Opportunistic pathogens in roof-captured rainwater samples, determined using quantitative PCR.

In this study, quantitative PCR (qPCR) was used for the detection of four opportunistic bacterial pathogens in water samples collected from 72 rainwater tanks in Southeast Queensland, Australia. Tank water samples were also tested for fecal indicator bacteria (Escherichia coli and Enterococcus spp.) using culture-based methods. Among the 72 tank water samples tested, 74% and 94% samples contained E. coli and Enterococcus spp., respectively, and the numbers of E. coli and Enterococcus spp. in tank water samples ranged from 0.3 to 3.7 log10 colony forming units (CFU) per 100 mL of water. In all, 29%, 15%, 13%, and 6% of tank water samples contained Aeromonas hydrophila, Staphylococcus aureus, Pseudomonas aeruginosa and Legionella pneumophila, respectively. The genomic units (GU) of opportunistic pathogens in tank water samples ranged from 1.5 to 4.6 log10 GU per 100 mL of water. A significant correlation was found between E. coli and Enterococcus spp. numbers in pooled tank water samples data (Spearman's rs = 0.50; P < 0.001). In contrast, fecal indicator bacteria numbers did not correlate with the presence/absence of opportunistic pathogens tested in this study. Based on the results of this study, it would be prudent, to undertake a Quantitative Microbial Risk Assessment (QMRA) analysis of opportunistic pathogens to determine associated health risks for potable and nonpotable uses of tank water.

Central venous catheters--the inability of 'intra-atrial ECG' to prove adequate positioning.

BACKGROUND: The classic increase in P wave size, known as 'P-atriale', is a widely accepted criterion for determination of proper positioning of central venous catheter tips. Recent transoesophageal echocardiography (TOE) studies did not confirm intra-atrial position despite advancing the central venous catheter further than indicated by ECG guidance. We postulate that the pericardial reflection rather than the entry into the right atrium corresponds to the ECG changes. In order to test our hypothesis we sought to determine the anatomical substrate for the electrical changes in an animal study. Subsequently, a modified version of the study was undertaken in man and is also reported. METHODS: In six juvenile pigs the left external jugular vein and right carotid artery were cannulated. A triple-lumen central venous catheter was positioned by ECG guidance using a Seldinger wire as an exploring electrode. The venous and arterial catheters were suture fixed 2 cm beyond the onset of an increase in P wave size. The corresponding anatomical catheter tip position was determined by open exploration of the vessels and the heart. Subsequently the catheter tip position (during advancement) of a pulmonary artery catheter and the corresponding electrical ECG changes were examined in 10 patients during open chest cardiac surgery. RESULTS: All catheters-arterial and venous, in animals and humans-revealed an increase in size of the P wave as well as the QRS complex. All venous catheters were positioned in the superior vena cava, beyond the pericardial reflection but outside the right atrium. All arterial catheters were positioned in the ascending aorta thus also beyond the pericardial reflection. CONCLUSIONS: The start of an increase in P wave size does not correspond with the entrance of the right atrium. The anatomic equivalent for the electrophysiological changes of the ECG is the pericardial reflection. ECG guidance is unable to distinguish between venous and arterial catheter position.

Post-operative hypoalbuminaemia and procalcitonin elevation for prediction of outcome in cardiopulmonary bypass surgery.

BACKGROUND: Because few studies have addressed postoperative hypoalbuminaemia in relation to hospital mortality, we evaluated this association and the prognostic value of increased procalcitonin (PCT) after cardiopulmonary bypass (CPB) surgery. METHODS: In 454 consecutive patients undergoing CPB, minimal serum albumin, colloid osmotic pressure (COP) and maximal PCT were retrospectively obtained from the 2nd to 10th postoperative day. Receiver operating characteristic (ROC) and multiple regression analyses determined independent predictive strength for 28-day mortality from preoperative albumin, Euroscore, postoperative minimal albumin and COP, and maximal PCT. Cut-off points for the four strongest predictors were calculated by the area under the curve (AUC) in the ROC for the 28-day mortality. RESULTS: Maximal PCT showed the largest AUC (0.85; 95% CI 0.79-0.90) and the highest relative risk (RR 12.17; 95%CI 5.26-28.16; P < 0.001), compared with postoperative albumin (AUC 0.72; 95% CI 0.62-0.81; RR 5.35; 95%CI 2.99-9.56; P < 0.001) and EuroSCORE (AUC 0.73; 95%CI 0.63-0.83; RR 4.48; 95%CI: 1.78-11.28; P < 0.01). By logistic regression, postoperative albumin was the strongest predictor of mortality (odds ratio 0.86; 95% CI 0.84-0.89). Cut-off values for predicting 28-day mortality were found for postoperative albumin and PCT at 17.8 g l(-1) and 2.5 ng l(-1), respectively. A slight but significant inverse correlation between PCT and albumin was found. Patients with albumin less than the cut-off showed significantly higher median values for PCT levels (2.5 vs. 1.0 g l-1), a higher 28-day mortality rate (20.8% vs. 4.5%), and a longer ICU stay (6 vs. 3 days) in comparison with patients with minimal albumin greater than 18 g l(-1). CONCLUSIONS: Post-operative serum albumin <18 g l(-1) and PCT >2.5 ng l(-1) are predictive for a higher 28-day mortality rate in cardiosurgical patients. Both peak PCT and minimal albumin were better outcome predictors than the Euroscore, which better represents the preoperative condition of the patient.

Comparison of pulsatile and nonpulsatile perfusion of the lung in an extracorporeal large animal model.

OBJECTIVE: Extracorporeal lung-perfusion models are widely used to evaluate pulmonary preservation techniques and reperfusion injury. However, these models mainly depend on nonpulsatile flow, which is not physiological and can subsequently lead to pulmonary edema. Observation in a standardized setting and reliability of functional and structural data assessment are therefore limited. To overcome these limitations we developed a new extracorporeal large animal lung perfusion model utilizing pulsatile flow to perfuse the pulmonary vasculature. METHODS: Lungs of juvenile domestic pigs were in situ preserved with 2 liters Perfadex and stored for 3 h at 10 degrees C. Thereafter, reperfusion of the lung was performed in an extracorporeal blood perfusion circuit employing either a modified roller pump with pulsatile module (300 ml/min; pulsation rate 90/min) or a standardized roller pump with continuous flow (30 ml/min). Ventilation was performed with physiologic room air (350 ml; 16/min) for 1 h. Pulsatile and nonpulsatile perfusion was performed in 2 groups (group NP: nonpulsatile; group P: pulsatile flow, n = 7) during reperfusion. Peak inspiratory pressure (PIP), mean pulmonary artery pressure (PAP), and oxygenation capacity (DeltaPO(2)) were continuously measured. For control of the effectiveness of the pulsatile perfusion pressure waveforms were obtained directly from the native pulmonary artery of both groups. Malondialdehyde (MDA) as a parameter for lipid peroxidation and endothelial cell damage was assessed at 10, 30 and 50 min reperfusion. At the end of the study, pulmonary water content was assessed by means of wet-to-dry ratio (W/D ratio). The tissue was further processed for microscopic analysis. RESULTS: PIP increased significantly in both groups during reperfusion. Mean PAP in both groups increased to 60 mm Hg after 20 min followed by a decrease after 60 min to 40 mm Hg. Pressure waveforms of the pulmonary artery showed sufficient pulsatility in the pulmonary vasculature with a systolic/diastolic pressure difference of 15 mm Hg whereas the pressure difference was 3-5 mm Hg in the nonpulsatile group. DeltaPO(2) was stable in groups NP and P during reperfusion (30 min: NP: 66.4 (62.2-88) mm Hg; P: 74.8 (65-81.7) mm Hg) without any statistically significant differences between the groups. MDA in group NP decreased over the reperfusion period from 6.2 (3.3-6.3) microM at 10 min to 5.2 (3.2-6.1) microM at 50 min, whereas in group P the level increased and was significantly higher after 50 min reperfusion compared to group NP [6.6 (6.1-9.2) microM at 50 min; p = 0.016]. W/D ratio was 6.7 (6.3-7.0) in group NP and 6.8 (6.3-7.6) in group P. Light microscopy evaluation showed no differences between both groups regarding severity of intra-alveolar and interstitial edema and numbers of intra-alveolar, intracapillary and interstitial granulocytes. CONCLUSION: Although effective pulsatile perfusion of the pulmonary vasculature was achieved by means of a modified roller pump, this measure obviously did not improve functional parameters nor did it significantly reduce the edema formation after 3 h ischemia in this extracorporeal lung perfusion model. The use of pulsatile perfusion is therefore not mandatory in the extracorporeal setting of a large animal lung perfusion model.

Redo- extirpation of a cardiac leiomyosarcoma to avoid transplantation.

Cardiac leiomyosarcoma is a very rare entity that is found in less than 0.2 % of all cardiac tumors. At the time of primary diagnosis, it often shows advanced local invasion or may even be metastasized. Thus, complete resection can not easily be achieved. Cardiac transplantation has been reported as a therapeutic option. Here, we report on a case of a leiomyosarcoma reoccurrence arising 2 years after initial surgery. We performed a radical redo-extirpation without the necessity of transplantation.

Influence of high molecular dextrans on lung function in an ex vivo porcine lung model.

BACKGROUND: Extracellular preservation solutions utilizing high molecular agents can reduce intracellular edema during ischemia/reperfusion in lung transplantation. A solution of 40,000 dalton molecular weight (DMW) has already been clinically established (Perfadex). However, it is unclear whether dextrans of this particular size represent the optimal additive for lung preservation solutions. MATERIALS AND METHODS: In a new ex vivo porcine lung model, lungs were each preserved with low-potassium solutions containing 5% dextran with 90,000 DMW (Dex 90) and 160,000 DMW (Dex 160) and with Perfadex (40,000 DMW). After 24 h of cold ischemia, reperfusion was performed employing a roller pump with a pulsatile module. Lungs were perfused with deoxygenated perfusate and ventilated with room air. The oxygenation capacity (Delta pO(2)), peak inspiratory pressure (PIP), and mean pulmonary artery pressure (PAP) were monitored for 60 min. Net weight gain (NWG) and wet-to-dry ratio (W/D ratio) were determined. Free-radical generation was assessed by measuring malondialdehyde (MDA) at 10, 30, and 50 min. RESULTS: PIP and PAP increased in all groups significantly during reperfusion. However, Dex 160-perfused lungs exhibited significantly higher values than those with Dex 90 and Perfadex. Perfadex showed the highest Delta pO(2) throughout the entire reperfusion, while Delta pO(2) was slightly reduced in Dex 160 and significantly lower in Dex 90. In Perfadex the lowest water content was observed assessed by NWG and W/D ratio. The highest MDA values were observed in Dex 90, followed by Dex 160, while the lowest values were seen in Perfadex. CONCLUSIONS: Preservation of the lung with Perfadex exhibited superior postischemic function in contrast to preservation solutions containing dextrans with a higher molecular weight.

Retrograde flush perfusion with low-potassium solutions for improvement of experimental pulmonary preservation.

BACKGROUND: Optimal preservation of post-ischemic organ function is a continuing challenge in clinical lung transplantation. Retrograde instillation of preservation solutions has the theoretic advantage of achieving homogeneous distribution in the lung because of perfusing both the pulmonary and the bronchial circulation. So far, we have seen no experimental studies that include stereologic analysis of intrapulmonary edema concerning the influence of retrograde preservation on post-ischemic lung function after preservation with Perfadex and Celsior. METHODS: In an extracorporeal rat model, we perfused 8 lungs, each, using either antegrade or retrograde perfusion technique with Celsior (CE(ant)/CE(ret)) and Perfadex (PER(ant)/PER(ret)). Results were compared with low-potassium Euro-Collins. Post-ischemic lungs were reventilated and reperfused mechanically. We continuously monitored relative oxygenation capacity (ROC), pulmonary artery pressure, flush time, and wet/dry ratio. Furthermore, we used stereologic means to evaluate edema formation. Statistics comprised different analysis of variance models. RESULTS: Relative oxygen capacity of CE(ant)-protected lungs was superior to that of PER(ant) preservation (p = 0.05). Use of PER(ret) resulted in significantly higher ROC as compared with PER(ant) (p < 0.001) and was comparable to results obtained with CE-preservation, which was not further improved with retrograde application. CONCLUSIONS: Celsior provides better lung preservation than does Perfadex when administered antegradely. Retrograde application of Perfadex results in significant functional improvement as compared with antegrade perfusion, which reaches the standard of Celsior-protected organs. Additional in vivo experiments in combination with ultrastructural analysis are warranted to further evaluate retrograde delivery of preservation solutions, which could be used in clinical lung transplantation to further optimize current results.

Roles and microenvironments of tryptophanyl residues of spinach phosphoribulokinase.

Phosphoribulokinase is one of several Calvin cycle enzymes that are light-regulated via the ferredoxin-thioredoxin system (R. A. Wolosiuk and B. B. Buchanan, 1978, Arch. Biochem. Biophys. 189, 97-101). Substitution of the only two Trp residues of the enzyme was prompted by the following goals: to identify each tryptophanyl residue with respect to prior classifications as exposed and buried (C. A. Ghiron et al., 1988, Arch. Biochem. Biophys. 260, 267-272); to explore the possible active-site location and function of conserved Trp155, as suggested by sequence proximity to catalytic Asp160 (H. A. Charlier et al., 1994, Biochemistry 33, 9343-9350); and to determine if fluorescence of a Trp residue can serve as a gauge of conformational differences between the reduced (active) and the oxidized (inactive) forms of the enzyme. Emission spectra and acrylamide quenching data demonstrate that Trp155 is solvent exposed, while Trp241 is buried. Kinetic parameters of the W241F mutant are not significantly altered relative to those of wild-type enzyme, thereby discounting any requirement for Trp at position 241. While substitution of Trp155 with Phe or Ala has little impact on Vmax, the Km for Ru5P and ATP are increased substantially; the diminished affinity for ATP is particularly pronounced in the case of the Ala substitution. In further support of an active-site location of Trp155, its fluorescence emission is subject to quenching by nucleotides. Fluorescence quenching of reduced W241F by ATP gives a dissociation constant (Kd) of 37 microM, virtually identical with its Km of 46 microM, and provides for the first time a direct measurement of the interaction of the kinase with product ADP (Kd of 1.3 mM). Fluorescence quenching of oxidized W241F by ATP reveals a Kd of 28 mM; however, this weakened binding does not reflect an altered microenvironment of Trp155, as its steady-state emission and fluorescence lifetimes are unaffected by the oxidation state.

Oxidation-reduction properties of the regulatory site of spinach phosphoribulokinase.

The oxidation-reduction midpoint potential (Em) of the regulatory disulfide, formed between Cys16 and Cys55, of spinach chloroplast phosphoribulokinase has been determined both for the wild-type enzyme and for a C244S-C250S double mutant, using enzymatic activity to monitor the oxidation-reduction state of the regulatory disulfide. At pH 7.0, Em values for the two-electron reduction of the regulatory disulfide of -295 +/- 10 and -290 +/- 10 mV were measured for the wild-type and mutant, respectively. In contrast to the dependence of activity on ambient potential (Eh) observed for the wild-type enzyme and the double mutant, which both followed the Nernst equation for a two-electron process, high and constant activity was exhibited by a C16S-C244S-C250 triple mutant of the enzyme at all Eh values tested. Em values for the wild-type enzyme were also measured at pH values of 6.7, 7.5, 7.7, and 8.2 and the Em vs pH data in this region give a good fit to a straight line with a slope of -60 mV/pH unit.

Potassium-reduced lung preservation solutions: a screening study.

In recent years several potassium-reduced solutions have been developed for improvement of pulmonary preservation. A comparison of these solutions in a single study, however, has not yet been performed. In an extracorporeal working rat heart-lung model (n = 7/group) lungs were preserved with 20 ml Euro-Collins (EC), low potassium EC (LPEC), low potassium 2% dextran (LPD), ET-Kyoto (ETK) or low potassium 5% dextran (Perfadex) solution, while hearts were arrested with 10 ml St. Thomas' cardioplegia. Lungs of controls were not perfused. The heart-lung blocks were stored for 2 h at 10 degrees C. Thereafter, heart-lung blocks were extracorporeally perfused with Krebs-Henseleit solution with washed bovine red blood cells. Coronaries were perfused with oxygenated perfusate. Lungs were perfused via the working right ventricle with deoxygenated perfusate and ventilated with room air. Oxygenation capacity (dPO2) and pulmonary vascular resistance (PVR) were measured. Reperfusion/ventilation was performed for 40 min. At the end of the experiment the wet to dry (W/D) ratio of lungs and light microscopic assessment of the degree of edema (0-4) were performed. All potassium-reduced solutions showed superior dPO2 and a lower PVR than EC and controls while LPEC exhibited the most stable dPO2 and lowest PVR after 30 min reperfusion. The W/D ratio of all potassium-reduced groups was lower than the ratio of EC and controls. In LPEC, ETK and Perfadex the least degree of edema was noted. All solutions used in this study are superior to regular EC. However, when compared directly, LPEC perfused lungs showed better functional preservation than all other alternative solutions.

Efficient expression of the gene for spinach phosphoribulokinase in Pichia pastoris and utilization of the recombinant enzyme to explore the role of regulatory cysteinyl residues by site-directed mutagenesis.

Phosphoribulokinase (PRK), unique to photosynthetic organisms, is regulated in higher plants by thioredoxin-mediated thiol-disulfide exchange in a light-dependent manner. Prior attempts to overexpress the higher plant PRK gene in Escherichia coli for structure-function studies have been hampered by sensitivity of the recombinant protein to proteolysis as well as toxic effects of the protein on the host. To overcome these impediments, we have spliced the spinach PRK coding sequence immediately downstream from the AOX1 (alcohol oxidase) promoter of Pichia pastoris, displacing the chromosomal AOX1 gene. The PRK gene is now expressed, in response to methanol, at 4-6% of total soluble protein, without significant in vivo degradation of the recombinant enzyme. This recombinant spinach PRK is purified to homogeneity by successive anion-exchange and dye-affinity chromatography and is shown to be electrophoretically and kinetically indistinguishable from the authentic spinach counterpart. Site-specific replacement of all of PRK's cysteinyl residues (both individually and in combination) demonstrates a modest catalytically facilitative role for Cys-55 (one of the regulatory residues) and the lack of any catalytic role for Cys-16 (the other regulatory residue), Cys-244, or Cys-250. Mutants with seryl substitutions at position 55 display non-hyperbolic kinetics relative to the concentration of ribulose 5-phosphate. Sulfate restores hyperbolic kinetics and enhances kinase activity, presumably reflecting conformational differences between the position 55 mutants and wild-type enzyme. Catalytic competence of the C16S-C55S double mutant proves that mere loss of free sulfhydryl groups by oxidative regulation cannot account entirely for the accompanying total inactivation.

The molecular pathway for the regulation of phosphoribulokinase by thioredoxin f.

Phosphoribulokinase (PRK) is one of several plant enzymes that is regulated by thiol-disulfide exchange as mediated by thioredoxin, which contains spatially vicinal, redox-active cysteinyl residues. In an earlier study (Brandes, H. K., Larimer, F. W., Geck, M. K., Stringer, C. D., Schürmann, P., and Hartman, F. C. (1993) J. Biol. Chem. 268, 18411-18414), our laboratory identified Cys-46 of thioredoxin f (Trx), as opposed to the other candidate Cys-49, as the primary nucleophile that attacks the disulfide of target proteins. The goal of the present study was to identify which of the two redox-active cysteinyl residues of PRK (Cys-16 or Cys-55) is paired with Cys-46 of Trx in the interprotein disulfide intermediate of the overall oxidation-reduction pathway. Incubation of a mixture of the C16S mutant of PRK and the C49S mutant of Trx with Cu2+ results in covalent complex formation as detected by SDS-polyacrylamide gel electrophoresis. Complexation is fully reversible by dithiothreitol and is retarded by ligands for PRK. Under the same conditions, Cu2+ induces very little complex formation between the following pairs of mutants: C16S PRK/C46S Trx, C55S PRK/C49S Trx, and C55S PRK/C46S Trx. When either 5-thio-2-nitrobenzoate-derivatized C16S or C55S PRK, as mimics of the oxidized (disulfide) form of the enzyme, is mixed with C49S Trx, stable covalent complex formation occurs only with the C16S PRK. Thus, two independent approaches identify Cys-55 of PRK in the intermolecular disulfide pairing with Trx.

Comparison of low potassium Euro-Collins solution and standard Euro-Collins solution in an extracorporeal rat heart-lung model.

OBJECTIVE: Euro-Collins solution (EC) is routinely used in lung transplantation. The high potassium of EC, however, may damage the vascular endothelium, thereby contributing to postischemic reperfusion injury. To assess the influence of the potassium concentration on lung preservation, we evaluated the effect of a "low potassium Euro-Collins solution" (LPEC), in which the sodium and potassium concentrations were reversed. METHODS: In an extracorporeal rat heart-lung model lungs were preserved with EC and LPEC. The heart-lung blocks (HLB) were perfused with Krebs-Henseleit solution containing washed bovine red blood cells and ventilated with room air. The lungs were perfused via the working right ventricle with deoxygenated perfusate. Oxygenation and pulmonary vascular resistance (PVR) were monitored. After baseline measurements, hearts were arrested with St. Thomas' solution and the lungs were perfused with EC or LPEC, or were not perfused (controls). The HLBs were stored for 5 min or 2 h ischemic time at 4 degrees C. Reperfusion and ventilation was performed for 40 min. At the end of the trial the wet/dry ratio of the lungs was calculated and light microscopic assessment of the degree of edema was performed. RESULTS: After 5 min of ischemia oxygenation was significantly better in both preserved groups compared to the controls. Pulmonary vascular resistance was elevated in all three groups after 30 min reperfusion at both ischemic times. After 2 h of ischemia PVR of the group preserved with LPEC was significantly lower than those of the EC and controls (LPEC-5 min: 184 +/- 65 dynes * sec * cm-5, EC-5 min: 275 +/- 119 dynes * sec * cm * cm-5, LPEC-2 h: 324 +/- 47 dynes * sec * m-5, EC-2 h: 507 +/- 83 dynes * sec * cm-5). Oxygenation after 2 h of ischemia and 30 min reperfusion was significantly better in the LPEC group compared to EC and controls (LPEC: 70 +/- 17 mmHg, EC: 44 +/- 3 mmHg). The wet/dry ratio was significantly lower in the two preserved groups compared to controls (LPEC-5 min: 5.7 +/- 0.7, EC-5 min: 5.8 +/- 1.2, controls-5 min: 7.5 +/- 1.8, LPEC-2 h: 6.7 +/- 0.4, EC: 6.9 +/- 0.4, controls-2 h: 7.3 +/- 0.4). CONCLUSIONS: We thus conclude that LPEC results in better oxygenation and lower PVR in this lung preservation model. A low potassium concentration in lung preservation solutions may help in reducing the incidence of early graft dysfunction following lung transplantation.

Influence of red blood cells on lung function in an ex vivo rat heart-lung model.

Crystalloid perfusates commonly are utilized for lung preservation in extracorporeal small animal lung models. However, the function of these grafts is limited. In a new ex-vivo rat heart-lung model the role of red blood cells added to the crystalloid perfusate was investigated. Heart-lung blocks were rapidly excised (n = 9, each group) and the blocks were connected to the extracorporeal perfusion circuit using Krebs-Henseleit solution (KH) or KH with washed bovine red blood cells (hematocrit 38%) (KHRBC). The lungs were ventilated with room air. The coronary system was perfused via the aortic root with oxygenated perfusate. The lungs were perfused via the right ventricle with deoxygenated perfusate (PO2 15 mm Hg). Venoarterial improvement of oxygenation, pulmonary vascular resistance (PVR), and peak inspiratory pressure (PIP) were continuously monitored for 50 min. At the end of the experiment the wet/dry (W/D) ratio was determined using the mediastinal lung lobe while the remaining lung was used for light microscopic (LM) evaluation. After 30 min of perfusion, lung function was significantly better in the KHRBC group (PVR (KH): 752 +/- 193 dyn*sec*cm-5; (KHRBC): 279 +/- 48 dyn*sec*cm-5; PIP (KH): 31 +/- 3.2 mm Hg; (KHRBC): 25.8 +/- 1.9 mm Hg). In addition, lungs perfused with KHRBC showed significantly less edema than those perfused with KH only (W/D ratio (KH): 7.8 +/- 1.2; (KHRBC) 5.1 +/- 0.6; LM (KH): 3.5 +/- 0.9; (KHRBC): 2.3 +/- 0.8). The use of red blood cells in KH perfusate improved functional and structural integrity.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct identification of the primary nucleophile of thioredoxin f.

Thioredoxin, by virtue of the proximal active-site sulfhydryls (Trp-Cys-Gly-Pro-Cys), catalyzes thiol-disulfide exchange with specific target enzymes. Considerable data (chemical modification, spectroscopic, and crystallographic) have implicated the cysteinyl residue nearest the N terminus of thioredoxin as the primary nucleophile; however, direct proof has been lacking. Proof is now provided by characterization of site-directed mutants of thioredoxin f with respect to activation of chloroplastic fructose-1,6-bisphosphatase (FBPase). The C49S mutant retains the capacity to activate FBPase, whereas the C46S mutant is totally lacking in this regard. Based on kinetics of FBPase activation, wild-type and C49S thioredoxins exhibit half-saturation values of 0.9 and 4 microM, respectively. Lack of activation by C46S is not because of failure to interact with FBPase, for it exhibits a Ki of 5 microM in competition with wild-type thioredoxin. Therefore, in the normal thioredoxin-catalyzed reduction pathway, Cys-46 is the nucleophile required to attack the disulfide of the substrate and Cys-49 serves to cleave the mixed disulfide intermediate, thus allowing for the release of oxidized thioredoxin and the reduced target enzyme.

[Imaging procedures in preoperative lymph node staging of invasive bladder cancer. Necessary or superfluous?]. / Bildgebende Verfahren zum präoperativen Lymphknotenstaging beim invasiven Harnblasenkarzinom.Sinnvoll oder überflüssig?

A staging programme for detection of lymph node involvement before radical cystectomy has been carried out in 22 patients. The programme includes: intravenous pyelography, chest X-ray, abdominal sonography, bone scan, CT and MRI. Also an immunoscintigraphic examination using monoclonal anti-CEA antibody (TUMAK BW 421/26) was done in every patient. Preoperative lymph node staging using CT, MRI and immunoscintigraphy was compared with post-operative histological staging: a total of 5 patients were found to have lymph node involvement. In none of them had lymph node involvement been predicted on the basis of CT, MRI or immunoscintigraphy.

Conformational flexibility of the regulatory site of phosphoribulokinase as demonstrated with bifunctional reagents.

Phosphoribulokinase (PRK) is one of several chloroplastic enzymes whose activity is regulated by thiol-disulfide exchange via thioredoxin. Activation entails reduction of an active-site disulfide bond between Cys16 and Cys55. Bifunctional cross-linking reagents have been used to approximate the interresidue distance between Cys16 and Cys55, an issue which impinges on the relative conformational states of the activated and deactivated forms of the enzyme. Spinach PRK is rapidly inactivated by stoichiometric levels of 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone (FNPS) or 1,5-difluoro-2,4-dinitrobenzene (DFNB), which span 9 and 3.5 A, respectively. ATP, but not ribulose 5-phosphate, retards the rate of inactivation, suggesting that modification has occurred at the nucleotide binding domain of the active site. Sulfhydryl modification is indicated by partial reversibility of inactivation as effected by exogenous thiols. Tryptic mapping by reverse-phase chromatography of [14C]carboxymethylated enzyme, subsequent to its reaction with either FNPS or DFNB, demonstrates modification of Cys16 and Cys55 by both reagents, and formation of only one major chromophoric peptide in each case. On the basis of the sequence analysis of the purified chromophoric peptides, Cys16 and Cys55 are cross-linked by both FNPS and DFNB. Thus, the intrasubunit distance between the beta-sulfhydryls of Cys16 and Cys55 is dynamic rather than static. Diminished conformational flexibility upon oxidation of the regulatory sulfhydryls to a disulfide may be partially responsible for the concomitant loss of enzymatic activity.