A novel liposome-based electrochemical biosensor for the detection of haemolytic microorganisms

Summary A biosensor strategy for rapid amperometric detection of organisms was investigated in which a redox mediator was entrapped within liposomes. The selective release of mediator by haemolytic bacteria, followed by signal generation, confers differentiation between haemolytic and non-haemolytic species. The potential of this approach is illustrated by results for various strains of Listeria monocytogenes, Listeria welshimeri and Escherichia coli.     

H+-ATPase-Mediated Cytoplasmic pH-Responses Associated with Elevation of Cytoplasmic Calcium in Cultured Rabbit Nonpigmented Ciliary Epithelium

Studies were conducted to test whether an increase of cytoplasmic calcium concentration influences H⁺-ATPase activity in cultured rabbit nonpigmented ciliary epithelium (NPE). Cytoplasmic calcium concentration or cytoplasmic pH was measured by a fluorescence ratio technique in cells loaded with either Fura-2 or BCECF. Cytoplasmic calcium was increased in three ways; by exposure to BAY K 8644 (1 μm), by exposure to a mixture of epinephrine (1 μm) + acetylcholine (10 μm) or by depolarization with potassium-rich solution. In each case cytoplasmic pH increased significantly. In all three cases 100 nm bafilomycin A₁, a specific H⁺-ATPase inhibitor, significantly inhibited the pH increase. These results suggest an increase of cytoplasmic calcium might initiate events that lead to activation of proton export from the cytoplasm by a mechanism involving H⁺-ATPase. This notion is supported by the observation that the pH increase was suppressed when either verapamil or nifedipine was used to prevent the cytoplasmic calcium increase in cells exposed to potassium-rich solution. Protein kinase C activation might also be involved in the mechanism of H⁺-ATPase stimulation since staurosporine suppressed the pH response to potassium-rich solution. A transient rise of cytoplasmic calcium concentration was observed when cytoplasmic acidification was induced by exposure to high pCO₂. This suggests a rise of cytoplasmic calcium might represent part of a physiological mechanism to stimulate H⁺-ATPase-mediated protein export under acid conditions. Received: 11 August 2000/Revised: 29 March 2001     

Fine structure of the vagina,accessory glands,uterus, oviducts and Gene's organ in the unfed tick,Ornithodoros (Pavlovskyella) erraticus (Ixodoidea: Argasidae)

The fine structure of the reproductive system of the unfed femaleOrnithodoros (Pavlovskyella) erraticus is described. The vagina consisting of vestibular (vv) and cervical (cv) regions is formed of a single epithelial layer lined with cuticle and surrounded by muscle layers. Epithelial cells ofvv show no signs of activity, while those ofcv have structural features of transporting epithelia. A pair of tubular accessory glands opening at the junction of the two vaginal regions consist of a layer of microvillate columnar cells containing rough endoplasmic reticulum; this is possibly involved in the formation of colloidal material which presumably coats the egg surface during its passage through the vagina. The bilobed uterus opening into the cervical vagina secretes material that is possibly responsible for the release of sperm from the endospermatophore. The paired oviducts joining the uterine lobes anteriorly have distinctive distal (do) and proximal (po) parts as well as ampullae (amp) at their junction.do cells contain many lysosome-like structures which probably play a role in breaking down some sperms ascending to the ovary. Cells ofamp are packed with rickettsia-likeWolbachia, while those ofpo contain a few granules. Gene's organ, present in female ticks only, consists of a stalk and two horns of epithelium lined internally by a cuticular layer. Bundles of microtubules are detected in the horn cell cytoplasm. This organ functions to waterproof the eggs with a waxy layer just before they are deposited.     

Purification and stabilization of phenylalanine ammonia lyase fromSporidiobolus pararoseus

The purification of phenylalanine ammonia-lyase (PAL) from S. pararoseus is described: PAL is recovered from cellular extracts by acid precipitation of contaminating proteins, nucleic acids and carotenoids; concentrated by ultrafiltration and purified by ion exchange chromatography in DEAE-Sephacel, with 80% yield and 32.6 fold purification. Elimination of carotenoids and stabilization with glycerol are introduced in the methodology to increase stability and purification yield.     

<Emphasis Type="SmallCaps">d</Emphasis>-amino acids for the enhancement of a binary biocide cocktail consisting of THPS and EDDS against an SRB biofilm

Biofilms of sulfate reducing bacteria (SRB) are often responsible for Microbiologically Influenced Corrosion (MIC) that is a major problem in the oil and gas industry as well as water utilities and other industries. This work was inspired by recent reports that some d-amino acids may be useful in the control of microbial biofilms. A d-amino acid mixture with equimolar d-tyrosine, d-methionine, d-tryptophan and d-leucine was tested in this work for their enhancement of a biocide cocktail containing tetrakis (hydroxymethyl) phosphonium sulfate (THPS) and ethylenediamine-N,N’-disuccinic acid (EDDS). Desulfovibrio vulgaris (ATCC 7757) was cultured in ATCC 1249 medium. Its biofilm was grown on C1018 carbon steel coupons. Experimental results indicated that the triple biocide cocktail consisting of 30 ppm THPS, 500 ppm EDDS and 6.6 ppm d-amino acid mixture (with equimolar d-tyrosine, d-methionine, d-tryptophan and d-leucine) was far more effective than THPS and EDDS alone and their binary combination. The triple biocide cocktail effectively prevented SRB biofilm establishment and removed the established SRB biofilm. The d-amino acid mixture alone did not show significant effects in the two tasks even at 660 ppm.     

Residual toxicity of some common acaricides in Brazil to the predacious miteAmblyseius brazilli

The direct toxicity of 5 common acaricides toAmblyseius brazilli El-Banhawy was tested. Omethoate proved to be the most toxic and Chlorobenzilate the least toxic. LC₅₀ were 1.2ppm, 220ppm, 720ppm and 1250ppm for Omethoate, Mexacarbate, Dicofol, Mitran and Chlorobenzilate respectively. The indirect toxicity was also studied by providing the female predators with the phytophagous miteAponicus spinosus (Banks) previously treated by Chlorobenzilate and Mexacarbate. The females were fed on the treated as well as the untreated prey. The eg laying was interrupted and almost stopped in the case of Mexacarbate.

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Résumé La toxicité directe de 5 acaricides communs surAmblyseius brazilli El-Banhawy a été examinée. L'Ométhoate est le plus toxique et le Chlorobenzilate le moins toxique. Les CL₅₀ sont respectivement de 1,2ppm, 4,7ppm, 220ppm, 720ppm et 1250ppm pour l'Ométhoate, le Mexacarbate, le Dicofol, le Mitran et le Chlorobenzilate. De même la toxicité indirecte des acaricides a été examinée en fournissant aux prédanteus femelles l'acarien phytophageAponicus spinosus (Banks) traité par le Chlorobenzilate et le Mexacarbate. Les femelles on été nourries de proies traitées et non traitées. La ponte a été interrompue; elle a été presque arrêtée dans le cas du Mexacarbate.

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This work was conducted as part of a program of research on the predadcious mites of Brazil, under support of a National Research Council grant to Dr.E. M. El-Banhawy.     

Electrophysiological Characterization of the Flounder Type II Na+/P i Cotransporter (NaPi-5) Expressed in Xenopus laevis Oocytes

The two electrode voltage clamp technique was used to investigate the steady-state and presteady-state kinetic properties of the type II Na⁺/P _i cotransporter NaPi-5, cloned from the kidney of winter flounder (Pseudopleuronectes americanus) and expressed in Xenopus laevis oocytes. Steady-state P _i -induced currents had a voltage-independent apparent K _m for P _i of 0.03 mm and a Hill coefficient of 1.0 at neutral pH, when superfusing with 96 mm Na⁺. The apparent K _m for Na⁺ at 1 mm P _i was strongly voltage dependent (increasing from 32 mm at −70 mV to 77 mm at −30 mV) and the Hill coefficient was between 1 and 2, indicating cooperative binding of more than one Na⁺ ion. The maximum steady-state current was pH dependent, diminishing by 50% or more for a change from pH 7.8 to pH 6.3. Voltage jumps elicited presteady-state relaxations in the presence of 96 mm Na⁺ which were suppressed at saturating P _i (1 mm). Relaxations were absent in non-injected oocytes. Charge was balanced for equal positive and negative steps, saturated at extremes of potential and reversed at the holding potential. Fitting the charge transfer to a Boltzmann relationship typically gave a midpoint voltage (V _0.5) close to zero and an apparent valency of approximately 0.6. The maximum steady-state transport rate correlated linearly with the maximum P _i -suppressed charge movement, indicating that the relaxations were NaPi-5-specific. The apparent transporter turnover was estimated as 35 sec⁻¹. The voltage dependence of the relaxations was P _i -independent, whereas changes in Na⁺ shifted V _0.5 to −60 mV at 25 mm Na⁺. Protons suppressed relaxations but contributed to no detectable charge movement in zero external Na⁺. The voltage dependent presteady-state behavior of NaPi-5 could be described by a 3 state model in which the partial reactions involving reorientation of the unloaded carrier and binding of Na⁺ contribute to transmembrane charge movement. Received: 11 March 1997/Revised: 3 June 1997     

<Emphasis Type="SmallCaps">l</Emphasis>-Arginine Suppresses Aggregation of Recombinant Growth Hormones in Refolding Process from <Emphasis Type="Italic">E. coli</Emphasis> Inclusion Bodies

l-Arginine was used to suppress the aggregation of recombinant mink and porcine growth hormones in the refolding process from E. coli inclusion bodies by solubilization–dilution protocol at high protein concentration and pH 8.0. The influence of l-arginine concentration on the renaturation yield of both proteins was investigated. l-Arginine effectively suppressed the precipitation of growth hormones during dilution, but did not inhibit soluble oligomers formation. The results of mink and porcine growth hormones purification from 4 g of biomass are presented.     

The effect of 2-chloro, 6-(trichloromethyl) pyridine on the chemoautotrophic metabolism of nitrifying bacteria

Studies were conducted to elucidate the mechanism of action of 2-chloro-6-(trichloromethyl)pyridine or Technical N-SERVE on the nitrification process brought about byNitrosomonas europaea. The growth ofNitrosomonas was completely inhibited in the presence of 0.2 ppm N-SERVE while 1.0 ppm of the chemical was effective in the complete inhibition of ammonia oxidation by fresh cell suspensions. Cells stored at 4 C for a period of three days required somewhat higher concentrations (1.5 ppm) of N-SERVE for the complete inhibition of their ammonia oxidizing ability while the cytochrome oxidase of these cells was inhibited to the extent of 65 to 70 percent in the presence of a corresponding amount of N-SERVE. A 45 – 70 percent reversal of the inhibition of ammonia oxidation caused by N-SERVE was obtained by the addition of 6×10^–4 M Cu⁺⁺. An equivalent concentration of Cu⁺⁺ was also effective for the complete reversal of the inhibition of cytochrome oxidase present in whole cells.Hydroxylamine oxidation by intactNitrosomonas cells was not affected by levels of N-SERVE ranging from 1 – 3 ppm. The cytochrome oxidase effective in hydroxylamine oxidation and present in cell-free extracts was not inhibited by even 100 ppm N-SERVE. Likewise, the hydroxylamine activating enzyme hydroxylamine cytochromec reductase was also not inhibited by such levels of the chemical. Raising the concentration to 170 ppm N-SERVE, however, caused a 90 percent inhibition of the enzyme.Although a 5×10^–6 M concentration of allylthiourea completely inhibited ammonia oxidation byNitrosomonas cells, concentrations up to 10^–3 M of this compound did not affect the cytochrome oxidase activity of whole cells or cell-free extracts. The inhibition of ammonia oxidation caused by 5×10^–6 M allythiourea, unlike the inhibition by N-SERVE, could not be reversed by the addition of 6×10^–4 M Cu⁺⁺.Evidence is presented that the action of N-SERVE is on that component of cytochrome oxidase which is involved in ammonia oxidation.     

Chiral resolution of phenylalanine by d-Phe imprinted membrane considering rejection property

An enantio-selective d-Phe imprinted P(AA-co-AN) membrane was prepared using the wet-phase inversion method. The membrane not only selectively adsorbed phenylalanine but also rejected it with a rejection selectivity of 0.82–0.64 and 0.91–0.63 during the filtration of 100 and 10 ppm (g m⁻³) racemate solutions, respectively. The fluxes of d-Phe and l-Phe during filtration of 10 ppm racemate solution were 0.0077–0.0229 and 0.0064–0.0208 mg m⁻² s⁻¹, respectively, and the fluxes of d-Phe and l-Phe during filtration of 100 ppm racemate solution were 0.1287–0.2522 and 0.1174–0.2458 mg m⁻² s⁻¹, respectively. The adsorption selectivity was higher at low concentration. The adsorption selectivities varied from 1.11 to 1.65 and from 1.64 to 2.78 during filtration of 100 and 10 ppm racemate solutions, respectively. In respect to desorption, the fractional difference between d-Phe and l-Phe in the recovered solution from 10 ppm was higher than that from 100 ppm.     

Production of l-alanine from ammonium fumarate using two immobilized microorganisms

Summary For the efficient production of l-alanine from ammonium fumarate using the aspartase activity of immobilized Escherichia coli cells and l-aspartate -decarboxylase activity of immobilized Pseudomonas dacunhae cells, alanine racemase and fumarase activities should be eliminated. We investigated various procedures to eliminate these side reactions, and found that both activities of intact E. coli cells could be eliminated by treating the culture broth at pH 5.0 and 45° C for 1 h, and those of intact P. dacunhae cells could be eliminated by treating the culture broth at pH 4.75 and 30° C for 1 h. Further, it was confirmed that l-alanine was efficiently produced using these two immobilized pH-treated microorganisms.     

Studies on Aspergilli XVI. Effect of pH,temperature, and carbon and nitrogen interaction

Summary The effect of pH, temperature, and carbon and nitrogen interaction on the growth and sporulation ofAspergillus nidulans (Eidam)Wint.,A. rugulosus Thom &Raper,A. variecolor (Berk. &Br.)Thom &Raper andA. quadrilineatus was studied. All the moulds could grow on a wide range of pH (2.0 to 12.0) but the growth was poor on too acid and too alkaline media. Best growth ofA. rugulosus, A. quadrilineatus, andA. violaceus was seen at pH 6.5 and that ofA. nidulans andA. variecolor at pH 7.0. In general maximum production of perithecia was recorded between pH 6.0 and 8.0.All the above species ofAspergillus under study could grow between a temperature range of 10° C–48° C, but the growth was poor at 10° C and 48° C. The present moulds showed good growth at 20° C, 25°C, and 30° C. At 40° CA. nidulans andA. rugulosus showed moderate growth while the rest of the Aspergilli attained good growth. Temperatures between 20° C–30° C favoured excellent perithecial production.In general, little improvement in growth was noted on media containing good carbon and nitrogen sources. Malic acid was found to be useless when supplied singly. But, poor growth was recorded when supplied in combination with amino acids, amide, and peptone. This was due to the fact that these N sources also supplied carbon for their metabolism.     

<Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> NAD-dependent, <Emphasis Type="SmallCaps">D</Emphasis>-fructose reducing,polyol dehydrogenases activity: screening,medium optimisation and application for enzymatic polyol production

Gluconobacter oxydans LMG 1489 was selected as the best strain for NAD(P)-dependent polyol dehydrogenase production. The highest enzyme activities were obtained when this strain was cultivated on a medium consisting of 30 g glycerol l^–1, 7.2 g peptone l–1 and 1.8 g yeast extract l^–1. Two D-fructose reducing, NAD-dependent intracellular enzymes were present in the G. oxydans cell-free extract: sorbitol dehydrogenase, and mannitol dehydrogenase. Substrate reduction occurred optimally at a low pH (pH 6), while the optimum for substrate oxidation was situated at alkaline pHs (pH 9.5–10.5). The mannitol dehydrogenase was more thermostable than the sorbitol dehydrogenase. The cell-free extract could be used to produce D-mannitol and D-sorbitol enzymatically from D-fructose. Efficient coenzyme regeneration was accomplished by formate dehydrogenase-mediated oxidation of formate into CO₂.     

Ethylenediamine uptake and metabolism in the cyanobacterium Anabaena variabilis

The cyanobacterium Anabaena variabilis showed a pH dependent uptake of ethylenediamine. No uptake of ethylenediamine was detected at pH 7.0. At higher pH values (e.g. pH 8.0 and pH 9.0) accumulation did occur and was attributed to diffusion of uncharged ethylenediamine in response to a pH gradient. A biphasic pattern of uptake was observed at these higher pH values. Treatment with l-methionine-d,l-sulphoximine (MSX) to inactivate glutamine synthetase (GS) inhibited the second slower phase of uptake without any significant alteration of the initial uptake. Therefore for sustained uptake, metabolism of ethylenediamine via GS was required. NH ₄ ⁺ did not alter the uptake of ethylenediamine. Ethylenediamine was converted in the second phase of uptake to an analogue of glutamine which could not be detected in uptake experiments at pH 7.0 or in uptake experiments at pH 9.0 following pretreatment of cells with MSX. Ethylenediamine treatment inhibited nitrogenase activity and this inhibition was greatest at high pH values.Abbreviations EDA 1,2-diaminoethane (ethylenediamine) - GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1 piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine - membrane potential - Tricine N-tris(hydroxymethyl) methylglycine     

Role of the bilayer in the shape of the isolated erythrocyte membrane

Summary The determinants of cell shape were explored in a study of the crenation (spiculation) of the isolated erythrocyte membrane. Standard ghosts prepared in 5mm NaP_i (pH 8) were plump, dimpled disks even when prepared from echinocytic (spiculated) red cells. These ghosts became crenated in the presence of isotonic saline, millimolar levels of divalent cations, 1mm 2,4-dinitrophenol or 0.1mm lysolecithin. Crenation was suppressed in ghosts generated under conditions of minimal osmotic stress, in ghosts from red cells partially depleted of cholesterol, and, paradoxically, in ghosts from red cells crenated by lysolecithin. The susceptibility of ghosts to crenation was lost with time; this process was potentiated by elevated temperature, low ionic strength, and traces of detergents or chlorpromazine.In that ghost shape was influenced by a variety of amphipaths, our results favor the premise that the bilayer and not the subjacent protein reticulum drives ghost crenation. The data also suggest that vigorous osmotic hemolysis induces a redistribution of lipids between the two leaflets of the bilayer which affects membrane contour through a bilayer couple mechanism. Subsequent relaxation of that metastable distribution could account for the observed loss of crenatability.     

Characterization of new <Emphasis Type="SmallCaps">l,d</Emphasis>-endopeptidase gene product CwlK (previous YcdD) that hydrolyzes peptidoglycan in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis has various cell wall hydrolases, however, the functions and hydrolase activities of some enzymes are still unknown. B. subtilis CwlK (YcdD) exhibits high sequence similarity with the peptidoglycan hydrolytic l,d-endopeptidase (PLY500) of Listeria monocytogenes phage and CwlK has the VanY motif which is a d-alanyl-d-alanine carboxypeptidase (Pfam: http://www.sanger.ac.uk/Software/Pfam/). The β-galactosidase activity observed on cwlK-lacZ fusion indicated that the cwlK gene was expressed during the vegetative growth phase, and Western blotting suggested that CwlK seems to be localized in the membrane. Truncated CwlK fused with a histidine-tag (h-ΔCwlK) was produced in Escherichia coli and purified on a nickel column. The h-ΔCwlK protein hydrolyzed the peptidoglycan of B. subtilis, and the optimal pH, temperature and NaCl concentration for h-ΔCwlK were pH 6.5, 37°C, and 0 M, respectively. Interestingly, h-ΔCwlK could hydrolyze the linkage of l-alanine-d-glutamic acid in the stem of the peptidoglycan, however, this enzyme could not hydrolyze the linkage of d-alanine-d-alanine, suggesting that CwlK is an l,d-endopeptidase not a d,d-carboxypeptidase. CwlK could not hydrolyze polyglutamate from B. natto or peptidoglycan of Staphylococcus aureus. This is the first report describing the characterization of an l,d-endopeptidase in B. subtilis and also the first report in bacteria of the characterization of a PLY500 family protein encoded in chromosomal DNA. Tatsuya Fukushima and Yang Yao contributed equally to this work.     

Isolation and characterization of a bacterium possessing L-phenylalanine dehydrogenase activity

The new enzyme phenylalanine dehydrogenase [L-phenylalanine: NAD⁺-oxidoreductase (deaminating)] was detected in the crude extract of a strain of Brevibacterium spec. The bacterium was isolated from a soil sample by enrichment with phenylalanine. This strain was the only one containing phenylalanine dehydrogenase out of 173 tested strains, among them 22 of the genus Brevibacterium, 74 strains from soil samples and 77 strains from a culture collection belonging to several genera. The enzyme is involved in the degradation of phenylalanine and could be induced by addition of L-, D-, D,l-phenylalanine or L-histidine, the optimum inducer concentration of phenylalanine being 1%.The reaction mechanism of a reductive amination was confirmed by demonstrating the close coupling between NADH-consumption and phenylalanine production; ammonia could not be replaced by L-glutamate or L-aspartate as amino donor. The -keto acid of L-tyrosine was converted too, while the corresponding compound of histidine was inactive. The optimum pH value for reductive amination in the crude extract was 8.5 and for oxidative desamination 10.5.     

Cloning,sequencing, overexpression and characterization of <Emphasis Type="SmallCaps">l</Emphasis>-rhamnose isomerase from <Emphasis Type="Italic">Bacillus pallidus</Emphasis> Y25 for rare sugar production

The l-rhamnose isomerase gene (L -rhi) encoding for l-rhamnose isomerase (l-RhI) from Bacillus pallidus Y25, a facultative thermophilic bacterium, was cloned and overexpressed in Escherichia coli with a cooperation of the 6×His sequence at a C-terminal of the protein. The open reading frame of L -rhi consisted of 1,236 nucleotides encoding 412 amino acid residues with a calculated molecular mass of 47,636 Da, showing a good agreement with the native enzyme. Mass-produced l-RhI was achieved in a large quantity (470 mg/l broth) as a soluble protein. The recombinant enzyme was purified to homogeneity by a single step purification using a Ni-NTA affinity column chromatography. The purified recombinant l-RhI exhibited maximum activity at 65°C (pH 7.0) under assay conditions, while 90% of the initial enzyme activity could be retained after incubation at 60°C for 60 min. The apparent affinity (K _m) and catalytic efficiency (k _cat/K _m) for l-rhamnose (at 65°C) were 4.89 mM and 8.36 × 10⁵ M⁻¹ min⁻¹, respectively. The enzyme demonstrated relatively low levels of amino acid sequence similarity (42 and 12%), higher thermostability, and different substrate specificity to those of E. coli and Pseudomonas stutzeri, respectively. The enzyme has a good catalyzing activity at 50°C, for d-allose, l-mannose, d-ribulose, and l-talose from d-psicose, l-fructose, d-ribose and l-tagatose with a conversion yield of 35, 25, 16 and 10%, respectively, without a contamination of by-products. These findings indicated that the recombinant l-RhI from B. pallidus is appropriate for use as a new source of rare sugar producing enzyme on a mass scale production.     

Certain aspects of the pH dependence of triggering inBacillus megaterium spores

Incubation of unactivatedBacillus megaterium 14581 spores in glucose, or in glucose plusl-alanine, at or below pH 3.6 resulted in germination arrested somewhere before onset of stainability. However, triggering continued at this reduced pH, and spores thus triggered were fully capable of completing the germination sequence in the absence of the germinants once the pH was neutralized. The same spores could be triggered either by a mixture of glucose andl-alanine or by a larger concentration of glucose alone. From this it was concluded that triggering results from an adequate stimulus which can be generated in different ways.l-alanine action in triggering has a pH profile distinct from that of glucose, suggesting that these two germinants have different receptor sites as well. At a level of acidity at which a weak glucose concentration triggered relatively few spores, a much larger fraction was found apparently distributed over a range of sub-triggering levels. Some of these could be made to trigger on transfer to a secondary reagent, or mixture of reagents, which by themselves are not very efficient germinants of the strain studied. The degree of additional triggering was found to depend on the nature of the complementary germinants, as well as on the pH at which glucose stimulated them. Evidence that spores may occupy stimulated states for finite lifetimes is presented and discussed.