Mutations in the gene encoding 21-hydroxylase detected by solid-phase minisequencing

We have developed an assay based on solid-phase minisequencing to screen for the following seven point mutations in the gene CYP21 encoding 21-hydroxylase: Pro30Leu, I2-splice, Ile172Asn, Cluster-E6, Val281Leu, Gln318Stop, and Arg356Trp. 5′-Biotinylated PCR products of CYP21 are bound to streptavidin-coated microtiter wells, where the minisequencing reaction takes place after denaturation of DNA. Depending on the sequence investigated, one specific ³H-labelled deoxyribonucleotide is incorporated to extend a detection primer. By using an appropriate set of detection primers, it is possible to screen the gene for several mutations within the same PCR amplificate. This fast and reliable method very clearly distinguishes between DNA from homozygous mutant, heterozygous, and normal individuals and is well suited for routine diagnosis of patients with 21-hydroxylase deficiency and for carrier detection. Received: 19 August 1996     

Fungal foraging behaviour and hyphal space exploration in micro-structured Soil Chips

How do fungi navigate through the complex microscopic maze-like structures found in the soil? Fungal behaviour, especially at the hyphal scale, is largely unknown and challenging to study in natural habitats such as the opaque soil matrix. We monitored hyphal growth behaviour and strategies of seven Basidiomycete litter decomposing species in a micro-fabricated “Soil Chip” system that simulates principal aspects of the soil pore space and its micro-spatial heterogeneity. The hyphae were faced with micrometre constrictions, sharp turns and protruding obstacles, and the species examined were found to have profoundly different responses in terms of foraging range and persistence, spatial exploration and ability to pass obstacles. Hyphal behaviour was not predictable solely based on ecological assumptions, and our results obtained a level of trait information at the hyphal scale that cannot be fully explained using classical concepts of space exploration and exploitation such as the phalanx/guerrilla strategies. Instead, we propose a multivariate trait analysis, acknowledging the complex trade-offs and microscale strategies that fungal mycelia exhibit. Our results provide novel insights about hyphal behaviour, as well as an additional understanding of fungal habitat colonisation, their foraging strategies and niche partitioning in the soil environment.Subject terms: Microbial ecology, Fungal ecology, Fungal biology, Microbial ecology     

Monoclonal antibodies specific for neutrophil proteinase 4. Production and use for isolation of the enzyme

Four stable hybridoma cell lines producing monoclonal antibodies specific for neutrophil proteinase 4 (NP4) were established and one monoclonal antibody was chosen to produce an immunoaffinity-resin for the purification of NP4. In a precipitation assay system these antibodies bound NP4 in a dose-dependent manner, but did so neither with neutrophil elastase nor with cathepsin G. NP4 was purified and electrophoresis of the affinity-purified enzyme in sodium dodecyl sulfate polyacrylamide gels resulted in a single Mr = 30,000 polypeptide. The purified enzyme digested fibrin but not elastin and it cleaved Boc-Ala-ONp readily (Km = 0.47mM) at neutral pH, but had no effect on Suc-[Ala]3 Nan and N-Suc-[Ala]2-Pro-Phe-pNA. The proteolytic activity was inhibited by DFP, alpha 1 PI and alpha 2 M with a Ki of 10(-9)M for the NP4-alpha 1 PI complex. The NH2-terminal sequence and the amino-acid composition of NP4 were distinct from those of elastase and cathepsin G. Neutrophils contain large amounts of NP4 as judged by the comparable amounts of elastase- and NP4-alpha 1 PI complexes present in inflammatory exudates.     

Degradation of 2-deoxyaldoses by alkaline hydrogen peroxide

Reaction of 2-deoxy-D-arabino-hexose, 2-deoxy-D-lyxo-hexose, and 2-deoxy-D-erythro-pentose with alkaline hydrogen peroxide in the presence of magnesium hydroxide afforded the corresponding 2-deoxyaldonic acid, the 1,4-lactone, and the 1-O-formyl derivative of the next lower alditol. The 2-deoxyaldonic acids were separated in 60–80% yields, as new, crystalline lithium salts. The 1,4-lactones were obtained under conditions that precluded intermidiate formation of the free acids: presumably, the reaction proceeded by way of an intermediate, furanosyl hydroperoxide, which was converted into the lactone by elimination of water. With an excess of alkaline hydrogen peroxide, in the absence of magnesium hydroxide, the substrates were degraded to formic acid, with concurrent decomposition of hydrogen peroxide. It is shown that decomposition of hydrogen peroxide is catalyzed by hydroperoxide anion, and that it takes place by both a chain, and a non-chain, process. The decomposition reactions afford an abundant source of hydroxyl radical capable of oxidizing a wide variety of compounds.     

IgE-mediated histamine release from nasal mucosa is inhibited by SLPI (secretory leukocyte protease inhibitor) to the level of spontaneous release.

The secretory leukocyte protease inhibitor (SLPI) is a low-molecular-weight inhibitor of proteases, such as elastase and cathepsin G which are released from leukocytes during phagocytosis. The purpose of this study was to determine whether or not SLPI is able to inhibit IgE-mediated histamine release. Nasal mucosa from 11 test subjects without atopic disposition was used for this in vitro study. We found that SLPI inhibited histamine release in a dose-dependent way but was without influence on the spontaneous release.     

Quantification of granulocyte elastase inhibitors in human mixed saliva and in pure parotid secretion

The predominant inhibitors of granulocyte elastase in plasma (alpha 1-proteinase inhibitor and alpha 2-macroglobulin) together with antileukoproteinase were quantified in parotid secretion and mixed saliva. Antileukoproteinase was the only inhibitor found in parotid saliva and was present in a concentration about 30 times the serum level, suggesting a local production. In mixed saliva, antileukoproteinase accounted for more than 70% of the molar concentration of the granulocyte elastase inhibitors studied. alpha 1-Proteinase inhibitor was measurable in about 1/3 of the specimens of mixed saliva. In parotid secretion, antileukoproteinase was present only as a free, active inhibitor. In mixed saliva about 15% of antileukoproteinase was in complex with granulocyte elastase, while the remaining amount of 85% was inhibitorily active. This suggests that antileukoproteinase has a biological function in a local defence mechanism directed towards the effects of granulocyte elastase in the oral cavity and salivary glands.     

Normal Binding and Reactivity of Copper in Mutant Superoxide Dismutase Isolated from Amyotrophic Lateral Sclerosis Patients

Abstract: In some families with amyotrophic lateral sclerosis (ALS), the disease is linked to mutations in the gene encoding CuZn-superoxide dismutase. The mutant CuZn-superoxide dismutases appear to cause motor neuron degeneration by a toxic property, suggested to be linked to an altered reactivity of the active-site Cu ions. Asp⁹⁰Ala mutant CuZn-superoxide dismutase was isolated from six patients with ALS, allowing properties of the mutant enzyme synthesized and conditioned in patients with ALS to be examined. The molecular mass of the Asp⁹⁰Ala mutant CuZn-superoxide dismutase was 45 Da lower than that of the wild-type enzyme, as expected from the amino acid exchange. The mobility after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was markedly increased, however, suggesting altered properties of the polypeptide. The mutant CuZn-superoxide dismutase showed a minimal reduction in stability but did not differ significantly from the wild-type enzyme in enzymic activity, in content and affinity for active-site Cu ions and in the propensity to catalyze formation of hydroxyl radicals. Our findings suggest that the deleterious effect of mutant CuZn-superoxide dismutases on motor neurons in ALS is not related to altered reactivity of active-site Cu ions, resulting in increased oxidant stress. Attention should therefore also be directed at other mechanisms and properties of the mutant polypeptides and their degradation products.