Simple Detection Methods for Antinutritive Factor β-ODAP Present in Lathyrus sativus L. by High Pressure Liquid Chromatography and Thin Layer Chromatography

Lathyrus sativus L. (Grass pea) is the source for cheap and nutritious food choice in drought and famine susceptible zones in greater part of North India and Africa. The non-protein amino acid β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP) has been known for decades for its potent neurotoxic effect, causing irreversible neurodegenerative disease “neurolathyrism”, present in both seed and leaf of Lathyrus sativus L. and other species in varying proportions. It is crucial to establish a rapid as well as reliable detection methodology for β-ODAP content in various Lathyrus plants. Currently available HPLC based methods involve multi-step derivatization of the sample. To overcome this, we have developed β-ODAP analysis method by HPLC without any prior derivatization. This method is statistically significant in the range of 2 to 100μg/ml and exhibited linear response with r ² > 0.99. Limit of detection and quantitation of the later method was determined to be 5.56 μg/ml and 16.86 μg/ml, respectively. In addition to this, a TLC based method has also been developed. The limit of detection of β-ODAP is 0.6μg and for its substrate, L-1,2-diaminopropionic acid is 5μg. Both HPLC and TLC methods were validated by conducting in-vitro bioconversion test to detect the presence of biocatalyst in plant extract. This method is economical, rapid and simple.     

Angiotensin I-Converting-Enzyme-Inhibitory and Antibacterial Peptides from Lactobacillus helveticus PR4 Proteinase-Hydrolyzed Caseins of Milk from Six Species

Sodium caseinates prepared from bovine, sheep, goat, pig, buffalo or human milk were hydrolyzed by a partially purified proteinase of Lactobacillus helveticus PR4. Peptides in each hydrolysate were fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest angiotensin I-converting-enzyme (ACE)-inhibitory or antibacterial activity were sequenced by mass spectrum and Edman degradation analyses. Various ACE-inhibitory peptides were found in the hydrolysates: the bovine α_S1-casein (α_S1-CN) 24-47 fragment (f24-47), f169-193, and β-CN f58-76; ovine α_S1-CN f1-6 and α_S2-CN f182-185 and f186-188; caprine β-CN f58-65 and α_S2-CN f182-187; buffalo β-CN f58-66; and a mixture of three tripeptides originating from human β-CN. A mixture of peptides with a C-terminal sequence, Pro-Gly-Pro, was found in the most active fraction of the pig sodium caseinate hydrolysate. The highest ACE-inhibitory activity of some peptides corresponded to the concentration of the ACE inhibitor (S)-N-(1-[ethoxycarbonyl]-3-phenylpropyl)-ala-pro maleate (enalapril) of 49.253 μg/ml (100 μmol/liter). Several of the above sequences had features in common with other ACE-inhibitory peptides reported in the literature. The 50% inhibitory concentration (IC₅₀) of some of the crude peptide fractions was very low (16 to 100 μg/ml). Some identified peptides were chemically synthesized, and the ACE-inhibitory activity and IC₅₀s were confirmed. An antibacterial peptide corresponding to β-CN f184-210 was identified in human sodium caseinate hydrolysate. It showed a very large spectrum of inhibition against gram-positive and -negative bacteria, including species of potential clinical interest, such as Enterococcus faecium, Bacillus megaterium, Escherichia coli, Listeria innocua, Salmonella spp., Yersinia enterocolitica, and Staphylococcus aureus. The MIC for E. coli F19 was ca. 50 μg/ml. Once generated, the bioactive peptides were resistant to further degradation by proteinase of L. helveticus PR4 or by trypsin and chymotrypsin.     

Nematicidal Activity of α -Chaconine: Effect of Hydrogen-ion Concentration

α-Chaconine, a steroid-glycoalkaloid from Solanum tuberosum L., was increasingly more toxic to a free-living nematode, Panagrellus redivivus, with decreasing acidity from about pH 5 to 7. A study of the toxicity to adult nematodes at three concentrations of α-chaconine in buffer from pH 4 to 7.5 indicated that the free base is the nematicidal form of the compound. The median effective doses (ED₅₀) of α-chaconine to inhibit the motility of P. redivivus were estimated as 85 μg/ml at pH 6.7, 170 μg/ml at pH 6.5, and 340 μg/ml at pH 6.2.     

In Vitro Evaluation of the Inhibitory Potential of Pharmaceutical Excipients on Human Carboxylesterase 1A and 2

Two major forms of human carboxylesterase (CES), CES1A and CES2, dominate the pharmacokinetics of most prodrugs such as imidapril and irinotecan (CPT-11). Excipients, largely used as insert vehicles in formulation, have been recently reported to affect drug enzyme activity. The influence of excipients on the activity of CES remains undefined. In this study, the inhibitory effects of 25 excipients on the activities of CES1A1 and CES2 were evaluated. Imidapril and CPT-11 were used as substrates and cultured with liver microsomes in vitro. Imidapril hydrolase activities of recombinant CES1A1 and human liver microsomes (HLM) were strongly inhibited by sodium lauryl sulphate (SLS) and polyoxyl 40 hydrogenated castor oil (RH40) [Inhibition constant (K_i) = 0.04±0.01 μg/ml and 0.20±0.09 μg/ml for CES1A1, and 0.12±0.03 μg/ml and 0.76±0.33 μg/ml, respectively, for HLM]. The enzyme hydrolase activity of recombinant CES2 was substantially inhibited by Tween 20 and polyoxyl 35 castor oil (EL35) (K_i = 0.93±0.36 μg/ml and 4.4±1.24 μg/ml, respectively). Thus, these results demonstrate that surfactants such as SLS, RH40, Tween 20 and EL35 may attenuate the CES activity; such inhibition should be taken into consideration during drug administration.     

Propofol Treatment Inhibits Constitutive Apoptosis in Human Primary Neutrophils and Granulocyte-Differentiated Human HL60 Cells

Apoptosis regulation is essential for neutrophil homeostasis. We previously demonstrated that a process involving glycogen synthase kinase (GSK)-3β determines neutrophil apoptosis. As for this apoptotic process, an overdose of propofol (2,6-Diisopropylphenol; 25 μg/ml or 140 μM) also causes GSK-3β-mediated macrophage apoptosis; however, the early deactivation of GSK-3β with low-dose propofol has been shown. Therefore, we hypothesize that low-dose propofol may induce neutrophil survival via GSK-3β inactivation. Following in vitro culture, the therapeutic concentration of propofol (10 μg/ml or 56 μM) treatment decreased constitutive apoptosis in isolated human primary neutrophils and in granulocyte-differentiated HL60 cells after all-trans retinoic acid (1 μM) treatment. The inactivation of phosphatidylinositol 3-kinase (PI3-kinase)/AKT and the activation of GSK-3β results in myeloid cell leukemia 1 (Mcl-1) down-regulation, the loss of the mitochondrial transmembrane potential, and caspase-3 activation in these cells, which is accompanied by apoptosis. Notably, propofol treatment attenuates these effects in a PI3-kinase-regulated manner. We found that propofol initiates PI3-kinase/AKT-mediated GSK-3β inactivation and Mcl-1 stabilization, rescuing the constitutive apoptosis in primary neutrophils and granulocyte-differentiated acute promyelocytic leukemia HL60 cells.     

Aqueous and Alcoholic Extracts of Triphala and Their Active Compounds Chebulagic Acid and Chebulinic Acid Prevented Epithelial to Mesenchymal Transition in Retinal Pigment Epithelial Cells,by Inhibiting SMAD-3 Phosphorylation

Epithelial to Mesenchymal Transition (EMT) of the retinal pigment epithelium is involved in the pathogenesis of proliferative vitreoretinopathy (PVR) that often leads to retinal detachment. In this study, Triphala, an ayurvedic formulation and two of its active ingredients, namely chebulagic acid and chebulinic acid were evaluated for anti-EMT properties based on in vitro experiments in human retinal pigment epithelial cell line (ARPE-19) under TGFβ1 induced conditions. ARPE-19 cells were treated with TGFβ1 alone or co-treated with various concentrations of aqueous extract (AqE) (30 - 300 μg/ml); alcoholic extract (AlE) (50 - 500 μg/ml) of triphala and the active principles chebulagic acid (CA) and chebulinic acid (CI) (CA,CI: 50 - 200 μM). The expression of EMT markers namely MMP-2, αSMA, vimentin and the tight junction protein ZO-1 were evaluated by qPCR, western blot and immunofluorescence. The functional implications of EMT, namely migration and proliferation of cells were assessed by proliferation assay, scratch assay and transwell migration assay. AqE, AlE, CA and CI reduced the expression and activity of MMP-2 at an ED50 value of 100 μg/ml, 50 μg/ml, 100 μM and 100 μM, respectively. At these concentrations, a significant down-regulation of the expression of αSMA, vimentin and up-regulation of the expression of ZO-1 altered by TGFβ1 were observed. These concentrations also inhibited proliferation and migration of ARPE-19 cells induced by TGFβ1. EMT was found to be induced in ARPE-19 cells, through SMAD-3 phosphorylation and it was inhibited by AqE, AlE, CA and CI. Further studies in experimental animals are required to attribute therapeutic potential of these extracts and their active compounds, as an adjuvant therapy in the disease management of PVR.     

Kinetics of growth retardant and hormone interactions in affecting cucumber hypocotyl elongation

The capacities of indole-3-acetic acid (IAA) and gibberellin A₃ (GA₃) to counteract the inhibitory effects of (2-chloroethyl) trimethylammonium chloride (CCC), 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride (Amo-1618), and N,N-dimethylaminosuccinamic acid (B-995) on hypocotyl elongation in light-grown cucumber (Cucumis sativus L.) seedlings were investigated. One μg of GA₃ applied to the shoot tip was sufficient to completely nullify the effect of 10 μg of Amo-1618 or 25 μg of B-995 applied simultaneously to the shoot tip, and 10 μg of GA₃ completely counteracted the effect of 10⁻³ m CCC added to the root medium. One μg of IAA counteracted the effect of 10⁻³ m CCC in the root medium, but IAA did not nullify the action of either Amo-1618 or B-995. Experiments were conducted using 2 growth retardants simultaneously, which indicated that Amo-1618 and CCC inhibit a common process, namely GA biosynthesis, essential to hypocotyl elongation. However, since the effect of CCC was overcome by applications of both GA and IAA, growth retardation resulting from treatment with CCC apparently is not due solely to inhibition of GA biosynthesis. B-995 did not interact additively with either Amo-1618 or CCC, which suggests that B-995 affects a process different from those affected by the other 2 retardants. Thus, while inhibition evoked by B-995 is reversible by applied GA, the action of B-995 does not appear to be inhibition of GA biosynthesis.     

Effect of Bacillus thuringiensis Cry1 Toxins in Insect Hemolymph and Their Neurotoxicity in Brain Cells of Lymantria dispar

Little information is available on the systemic effects of Bacillus thuringiensis toxins in the hemocoel of insects. In order to test whether B. thuringiensis-activated toxins elicit a toxic response in the hemocoel, we measured the effect of intrahemocoelic injections of several Cry1 toxins on the food intake, growth, and survival of Lymantria dispar (Lepidoptera) and Neobellieria bullata (Diptera) larvae. Injection of Cry1C was highly toxic to the Lymantria larvae and resulted in the complete inhibition of food intake, growth arrest, and death in a dose-dependent manner. Cry1Aa and Cry1Ab (5 μg/0.2 g [fresh weight] [g fresh wt]) also affected growth and food intake but were less toxic than Cry1C (0.5 μg/0.2 g fresh wt). Cry1E and Cry1Ac (5 μg/0.2 g fresh wt) had no toxic effect upon injection. Cry1C was also highly toxic to N. bullata larvae upon injection. Injection of 5 μg/0.2 g fresh wt resulted in rapid paralysis, followed by hemocytic melanization and death. Lower concentrations delayed pupariation or gave rise to malformation of the puparium. Finally, Cry1C was toxic to brain cells of Lymantria in vitro. The addition of Cry1C (20 μg/ml) to primary cultures of Lymantria brain cells resulted in rapid lysis of the cultured neurons.     

In Vitro Sensitivity of Torulopsis glabrata to Amphotericin B, 5-Fluorocytosine, and Clotrimazole (Bay 5097)

Thirty-five strains of Torulopsis glabrata were tested by a tube dilution method for their susceptibility to amphotericin B, 5-fluorocytosine, and clotrimazole (Bay 5097). Amphotericin B was the most active in vitro, inhibiting all strains at a concentration of 1 μg/ml and killing all strains at 2 μg/ml. 5-Fluorocytosine inhibited over 80% of strains at 0.24 μg/ml, but three strains required ≥7.8 μg/ml for killing. A concentration of 2 μg of clotrimazole per ml inhibited less than 50% of strains, and 8 μg/ml killed only 10% of strains. Most strains of T. glabrata were killed by therapeutically achievable concentrations of amphotericin B and 5-fluorocytosine, but not clotrimazole.     

Effect of Monensin and Lasalocid-Sodium on the Growth of Methanogenic and Rumen Saccharolytic Bacteria

It is thought that monensin increases the efficiency of feed utilization by cattle by altering the rumen fermentation. We studied the effect of monensin and the related ionophore antibiotic lasalocid-sodium (Hoffman-LaRoche) on the growth of methanogenic and rumen saccharolytic bacteria in a complex medium containing rumen fluid. Ruminococcus albus, Ruminococcus flavefaciens, and Butyrivibrio fibrisolvens were inhibited by 2.5 μg of monensin or lasalocid per ml. Growth of Bacteroides succinogenes and Bacteroides ruminicola was delayed by 2.5 μg of monensin or lasalocid per ml. Populations of B. succinogenes and B. ruminicola that were resistant to 20 μg of either drug per ml were rapidly selected by growth in the presence of each drug at 5.0 μg/ml. Selenomonas ruminantium was insensitive to 40 μg of monensin or lasalocid per ml. Either antibiotic (10 μg/ml) inhibited Methanobacterium MOH, Methanobacterium formicicum, and Methanosarcina barkeri MS. Methanobacterium ruminantium PS was insensitive to 40 μg of monensin or 20 μg of lasalocid per ml. The methanogenic strain 442 was insensitive to 40 μg of monensin but sensitive to 10 μg of lasalocid per ml. The results suggest that monensin or lasalocid acts in the rumen by selecting for succinate-forming Bacteroides and for S. ruminantium, a propionate producer that decarboxylates succinate to propionate. The selection could lead to an increase in rumen propionate formation. Selection against H₂ and formate producers, e.g. R. albus, R. flavefaciens, and B. fibrisolvens, could lead to a depression of methane production in the rumen.     

Effect of Water Extracts of Carob Pods, Tannic Acid, and Their Derivatives on the Morphology and Growth of Microorganisms

The effect of aqueous extracts of carob (Ceratonia siliqua) pods, gallotannic acid, gallic acid, and catechol on several microorganisms was studied. Carob pod extract and tannic acid showed a strong antimicrobial activity toward some cellulolytic bacteria. On the basis of tannin content, to which antimicrobial effect was related, carob pod extracts inhibited Cellvibrio fulvus and Clostridium cellulosolvens at 15 μg/ml, Sporocytophaga myxococcoides at 45 μg/ml, and Bacillus subtilis at 75 μg/ml. The inhibiting concentrations for tannic acid were found to be 12, 10, 45, and 30 μg/ml, respectively. Gallic acid and catechol were much less effective. Tannic acid and the tannin fraction of carob extract exerted both bacteriostatic and bactericidal effects on C. fulvus. Respiration of C. fulvus in the presence of bactericidal concentrations of tannic acid or tannin fraction of carob extract was inhibited less than 30%. A partial formation of “protoplasts” by C. fulvus was obtained after 2 hr of incubation in a growth medium to which 20% sucrose, 0.15% MgSO₄·7H₂O, and 10 to 50 μg/ml of tannic acid or 500μg/ml of penicillin, or both, had been added. Tannic acid and the tannin fraction of carob extract protected C. fulvus from metabolic lysis in sucrose solution. Although the growth of other microorganisms tested was only slightly affected, the morphology of some of them was drastically changed in the presence of subinhibitory concentrations of carob pod extracts of tannic acid. It is suggested that the site of action of tannins on sensitive microorganisms is primarily the cell envelope.     

Correspondence of High Levels of Beta-Exotoxin I and the Presence of cry1B in Bacillus thuringiensis

Examination of 640 natural isolates of Bacillus thuringiensis showed that the 58 strains (9%) whose supernatants were toxic to Anthonomus grandis (Coleoptera: Curculionidae) produced between 10 and 175 μg of β-exotoxin I per ml. We also found that 55 (46%) of a sample of 118 strains whose culture supernatants were not toxic to A. grandis nevertheless produced between 2 and 5 μg/ml. However, these amounts of β-exotoxin I were below the threshold for detectable toxicity against this insect species. Secretion of large amounts of β-exotoxin I was strongly associated with the presence of cry1B and vip2 genes in the 640 natural B. thuringiensis isolates studied. We concluded that strains carrying cry1B and vip2 genes also possess, on the same plasmid, genetic determinants necessary to promote high levels of production of β-exotoxin I.     

Susceptibility of Phytopathogenic Bacteria to Wheat Purothionins In Vitro

Purothionins are basic polypeptides with antimicrobial properties that are present in the endosperm of wheat and other cereal species. Susceptibility to wheat purothionins among phytopathogenic bacteria of the genera Pseudomonas, Xanthomonas, Agrobacterium, Erwinia, and Corynebacterium has been investigated. Sensitive strains have been found in all of these genera except Agrobacterium (the only strain of A. tumefaciens available proved to be resistant). Minimal inhibitory concentrations (MIC) with partially purified crude purothionins ranged from 1 μg/ml for C. sepedonicum (C.5) to 540 μg/ml for E. amylovora (E.3). Minimal bactericidal concentrations (MBC) were not higher than twice the MIC value, except for C. poinsettiae (C.4) (MBC/MIC = 8). Purothionins α and β, obtained by carboxymethyl-cellulose column chromatography, were tested against P. solanacearum (P.2) and X. phaseoli (X.2); α purothionin was more active than β against X.2, and β more active than α against P.2. This suggests a relationship between polypeptide sequence and specificity of action.     

Growth of Potato and Control of Pratylenchus penetrans with Oxamyl-treated Seed Pieces in Greenhouse Studies

Oxamyl was applied to both uncut and cut potato tubers in aqueous solutions of 1,000 to 32,000 μg/ml. Emergence in greenhouse pots was delayed for a day or more after soaking cut tuber pieces in 32,000 μg/ml. After 10 weeks plant growth was greater, relative to the control, when Pratylenchus penetrans-infested soil was planted with cut tubers soaked for 20 minutes in 32,000 μg/ml. Soaking for 40 minutes did not increase nematode control nor affect plant growth. Oxamyl applied to tubers at 1,000 μg/ml reduced the numbers of P. penetrans in the soil by 20% and in the roots by 35%; at 32,000 μg/ml, the numbers of P. penetrans in the soil were reduced by 73-86% and in the roots by 86-97%. The numbers of P. penetrans did not increase in the roots of plants developed from cut tubers soaked in 32,000 μg/ml over a period of 10 weeks, but numbers of lesion nematodes had begun to increase in the soil.     

Carprofen inhibits the release of matrix metalloproteinases 1, 3, and 13 in the secretome of an explant model of articular cartilage stimulated with interleukin 1β

Introduction

Arthritic diseases are characterized by the degradation of collagenous and noncollagenous extracellular matrix (ECM) components in articular cartilage. The increased expression and activity of matrix metalloproteinases (MMPs) is partly responsible for cartilage degradation. This study used proteomics to identify inflammatory proteins and catabolic enzymes released in a serum-free explant model of articular cartilage stimulated with the pro-inflammatory cytokine interleukin 1β (IL-1β). Western blotting was used to quantify the release of selected proteins in the presence or absence of the cyclooxygenase-2 specific nonsteroidal pro-inflammatory drug carprofen.

Methods

Cartilage explant cultures were established by using metacarpophalangeal joints from horses euthanized for purposes other than research. Samples were treated as follows: no treatment (control), IL-1β (10 ng/ml), carprofen (100 μg/ml), and carprofen (100 μg/ml) + IL-1β (10 ng/ml). Explants were incubated (37°C, 5% CO₂) over twelve day time courses. High-throughput nano liquid chromatography/mass spectrometry/mass spectrometry uncovered candidate proteins for quantitative western blot analysis. Proteoglycan loss was assessed by using the dimethylmethylene blue (DMMB) assay, which measures the release of sulfated glycosaminoglycans (GAGs).

Results

Mass spectrometry identified MMP-1, -3, -13, and the ECM constituents thrombospondin-1 (TSP-1) and fibronectin-1 (FN1). IL-1β stimulation increased the release of all three MMPs. IL-1β also stimulated the fragmentation of FN1 and increased chondrocyte cell death (as assessed by β-actin release). Addition of carprofen significantly decreased MMP release and the appearance of a 60 kDa fragment of FN1 without causing any detectable cytotoxicity to chondrocytes. DMMB assays suggested that carprofen initially inhibited IL-1β-induced GAG release, but this effect was transient. Overall, during the two time courses, GAG release was 58.67% ± 10.91% (SD) for IL-1β versus 52.91% ± 9.35% (SD) with carprofen + IL-1β.

Conclusions

Carprofen exhibits beneficial anti-inflammatory and anti-catabolic effects in vitro without causing any detectable cytotoxicity. Combining proteomics with this explant model provides a sensitive screening system for anti-inflammatory compounds.     

The Potential Regimen of Target-Controlled Infusion of Propofol in Flexible Bronchoscopy Sedation: A Randomized Controlled Trial

Objectives

Target-controlled infusion (TCI) provides precise pharmacokinetic control of propofol concentration in the effect-site (Ce), eg. brain. This pilot study aims to evaluate the feasibility and optimal TCI regimen for flexible bronchoscopy (FB) sedation.

Methods

After alfentanil bolus, initial induction Ce of propofol was targeted at 2 μg/ml. Patients were randomized into three titration groups (i.e., by 0.5, 0.2 and 0.1 μg/ml, respectively) to maintain stable sedation levels and vital signs. Adverse events, frequency of adjustments, drug doses, and induction and recovery times were recorded.

Results

The study was closed early due to significantly severe hypoxemia events (oxyhemoglobin saturation <70%) in the group titrated at 0.5 μg/ml. Forty-nine, 49 and 46 patients were enrolled into the 3 respective groups before study closure. The proportion of patients with hypoxemia events differed significantly between groups (67.3 vs. 46.9 vs. 41.3%, p = 0.027). Hypotension events, induction and recovery time and propofol doses were not different. The Ce of induction differed significantly between groups (2.4±0.5 vs. 2.1±0.4 vs. 2.1±0.3 μg/ml, p = 0.005) and the Ce of procedures was higher at 0.5 μg/ml titration (2.4±0.5 vs. 2.1±0.4 vs. 2.2±0.3 μg/ml, p = 0.006). The adjustment frequency tended to be higher for titration at 0.1 μg/ml but was not statistically significant (2 (0∼6) vs. 3 (0∼6) vs. 3 (0∼11)). Subgroup analysis revealed 14% of all patients required no further adjustment during the whole sedation. Comparing patients requiring at least one adjustment with those who did not, they were observed to have a shorter induction time (87.6±34.9 vs. 226.9±147.9 sec, p<0.001), a smaller induction dose and Ce (32.5±4.1 vs. 56.8±22.7 mg, p<0.001; 1.76±0.17 vs. 2.28 ±0.41, p<0.001, respectively), and less hypoxemia and hypotension (15.8 vs.56.9%, p = 0.001; 0 vs. 24.1%, p = 0.008, respectively).

Conclusion

Titration at 0.5 μg/ml is risky for FB sedation. A subgroup of patients required no more TCI adjustment with fewer complications. Further studies are warranted to determine the optimal regimen of TCI for FB sedation.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01101477","term_id":"NCT01101477"}}NCT01101477     

Supplemental Substrate Enhancement of 2,4-Dinitrophenol Mineralization by a Bacterial Consortium

A Janthinobacterium sp. and an actinomycete, both capable of mineralizing 2,4-dinitrophenol (DNP), were used to construct a consortium to mineralize DNP in nonaxenic bench-scale sequencing batch reactors (SBRs). Average K_m values for DNP mineralization by pure cultures of the Janthinobacterium sp. and the actinomycete were 0.01 and 0.13 μg/ml, respectively, and the average maximum specific growth rate (μ_max) values for them were 0.06 and 0.23/h, respectively. In the presence of NH₄Cl, nitrite accumulation in pure culture experiments and in the SBRs was stoichiometric to initial DNP concentration and the addition of nitrogen enhanced DNP mineralization in the SBRs. Mineralization of 10 μg of DNP per ml was further enhanced in SBRs by the addition of glucose at concentrations of 100 and 500 μg/ml but not at 10 μg/ml. Possible mechanisms for this enhanced DNP mineralization in SBRs were suggested by kinetic analyses and biomass measurements. Average μ_max values for DNP mineralization in the presence of 0, 10, 100, and 500 μg of glucose per ml were 0.33, 0.13, 0.42, and 0.59/h, respectively. In addition, there was greater standing biomass in reactors amended with glucose. At steady-state operation, all SBRs contained heterogeneous microbial communities but only one organism, an actinomycete, that was capable of mineralizing DNP. This research demonstrates the usefulness of supplemental substrates for enhancing the degradation of toxic chemicals in bioreactors that contain heterogeneous microbial communities.     

Benomyl Tolerance of Ten Fungi Antagonistic to Plant-parasitic Nematodes

Ten strains of fungi were tested for tolerance to the fungicide benomyl. Verticillium chlamydosporium strain 2 did not grow in the presence of benomyl; Drechraeria coniospora strains 1 and 2 and Chaetomium sp. tolerated only 0.1 μg benomyl/ml medium; Acremonium bacillisporum, an unidentified fungus, and Phoma chrysanthemicola uniformly grew at 1 μg/ml, but some hyphae grew at higher benomyl concentrations; Fusarium sp. tolerated 475 μg/ml, but some hyphae grew on medium amended with 1,000 μg/ml; Verticillium lecanii and V. chlamydosporium strain 1 routinely tolerated 1,000 μg/ml. Fungi generally grew more slowly at higher than at lower benomyl concentrations. Strains with elevated tolerance to benomyl were selected from Acremonium bacillisporum, Drechmeria coniospora, Fusarium sp., and an unidentified fungus. These strains retained the increased tolerance after repeated transfers on unamended medium.