Effects of phenolic acids on ammonia oxidation by terrestrial autotrophic nitrifying microorganisms

Abstract It has been hypothesized that vegetation in certain ecosystems inhibits nitrification in soil by producing phenolic compounds that inhibit oxidation of ammonia by nitrifying microorganisms. This hypothesis is based largely on a report that very low concentrations (10⁻⁶ M–10⁻⁸ M) of several phenolic acids (notably ferulic acid) completely inhibited NO₂⁻ production in an aqueous suspension of soil treated with (NH₄)₂SO₄ and a nutrient solution suitable for growth of Nitrosomonas and other autotrophic nitrifying microorganisms. To evaluate this hypothesis, we determined the effects of three ohenolic acids (ferulic acid, caffeic acid, and p -coumaric on nitrite production by representatives of three genera of terrestrial autotrophic nitrifying microorganisms ( Nitrosospira, Nitrosomonas , or Nitrosolubos ) grown on a defined medium containing NH₄⁺. We found that nitrite production by the Nitrososspira was not inhibited by ferulic acid, caffeic acid, or p -coumaric acid at concentrations of 10⁻⁶ or 10⁻⁵ M and was only slightly inhibited when these acids were at a concentration of 10⁻⁴ M. We also found that ferulic acid did not markedly inhibit nitrite production by the three genera of nitrifying microorganisms studied, even when its concentration was as high as 10⁻³ M. These observations invalidate the hypothesis tested because the phenolic acids studied did not significantly retard ammonia oxidation by autotrophic microorganisms even when their concentration in cultures of these microorganisms greatly exceeded their concentrations in soils.     

The influence of pre-treatment temperature on the thermal sensitivity of a mouse tumour

The response of tumours to hyperthermia was tested by giving graded heat treatments and assessing local control at 90 days. Mice were divided into three groups which were pre-treated for 3 days in ambient temperatures of 4, 21 or 35 degrees C. This enabled the mean tumour resting temperature to be varied by up to 11 degrees C, before subsequent heat treatment. For the heat treatments, the tumours were clamped in order to eliminate blood flow, resulting in uniform temperature distributions and hence more uniform thermal sensitivity. TCD50 values were used to construct Arrhenius plots. For all three pre-treatment temperatures, these plots demonstrated a factor of 1.6 increase in heating time per degree Celsius reduction in heating temperature. However, tumours kept in a 4 degrees C environment before treatment were more thermally sensitive than those kept in 21 degrees C conditions, while those in a 35 degrees C environment were more resistant. Pretreatment at 4 degrees C was equivalent to an increase of either 0.5 degree C in heating temperature or 28 per cent in heating time, compared with pre-treatment at 21 degrees C. Pre-treatment at 35 degrees C was equivalent to a reduction of either 0.6 degree C in heating temperature or 25 per cent in heating time. These data indicate that the pre-treatment tumour temperature is an important parameter, but the effect of heat treatment is more closely related to absolute heating temperature rather than to the increase in temperature above the normal resting level.     

Genetic changes in skin tumor progression: correlation between presence of a mutant ras gene and loss of heterozygosity on mouse chromosome 7

Initiation of tumorigenesis in mouse skin can be accomplished by mutagenesis of the H-ras gene by treatment with chemical carcinogens. A mouse model system has been developed to study the additional genetic events that take place during tumor progression. Skin carcinomas were induced in F1 hybrid mice exhibiting restriction fragment length polymorphisms at multiple chromosomal loci. Analysis of loss of heterozygosity in such tumors showed that imbalance of alleles on mouse chromosome 7, on which the H-ras gene is located, occurs very frequently in skin carcinomas. The chromosomal alterations detected, which included both nondisjunction and mitotic recombination events, were only seen in tumors that have activated ras genes. We conclude that gross chromosomal alterations that elevate the copy number of mutant H-ras and/or lead to loss of normal H-ras are a consistent feature of mouse skin tumor development.     

Species differences in the occurrence of copper-metallothionein in the particulate fractions of the liver of copper-loaded animals.

Large amounts of Cu-metallothionein were obtained by 2-mercaptoethanol and sodium dodecyl sulphate extractions of the particulate fractions of the liver of pigs given high-Cu2+ diets or rats injected with Cu2+. Three isoproteins were purified from pig liver and characterized on the basis of their physicochemical properties, metal content and amino acid composition. No such pool of Cu-metallothionein was present in the liver of Cu2+-loaded sheep or of rats given Cu2+-supplemented diets.     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

An Automated Technique for the Radiographic Determination of Bone Age

We have developed an automated procedure for determining bone-age in the monkey, Macaca fascicularis. The status of epiphysial junctions and sesamoids were determined from radiographs. Epiphyses were scored as either open or closed, and sesamoids were scored as either present or absent. Bone-age was calculated by a computer program that stored and used a data base of probabilities for epiphysial closure and sesamoid appearance. The simplicity of scoring, combined with the speed and accuracy of the calculations, make this technique potentially adaptable for use in a variety of primate species.     

Characterization of plasmids from antibiotic-resistant Shigella isolates by agarose gell electrophoresis.

Gel electrophoresis of DNA from 95 clinical isolates of Shigella sonnei and Shigella flexneri resistant to antibiotics revealed a heterogeneous plasmid population. Most of the plasmids were smaller than 6 megadaltons (Mdal). Six S. sonnei isolates with the most common antibiotic resistance pattern were characterized. They had two plasmids in common: one was a self-transmissible Fi+ plasmid of 46 Mdal encoding resistance to streptomycin and sulphafurazole. In addition, several cryptic plasmids ranging in size from 1.0 to 24.5 Mdal were present. Mobilization of the 5.5 Mdal SuSm plasmid and a 1.0 Mdal cryptic plasmid was demonstrated with all six S. sonnei isolates during conjugation. This mobilization was mediated by the 46 Mdal self-transmissible Fi+ R plasmid and also by a 24.5 Mdal Fi- plasmid carrying no known drug resistance determinants.     

The protective role of metallothionein in copper overload: I. Differential distribution of immunoreactive metallothionein in copper-loaded rat liver and kidney

The cytotoxicity of copper is probably determined by its molecular association and subcellular localisation rather than its concentration within tissues. Metallothionein (MT) is a copper binding protein distributed between the particulate and soluble cellular components. The role of MT in conferring protection to the copper-loaded rat has been investigated by comparing the distribution of the immunoreactive protein between the soluble and particulate fractions of liver and kidney during the development of copper tolerance. Young male Wistar rats were fed a high copper (1 g/kg) diet for 16 weeks and killed sequentially during this period; liver and kidneys were retained. Pellet and supernatant preparations from homogenised, pooled samples of liver and kidney were subjected to chromatographic separation. Copper and zinc were analysed in whole tissue, homogenates and eluant fractions and MT identified likewise using an enzyme-linked immunoassay. Copper accumulated for 5 weeks in the liver falling subsequently accompanied by similar changes in MT content. Kidney copper and MT rose to maximum concentrations at 8 weeks and were maintained thereafter. Substantial differences were apparent in the relative distribution of MT between the two organs. MT was the major, predominantly cytosolic, copper-binding protein in the kidney but in the liver immunoreactive MT was pelleted and present in lower concentration than the high molecular weight cuproproteins. It was concluded that whilst MT plays a role in the detoxification and adaptation of rats to copper-loading the regulatory functions of liver and kidney may differ significantly in this respect.     

Aggregation-dependent turnover of flagellar adhesion molecules in chlamydomonas gametes

Previous studies on flagellar adhesion in chlamydomonas (Snell, W. and S. Roseman. 1979. J. Biol. Chem. 254:10820-10829.) have shown that as gametes adhere to flagella isolated from gametes of the opposite mating type, the adhsiveness of the added flagella but not of the gametes is lost. The studies reported here show that the addition of protein synthesis inhibitors (cycloheximide [CH] or anisomycin) to the medium of such cell- flagella mixtures causes the cells to lose their adhesiveness. This loss, however, occurs only after the cells have interacted with 4-8 flagella/cell and does not occur if the cells are kept in CH (7 h) without aggregating. The availability of an impotent (imp) mating type plus (MT(+)) mutant (provided by U.W. Goodenough), which adheres but is unable to undergo the fusion that normally follows adhesion, made it possible to determine whether a similar loss of adhesiveness occurs in mixtures of matting type minus (mt(-)) and imp mt(+) gametes. In the absence of inhibitor, mt(-) and imp mt(+) gametes adhered to each other (without fusing) for several hours; however, in the presence of CH or anisomycin, the gametes began to de-adhere 35 min after mixing, and, by 90 min, 100 percent of the cells were single again. This effect was reversible, and the rapid turnover of cells were single again. This effect was reversible, and the rapid turnover of molecules involved in adhesion occurred only during adhesion inasmuch as gametes pretreated for 4 h with CH were able to aggregate in CH for the same length of time as nonpretreated cells aggregated in CH. By the addition of CH at various times after the mt(-) and imp mt(+) gametes were mixed, measurements were made of the “pool size” of the molecules involved in adhesion. The pool reached a minimum after 25 min of aggregation, rapidly increased for the next 25 min, and then leveled off at the premixing level. These results suggest that flagellar adhesion in chlamydomonas causes modification of surface molecules (receptors, ligands), which brings about their inactivation and stimulates their replacement.     

Nucleoside phosphorylase 2 (Np-2) of mice

Isozyme patterns of nucleoside phosphorylase (NP) in 16 inbred strains, two recombinant inbred, one congenic, and three species of wild mice were studied. Evidence is provided for a genetic locus, Np-2, encoding an electrophoretic variant which is expressed exclusively in erythrocytes of certain inbred strains. This finding establishes the occurrence of genetic polymorphism of NP among inbred strains of mice. In addition, the Npla allele previously reported only in inbred strains has been observed in one of the species of wild mice (Mus musculus castaneus) studied.