Long-term study of indomethacin and alclofenac in treatment of rheumatoid arthritis.

Indomethacin and alclofenac were compared for 13 months under double-blind conditions in 109 patients with active, classical, or definite rheumatoid arthritis at a relatively early stage of the disease. Both indomethacin and alclofenac were clearly effective: most patients either improved or remained as well controlled as on entry. Alclofenac proved the more effective drug, however, producing a significantly greater reduction in morning stiffness, articular index, and erythrocyte sedimentation rate, and only in the alclofenac-treated group did functional capacity improve and latex-agglutination titres diminish. Comprehensive laborabory tests showed no significant deviation from normal which could have been attributed to either drug.     

Biochemical characterization of a variant form of cytosolic epoxide hydrolase induced by parental exposure to N-ethyl-N-nitrosourea

1. ENU4 mice express a protein variant originally detected in a CBF1 mouse sired by a C57BL/6 mouse exposed to N-ethyl-N-nitrosourea. It appears to be an isolelectric point variant of cytosolic epoxide hydrolase. Affinity purified cytosolic epoxide hydrolase from ENU4 mice has a pI of approximately 5.1 compared to 5.6 in other mouse strains.2. Clofibrate induced cytosolic epoxide hydrolase to similar levels in five strains of mice. However, CBF1 and ENU4 mice were more sensitive to the induction of palmitoyl CoA oxidase activity.3. Except for isoelectric point, the physico- and immunochemical properties of cytosolic epoxide hydrolase from ENU4 mice were similar to those of the other mouse strains. Substrate specificities for five of six substrates tested were also similar.     

The metabolism of drugs and carcinogens in isolated subcellular fractions of Drosophila melanogaster. II. Enzyme induction and metabolism of benzo[a]pyrene

The effect of various pretreatments on the activities of several drug metabolizing enzymes was investigated in microsomes and postmicrosomal supernatant fractions isolated from whole body homogenates of Drosophila melanogaster larvae of different strains. Pretreatments of larvae with either phenobarbital (PB), β-naphthoflavone (BNF) or a mixture of polychlorinated biphenyls (Aroclor 1254, PCB) for 24 h increased microsomal benzo[a]pyrene (BP) monooxygenase activity 2- to 6-fold in all strains as compared to untreated larvae. A simultaneous increase in the contents of cytochrome P-450 occurred after pretreatment with PB and PCB. Comparison of the turnover rates of BP per molecule of cytochrome P-450 indicated that BP was a poor substrate for control cytochrome P-450 whereas BNF induced a most active hemoprotein for this metabolism. Marked differences in the qualitative pattern of BP metabolites were obtained between microsomes isolated from BNF-treated larvae or rat liver microsomes. 3-Hydroxy-BP (3-OH-BP) was the dominating metabolite with both preparations, while the BP dihydrodiols were formed in minor quantities in Drosophila as compared to rat liver. Metyrapone and SKF 525-A inhibited BP metabolism in microsomes isolated from untreated and BNF treated larvae of all strains. In contrast, α-naphthoflavone (ANF) stimulated the BP monooxygenase activity of microsomes isolated from untreated larvae approx. 3-fold but only slightly influenced the activity of microsomes from BNF treated larvae indicating that the latter species of cytochrome P-450 was less sensitive to ANF.In all strains, PCB and PB treatments approximately doubled microsomal epoxide hydrolase activity and increased cytosolic glutathione-S-transferase activity 25–60%, significant only in strain Berlin K after PB treatment. The activities of epoxide hydrolase and glutathione-S-transferase in control larvae were comparable in the different strains, whereas the content of cytochrome P-450 and BP monooxygenase activity was higher in the Hikone R strain. Variability in the induction response to the various pretreatment was observed among the three strains.     

Evidence for modified mechanisms of chloroethene oxidation in Pseudomonas butanovora mutants containing single amino acid substitutions in the hydroxylase alpha-subunit of butane monooxygenase

The properties of oxidation of dichloroethene (DCE) and trichloroethylene (TCE) by three mutant strains of Pseudomonas butanovora containing single amino acid substitutions in the α-subunit of butane monooxygenase hydroxylase (BMOH-α) were compared to the properties of the wild-type strain (Rev WT). The rates of oxidation of three chloroethenes (CEs) were reduced in mutant strain G113N and corresponded with a lower maximum rate of butane oxidation. The rate of TCE degradation was reduced by one-half in mutant strain L279F, whereas the rates of DCE oxidation were the same as those in Rev WT. Evidence was obtained that the composition of products of CE oxidation differed between Rev WT and some of the mutant strains. For example, while Rev WT released nearly all available chlorine stoichiometrically during CE oxidation, strain F321Y released about 40% of the chlorine during 1,2-cis-DCE and TCE oxidation, and strain G113N released between 14 and 25% of the available chlorine during oxidation of DCE and 56% of the available chlorine during oxidation of TCE. Whereas Rev WT, strain L279F, and strain F321Y formed stoichiometric amounts of 1,2-cis-DCE epoxide during oxidation of 1,2-cis-DCE, only about 50% of the 1,2-cis-DCE oxidized by strain G113N was detected as the epoxide. Evidence was obtained that 1,2-cis-DCE epoxide was a substrate for butane monooxygenase (BMO) that was oxidized after the parent compound was consumed. Yet all of the mutant strains released less than 40% of the available 1,2-cis-DCE chlorine, suggesting that they have altered activity towards the epoxide. In addition, strain G113N was unable to degrade the epoxide. TCE epoxide was detected during exposure of Rev WT and strain F321Y to TCE but was not detected with strains L279F and G113N. Lactate-dependent O₂ uptake rates were differentially affected by DCE degradation in the mutant strains, providing evidence that some products released by the altered BMOs reduced the impact of CE on cellular toxicity. The use of CEs as substrates in combination with P. butanovora BMOH-α mutants might allow insights into the catalytic mechanism of BMO to be obtained.     

Mutagenicity of 2-methylpropene (isobutene) and its epoxide in a modified Salmonella assay for volatile compounds.

The mutagenic properties of 2-methylpropene (MP) and 2-methyl-1,2- epoxypropane (MEP) were investigated in the Salmonella assay. A simple exposure system, consisting of gastight tissue culture flasks, was used. This method has the advantage that the volatile test chemical is present during the entire incubation period and that several concentrations of the investigated compound can be tested on a single day. MP is not mutagenic in strains TA100, TA102 and TA1535, and in the latter strain not even in the presence of metabolizing S9 mix. MEP is mutagenic in all the strains tested, as demonstrated by a clear dose-response relationship. Strain TA1535 seems to be most sensitive to MEP compared with the other bacterial strains studied. For this strain, the mutagenic activity of MEP decreased significantly in the presence of S9 mix, compatible with the epoxide being inactivated by epoxide hydrolase and by glutathione S-transferase, as reported previously. From the present study it can be concluded that the parent compound MP is not mutagenic, but that its primary metabolite MEP is a mutagenic substance. However, very high concentrations are necessary to induce a mutagenic effect and the epoxide is efficiently detoxified by different liver enzymes.     

Development of gallic acid formazans as novel enoyl acyl carrier protein reductase inhibitors for the treatment of tuberculosis

The enoyl acyl carrier protein reductase (InhA) of Mycobacterium tuberculosis (MTB) is an attractive target for developing novel antitubercular agents. A series of gallic acid formazans, were computationally designed and docked into the active site of InhA to understand their binding mode and potential to inhibit InhA. Nine compounds from the designed series were identified as potential InhA inhibitors, on the basis of good Glide score. These compounds were synthesized in the laboratory and evaluated for in vitro antitubercular activity against drug-sensitive and multi-drug resistant strains of MTB. Out of nine compounds, three compounds exhibited the most promising MIC of <2 μM against the sensitive strain of MTB, H37Rv. The compounds were evaluated against five resistant strains of MTB. Most of the compounds exhibited activity superior to the standard, linezolid, against all these resistant strains. The mechanism of action of these compounds was concluded to be InhA inhibition, through InhA enzyme inhibition study. Insignificant cytotoxicity of these compounds was observed on RAW 264.7 cell line. Inactivity of all these compounds against gram positive and gram negative bacteria indicated their specificity against MTB. The compounds were further analyzed for ADME properties and showed potential as good oral drug candidates. The results clearly identified some novel, selective and specific InhA inhibitors against sensitive and resistant strains of MTB.     

Identification and Characterization of Epoxide Carboxylase Activity in Cell Extracts of Nocardia corallina B276

The metabolism of aliphatic epoxides (epoxyalkanes) by the alkene-utilizing actinomycete Nocardia corallina B276 was investigated. Suspensions of N. corallina cells grown with propylene as the carbon source readily degraded propylene and epoxypropane, while suspensions of glucose-grown cells did not. The addition of propylene and epoxypropane to glucose-grown cells resulted in a time-dependent increase in propylene- and epoxypropane-degrading activities that was prevented by the addition of rifampin and chloramphenicol. The expression of alkene- and epoxide-degrading activities was correlated with the high-level expression of several polypeptides not present in extracts of glucose-grown cells. Epoxypropane and epoxybutane degradation by propylene-grown cell suspensions of N. corallina was stimulated by the addition of CO₂ and inhibited by the depletion of CO₂. Cell extracts catalyzed the carboxylation of epoxypropane to form acetoacetate in a reaction that was dependent on the addition of CO₂, NAD⁺, and a reductant (NADPH or dithiothreitol). In the absence of CO₂, epoxypropane was isomerized by cell extracts to form acetone at a rate approximately 10-fold lower than the rate of epoxypropane carboxylation. Methylepoxypropane was found to be a time-dependent, irreversible inactivator of epoxyalkane-degrading activity. These properties demonstrate that epoxyalkane metabolism in N. corallina occurs by a carboxylation reaction forming β-keto acids as products and provide evidence for the involvement in this reaction of an epoxide carboxylase with properties and cofactor requirements similar to those of the four-component epoxide carboxylase enzyme system of the gram-negative bacterium Xanthobacter strain Py2 (J. R. Allen and S. A. Ensign, J. Biol. Chem. 272:32121–32128, 1997). The addition of epoxide carboxylase component I from Xanthobacter strain Py2 to methylepoxypropane-inactivated N. corallina extracts restored epoxide carboxylase activity, and the addition of epoxide carboxylase component II from Xanthobacter Py2 to active N. corallina extracts stimulated epoxide isomerase rates to the same levels observed with the purified Xanthobacter system. Antibodies raised against Xanthobacter strain Py2 epoxide carboxylase component I cross-reacted with a polypeptide in propylene-grown N. corallina extracts with the same molecular weight as component I but did not cross-react with glucose-grown extracts. Together, these results suggest a common pathway of epoxyalkane metabolism for phylogenetically distinct bacteria that involves CO₂ fixation and the activity of a multicomponent epoxide carboxylase enzyme system.     

Killer activity of Saccharomyces cerevisiae strains: partial characterization and strategies to improve the biocontrol efficacy in winemaking

Killer yeasts are considered potential biocontrol agents to avoid or reduce wine spoilage by undesirable species. In this study two Saccharomyces cerevisiae strains (Cf8 and M12) producing killer toxin were partially characterized and new strategies to improve their activity in winemaking were evaluated. Killer toxins were characterized by biochemical tests and growth inhibition of sensitive yeasts. Also genes encoding killer toxin were detected in the chromosomes of both strains by PCR. Both toxins showed optimal activity and production at conditions used during the wine-making process (pH 3.5 and temperatures of 15–25 °C). In addition, production of both toxins was higher when a nitrogen source was added. To improve killer activity different strategies of inoculation were studied, with the sequential inoculation of killer strains the best combination to control the growth of undesired yeasts. Sequential inoculation of Cf8–M12 showed a 45 % increase of killer activity on sensitive S. cerevisiae and spoilage yeasts. In the presence of ethanol (5–12 %) and SO₂ (50 mg/L) the killer activity of both toxins was increased, especially for toxin Cf8. Characteristics of both killer strains support their future application as starter cultures and biocontrol agents to produce wines of controlled quality.     

A rapid assay for epoxide hydratase activity with benzo(a)pyrene 4,5-(K-region-)oxide as substrate

A rapid radiometric assay for epoxide hydratase activity has been developed using the highly mutagenic [³H]benzo(a)pyrene 4,5-(K-region-)oxide as substrate. By addition of dimethylsulfoxide after the incubation, conditions were found where the unreacted substrate could be separated from the product benzo(a)pyrene-4,5-dihydrodiol(trans) simply by extraction into petroleum ether. The product is then extracted into ethyl acetate and, radioactivity is measured by scintillation spectrometry. This assay allows a rapid measurement of epoxide hydratase activity with an epoxide derived from a carcinogenic polycyclic hydrocarbon as substrate and is at the same time sensitive enough for accurate determination of epoxide hydratase activity in preparations with extremely low enzyme levels such as rat skin homogenate (8–14 pmol of product/mg of protein/min).     

Mutagenic effect of furocoumarin monoadducts and cross-links on bacteriophage lambda

The mutagenicities of 17 closely related oxiranes were determined in 4 tester strains (Salmonella typhimurium TA98, TA100, TA1535, TA1537). The test compounds comprised all possible oxides of benzene and its partially hydrogenated congeners. In TA100 and TA1535, 12 of the tested oxiranes were weak to moderate mutagens. 4 of these were also active in TA98. No mutagenicity was observed with the remaining 5 compounds in any of the 4 strains.The presence of a double bond in formal conjugation with the epoxide ring increased the mutagenicity relative to that of the saturated oxirane. Interestingly, additional epoxide rings within the same molecule did not markedly increase the mutagenic activity, and for the oxiranes that are not activated by a double bond, the relationship between mutagenic activity and the number of epoxide rings in the molecule was even inverse.The influence of bromo and hydroxyl substitution on oxirane mutagenicity is discussed. Most notably, a compound having a 4-hydroxyl group in syn position to a 1,2-epoxide ring fused to the cyclohexane ring, a structure which has been suggested to increase the electrophilic reactivity of dihydrodiol epoxides through hydrogen bonding, was almost inactive.     

Identification and characterization of epoxide hydrolase activity of polycyclic aromatic hydrocarbon-degrading bacteria for biocatalytic resolution of racemic styrene oxide and styrene oxide derivatives

A novel epoxide hydrolase (EHase) from polycyclic aromatic hydrocarbon (PAH)-degrading bacteria was identified and characterized. EHase activity was identified in four strains of PAH-degrading bacteria isolated from commercial gasoline and oil-contaminated sediment based on their growth on styrene oxide and its derivatives, such as 2,3- and 4-chlorostyrene oxides, as a sole carbon source. Gordonia sp. H37 exhibited high enantioselective hydrolysis activity for 4-chlorostyrene oxide with an enantiomeric ratio of 27. Gordonia sp. H37 preferentially hydrolyzed the (R)-enantiomer of styrene oxide derivatives resulting in the preparation of a (S)-enantiomer with enantiomeric excess greater than 99.9 %. The enantioselective EHase activity was identified and characterized in various PAH-degrading bacteria, and whole cell Gordonia sp. H37 was employed as a biocatalyst for preparing enantiopure (S)-styrene oxide derivatives.     

Rifamycin: XXXIII. Isolation of Actinophages Active on Streptomyces mediterranei and Characteristics of Phage-Resistant Strains

Five actinophages highly specific for Streptomyces mediterranei were isolated from lysed broth cultures. Studies were performed on the effect of plating conditions on plaque formation. The development of phage-resistant strains of S. mediterranei not only eliminated the phage but also significantly increased rifamycin yields. The phage-resistant cultures proved to be more unstable than the original sensitive strain. Maintenance of the cultures as frozen vegetative mycelium assured culture stability and reproducibility of the results. Strict aseptic precautions throughout the laboratories and fermentation areas did not eliminate the danger of phage infection; effective control was obtained only with the introduction of resistant strains. S. mediterranei phages proved to be highly specific for calcium as an adsorption cofactor; addition of calcium-sequestering agents to sensitive mycelium completely prevented its lysis by the phage. The resistant strains developed were capable of adsorbing the phage and of releasing it without multiplication upon aging of the mycelium. No marked morphological, cultural, or biochemical differences were found among the various phage-resistant strains.     

Rifamycin: XXXIV. Physicochemical Characterization of Actinophages Active on Streptomyces mediterranei

Five actinophages highly specific for Streptomyces mediterranei were isolated from lysed broth cultures. Studies were performed on the effect of plating conditions on plaque formation. The development of phage-resistant strains of S. mediterranei not only eliminated the phage but also significantly increased rifamycin yields. The phage-resistant cultures proved to be more unstable than the original sensitive strain. Maintenance of the cultures as frozen vegetative mycelium assured culture stability and reproducibility of the results. Strict aseptic precautions throughout the laboratories and fermentation areas did not eliminate the danger of phage infection; effective control was obtained only with the introduction of resistant strains. S. mediterranei phages proved to be highly specific for calcium as an adsorption cofactor; addition of calcium-sequestering agents to sensitive mycelium completely prevented its lysis by the phage. The resistant strains developed were capable of adsorbing the phage and of releasing it without multiplication upon aging of the mycelium. No marked morphological, cultural, or biochemical differences were found among the various phage-resistant strains.     

A rapid and reproducible assay for collagenase using [1-14C]acetylated collagen.

A rapid, continuous, and highly sensitive fluorescence assay is described for the measurement of epoxide hydrase activity. The method is based on the large differences between the fluorescence spectra of certain K-region arene oxides and their corresponding trans-dihydrodiols. Enzymatic hydration of K-region arene oxides of phenanthrene, pyrene, benzo[a]pyrene, and 7,12-dimethylbenzo[a]anthracene was studied. The assay was most sensitive with benzo[a]pyrene-4,5-oxide as substrate. With 10 μm benzo[a]pyrene-4,5-oxide, enzymatic rates of 30 pmol of dihydrodiol/min/mg of protein are three to five times those of the blank without enzyme. The fluorometric method described has been used to study site-directed inhibitors of epoxide hydrase and the stereoselective hydration of racemic arene oxides.     

Purification and specificity of a human microsomal epoxide hydratase

Epoxide hydratase was solubilized from human liver microsomal fractions and purified to an extent where the specific activity was 40-fold greater than that of the liver homogenate. Combination of homogenate and purified preparation showed that the increase in activity was not due to the removal of an inhibitor. Monosubstituted oxiranes with a lipophilic substituent larger than an ethyl group (isopropyl, t-butyl, n-hexyl, phenyl) readily interacted as substrates or inhibitors with this purified human epoxide hydratase, whereas those with a small substituent (methyl, ethyl, vinyl) were inactive, probably reflecting greater affinity of the former epoxides owing to lipophilic binding sites near the active site of the enzyme. In a series of oxiranes having a lipophilic substituent of sufficient size (styrene oxides), monosubstituted as well as 1,1- and cis-1,2-disubstituted oxiranes readily served as substrates or inhibitors of the enzyme, but not the trans-1,2-disubstituted, tri- or tetra-substituted oxiranes. trans-Substitution at the oxirane ring apparently prevents access of the oxirane ring to the active site by steric hindrance. Epoxide hydratase was also solubilized from microsomal fractions of rat and guinea-pig liver and purified by the same procedure. Structural requirements for effective interaction of substrates, inhibitors and activators were qualitatively identical for epoxide hydratase from the three sources. However, several quantitative differences were observed. Thus human hepatic epoxide hydratase seems to be very similar to, although not identical with, the enzyme from guinea pig or rat. Studies with epoxide hydratase from the latter two species therefore appear to be significant with respect to man. In addition, knowledge of structural requirements for epoxides to serve as substrates for human epoxide hydratase may prove useful for drug design. Compounds which need aromatic or olefinic moieties for their desired effect would not be expected to lead to accumulation of epoxides if their structure was such as to allow for a metabolically produced epoxide to be rapidly consumed by epoxide hydratase.     

Epoxide hydrase and mixed-function oxidase activities of rat liver nuclear membranes.

Rat liver nuclei have 2 to 12% of the corresponding microsomal aryl hydrocarbon hydroxylase, aminopyrine and benzphetamine N-demethylase, NADPH-cytochrome c reductase, and epoxide hydrase activities. Nuclear membranes were prepared from isolated liver nuclei by a sucrose density centrifugation technique. A 2.5- to 10.2-fold increase in the specific enzyme activities was observed in nuclear membrane as compared to intact nuclei. Several properties of the rat liver nuclear membrane and microsomal epoxide hydrase have been compared. Nuclear epoxide hydrase was similar to the corresponding microsomal enzyme in being induced by phenobarbital whereas 3-methylcholanthrene did not produce any effects. Nuclear membrane and microsomal epoxide hydrase were inhibited to a similar degree by 1,1,1-trichloropropene oxide, cyclohexene oxide, an trans-stilbene oxide. The apparent K_m value of nuclear membrane epoxide hydrase was 20 μm for benzo(a)pyrene 4,5-oxide, which is 5.5-fold lower than the corresponding microsomal K_m value (112 μm). Nuclear membranes were prepared from isolated nuclei of rat kidney, lung, spleen, and heart by the DNase digestion method. Epoxide hydrase activity in intact nuclei was in the following order: kidney > lung ? spleen, or heart. Increases of 2.2- and 2.5-fold in specific epoxide hydrase activity were observed in kidney and lung when nuclear membranes were compared to intact nuclei. DMSO, dimethylsulfoxide     

Design,synthesis and evaluation of hydrazine and acyl hydrazone derivatives of 5-pyrrolidin-2-one as antifungal agents

Twenty-eight 5-pyrrolidine-2-ones decorated by hydrazine or acyl hydrazones groups have been designed, synthesized and evaluated as antifungal agents on a panel of twelve fungal strains and three non albicans candida yeasts species which have demonstrated reduced susceptibility to commonly used antifungal drugs. Half of the target compounds exhibited good to high antifungal activities on at least one strain with MIC₅₀ lower than the control antifungal agent – hymexazol or ketoconazole. 5-Arylhydrazino-pyrrolidin-2-ones were found active and the –NH-NH- linker proved to be essential to maintain the antifungal potential. Compound 2a is a broad-spectrum antifungal, active on 60% of the tested strains. Replacing the hydrazine linker by an acylhydrazone one narrowed the spectrum of activity but pyroglutamylaryl hydrazones, mainly aromatic ones, exhibited good activity, adequate “fungicide-like” properties and were devoted of cytotoxicity.     

Isolation and characterization of Xenopus soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) contributes to cell growth, but the contribution of sEH to embryonic development is not well understood. In this study, Xenopus sEH cDNA was isolated from embryos of Xenopus laevis. The Xenopus sEH was expressed in Escherichia coli and was purified. The epoxide hydrolase and phosphatase activities of purified sEH were investigated. The Xenopus sEH did not show phosphatase activity toward 4-methylumbelliferyl phosphate or several lysophosphatidic acids although it had EH activity. The amino acid sequence of Xenopus sEH was compared with that reported previously. We found amino acid substitutions of the 29th Thr to Asn and the 146th Arg to His and prepared a sEH mutant (N29T/H146R), designed as mutant 1. Neither wild-type sEH nor mutant 1 had phosphatase activity. Additional substitution of the 11th Gly with Asp was found by comparison with human sEH which has phosphatase activity, but the Xenopus sEH mutant G11D prepared as mutant 2 did not have phosphatase activity. The epoxide hydrolase activity of sEH seemed to be similar to that of human sEH, while Xenopus sEH did not have phosphatase activity toward several substrates that human sEH metabolizes.     

Epoxide hydrolases from yeasts and other sources: versatile tools in biocatalysis

Major characteristics, substrate specificities and enantioselectivities of epoxide hydrolases from various sources are described. Epoxide hydrolase activity in yeasts is discussed in more detail and is compared with activities in other microorganisms. Constitutively produced bacterial epoxide hydrolases are highly enantioselective in the hydrolysis of 2,2- and 2,3-disubstituted epoxides. A novel bacterial limonene-1,2-epoxide hydrolase, induced by growth on monoterpenes, showed high activities and selectivities in the hydrolysis of several substituted alicyclic epoxides. Constitutively produced epoxide hydrolases are found in eukaryotic microorganisms. Enzymes from filamentous fungi are useful biocatalysts in the resolution of aryl- and substituted alicyclic epoxides. Yeast epoxide hydrolase activity has been demonstrated for the enantioselective hydrolysis of various aryl-, alicyclic- and aliphatic epoxides by a strain of Rhodotorula glutinis. The yeast enzyme, moreover, is capable of asymmetric hydrolysis of meso epoxides and performs highly enantioselective resolution of unbranched aliphatic 1,2-epoxides. Screening for other yeast epoxide hydrolases shows that high enantioselectivity is restricted to a few basidiomycetes genera only. Resolution of very high substrate concentrations is possible by using selected basidiomycetes yeast strains.     

In Vitro Nitrogen Fixation by Two Actinomycete Strains Isolated from Casuarina Nodules

Acetylene reduction activity was demonstrated in pure cultures of two actinomycete strains isolated from nodules of Casuarina equisetifolia. This activity was comparable to that of free-living Rhizobium strains, but appeared to be less sensitive to pO₂ and more sensitive to the presence of combined nitrogen.