Thermodynamic resolution of cytochrome P450 and characterization of an iron-sulfur center in rat liver microsomal preparations.

Binding of 8-anilinonaphthalene sulfonate (ANS) to glutamate dehydrogenase results in enzyme inhibition and a marked increase in the fluorescence of ANS. Perphenazine and GTP increase the fluorescence of ANS-glutamate dehydrogenase secondary to their known ability to alter the conformation of this enzyme. Aspartate aminotransferases, which form enzyme-enzyme complexes with glutamate dehydrogenase, produce a slight decrease in the fluorescence of ANS-glutamate dehydrogenase.While ANS and perphenazine are allosteric inhibitors of reactions catalyzed by free glutamate dehydrogenase, they do not inhibit reactions catalyzed by aminotransferaseglutamate dehydrogenase complexes. This is in spite of the fact that the aminotransferase does not prevent either ANS or perphenazine from being bound to glutamate dehydrogenase. Therefore, reactions catalyzed by the enzyme-enzyme complex are apparently not inhibited by ANS or perphenazine because binding of the aminotransferase to glutamate dehydrogenase prevents these ligands from altering the conformation of glutamate dehydrogenase. This is consistent with the fact that the aminotransferase also prevents perphenazine from enhancing the fluorescence of ANS-glutamate dehydrogenase.Reactions catalyzed by the enzyme-enzyme complex are inhibited by GTP and the aminotransferase does not prevent GTP from enhancing the fluorescence of ANS-glutamate dehydrogenase. Therefore, binding of the aminotransferase to glutamate dehydrogenase does not prevent GTP from altering the conformation of glutamate dehydrogenase.The fact that the aminotransferase completely prevents perphenazine from increasing the fluorescence of ANS-glutamate dehydrogenase suggests that in the enzymeenzyme complex each glutamate dehydrogenase polypeptide chain can be bound to an aminotransferase polypeptide chain. This would mean that three aminotransferase molecules can be bound to each monomeric unit (M_r 3 × 10⁵) of glutamate dehydrogenase.     

Electron nuclear double resonance (ENDOR) of the Qc.- ubisemiquinone radical in the mitochondrial electron transport chain

We present an electron nuclear double resonance (ENDOR) study of the bound Qc.- ubisemiquinone in the mitochondrial quinol cytochrome c reductase complex. An ENDOR probe specifically modified for insertion into our electron paramagnetic resonance cavity was used for this study. We observed strongly hyperfine-coupled protons whose exchangeable nature indicated they were hydrogen-bonded to the quinone oxygen(s). It is thought that such hydrogen bonds are critical in binding the ubiquinone to protein, in stabilizing its semiquinone form, and in modulating the thermodynamic properties of the bound ubiquinone in the mitochondrial quinol cytochrome c reductase complex. Additional ENDOR features were assigned to protons of the quinone ring itself and to weakly coupled protons that may be associated with nearby amino acids. From very weakly hyperfine-coupled, distant, exchangeable protons there was also ENDOR evidence to suggest proximity and accessibility of the ubiquinone site to the solvent.     

A mitochondrial alkaline/neutral invertase isoform (A/N-InvC) functions in developmental energy-demanding processes in Arabidopsis

Recent findings demonstrate that alkaline/neutral invertases (A/N-Invs), enzymes that catalyze the breakdown of sucrose into glucose and fructose, are essential proteins in plant life. The fact that different isoforms are present in multiple locations makes them candidates for the coordination of metabolic processes. In the present study, we functionally characterized the encoding gene of a novel A/N-Inv (named A/N-InvC) from Arabidopsis, which localizes in mitochondria. A/N-InvC is expressed in roots, in aerial parts (shoots and leaves) and flowers. A detailed phenotypic analysis of knockout mutant plants (invc) reveals an impaired growth phenotype. Shoot growth was severely reduced, but root development was not affected as reported for A/N-InvA mutant (inva) plants. Remarkably, germination and flowering, two energy demanding processes, were the most affected stages. The effect of exogenous growth regulators led us to suggest that A/N-InvC may be modulating hormone balance in relation to the radicle emergence. We also show that oxygen consumption is reduced in inva and invc in comparison with wild-type plants, indicating that both organelle isoenzymes may play a fundamental role in mitochondrion functionality. Taken together, our results emphasize the involvement of mitochondrial A/N-Invs in developmental processes and uncover the possibility of playing different roles for the two isoforms located in the organelle.     

Royal College of Physicians and Surgeons of Canada: annual meeting draws over 2000 physicians

In a review of 19 years'' experience with inhalation of foreign bodies by children the 33 patients (mean age 28 months) were found to have presented most frequently with wheezing or coughing, or both, of recent onset, and to have decreased air entry, rhonchi or respiratory stridor, or a combination of these signs. Eighteen children had inhaled a nut, a pea or a bean. The other 15 had inhaled various organic and inorganic objects. All the children underwent bronchoscopy, and the foreign body was completely removed in 19 during the first procedure; the remainder required repeated bronchoscopy or direct surgical removal of the foreign body, or both. Permanent disability or death was not encountered. The findingsof the study indicate that early bronchoscopic removal is the preferred treatment when a child inhales a foreign body.     

Novel fluorescent triazinobenzimidazole derivatives as probes for labelling human A1 and A2B adenosine receptor subtypes

The expression levels and the subcellular localization of adenosine receptors (ARs) are affected in several pathological conditions as a consequence of changes in adenosine release and metabolism. In this respect, labelled probes able to monitor the AR expression could be a useful tool to investigate different pathological conditions. Herein, novel ligands for ARs, bearing the fluorescent 7-nitrobenzofurazan (NBD) group linked to the N¹ (1,2) or N¹⁰ (3,4) nitrogen of a triazinobenzimidazole scaffold, were synthesized. The compounds were biologically evaluated as fluorescent probes for labelling A₁ and A_2B AR subtypes in bone marrow-derived mesenchymal stem cells (BM-MSCs) that express both receptor subtypes. The binding affinity of the synthetized compounds towards the different AR subtypes was determined. The probe 3 revealed a higher affinity to A₁ and A_2B ARs, showing interesting spectroscopic properties, and it was selected as the most suitable candidate to label both AR subtypes in undifferentiated MSCs.Fluorescence confocal microscopy showed that compound 3 significantly labelled ARs on cell membranes and the fluorescence signal was decreased by the cell pre-incubation with the A₁ AR and A_2B AR selective agonists, R-PIA and BAY 60-6583, respectively, thus confirming the specificity of the obtained signal. In conclusion, compound 3 could represent a useful tool to investigate the expression pattern of both A₁ and A_2B ARs in different pathological and physiological processes. Furthermore, these results provide an important basis for the design of new and more selective derivatives able to monitor the expression and localization of each different ARs in several tissues and living cells.     

Heat shock proteins in <Emphasis Type="Italic">Varroa destructor</Emphasis> exposed to heat stress and in-hive acaricides

Varroa destructor is one of the major pests that affect honeybees around the world. Chemical treatments are common to control varroosis, but mites possess biochemical adaptive mechanisms to resist these treatments, enabling them to survive. So far, no information is available regarding whether these pesticides can induce the expression of heat shock protein (Hsp) as a common protective mechanism against tissue damage. The aims of this study were to determine differences in heat shock tolerance between mites collected from brood combs and phoretic ones, and to examine patterns of protein expression of Hsp70 that occur in various populations of V. destructor after exposure to acaricides commonly employed in beekeeping, such as flumethrin, tau-fluvalinate and coumaphos. Curiously, mites obtained from brood cells were alive at 40 °C, unlike phoretic mites that reached 100% mortality, demonstrating differential thermo-tolerance. Heat treatment induced Hsp70 in mites 4?×?more than in control mites and no differences in response were observed in phoretic versus cell-brood-obtained mites. Dose–response assays were carried out at increasing acaricide concentrations. Each population showed a different stress response to acaricides despite belonging to the same geographic region. In one of them, coumaphos acted as a hormetic stressor. Pyrethroids also induced Hsp70, but mite population seemed sensitive to this treatment. We concluded that Hsp70 could represent a robust biomarker for measuring exposure of V. destructor to thermal and chemical stress, depending on the acaricide class and interpopulation variability. This is relevant because it is the first time that stress response is analyzed in this biological model, providing new insight in host-parasite-xenobiotic interaction.     

Physical and Kinetic Evidence for an Association between Sucrose-Phosphate Synthase and Sucrose-Phosphate Phosphatase

The possible formation of a multienzyme complex between sucrose (Suc)-phosphate synthase (SPS) and Suc-phosphate phosphatase (SPP) was examined by measuring the rates of Suc-6-phosphate (Suc-6-P) synthesis and hydrolysis in mixing experiments with partially purified enzymes from spinach (Spinacia oleracea) and rice (Oryza sativa) leaves. The addition of SPP to SPS stimulated the rate of Suc-6-P synthesis. SPS inhibited the hydrolysis of exogenous Suc-6-P by SPP when added in the absence of its substrate (i.e. UDP-glucose) but stimulated SPP activity when the SPS substrates were present and used to generate Suc-6-P directly in the reaction. Results from isotope-dilution experiments suggest that Suc-6-P was channeled between SPS and SPP. A portion of the SPS activity comigrated with SPP during native polyacrylamide gel electrophoresis, providing physical evidence for an enzyme-enzyme interaction. Taken together, these results strongly suggest that SPS and SPP associate to form a multienzyme complex.     

Elastic moduli of excised constricted rat lungs

When airways constrict, the surrounding parenchyma undergoesstretch and distortion. Because of the mechanical interdependence between airways and parenchyma, the material properties of the parenchyma are important factors that modulate the degree ofbronchoconstriction. The purpose of this study was to investigate theeffect of changes in transpulmonary pressure (Ptp) and inducedconstriction on parenchymal bulk (k)and shear (µ) moduli. In excised rat lungs, pressure was measured atthe airway opening, and pressure-volume curves were obtained byimposing step decreases in volume with a calibrated syringe from totallung inflation. Calculation was made ofk during small-volume oscillations (1 Hz). Absolute lung volume at 0 cmH₂O Ptp was obtained bysaline displacement. To calculate µ, a lung-indentation test wasperformed. The lung surface was deformed with a cylindrical punch(diameter = 0.45 cm) in 0.25-mm increments, and the force required toeffect this displacement was measured by a weight balance. Measurementsof k and µ were obtained at 4 and 10 cmH₂O Ptp, and again at 4 cmH₂O Ptp, after delivery ofmethacholine aerosol (100 mg/ml) into the trachea. Values ofk and µ in rat lungs were similar tothose reported in other species. In addition, k and µ were dependent on Ptp. Afterinduced constriction, k and µ increased significantly. That k and µ can increase after induced constriction has important implicationsvis a vis the factors modulating airway narrowing.