Acriflavin Inhibition of Morphogenesis in Naegleria gruberi*

SYNOPSIS. When amoeboid organisms of Naegleria gruberi are treated with acriflavin at concentrations under 70 μg/ml, they still can form flagellates. Acriflavin between 70 μg/ml and 110 μg/ml increasingly inhibits morphogenesis. The effect of acriflavin is specific for the morphogenetic system since amoeboid organisms treated with acriflavin between 110 and 200 μg/ml are not otherwise inhibited and behave like the controls.     

Studies on root nodules of leguminous plants bioproduction of indole acetic acid by a Rhizobium sp. from a twiner Clitoria ternatea L.

The Rhizobium sp. isolated from the root nodules of Clitoria ternatea L., a leguminous twiner, produced a high amount of IAA (16.4 μg/ml) from tryptophan in an unsupplemented basal medium. The production of IAA started simultaneously with the growth and had no different growth and production phase. The growth and production were parallel and increased up to 45–50 h. The IAA production by the Rhizobium sp. was increased by 520% when the medium was supplemented with fructose (1.5%), MnSO₄ (1.0 μg/ml), riboflavin (0.10 μg/ml) and Triton X-100 (0.01%). The possible role of the rhizobial production of IAA on the rhizobia-legume symbiosis is discussed.     

Acriflavin-Induced Dyskinetoplastic Leishmania donovani Grown in Monkey Kidney Cell Culture

SYNOPSIS. Monkey kidney cells (LLC-MK₂) grown in flasks and on coverslips in Leighton tubes were used as host cells for the growth of the intracellular stage, Leishman-Donovan bodies (LDs), of Leishmania donovani obtained from hamster spleen. These parasitized cultures were then used to determine the ability of acriflavin to induce dyskinetoplastic LDs.
LD-infected cells were somewhat fewer in number than uninfected cells at all times except for the 1st day after infection. The parasites attained their maximum numbers on the 5th day after infection of the cultures having a 1.9-fold increase at that time.
When acriflavin was added to the cell culture medium (250 mμ/ml) the numbers of monkey kidney cells did not differ greatly from non-treated cultures until 6–7 days after treatment with acriflavin. Similarly, the numbers of LDs in acriflavin-treated cell cultures, altho somewhat below those of untreated cultures, did not differ greatly from them.
The combined effect of acriflavin and LDs reduced the numbers of monkey kidney cells in treated, LD-infected cell cultures more than either alone.
Dyskinetoplastic LDs appeared in considerable numbers in acriflavin-treated, LD-infected cell cultures. Dyskinetoplastic and normal LDs harvested from cell cultures were inoculated into NIH medium and incubated at 27 C for transformation into leptomonads. There was no indication that dyskinetoplastic LDs were capable of transforming into leptomonads.     

Biomethanation of spent wash: Heavy metal inhibition of methanogenesis in synthetic medium

Five heavy metals detected in distillery waste were lead (1.0–8.8 μg/ml), copper (1.7–15.7 μg/ml), zinc (3.1–11.8 μg/ml), iron (36.0–43.5 μg/ml), and manganese (3.0–5.1 μg/ml). Their toxicity to biomethanogenesis in a synthetic medium containing 1% sodium acetate, propionate, or butyrate was measured by batch fermentation, after cultivating the bacterial biomass semicontinuously. Lead, copper, and zinc in decreasing order were found to be toxic to biomethanogenesis. Lead at the concentration of 10 μg/ml completely stopped methane production. Iron did not produce any notable change in the process while manganese stimulated the rate of methane production. The toxicity of lead, copper, and zinc to methanogenic bacteria and methane production was dose-dependent but the growth of acetogenic bacteria was impaired at higher concentrations (2.5–10.0 μg/ml) of lead, copper, and zinc. Manganese stimulated the growth of only methanogenic bacteria, but not that of non-methanogenic bacteria or acetic acid production. The reduction in the synthesis of acetic acid via butyrate was more in the presence of these three metals than the synthesis of this acid via propionate.     

Metal Tolerance and Accumulation Ability of the Ni Hyperaccumulator Streptanthus polygaloides Gray (Brassicaceae)

High concentrations of metals occur in some plant species (termed hyperaccumulators), such as the Ni hyperaccumulator Streptanthus polygaloides. We determined the tolerance of S. polygaloides to, and its accumulation abilities for, six metals (Ni, Zn, Cu, Co, Mn, and Pb). Potting mix concentrations used for all metals ranged from 0 to 1200 μg/g dry weight. For Ni, a treatment of 1600 μg/g was included. For Mn, treatments of 1600, 2000, and 2500 μg/g also were used, and for Pb these concentrations plus 3500 μg/g were included. Germination, plant number per pot, and size at days 30 and 39, number of plants at the end of the experiment (day 49), flower production, and metal concentration in the aboveground biomass were documented. Lead and Ni showed no consistent effects on plant performance, but yielded increased tissue metal concentrations. Streptanthus polygaloides was more sensitive to Co, Cu, and Zn, as ≥ 400 mg/g significantly suppressed plant growth, survival, and flower production. Tissue metal concentrations also were increased to maxima of 1500 μg Co/g, 120 μg Cu/g, and 6000 μg Zn/g. Manganese affected S. polygaloides less markedly, as ≥ 800 mg/kg decreased growth, survival, and flower production. Maximum tissue Mn concentration was 2900 μg/g. We concluded that S. polygaloides would be an appropriate phytoextractor for soils contaminated with Ni or low levels of Co but would not be useful for Cu, Zn, Mn, and Pb.     

Production of indole acetic acid in root nodules and culture by a Rhizobium species from root nodules of the fodder legume Melilotus alba DESR.

The root nodules of Melilotus alba DESR ., a fodder legume, contained high amounts of IAA. A tryptophan pool present in the nodule might serve as a source of IAA production. Presence of IAA oxidase and peroxidase in the nodules indicated the metabolism of IAA, at least in part, in the nodules. The Rhizobium species isolated from the root nodules produced a high amount of IAA (190 μg/ml) from L-tryptophan supplemented basal medium. IAA production and microbial growth were coincident. The production of IAA by the Rhizobium sp. was increased by 315% when the medium was supplemented with lactose (1%), NiCl₂ (10 μg/ml), cetyl pyridinium chloride (0.5 μg/ml) and glutamic acid (0.4%), in addition to L-tryptophan (3 mg/ml). The possible role of the rhizobial production of IAA on the rhizobia-legume symbiosis is discussed.     

Variation in the sensitivity to metalaxyl of Pythium spp. isolated from carrot and other sources

Over a 3-yr period 261 isolates of 17 species of Pythium were tested for sensitivity to metalaxyl at concentrations of 5, 50 or 100 μ/ml. A wide range of responses was observed, from isolates where growth ceased at 5 μg/ml to those where growth at 100 μg/ml was similar to that of the untreated controls. In further tests isolates of 11 different species had ED50's < 1 μg/ml. A lower sensitivity was detected in isolates of six Pythium spp. where values in the range 1–10 μg/ml were obtained. This lower sensitivity was not related to previous known use of metalaxyl. Three isolates of Pythium dissotocum from sites where the fungicide had been used repeatedly had ED50's > 100 μg/ml and were considered resistant. The resistance was stable over a 2-yr period and isolates were cross-resistant to furalaxyl, benalaxyl, ofurace, cyprofuram and oxadixyl. Increasing concentrations of metalaxyl reduced or prevented the production of zoospores by four species of Pythium, although when zoospores were produced, this was followed by the normal processes of encystment and germination. Culturing P. dissotocum on different sub-lethal concentrations of metalaxyl for 18 wk did not induce a high level of resistance to the fungicide.     

Morphology and Physiology of Lyngbya nigra with Reference to Copper Toxicity

The effect of copper sulphate on morphology and physiology of Lyngbya nigra has been studied. The growth was inhibited in all treatments (0.4 to 80.0 μM) of copper sulphate. There were no apparent morphological changes up to 0.8 μM and during the first two days of treatment even in the higher concentrations of copper sulphate. In concentrations above 0.8 μM the first symptom of toxicity was the formation of separation discs in large numbers. The trichomes contracted longitudinally and the cells became swollen and constricted at the cross walls. The cells also became yellowish due to loss of photosynthetic pigments. Finally, in 4 μM and above, vacuoles appeared in large number indicating the moribund state of the cells. Copper sulphate increased respiration at 2 μM, and optimum effect was observed in 8 μM after 96 h. Inhibition of photosynthesis was detectable in 0.8 μM, and 100% inhibition took place in 8 μM after 96 h. In higher concentrations the effect was immediate, and a conspicuous inhibition of photosynthesis could be observed within 10 min. The copper content of the alga increased with increased concentration of copper sulphate while potassium content decreased. With rise in outside concentrations of copper, there was a comparatively great increase of absorption in 2 and 4 μM, while further increases were gradually less. The observations indicate that changes in the physiological activity of the alga under treatment are closely interlinked with marked changes in morphology.     

The effects of cyclosporin and hydrocortisone on human T lymphocyte colony formation

The immunosuppressive activities of two compounds, cyclosporin A and hydrocortisone, were evaluated using a human T lymphocyte colony assay. Both compounds produced a dose-related reduction in colony formation. With cyclosporin, concentrations of 1.0–100 μg/ml virtually abolished PHA-induced lymphocyte growth; as little as 0.01 μg/ml decreased clonal growth by 48%. Suppression was observed as late as 4 days after the addition of PHA. The addition of exogenous IL-2 did not completely restore clonal growth to normal. Similarly, hydrocortisone, in concentrations of 0.4 μg/ml, produced a 96% inhibition in clonal growth. Partial suppression could also be obtained if the drug was added as late as 4 days after PHA. Exogenous IL-2 completely reversed the inhibitory effects of hydrocortisone. By contrast, IL-1 was able to only partially restore growth potential. Parallel studies using TPA indicated that both immunosup-pressants inhibited responses to this mitogen. However, in plates containing both TPA and PHA, hydrocortisone failed to impair clonal growth.     

Technical Note: Bioluminescence Measurements of the Antilisterial Activity of Nisin: Comparison with Ampicillin and Streptomycin

The influence of nisin on intracellular ATP and cell numbers of Listeria monocytogenes strain Scott A was determined and compared with the effect of ampicillin and streptomycin under similar conditions. In the presence of nisin (3–12 μg/ml), intracellular ATP and cell numbers decreased rapidly during the first hour at 35°C and extracellular ATP increased. Cell numbers and intracellular ATP increased after 3 h of incubation. No effect was observed in cells treated with ampicillin (3–12 μg/ml) and streptomycin (15–60 μg/ml) during the first hour. However, concentrations of ≥3 μg/ml ampicillin and ≥30 μg/ml streptomycin were listeriostatic after 3 h of incubation. Progressive loss of viability and reduction of intracellular ATP were observed in resting cells in PBS (pH 7.2) containing increasing concentrations of the antimicrobials. Rapid accumulation of extracellular ATP, observed immediately after treatment with nisin but not with the antibiotics, supports the reported collapse of proton motive force in L. monocytogenes by nisin.     

Relaxing effects of beta-receptor stimulators in isolated, gravid human myometrium

The effects of isoprenaline and the selective β₂-receptor terbutaline were investigated on strips from human myometrium obtained at caesarean sections. In normally polarized preparations both amines (isoprenaline 0.02 – 0.4 μg/ml, terbutaline 0.2 – 8 μg/ml) decreased the frequency and the amplitude of the spontaneous contractile activity. This action was not affected by phenoxybenzamine, 0.5 μg/ml, but could be blocked by propranolol, 0.1 μg/ml. In myometrial strips, depolarized by increasing the extracellular potassium concentration, isoprenaline, 0.004 – 0.4 μg/ml, and terbutaline, 0.008 – 8 μg/ml, had relaxing effects that were unaffected by phenoxybenzamine, 0.5 μg/ml, but could be blocked by propranolol, 0.1 gm/ml. It is concluded that the effects of the tested amines are mediated by actions on β-adrenoceptors, probably β₂-receptors.     

Effects of surfactants on cell growth and pigment production in suspension cultures ofPerilla frutescens

The effects of surfactants, adecanol LG-294 and silicone A, on anthocyanin accumulation and the growth ofPerilla frutescens cells in suspension cultures were studied. Production of the red pigment was remarkably reduced from about 1.9 g/l to 0.4 g/l by adecanol LG-294 at 0.06 ml/l but not by silicone A up to 0.4 ml/l. Several repeated shake-flask cultures also demonstrated no adverse effects of silicone A on the metabolite accumulation by the suspended cells. Furthermore, the addition of silicone A to a culture in a stirred bioreactor produced a three-fold higher growth rate and a seven-fold increase in anthocyanin compared with surfactant-free cultures. The improvement was due to the substantial reduction or prevention of foaming and of cell adhesion to the bioreactor wall.     

Overexpression of glucose-6-phosphate dehydrogenase enhances riboflavin production in Bacillus subtilis

Carbon flow in Bacillus subtilis through the pentose phosphate (PP) pathway was modulated by overexpression of glucose-6-phosphate dehydrogenase (G6PDH) under the control of the inducible Pxyl promoter in B. subtilis PY. Alteration of carbon flow into the PP pathway will affect the availability of ribulose-5-phosphate (Ru5P) and the riboflavin yield. Overexpression of G6PDH resulted in the glucose consumption rate increasing slightly, while the specific growth rate was unchanged. An improvement by 25% ± 2 of the riboflavin production was obtained. Compared to by-products formation in flask culture, low acid production (acetate and pyruvate) and more acetoin were observed. Metabolic analysis, together with carbon flux redistribution, indicated that the PP pathway fluxes are increased in response to overexpression of G6PDH. Moreover, increased flux of the PP pathway is associated with an increased intracellular pool of Ru5P, which is a precursor for riboflavin biosynthesis. The high concentrations of Ru5P could explain the increased riboflavin production.     

Acquired resistance of Alternaria solani and Sclerotium rolfsii to Polyoxin-D

Acquired resistance to the antibiotic polyoxin-D was studied in two phytopathogenic fungi, Alternaria solani Sorauer, and Sclerotium rolfsii Sacc. The ED₅₀ value of the antibiotic for A. solani was 100 μg/ml and S., rolfsii 200 μg/ml. A. solani and S. rolfsii could be trained to tolerate concentrations upto 1.000 μg/ml and 2.200 μg/ml respectively. The acquired resistance in both cases was not lost on continued subculturing in fungicide-free, media. On transfer to fungicide-free media, Polyoxinresistant strains of both the fungi showed faster growth rate and appreciable reduction in sporulation compared to the original strains. The adapted strain of A. solani showed cross-resistance to Cycloheximide and Difolatan but not to Hinosan and Bayleton.     

The Sterol Requirement of Tetrahymena setosa HZ-11

The growth rate of Tetrahymena setosa cells is stimulated significantly by as little as 0.1 μg and optimally by about 1 μg of ergosterol per ml of medium. Cell yields in the stationary phase are, however, not perceptibly affected by increasing sterol concentrations. Ergosterol, in concentrations that stimulate growth optimally, does not cause a reduction of tetrahymanol synthesis. The latter process is impaired only at much higher ergosterol concentrations. Epicholesterol and coprostanol inhibit ergosterol-stimulated growth competitively. It is concluded that the trace amounts of sterol needed by T. setosa do not serve to replace tetrahymanol but function in some other manner, probably unrelated to the control of membrane fluidity. This conclusion supports the views advanced earlier by Holz, Erwin, Wagner & Rosenbaum.     

Effects of Lectins on the Hemolysis of Rabbit Erythrocytes by Staphylococcal Alpha Toxin

When concanavalin A (1 μg/ml) or wheat germ agglutinin (2 μg/ml) was preincubated with a suspension of 2% rabbit erythrocytes for 5 min at 20 C, the binding of [¹²⁵I]-labeled staphylococcal alpha toxin to these erythrocytes was greatly inhibited and the hemolytic action of alpha toxin was decreased. The inhibitory effect of concanavalin A on hemolysis by alpha toxin was completely reversed in the presence of 0.1 M α-methyl-D -glucoside or α-methyl-D -mannoside. Phytohemagglutinin-P from Phaseolus vulgaris and soybean agglutinin inhibited hemolysis by the toxin at concentrations exceeding 20 μg/ml. The effect of concanavalin A on alpha-toxin hemolysis was studied further to ascertain the nature of the inhibition. Double reciprocal plots were made of hemolysis against alpha toxin concentrations, and the data suggested that inhibition of the initial rate of the hemolysis by concanavalin A is competitive in nature. This was probably due to an interaction with the alpha toxin binding sites on the cell membrane surface.     

Over-expression of glucose dehydrogenase improves cell growth and riboflavin production in Bacillus subtilis

Ribulose 5-phosphate is a precursor for riboflavin biosynthesis. Alteration of carbon flow into the pentose phosphate pathway will affect the availability of ribulose 5-phosphate and the riboflavin yield. We have modulated carbon flow in Bacillus subtilis through the gluconate bypass by over-expression of glucose dehydrogenase under the control of the constitutively expressed P43 promoter. Over-expression of glucose dehydrogenase resulted in low acid production (acetate and pyruvate). The substantial reduction in acid production is accompanied by increased riboflavin production and an increased rate of growth while glucose consumption remained unchanged. Metabolic analysis indicated that over-expression of glucose dehydrogenase increased intracellular pool of ribulose 5-phosphate. The high concentrations of ribulose 5-phosphate could explain the increased riboflavin production.     

Effects of Thymol on Ruminal Microorganisms

Thymol (5-methyl-2-isopropylphenol) is a phenolic compound that is used to inhibit oral bacteria. Because little is known regarding the effects of this compound on ruminal microorganisms, the objective of this study was to determine the effects of thymol on growth and lactate production by the ruminal bacteria Streptococcus bovis JB1 and Selenomonas ruminantium HD4. In addition, the effect of thymol on the in vitro fermentation of glucose by mixed ruminal microorganisms was investigated. Neither 45 nor 90 μg/ml of thymol had any significant effect on growth or lactate production by S. bovis JB1, but 180 μg/ml of thymol completely inhibited growth and lactate production. In the case of S. ruminantium HD4, 45 μg/ml of thymol had little effect on growth and lactate production; however, 90 μg/ml of thymol completely inhibited growth of S. ruminantium HD4. Thymol also decreased glucose uptake by whole cells of both bacteria. When mixed ruminal microorganisms were incubated in medium that contained glucose, 400 μg/ml of thymol increased final pH and the acetate to propionate ratio and decreased concentrations of methane, acetate, propionate, and lactate. In conclusion, thymol was a potent inhibitor of glucose fermentation by S. bovis JB1 and S. ruminantium HD4. Even though thymol treatment decreased methane and lactate concentrations and increased final pH in mixed ruminal microorganism fermentations of glucose, concentrations of acetate and propionate were also reduced. Received: 13 May 2000 / Accepted: 14 June 2000     

Use of natural polymers as immobilizing agents and effects on the growth of Dunaliella salina and its glycerol production

Agar-agar, agarose, carrageenan and calcium alginate were used for the immobilization of Dunaliella salina cells. Out of the four, agar-agar was found to be the most effective and therefore the study was carried out on it using different pH values ranging from 6 to 10 and cell densities from 0.1 to 0.8 μg chlorophyll (chl, a) per bead to find which are is best suited for glycerol production. The maximum glycerol production of 9.2 μM/mg chl a was recorded in agar-agar immobilized algae and this was followed by 8.4 μM/mg chl a in calcium alginate. The maximum cell number 6.2 × 10⁹/ml and the specific growth rate (μ) of 0.80 l/day were reached at pH 8 in agar-agar immobilized algae. It was shown that the maximum amount of glycerol was produced when the cell density was 0.8 μg chl a/ block. Changing the medium after 24 hours affected the rate of glycerol production at different pH values. Using a cell density of 0.8 μg chl a/block at 16 W/m² light intensity increased the glycerol production in comparison with the use of free living cells.     

Production of extracellular polysaccharides by a Rhizobium species from root nodules of Cajanus cajan

The ability of the Rhizobium sp., isolated from the root nodules of the leguminous pulse yielding shrub Cajanus cajan, to produce extracellular polysaccharides (EPS) was checked. A large amount of EPS (1, 128 μg/ml) was produced by the bacteria in yeast extract mannitol medium. Growth and EPS production started simultaneously, but the production reached its maximum level in the stationary phase of growth at 28 h. The EPS production by this Rhizobium sp. was much higher than by many other strains from nodules of Cajanus cajan which took a much longer time to reach maximum EPS production than this strain. The maximum EPS production (2,561 μg/ml) was obtained when the medium was supplemented with mannitol (1%), cetyl pyridinium chloride (2 μg/ml) and KNO₃ (0.2%), in which the production was increased by 276% compared to the control. The EPS production rose in the period up to 65 h with increased mannitol concentration. The EPS contained arabinose, xylose and rhamnose monomers. The possible role of rhizobial EPS production in root nodule symbiosis is discussed.