Inhibition of the tempe mould, Rhizopus oligosporus, by ammonia

The hyphal extension rate of Rhizopus oligosporus NRRL 2710 was slowed in the presence of 0·42 and 0·84 mmol NH3 l⁻¹ and inhibited by 1·3 mmol l⁻¹. Sporulation was prevented at NH3 concentrations of 0·42 mmol l⁻¹ andabove. There was no evidence of toxicity due to NH⁺₄ at concentrations up to 300 mmol l⁻¹.Independent of the concentrations of NH3 or NH⁺₄, the lower the pH value, in therange 6·0–9·0, the higher was the rate of hyphal extension. It is suggested that accumulationof toxic levels of NH3 could be responsible for the cessation of mould growth in tempe.     

Kinetics of FeS-mediated denitrification in sediments from the Camargue (Rhone delta, southern France)

Abstract Denitrification rates were measured in sediments after the addition of different concentrations of FeS. A decrease of the denitrification rate was observed when high concentrations of ferrous iron (> 10 mM) were present. In the experiments with no significant concentrations of free Fe²⁺, the relationship between NO₃⁻ reduction and FeS concentration followed Michaelis and Menten kinetics. The maximum rate was 0.273 mmol l⁻¹ d⁻¹, 6 times as much as the basal rate 0.046 mmol l⁻¹ d⁻¹, which was attributed to organic matter; the K_s was 1.45 mM FeS. The stoichiometry of the overall reaction involving NO₃⁻ reduction and the concomitant S²⁻ oxidation was also investigated. Measured ΔS/ΔN ratios ranged between 0.55 and 0.64, with ΣH₂S + SO₄²⁻ changing less than 10%. These values agree with the theoretically expected value of 0.56.     

Intracellular ion levels in erythrocytes and hepatocytes isolated from three teleost species

Intracellular concentrations of Na⁺ and K⁺ were similar (∼75 mmol l⁻¹) in rainbow trout Oncorhynchus mykiss hepatocytes directly following isolation by collagenase digestion, but partial recovery occurred over 6 h with K⁺ levels increasing to 110 mmol l⁻¹ and Na⁺ levels decreasing to 42 mmol l⁻¹. Black bullhead Ameiurus melas hepatocytes exhibited higher intracellular concentrations of K⁺ (90 mmol l⁻¹) than Na⁺ (55 mmol l⁻¹) with no recovery occurring over 6 h following cell isolation. Concentrations of Na⁺, K⁺ and Cl⁻ in eel Anguilla rostrata hepatocytes were similar (∼ 55 mmol l⁻¹) following isolation, with no recovery occurring over time. Erythrocytes from all species apparently did not experience an intracellular ion imbalance following isolation as indicated by high K⁺ levels (<140 mmol l⁻¹) and low Na⁺ levels (<40 mmol l⁻¹) during the entire 24-h monitoring period. Although hepatocytes from all species exhibited an ion imbalance post-isolation, comparison of their in vitro intracellular Na⁺ and K⁺ concentrations with those in plasma demonstrated that directionally correct ion gradients still exist across the cell membrane, albeit differing from those that would be found in the tissue in vivo .     

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.     

Bactericidal activity of catechin-copper (II) complexes on Escherichia coli ATCC11775 in the absence of hydrogen peroxide

Washed Escherichia coli ATCC11775 cells were killed by (–)-epigallocatechin (EGC) in the presence of a non- lethal concentration of Cu²⁺ (1 μmol l⁻¹) without additional H₂O₂, but not by (–)-epicatechin (EC). EGC alone (< 0·1 mmol l⁻¹) did not reduce the viability of the cells. The survival curve obtained in the presence of EGC and Cu²⁺ was similar to that obtained in the presence of (–)-adrenaline (EN) and Cu²⁺.     

Pathogenesis-related protein b1' in plants and in cell suspension cultures of Nicotiana glutinosa Nicotiana debneyi and an amphidiploid cross (N. glutinosa×N. debneyi)

The expression of PR-protein b₁' in plants and cell suspension cultures of Nicotiana glutinosa L., Nicotiana debneyi Domin, and an amphidiploid cross of these two species, a hybrid, has been investigated. An enzyme linked immunosorbent assay has been employed to determine the concentration of PR-protein b₁' in extracts. The PR-Protein b₁' was constitutively produced in intact plants of the hybrid (around 25 μg g⁻¹ leaf tissue), while only trace amounts of the protein (< 50 ng g⁻¹ leaf tissue) were found in plants of the two parents. In suspension culture, the concentrations of PR-protein b₁' were 8, 0.4 and less than 0.1 mg l⁻¹ medium for the hybrid. N. debneyi and N. glutinosa , respectively. Only trace amounts of the protein were found in extracts from cells. Seven days after infection by tobacco mosaic virus (TMV) the concentration of PR-protein b₁' in leaves of N. glutinosa was 22.5 μg g⁻¹ leaf tissue. In N. debneyi and the hybrid a relatively limited induction of PR-protein b₁' by TMV was observed. The influence of various phenoxyacetic acids on the expression of PR-protein b₁' in the 3 cell cultures has been investigated. Cultures of N. glutinosa responded to treatments with 2,4-D and 2,4,5-T while cultures of N. debneyi and the hybrid were essentially unaffected. In the former case a concentration of 5–10 mg l⁻¹ 2,4,5-T was optimal and cells were most responsive to the treatment 4 days after subcultivation. The concentration of PR-protein b₁' in elicited cell cultures of N. glutinosa was 2 to 4 mg l⁻¹ medium.     

Cell proliferation and polyamine metabolism in 9L cells treated with (2R,5R)-6-heptyne-2,5-diamine or α-difluoromethylornithine

Abstract We studied the effects of the ornithine decarboxylase inhibitors (2R,5R)-6-heptyne-2,5-diamine (R,R,-MAP) and α-difluoromethylornithine (DFMO) on cell proliferation and polyamine metabolism in 9L rat brain tumour cells. Treatment with 5 μM R,R-MAP inhibited cell proliferation to the same extent as did treatment with 1 mM DFMO. Both inhibitors depleted putrescine and spermidine concentrations to less than detectable levels within 24 h and 48 h of drug treatment, respectively; spermine levels were not affected significantly by either inhibitor. The effects of DFMO on 9L cell cycle kinetics were similar to those of R,R-MAP. During the first 3 days of treatment, both drugs caused an accumulation of cells in G₁ and a reduction of cells in S phase, as compared with control cells with a slowing in the rate of cell cycle traverse. In cultures seeded at low (1 × 10⁵), medium (5 × 10⁵), or high (2 × 10⁶) cell densities in a 25 cm² flask, inhibition of cell proliferation and polyamine depletion by both R,R-MAP and DFMO was more pronounced at the lower densities relative to the density-matched control cells. Thus, R,R-MAP was a more potent inhibitor of ornithine decarboxylase than was DFMO in 9L cells, and the inhibitory effects of both compounds on cell proliferation and polyamine biosynthesis were greater in actively proliferating cells.     

Note: Purification of amylase secreted from Bifidobacterium adolescentis

Bifidobacterium adolescentis Int-57 isolated from human faeces produced extracellular amylase. The enzyme was purified from the culture supernatant fluids by ammonium sulphate precipitation, gel-filtration chromatography (Sephadex-G-75), ion-exchange chromatography (CM-cellulose) and FPLC. SDS-PAGE of the purified enzyme revealed a major band with an apparent molecular weight of 66 kDa. The pI was 5·2. Enzyme activity was optimal at 50°C, and at pH 5·5. The enzyme was stable at 20–40°C, and at pH 5–6 with a K _m value of 2·4 g l⁻¹ soluble starch. The activation energy was 42·3 kJ mol⁻¹. The enzyme was significantly inhibited by maltose (10%), glucose (10%), Cu²+ (5 mmol l⁻¹), Zn²+ (5 mmol l⁻¹), N- bromosuccinimide (5 mmol l⁻¹), EDTA (5 mmol l⁻¹), I₂ (1 mmol l⁻¹) and activated by β-mercaptoethanol (10 mmol l⁻¹).     

Methylxanthines as inducers of pectin lyase in Penicillium griseoroseum cultured on sucrose

Pectin lyase (PL) from Penicillium griseoroseum can be induced by xanthine, theobromine, theophylline and especially by caffeine and hypoxanthine (5 mmol l⁻¹ with 0·01% yeast extract (YE)). For caffeine and hypoxanthine, PL activity was, respectively, 5·2 and 3·7 times higher than with YE alone. The simultaneous addition of caffeine or hypoxanthine (5 mmol l⁻¹) and YE (0·1%) had a synergistic effect on PL activity as compared to the addition of these substances alone (0·2% YE; 10 mmol l⁻¹ caffeine; 10 mmol l⁻¹ hypoxanthine). Increasing caffeine concentrations (0–10 mmol l⁻¹) for a constant YE content of 0·01%, resulted in an increase in PL activity and a decrease in mycelial mass. For a constant caffeine concentration (5 mmol l⁻¹) and increasing YE contents (0–0·2%), a higher PL activity and mycelial mass were detected. The addition of caffeine (10 mmol l⁻¹) at the beginning of incubation increased PL activity and decreased mycelial mass, while caffeine added after 12 and 24 h resulted in decreases in PL activity and increases in mycelial mass. The results presented here indicate that methylxanthines, especially caffeine, can induce PL in P. griseoroseum .     

Effects of cultural conditions on denitrification by Pseudomonas stutzeri measured by Membrane Inlet Mass Spectrometry

Denitrification is a globally important process leading to loss of fertiliser efficiency and the production of the greenhouse gas nitrous oxide and nitric oxide, an ozone depleter. Membrane inlet mass spectrometry (MIMS) was employed to study the effect of different variables on the process of denitrification by Pseudomonas stutzeri in a defined salts medium. MIMS was used for concomitant measurements of nitrous oxide, nitrogen and oxygen and showed that denitrification occurred in the presence of dissolved oxygen. A nitrate concentration of 15 mmol l⁻¹ and a nitrite concentration of 5 mmol l⁻¹ were found to be optimum for complete denitrification of nitrate or nitrite to nitrogen and varying these concentrations had a marked effect on the ratio of gaseous products released. Denitrification products were also dependant on pH with neutral or alkaline conditions being best for production of gaseous end products. Our results suggest that under nutrient rich conditions the most important factor in the regulation of denitrification by Ps. stutzeri is the amount of nitrite generated at the first enzymatic stage of the process. This appears to cause inhibition of the denitrification pathway above 5 mmol l⁻¹ and at high enough concentrations (15 mmol l⁻¹) restricts growth.     

Physiological responses of a methylotrophic bacterium after sudden shifts from C-limited chemostat to C-excess batch growth conditions

The response of steady-state continuous cultures of Methylobacterium sp. RXM to the addition of methanol pulses was studied. The increase of methanol concentration in the medium did not result in cell death under any of the conditions tested. When the growth rate of the steady-state cultures was low ( D = 0.046 h⁻¹), the specific growth rate increased. When the concentration of methanol in the pulse was increased from 36 mmol l⁻¹ to 280 mmol l⁻¹, uncoupled growth occurred and the molar cell yield decreased. Conversely, steady-state cultures at high growth rate ( D = 0.2 h⁻¹) showed a decrease in both specific growth rate and molar cell yield after the addition of the methanol pulses (32 and 164 mmol 1⁻¹). For all conditions, formaldehyde and formate were excreted into the medium but the levels did not exceed 1.13 mmol 1⁻¹ Slow-growing cultures were characterized by cells with high derepressed specific activities of methanol dehydrogenase and low specific activities of formaldehyde and formate dehydrogenases, fast-growing cells had lower specific activity for methanol dehydrogenase and higher activities of formaldehyde and formate dehydrogenases, resulting in the excretion of lower concentrations of formaldehyde and formate.
It is concluded that slow-growing cultures are more stable than fast-growing cultures for low methanol concentration fluctuations, and it is expected that maximum growth yields throughout the fermentation time are better achieved under the former conditions. However, for large fluctuations in the substrate concentration, the bacterial metabolic responses were identical both for slow-growing and fast-growing cultures.     

Research note: In vitro inhibition of Listeria monocytogenes growth by veillonellae cultures grown on tartrate media

Veillonellae cultures were grown on agar media supplemented with tartrate and examined for inhibitory effects on the growth of Listeria monocytogenes . Veillonellae cultures grown on media supplemented with 0 or 50 mmol l⁻¹ of tartrate did not inhibit the growth of L. monocytogenes ; however, veillonellae grown on media supplemented with 100, 150 or 200 mmol l⁻¹ of tartrate did inhibit the growth of L. monocytogenes . The inhibition of the growth of L. monocytogenes by the veillonellae was correlated with the increased production of acetate and propionate from tartrate by veillonellae and with the reduction of the pH of the media by L. monocytogenes .     

Inhibition of electron transport activities in mitochondria from avocado and pepper fruit by naturally occurring polyamines

The effects of the naturally occurring polyamines, spermine, putrescine, and spermidine were explored on mitochondrial state 3. state 4, and uncoupled respiration activities, ADP/O ratio, respiratory control ratio of pepper ( Capsicum annuum L. cv. Early Cal Wonder) and avocado ( Persea americana Mill. cv. Booth-8 or Simmonds) mitochondria oxidizing either succinate, external NADH, malate, α-ketoglutarate or tetramethyl- p -phenylenediamine. Abnormally high concentrations of spermine and spermidine such as might occur during chilling stress of these chilling-sensitive fruits were detrimental to several oxidase activities, especially to external NADH oxidase. State 3 respiration for NADH oxidase was inhibited more than 70% by 10 m M spermine. The spermine inhibition of uncoupled NADH oxidase was not reversed by the presence of divalent cations including Ca²⁺, Mg²⁺, Mn²⁺, and Sr²⁺ at concentrations up to 10 m M or by 100 m M KCl. The inhibition primarily affected the V_max. Other possible sites of polyamine interactions are discussed.     

The spermatozoon of the African catfish: fine structure, motility, viability and its behaviour in seminal vesicle secretion

The spermatozoon of the African catfish Clarias gariepinus is a simple organized aquasperm although it reveals very unique characteristics: the cytoplasmic channel is lacking, the mitochondria form a complex structure and the arrangement of the centriolar complex is species specific. Semen has high initial motility rates ( c. 70–90%) and swimming velocities ( c. 120–140 μm s⁻¹), the main swimming type is linear. Motility duration in water is 30 s and is prolonged only to 40 s in NaCl solutions or more complex bu ered motility activating saline solutions. A pH between 7.0 and 9.0 has no e ect on the sperm motility parameters. Motility is completely and reversibly suppressed in electrolyte and non-electrolyte solutions with an osmolality of 200 mosmol kg⁻¹. During immotile storage the sperm viability is influenced by the osmolality and the potassium levels of the storage medium, by the temperature and by the dilution. At optimal conditions (bu ered sperm motility inhibiting saline solution: 150 mmol l⁻¹ NaCl, 2.5 mmol l⁻¹ KCl, 1 mmol l⁻¹ CaCl₂, 1 mmol l⁻¹ MgSO₄, 20 mmol l⁻¹ Tris solution, pH 8.5; dilution rate 1: 5; storage temperature, 4°C) sperm viability persists for >7 days. High viscosity of the pure seminal vesicle secretion completely inhibits the sperm motility. When the seminal vesicle secretion is diluted in water the viscosity decreases and the motility suppressing e ect is neutralized. When semen is mixed with seminal vesicle secretion the sperm viability decreases to zero within 10 min.     

Exoprotease activity of Leuconostoc oenos in stress conditions

Exoprotease activity during 48 h of total energy and nutrient starvation was examined in Leuconostoc oenos X₂L isolated from wine. Starved cells after 2 h of incubation at 30 °C in citrate buffer, 0.05 mmol 1⁻¹ pH 5, showed greater extracellular proteolytic activity than at the onset of starvation. In the presence of 60 mg l⁻¹ SO₂ and 8% or 12% ethanol, the proteolytic activity was higher ; 10 mmol l⁻¹ Ca²⁺ and Mg²⁺ produced an increase in protease activity during starvation. Glucose and 2-deoxyglucose (2-DOG) were found to repress synthesis by 80% and 100%, respectively. Cyclic adenosine 3'-5'-phosphate increased the exoprotease activity and reverted the repression by glucose and 2-DOG. De novo synthesis of proteins was required for the exoprotease activity by cells submitted to stress conditions. The absence of protease activity in the supernatant fluids from chloramphenicol-treated cells indicated that the activity is a result of deliberate release and not of passive cell lysis.     

Isolation and characterization of an intracellular esterase from Lactobacillus casei subsp. casei IFPL731

An intracellular esterase from Lactobacillus casei subsp. casei IFPL731 was purified 1000-fold by ion exchange chromatography and gel filtration chromatography. The relative molecular mass of the native enzyme was 105 kDa, while the subunit molecular mass was estimated to be 38 kDa. The esterase hydrolysed tributyrin and had a preference for esters of short-chain fatty acids (butyrate, caproate and caprylate), while it did not hydrolyse palmitate and sterate esters. The apparent Michaelis-Menten constant of the enzyme on p -nitrophenyl butyrate was 0·3 mmol l⁻¹ while on p -nitrophenyl caprylate, it was 0·04 mmol l⁻¹. The esterase was active over a broad range of pH and temperature values, and retained about 50% of maximal activity at pH 5·0 and 12 °C. Activity was strongly inhibited by 5 mmol l⁻¹ phenylmethylsulphonyl fluoride, β-mercaptoethanol and N -ethylmaleimide, and was stimulated by Zn²⁺ at 1 mmol l⁻¹.     

The effects of variable nitrogen and phosphorous concentrations on Eriophorum vaginatum tillers grown in nutrient solutions

Eriophorum vaginatum tillers were collected at Eagle Creek, Alaska and cultivated in aerated solutions under controlled environmental conditions. The nutrient solutions contained traces, 1.05 and 21 mg l⁻¹ N (nitrate) and traces, 0.15 and 3.10 mg l⁻¹ P (phosphate), pH was maintained at 5.5. The high N, 21 mg l⁻¹, and P, 3.18 mg l⁻¹, nutrient solution produced significant biomass increases. Functional leaf areas were significantly enhanced by high N and P doses in the solutions. Root surface areas varied considerably between treatments; however, significant differences were not found. The mean root surface area of a tiller reached 126 cm² (range 35–290 cm²), whereas the functional leaf area was 6.8 cm² (range 3.3–20.3 cm²). Tillers growing in the highest N + P solutions produced twice the number of daughter tillers as tillers growing in solutions with trace amounts of N and P.     

Characterization of acetylcholinesterase from Arthrobacter ilicis associated with the marine sponge (Spirastrella sp.)

The bacterium Arthrobacter ilicis isolated from the marine sponge Spirastrella sp. produces extracellular serine type acetylcholinesterase. The maximum enzyme activity was found at 45 °C and pH 8·0. The activation and deactivation energies, calculated from an Arrhenius plot, were 13·68 and 36·96 kcal mol⁻¹, respectively. The enzyme was not affected by the addition of the major cations of sea water, such as Ca²⁺ and Mg²⁺ at 25 mmol l⁻¹, and was strongly inhibited by EDTA and different organophosphorus and carbamate compounds at 5 mmol l^−1.     

Competition between anoxygenic phototrophic bacteria and colorless sulfur bacteria in a microbial mat

Abstract The populations of chemolithoautotrophic (colorless) sulfur bacteria and anoxygenic phototrophic bacteria were enumerated in a marine microbial mat. The highest population densities were found in the 0–5 mm layer of the mat: 2.0 × 10⁹ cells cm⁻³ sediment, and 4.0 × 10⁷ cells cm⁻³ sediment for the colorless sulfur bacteria and phototrophs, respectively. Kinetic parameters for thiosulfate-limited growth were assessed for Thiobacillus thioparus T5 and Thiocapsa roseopersicina M1, both isolated from microbial mats. For Thiobacillus T5, growing at a constant oxygen concentration of 43 μmol l⁻¹, μ_max was 0.336 h⁻¹ and K _s 0.8 μmol l⁻¹. Phototrophically grown Thiocapsa strain M1 displayed a μ_max of 0.080 h⁻¹ and a K _s of 8 μmol l⁻¹ when anoxically grown under thiosulfate limitation. In a competition experiment with thiosulfate as electron donor, Thiocapsa became dominant during a 10-h oxic/14-h anoxic regimen at continuous illumination, despite the higher affinity for thiosulfate of Thiobacillus .     

Effect of changes in nitrogen nutrition on the polyamine content of Alnus glutinosa

Abstract. The principal polyamines in Alnus glutinosa roots, nodules and root pressure sap, putrescine, spermidine and spermine, were quantified by reversed-phase, high-performance liquid chromatography with fluorescence detection following precolumn derivatization with 9-fluorenylmethyl chloroformate and 1-ada-mantanamine. Putrescine was the major component of all tissues and sap. It comprised 70% or more of the polyamine pool except in roots of KNO₃-fed plants, in which similar amounts of putrescine and spermidine were present at levels five-fold lower than plants fed (NH₄)₂SO₄. Polyamine levels in nodules were 50% greater than in roots. The polyamine content of roots and nodules was not altered significantly when the nitrogen nutrition was changed from sole reliance on nitrogen fixation to partial or complete utilization of (NH₄)₂SO₄. However, the polyamine content of root pressure sap from nodulated plants increased almost four-fold when they were fed with increasing concentrations of NH₄NO₃, although the total polyamine content remained low (5mmol m⁻³ sap). The polyamine content of the Alnus root system changed with plant age. In particular, the spermidine content of both roots and nodules was higher in 10- as compared to 16-week-old plants.