Seed dimorphism in Salicornia europaea: Nutrient reserves

Median and lateral seeds of Salicornia europaea L. were separately analysed for their sizes and nutrient reserves. The mean air-dry weight of a single median and lateral seed was 0.31 and 0.25 mg, respectively. The composition as well as the concentration of the nutrient reserves were similar in both seed types. The bulk of the cations was derived from K⁺, followed by Mg²⁺, Na⁺ and Ca²⁺. The chloride content was somewhat higher than the sodium content, and phosphate was equalled by acid soluble Ca²⁺ and Mg²⁺. Starchy compounds and sucrose were present in equal amounts, each of them accounted for about 50% of the carbohydrates. Glucose and fructose were less than 1%. Protein-nitrogen (ethanol-insoluble N) was about 34 g (kg dry seeds)⁻¹. About 7 g (kg dry seeds)⁻¹ was ethanol-soluble nitrogen, of which 10% was derived from amino acids. The total lipid content was more than 290 g (kg dry seeds)⁻¹, 65% were calculated to be glycerides. More than 90% of the fatty acids consisted of linoleic and oleic acids, the majority (72%) of which was linoleic acid.     

Identification of 4-chloroindole-3-acetic acid and indole-3-aldehyde in seeds of Pinus sylvestris

4-Chloroindole-3-acetic acid (4-Cl-IAA) and indole-3-aldehyde (IAId) have been characterized as endogenous constituents in seeds of Pinus sylvestris L. by gas chromatography-mass spectrometry. Quantitative estimates indicate that immature seeds contained 640 pg 4-Cl-IAA (g fresh weight)^-1 while mature seeds contained 340 pg (g dry weight)^-1. 4-Cl-IAA could not be detected in seeds five days after germination. The content of IAld increased from 127 pg (g dry weight)^-1 in mature seeds to 315 pg (g dry weight)^-1 after five days of germination.     

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 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.     

The use of fed batch cultivation for achieving high cell densities in the production of a recombinant protein in Escherichia coli

Abstract: The production of the fusion protein staphylococcal protein A/E. coli β-galactosidase in Escherichia coli was studied in batch and fed batch cultivations. Batch cultivation of a recombinant E. coli strain yielded a final cell dry weight of 16.4 g 1^-1 with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the cell dry weight. Fed batch cultivation made it possible to increase the final cell dry weight to 77.0 g 1^-1. The intracellular product concentration (25%) was lower as compared to batch cultivation resulting in a total concentration of recombinant protein of 19.2 g 1^-1.     

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.     

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.     

Effects of glyphosate on nitrogen fixation of free-living heterotrophic bacteria

The effect of the herbicide glyphosate ( N -(phosphonomethyl)glycine) on the growth, respiration and nitrogen fixation of Azotobacter chroococcum and A. vinelandii was studied. Azotobacter vinelandii was more sensitive to glyphosate toxicity than A. chroococcum. Recommended dosages of glyphosate did not affect growth rates. More than 4 kg ha^-1 is needed to find some inhibitory effect. Specific respiration rates were 19.17 mmol O₂ h^-1 g^-1 dry weight for A. chroococcum and 12.09 mmol h^-1 g^-1 for A. vinelandii. When 20 kg ha^-1 was used with A. vinelandii , respiration rates were inhibited 60%, the similar percentage inhibition A. chroococcum showed at 28 kg ha^-1. Nitrogen fixation dropped drastically 80% with 20 kg ha^-1 in A. vinelandii and 98% with 28 kg ha^-1 in A. chroococcum. Cell size as determined by electron microscopy decreased in the presence of glyphosate, probably because glyphosate induces amino acid depletion and reduces or stops protein synthesis.     

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.     

Effects of substrates and phosphate on INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride) and CTC (5-cyano-2,3-ditolyl tetrazolium chloride) reduction in Escherichia coli

The effects of substrates of primary aerobic dehydrogenases, and inorganic phosphate on aerobic INT and CTC reduction in Escherichia coli were examined. In general, INT produced less formazan than CTC, but INT (+) cell counts remained near values of CTC (+) cells. INT and CTC (+) cell numbers were higher than plate counts on R2A medium using succinate, formate, lactate, casamino acids, glucose, glycerol (INT only) and no substrate. Formate resulted in the greatest amount of INT and CTC formazan. Reduction of both INT and CTC was inhibited above 10 mmol 1^-1 phosphate, and this appeared to be related to decreased rates of O₂ consumption. Formation of fluorescent CTC (+), but not INT (+) cells was also inhibited in a concentration dependent manner by phosphate above 10 mmol 1^-1. From light microscopic observations it appeared CTC formed increasing amounts of poorly or non-fluorescent formazan with increasing phosphate. Therefore, use of phosphate buffer in excess of 10 mmol 1^-1 may not be appropriate in CTC and INT reduction assays.     

Citrate metabolism in 16 Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum strains

Citrate metabolism was studied in non-growing cells of Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum with respect to energetics, formation of degradation products and stoichiometry. The use of selective ionophores and uncoupler showed that citrate utilization was coupled to the proton motive force generated by ATP hydrolysis. Differences in citrate metabolism observed in 20 Leuconostoc strains were related to strains but not to the species or subspecies studied. Citrate metabolism was stimulated by glucose up to a concentration of 25 mmol 1^-1 and decreased at higher concentrations. The main degradation products resulting from the co-metabolism of citrate (10 mmol 1^-1) and glucose (2 mmol 1^-1) were acetate, lactate and pyruvate. Only four Leuconostoc strains produced low levels of acetoin and diacetyl. No strains produced ethanol or acetaldehyde. Citrate degradation ability was stable for at least 130 generations in 81% of the Leuconostoc strains.     

Content of low-molecular-weight thiols during the imbibition of Pea seeds

The metabolism of low-molecular-weight thiols was investigated in seeds of Pisum sativum L. cv. Kleine Rheinländerin during imbibition in water for 14 h. The amount of oxidized glutathione (GSSG) decreased from 319 nmol (g dry weight)⁻¹ in dry seeds to 38 nmol (g dry weight)⁻¹ within the first 14 h of imbibition. The decrease may have been due to the reduction of GSSG to reduced glutathione (GSH), catalyzed by the enzyme glutathione reductase (GR; EC 1.6.4.2). The enzyme activity was high in dry seeds [25 nkat (g dry weight)⁻¹] and decreased to 20 nkat (g dry weight)⁻¹ within 14 h of imbibition. The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) decreased from 100 nkat (g dry weight)⁻¹ in dry seeds to 67 nkat (g dry weight)⁻¹ after 14 h of imbibition. Within 14 h the amount of γ-glutamyl-cysteine (γ-GC) decreased from 135 to 38 nmol (g dry weight)⁻¹, whereas the cysteine content rose from 81 nmol (g dry weight)⁻¹ in dry seeds to a maximum of 170 nmol (g dry weight)⁻¹ after 12 h of imbibition, which may be due to the degradation of γ-GC into cysteine.     

Involvement of potassium ions in the action of various antineoplastic drugs on the growth of Saccharomyces cerevisiae

The involvement of potassium ions in the action of some antineoplastic drugs on the growth of Saccharomyces cerevisiae was studied by incubating yeast cells in the presence of drugs at various concentrations and KC1 at concentrations of 50 mmol 1^-1 and 100 mmol 1^-1. The presence of 6.25–50 μg m1^-1 amsacrine or melphalan alone in the culture medium had no significant effect on yeast growth. Addition of KC1 significantly increased the sensitivity to these drugs. On the contrary, incubation of yeast cells with KC1 had no effect on the cytotoxic action of doxorubicin, methotrexate or 5-fluorouracil.     

Transfer of plasmids between strains of Escherichia coli under rumen conditions

K. P. SCOTT AND H.J. FLINT. 1995. Strains of Escherichia coli originally isolated from the rumen of sheep were shown to be capable of exchanging a 60kb plasmid, conferring resistance to tetracycline and ampicillin, at low frequencies (below 10^-6 per recipient) under anaerobic conditions in the presence of (a) autoclaved and clarified rumen fluid, (b) raw clarified rumen fluid, or (c) whole rumen fluid. Under anaerobic conditions the two rumen strains showed no inhibition of growth rate when 50 mmol 1^-1 volatile fatty acids were added to LB medium at pH 7, although significant inhibition resulted with 100 mmol 1^-1 VFA. The two rumen strains, and four strains from the pig gut, showed less inhibition of anaerobic growth by volatile fatty acids than did three laboratory strains examined for comparison. These findings indicate that plasmid transfer between certain E. coli strains can occur under conditions that closely simulate an anaerobic gut environment.     

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.     

Purification and characterization of diacetyl reductase from Kluyveromyces marxianus

Diacetyl reductase from Kluyveromyces marxianus NRRL Y-1196 was purified 27.5-fold with a yield of 13% by ammonium sulphate fractionation, DEAE-anion exchange chromatography, hydroxyapatite chromatography and chromatofocusing. The purified enzyme was most active at pH 7.0 and exhibited optimal activity at 40°C. The K _m and V _max values for diacetyl were 2.5 mmol 1^-1 and 0.026 mmol 1^-1 min^-1, respectively. The enzyme did not react with monoaldehydes or monoketones, but reduced acetoin, diacetyl and methylglyoxal with NADH as a cofactor. The enzyme had an isoelectric point (pl) of pH 5.8, and its molecular weight was 50 kDa.     

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.     

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.     

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.     

Digestibility and retension of food components in caged mountain hares Lepus timidus during the winter

Adult mountain hares in outdoor cages were studied in order to evaluate the consumption and the digestibility of natural winter foods. Food, feces and urine were analysed for nitrogen, phosphorus, calcium, potassium, magnesium and sodium, and food and feces for fiber as well. The metabolism of the different nutrients are presented in this paper.
When energy equilibrium was maintained hares assimilated as an average 21.6 g dry matter per kg body weight and day (29.5 g kg^0-75). The fiber fraction showed a low digestibility ranging from 10 to 30% between different food species.
At energy equilibrium nitrogen equilibrium was usually also achieved; the hares then digested approximately 0.20–0.25. g nitrogen per kg body weight and day (0.27–0.34 kg ^-0.75). The apparent nitrogen digestibility was low, usually below 40%.
The demand for calcium, potassium and magnesium seemed to be well covered via the food. Phosphorus on the other hand was absorbed in small amounts only, which may sometimes have been insufficient. A negative sodium balance occurred when the hares were fed heather or the finest twigs of birch. Due to the low concentrations in the food this could create problems in the supply of sodium.