Pig heart fumarase contains two distinct substrate-binding sites differing in affinity

A eukaryotic fumarase is for the first time unequivocally shown to contain two distinct substrate-binding sites. Pig heart fumarase is a tetrameric enzyme consisting of four identical subunits of 50 kDa each. Besides the true substrates L-malate and fumarate, the active sites (sites A) also bind their analogs D-malate and oxaloacetate, as well as the competitive inhibitor glycine. The additional binding sites (sites B) on the other hand also bind the substrates and their analogs D-malate and oxaloacetate, as well as L-aspartate which is not an inhibitor. Depending on the pH, the affinity of sites B for ligands (Kd being in the millimolar range) is 1-2 orders of magnitude lower than the affinity of sites A (of which Kd is in the micromolar range). However, saturating sites B results in an increase in the overall activity of the enzyme. The benzenetetracarboxyl compound pyromellitic acid displays very special properties. One molecule of this ligand is indeed able to bind into a site A and a site B at the same time. Four molecules of pyromellitic acid were found to bind per molecule fumarase, and the affinity of the enzyme for this ligand is very high (Kd = 0.6 to 2.2 microM, depending on the pH). Experiments with this ligand turned out to be crucial in order to explain the results obtained. An essential tyrosine residue is found to be located in site A, whereas an essential methionine residue resides in or near site B. Upon limited proteolysis, a peptide of about 4 kDa is initially removed, probably at the C-terminal side; this degradation results in inactivation of the enzyme. Small local conformational changes in the enzyme are picked up by circular dichroism measurements in the near-UV region. This spectrum is built up of two tryptophanyl triplets, the first one of which is modified upon saturating the active sites (A), and the second one upon saturating the low affinity binding sites (B).     

Insertional re-activation of a chloramphenicol acetyltransferase misfolding mutant protein

The deletion of nine residues from the C-terminus of the bacterialchloramphenicol acetyltransferase (CAT) results in depositionof the mutant protein in cytoplasmic inclusion bodies and lossof chloramphenicol resistance in Escherichia coli. This foldingdefect is relieved by C-terminal fusion of the polypeptide withas few as two residues. Based on these observations, efficientpositive selection for the cloning of DNA fragments has beendemonstrated. The cloning vector encodes a C-terminally truncatedCAT protein. Restriction sites in front of the stop codon allowthe insertion of target DNA, resulting in the production ofproperly folded CAT fusion proteins and regained chloramphenicolresistance. The positive selection of recombinants is accomplishedby growth of transformants on chloramphenicol-containing agarplates. The method appears particularly convenient for the cloningof DNA fragments amplified by the PCR because minimal informationto restore CAT folding can be included in the primers. The cloningof random sequences shows that the folding defect can be relievedby fusion to a wide variety of peptides, providing great flexibilityto the positive selection system. This vector may also contributeto the determination of the role of the C-terminus in CAT folding.     

Unexplained physical symptoms: outcome, utilization of medical care and associated factors

BACKGROUND: Measurement of intracardiac hemodynamic parameters has been limited to brief periods in the acute care setting. We developed and evaluated an implantable hemodynamic monitor that is capable of measuring chronic right ventricular oxygen saturation and pulmonary artery pressure. METHODS AND RESULTS: The device consists of an electronic controller placed subcutaneously and two transvenous leads placed in the right ventricle (reflectance oximeter) and pulmonary artery (variable capacitance pressure sensor). Implantation was performed in 10 patients with severe left ventricular dysfunction. Average implant pulmonary artery pressures were systolic, 52 +/- 16 mm Hg; diastolic, 29 +/- 11 mm Hg; and mean, 40 +/- 12 mm Hg. The mean right ventricular oxygen saturation at implant was 51%. Provocative maneuvers, including postural changes, sublingual nitroglycerin, and bicycle exercise, demonstrated expected changes in measured oxygen saturation and pulmonary artery pressures over time. At follow-up of 0.5 to 15.5 months, there were no significant differences between pulmonary artery pressures or oxygen saturation values transmitted from the device and simultaneous measurement with balloon flotation catheters. Four of the pulmonary artery leads dislodged and three demonstrated sensor drift, whereas two of the oxygen saturation sensors failed. Four patients died and four received transplants. Pathological study did not demonstrate injury to the right ventricular outflow tract or pulmonic valve. CONCLUSIONS: Chronic measurement of hemodynamic parameters in the outpatient setting with implantable sensor technology appears to be feasible. The devices are well tolerated without significant untoward effects, and the sensors generally function well over time, providing reliable information. Clinical usefulness remains to be established.     

A novel cyclic process using CaSO<Subscript>4</Subscript>/CaS pellets for converting sulfur dioxide to elemental sulfur without generating secondary pollutants: Part I. Feasibility and kinetics of the reduction of sulfur dioxide with calcium-sulfide pellets

Certain new sulfide-smelting processes and coal-gasification processes generate high-strength sulfur dioxide streams, for which a new process for converting sulfur dioxide to elemental sulfur needs to be developed because no process exists that is generally and economically applicable to the treatment of such streams. A thermodynamic and experimental investigation to develop a new process for converting sulfur dioxide to elemental sulfur by a cyclic process involving calcium sulfide and calcium sulfate without generating secondary pollutants was carried out. In this process, the starting raw material, calcium sulfate, is reduced by a suitable reducing agent, such as hydrogen, to produce calcium sulfide, which is used to reduce sulfur dioxide to elemental sulfur vapor and calcium sulfate. The latter is, in turn, reduced to regenerate calcium sulfide. In this Part I, detailed experimental results are presented on the kinetics of the reaction between sulfur dioxide and calcium-sulfide pellets, which produces elemental sulfur and calcium sulfate. The experiments were carried out at temperatures between 1023 and 1088 K and sulfur-dioxide partial pressures between 9 and 60 kPa by the use of a thermogravimetric analysis (TGA) technique. The rate of this reaction was demonstrated by the conversion of 40 pct calcium-sulfide pellets obtained from the hydrogen reduction of fresh calcium sulfate in 10 minutes at 1073 K under a sulfur-dioxide partial pressure of 43 kPa. The reactivity decreased somewhat during the first three cycles but remained largely unchanged thereafter up to the tenth cycle. This characteristic of the pellets is important because the solids must be reusable for repeated cycles to avoid generating secondary pollutants. A pore-blocking model was found to fit the reaction rate. The reaction is first order with respect to sulfur-dioxide partial pressure and has an activation energy of 101 to 134 kJ/mol (24 to 32 kcal/mol) for calcium-sulfide pellets reacted and regenerated several different times. Sulfur dioxide-containing streams from certain sources, such as the regenerator off-gas from an integrated-gasification, combined-cycle, desulfurization unit and new sulfide-smelting plants, contain much higher partial pressures of SO₂. In these cases, the rate of the first reaction is expected to be proportionally higher than in the test conditions reported in this article. The reduction kinetics of calcium-sulfate pellets with hydrogen gas is reported in the accompanying Part II.     

Microbiological denitrification of red beet juice

The purpose of study was the determination of optimal conditions for red beet juice denitrification by Paracoccus denitrificans bacteria as well as the evaluation of the usefulness of microbiologically treated juice for the production of a natural colouring preparation for foods. Total reduction of nitrates was found during bacterial culturing at 25 and 30 °C, at an initial pH of 7.0-8.0 and juice osmotic pressure of 900-1100 mOsm/kg. Microbiological denitrification procedure affected colour, taste and aroma of the juice. However, the use of juice as food colouring and component was made feasible by acidification and partial evaporation of water together with volatile substances.     

Suspension force and mechanism of debris flows

The paper discusses the postulated suspension force, by which it is possible to explain some enigmatic phenomena observed in different types of mass movements. This force is generated in thick suspensions as a result of friction between sinking solid particles and static water. The suspension force is related to the seepage force but they have opposite directions. Both forces form flow networks. Having explained the suspension force, the paper describes how this influences the movement of material, particularly in debris flows. Enigmatic features and the mechanism of debris flows and lahars are explained by virtue of the suspension force. Electronic Publication     

Scheduling railway traffic at a construction site

We consider the problem of rescheduling trains in the case where one track of a railway section consisting of two tracks in opposing directions is closed due to construction activities. After presenting an appropriate model for this situation we derive a polynomial algorithm for the subproblem of finding an optimal schedule with minimal latenesss if the subsequences of trains for both directions outside the construction site are fixed. Based on this algorithm we propose a local search procedure for the general problem of finding good schedules and report test results for some real world instances. Received: December 8, 1999 / Accepted: May 2, 2001     

Replacement of beef fat in Frankfurters by interesterified palm,cottonseed and olive oils

Three interesterified vegetable oils (IVOs) prepared from palm, cottonseed, and olive oil, respectively, have been studied for application to Frankfurters. Beef fat (10%) was replaced by one of the IVOs at 20, 60 or 100%. Fatty acid composition of the lipid fraction in the Frankfurters and saturated/unsaturated ratios were established for all batches. We found that total or partial replacement of beef fat by IVOs did not lead to significant changes in appearance, colour, texture, flavour or sensory score. We conclude that IVOs may be successfully applied as beef fat substitutes in Frankfurters.