Purification of arogenate dehydrogenase from Phenylobacterium immobile

Phenylobacterium immobile, a bacterium which is able to degrade the herbicide chloridazon, utilizes for L-tyrosine synthesis arogenate as an obligatory intermediate which is converted in the final biosynthetic step by a dehydrogenase to tyrosine. This enzyme, the arogenate dehydrogenase, has been purified for the first time in a 5-step procedure to homogeneity as confirmed by electrophoresis. The Mr of the enzyme that consists of two identical subunits amounts to 69000 as established by gel electrophoresis after cross-linking the enzyme with dimethylsuberimidate. The Km values were 0.09 mM for arogenate and 0.02 mM for NAD+. The enzyme has a high specificity with respect to its substrate arogenate.     

Amino acid sulfur as a source of sulfate for sulfated proteoglycans produced by Swiss mouse 3T3 cells

The uptake of sulfate by Swiss mouse 3T3 cells is blocked in the presence of 1 mM 4-isothiocyano-4'-acetamido-stilbene-2,2-disulfonic acid (SITS). In the absence of an exogenous source of sulfate, glycosaminoglycans produced by cells in the presence of the inhibitor are sulfated to the same extent as those produced by cells grown in its absence. The sulfate utilized in the absence of medium sulfate has been identified as that produced by the oxidation of the sulfur present in the amino acids cysteine and methionine. This finding indicates that, under conditions of restricted exogenous sulfate, caution is needed in the interpretation of data obtained with the use of [35S]methionine and/or [35S]cysteine as a general protein label, since both tyrosine and a variety of types of protein-linked carbohydrate chains may be modified by sulfation.     

Viscoelasticity of packed erythrocyte suspensions subjected to low amplitude oscillatory deformation.

Concentrated adult erythrocyte suspensions were subjected to low amplitude oscillatory shear in a Weissenberg rheogoniometer equipped with a cone-and-plate assembly. The dynamic viscoelastic properties of the suspension were measured over a broad range of frequency by a numerical solution that accounted for fluid inertia. Variation of shear amplitude and cell volume percent, and comparison of buffered saline, plasma, and dextran as suspending media showed that the cellular elements had undergone small bending and shearing deformations. Studies of normal adult erythrocytes, hypotonically swollen cells, temperature-altered cells, and erythrocyte ghosts suggested that the method was evaluating membrane material properties. The normal membrane was found to exhibit a shear rate dependent elastic modulus that increased by more than a factor of 20 over a frequency range from 0.0076 Hz to 60 Hz. The membrane viscosity showed a substantial drop with frequency indicative of a frequency thinning phenomenon. At high frequency of deformation the viscous response of normal erythrocytes was no longer indicative of a membrane property due to the dominant influence of the internal hemoglobin solution. The studies generally supported the ability of the method to quantify relative membrane material properties and detect changes in membrane structure.     

Cortinarius sarcoflammeus sp. nov, a new species of subgenus Dermocybe (Agaricales) growing in Sphagnum bogs

 Cortinarius sarcoflammeus is proposed as a new species belonging to subgenus Dermocybe, on the basis of its morphological, chemical and ecological characters. The strong red-orange colour of the context and stipe base, large spores, sphagnicolous habitat and high dermorubin content are characteristic for the new species. Holotypes of C. huronensis and C. huronensis var. olivaceus have been examined for comparison, and their differences discussed. Photographs and line drawings of C. sarcoflammeus are added. Received February 13, 2001 Accepted March 23, 2001     

Cell adhesion molecule in the hexactinellid Aphrocallistes vastus

Abstract. The Hexactinellida sponge Aphrocallistes vastus contains a soluble aggregation factor (AF) whose purification has been described in this communication. It is characterized by a S°_20.w value of 37 and a buoyant density of 1.45 g/cm³. The AF is a glycoporteinaceous particle composed of three major protein species; no core structure could be visualized. In the presence of Ca²⁺, the AF causes secondary aggregation of single cells. The aggregation process is temperature, pH, and ionic strength independent within a broad range. Evidence is presented indicating that two (or more) AF molecules are required for the establishment of a stable cell: cell interaction. In contrast to the AFs from demosponges, the hexactinellid AF functions species-unspecifically.     

A familial mutation in the testis-determining gene SRY shared by both sexes

A familial mutation in SRY, the gene coding for the testis-determining factor TDF, was identified in an XY female with gonadal dysgenesis, her father, her two brothers and her uncle. The mutation consists of a T to C transition in the region of the SRY gene coding for a protein motif known as the high mobility group (HMG) box, a protein domain known to confer DNA-binding specificity on the SRY protein. This point mutation results in the substitution, at amino acid position 109, of a serine residue for phenylalanine, a conserved aromatic residue in almost all HMG box motifs known. This F109S mutation was not found in 176 male controls. When recombinant wildtype SRY and SRY^F109S mutant protein were tested in vitro for binding to the target site AAC AAAG, no differences in DNA-binding activity were observed. These results imply that the F109S mutation either is a rare neutral sequence variant, or produces an SRY protein with slightly altered in vivo activity, the resulting sex phenotype depending on the genetic back-ground or environmental factors.This paper is dedicated by G. S. to Professor Ulrich Wolf on the occasion of his 60th birthday