapnia; n = 7), PCV was given at 20 (T1), 25 (T2), and 30 (T3) cm H(2)O. The targeted PCO(2) was 40 mm Hg in Groups A and B. Group C (high PCV/hypercapnia; n = 7) was ventilated in the same way as Group B, but the targeted PCO(2) was approximately equal to 70 to 100 mm Hg. The changes (from Baseline to T3) in weight gain (Delta WG: g) and in the ultrafiltration coefficient (Delta K(f) = gr/min/ cm H(2)O/100g) and the protein and hemoglobin concentrations in bronchoalveolar lavage fluid (BALF) were used to assess injury. Group B experienced a significantly greater Delta WG (14.85 +/- 5.49 [mean +/- SEM] g) and Delta K(f) (1.40 +/- 0.49 g/min/cm H(2)O/100 g) than did either Group A (Delta WG = 0.70 +/- 0.43; Delta K(f) = 0.01 +/- 0.03) or Group C (Delta WG = 5.27 +/- 2.03 g; Delta K(f) = 0.25 +/- 0.12 g/min/cm H(2)O/ 100 g). BALF protein and hemoglobin concentrations (g/L) were higher in Group B (11.98 +/- 3.78 g/L and 1.82 +/- 0.40 g/L, respectively) than in Group A (2.92 +/- 0.75 g/L and 0.38 +/- 0.15 g/L) or Group C (5.71 +/- 1.88 g/L and 1.19 +/- 0.32 g/L). We conclude that respiratory acidosis decreases the severity of VILI in this model.

Acidosis, Respiratory↗
PubMed2001

Biochemical signals and biological responses elicited by the focal adhesion kinase.

The focal adhesion kinase, FAK, is an important component of an integrin-dependent signaling pathway, which functions to transmit signals from the extracellular matrix into the cytoplasm. FAK is an essential gene product, since the fak-/- mouse exhibits embryonic lethality. A number of important biological processes, including cell motility and cell survival, are controlled by integrin-dependent signals and FAK has been implicated in regulating these processes. This review will focus upon recent findings providing insight into the mechanisms by which FAK transmits biochemical signals and elicits biological effects.

Animals↗
PubMed2001

Inhibition of the catalytic activity of cell adhesion kinase beta by protein-tyrosine phosphatase-PEST-mediated dephosphorylation.

Protein-tyrosine phosphatase (PTP)-PEST is a cytoplasmic tyrosine phosphatase that can bind and dephosphorylate the focal adhesion-associated proteins p130(CAS) and paxillin. Focal adhesion kinase (FAK) and cell adhesion kinase beta (CAKbeta)/PYK2/CADTK/RAFTK are protein-tyrosine kinases that can colocalize with, bind to, and induce tyrosine phosphorylation of p130(CAS) and paxillin. Thus, we considered the possibility that these kinases might be substrates for PTP-PEST. Using a combination of substrate-trapping assays and overexpression of PTP-PEST in mammalian cells, CAKbeta was found to be a substrate for PTP-PEST. Both the major autophosphorylation site of CAKbeta (Tyr(402)) and activation loop tyrosine residues, Tyr(579) and Tyr(580), were targeted for dephosphorylation by PTP-PEST. Dephosphorylation of CAKbeta by PTP-PEST dramatically inhibited CAKbeta kinase activity. In contrast, FAK was a poor substrate for PTP-PEST, and treatment with PTP-PEST had no effect on FAK kinase activity. Tyrosine phosphorylation of paxillin, which is greatly enhanced by CAKbeta overexpression, was dramatically reduced upon coexpression of PTP-PEST. Finally, endogenous PTP-PEST and endogenous CAKbeta were found to localize to similar cellular compartments in epithelial and smooth muscle cells. These results suggest that CAKbeta is a substrate of PTP-PEST and that FAK is a poor PTP-PEST substrate. Further, PTP-PEST can negatively regulate CAKbeta signaling by inhibiting the catalytic activity of the kinase.

Catalysis↗
PubMed2001

CD14 expression on monocytes and TNF alpha production in patients with septic shock, cardiogenic shock or bacterial pneumonia.

OBJECTIVES: In patients with septic shock, circulating monocytes become refractory to stimulation with microbial products. Whether this hyporesponsive state is induced by infection or is related to shock is unknown. To address this question, we measured TNF alpha production by monocytes or by whole blood obtained from healthy volunteers (controls), from patients with septic shock, from patients with severe infection (bacterial pneumonia) without shock, and from patients with cardiogenic shock without infection. MEASUREMENTS: The numbers of circulating monocytes, of CD14+ monocytes, and the expression of monocyte CD14 and the LPS receptor, were assessed by flow cytometry. Monocytes or whole blood were stimulated with lipopolysaccharide endotoxin (LPS), heat-killed Escherichia coli or Staphylococcus aureus, and TNF alpha production was measured by bioassay. RESULTS: The number of circulating monocytes, of CD14+ monocytes, and the monocyte CD14 expression were significantly lower in patients with septic shock than in controls, in patients with bacterial pneumonia or in those with cardiogenic shock (p < 0.001). Monocytes or whole blood of patients with septic shock exhibited a profound deficiency of TNF alpha production in response to all stimuli (p < 0.05 compared to controls). Whole blood of patients with cardiogenic shock also exhibited this defect (p < 0.05 compared to controls), although to a lesser extent, despite normal monocyte counts and normal CD14 expression. CONCLUSIONS: Unlike patients with bacterial pneumonia, patients with septic or cardiogenic shock display profoundly defective TNF alpha production in response to a broad range of infectious stimuli. Thus, down-regulation of cytokine production appears to occur in patients with systemic, but not localised, albeit severe, infections and also in patients with non-infectious circulatory failure. Whilst depletion of monocytes and reduced monocyte CD14 expression are likely to be critical components of the hyporesponsiveness observed in patients with septic shock, other as yet unidentified factors are at work in this group and in patients with cardiogenic shock.

Antigens, CD↗
PubMed2001

The protein tyrosine phosphatase-PEST is implicated in the negative regulation of epidermal growth factor on PRL signaling in mammary epithelial cells.

Treatment of HC11 mammary epithelial cells with the lactogenic hormone PRL promotes differentiation and induction of milk protein gene expression via stimulation of the Janus kinase (JAK)/signal transducer and activator of transcription pathway. We have previously shown that autocrine activation of epidermal growth factor (EGF) receptor interferes with normal PRL-induced differentiation. Here we show that PRL activation of JAK2 was dramatically reduced in HC11 cells pretreated with EGF, demonstrating that the target of EGF receptor activation is JAK2 kinase. Using an in-gel protein tyrosine phosphatase (PTP) assay, we observed that the activity of a 125-kDa PTP was up-regulated in HC11 cells in response to EGF. A specific antiserum was used to demonstrate that the 125-kDa PTP was PTP-PEST and to show that EGF treatment of HC11 cells led to an increase in the level of PTP-PEST. In intact HC11 cells, PTP-PEST was constitutively associated with JAK2, and in response to EGF treatment there was an increased level of PTP-PEST in JAK2 complexes. An in vitro phosphatase assay, using PRL-activated JAK2 as the substrate and lysates from HC11 cells as the source of PTP-PEST, revealed that JAK2 could serve as a PTP-PEST substrate. However, in intact cells the regulation of JAK2 by PTP-PEST was complex, since transient overexpression of PTP-PEST had a negligible effect on PRL-induced JAK2 activation. EGF's negative influence on JAK2 activity was blocked by actinomycin D treatment of HC11 cells, suggesting that EGF induced a protein that mediated the effects of PTP-PEST on JAK2. In support of this model, PTP-PEST-containing lysates from EGF-treated HC11 cells dephosphorylated JAK2 to a greater extent than lysates prepared from control cells.

Animals↗
PubMed2001

[Endocarditis due to HACEK bacteria. A case report of endocarditis due to Kingella kingae].

Endocarditis is a common disease in hospital practice. Identification of the microorganism responsible for the valvular damage is essential to establish the prognosis and to determine the optimal antibiotic treatment. In some cases of endocarditis the diagnosis is laborious, especially when the responsible microorganism is difficult to detect using standard culture techniques. Here we report a case of native aortic valve endocarditis due to Kingella kingae, a Gram negative organism of the HACEK group. In addition we review 6 other cases of endocarditis caused by organism belonging to this group, treated in our hospital between 1983 and 1999. Epidemiological studies show that less than 5% of all cases of endocarditis are caused by organisms of the HACEK group. The diagnosis is often delayed because their slow growth on a standard culture medium. We describe clinical and microbiological characteristics of this group of endocarditis.

Adult↗
PubMed2000

The noncatalytic domain of protein-tyrosine phosphatase-PEST targets paxillin for dephosphorylation in vivo.

The noncatalytic domain of protein-tyrosine phosphatase (PTP)-PEST contains a binding site for the focal adhesion-associated protein paxillin. This binding site has been narrowed to a 52-residue sequence that is composed of two nonoverlapping, weak paxillin binding sites. The PTP-PEST binding site on paxillin has been mapped to the two carboxyl-terminal LIM (lin11, isl-1, and mec-3) domains. Transient expression of PTP-PEST reduced tyrosine phosphorylation of p130(cas), as anticipated. A PTP-PEST mutant defective for binding p130(cas) does not cause a reduction in its tyrosine phosphorylation in vivo. Expression of PTP-PEST also caused a reduction of phosphotyrosine on paxillin. Expression of mutants of PTP-PEST with deletions in the paxillin-binding site did not associate with paxillin in vivo and failed to cause a reduction in the phosphotyrosine content of paxillin. These results demonstrate that paxillin can serve as a PTP-PEST substrate in vivo and support the model that a noncatalytic domain interaction recruits paxillin to PTP-PEST to facilitate its dephosphorylation.

Animals↗
PubMed2000

Paxillin binding is not the sole determinant of focal adhesion localization or dominant-negative activity of focal adhesion kinase/focal adhesion kinase-related nonkinase.

The carboxy-terminal 150 residues of the focal adhesion kinase (FAK) comprise the focal adhesion-targeting sequence, which is responsible for its subcellular localization. The mechanism of focal adhesion targeting has not been fully elucidated. We describe a mutational analysis of the focal adhesion-targeting sequence of FAK to further examine the mechanism of focal adhesion targeting and explore additional functions encoded by the carboxy-terminus of FAK. The results demonstrate that paxillin binding is dispensable for focal adhesion targeting of FAK. Cell adhesion-dependent tyrosine phosphorylation strictly correlated with the ability of mutants to target to focal adhesions. Focal adhesion targeting was also a requirement for maximal FAK-dependent tyrosine phosphorylation of paxillin and FAK-related nonkinase (FRNK)-dependent inhibition of endogenous FAK function. However, there were additional requirements for these latter functions because we identified mutants that target to focal adhesions, yet are defective for the induction of paxillin phosphorylation or the dominant-negative function of FRNK. Furthermore, the paxillin-binding activity of FRNK mutants did not correlate with their ability to inhibit FAK, suggesting that FRNK has other targets in addition to paxillin.

3T3 Cells↗
PubMed2000

Tissue oxygenation and hemodynamic response to NO synthase inhibition in septic shock.

The objective of the study was to evaluate the tissue oxygenation and hemodynamic effects of NOS inhibition in clinical severe septic shock. Eight patients with septic shock refractory to volume loading and high level of adrenergic support were prospectively enrolled in the study. Increasing doses of NOS inhibitors [N(G)-nitro-L-arginine-methyl ester (L-NAME) or N(G)-monomethyl-L-arginine (L-NMMA)] were administered as i.v. bolus until a peak effect = 10 mmHg on mean blood pressure was obtained or until side effects occurred. If deemed clinically appropriate, a continuous infusion of L-NAME was instituted and adrenergic support weaning attempted. The bolus administration of NOS inhibitors transiently increased mean blood pressure by 10 mm Hg in all patients. Seven out of eight patients received an L-NAME infusion, associated over 24 h with a progressive decline in cardiac index (P < 0.001) and an increase in systemic vascular resistance (P < 0.01). Partial or total adrenergic support weaning was rapidly possible in 6/8 patients. Oxygen transport decreased (P < 0.001), but oxygen consumption remained unchanged in those patients in whom it could be measured by indirect calorimetry (5/8). Blood lactate and the difference between tonometric gastric and arterial PCO2 remained unchanged. There were 4/8 ICU survivors. We conclude that nitric oxide synthase inhibition in severe septic shock was followed with a progressive correction of the vasoplegic hemodynamic disturbances with finally normalization of cardiac output and systemic vascular resistances without any demonstrable deterioration in tissue oxygenation.

Adrenergic Agonists↗
PubMed2000

Permissive hypercapnia impairs pulmonary gas exchange in the acute respiratory distress syndrome.

Current recommendations for mechanical ventilation in the acute respiratory distress syndrome (ARDS) include the use of small tidal volumes (VT), even at the cost of respiratory acidosis. We evaluated the effects of this permissive hypercapnia on pulmonary gas exchange with the multiple inert gas elimination technique (MIGET) in eight patients with ARDS. After making baseline measurements, we induced permissive hypercapnia by reducing VT from 10 +/- 2 ml/kg to 6 +/- 1 ml/kg (mean +/- SEM) at constant positive end-expiratory pressure. After restoration of initial VT, we infused dobutamine to increase cardiac output (Q) by the same amount as with hypercapnia. Permissive hypercapnia increased Q by an average of 1.4 L. min(-)(1). m(2), decreased arterial oxygen tension from 109 +/- 10 mm Hg to 92 +/- 11 mm Hg (p < 0.05), markedly increased true shunt (Q S/Q T), from 32 +/- 6% to 48 +/- 5% (p < 0.0001), and had no effect on the dispersion of VA/Q.VA/Q. On reinstatement of baseline V T with maintenance of a high Q, Q S/Q T remained increased, to 38 +/- 6% (p < 0.05), and Pa(O(2 ))remained decreased, to 93 +/- 4 mm Hg (p < 0. 05). These results agreed with effects of changes in VT and Q predicted by the mathematical lung model of the MIGET. We conclude that permissive hypercapnia increases pulmonary shunt, and that deterioration in gas exchange is explained by the combined effects of increased Q and decreased alveolar ventilation.

Adolescent↗
PubMed2000

Use of botulinum toxin type A to avoid tracheal intubation or tracheostomy in severe paradoxical vocal cord movement.

Paradoxical vocal cord movement (PVCM) is characterized by paradoxical adduction of the vocal cords during inspiration and/or expiration. Patients with severe forms of PVCM can present with acute dyspnea. In this article, we describe a patient with severe PVCM who had required tracheal intubation or tracheostomy at multiple occasions and who presented with acute hypercapnic respiratory failure. Using sedation and intralaryngeal injection of botulinum toxin type A, we could avoid more invasive intervention. Our observation shows that botulinum toxin type A should be considered in the acute care setting for severe PVCM.

Adult↗