Activity of mushroom polyphenol oxidase in organic medium

A kinetic study of the activity of mushroom polyphenol oxidase in an organic system was carried out to obtain detailed enzyme kinetic data in relation to optimization of reaction conditions and substrate specificity. A simple method for consistent measurement of reaction rates in the heterogeneous enzyme/organic solvent system (consisting of immobilized polyphenol oxidase and a hydrated solution of the substrate in chloroform) was designed. The aqueous content of the system was optimized using p-cresol as the substrate. With this system, a crude extract of Agaricus bisporus was used to hydroxylate and oxidize a range of selected p-substituted phenolic substrates, yielding o-quinone products. Michaelis-Menten kinetics were used to obtain apparent K(M) and V(max) values with respect to each of these substrates. Results from this analysis indicated a correlation between the enzymic kinetic parameters obtained and the steric requirements of the substrates, which could be rationalized in terms of the restricted flexibility of the enzyme when it is in chloroform and also in terms of substrate and solvent hydrophobicity. In the course of the investigation UV molar absorption coefficients of several o-quinones were measured by a novel method: (1)H nuclear magnetic resonance (NMR) spectroscopy was employed to determine component concentrations in reaction mixtures resulting from the transformation of phenols by polyphenol oxidase in chloroform. Thus the UV molar absorption coefficients could be obtained directly, avoiding the necessity to isolate the water-sensitive, unstable o-quinones. (c) 1993 John Wiley & Sons, Inc.     

A New Method for Finding Small Vertebrate Fossils: Ultraviolet Light At Night

Vertebrate fossils from many different formations fluoresce when exposed to ultraviolet (UV) light. In this paper field observations and controlled experiments in the Chadron Formation (White River Group, late Eocene) of Wyoming are used to assess the utility of searching for fossils at night using ultraviolet light. The results indicate that, especially for very small teeth and egg-shell fragments, searching with ultraviolet light at night can result in significantly more specimens than searching during daylight hours. This method has the potential to increase sample sizes for small vertebrate specimens that are often overlooked when using standard collecting techniques.     

Characterization of the amino acids of bovine fibrinogen involved in the fibrinogen-thrombin interaction of the blood clotting process. Comparison with the milk clotting process

Summary Bovine fibrinogen and the A and B chains of bovine fibrinogen have been subjected to chemical modification by a number of reagents and the effects of these procedures on the susceptibility of the proteins to thrombin hydrolysis is described. The reagents used were rose bengal (for photo-oxidation), 2-hydroxy-5-nitrobenzyl bromide, N-acetylimidazole, iodoacetic acid and diethyl pyrocarbonate. Evidence is presented which indicates that the tryptophan and tyrosine residues of fibrinogen are not involved to any great extent in the interaction of this protein with thrombin. Modification with iodoacetic acid suggests that methionine residues play a major role in such interactions, but the fibrinogen chains on which the important residues reside remain uncertain. The use of diethyl pyrocarbonate indicates the participation also of histidine in fibrinogen-thrombin interactions and that, whereas the histidine residues of the B chain are involved to a great extent, it appears that those of the Aa chain are not. The similarities which exist between the fibrinogen-thrombin and the -casein-chymosin systems are discussed.Abbreviations used DEP diethyl pyrocarbonate (ethoxyformic anhydride) - HNBB 2-hydroxy-5-nitrobenzyl bromide - N-Acl N-acetylimidazole - PTC phenylthiocarbamyl - PTH 3-phenyl-2-thiohydantoin.     

IN VIVO RNA SYNTHESIS WITHIN THE RAT NODOSE GANGLIA

Abstract— The in vivo synthesis of RNA in the rat nodose ganglia has been studied following the intravenous introduction of ortho-[³²P]phosphate. Analysis of the labelled RNA upon 2.2% polyacrylamide gels was performed. Rapidly-labelled heterodisperse RNA. with a size range of 10S-30S was detected after 3 h exposure to the isotope. Two peaks, of size 28S and 12S-14S. were also observed. The former is thought to be processed 28S rRNA whereas the latter is consistent with its designation as 'message-like' RNA (mlRNA). A rapidly turning-over phosphorylated non-nucleic acid contaminant prevented clear interpretation of the 4S region of the gel at short labelling times. However, this material was not present when the exposure time was increased to 24 h. At this time, only the stable RNA species 28S and 18S rRNA and 4S tRNA, were detected.     

Colonization factors andEscherichia coli belonging to enterotoxin-associated serotypes

Hemagglutination (HA) tests using human and bovine erythrocytes and microagglutination tests using pili-specific antisera (PSA) were performed to examine 168 strains ofEscherichia coli belonging to enterotoxin-associated serotypes for colonization factors (CFs). Seventy-one (42%) of these 168 strains possessed at CF, but only 10 (6%) were found positive by both HA and PSA tests. Groups of test strains from different sources (feces, urine, blood, and wounds) were not found to contain statistically different percentages of CF-positive strains. Strains producing heat-stable enterotoxin alone were less frequently associated with a CF than were other enterotoxigenic and nonenterotoxigenic strains. Strains showing heat-labile hemolytic activity and belonging to serotype O6: H—were less likely (P=0.014, Fisher's exact probability) to contain a CF than were similarly hemolytic strains belonging to other serotypes.     

Plasmids and serotypes of hemolytic fecalEscherichia coli

Twelve of 21 human, hemolytic, fecal isolates ofEscherichia coli produced type 1 hemolysin (HLY1), an extracellular, heat-labile molecule (alpha-hemolysin). Although no common plasmid species was apparent, 11 of 12 HLY1 strains possessed a plasmid60 megadaltons (Mdal); 5 of 9 strains with other hemolysins possessed a plasmid of comparable molecular mass (Fisher's exact probability=0.0805). One derivative of an HLY1+strain, which contained a 125 Mdal plasmid, no longer expressed HLY1 and contained a single 102 Mdal plasmid. The presence of large plasmids of varying size and an apparent deletion mutation in HLY1 strains suggest that HLY1 determinants are located on a small, unstable genetic element. In an initial survey of 224 human fecal isolates ofE. coli, the predominant hemolytic serotype was 06:H-, and conversely most (85%) 06:H-isolates were HLY1+. Serotype appears to play an important role in HLY1 expression.     

Regulation of proliferation of rat cartilage and bone by sex steroid hormones

We have demonstrated previously that 17β-estradiol (E₂) stimulates proliferation of skeletal tissues, both in vivo and in vitro, as measured by increased DNA synthesis and creatine kinase (CK) specific activity. The effect of E₂ on bone is sex specific. E₂ is active only in females and androgens only in males. By contrast, in cartilage of both sexes, dihydrotestosterone (DHT) as well as E₂ stimulates CK specific activity and DNA synthesis. In bone, we find that sex steroids stimulate skeletal cell proliferation in gonadectomized as well as in immature rats. Ovariectomized (OVX) rats, between 1 and 4 weeks after surgery, show stimulation of CK by E₂. The basal activity and response of CK changes with the varying endogenous levels of E₂ in cycling rats, in which the highest basal activity is at proestrus and estrus and the highest response is in diestrus. In rats of all ages tested, both the basal and stimulated specific activity of CK is higher in diaphysis and epiphysis than in the uterus, or in the adipose tissue adjacent to the uterus, which has a response similar to that of the uterus itself. The effect of E₂ in vivo, and in chrondroblasts and osteoblasts in vitro, is inhibited by high levels of the antiestrogen tamoxifen which, by itself, in similar high concentrations, shows stimulatory effects. In addition to the sex steroids, skeletal cells are also stimulated by secosteroid and peptide calciotrophic hormones. The interactions of the sex steroids and the other calciotrophic hormones. These results provide the first steps towards understanding the regulation of bone cell proliferation and growth by the concerted action of a variety of hormones and growth factors.     

Human vaginal epithelial multilayer tissue culture

Summary Fragments of normal human adult vagina, when explanted onto glass slides gave rise to outgrowing sheets of pure epithelium, which had microscopic morphological features in common with normal vaginal epithelium. Infrequent fibroblast contamination was observed. Proliferating epithelial cells formedmultilayers of stratified squamous epithelium and demonstrated a progressive decrease in proliferative activity after 14 days. Continuous lines of epithelial cells were not obtained. Even in the absence of estrogens, transmission electron microscopy revealed evidence of keratinization of the superficial cells of the multilayer. Scanning electron microscopy of the surface of mature epithelial cells in culture revealed ultrastructural features that closely resembled those present on the surface of exfoliated cells obtained by scraping the vagina in vivo. This in vitro tissue culture model of human vaginal epithelium may provide a simple method of studying factors that influence vaginal epithelium growth, maturation and function.