Effects of pollutants in fresh waters on European non-salmonid fish I: Non-metals

As part of a programme of acquiring data for preparing standards to safeguard European, nonsalmonid, freshwater fish from pollution, toxicity tests were carried out in hard, well-aerated water. Asymptotic median lethal concentrations (LC50s) of undissociated ammonia, cyanide, nitrite and phenol to one or more of three species were determined. The LC50s were as follows: to common carp 16mg1^-1 as NO₂–N; to perch 0.1 mg1^-1 as HCN; to roach 0.35 mg1^-1 as NH₃-N, 0-11mg1^-1 as HCN, 10.1 mg1^-1 asNO₂-Nand 10mg^-1 as phenol. In order to define these LC50s, exposure periods within the range 0.3 days (phenol) to 14 days (nitrite) were required. Comparisons are made with other data and the tentative water quality 'criteria' proposed by the European Inland Fisheries Advisory Commission.     

The effect of calcium concentration on the toxicity of copper, lead and zinc to yolk-sac fry of brown trout, Salmo trutta L., in soft, acid water

Yolk-sac fry of brown trout were exposed to three levels of single trace metals (Cu, 20,40,80 nmol 1^-1; Pb, 12·5,25,50 nmol 1^-1; Zn, 75,150,300 nmol 1^-1) typical of concentrations reported for acid soft waters, in flowing, artificial, soft water media maintained at pH 4·5 and [Ca] of 20 or 200 μmol 1^-1for 30 days.
Mortalities were high in fry subjected to all levels of the three trace metals at [Ca] 20 μmol 1^-1, with 80% of the total deaths occurring between days 11 and 15 of the experiment. 25% mortality occurred at low [Ca] and pH 4·5 in the absence of trace metals, with only one death recorded at [Ca] 200 μmol1^-1'(Cu, 80 nmol 1^-1). At high [Ca] all three levels of Cu and Pb impaired net Na and K uptake; Cu was the only metal to reduce the uptake of Ca. The Zn treatments had no significant effect on mineral uptake. Calcification of centra was reduced by all three Cu treatments at [Ca] 200 μmol 1^-1. The lowest Zn concentration (75 nmol 1^-1) was the only other treatment to impair skeletal development. In the absence of trace metals, low [Ca] significantly reduced Ca, Na and K uptake, skeletal calcification and dry mass at pH 4·5.
The deleterious effects of low Cu, Pb and Zn concentrations at low pH and low [Ca], and the ameliorative effect of higher ambient [Ca], are discussed in relation to fishery status in soft, acid waters.     

Effect of pH, temperature and culture medium composition on the production of an extracellular cysteine proteinase by Micrococcus sp. INIA 528

Growth and proteinase production by Micrococcus sp. INIA 528 in a batch-operated laboratory fermentor were investigated, with trypticase soy broth as the basal medium for studies on optimum temperature, pH and medium composition. Maximum growth was recorded at 34°C and pH 715, whereas optimum temperature and pH for proteinase production were 31°C and pH 6.25. Maximum rate of enzyme production occurred during the late log and early stationary phases of growth. Addition of 5.0 g 1^-1 yeast extract, 1.0 g 1^-1 glucose, 1.0 g 1^-1 MgSO₄ or 1.0 g 1^-1 K₂HPO₄ to basal medium resulted in a lower enzyme yield, but supplementation of basal medium with 2.5 g 1^-1 (NH₄)₂SO₄ increased enzyme production by 45%. A high initial biomass added to fresh broth supplemented with 2.5 g 1^-1 (NH₄)₂SO₄ only increased enzyme activity by 19%, compared to the maximum enzyme activity achieved with the standard inoculum.     

Ecology of Peridinium willei and P. volzii (Dinophyceae) in Danish lakes

The distribution of Peridinium willei and P. volzii was studied in Danish lakes. Both species were confined to lakes with concentrations of Total P < 0.15 mg 1^-1, with the majority of occurrences at Total P concentration between 0.020–0.040 mg 1^-1 and concentrations of PO₄ P between detection limit and 0.040 mg 1^-1. The occurrence of the species in relation to inorganic N compounds (NH₄ N and NO₂+ NO₃ N) was significantly broader for P. willei than for P. volzii: P. willei had an almost even distribution within a wide range of NH₄ N, whereas P. volzii mainly occurred between 0.001 and 0.10 NH₄ N 1^-1. P. willei had an almost even distribution at values beween 0.005 and 0.42 mg NO₂+ NO₃ N 1^-1, whereas P. volzii mainly occurred below 0.050 mg NO₂+ NO₃ N 1¹. P. willei was found at pH values between 4.2 and 8.5, whereas P. volzii was confined to lakes with a slightly basic pH. The study confirmed the broad limits of P. willei and the much more narrow limits of P. volzii in relation to seasonal occurrence and pH, as well as an affinity of the former to ponds and lakes with a rich bottom vegetation. The study also showed, however, that the species were not as widespread and common in recent Danish lake phytoplankton as generally stated by previous authors. The use of different ecological factors to give weight to species separation is discussed. The inclusion of P. volzii in P. willei proposed by Popovsky & Phiester is not supported by the present study, as the two taxa appear to have different ecological tolerances.     

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).     

Fast locomotion of some African ungulates

Ten species of ungulate were filmed, galloping in their natural habitat. They ranged in size from Thomson's gazelle (about 20 kg) to giraffe (about 1000 kg). They were pursued to make them run as fast as possible. The films have been analysed to determine speed, stride frequency, stride and step lengths, and duty factors. The dependence of these quantities on body size is discussed.  

Summary:

Fast locomotion of zebra, giraffe, warthog and seven species of Bovidae has been studied. The animals were filmed from a pursuing vehicle while galloping in their natural habitat.
Stride frequency was more closely correlated with limb length (represented by hip height) than with body mass. Mean stride frequency was proportional to (hip height)^-0·51 and maximum stride frequency to (hip height) ^-0·63.
Maximum speed was between 10 and 14 m s ^-1 for all species except buffalo (7 m s ^-1). It was not significantly correlated with body mass.
Since the small species ran at least as fast as the large ones they attained higher Froude numbers. Relative stride length was approximately 1·8 (Froude number)^0·39 for all species, irrespective of size. Relative step length was approximately 0·65 (Froude number)^0·2, both for the fore feet and for the hind ones. The vertical forces exerted by the feet are proportional to (body weight)×(Froude number)^0·2 so the forces at maximum speed are larger multiples of body weight for small species than for large ones.     

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.     

Kinetic and metabolic effects of nitrogen, magnesium and sulphur restriction in Xanthomonas campestris batch cultures

By reducing the concentration of nitrogen (from 5.0 to 2.5 mmol 1^-1), batch cultures of Xanthomonas campestris induced the enzyme UDP-glucose dehydrogenase and stimulated the Entner-Doudoroff pathway enzyme glucose-6-P dehydrogenase. The surplus energy generation was directed to xanthan biosynthesis resulting in a 10% polysaccharide increase. The nitrogen restriction led to a higher consumption of nitrogen (93%) whereas glucose consumption did not surpass 75% utilization. Low concentrations of both magnesium and sulphur exerted a negative effect on xanthan formation. Both restrictions reduced the phosphomannose isomerase enzyme activity by 10-fold turning the mannose transference presumably into the rate-limiting step for xanthan biosynthesis. Conversely, the rate of synthesis of glucuronic acid residues did not affect the rate of xanthan biosynthesis. Polysaccharide synthesis in magnesium and sulphur cultures was negatively affected in comparison with cell formation as the cell volumetric production rate increased from 0.037 to 0.091 g 1^-1 h^-1 and the xanthan volumetric production rate dropped from 0.133 g 1^-1 h^-1 to the minimum obtained at 0.083 g 1^-1 h^-1. The efficiency of the carbon substrate conversion was also greatly changed.     

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.     

A β-N-acetylhexosaminidase from Penicillium oxalicum implicated in its cell-wall degradation

High β- N -acetylhexosaminidase (EC.3.2.1.52) activity was detected during autolysis of Penicillium oxalicum . Purification of the enzyme to homogeneity yielded an enzyme with a molecular weight of 132 000 Da by gel filtration and 71 900 Da by SDS polyacrylamide gel electrophoresis, suggesting a dimeric structure. The enzyme is an acidic protein with a pl of 5.0. Optimal activity was at pH 4.0 and 40°C, with a K _m of 0.80 mmol 1^-1 for p -nitrophenyl-β- N -acetylglucosaminide and 1.03 mmol 1^-1 for p -nitrophenyl-β- N -acetylgalactosaminide. The K _i with the competitive inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino- N -phenylcarbamate was 1 μmol 1^-1. Hg²⁺, Ag⁺ and Fe³⁺ were effective inhibitors. β- N -acetylhexosaminidase hydrolysed chitobiose, chitotriose, chitotetrose and chitopentose to monomer to an extent of 92, 74, 44 and 17% respectively in 40 min. This enzyme, in conjunction with a purified endochitinase from P. oxalicum , hydrolysed a cell-wall chitin fraction isolated from this fungus, with the production of N -acetylglucosamine.     

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.     

The effects of two synthetic putrescine analogues on germination and appressorium formation by uredospores of Uromyces viciae-fabae on artificial membranes

M. REITZ, D.R. WALTERS, B. MOERSCHBACHER AND D.J. ROBINS. 1995. An examination was made of the effects of two synthetic putrescine analogues, ( E )-1,4-diaminobut-2-ene (E-BED) and ( E )-( N, N, N, N -tetraethyl)-1,4-diaminobut-2-ene (E-TED), on germination and appressorium formation by uredospores of the rust fungus Uromyces viciaefabae on artificial membranes. E-BED reduced germination by just 11% at 0.1 mmol 1^-1and by 24% at 1 mmol 1^-1, while appressorium formation was reduced by 37% at 0.05 mmol 1^-1and was completely prevented at 1 mmol 1^-1E-BED. The E-BED derivative E-TED reduced uredospore germination by 45% at 0.05 mmol 1^-1, while no appressoria were formed when uredospores were exposed to 0.05 mmol 1^-1E-TED. These results support previous suggestions that E-BED and E-TED exert their main effect on fungal development on the leaf surface.     

α-Galactosidase from the yeast Torulaspora delbrueckii IFO 1255

The yeast Torulaspora delbrueckii IFO 1255 was selected as the strain fermenting melibiose from 35 strains of Torulaspora species. The strain IFO 1255 produced extracellular and cell-associated forms of α-galactosidase when grown on either melibiose or galactose as the sole carbon source. Most of the enzyme was located outside of the cell membrane: the periplasmic space, or cell walls, or both. α-Galactosidase was purified to homogeneity from the cell-free extract of the strain IFO 1255 by acid treatment and column chromatography on DEAE-Toyopearl 650M and Butyl-Toyopearl 650M. The molecular weight of the purified enzyme was estimated to be 88 000 by SDS-polyacrylamide gel electrophoresis and 530 000 by gel filtration. The enzyme contained 50% of its molecular weight as carbohydrate. Optimum pH and temperature were 4.5–5.5 and 55°C, respectively. The enzyme was inhibited strongly by Ag²⁺, Hg²⁺ and Cu²⁺ each at 1 mmol 1^-1. The K _m (μmol 1^-1) for p -, o -, m -nitrophenyl α-D-galactopyranoside, melibiose, raffinose and stachyose were 2.8, 1.3, 2.8, 4.2, 170 and 230, respectively, and V _max (μmol min^-1 mg protein^-1) for those substrates were 310, 140, 21, 22, 30 and 44, respectively. The properties of α-galactosidase from T. delbrueckii IFO 1255 were similar to those from the related species, Saccharomyces cerevisiae.     

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.     

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.     

Inhibition of Salmonella typhimurium by the products of tartrate metabolism by a Veillonella species

The inhibition of the growth of Salmonella typhimurium by a Veillonella species grown on media supplemented with tartrate was examined. Growth of Salmonella typhimurium was not inhibited by the concentrations of products metabolized by Veillonella cultures on media supplemented with 0 or 50 mmol 1^-1 of tartrate, but was inhibited on media supplemented with 100 or 150 mmol 1^-1 of tartrate. Inhibition of Salm. typhimurium was correlated with the increased production of acetate and propionate from tartrate by the Veillonella species.     

A behavioural test of the sensitivity of a nocturnal mosquito, Anopheles gambiae, to dim white, red and infra-red light

Abstract. A behavioural test was used to determine the light sensitivity of the nocturnal mosquito Anopheles gambiae Giles s.s. to low intensities of 'white' light (tungsten filament), 'red' light (white light filtered by a darkroom safelight filter) and 'infra-red' light) of two types (white light filtered by a λ>700 nm filter, and light-emitting diodes with λ>900 nm). Mosquitoes were placed in a 20 cm diameter flight-tunnel and their 'optomotor' response to a pattern of stripes moving across their visual field (at 14.5 cm s^-1) was recorded with infra-red-sensitive video. In free-flight, with ample light, the mosquitoes controlled their flight speed and direction in relation to the stripe movement, so that the stripes always appeared to move across their visual field from front to back. They did this by flying either with the moving stripes fast enough to overtake them (19.5 ± 0.7 cm s^-1), or against them more slowly (10.3 ± 0.7 cm s^-1)- The net ground speed of the mosquitoes was thus c. 4–5 cm s^-1. This response was significant down to 10^-5 W m^-2 in 'white' light, and 10^-3 W m^-2 in 'red' light. At light intensities below threshold and in infra-red light, however, they appeared to fly at random with respect to the stripe movement. The assumption commonly made, that mosquitoes do not 'see' in red light, may thus have to be revised.     

Nutrient reserves of Salicornia europaea seeds

Seeds of Salicornia europaea L. were analyzed for their nutrient reserves. The content of potassium and sodium was 216 and 39 mmol (kg dry seeds)^-1, respectively. Calcium and magnesium accounted for 30 and 138 mmol (kg dry seeds)^-1, respectively. Whereas most of the alkali metals were water soluble, the alkaline earth metals were mostly acid soluble. The acid-soluble calcium plus magnesium corresponded well with the acid-soluble phosphate. Chloride was accumulated to a level equivalent to that of sodium. Carbonate was present at a concentration of 9 mmol (kg dry seeds)^-1. Carbohydrates accounted for 93 g (kg dry seeds)^-1, nearly half of which was derived from sucrose. Fructose and glucose were present only in traces. Total nitrogen was determined to be 55 g (kg dry seeds)^-1, 16% of which was diethylether soluble. The remaining nitrogen was separated into 39 g (kg dry seeds)^-1 ethanol-insoluble and 8 g (kg dry seeds)^-1 ethanol-soluble nitrogen. About 10% of the ethanol-soluble nitrogen were derived from amino acids. Total lipid content was about 280 g (kg dry seeds)^-1. The alcoholic component of the storage lipids was glycerol and the glycerides were calculated from gas chromatography to be 66% of the total lipids. About 90% of the fatty acids consisted of unsaturated acids, linoleic and oleic acid, the majority (77%) of which was linoleic acid.     

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 alcohols on the association of photosynthetic fructose-1,6-bisphosphatase to thylakoid membranes

Aliphatic alcohols have a positive effect on the assoociation of pea ( Pisum sativum L. cv. Lincoln) chloroplast fructose- 1,6-bisphosphatase (FBPase; EC 3.1.3.11) with thylakoid membranes. The alcohol concentration needed to obtain a fixed percentage of enzyme association decreased with increased length of the aliphatic chain of the alcohol; maximum binding was obtained when the lysis medium contained, in molar fractions (or v/v percentages): 48×10^-4(T4 (2.4%), 26×10^-3 (10%), 40×10^-3 (15%), 76×10^-3 (21%), and 13×10^-2 (24%), of 1-butanol, 1-propanol, 2-propanol, ethanol, and methanol, respectively. A good correlation of binding with the octanol/water partition coefficient was observed. Since this coefficient constitutes a measure of hydrophobicity, we suggest that the binding of FBPase to the membranes occurs via hydrophobic clusters of both components.