Isolation,partial purification,and some properties of protease I from a thermophilic mold Thermoascus aurantiacus var. levisporus

When the thermophilic mold Thermoascus aurantiacus var. levisporus was grown in a modified Czapek Dox medium containing casein the filtrate was found to contain proteolytic activity. The maximum production of activity occurred at 50 ° C in a medium containing 8% casein. The filtrate was subjected to ammonium sulfate fractionation and chromatography on DEAE-cellulose. Two proteases were separated. No further work was done on protease II. Protease I was further purified by gel filtration on Sephadex G 100–200. It showed a 40-fold purification with a final recovery of approximately 25%. It is a neutral protease with a pH optimum at 7.0. The optimal activity of the enzyme occurred in 0.02 M phosphate buffer but was completely inhibited at a concentration of 0.1 M. The optimum temperature for casein hydrolysis was found to be 55 ° C. The enzyme was inhibited by Hg⁺⁺ but was greatly stimulated by Cu⁺⁺ and mercaptoethanol. Metallo and sulfhydryl agents had no significant effect on enzyme activity.     

Transformation of o-toluate in Pseudomonas putida isolate 1065 and Rhizopus japonicus ATCC 24794

Summary Among various soil microorganisms tested only Pseudomonas putida isolate 1065 and Rhizopus japonicus ATCC 24794 were able to transform o-toluate. In P. putida o-toluate was quantitatively hydroxylated to give 2-hydroxymethyl-benzoate and in R. japonicus it was reduced to 2-hydroxymethyltoluene. Both compounds, which were identified on the basis of their physical properties, accumulated during a one week growth period.     

Access to glacial and subglacial environments in the Solar System by melting probe technology

A key aspect for understanding the biological and biochemical environment of subglacial waters, on Earth or other planets and moons in the Solar system, is the analysis of material embedded in or underneath icy layers on the surface. In particular the Antarctic lakes (most prominently Lake Vostok) but also the icy crust of Jupiter’s moon Europa or the polar caps of Mars require such investigation. One possible technique to penetrate thick ice layers with small and reliable probes is by melting, which does not require the heavy, complex and expensive equipment of a drilling rig. While melting probes have successfully been used for terrestrial applications e.g. in Antarctic ice, their performance in vacuum is different and theory needs confirmation by tests. Thus, a vacuum chamber has been used to perform a series of melting tests in cold (liquid nitrogen cooled) water ice samples. The feasibility of the method was demonstrated and the energy demand for a space mission could be estimated. Due to the high energy demand in case of extraterrestrial application (e.g. Europa or polar caps of Mars), only heating with radioactive isotopes seems feasible for reaching greater depths. The necessary power is driven by the desired penetration velocity (approximately linearly) and the dimensions of the probe (proportional to the cross section). In comparison to traditional drilling techniques the application of a melting probe for exploration of Antarctic lakes offers the advantage that biological contamination is minimized, since the Probe can be sterilized and the melting channel freezes immediately after the probe’s passage, inhibiting exchange with the surface layers and the atmosphere. In order to understand the physical and chemical nature of the ice layers, as well as for analysing the underlying water body, a melting probe needs to be equipped with a suite of scientific instruments that are capable of e.g. determining the chemical and isotopic composition of the embedded or dissolved materials.     

The three-dimensional structure of the regular surface protein of Comamonas acidovorans derived from native outer membranes and reconstituted two-dimensional crystals

The three-dimensional structure of the regular surface protein (p4 symmetry, lattice constant a = b = 10.5 nm) of Comamonas acidovorans has been determined to a resolution of about 1.5 nm by means of electron microscopy and image processing. Three-dimensional reconstructions were performed using native outer membranes and artificial two-dimensional crystals of the surface protein, which was selectively solubilized by deoxycholate and recrystallized on carbon films. The two-fold symmetric morphological complex is composed of two identical monomers which are in tight contact with the outer membrane and presumably anchored to it by a small hydrophobic domain.     

Hydrocarbon metabolism and cortisol balance in oil-exposed ringed seals, Phoca hispida

1. Ringed seals were exposed experimentally to oil contamination, by feeding of a [14C]naphthalene marked crude oil in fish for up to 4 days at a rate of 5 ml/day. 2. Mixed function oxygenase (MFO) activity, measured as aryl hydrocarbon hydroxylase in liver and kidney, was found to be induced, in particular in kidney tissue where the activity increased 3-fold. 3. MFO induction correlated with a high degree of conversion of crude oil hydrocarbons to water-soluble metabolites. Most of the radioactivity was found in the polar fraction of plasma and urine. 4. Plasma cortisol levels were somewhat elevated by captive holding, and increased markedly after oil-exposure. Cortisol half-life decreased after oil exposure from 1 3/4 to 1 hr.     

Simplified method for the discrimination of acute myelogenous and non-myelogenous leukemias

Rapid discrimination of acute myelogenous and non-myelogenous leukemias is of great importance when chemotherapy is urgently needed in severely ill patients. For decades the most reliable cytochemical method for this classification is the demonstration of myeloperoxidase in blast cells [1-4, 6, 7]. We combined the simplified myeloperoxidase stain as described by Kaplow with a brief stain similar to Pappenheim's procedure or with commercially available Hemacolor rapid blood smear: this proved to be a simple staining method that permits good morphological judgment of the cells as well as reliable demonstration of peroxidase activity. This procedure takes less than 10 min using Hemacolor and can easily be done with prepared solutions without technical assistance.     

Photorespiratory 2-phosphoglycolate metabolism and photoreduction of O<Subscript>2</Subscript> cooperate in high-light acclimation of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. strain PCC 6803

In cyanobacteria, photorespiratory 2-phosphoglycolate (2PG) metabolism is mediated by three different routes, including one route involving the glycine decarboxylase complex (Gcv). It has been suggested that, in addition to conversion of 2PG into non-toxic intermediates, this pathway is important for acclimation to high-light. The photoreduction of O₂ (Mehler reaction), which is mediated by two flavoproteins Flv1 and Flv3 in cyanobacteria, dissipates excess reductants under high-light by the four electron-reduction of oxygen to water. Single and double mutants defective in these processes were constructed to investigate the relation between photorespiratory 2PG-metabolism and the photoreduction of O₂ in the cyanobacterium Synechocystis sp. PCC 6803. The single mutants Δflv1, Δflv3, and ΔgcvT, as well as the double mutant Δflv1/ΔgcvT, were completely segregated but not the double mutant Δflv3/ΔgcvT, suggesting that the T-protein subunit of the Gcv (GcvT) and Flv3 proteins cooperate in an essential process. This assumption is supported by the following results: (1) The mutant Δflv3/ΔgcvT showed a considerable longer lag phase and sometimes bleached after shifts from slow (low light, air CO₂) to rapid (standard light, 5% CO₂) growing conditions. (2) Photoinhibition experiments indicated a decreased ability of the mutant Δflv3/ΔgcvT to cope with high-light. (3) Fluorescence measurements showed that the photosynthetic electron chain is reduced in this mutant. Our data suggest that the photorespiratory 2PG-metabolism and the photoreduction of O₂, particularly that catalyzed by Flv3, cooperate during acclimation to high-light stress in cyanobacteria. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.