Recent developments in in vivo neutron activation analysis facilities

Two new facilities for in vivo activation analysis of patients have been designed, developed, and constructed at Toronto General Hospital. One of these is for the determination of body calcium for the diagnosis of osteoporosis and other diseases associated with bone loss. The other is for the measurement of total body nitrogen for the determination of protein status. These facilities replace old university facilities and take into account the comfort and management of patients. In addition, in the case of the calcium facility, the precision of the measurements has been improved because of larger detector volume and increased neutron source strength. Both the facilities are now in routine hospital clinical use.     

Photosynthetic induction in wheat protoplasts and chloroplasts. Autocatalysis and light activation of enzymes

Abstract Unlike wheat chloroplasts, wheat protoplasts showed a pronounced restoration of the induction phase after a short period of darkness. This difference was used to investigate the relative roles of light-induced reductive activation of enzymes and the auto-catalytic increase in the level of substrates in the control of the rate of photosynthesis during induction. Light activation and dark inactivation of ribulose 5-phosphate kinase, fructose 1,6-biphosphatase and NADP⁺-specific glyceraldehydephosphate dehydrogenase were measured. In this respect there was no appreciable difference between protoplasts and chloroplasts. In contrast, the level of photosynthetic intermediates remained constant in darkened isolated chloroplasts, but declined rapidly in chloroplasts isolated from darkened protoplasts. When fructose 1,6-bisphosphatase was pre-activated by treating protoplasts with dithiothreitol the lag was only slightly shortened. These results are discussed in terms of control of the rate of the photosynthesis during the lag by substrates rather than limitation imposed by activity of any of the enzymes measured.     

Targeted alteration of the substrate specificity of peptide synthetases by rational module swapping

Analysis of the primary structure of peptide synthetases involved in the non-ribosomal synthesis of peptide antibiotics has revealed a highly conserved and ordered modular arrangement. A module contains at least two domains, involved in ATP-dependent substrate activation and thioester formation. The occurrence and arrangement of these functional building blocks is associated with the number and order of the amino acids incorporated in the peptide product. In this study, we present data on the targeted exchange of the leucine-activating module within the three-module surfactin synthetase 1 (SrfA-A) of Bacillus subtilis. This was achieved by engineering several hybrid srfA-A genes, which were introduced into the surfactin biosynthesis operon by in vivo recombination. We examined the hybrid genes for expression and investigated the enzymatic activities of the resulting recombinant peptide synthetases. For the first time, we demonstrate directly that an individual minimal module, of bacterial or fungal origin, confers its amino acid-specific activity on a multi-modular peptide synthetase. Furthermore, it is shown that directed incorporation of ornithine at the second position of the peptide chain induces a global alteration in the conformation of surfactin and may result in premature cyclization or a branched cyclic structure. Received: 10 July 1997 / Accepted: 11 September 1997     

In Vivo Substitution of Choline for Sodium Evokes a Selective Osmoinsensitive Increase of Extracellular Taurine in the Rat Hippocampus

Recent investigations have demonstrated that taurine and phosphoethanolamine (PEA) are the amino acids most sensitive to microdialysis-perfusion with reduced concentrations of NaCl. The aim of the present work was to assess the importance of Na+ deficiency in evoking this response. Further, the previously described selectivity of replacement of Cl- with acetate with respect to amino acid release was reinvestigated. The hippocampus of urethane-anesthetized rats was dialyzed with Krebs-Ringer bicarbonate buffer, and amino acid concentrations of the perfusate were determined. Choline chloride was then stepwise substituted for NaCl, and, in some cases, mannitol (122 mM) was included in low sodium-containing media. In other experiments, NaCl was replaced with sodium acetate. The dialysate levels of taurine increased selectively in response to Na+ substitution. The elevation of taurine was linearly related to the increase in choline chloride, and maximal levels amounted to 335% of basal levels. The increase in extracellular taurine was not inhibited by perfusion with medium made hyperosmotic with mannitol. Replacement of Cl- with acetate stimulated the release of taurine to 652% of resting levels. In addition, PEA levels increased to 250% of control concentration. Other amino acids were unaffected by Cl- substitution. The results show that taurine transport is considerably more sensitive to Na+ depletion than glutamate transport, which also is known to be Na+ dependent. The taurine increase evoked by low Na+ is not caused by cellular swelling as it was unaffected by hyperosmolar medium. Finally, substitution of acetate for Cl- causes a specific elevation of extracellular taurine and PEA, possibly as a result of cytotoxic edema.     

Study of physiopathological phenomena in dental enamel by neutron activation analysis

An epithermal neutron activation method is used to determine the concentration of mineral elements in human dental enamel. A large number (252) of samples from ancient and modern origins are analyzed. The analytical results are mathematically processed using a statistical multivariant method. This allows to differentiate deciduous from permanent teeth and decayed from sound enamel. It is also possible to distinguish the teeth coming from two different necropoles. The origin and the localization of determined elements in the mineralized part, or in the aqueous-organic part, of enamel is suggested. Their role, as witnessed in the physiopathological phenomena of dental enamel, is discussed.     

Crystal structures of the G139A, G139A–NO and G143H mutants of human heme oxygenase-1. A finely tuned hydrogen-bonding network controls oxygenase versus peroxidase activity

Conserved glycines, Gly139 and Gly143, in the distal helix of human heme oxygenase-1 (HO-1) provide the flexibility required for the opening and closing of the heme active site for substrate binding and product dissociation during HO-1 catalysis. Earlier mutagenesis work on human HO-1 showed that replacement of either Gly139 or Gly143 suppresses heme oxygenase activity and, in the case of the Gly139 mutants, increases peroxidase activity (Liu et al. in J. Biol. Chem. 275:34501, 2000). To further investigate the role of the conserved distal helix glycines, we have determined the crystal structures of the human HO-1 G139A mutant, the G139A mutant in a complex with NO, and the G143H mutant at 1.88, 2.18 and 2.08 Å, respectively. The results confirm that fine tuning of the previously noted active-site hydrogen-bonding network is critical in determining whether heme oxygenase or peroxidase activity is observed.     

Steps in reductive activation of the disulfide-generating enzyme Ero1p

Ero1p is the primary catalyst of disulfide bond formation in the yeast endoplasmic reticulum (ER). Ero1p contains a pair of essential disulfide bonds that participate directly in the electron transfer pathway from substrate thiol groups to oxygen. Remarkably, elimination of certain other Ero1p disulfides by mutation enhances enzyme activity. In particular, the C150A/C295A Ero1p mutant exhibits increased thiol oxidation in vitro and in vivo and interferes with redox homeostasis in yeast cells by hyperoxidizing the ER. Inhibitory disulfides of Ero1p are thus important for enzyme regulation. To visualize the differences between de-regulated and wild-type Ero1p, we determined the crystal structure of Ero1p C150A/C295A. The structure revealed local changes compared to the wild-type enzyme around the sites of mutation, but no conformational transitions within 25 Å of the active site were observed. To determine how the C150—C295 disulfide nonetheless participates in redox regulation of Ero1p, we analyzed using mass spectrometry the changes in Ero1p disulfide connectivity as a function of time after encounter with reducing substrates. We found that the C150—C295 disulfide sets a physiologically appropriate threshold for enzyme activation by guarding a key neighboring disulfide from reduction. This study illustrates the diverse and interconnected roles that disulfides can play in redox regulation of protein activity.     

The promoter of the pepper pathogen-induced membrane protein gene <Emphasis Type="Italic">CaPIMP1</Emphasis> mediates environmental stress responses in plants

The promoter of the pepper pathogen-induced membrane protein gene CaPIMP1 was analyzed by an Agrobacterium-mediated transient expression assay in tobacco leaves. Several stress-related cis-acting elements (GT-1, W-box and ABRE) are located within the CaPIMP1 promoter. In tobacco leaf tissues transiently transformed with a CaPIMP1 promoter-β-glucuronidase (GUS) gene fusion, serially 5′-deleted CaPIMP1 promoters were differentially activated by Pseudomonas syringae pv. tabaci, ethylene, methyl jasmonate, abscisic acid, and nitric oxide. The −1,193 bp region of the CaPIMP1 gene promoter sequence exhibited full promoter activity. The −417- and −593 bp promoter regions were sufficient for GUS gene activation by ethylene and methyl jasmonate treatments, respectively. However, CaPIMP1 promoter sequences longer than −793 bp were required for promoter activation by abscisic acid and sodium nitroprusside treatments. CaPIMP1 expression was activated in pepper leaves by treatment with ethylene, methyl jasmonate, abscisic acid, β-amino-n-butyric acid, NaCl, mechanical wounding, and low temperature, but not with salicylic acid. Overexpression of CaPIMP1 in Arabidopsis conferred hypersensitivity to mannitol, NaCl, and ABA during seed germination but not during seedling development. In contrast, transgenic plants overexpressing CaPIMP1 exhibited enhanced tolerance to oxidative stress induced by methyl viologen during germination and early seedling stages. These results suggest that CaPIMP1 expression may alter responsiveness to environmental stress, as well as to pathogen infection. The nucleotide sequence data reported here has been deposited in the GenBank database under the accession number DQ356279.