[Basic algorithm for Point-of-Care based hemotherapy: perioperative treatment of coagulopathic patients]. / Basisalgorithmus für "Point-of-Care" basierte Hämotherapie : Perioperative Versorgung koagulopathischer Patienten.

During perioperative treatment of coagulopathic patients the so-called Point-of-Care (POC) analyses enable more rapidly available and more comprehensive hemostatic analyses compared to routinely performed conventional coagulation testing, such as activated partial thromboplastin time (aPTT), international normalized ratio (INR), fibrinogen concentration and platelet count. In this review article a hemotherapy algorithm is presented which is based on viscoelastic and aggregometric POC measurements. The algorithm was designed double sided and consists of a general and a special part. The general part contains boxes and fields for sociodemographic data and gives general recommendations for coagulation management and therapy specifications for particular patient collectives and presents proposals for emergency reversal of anticoagulation therapy. The special part refers to basic physiological conditions for hemostasis and asks for measurement results of clot initiation, clot firmness, clot stability and platelet function analyses. Reference values were defined for each parameter and therapeutic options are presented. In cases of persistent coagulopathy despite algorithm-conform therapy, the algorithm could be run through once again. Finally, the algorithm presents therapeutic options for an ultima ratio therapy approach.

[Less pain on injection by a new formulation of propofol? A comparison with propofol LCT]. / Weniger Injektionsschmerz durch Propofol-MCT/LCT? Ein Vergleich mit Propofol-LCT.

BACKGROUND: Pain on injection is a major disadvantage of propofol, experienced by the vast majority of patients. Since the traditional formulation has almost normal osmolality and pH, it is hypothesised that the concentration of free propofol in the aqueous phase of the emulsion is responsible for the pain and that reducing the amount of free propofol would also reduce the frequency and intensity of pain on injection. This study was designed to investigate whether pain on injection can be reduced in frequency and intensity by a new formulation of propofol. METHODS: We performed a monocentre, controlled, randomised, double-blind study to compare the pain produced by intravenous injection of a new propofol preparation (propofol-MCT/LCT) with standard propofol in patients undergoing elective surgical procedures. A total of 184 non-premedicated patients received either 1% propofol prepared in a mixture of medium and long chain triglycerides (Propofol-MCT/LCT, Propofol- Lipuro, B. Braum Melsungen AG) or standard 1% propofol prepared exclusively in long chain triglycerides (Propofol-LCT; Disoprivan, AstraZeneca) into a vein of the dorsal hand for induction of anaesthesia. Anaesthesia was maintained by TIVA with propofol and remifentanil. Pain on injection was recorded and graded as none, mild, moderate or severe. RESULTS: Patients receiving propofol-MCT/LCT had a significantly lower incidence of pain on injection compared to the standard propofol group (37% vs 64%) with the intensity of pain also being less severe. There were no differences between both groups in propofol dosage for induction (3.2 +/- 0.8 mg/kg vs 3.3 +/- 0.9 mg/kg) and maintenance of anaesthesia (3.4 +/- 0.6 mg/kg/h vs 3.2 +/- 0.5 mg/kg/h), remifentanil dosage (25 +/- 6 micrograms/kg/h vs. 24 +/- 6 micrograms/kg/h), intraoperative hemodynamics, recovery parameters and postoperative patient satisfaction. Postoperative thrombophlebitis at the injection site for propofol was not observed in any of the patients. CONCLUSIONS: Propofol-MCT/LCT produced significantly less pain on injection when compared to standard propofol in ASA I and II patients undergoing elective surgery. Pain was also significantly less severe, with both effects presumably being due to the lower concentration of free propofol in the MCT/LCT-preparation. With regard to injection pain propofol-MCT/LCT offers significant a advantage over standard propofol.

Identification and cloning of a human urea transporter HUT11, which is downregulated during adipogenesis of explant cultures of human bone.

Bipotential cells in human trabecular bone explant cultures that express osteoblast characteristics are able to undergo adipogenesis in the presence of 3-isobutyl-1-methylxanthine plus dexamethasone (Nuttall et al. [1998] J Bone Miner Res 13:371-382). The initial studies of these bipotential cells in explant cultures have been extended to examine differential gene expression during osteoblast/adipocyte transdifferentiation. Using differential display, we have identified a gene expressed in trabecular bone explant cultures that is downregulated as these cells differentiate from an osteoblast to an adipocyte phenotype. Homology searching identified this gene as the human urea transporter HUT11. The expression and downregulation of HUT11 have been observed in multiple patient bone explant cultures. The size of the bone explant-derived HUT11 mRNA is approximately 4.4 kb, which is identical to the largest splice variant reported. In this article, we report the cloning and sequencing of this gene from primary human osteoblasts. In addition, we report tissue distribution for the bone explant-derived form of HUT11 mRNA and show a reciprocal relationship between the expression of HUT11 and the nuclear hormone receptor peroxisome proliferator-activated receptor gamma 2, which is a marker of adipocyte differentiation. Because the control of osteoblast/adipocyte transdifferentiation is unknown, selective downregulation of HUT11 during adipogenesis suggests that HUT11 expression may be a marker of the switch from an osteoblast to an adipocyte phenotype. Understanding the role of HUT11 in osteoblasts may provide insights into the mechanism controlling osteoblast and adipocyte differentiation.

Cloning and functional characterization of a sodium-dependent phosphate transporter expressed in human lung and small intestine.

A cDNA clone with 53% amino acid identity to the human type II sodium-dependent phosphate transporter (NaPi-3) was isolated from human small intestine and lung. Functional characterization in Xenopus laevis oocytes showed this cDNA to encode a sodium-dependent phosphate transporter. The electrogenic response is similar to that found in other type II transporters but an inverse pH dependence was observed. By Northern blot, a 4.2-kb transcript was found to be abundantly expressed in lung and, to a lesser degree, in several other tissues of epithelial origin including small intestine, pancreas, prostate, and kidney. This transcript encompasses a 2.073-kb open reading frame which is most closely related (78% amino acid identity) to the mouse sodium-dependent phosphate transporter IIb isoform. This novel transporter, designated human NaPi-3b (Genbank AF111856), appears to be an isoform of the mammalian renal type II co-transporter family.

Expression in Escherichia coli, refolding, and purification of human procathepsin K, an osteoclast-specific protease.

We have constructed and optimized a high yielding Escherichia coli expression system to produce glycosylation-free human procathepsin K and have developed conditions for refolding this enzyme. Recombinant human procathepsin K (EC 3.4.22.38) was expressed in E. coli, refolded from inclusion bodies, and further purified by Superdex 75 size-exclusion chromatography. Purified procathepsin K had a [MH]+ of 35,063 Da which is in agreement with the predicted mass of the construct. Amino-terminal sequence analysis matched the predicted sequence with no secondary sequence detected. Purified procathepsin K activated under autocatalytic conditions to a final specific activity of 23 micromol 7-amido-4-methylcoumarin liberated/min/mg of enzyme using the fluorescent peptide substrate benzyloxycarbonyl-phenylalanine-arginine-7-amido-4-methylcoumarin. This expression and refolding procedure yielded 50 mg of purified, glycosylation-free human procathepsin K from 1 liter of E. coli cell culture and enabled the determination of the structure of human procathepsin K at 2.6 A resolution.

Site-directed mutagenesis probing the catalytic role of arginines 165 and 166 of human cytomegalovirus protease.

Human cytomegalovirus (CMV) is a member of the Herpesviridae family of viruses that also includes herpes simplex viruses (HSV-1 and HSV-2), varicella-zoster virus (VZV), human herpes virus-6, 7, and 8 (HHV-6, HHV-7, and HHV-8), and Epstein-Barr virus (EBV). Each member of this family encodes a serine protease that is a potential target for antiviral therapeutic intervention. We recently reported the crystal structure of CMV proteases [Qiu, X., Culp, J. S., DiLella, A. G., Hellmig, B., Hoog, S. S., Janson, C. A., Smith, W. W., and Abdel-Meguid, S. S. (1996) Nature 383, 275-279] and proposed that the highly conserved Arg165 and Arg166 residues are involved in stabilizing the oxyanion intermediate in human herpes protease catalyzed reactions through the backbone NH and side chain, respectively. In the current study, site-directed mutagenesis was carried out to probe the catalytic function of these two amino acid residues. Substitution of Arg166 with an alanine has led to ablation of enzymatic activity without detectable change in CMV protease conformation, supporting suggestions from the crystal structure that Arg166 side chain plays a major role in catalysis. The wild-type has a Km = 138 +/- 17 microM and kcat = 19.9 +/- 1.1 min-1, while R166A has only residual activity, with a kcat = 0.012 +/- 0.001 min-1 and an unaltered Km = 145 +/- 18 microM. In the crystal structure, the side chain of Arg166 was shown previously to hold a water molecule that can act as a hydrogen-bond donor to the oxyanion and was thus proposed to stabilize the oxyanion intermediate. However, kinetic characterization of the mutant R165A only reveals a 2.7-fold lower activity than wild-type, with a Km = 166 +/- 19 microM and a kcat = 7.4 +/- 0.4 min-1. These results confirm that Arg165 side chain is not involved in the stabilization of the oxyanion. It is likely that Arg165 only utilizes the backbone NH for catalysis as suggested by the crystal structure.

Cynomolgus monkey (Macaca fascicularis) cathepsin K: cloning, expression, purification, and activation.

Methodology for the production of recombinant active cynomolgus monkey (Macaca fascicularis) cathepsin K (EC 3.4.22.38) was elucidated. The cDNA encoding the cathepsin K was cloned from female M. cynomolgus monkey mRNA. The deduced amino acid sequence of M. cynomolgus preprocathepsin K from the cDNA sequence showed 94.2% identity to human preprocathepsin K. Sequence differences occurred only in the prepro- domains; the mature domains were identical. The recombinant M. cynomolgus cathepsin K was expressed as a secreted proenzyme using baculovirus-infected SF21 insect cells having the predicted N-terminus (LYPEEILDTH ellipsis ), indicating proper cleavage of the secretion sequence. Purified monkey procathepsin K was activated under autocatalytic conditions at pH 4.0. The mature enzyme was composed of mixture of enzymes having N-termini of Gly113 and Arg114. The molecular weight was determined to be 23,668.3 Da by MALDI-TOF-MS which is consistent with the absence of carbohydrate on the mature enzyme. These results indicate that monkey procathepsin K is able to autoactivate and produces a mature enzyme which is identical to that of human cathepsin K. Since the sequence of monkey and human mature cathepsin K are identical and the in vitro activation mechanisms appear to be indistinguishable, monkeys are predicted to be a good animal model for evaluating cathepsin K inhibitors in vivo as therapeutic agents for diseases characterized by excessive bone loss, such as osteoporosis.

[Remifentanil with propofol or isoflurane. A comparison of the recovery times after arthroscopic surgery]. / Remifentanil mit Propofol oder Isofluran. Ein Vergleich des Aufwachverhaltens bei arthroskopischen Eingriffen.

OBJECTIVES: Due to its unique pharmacokinetics, the new esterase-metabolised opioid remifentanil results in rapid post-anesthesia recovery. The aim of this clinical investigation was to compare recovery times after remifentanil anaesthesia in combination with hypnotic concentrations of either propofol or isoflurane. Dosages used in the study protocol were based on recommendations by the pharmaceutical manufacturer. METHODS: Patients (ASA status I-II) scheduled for elective arthroscopy were included in this trial. Without premedication in the morning, anaesthesia was induced identically in both groups: remifentanil bolus (1 microgram/kg), start of remifentanil-infusion (0.5 micrograms/kg/min), followed immediately by propofol (ca. 2 mg/kg). For maintenance of anaesthesia remifentanil (0.25 micrograms/kg/min) was combined with either a propofol infusion of 0.1 mg/kg/min or 0.5 MAC isoflurane (= 0.6 vol. %) in O2/air. Anaesthetic delivery was discontinued simultaneously with termination of surgery and recovery times were recorded. RESULTS: A total of 40 patients were studied at random in two groups of 20 each with comparable demographic data and anaesthetic technique (Tables 1 and 2). In both groups emergence was very rapid. Recovery times were significantly shorter for remifentanil-isoflurane than for remifentanil-propofol (Table 3): spontaneous ventilation 5.1 vs 8.1 min (P < 0.05), extubation 5.5 vs 8.6 min (P < 0.02), post-anaesthesia recovery score > or = 9 of 10 points 6.2 vs 11.3 min (P < 0.01), and arrival at PACU 16.2 vs 19.2 min (P < 0.01). Mild to moderate shivering was noted in 40% of all patients (9 cases following isoflurane, 7 following propofol). CONCLUSIONS: Using the manufacturer's recommended dosages, emergence after remifentanil anaesthesia is more rapid with 0.5 MAC isoflurane than with 0.1 mg/kg/min propofol. These results are most probably due to the different pharmacological properties of both co-anaesthetics, especially the applied dosages, and to different interactions with remifentanil. Present clinical experience suggests that a further dose reduction, especially for propofol, is possible. For both remifentanil groups emergence was remarkably rapid between return of consciousness and the awake state (on-off phenomenon), which might contribute to post-anaesthesia safety.

Cloning and characterization of a novel endothelin receptor from Xenopus heart.

Endothelin (ET) receptors display subtype heterogeneity and so far three subtypes of ET receptors, namely ETA, ETB, and ETC, have been identified, cloned, sequenced, and characterized. Based on the binding profile of ET and related peptides, a novel ET receptor (ETAX) was identified in the follicular membranes of Xenopus laevis oocytes (Kumar, C. S., Nuthulaganti, P., Pullen, M., and Nambi, P. (1993). Mol. Pharmacol. 44, 153-157). Here we report the cloning and characterization of this ETAX subtype from X. laevis heart. A cDNA was isolated that encodes a protein of 415 amino acids that shares 74, 60, and 51% identities with human ETA, human ETB, and Xenopus ETC receptors, respectively. Competition binding studies of the cloned receptor expressed in COS cells using ET-related peptides suggested that this receptor is pharmacologically identical to that expressed in Xenopus oocyte follicular, heart, and lung membranes. Phosphoinositide turnover and oocyte electrophysiological studies indicated that the cloned receptor is functionally coupled to a second messenger system.