Ex vivo analysis of synergistic anion binding to FbpA in Gram-negative bacteria

Ferric binding protein, FbpA, is a member of the transferrin superfamily whose function is to move an essential nutrient, iron, across the periplasm and into the cytosol through formation of a ternary complex containing Fe (3+) and a synergistic anion, X. Here we utilize SUPREX ( stability of unpurified proteins from rates of H/D exchange) to determine the identification and distribution of the synergistic anion in FeFbpA-X species in periplasmic preparations from Gram-negative bacteria. SUPREX is a mass spectrometry-based technique uniquely suited for thermodynamic analyses of protein-ligand complexes in complex biological mixtures such as periplasmic preparations. Model binary mixtures of FeFbpA-Cit and FeFbpA-PO 4 were initially characterized by SUPREX due to the likely presence of citrate and phosphate ions in the periplasm. Ex vivo SUPREX analyses were performed on FeFbpA-X species overexpressed in an Escherichia coli cell line and on endogenous FeFbpA-X species in Neisseria gonorrheae. Detected in the E. coli periplasmic extract were two distinct populations of FbpA, including one in which the protein was unliganded (i.e., apoFbpA) and one in which the protein was bound to iron and the synergistic anion, phosphate (i.e., FeFbpA-PO 4). FeFbpA-PO 4 was the only population of FbpA molecules detected in the N. gonorrheae periplasmic extract. This work provides the first determination of the identity of the in vivo anion bound to FeFbpA-X in the periplasm and substantiates the hypothesis that the synergistic anion plays a structural and functional role in FbpA-mediated transport of iron across the periplasm and into the cytosol.     

Regional VA, Q, and VA/Q during PLV: effects of nitroprusside and inhaled nitric oxide.

Partial liquid ventilation (PLV) with high-specific-weight perfluorocarbon liquids has been shown to improve oxygenation in acute lung injury, possibly by redistributing perfusion from dependent, injured regions to nondependent, less injured regions of the lung. Our hypothesis was that during PLV in normal lungs, a shift in perfusion away from dependent lung zones might, in part, be due to vasoconstriction that could be reversed by infusing sodium nitroprusside (NTP). In addition, delivering inhaled NO during PLV should improve gas exchange by further redistributing blood flow to well-ventilated lung regions. To examine this, we used a single transverse-slice positron emission tomography camera to image regional ventilation and perfusion at the level of the heart apex in six supine mechanically ventilated sheep during five conditions: control, PLV, PLV + NTP, and PLV + NO at 10 and 80 ppm. We found that PLV shifted perfusion from dependent to middle regions, and the dependent region demonstrated marked hypoventilation. The vertical distribution of perfusion changed little when high-dose intravenous NTP was added during PLV, and inhaled NO tended to shift perfusion toward better ventilated middle regions. We conclude that PLV shifts perfusion to the middle regions of the lung because of the high specific weight of perflubron rather than vasoconstriction.     

Prophage F116: Evidence for Extrachromosomal Location in Pseudomonas aeruginosa Strain PAO

F116 is a temperate-generalized transducing phage of Pseudomonas aeruginosa. Genetic evidence leads to the conclusion that F116 prophage DNA is maintained extrachromosomally as a plasmid. Preliminary physical evidence is presented to support this hypothesis.     

Effect of enzymatic methylation on the chemical,physical, and biological properties of DNA

DNA methylase was partially purified from Escherichia coli W and used to methylate DNA from Bacillus subtilis and bacteriophage φ105. The former DNA was methylated 1.17% and the latter 0.87%. The products were 6-methyladenine (85%) and 5-methylcytosine (15%) in both cases. The methylated DNA was stable toward depurination and viscosity loss at elevated temperatures. Methylation led to a 50% decrease in transforming activity in two strains of B. subtilis and no change in a third strain. The ability of phage φ105 DNA to rescue a defective phage strain was decreased 50% by methylation. No changes were observed in the ability of methylated DNA to serve as a template for DNA polymerase or RNA polymerase. The pattern of cleavage of DNA by a variety of restriction endonucleases was not affected by methylation. There were no changes in the physicochemical properties of DNA on methylation as measured by hyperchromicity on heating, formaldehyde denaturation, viscosity, and sedimentation.     

Voluntary control of cardiovascular reactions to demanding tasks

Three groups of 10 normotensives participated in a study exploring voluntary control of cardiovascular reactions to cognitive tasks. Pulse transit time (TT), interbeat interval (IBI), skin conductance level, and respiratory variables were monitored throughout one introductory and four experimental sessions. The groups were matched on the basis of initial TT and IBI responses to the taxing arithmetic and reasoning tasks. During experimental sessions, one group (FB) was provided with visual analogue TT feedback, while the REL group was given detailed relaxation instructions. A third group (CON) performed the same sequence of tasks but was not instructed to modify cardiovascular reactions at any point. Training in voluntary control was carried out in resting (no-task) conditions, and during task administration. Both treatment groups showed lower cardiovascular reactions than controls in task trials. Only REL subjects lengthened TT in no-task conditions, while both FB and REL groups showed smaller cardiovascular reactions than CON during the tasks. Effects were confined to cardiovascular variables, since respiratory and electrodermal reactions during task administration were similar in all groups. The effects of treatments were especially marked among the most reactive subjects.This work was supported by the Medical Research Council, U.K. We are grateful to Derek Johnston for his comments on an earlier draft of this paper.     

Effect of short- and medium-term toxicity of doxorubicin on spermatogenesis in adult Wistar rats

Doxorubicin (DXR) is a widely used chemotherapeutic anticancer agent that has potent activity against several solid and non-solid human malignant tumors, including childhood malignancies. However, DXR has serious toxic effects on tissues with rapid cell cycles, such as myeloid and lymphatic tissues, intestinal mucosa, testes and ovaries. In the present study, the short- and medium-term toxic effects of DXR on the reproductive system of male Wistar rats were evaluated using morphometric and stereological tools to quantify damage to the seminiferous epithelium. Adult male Wistar rats were treated with dose of 7.5?mg/kg of DXR and were sacrificed at seven, 14, 21 and 28?days after treatment. The testes were fixed in glutaraldehyde solution, routinely processed and embedded in plastic for evaluation under a light microscope. A significant reduction in testis weight was found as a result of massive germ cell apoptosis. Differences in comparison to the control group were found in the relative frequency of all stages of the seminiferous epithelium cycle, with significant differences for stages VIII–XI. Apoptosis significantly decreased the number of pachytene spermatocytes in the stages evaluated (I, II–III and VIII) at seven and 14?days. At 21 and 28?days after treatment, the testes exhibited the massive loss of germ cells that resulted in a missing cell layer. Moreover, reductions in the height of seminiferous tubules, tubular diameter and tubular compartment as well as an increase in the intertubular compartment were found in the period studied.     

Vitamin E up-regulates arachidonic acid release and phospholipase A2 in megakaryocytes

The alteration in calcium transport in the liver nuclei of rats orally administered carbon tetrachloride (CCl4) was investigated. Rats received a single oral administration of CCl₄(5, 10, and 25%, 1.0ml/100 g body weight), and 5, 24 and 48 h later the animals were sacrificed. The administration of CCl₄ (25%) caused a remarkable elevetion of calcium content in the liver tissues and the nuclei of rats. Liver nuclear Ca2+-ATPase activity was markedly decreased by CCl₄ (25%) administration. The presence of dibutyryl cyclic AMP(10^-4 and 10^-3 M) or inositol 1,4,5-trisphosphate (10^-6 and 10^-5 M) in the enzyme reaction mixture caused a significant decrease in Ca²⁺-ATPase activity in the liver nuclei obtained from normal rat, while the enzyme activity was significantly increased by calmodulin (1.0 and 2.0 g/ml). These signaling factor's effects were completely impaired in the liver nuclei obtained from CCl₄ (25%)-administered rats. DNA fragmentation in the liver nuclei obtained from CCl₄ -administered rats was significantly decreased by the presence of EGTA (2 mM) in the reaction mixture, suggesting that the endogenous calcium activates nuclear DNA fragmentation. The present study demonstrates that calcium transport system in the liver nuclei is impaired by liver injury with CCl₄ administration in rats.     

Development of a Paper Strip Test for Detection of Niacin Produced by Mycobacteria

The development of a reagent-impregnated paper strip test for niacin is described. The test system is based on the formation of cyanogen chloride by the reaction of chloramine-T and potassium thiocyanate in the presence of citric acid. Rupture of the pyridine ring of niacin by cyanogen chloride yields gamma-carboxy glutaconic aldehyde and coupling with a primary aromatic amine produces a yellow color. Sensitivity to niacin, both in known solutions and from extracts of 378 clinical mycobacteria isolates, equalled or exceeded that of other methods for detection of niacin. Correlation with other tests for mycobacterial niacin was excellent.