To find whether the liver can be procured after exclusive aortic perfusion, three organ perfusion models were used in three groups of donor rats. Group 1 underwent liver wash-out via the portal vein; in group 2, the kidneys alone were perfused via the aorta; and group 3 underwent simultaneous aortic perfusion of liver and kidneys. All perfusion flow rates in the three groups were adjusted to physiological values. Harvested organs were transplanted and recipient animals were killed 4 h after transplantation to study liver and kidney viability by using intracellular ATP measurement. Liver ATP was lower (P < 0.005) in the portal perfusion group (group 1: 1.396 +/- 0.412) than in the aortic perfusion group (group 3: 2.181 +/- 0.061). Kidney ATP was comparable in groups 2 and 3:1.066 +/- 0.09 vs 1.059 +/- 0.273 (mumol/g) tissue). Liver cooling was quicker with portal perfusion than with the aortic flush (20 degrees C in 20 s vs 15 degrees C in 60 s). Aortic perfusion at a physiologic flow rate has no detrimental effect on renal viability studied by intracellular ATP measurement. We conclude that liver cooling via the aortic route only is a good alternative to portal perfusion and seems to give good preservation. Application of this observation to emergency procurement in humans is still the subject of controversy.

Bacterial UMP kinases do not exhibit any sequence homology with other nucleoside monophosphate kinases described so far, and appear under oligomeric forms, submitted to complex regulation by nucleotides. We propose here a structural model of UMP kinase from Escherichia coli based on the conservation of the fold of carbamate kinase whose crystal structure was recently solved. Despite sequence identity of only 18% over 203 amino acids, alignment of UMP kinase from E. coli with carbamate kinase from Enterococcus faecalis by hydrophobic cluster analysis and threading suggested the conservation of the overall structure, except for a small subdomain (absent in UMP kinase). The modelled dimer suggested conservation of the dimer interface observed in carbamate kinase while interaction of UMP kinase with a monoclonal antibody (Mab 44-2) suggests a three in-plane dimer subunit arrangement. The model was analyzed in light of various modified forms of UMP kinase obtained by site-directed mutagenesis.

We have developed a WWW server for the rapid structural analysis of protein sequence/structure alignments. Users can submit an amino acid sequence to perform structural predictions and fold recognition. Pairwise 1D/3D alignments are then visualized as colored alignments to highlight sequence similarities and the relative positions of secondary structure elements and insertions/deletions. A pseudo-energy score is computed for each block in the proposed structural alignment to help manual refinement of the threading if necessary. Modelling is then performed using MODELLER and directly validated using Verify3D. The Web interfaces, we have developed, allow rapid and automatic molecular modelling even in the case of low sequence identity level (20-30%).Here, we report a structural analysis of the Thymidine Monophosphate Kinase (TMPK) from Campylobacter jejuni (hereafter, TMPKcj), a Gram-negative bacterium responsible for human diarrhea. Limited but significant sequence similarities could be detected with other nucleotide monophosphate kinases. The comparison of the TMPK with the crystal structure of other TMPK, strongly suggested that it might adopt a similar fold and revealed the conservation of the monomer-monomer interface. We propose a structural model of TMPKcj and analyze the slight differences at the levels of primary and 3D-structure suggesting putative variations in the binding of substrate analogs. (C) 2002 Elsevier Science Inc. All rights reserved.

Diacylglyceride kinases, sphingosine kinases, NAD kinases and 6-phosphofructokinases are thought to be related despite large evolution of their sequences. Discovery of a common signature has led to the suggestion that they possess a similar phosphate-donor-binding site and a similar phosphorylation mechanism. The substrate- and allosteric-binding sites are much more divergent and their delineation remains to be determined experimentally.

UNLABELLED:We have developed a WWW server for the integration and comparison of protein structure predictions performed by five different servers. Users submit an amino acid sequence to a selected set of these prediction methods. Results are gathered on a web-based page in order to facilitate comparison and analysis. All the alignments are further evaluated through a common threading tool making their comparisons easier.AVAILABILITY:The meta-server is available free at http://www.infobiosud.cnrs.fr/bioserverSUPPLEMENTARY INFORMATION:http://www.infobiosud.cnrs.fr/bioserver/hah1.html

OBJECTIVETo assess the effects of various catecholaminergic agents on pulmonary venous tone.DESIGNProspective, randomized, controlled, experimental study.SETTINGPhysiology laboratory of a university hospital.SUBJECTSThirty anesthetized, mechanically ventilated adult sheep.INTERVENTIONSFour groups of six animals received 1-hr infusions of norepinephrine (0.5 microg/kg/min), epinephrine (0.5 microg/kg/ min), dopamine (10 microg/kg/min), or dobutamine (10 microg/kg/min).MEASUREMENTS AND MAIN RESULTSA 7-Fr pulmonary artery catheter was placed in a proximal location to measure cardiac output and pressure in a large pulmonary vein (Ppw) after balloon inflation. Another catheter wedged in a small pulmonary artery measured pressure in a small pulmonary vein (Pdw). A third catheter measured left atrial pressure (PLA ). This method was able to detect the pulmonary venoconstrictive effects of histamine in a separate group of six animals. Pdw-PLA increased from a mean of 2.0+/-1.7 to 3.0+/-1.5 (SD) cm H2O (p < .01), 2.3+/-1.6 to 4.4+/-1.3 cm H2O (p < .01), and 1.7+/-1.0 to 3.5+/-2.2 cm H2O (p < .05) with norepinephrine, epinephrine, and dopamine, respectively. All of these drugs increased Pdw-Ppw, but only norepinephrine and epinephrine increased Ppw-PLA . No change in either pressure difference was observed with dobutamine. Elevation of cardiac output alone could not account for these findings since the increase in cardiac output induced by fluid infusion did not change the pressure differences.CONCLUSIONNorepinephrine, epinephrine, and dopamine at doses commonly used in humans increase pulmonary venous tone in sheep.

We validated experimentally the ability of hood indirect calorimetry to measure accurately VO2. For this purpose we compared cardiac output calculated from the Fick equation Q = VO2/(Ca(O2) - CV(O2)), in which VO2 was obtained by hood indirect calorimetry, to thermodilution cardiac output (Qth) measured simultaneously during cardiac catheterization in children (n = 16). Because FI(CO2) is a critical factor in hood indirect calorimetry calculations, we also assessed the consequence of taking into account measured FI(CO2) rather than using the usual standard value of 0.0004. We found a good agreement between Q and Qth whether we used experimentally measured FI(CO2) in ambient air (Qth = 0.89 Q + 0.39, r = 0.941) or standard FI(CO2) (Qth = 0.84 Q + 0.55, r = 0.930). However, VCO2 and R computed from standard FI(CO2) differed significantly (p < 0.001) from values derived from measured FI(CO2). This demonstrates that indirect calorimetry allows reasonable estimates of Q, VO2, VCO2, and R provided that the actual values of FI(CO2) are used.

OBJECTIVES:In order to prolong the cold ischaemia time and to improve the quality of donor kidneys, we have designed and developed a renal perfusion machine allowing the control of perfusion parameters (temperature, pressure, flow rate, resistance) during the various phases of a kidney perfusion and storage protocol at -4 degrees C.ANIMALS, MATERIALS AND METHODS:Twenty four rat kidneys were removed and the effects of perfusion and storage at -4 degrees C were studied using a perfusion/storage machine allowing the controlled addition of 2,3 butanediol in University of Wisconsin (UW solution). The kidneys were stored for 96 hours at -4 degrees C and were studied in terms of perfusion parameters (pressure, resistance) and according to their histological appearance.RESULTS:The machine allows controlled perfusion of a cryoprotective agent and preservation of kidneys at -4 degrees C for 96 hours.CONCLUSION:In animals, it is possible to store kidneys at a temperature of -4 degrees C for 96 hours by using a vecor solution (UW solution) and a cryoprotective agent (2,3 butanediol). The perfusion and storage of organs under these conditions must be performed by a computer-assisted machine, allowing monitoring and control of the various steps of perfusion/storage.

Interleukin-2 (IL-2) is reputed to cause a "vascular leak syndrome." We studied pulmonary hemodynamics and lymph dynamics in six sheep treated for 7 days with IL-2 (1.8 million IU/kg twice daily or 1.8 million IU/kg each day as a continuous infusion). Lung lymph flow increased from 4.8 +/- 2 ml/15 min pre-IL-2 to 14.4 +/- 6.8 ml/15 min on the seventh day of IL-2. The lymph-to-plasma protein concentration ratio was unchanged (0.70 +/- 0.06 vs. 0.63 +/- 0.13). The plasma-to-lymph equilibration half-time of radiolabeled albumin was 2.0 +/- 0.6 h pre-IL-2 and 1.0 +/- 0.7 h on day 7 of IL-2. Pulmonary arterial pressure was 24 +/- 7 cmH2O pre-IL-2, increased to 32 +/- 4 cmH2O on the fourth day of IL-2, and returned to 29 +/- 5 cmH2O on the seventh day of IL-2. Extravascular lung water was normal (4.07 +/- 0.25 g/g dry lung). To clearly determine whether the increase in lung lymph flow was due to hemodynamic changes or to increased leakiness of the microvascular barrier, we volume loaded six sheep with lactated Ringer solution before and after 3 days of IL-2 treatment (1.8 million IU/kg twice daily). Lung lymph flows increased fivefold during 4 h of crystalloid infusion compared with baseline and were higher after 3 days of IL-2. However, lymph-to-plasma protein concentration ratios decreased to the same low levels pre-and post IL-2 (0.39 +/- 0.06 vs. 0.41 +/- 0.10), indicating and intact microvascular barrier. Extravascular lung water was elevated (5.56 +/- 0.39 g/g dry lung) but was not different from lung water in three volume-loaded control sheep (4.87 +/- 0.53 g/G dry lung). We conclude that IL-2 causes minimal or no injury to the pulmonary microvascular barrier and that volume expansion during IL-2 treatment can cause hydrostatic pulmonary edema.

Abstract: In order to study whether sheep lung could be used as a suitable oxygenator for human blood, the left lungs of two groups of five sheep were used in an “isolated perfused working lung model system.” A desaturator was inserted in the circuit and the lung was ventilated with ambient air. The lung was perfused in group 1 with heparinated autologous sheep blood and in group 2 with washed human erythrocytes suspended in buffered gelatin. Blood oxygenation as well as hemodynamics and airway parameters followed similar time course in both groups.

Interleukin-2 (IL-2) is reputed to cause pulmonary microvascular injury. We studied the pulmonary and splanchnic microcirculation of anesthetized sheep after one dose (1.8 x 10(6) IU/kg) of IL-2 (n = 9) and after six doses (1.8 x 10(6) IU.kg-1.dose-1) of IL-2 over 3 days (n = 9). Seven control sheep received only 5% dextrose diluent. We measured hemodynamics and lymph dynamics in anesthetized sheep after the final dose of IL-2 or diluent. After one dose of IL-2, caudal mediastinal node (mainly pulmonary) lymph flow was stable, whereas thoracic duct lymph flow increased from a baseline of 54 +/- 6 to 124 +/- 22 ml/h. After 3 days of IL-2, the caudal mediastinal node lymph flow increased from 7.7 +/- 5.5 to 19.0 +/- 14.8 ml/h 5-6 h after the final dose of IL-2, and thoracic duct lymph flow increased from 84 +/- 43 to 143 +/- 42 ml/h. The lymph-to-plasma protein concentration ratio increased after IL-2 for thoracic duct but not for caudal mediastinal node lymph. The equilibration rate of 125I-albumin from plasma to caudal mediastinal node lymph did not change, whereas plasma-to-thoracic duct lymph equilibration was faster after both one dose and 3 days of IL-2. Positron emission tomography showed no increase in the pulmonary transcapillary escape rate for 68Ga-labeled transferrin or in extravascular lung water (n = 4). We conclude that IL-2 at doses two to three times those used clinically does not significantly injure the pulmonary microcirculation of sheep.

Two non-randomized groups of 100 kidney transplants each were compared in relation to the flush-out solution used. The two groups were similar with the exceptions of the number of multiple artery kidneys and the proportion of kidneys procured in our center. The rate of vascular complications such as arterial venous thrombosis and renal artery stenosis was higher in the Eurocollins (19) group than in the UW group (4) p < 0.01, even if the multiple artery kidneys are excluded. These results need to be confirmed by a randomized study but it is clear that the use of the UW solution leads to a better arterial flow with fewer vascular complications.