Meningococcal meningitis with Arnold-Chiari malformation.
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Acute Disease↗
Biomedical subjects
R M Jackson
Publications and source records attributed to R M Jackson.
Epidemiological typing of Klebsiella pneumoniae by pyrolysis mass spectrometry.
Thirteen isolates of ceftazidime-resistant Klebsiella pneumoniae from a suspected cross-infection outbreak involving patients on an intensive care unit and a haematology ward were examined in pyrolysis-mass spectrometry (Py-MS), along with eight concurrent non-outbreak-associated clinical isolates of klebsiellae as controls. Py-MS showed tight clustering of the suspected outbreak isolates, suggesting cross-infection with a single strain. Non-outbreak isolates were clearly distinct from one another and from the outbreak strain. The results confirm that Py-MS is a powerful tool for rapid strain comparison in investigations of cross-infection incidents.
Bacterial Typing Techniques↗
Effects of hypoxia and hyperoxia on lung prostaglandin E1 metabolism.
Exposure to hypoxia (10% O2 for 5 to 7 days) results in increased survival and decreased pulmonary toxicity of adult rats subsequently exposed to hyperoxia (> 97% O2). These experiments tested whether hypoxia preexposure minimized the decrease in lung metabolism of prostaglandin E1 (PGE1), a vasoactive and antiinflammatory prostaglandin, caused by hyperoxia. Transpulmonary PGE1 clearance was measured as fractional metabolism of PGE1 (2 microM to 30 microM) infused during a 45-second period in an isolated, buffer-perfused rat lung preparation after exposure of rats to one of the following conditions: (1) hyperoxia (> 97% O2 for 48 hours), (2) hypoxia (10% O2 for 120 hours), or (3) hypoxia followed by hyperoxia. Hyperoxia exposure decreased both lung PGE1 metabolism and lung prostaglandin dehydrogenase activity (PGDH). Hypoxia also decreased lung PGE1 metabolism but, in contrast, increased lung PGDH activity. Hypoxia preexposure did not prevent the depression of PGE1 metabolism or PGDH activity caused by hyperoxia, which indicates that survival in hyperoxia did not depend on lung PGE1 metabolism. Hypoxia itself impaired transpulmonary metabolism of PGE1 despite increasing PGDH activity, which suggests possible interference with substrate delivery.
Alprostadil↗
Biophysical and molecular properties of amiloride-inhibitable Na+ channels in alveolar epithelial cells.
The recent immunopurification and cloning of various lung Na+ channel proteins has provided the necessary tools to study Na+ transport at a fundamental level across a number of epithelial tissues. Various macroscopic measurements of Na+ transport have shown that Na+ ions enter the cytoplasm of alveolar cells mainly through amiloride-inhibitable Na+ channels. Molecular biology studies have shown the existence of three Na+ channel subunit mRNAs (alpha-, beta-, and gamma-rENaC) in mature fetal (FDLE) and adult alveolar type II (ATII) cells. Patch-clamp studies have demonstrated the existence of various types of amiloride-inhibitable Na+ channels, located in the apical membranes of FDLE and ATII cells. beta-Agonists and agents that enhance intracellular adenosine 3',5'-cyclic monophosphate levels increase the open probability of these channels, leading to increased Na+ transport across the alveolar epithelium in vivo. Immunopurification of a putative channel protein from adult ATII cells showed that it contains an amiloride-binding subunit with a molecular mass of 150 kDa. When this protein was reconstituted in planar lipid bilayers, it exhibited single channels with a conductance of 25 pS, which were moderately selective for Na+ over K+. The open probability of these channels was increased by the addition of protein kinase A (PKA) and ATP, and was decreased to the same extent by addition of [N-ethyl-N-isopropyl]-2'-4'-amiloride (EIPA) and amiloride (1 microM each) in the apical side of the bilayer, in agreement with the results of patch-clamp studies in ATII cells. Exposure of rats to sublethal hyperoxia increased alpha-rENaC mRNA and the functional expression of Na+ channels in alveolar epithelial cells and limited alveolar edema. These findings indicate that alveolar epithelial channels contain at least one family of amiloride-sensitive Na+ channel proteins, which displays a number of unique properties, including sensitivity to EIPA.
Amiloride↗
Effects of hypoxia on expression of superoxide dismutases in cultured ATII cells and lung fibroblasts.
This study investigated whether hypoxia affected the expression of mitochondrial manganese-containing superoxide dismutase (Mn-SOD) and the cytosolic copper and zinc-containing superoxide dismutase (Cu,Zn-SOD) in alveolar type II epithelial (ATII) cells and lung fibroblasts. Cells were exposed in vitro to air (controls) or to 2.5% oxygen (hypoxia) for 24 h. Mn-SOD and Cu,Zn-SOD mRNA expression was measured by quantitative reverse transcriptase-polymerase chain reaction. Both Mn-SOD and Cu,Zn-SOD mRNA expression in ATII cells decreased significantly after 1 day in hypoxic conditions. The decrease in Mn-SOD mRNA (-69%) was greater than that in Cu,Zn-SOD mRNA (-48%). ATII cell surfactant protein A transcript expression remained constant. Mn-SOD (-52%) and Cu,Zn-SOD (-54%) mRNA expression decreased similarly in lung fibroblasts cultured during hypoxia. The half-life of the Mn-SOD mRNA measured in lung fibroblasts exposed to air or hypoxia for 24 h decreased significantly from 5.8 +/- 0.1 to 3.8 +/- 0.7 h (-34%). The half-life for the Cu,Zn-SOD decreased significantly from 4.0 +/- 0.3 to 2.4 +/- 0.1 h (-40%). Neither Mn-SOD nor Cu,Zn-SOD protein expression in ATII cells changed significantly during hypoxia. Hypoxia decreases expression of Mn-SOD and Cu,Zn-SOD mRNA in ATII cells and lung fibroblasts in part by decreasing stability of the mRNA transcripts.
Animals↗
Increased expression and activity of sodium channels in alveolar type II cells of hyperoxic rats.
We investigated the cellular and molecular events associated with the increase in sodium transport across the alveolar epithelium of rats exposed to hyperoxia (85% O2 for 7 days followed by 100% O2 for 4 days). Alveolar type II (ATII) cell RNA was isolated and probed with a cDNA for one of the rat colonic epithelial sodium channel subunits (alpha rENaC). The alpha rENaC mRNA (3.7-kb transcript) increased 3-fold in ATII cell RNA isolated from rats exposed to 85% O2 for 7 days and 6-fold after 4 days of subsequent exposure to 100% O2. In situ hybridization revealed increased expression of alpha rENaC mRNA transcripts in both airway and alveolar epithelial cells of hyperoxic rats. When immunostained with a polyclonal antibody to kidney sodium channel protein, ATII cells from hyperoxic rats exhibited a significant increase in the amount of immunogenic protein present in both the plasma membrane and the cytoplasm. When patched in the whole-cell mode, ATII cells from hyperoxic rats exhibited amiloride and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride (EIPA)-sensitive currents that were 100% higher compared with those obtained from air-breathing rats. Single-channel sodium currents (mean conductance of 25 pS) were seen in ATII cells patched in both the inside-out and cell-attached modes. The number and open probability of these channels increased significantly during exposure to hyperoxia. Exposure to sublethal hyperoxia up-regulated both alpha rENaC mRNA and the functional expression of sodium channels in ATII cells.
Animals↗
A continuum model for protein-protein interactions: application to the docking problem.
The prediction of protein-protein interactions in solution is a major goal of theoretical structural biology. Here, we implement a continuum description of the thermodynamic processes involved. The model differs considerably from previous models in its use of "molecular surface" area to describe the hydrophobic component to the free energy of conformational change in solution. We have applied this model to a data set of alternative docked conformations of protein-protein complexes which were generated independently of this work. It was found previously that commonly used energy evaluation techniques fail to distinguish between near-native and certain non-native complexes in this data set. Here, we found that an energy function that takes into account (1) total electrostatic free energy, (2) hydrophobic free energy and (3) loss in side-chain conformational energy was able to reliably discriminate between near-native and non-native configurations but only when molecular surface is used as a descriptor of the hydrophobic effect. It is shown that the molecular surface and the more conventional surface descriptor "solvent accessible surface" give very different quantitative measures of hydrophobicity. In terms of the contribution of different energy components to the free energy of complex formation it was found that loss in side-chain conformational entropy is a second order effect. Electrostatic interaction energy (which is commonly used to score docked conformations) was a poor indicator of complementarity when starting from unbound conformations. It was found that electrostatic desolvation energy and the hydrophobic contribution (based on a molecular surface area descriptor) are much less sensitive to local fluctuations in atomic structure than point-to-point interaction energies and thus may be more suited for use as a scoring function when docking unbound conformations, where atomic complementarity is much less apparent. Whilst a combined energy function was able to distinguish near-native from non-native conformations in the six systems studied here, it remains to be determined to what extent more sizeable conformational changes would influence the results.
Algorithms↗
Effects of hypoxia on MnSOD expression in mouse lung.
Mitochondrial manganese-containing SOD (MnSOD) is located at the primary site of O2 metabolism, and its expression may be regulated by changes in O2 level. We hypothesized that lung MnSOD expression and promoter activity would decrease in response to hypoxia. We tested effects of hypoxia (10% O2 at sea level for 7 days) on chloramphenicol acetyltransferase (CAT) reporter and MnSOD gene expression in transgenic mice. The transgene consisted of a 3.3-kb portion of the rat MnSOD gene 5' flanking region coupled to a CAT reporter gene. Lung MnSOD activity in male (but not female) mice decreased significantly after hypoxia exposure. The decrease in MnSOD enzymatic activity in male mice was specific. Neither total SOD nor glucose-6-phosphate dehydrogenase (G-6-PDH) activity decreased significantly in hypoxia. CAT protein expression decreased in transgenic males exposed to hypoxia, while CAT protein expression in hypoxic transgenic females remained comparable with controls. The mRNA for both the native MnSOD and the MnSOD-CAT reporter genes remained constant after hypoxia, as did CuZnSOD and G-6-PDH mRNAs.
Animals↗
Application of scaled particle theory to model the hydrophobic effect: implications for molecular association and protein stability.
The energetics of alkane dissolution and partition between water and organic solvent are described in terms of the energy of cavity formation and solute-solvent interaction using scaled particle theory. Thermodynamic arguments are proposed that allow comparison of experimental measurements of the surface area with values calculated from an all-atom representation of the solute. While the surface tension relating to the accessible surface is shape dependent, it is found that for the molecular surface it is not. This model rationalizes the change in surface tension between the microscopic (20-30 cal/mol/A2) and macroscopic (70-75 cal/mol/A2) regimes without the need to invoke Flory-Huggins theory or to apply other corrections. The difference in the values arises (i) to a small extent as a result of the curvature dependence of surface tension and (ii) to a large extent due to the difference in the molecular surface derived from the experiment and that calculated from an extended all-atom model. The model suggests that the primary driving force for alkane association in water is due to the tendency of water to reduce the solute cavity surface. It is argued that to model the energetics of alkane association, the surface tension should be related to the molecular surface (rather than the accessible surface) with a surface tension near the macroscopic limit for water. This model is compared with results from theoretical simulations of the hydrophobic effect for two well-studied systems. The implications for antibody-antigen interactions and the effect of hydrophobic amino acid deletion on protein stability are discussed. The approach can be used to model the solute cavity formation energy in solution as a first step in the continuum modelling of biomolecular interactions.
Antigen-Antibody Reactions↗
Hydrogen peroxide release by mitochondria from normal and hypoxic lungs.
Ischemia/reperfusion mechanisms contribute to lung injury after transplantation, pulmonary embolism, and resolution of atelectasis. Alveolar tissue becomes hypoxic and deprived of substrate only when both ventilation and perfusion are interrupted, a situation modeled in vivo by complete, unilateral lung collapse. Because previously hypoxic mitochondria may be an important intracellular source of superoxide and hydrogen peroxide (H2O2) during reperfusion and re-oxygenation, the authors, in this study, investigated whether mitochondrial H2O2 release changed as a result of lung hypoxia/hypoperfusion resulting from collapse. Mitochondria were isolated from hypoxic (previously collapsed) right or contralateral left rabbits' lungs and from control rabbits' lungs. Mitochondrial H2O2 release, a marker of superoxide production, was measured fluorometrically after incubation with or without 1 mmol/L cyanide and 0.1 mmol/L nicotinamide adenine dinucleotide. Mitochondrial recovery was determined by assaying succinate dehydrogenase activity in mitochondrial preparations and lung homogenates. Lung succinate dehydrogenase activity and mitochondrial recovery were comparable among groups. Calculated lung mitochondrial content did not change (control subjects: left 7.9 +/- 0.5, right 13.8 +/- 1.7; hypoxic: left 10.3 +/- 1.3, right 10.5 +/- 2.4, all mg mitochondrial protein/lung). Mitochondria released hydrogen peroxide at approximately 5.6 nmol/h/mg pro in buffer alone and 14.8 nmol/h/mg pro in buffer with cyanide and nicotinamide adenine dinucleotide. However, lung collapse and resulting hypoxia caused no change in mitochondrial number or capacity to release H2O2 in vitro. Based on these findings, it is suggested that other sources of reactive oxygen metabolites, including xanthine oxidase and activated neutrophils, contribute to the oxidant injury observed in this model.
Animals↗
Manganese superoxide dismutase expression in alveolar type II epithelial cells from nonventilated and hypoperfused lungs.
Lungs that have been hypoxic and hypoperfused because of atelectasis and the resulting decrease in pulmonary arterial blood flow develop specific decreases in manganese superoxide dismutase (MnSOD) activity and are sensitive to oxidant injury during reoxygenation. Since the MnSOD protein is concentrated in mitochondria of alveolar epithelial type II cells (ATII), we hypothesized that expression of MnSOD would be decreased in these cells also as a result of hypoxia. To investigate whether regulation of MnSOD expression occurred before or after transcription, we determined whether MnSOD protein content or steady-state mRNA level changed after hypoxia as well. ATII cells were isolated by elastase digestion from lungs of adult rabbits after right lungs had been hypoxic and hypoperfused for 7 days because of unilateral atelectasis. MnSOD activity was measured by inhibition of cytochrome c reduction in the presence of 1 mM KCN, MnSOD protein content was measured on immunoblots, and MnSOD mRNA was quantified on slot blot autoradiograms. MnSOD activity was 8.4 +/- 1.9 U/mg protein in ATII cells from control lungs and 6.8 +/- 1.5 U/mg protein in ATII cells from hypoxic and hypoperfused lungs (n = 9, P = 0.037). MnSOD protein content was 5.1 +/- 1.4 micrograms/mg protein in ATII cells from control and 4.1 +/- 1.2 micrograms/mg protein in ATII cells from hypoxic and hypoperfused lungs (P = 0.021). ATII cell MnSOD mRNA/18S ribosomal RNA (ratio of arbitrary absorbance units) determined by RNA slot blots was 2.18 +/- 1.26 in ATII cells from control lungs and 2.94 +/- 0.88 in ATII cells from hypoxic lungs (n = 7, P > 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
MnSOD protein content changes in hypoxic/hypoperfused lung tissue.
Previous studies using an in vivo rabbit model in which lung tissue hypoxia/hypoperfusion was created by unilateral lung collapse for 7 days demonstrated a decrease in MnSOD activity in previously hypoxic/hypoperfused lungs. In the present study, we determined whether tissue hypoxia/hypoperfusion decreased MnSOD protein concentration or mRNA expression in the lung as well, changes that would suggest pretranslational regulation of enzyme activity. Expression of MnSOD may be critical in determining the degree of tissue injury during re-oxygenation because the mitochondrial electron transport system produces reactive oxygen species (ROS) both during hypoxia and re-oxygenation. We purified MnSOD protein from rabbit livers to a specific activity of approximately 3,500 U/mg protein and found the amino terminal sequence nearly identical to those of the rat and human MnSOD proteins. Lung MnSOD protein content was quantitated by immunoassay, and MnSOD mRNA content was determined by slot blotting. Results from five control and six experimental rabbits, the right lungs of which had been hypoxic/hypoperfused because of collapse for 7 days, demonstrated a 32% decrease (P < 0.03) in MnSOD protein content (42 +/- 8 micrograms/mg DNA in hypoxic lungs compared with 61 +/- 3 micrograms/mg DNA in contralateral lungs) that was not due to decreased numbers of mitochondria. Lung succinate dehydrogenase activity, a mitochondrial marker, did not change in hypoxic/hypoperfused lungs. The mRNA for MnSOD did not change relative to B-actin mRNA in lungs that had been hypoxic and hypoperfused for 7 days.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Construction of a stable dimer of Bacillus stearothermophilus lactate dehydrogenase.
A molecular graphics analysis of the features which prevent cytosolic malate dehydrogenase dimers from forming tetramers was evaluated by its success in predicting the synthesis of a version of the LDH framework which is a stable dimer. Surface residues responsible for malate dehydrogenases being dimers were revealed by superimposing the structures of two dimers of pig cytosolic malate dehydrogenase on one homologous tetramer of L-lactate dehydrogenase from Bacillus stearothermophilus. Four regions were identified as composing the P-axis dimer-dimer interface. Two regions of the dimer were surface loops that collided when built as a tetramer: a large loop (residues 203-207, KNOBI) and a small loop (residues 264-269, KNOBII), and these were candidates to explain the dimeric character of malate dehydrogenase. The analysis was tested by constructing a synthetic B. stearothermophilus lactate dehydrogenase (KNOBI) containing the large malate dehydrogenase loop (residues 203-207 being AYIKLQAKE, and extra four amino acids). The new construct was thermotolerant (90 degrees C) and enzymically active with kcat and KM (pyruvate) values similar to those of the wild-type enzyme. However, whereas the allosteric activator fructose 1,6-bisphosphate decreased KM 100 times for wild type, it had no influence on KNOBI. The molecular volumes of 1-120 microM concentrations of the construct were measured by time-resolved decay of tryptophan fluorescence anisotropy and by gel filtration. Both methods showed the molecular weight of wild type increased from dimer to tetramer with Kd about 20 microM dimer. KNOBI remained a dimer under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Genetic evidence for an androgen-regulated epididymal secretory glutathione peroxidase whose transcript does not contain a selenocysteine codon.
Epididymal glutathione peroxidase (GPX) has been suggested as a major factor in combating loss of fertility of spermatozoa due to lipid peroxidation. We report here the isolation and sequence of putative GPX cDNAs from rat (Rattus rattus) and cynomolgus-monkey (Macaca fascicularis) epididymis, which exhibit marked sequence identity with known GPXs. In both species the cDNAs encode predicted preproteins containing 221 amino acid residues. Unlike other characterized GPX sequences, epididymal GPX mRNA does not contain a selenocysteine codon (UGA). However, sequence comparison and molecular-modelling studies suggest a high degree of structural conservation between epididymal and other GPXs. Transcripts corresponding to epididymal GPX are not detected in a variety of other tissues (liver, spleen, kidney and testis) and appear to be androgen-regulated in the epididymis.
Amino Acid Sequence↗
Charge balance in the alpha-hydroxyacid dehydrogenase vacuole: an acid test.
The proposal that the active site vacuole of NAD(+)-S-lactate dehydrogenase is unable to accommodate any imbalance in electrostatic charge was tested by genetically manipulating the cDNA coding for human muscle lactate dehydrogenase to make a protein with an aspartic acid introduced at position 140 instead of the wild-type asparagine. The Asn 140-Asp mutant enzyme has the same kcat as the wild type (Asn 140) at low pH (4.5), and at higher pH the Km for pyruvate increases 10-fold for each unit increase in pH up to pH 9. We conclude that the anion of Asp 140 is completely inactive and that it binds pyruvate with a Km that is over 1,000 times that of the Km of the neutral, protonated aspartic-140. Experimental results and molecular modeling studies indicate the pKa of the active site histidine-195 in the enzyme-NADH complex is raised to greater than 10 by the presence of the anion at position 140. Energy minimization and molecular dynamics studies over 36 ps suggest that the anion at position 140 promotes the opening of and the entry of mobile solvent beneath the polypeptide loop (98-110), which normally seals off the internal active site vacuole from external bulk solvent.
Amino Acid Sequence↗
A prediction of the three-dimensional structure of maize NADP(+)-dependent malate dehydrogenase which explains aspects of light-dependent regulation unique to plant enzymes.
A model has been built for the plant NADP-malate dehydrogenase from Zea mays, a key enzyme in photosynthesis, which undergoes light-dependent regulation. The model was based on sequence and presumed structural homology to the known three-dimensional structure of mammalian porcine cytosolic NAD-malate dehydrogenase. A cystine-loop present in an extended C-terminal region of plant NADP-malate dehydrogenases was modelled using molecular mechanics and computer graphical methods, based on the assumption that a disulphide bridge exists in the inactive form of the enzyme between Cys351 and Cys363. The predicted conformation of the intact C-terminal cystine-loop suggests that the extended polypeptide will bind in the active centre and inhibit enzyme activity. Another ionizable cysteine residue in the active site is predicted to control the charge of the catalytic His215 and might be responsible for the uniquely tight binding of the positively charged nicotinamide ring of NADP+ in this and other C4 and C3 plant NADP-malate dehydrogenases.
Amino Acid Sequence↗
Endogenous and exogenous catalase in reoxygenation lung injury.
Reexpansion pulmonary edema parallels reperfusion (reoxygenation) injuries in other organs in that hypoxic and hypoperfused lung tissue develops increased vascular permeability and neutrophil infiltration after reexpansion. This study investigated endogenous lung catalase activity and H2O2 production during hypoxia (produced by lung collapse) and after reoxygenation (resulting from reexpansion), in addition to assessing the effects of exogenous catalase infusion on the development of unilateral pulmonary edema after reexpansion. Lung collapse resulted in a progressive increase in endogenous catalase activity after 3 (14%) and 7 days (23%), while activities in contralateral left lungs did not change (normal left lungs averaged 180 +/- 11 units/mg DNA). Tissue from control left lungs released H2O2 into the extracellular medium at a rate calculated to be 242 +/- 34 nmol.h-1.lung-1. No significant change in extracellular release of H2O2 occurred after 7 days of right lung collapse. However, after reexpansion of the previously collapsed right lungs for 2 h, H2O2 release from both reexpanded right and contralateral left lungs significantly increased (88 and 60%, respectively) compared with controls. Infusion of exogenous catalase significantly increased plasma and lung catalase activities. Exogenous catalase infusion prevented neither the increase in lung permeability nor the infiltration with neutrophils that typically occurs in reexpanded lungs. These data indicate that lung hypoxia/reoxygenation, induced by sequential collapse and reexpansion, has specific effects on endogenous lung catalase activity and H2O2 release. However, exogenous catalase does not prevent reexpansion pulmonary edema, eliminating extracellular (but not intracellular) H2O2 as an important mediator of unilateral lung injury in this model.
Animals↗
Activity and specificity of human aldolases.
The structure of the type I fructose 1,6-bisphosphate aldolase from human muscle has been extended from 3 A to 2 A resolution. The improvement in the resulting electron density map is such that the 20 or so C-terminal residues, known to be associated with activity and isozyme specificity, have been located. The side-chain of the Schiff's base-forming lysine 229 is located towards the centre of an eight-stranded beta-barrel type structure. The C-terminal "tail" extends from the rim of the beta-barrel towards lysine 229, thus forming part of the active site of the enzyme. This structural arrangement appears to explain the difference in activity and specificity of the three tissue-specific human aldolases and helps with our understanding of the type I aldolase reaction mechanism.
Amino Acid Sequence↗