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.     

Development of an Enzymic Time Temperature Integrator for Sterilization Processes Based on Bacillus licheniformis α-amylase at Reduced Water Content

ABSTRACT: The thermal stability of Bacillus licheniformis α-amylase at low moisture content was studied, based on isothermal experiments performed in a temperature range 113 to 125 °C. The thermal inactivation was monitored by measuring the decrease in thermal denaturation enthalpy and/or the decrease in enzymic activity on pnitrophenyl-α-D-maltoheptaoside, or on starch as a substrate. Based on enthalpy readings, an enzymic system with a z-value of 10.4 °C was observed when using a relative humidity of 81% at 4 °C. A theoretical study showed that this system could be used as a Time Temperature Integrator (TTI) to monitor the safety of sterilization processes of numerous food products.     

Formation of linear aldehydes during surface water preozonization and their removal in water treatment in relation to mutagenic activity and sum parameters

Low molecular weight aldehydes were formed during surface water preozonization, their levels showing a positive correlation with increasing ozone dose applied and with increasing water temperature. A strong negative correlation was observed between aldehyde levels and U.V. absorbance at 254 nm.Coagulation had no influence on the aldehydes present and the influence of rapid double layer filtration varied strongly with temperature : significant removals were only observed above 10°C.Mutagenic activity generated by preozonization in Salmonella typhimurium TA98 shows an ozone dose depending relationship different from the formation of linear aldehydes. Its removal by coagulation is not effective but rapid double layer filtration reduces mutagenic activity to marginal levels.In this respect too no clear parallel can be drawn between the presence of low molecular weight aldehydes and mutagenic activity.     

Quality of Crude Fish Oil Extracted from Herring Byproducts of Varying States of Freshness

ABSTRACT: Herring byproducts were stored at 2 and 15 °C for up to 72 h. Over time, significant increases of total volatile bases (TVB), histamine, putrescine, cadaverine, and tyramine were detected. However, only tyramine and TVB levels were temperature-dependent. The level of total polyunsaturated fatty acids (PUFAs) was constant. Longer byproducts storage gave rise to an oil with higher levels of free fatty acids, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and total PUFAs, while fluorescent compounds were lower. A higher storage temperature led to oil higher in α-tocopherol and EPA levels and lower in anisidine value. Surprisingly, the oil with the highest content of PUFAs was not produced from the freshest byproducts, and oil with low oxidation products can be extracted from stored byproducts.     

Instructional texts rewritten by five expert teams: Revisions and retention improvements.

700 undergraduates were tested in 3 experiments on original or rewritten versions of 52 instructional texts about Army job tasks, general science, philosophy, and history. Five experts had rewritten various sets of the texts and stated hypotheses about the efficacious features of their revisions. We tested their hypotheses and several others. Recall and recognition tests were given immediately and after a 24-hr delay. Results showed that revisions made by 3 of the 5 experts improved retention of text information. The kind and number of revisions and improvements varied across the text sets. Most expert hypotheses were not supported, and they made many revisions they were declaratively unaware of. Some of our hypotheses about the revision features were supported, but different features were effective for different sets of texts. It was concluded that some experts have effective knowledge about improving instructional text, but it exists primarily in procedural form. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Effect of orienting fields on agglomeration in dilute dispersions of magnetic fine particles

Static light scattering was used to investigate dilute dispersions of fine gamma -Fe/sub 2/O/sub 3/ magnetic particles. The particles were acicular and on average 0.39 mu m long and 0.056 mu m in diameter. They were dispersed in organic solvents at concentrations of approximately 10/sup -4/ vol%, corresponding to an average interparticle separation of 9 mu m. Computer programs using a coupled dipole algorithm to predict the scattering from the particles were written. Experimental results do not agree with computer calculations of the light scattering from single particles, but are consistent with scattering from thick ropelike agglomerates of particles. The size of these agglomerates was estimated after 10 s to be less than 100 particles, while the thickness of the ropes is at least 5 particles.<>     

Hydrogen adsorption studies in micro-size cobalt dots

Hydrogen desorption curves were obtained from a sample composed of a square arrangement of Co dots with average diameter of 4.4 μm, separated by a distance of 11.6 μm. A macroscopic sample of Co dots grown on a 2.5 × 2.5 cm Si substrate was made by standard lithographic techniques and used in these experiments. Thermal programmed desorption (TPD) was performed under ultra-high vacuum conditions. Hydrogen TPD curves were obtained from a 1 × 1 cm Co dots samples displaying a maximum of intensity at 425 K. Hydrogen TPD curve was also obtained from 1 cm× 1 cm samples of Co films and Co foils for comparison. The hydrogen TPD curves have decreasing intensity from the Co foils to the Co dots and finally to the Co films. This indicates that there are more sites for hydrogen adsorption on the Co dots than in the Co films. This is a surprising result because there is approximately 8.7 times less Co atoms exposed in the Co dots that in the Co film sample. A desorption energy of 27 kcal/mol was obtained for the Co dots suggesting that hydrogen is adsorbed on an hcp hollow site of the Co dot crystalline structure.