Hydrolysis of dietary flavonoid glycosides by strains of intestinal Bacteroides from humans.

Rutin and quercitrin are hydrolysed to quercetin, and robinin is hydrolysed to kaempferol, by faecal flora from healthy subjects. The enzymes required for these hydrolyses, namely alpha-rhamnosidase and beta-galactosidase, were produced by some strains of Bacteroides distasonis; other strains, however, synthesized beta-glucosidase. The last-named enzyme was also elaborated by Bacteroides uniformis and Bacteroides ovatus. All the enzymes were produced constitutively. A cell-free extract of B. distasonis containing beta-glucosidase displayed an enzymic activity of 1 mumol/10 min per 10 mg of protein.     

Soil N mineralization and nitrification in relation to nitrogen solution chemistry in a small forested watershed

Spatial variations in soil processes regulating mineral N losses to streams were studied in a small watershed near Toronto, Ontario. Annual net N mineralization in the 0–8 cm soil was measured in adjacent upland and riparian forest stands using in situ soil incubations from April 1985 to 1987. Mean annual rates of soil N mineralization and nitrification were higher in a maple soil (93.8 and 87.0 kg.ha^–1) than in a pine soil (23.3 and 8.2 kg.ha^–1 ). Very low mean rates of mineralization (3.3 kg.ha^–1) and nitrification (3.4 kg.ha^–1) were found in a riparian hemlock stand. Average NO₃-N concentrations in soil solutions were 0.3–1.0 mg.L^–1 in the maple stand and >0.06mg.L^–1 in the pine stand. Concentrations of NO₃–N in shallow ground water and stream water were 3–4× greater in a maple subwatershed than in a pine subwatershed. Rapid N uptake by vegetation was an important mechanism reducing solution losses of NO₃–N in the maple stand. Low rates of nitrification were mainly responsible for negligible NO₃–N solution losses in the pine stand.     

Synthesis and evaluation of some novel phosphate and phosphinate derivatives of araA. Studies on the mechanism of action of phosphate triesters.

A number of novel phosphinate and phosphate triester derivatives of the anti-viral nucleoside analogue araA have been prepared. Spectroscopic and analytical data have been collected on both the reagents and the nucleotides. An in vitro assay indicated inhibition of DNA synthesis by mammalian cells, by each of the nucleotide derivatives, in the range 3-30 microM. Inhibition was reduced, but not abolished, for the phosphinates relative to the phosphates. These results are consistent with a mode of action involving release of the free nucleoside araA and the nucleotide araAMP.     

Conversion of 11-deoxycorticosterone and corticosterone to aldosterone by cytochrome P-450 11 beta-/18-hydroxylase from porcine adrenal

Highly purified cytochrome P-450 11 beta-/18-hydroxylase and the electron carriers adrenodoxin and adrenodoxin reductase were prepared from porcine adrenal. When the enzyme was incubated with the electron carriers, 11-deoxycorticosterone (DOC) and NADPH, the following products were isolated and measured by HPLC: corticosterone, 18-hydroxy-11-deoxycorticosterone (18-hydroxyDOC), 18-hydroxycorticosterone and aldosterone. All of the DOC consumed by the enzyme can be accounted for by the formation of these four steroids. Aldosterone was identified by mass spectroscopy and by preparing [3H]aldosterone from [3H]corticosterone followed by recrystallization at constant specific activity after addition of authentic aldosterone. Corticosterone and 18-hydroxycorticosterone were also converted to aldosterone. Conversion of corticosterone and 18-hydroxycorticosterone to aldosterone required P-450, both electron carriers, NADPH and substrate. The reaction is inhibited by CO and metyrapone. Moreover, all three activities of the purified enzyme decline at the same rate when the enzyme is kept at room temperature for various periods of time and when the enzyme is treated with increasing concentrations of anti-11 beta-hydroxylase (IgG) before assay. It is concluded that cytochrome P-450 11 beta-/18-hydroxylase can convert DOC to aldosterone via corticosterone and 18-hydroxycorticosterone. The stoichiometry of this conversion was found to be 3 moles of NADPH, 3 moles of H+ and 3 moles of oxygen per mole of aldosterone produced.     

Induction of endogenous xenotropic retrovirus expression by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate

Summary The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined for its ability to induce endogenous retrovirus from a high-passage clone of Kirsten sarcoma virus-transformed Balb/c (K-Balb) mouse cells. TPA activated virus in a concentration-dependent manner (0.0016 to 4.0 μM). Exposure to 1mM actinomycin D inhibited virus induction, suggesting that cellular RNA synthesis is required de novo by this inducer. A broad-spectrum neutralizing antibody to murine type C virus, gp70, was shown to neutralize the infectivity of the induced virus. The activated virus had the host range of the xenotropic Balb virus:2, and after removal of the inducer, the activated state decayed rapidly. TPA stimulated DNA, RNA, and protein synthesis in K-Balb cells, indicating that the mechanism of inducation may be different from that of previously identified virus inducers. The effects observed using the well-defined K-Balb system offer an opportunity to study the modulation of retrovirus gene expression by TPA. This work was conducted while the authors were with the Biological Carcinogenesis Program, Frederick Cancer Research Facility, Frederick, MD 21701, and was supported under Contract NO1-CO-75380 with the National Cancer Institute, National Institutes of Health, Bethesda, MD 20205.     

Equol: a contributor to enigmatic immunoassay measurements of estrogen

The efficacy of radioimmunoassay (RIA) for the measurement of estradiol-17 beta (E2) in murine plasma was investigated. When Sephadex LH-20 or celite column chromatography was used to separate E2 from estrone (E1) and other cross-reacting compounds, the results were erratic if small volumes of mouse plasma were resolved. Assay of a diethyl ether extract of plasma (500 microL) was the most practical method for estimating the concentration of estradiol-17 beta in mice. This method was used to determine the pattern of estrogen secretion during the estrous cycle, on the day of implantation and during pregnancy. No convincing change in estrogen secretion was observed in the diestrous/proestrous mouse. By comparison, estrogen levels were elevated during pregnancy. Taken together, these results implied that cross-reactive components in plasma masked low levels of endogenous estrogen. Further evaluation of mouse plasma and urine using a co-chromatography technique to examine estrogen elution from a reverse-phase HPLC system followed by GC/MS analysis indicated the presence of equol [7-hydroxy-3-(4-hydroxyphenyl)chroman], a phytoestrogen metabolite with a ring structure similar to estradiol-17 beta. Equol and possibly other cross-reactive components of plasma may account for the apparent lack of increased estrogen secretion during the mouse estrous cycle and on the day of implantation as determined by the radioimmunoassay of ether extracts of plasma.     

The 21-acetylation of corticosteroids by Clostridium sporogenes

A strain of Clostridium sporogenes, an anaerobic bacterium, isolated from sewage in New York City synthesizes two constitutive enzymes with action on steroid molecules: (i) an enzyme capable of selectively acetylating the 21-hydroxyl function of certain steroids and (ii) the corresponding esterase. Under our experimental conditions the enzymes have a strict structural requirement for 3-keto-4-ene and C-20-keto or 20 alpha-hydroxyl group and convert their respective substrates to a mixture of free and acetylated products.     

Identification of indolepyruvic acid as an intermediate of rebeccamycin biosynthesis

Summary Experimental evidence is presented to demonstrate that indolepyruvic acid is an intermediate in the rebeccamycin biosynthetic pathway. [3-¹⁴C]Indolepyruvic acid was prepared and efficiently incorporated (8%) into rebeccamycin bySaccharothrix aerocolonigenes.     

Targeting of proteins to the thylakoids by bipartite presequences: CFoII is imported by a novel, third pathway.

The CFoII subunit of the ATP synthase is an integral component of the thylakoid membrane which is synthesized in the cytosol with a bipartite, lumen-targeting presequence similar in structural terms to those of imported lumenal proteins such as plastocyanin. This presequence is shown to possess a terminal cleavage site for the thylakoidal processing peptidase, but no intermediate site for the stromal processing peptidase. The integration of CFoII into the thylakoid membrane of Pisum sativum has been analysed using in vitro assays for the import of proteins into intact chloroplasts or isolated thylakoids. Efficient integration into thylakoids is observed in the light and dark, and the integration process does not require the presence of either stromal extracts or nucleoside triphosphates. The uncoupler nigericin inhibits integration only very slightly, indicating that the thylakoidal delta pH does not play a significant role in the integration mechanism. In each of these respects, the requirements for CFoII integration differ notably from those determined for integration of the light-harvesting chlorophyll-binding protein of photosystem II. The integration mechanism also differs significantly from the two mechanisms involved in the translocation of lumenal proteins across the thylakoid membrane, since one of these processes requires the presence of stromal protein factors and ATP, and the other mechanism is dependent on the thylakoidal delta pH. This conclusion is reinforced by the finding that saturation of the translocation system for the precursor to the lumenal 23 kDa oxygen-evolving complex protein does not affect integration of CFoII into thylakoids.(ABSTRACT TRUNCATED AT 250 WORDS)