Modulation of stability properties of bovine trypsin after in vitro structural changes with a variety of chemical modifiers

Controlled chemical modification of enzymes, targeting groups not involvedin the active site, can lead to modified catalysts that are intrinsicallymore efficient and resistant to heat and denaturing agents. Bovinepancreatic trypsin was covalently modified up to 75-85% with monomericglutaraldehyde (MGA), polymeric glutaraldehyde (PGA), oxidized sucrose andoxidized sucrose polymers (OSP 70 and OSP 400). Virtually no loss inactivity occurred upon modification. Temperature optima of trypsin shiftsfrom 45-76 degrees C and T50 from 54-76 degrees C for the best modifiedsample made with OSP. The efficiency of the modifiers in stabilization wasranked in the order: OSP 400-T > OSP 70-T > PGA-T > MGA-T >Sucrose-T. Half-life of modified enzymes also followed the same trend. Bothstabilization factor and t1/2 decreased with increasing temperatures. Thefree energy of activation for inactivation delta(deltaG*) varies from 12-20kJ/mol and the activation enthalpy delta(deltaH*) of the modified trypsinby 80-120 kJ/mol indicating stabilization. Inactivation of modified trypsinby urea is less noticeable. The character of the two-step inactivationprocess of trypsin changes with the degree of stabilization in that theduration of phase I one increased noticeably as stabilization increases.Native trypsin fluoresces less intensely showing a red shift under theinfluence of denaturation. Such a fluorescence change is not so obvious forthe modified enzymes indicating conformational stability acquired bymodification.     

Towards modeling gadolinium-lead-borate glasses

Infrared spectra of gadolinium-lead-borate glasses of the xGd₂O₃·(100 − x)[3B₂O₃·PbO] system, where x = 0, 5, 10, 15, 25, 35 and 50 mol.%, have been recorded to explore the role of content of gadolinium ions behaving as glass modifier.The FTIR spectroscopy data for the xGd₂O₃·(1 − x)[3B₂O₃·PbO] glasses show the structural role of lead ions as a network-formers and of the gadolinium ions network modifiers. Adding of the rare earth ion up to 35 mol.% into the glass matrix, the IR bands characteristic to the studied glasses become sharper and more pronounced.Structural changes, as recognized by analyzing band shapes of IR spectra, revealed that Gd₂O₃ causes a change from the continuous borate network to the continuous lead-borate network interconnected through Pb-O-B and B-O-B bridges and the transformation of some tetrahedral [BO₄] units into trigonal [BO₃] units. Then, gadolinium ions have affinity towards [BO₃] structural units which contain non-bridging oxygens necessary for the charge compensation because the more electronegative [BO₃] structural units were implied in the formation of B-O-Gd bonds and the transformation of glass network into a glass ceramic.We propose a possible structural model of building blocks for the formation of continuous random 3B₂O₃·PbO network glass used by density functional theory (DFT) calculations.DFT calculations show that lead atoms occupy three different sites in the proposed model. The first is coordinated with six oxygen atoms forming distorted octahedral geometries. The second lead atom has an octahedral oxygen environment and the five longer Pb-O bonds are considered as participating in the metal coordination scheme. The third lead atom has ionic character. In agreement with the results offered by the experimental FTIR data, the theoretical IR data confirm that our proposed structure is highly possible.     

The improvement of the amorphous environment of the germanate–tellurate glasses in the presence of the gadolinium ions

Glasses in the system xGd₂O₃·(100 − x)[TeO₂·GeO₂] with 0 ≤ x ≤ 50 mol% have been prepared from melt quenching method. In this paper, we investigated changes of the coordination numbers of germanium, tellurium, and gadolinium ions by investigations of FTIR, EPR, and UV–VIS spectroscopy. By analyzing the structural changes resulted from the IR spectra we found that the bending modes of [GeO₄] structural units and the deformed modes of the Te–O–Te linkages produce intercalation of the [GdO _n ] entities in the germanate–tellurate chain network and densification of the glasses by increasing the number of [GeO₆] structural units. EPR spectra of the studied samples reveal that the gadolinium ions play a role of network former. The UV–VIS spectra show broad UV absorption bands located in the 250–350 nm region. Their intensity increase with the increasing of Gd₂O₃ content showing that these stronger transitions can be due to the presence of the O=Ge bonds (n–π* excitations) of [GeO₅] structural units. The [GeO₅] structural units are more stable thermodynamically than their analogues and the [GeO₆] structural units produce the improvement of the amorphous character of these glasses.     

Physiologically based indices of volumetric capnography in patients receiving mechanical ventilation

Several indices of ventilatory heterogeneity can be identified from the expiratory CO2 partial pressure or CO2 elimination versus volume curves. The aims of this study were: 1) to analyse several computerizable indices of volumetric capnography in order to detect ventilatory disturbances; and 2) to establish the relationship between those indices and respiratory system mechanics in subjects with normal lungs and in patients with acute respiratory distress syndrome (ARDS), both receiving mechanical ventilation. We studied six normal subjects and five patients with early ARDS mechanically ventilated at three levels of tidal volume (VT). Respiratory system mechanics were assessed by end-expiratory and end-inspiratory occlusion methods, respectively. We determined Phase III slopes, Fletcher's efficiency index, Bohr's dead space (VD,Bohr/VT), and the ratio of alveolar ejection volume to tidal volume (VAE/VT) from expiratory capnograms, as a function of expired volume. Differences between normal subjects and ARDS patients were significant both for capnographic and mechanical parameters. Changes in VT significantly altered capnographic indices in normal subjects, but failed to change ventilatory mechanics and VAE/VT in ARDS patients. After adjusting for breathing pattern, VAE/VT exhibited the best correlation with the mechanical parameters. In conclusion, volumetric capnography, and, specifically, the ratio of alveolar ejection volume to tidal volume allows evaluation and monitoring of ventilatory disturbances in patients with adult respiratory distress syndrome.     

Interaction of the S phase regulator cdc18 with cyclin-dependent kinase in fission yeast

The fission yeast gene cdc18(+) is required for entry into S phase and for coupling mitosis to the successful completion of S phase. Cdc18 is a highly unstable protein that is expressed only once per cell cycle at the G1/S boundary. Overexpression of Cdc18 causes a mitotic delay and reinitiation of DNA replication, suggesting that the inactivation of Cdc18 plays a role in preventing rereplication within a given cell cycle. In this paper, we present evidence that Cdc18 is associated with active cyclin-dependent kinase in vivo. We have expressed Cdc18 as a glutathione S-transferase fusion in fission yeast and demonstrated that the fusion protein is functional in vivo. We find that the Cdc18 fusion protein copurifies with a kinase activity capable of phosphorylating histone H1 and Cdc18. The activity was identified by a variety of methods as the cyclin-dependent kinase containing the product of the cdc2(+) gene. The amino terminus of Cdc18 is required for association with cyclin-dependent kinase, but the association does not require the consensus cyclin-dependent kinase phosphorylation sites in this region. Additionally, both G1/S and mitotic forms of cyclin-dependent kinase phosphorylate and interact with Cdc18. These interactions between Cdc18 and cyclin-dependent kinases suggest mechanisms by which cyclin-dependent kinases could activate the initiation of DNA replication and could prevent rereplication.     

Behavioral, situational, and temporal effects of treatment of ADHD with methylphenidate

OBJECTIVE: To determine the behavioral, situational, and temporal effects of 4 months of methylphenidate (MPH) treatment for attention-deficit hyperactivity disorder (ADHD). METHOD: Ninety-one children with ADHD were randomly assigned to receive either MPH (titrated to a target dose of 0.7 mg/kg twice a day) or a placebo. Treatment effects were investigated with measures sensitive to various behaviors (core and associated symptoms), situations (home and school), time periods (morning and afternoon, after reaching the target dose, and after 4 months of treatment), and side effects. RESULTS: MPH treatment improved symptoms of ADHD and oppositional behavior at school, both in the morning and afternoon, but not at home. Side effects (increase in physiological and effective symptoms, lack of weight gain) were significantly more frequent with MPH than with placebo treatment. Benefit was evident after titration, but the onset of some side effects was delayed. Side effects were reported by parents but not by teachers. CONCLUSIONS: Positive effects of MPH on behavior are evident in the classroom, but with MPH given twice daily, parents do not report that MPH improves behavior at home. Greater impact on home behavior may require three times daily MPH and combined treatments.