Kinetics and in vitro origin of the temperature-dependent transition of the estrogen receptor monomer

Partitioning of estrogen receptors in aqueous two-phase polymer systems has provided the basis for a detailed kinetic analysis of the effects of temperature on estrogen receptor (ER) structure in vitro. Exposure to temperatures of 0-30 degrees C increased the rate of change in ER partition coefficients by up to 100-fold but did not affect the final extent of the process. The temperature-dependent change in ER partition coefficients was characterized by a linear Arrhenius plot and an activation energy of 25 kcal/mol. The rate of the temperature-dependent ER transition (28 degrees C) was found to be unaffected by greater than 50-fold changes in receptor concentration, which indicates that the temperature-dependent change in partition coefficients reflects a first-order process. The partition coefficients of heated ER were unaffected by subsequent 18-h incubations at 0 degree C, indicating that the temperature-dependent ER transition is irreversible in vitro. Direct heating of the unoccupied ER resulted in both a change in ER partition coefficients and a loss of ER binding sites. The temperature-dependent change in unoccupied ER partition coefficients was complete within 30 min at 28 degrees C and yielded a first-order rate constant that was the same as that obtained for heating the receptor-estradiol complex at 28 degrees C. In contrast, the loss of unoccupied ER binding sites that occurred during 28 degrees C incubations did not reach completion after 150 min of heating and was found to behave as a second-order process.(ABSTRACT TRUNCATED AT 250 WORDS)     

A rod-shaped,gram-negative,propionigenic bacterium with a wide substrate range and the ability to fix molecular nitrogen

From estuarine mud a rod-shaped, motile, gram-negative, anaerobic bacterium was isolated (strain asp 66). Asp 66 fermented several substrates including glucose, fructose, malate, fumarate, citrate and aspartate. Fermentation products were acetate, propionate and presumably CO₂. Hydrogen was never formed nor utilized. Succinate conversion to propionate was catalyzed by cell suspensions but did not support growth. Asp 66 did not require vitamins and grew well in mineral media with a fermentable substrate. The pH range for growth was from 6.5 to 8.5. Temperature optimum was 27 to 30°C. The strain was able to fix N₂ as evidenced by its growth with N₂ as sole nitrogen source and its ability to reduce acetylene to ethylene. Cell-free extracts of cultures grown under air without shaking contained cytochrome(s) with absorption peaks at 523 nm and at 553 nm. The G+C content of the DNA was 60.8+-1 mol%. The taxonomic position of strain asp 66 is discussed.     

In vivo genetic engineering: homologous recombination as a tool for plasmid construction

This paper describes a novel method for creating exact DNA fusions between any two points in a plasmid carried in Bacillus subtilis. It exploits the homologous in vivo recombination between directly repeated sequences that can be established by insertion of a synthetic oligodeoxyribonucleotide. The method was used to enhance the productivity in B. subtilis of a cloned alpha-amylase (Amy)-encoding gene originating from Bacillus stearothermophilus. Thus, an exact fusion between nucleotide sequences encoding the expression signals, including the signal peptide, of a Bacillus licheniformis Amy-encoding gene and the mature Amy of B. stearothermophilus, was created. The resulting hybrid translational product was processed correctly in B. subtilis during secretion, giving rise to an Amy identical to the mature Amy secreted by B. stearothermophilus.     

Prevalence of the 281 (Gly→Glu) mutation in hepatoerythropoietic porphyria and porphyria cutanea tarda

Summary The prevalence of the 281 (GlyGlu) mutation in hepatoerythropoietic porphyria (HEP) was investigated by the use of hybridization with a synthetic oligonucleotide probe. The mutation was found in HEP-affected members of two unrelated families from Spain, but was absent in two other patients from Italy and Portugal who also had HEP. Moreover, this mutation was not detected in 13 unrelated cases of familial (type II) porphyria cutanea tarda.     

Production and immunohistochemical characterization of monoclonal antibodies directed against renal basement membranes of rats

Basement membranes were separated from rat glomeruli and purified by mild procedures, which led to a highly enriched basement membrane fraction. Here, the production and characterization of five monoclonal antibodies against tubular and glomerular basement membranes are described. These antibodies were analyzed immunohistochemically on frozen sections of rat, bovine, and human kidneys as well as on rat embryos. One monoclonal antibody (BM O II) exclusively recognized the glomerular basement membranes, another one (BM O VII) bound to tubular basement membranes and to Bowman's capsule. Three antibodies (BM O IV, BM M II, BM M III) recognized their antigens in both glomerular and tubular basement membranes as well as in mesangial cells. The BM O II antibody showed a stringent species specificity and bound only to glomerular basement membranes of the rat. The other four antibodies cross-reacted with human and bovine glomerular basement membrane and mesangial antigens; they also bound to other tissues in the developing rat embryo. Antibody binding to specific purified components of the basement membranes such as collagen type IV, laminin, heparan sulphate proteoglycan, and fibronectin was investigated by enzyme-linked immunosorbent assay (ELISA). None of these antibodies reacted with any of these known basement membrane components, indicating that the antibodies may serve as useful tools in future investigations of so far unidentified components of basement membranes.     

Effects of CaSO4 and NaCl on growth and nitrogen fixation ofLeucaena leucocephala

Effects and interactions of varying CaSO₄ and NaCl levels on growth and nitrogen fixation ofLeucaena leucocephala K8 were examined. Leucaena was grown in nutrient solution at four levels of CaSO₄ (0.5, 1.0, 2.5 and 5.0 mM) and NaCl (1, 25, 50 and 100 mM) in randomized blocks with five replications. While NaCl significantly reduced plant growth, additions of CaSO₄ increased plant height, leaf number, and biomass of salt treated plants. For the nonsaline treatments, high CaSO₄ levels slightly depressed growth, which contradicts suggestions that Leucaena has a high calcium requirement. A significant calcium/sodium interaction was not seen for nodule number or weight. Nodule number was significantly depressed by 100 mM NaCl and nodule weight of the salt stressed plants significantly increased as CaSO₄ concentration increased from 0.5 to 2.5 mM. Effects of NaCl and CaSO₄ on nitrogen content of plant parts were inconclusive. The promotion of Leucaena salinity tolerance by addition of CaSO₄ may be attributed to the effect of calcium in maintaing the selective permeability of membranes.     

Polar hydrogen positions in proteins: Empirical energy placement and neutron diffraction comparison

A method for the prediction of hydrogen positions in proteins is presented. The method is based on the knowledge of the heavy atom positions obtained, for instance, from X-ray crystallography. It employs an energy minimization limited to the environment of the hydrogen atoms bound to a common heavy atom or to a single water molecule. The method is not restricted to proteins and can be applied without modification to nonpolar hydrogens and to nucleic acids. The method has been applied to the neutron diffraction structures of trypsin ribonuclease A, and bovine pancreatic trypsin inhibitor. A comparison of the constructed and the observed hydrogen positions shows few deviations except in situations in which several energetically similar conformations are possible. Analysis of the potential energy of rotation of Lys amino and Ser, Thr, Tyr hydroxyl groups reveals that the conformations of lowest intrinsic torsion energies are statistically favored in both the crystal and the constructed structures.