Biodegradation of 2,4,6-trinitrotoluene byPhanerochaete chrysosporium: Identification of initial degradation products and the discovery of a TNT metabolite that inhibits lignin peroxidases

Biodegradation of 2,4,6-trinitrotoluene (TNT) by the wood-rotting BasidiomycetePhanerochaete chrysosporium was studied in a fixed-film silicone membrane bioreactor and in agitated pellected cultures. The initial intermediate products of TNT biodegradation were shown to be 2-amino-4,6-dinitrotoluene (2amDNT) and 4-amino-2,6-dinitrotoluene (4amDNT). These intermediates were also degraded byP. chrysosporium. However, their rates of degradation were slow and appeared to represent rate-limiting steps in TNT degradation. The fact that 2amDNT and 4amDNT were further degraded is of importance. In most other microbial systems these compounds are typically not further degraded or are dimerized to even more persistent azo and azoxydimers. Similar to previous studies performed in stationary cultures, it was shown that substantial amounts of [¹⁴C]-TNT were degrade to [¹⁴C]-carbon dioxide in agitated pelleted cultures. Lignin peroxidase activity (assayed by veratryl alcohol oxidation) virtually disappeared upon addition of TNT to ligninolytic cultures ofP. chrysosporium. However, TNT, 2amDNT, and 4amDNT did not inhibit lignin peroxidase activity, nor were they substrates for this enzyme. Subsequent studies revealed that 4-hydroxylamino-2,6-dinitrotoluene, an intermediate in TNT reduction, was a potent lignin peroxidase inhibitor. Further studies revealed that this compound was also a substrate for lignin peroxidase H8.     

Rat ovarian angiotensin II receptors. Characterization and coupling to estrogen secretion

Angiotensin II receptor agonist (125I-angiotensin II) and antagonist (125I-[Sar1,Ile8]angiotensin II) bind in a specific and saturable manner to rat ovarian membranes. Agonist and antagonist binding affinity (KD approximately 0.5 nM) and the number of sites estimated (Bmax approximately 60 fmol/mg of protein) were similar. Dissociation of receptor-bound agonist was more rapid than the dissociation of receptor-bound antagonist, and agonist, but not antagonist, dissociation from the receptor was accelerated by GTP gamma S. A 0-150 mM increase in Na+ produced a 27% increase in the KD of agonist binding. Antagonist binding was not modified by Na+. These studies suggest that both agonist and antagonist identify putative angiotensin II receptors in the ovary but that the properties of agonist and antagonist binding are distinct. Angiotensin II antagonist binding sites are present on the granulosa cell layer of rat ovarian follicles (Speth, R. C., Bumpus, F. M., and Husain, A. (1986) Eur. J. Pharmacol. 130, 351-352). To determine the role of angiotensin II in ovarian function, we examined angiotensin II receptors and function during the onset of puberty. High affinity and low capacity angiotensin II receptors were present in ovaries from immature rats. After pregnant mare's serum gonadotropin induced ovulation in immature rats, antagonist binding to total ovarian membranes increased over 3-fold. In vitro incubation of peripubertal ovaries with 1 microM angiotensin II produced a stimulation of estrogen, but not progesterone, secretion. This steroidogenic effect of angiotensin II was most pronounced in the luteal phase of the estrus cycle. These studies point toward the involvement of angiotensin II in the regulation of ovarian function, possibly through modulation of follicular estrogen levels.     

Biodegradation of pentachlorophenol by the white rot fungus Phanerochaete chrysosporium

Extensive biodegradation of pentachlorophenol (PCP) by the white rot fungus Phanerochaete chrysosporium was demonstrated by the disappearance and mineralization of [14C]PCP in nutrient nitrogen-limited culture. Mass balance analyses demonstrated the formation of water-soluble metabolites of [14C]PCP during degradation. Involvement of the lignin-degrading system of this fungus was suggested by the fact the time of onset, time course, and eventual decline in the rate of PCP mineralization were similar to those observed for [14C]lignin degradation. Also, a purified ligninase was shown to be able to catalyze the initial oxidation of PCP. Although biodegradation of PCP was decreased in nutrient nitrogen-sufficient (i.e., nonligninolytic) cultures of P. chrysosporium, substantial biodegradation of PCP did occur, suggesting that in addition to the lignin-degrading system, another degradation system may also be responsible for some of the PCP degradation observed. Toxicity studies showed that PCP concentrations above 4 mg/liter (15 microM) prevented growth when fungal cultures were initiated by inoculation with spores. The lethal effects of PCP could, however, be circumvented by allowing the fungus to establish a mycelial mat before adding PCP. With this procedure, the fungus was able to grow and mineralize [14C]PCP at concentrations as high as 500 mg/liter (1.9 mM).     

The key role of residue 5 in angiotensin II

The conformation–biological activity relationships in a series of angiotensin II analogs substituted in position 5 were studied. Results indicated that only analogs with β-branched residue in position 5 possess spectral and biological properties identical to that of parent angiotensin II.     

A cardiac specific analog of angiotensin I potentiated by converting enzyme inhibition

[1-Sarcosine, 7-Alanine] angiotensin I [( 1-Sar, 7-Ala] AI) and closely related analogs were tested for inotropic activity in the isolated cat heart, and for pressor activity in the intact conscious sheep both before and during converting enzyme inhibition (CEI). [1-Sar, 7-Ala] AI exhibited potent inotropic activity but was only weakly pressor. [1-Sar] AI, [1-Sar, 5-Val] AI, [1-Sar, 7-alpha MeAla] AI [1-Sar, 5-Val, 7-NMeAla] AI and [1-Sar, 5-Val, 7-Sar] were all potent agonists in both preparations. The action of [1-Sar, 7-Ala] AI was potentiated by CEI in both the isolated heart and the intact sheep. The activity of the remaining analogs was either partially or completely blocked by CEI. The activity of all analogs was inhibited by AII receptor blockade. These data indicate that the nature of the substitution in position 7 determines the affinity of the analog for converting enzyme. The [7-Ala] substitution appears to decrease the effect of the analog upon vascular receptors.     

Biodegradation of azo and heterocyclic dyes by Phanerochaete chrysosporium.

Biodegradation of Orange II, Tropaeolin O, Congo Red, and Azure B in cultures of the white rot fungus, Phanerochaete chrysosporium, was demonstrated by decolarization of the culture medium, the extent of which was determined by monitoring the decrease in absorbance at or near the wavelength maximum for each dye. Metabolite formation was also monitored. Decolorization of these dyes was most extensive in ligninolytic cultures, but substantial decolorization also occurred in nonligninolytic cultures. Incubation with crude lignin peroxidase resulted in decolorization of Azure B, Orange II, and Tropaeolin O but not Congo Red, indicating that lignin peroxidase is not required in the initial step of Congo Red degradation.