The cytotoxic and photodynamic inactivation of micro-organisms by Rose Bengal

Rose Bengal was cytotoxic to the following bacteria at the concentrations given in parentheses (highest concentrations of dye in mol/1 at which growth occurred on nutrient medium): Brochothrix thermosphacta and Deinococcus radiodurans (1 times 10^-6 or less); Streptococcus, Micrococcus, Staphylococcus, Bacillus, Arthrobacter and Kurthia spp. (1 times 10^-5–1 x 10^-4), and Pseudomonas spp. and Enterobacteriaceae (5 times 10^-3–1 x 10^-2 or greater). These organisms were killed rapidly when suspended in illuminated (170 μE/m²/s) solutions of Rose Bengal (1 times 10^-4 mol/1) providing oxygen was present. Singlet oxygen was identified as the lethal agent, because the rate of killing was increased by dissolving the dye in deuterium oxide while the organisms were protected against photoinactivation by L-histidine or crocetin. Yeasts from chilled foods were killed in illuminated solutions of Rose Bengal but a light intensity of 315 μE/m²/s was needed for a death rate comparable with that of bacteria. The yeasts present in a range of chilled meat and dairy products failed to form colonies on Rose Bengal (5 times 10^-5 mol/1) media exposed continuously to modest illumination (55–80 μE/m²/s).     

Effect of 2-chloroethylphosphonic acid on capsule formation and alkaloid content in Papaver somniferum

Application of different concentrations of ethephon (2-chloroethylphosphonic acid) to Papaver somniferum L. at the times of stem elongation, bud, and capsule formation produced different effects. Ethephon (10^-2 M ) retarded growth of the plant and inhibited capsule formation during stem elongation, significantly reduced capsule size during the flowering period, but did not alter capsule development during capsule formation. When applied during the period of stem elongation, ethephon (10^-3 M and 10^-4 M ) reduced capsule size; alkaloid accumulation was reduced by ethephon at a concentration of 10^-3 M , but slightly increased by 10^-4 M . Ethephon (10^-3 M and 10^-4 M ) did not alter capsule development or alkaloid content significantly when applied during bud formation, but stimulated capsule size and alkaloid content when applied during capsule formation. Pretreating the plants with Ag⁺ (silver nitrate) did not reverse the ethephon effect. The results suggest that capsule maturation and alkaloid accumulation in P. somniferum are modified by ethylene, which is produced as a result of exogenous ethephon treatment.     

Neuroendocrine stimulation of uterine gland protein synthesis in the tsetse fly, Glossina morsitans

ABSTRACT. The uterine gland of the tsetse fly Glossina morsitans morsitans Westw. synthesizes a secretion which nourishes the developing larva in the uterus. Aqueous extracts of the brain have been shown to stimulate the synthesis of the protein and amino acid components of this secretion from L- [U-¹⁴C]leucine by uterine gland tubules in vivo and in vitro. A linear dose response relationship was demonstrated in vitro with extract concentrations ranging from 1 × 10^-4 to 1 × 10^-2 brains μl^-1. The maximum response, a > 300% increase in the rate of protein and amino acid synthesis, was achieved with as little as 1 × 10^-2 brains μl^-1 The concentration of active factor(s) in the brain declined during a single interlarval period coincident with the period of release of secretion associated with larval growth. The stimulatory activity in brain extracts was destroyed by proteolytic enzymes indicating that it is probably a protein or peptide. Results suggest that the active factor(s) is a hormone responsible for the stimulation of uterine gland protein synthesis essential for larval nutrition.     

Studies on the effect of mercury and organomercurial on the growth and nitrogen fixation by mercury-resistant Azotobacter strains

Five nitrogen-fixing Azotobacter strains isolated from agricultural farms in West Bengal, India, were resistant to mercuric ion and organomercurials. Resistance of Hg-resistant bacteria to mercury compounds is mediated by the activities of mercuric reductase and organomercurial lyase in the presence of NADPH and GSH as cofactors. These bacteria showed an extended lag phase in the presence of 10–50 μmol 1^-1 HgCl₂. Nitrogen-fixing ability of these isolates was slightly inhibited when the mercuryresistant bacterial cells were preincubated with 10 μmol 1^-1 HgCl₂. Acetylene reduction by these bacteria was significantly inhibited (91-97%) by 50 μmol 1^-1 HgCl₂. However, when GSH and NADPH were added to the acetylene reduction assay mixture containing 50 μmol 1^-1 HgCl₂, only 42–50% inhibition of nitrogenase activity was observed. NADPH and GSH might have a role in suppressing the inhibition of N₂-fixation in the presence of Hg compounds either by assisting Hg-detoxifying enzymes to lower Hg concentration in the assay mixture or by formation of adduct comprising Hg and GSH which is unable to inhibit nitrogen fixation.     

Studies of the extracellular proteolytic activity produced by Propionibacterium acnes

Of several commercial media tested, trypticase soya broth containing 0.4% (w/v) D-sorbitol was superior as a growth medium for the production of extracellular proteinase by Propionibacterium acnes (strain P-37). Extracellular proteinase, production of which was shown to be growth-associated by both batch and continuous culture studies, was partially purified by 70% (NH₄)₂SO₄ saturation, Sephadex G-75 chromatography and ion exchange on DEAE-Sephadex A-50. It was shown to be a heterogeneous mixture of at least three molecular species of enzyme. Proteinase I was inhibited by EDTA (10^-3 mol/l) and PMSF (5 millimol/l) and stimulated by CaCl₂ (190% at 10^-3 mol/l). It had a molecular weight of 20 to 30000 and a broad pH optimum from 6.5 to 7.5. Proteinase II was an alkaline proteinase with a molecular weight of 30 to 40000 which was not significantly inhibited by EDTA (10^-2 mol/l) nor stimulated by CaCl₂. Proteinase III represented a minor proportion of the recovered proteolytic activity, had a molecular weight of 20 to 30000 and was most active in the alkaline pH range. This enzyme was inhibited by EDTA (10^-4 mol/l) and PMSF (5 millimol/l), and stimulated by CaCl₂ (250% at 10^-2 mol/l).     

2,4-Dichlorophenoxyacetic acid inhibition of potassium transport in barley seedlings

Growth, potassium uptake and translocation as well as transpiration rates were measured in intact low-salt barley seedlings ( Hordeum vulgare L. cv. Union) in the presence of different 2,4-D concentrations at pH 6.5. Growth was only affected at 10^-3 M .
Above 10^-7 M 2,4-D both uptake by the roots and transport to the shoots were inhibited. The inhibition at 10^-5 M remained constant for at least 24 h. Furthermore inhibition of uptake was measurable within 1 h. Excised roots and roots of intact plants showed the same uptake pattern.
It is suggested that the observed effects were caused by 2,4-D-induced changes in uptake and translocation systems in the roots. Pre-treatment with 10^-5 M 2,4-D had no effect upon subsequent potassium uptake. Transpiration was reduced within 1 h in 10^-4 or 10^-3 M 2,4-D, probably due to changes in water transport or root permeability.     

Effect of titanium dioxide concentration on the survival of Pseudomonas stutzeri during irradiation with near ultraviolet light

Cells of Pseudomonas stutzeri in suspensions of TiO₂ ranging in concentration from 0.5 to 4.0 g l^-1 were irradiated with a blacklight blue u.v. source displaying peak emissivity at approximately 370 mm. Irradiation under these conditions is known to result in the generation of lethal free radicals. During irradiation the suspensions were agitated, using a specially modified laboratory shaker, to ensure efficient exposure of the TiO₂. A u.v. radiation dose of 175 kJ m^-2 resulted in cell fractional survival ranging from 5.5 times 10^-5, at the lowest TiO₂ concentration, to 1.0 times 10^-6, at the highest TiO₂ concentration. The advantages of contactors employing TiO₂ suspensions are briefly compared to immobilized TiO₂ systems.     

Sterilization of soybean cotyledons by boiling in lactic acid solution

F. RUÍZ-TERÁN AND J.D. OWENS. 1996. The effect of pH on the heat resistance of Bacillus stearothermophilus spores at 100°C in the presence of 0.11 mol 1^-1 lactic acid and 0.2 mol 1^-1 sodium phosphate buffer was examined. At pH values of 7.0 and 6.0 spores survived 60 min exposure unharmed but at pH 4.3 and 3.0 they died with decimal reduction times (DRTs) of 27 min and 2.8 min, respectively. Death rates were similar in the presence or absence of hydrated soybean cotyledons. In the presence of phosphate buffer and cotyledons at mean pH 3.6 the DRT was 118 min but in the presence, in addition, of lactic acid it was 11 min. It is suggested that the enhanced death rate was due to toxic effects of undissociated lactic acid. Rhizopus oligosporus NRRL 2710 grew well on cotyledons, having pH values from 7.0 to 3.7, prepared by boiling for 60 min in the presence of 0.11 mol 1^-1 lactic acid and 0.2 mol 1^-1 phosphate buffer.     

3,5-Dichlorophenoxyacetic acid as a growth retardant

Tomato seedlings, grown in the glasshouse, were sprayed with solutions of 3,5-dichlorophenoxyacetic acid as sodium salt at 2 times 10^-5, 4 times 10^-5 and 10^-4 M. The treated plants became dark-green, dwarfed, and compact. After 6–7 weeks normal growth was resumed. Measurements and analytical data on treated and control plants are presented.     

A Simple Cranial Window Technique for Optical Monitoring of Cerebrocortical Microcirculation and NAD/NADH Redox State. Effect of Mitochondrial Electron Transport Inhibitors and Anoxic Anoxia

Abstract: Fluorescence of NADH and vascular volume of the brain cortex of chloralose-anesthetized cats were measured by surface fluororeflectometry. A cranial window and superfusion technique was elaborated for the topical inhibition of mitochondrial electron transport in the brain cortex by amytal (inhibits at site I) and cyanide (inhibits at site III). The changes in NAD/NADH redox state and CVV evoked by these electron transport inhibitors were compared with those elicited by anoxic anoxia. Amytal (10^-3-10^-1 M ) and cyanide (10^-5-10^-2 M ) resulted in a concentration-dependent and reversible increase in cortical NAD reduction and vascular volume, but the cerebrocortical vessels were almost completely dilatated long before maximum NAD reduction was reached. Cyanide at 10^-2 M increased cortical NAD reduction and vascular volume as much as anoxic anoxia. Amytal at 10^-1 M induced approximately half of the NAD reduction evoked by 10^-2 M cyanide or anoxic anoxia, but resulted in only slightly less vasodilatation than that following cyanide and anoxic anoxia. Since amytal inhibits mitochondrial electron transport at site I—and cyanide and anoxia at site III—but induces a comparable degree of vasodilatation, it is concluded that cytochrome oxidase cannot be the single molecular oxygen sensor in the brain cortex.     

Effects of cytosine arabinoside and daunorubicin on survival and cell cycle progression of chinese hamster ovary cells

Abstract. Kinetic and cytotoxic effects of cytosine arabinoside (Ara-C) and daunorubicin (DNR) on exponentially growing Chinese hamster ovary (CHO) cells were measured by flow cytometry and by a colony-forming assay, respectively. With Ara-C alone, increasing drug concentrations between 10^-7 M, for up to 27 hr, were associated with increased inhibition of cell progression through the S phase. Even at the very toxic concentration of 10^-4 M, however, cells were able to enter and progress slowly through S. DNR, which appears to enter these cells relatively slowly, was highly toxic even at 2 times 10^-7 M. It decreased the rate of progression through S phase and caused cells to accumulate in G₂, except at the highest concentration (2 times 10^-5 M), at which progression was inhibited throughout the cycle. Simultaneous exposure of the cells to Ara-C and DNR yielded cell cycle distributions similar to those of the former drug alone. When cells were exposed to a non-lethal dose of Ara-C and to a dose of DNR which was lethal to a fraction of the cell population (or conversely), either simultaneously or separated by a drug-free interval, small, but in some cases significant, drug interactions were observed. These effects were not caused by druginduced redistribution of cells within the cell cycle, but may have been related to the effects of the non-lethal drug on DNA synthesis rate.     

Interaction of triacontanol with gibberellin in control of lettuce seed germination

Triacontanol at concentrations from 2.3 × 10^-9 M to 2.3 × 10^-7 M did not affect the germination of lettuce ( Lactuca sativa L., cv. Grand Rapids) seeds in darkness, stimulated by light at 25°C or by benzyladenine at 31°C. Stimulation of seed germination by gibberellin A₃ (10^-5 M ) was significantly inhibited by triacontanol; the most effective concentration was 4.6 × 10^-8 M. Pulse experiments demonstrated that triacontanol was ineffective when applied later than gibberellin, whereas an inverse sequence of treatment caused an inhibition comparable to that resulting from continuous treatment of seeds with both factors. Possible interaction of triacontanol with gibberellin receptor is discussed.     

Nickel and rubidium uptake by whole oat plants in solution culture

Nickel and rubidium uptake by oat plants ( Avena sativa L. cv. Victory) were examined in relation to solution temperature, solution concentrations, metabolic inhibitors, anaerobic root conditions, transpiration and time. Over a 4-h period, uptake rates for both Ni²⁺ and Rb⁺ remained constant at 23°C. Decreasing temperatures to 2°C, 20 μ M concentrations of 2,4-dinitrophenol (DNP), or anaerobic root conditions decreased Ni²⁺ and Rb⁺ uptake rates by 97 to 86% in whole plants. Treatment of excised roots with 20 μ M DNP decreased Ni²⁺ uptake by 93%. Nickel and Rb⁺ uptake rates measured as a function of the external solution concentration followed a typical parabolic curve. K_m (0.012 m M ) and V_max [2.72 μmol (g dry weight)^-1 h^-1] values for Ni²⁺ were nearly 7 times lower than those for Rb⁺ [0.09 m M and 19.2 μmol (g dry weight)^-1 h^-1]. In all experiments, Ni²⁺ and Rb⁺ showed qualitatively similar uptake patterns, but Rb⁺ uptake was quantitatively more sensitive than Ni²⁺ to experimental manipulations.     

Comparison of some decontamination methods and growth media for isolation of mycobacteria from northern brook waters

Two decontamination methods and five media were compared for the isolation of mycobacteria from brook waters of different physical, chemical and bacteriological characteristics. The decontaminants used were: 0.7 mol 1^-1 NaOH followed by 50 g 1^-1 oxalic acid and 0.9 mol 1^-1 H₂SO₄ combined with 0.5 g 1^-1 cycloheximide. The media compared were: Mycobacteria 7H11 agar with OADC enrichment (pH 6.6), glycerol egg (pH 6.5 and 5.5), and pyruvate egg (pH 6.5 and 5.5). All media contained cycloheximide, 0.5 g 1^-1. The NaOH—oxalic acid method generally resulted in lower contamination and higher isolation of mycobacteria than the H₂SO₄-cycloheximide method. With the NaOH—oxalic acid method, all five media were equal in positivity rates but contamination was a problem on Mycobacteria 7H11 agar. Of the four egg media tested, the highest positivity rate (92% of the samples) was obtained on the pyruvate modification (pH 6.5), and the highest mean colony count of mycobacteria (900 cfu 1^-1) on the glycerol modification (pH 6.5). Characteristics of water and sampling site had similar effects on the isolation frequencies of mycobacteria obtained by different combinations.     

Effect of some factors on determination of viable microbial cells by peroxyoxalate chemiluminescence method

The effects of physical and chemical factors on the production of H₂O₂ from Escherichia coli cells were studied. When 20 mmol 1^-1 Tris-HCl buffer was used for this purpose the electron transport system (ETS) showed the highest activity at pH 7.6-8.2. KCN promoted the production of H₂O₂ from E. coli cells, and the optimum concentration was changed in different reaction times and pH values. Glucose, 5 mg ml^-1, increased the ETS activity about twofold. The other substrates and surfactants did not increase the chemiluminescence intensity. NaNO₂ and Na₂SO₄ in inorganic salts significantly reduced the ETS activity above 70%. In addition, the optimum temperature for the production of H₂O₂ was 30°C in this study. When glucose (5 mg ml^-1) and KCN (0.2 mmol 1^-1) were added to the reaction buffer containing 0.5 mmol 1^-1 menadione, the detectable minimum cell densities (averages of triplicate assay) of E. coli, Enterobacter cloacae and Serratia marcescens were 5 times 10³ cells ml^-1, 10⁴ cells ml^-1 and 10⁴ cells ml^-1 respectively.     

Properties and significance of an α-amylase produced by Myxococcus coralloides D

M.E.FÁREZ-VIDAL, A. FERNÁNDEZ-VIVAS, F. GONZÁLEZ AND J.M. ARIAS. 1995. The extracellular amylase activity from Myxococcus coralloides D was purified by Sephacryl S-200 gel filtration and by ion-exchange chromatography on DEAE-Sephadex A-25. The molecular weight was estimated by SDS-PAGE and by gel filtration as 22.5 kDa. The optimum temperature was 45°C. The pH range of high activity was between 6.5 and 8.5, with an optimum at pH 8.0. Activity was strongly inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Ag⁺, Pb²⁺, Fe²⁺ and Fe³⁺, EDTA and glutardialdehyde, but was less affected by Ni²⁺ and Cd²⁺. Li⁺, Mg²⁺, Ba²⁺, Ca²⁺, N -ethylmaleimide, carbodiimide and phenyl methyl sulphonyl fluoride had almost no affect. The K _m (45°C, pH 8) for starch hydrolysis was 2.0 times 10^-3 gl^-1. Comparison of the blue value-reducing curves with the time of appearance of maltose identified the enzyme produced by M. coralloides D as an α-amylase.     

The acute lethal toxicity of mixtures of cyanide and ammonia to smolts of salmon, Salmo salar L. at low concentrations of dissolved oxygen

The survival of Atlantic salmon smolts on exposure to constant concentrations of cyanide and ammonia, singly and together, has been measured under laboratory conditions at a concentration of 5 mgl^-1 of carbon dioxide. The 24-h LC50 values of cyanide and of un-ionised ammonia, in fresh water, were 0·073 mg HCN l^-1 and 0·20 mg NH₃l^-1 respectively at a concentration of dissolved oxygen of 10 mg l^-1, and 0·024 mg HCN l^-1 and 0·08 mgNH₃l^-1 respectively at a concentration of dissolved oxygen of 3·5 mg l^-1. In 30% sea water the corresponding values were similar for cyanide but markedly higher for ammonioa. In 80% sea water the values were intermediate between those of fresh water and 30% sea water. Prior acclimation of the fish to the respective toxicant increased the resistance of the fish only slightly to cyanide, but with ammonia the 24-h LC50 was increased between 1·4 and 2-fold after acclimation for 1–3 days to between 0·2 and 0·5 of the 24-h LC50 value. Mixtures of cyanide and ammonia were between 0·6 and 1·25 times as toxic as expected, assuming simple additivity of toxicity.     

Effects of di-n-butyl phthalate on growth and photosynthesis in algae and on isolated organelles from higher plants

Di- n -butyl phthalate (DBF) is widely used as a plasticizer and has been found in all types of ecosystems. It inhibits growth and photosynthesis of green algae ( Chlorella emersonii CCAP strain 211/8 h and Selenastrum capricornutum CCAP strain 278/4) at concentrations higher than 10-⁵ M . The IC₅₀ value for CO₂-dependent oxygen evolution in algae was 3 × 10^-4M. The CO₂-reduction in isolated protoplasts prepared from barley ( Hordeum vulgare L. cv. Simba) was also inhibited by phthalate. The IC₅₀ value was 2 × 10^-4 M . The electron transport in isolated thylakoids prepared from spinach was inhibited with an IC₅₀ value of 3 × 10^-4 M . The IC₅₀ value for uncoupled electron transport extrapolated to zero chlorophyll concentration was 2.5 × 10^-5 M . The effect of di-n-butyl phthalate was localized to reactions in photosystem II. Di-n-butyl phthalate could thus be a pollutant which affects growth and photosynthesis of plants. The reported IC₅₀ values may be underestimated since di- n -butyl phthalate can attach to surfaces. The results are discussed in relation to observed effects of di- n -butyl phthalate on other organisms.     

THE IRREVERSIBLE INHIBITION OF BRAIN L-GLUTAMATE-I-DECARBOXYLASE BY (2RS,3E)-2-METHYL-3,4-DIDEHYDROGLUTAMIC ACID

Abstract— Incubation of chick embryo brain l -glutamate-1-dccarboxylase (GAD, EC 4.1.1.15) with (2RS,3E)-2-methyl-3,4-didehydroglutamic acid (MDG), a substrate analog of l -glutamic acid, results in a time-dependent irreversible inhibition of the enzymic activity. In the presence of 2.0 ± 10^-3 m inhibitor the half-life for inactivation is 11.6min. The inhibitor is a substrate for GAD and requires turnover prior to inactivating the enzyme and is therefore another example of the k _cat class of inactivator. The measured K _l is 6.6 ± 10^-4 m and the k _cat for its turnover is 1.01 ± 10^-3 s-¹ at 37°C (pH 7.2). The inhibitor has no effect on the apoenzymc or the holoenzyme treated with 1.0 ± 10^-3 m hydrazinc. Both l -and d -glutamate, but not mercaptoethanol, reduce the rate of enzymie inactivation by the inhibitor. The exceedingly high specificity implicit in the design of this inhibitor should render it useful in studies designed to uncover the physiological role of GABA.     

Effects of culture conditions on Pectinatus cerevisiiphilus and Pectinatus frisingensis metabolism: a physiological and statistical approach

The genus Pectinatus has been often reported in beer spoilage with off-flavours. The bacteria are strictly anaerobic, Gram-negative rods. Propionate and acetate are the main fermentation products from glucose in the two species belonging to the genus, P. cerevisiiphilus and P. frisingensis. Amino acids routinely present at a high level in beer were not growth substrates for both species, and a significant accumulation of succinate was observed with lactate as growth substrate. Both Pectinatus ssp. showed almost identical fermentation balances on glucose. Growth kinetics of both glucose-grown species were unchanged under a N₂, H₂ or 20% CO₂-containing atmosphere. Combinations of culture medium pH values from pH 3·9 to pH 7·2, of glucose levels between 5 and 55 mmol l^-1, and of lactate concentrations varied from 4 to 40 mmol l^-1 demonstrated that biomass and volatile fatty acids production were proportional to glucose concentration for both Pectinatus species. A significant increase of volatile fatty acid production was measured for both species at the lowest pH values with a lactate or a glucose concentration increase. The maximum biomass production was observed at pH 6·2 for P. cerevisiiphilus , and between pH 4·5 and pH 4·9 for P. frisingensis. Glucose and lactate or pH value were dependent with regard to propionate and acetate production in P. frisingensis. On the other hand, the variations of these three parameters were independent with regard to biomass production for both strains, and to volatile fatty acids production for P. cerevisiiphilus. Addition of ethanol to glucose-grown cultures completely inhibited growth at 1·3 mol l^-1 ethanol for P. cerevisiiphilus , and at 1·8 mol l^-1 for P. frisingensis.