Inactivation by bromine of single poliovirus particles in water.

Quantitative electron microscopy shows that Freon-extracted poliovirus, velocity banded in a sucrose gradient, contains over 95% single particles. This well-dispersed virus reacts quite rapidly with bromine in turbulent flowing water, losing plaque titer at the rate of one log10 unit in 10s at pH 7, 2 C, and at a bromine concentration of 2.2 muM. At 10 and 20 C the rate of disinfection (log10 plaque-forming units per second) is faster, and at both temperatures it increases in approximately linear fashion with increasing bromine concentration. At 2 C such a linear relationship is not observed.     

Initial fast reaction of bromine on reovirus in turbulent flowing water.

An apparatus is described for precise observation of the kinetics of the initial fast reaction of bromine with reovirus in turbulent flowing water. When quantitative electron microscopy shows that virus suspensions are essentially all single particles, the loss of infectivity follows first-order kinetics, the plaque titer falling at the rate of 3 log10 units/s at pH 7, 2 C, and at a 3-muM bromine concentration. Virus suspensions containing small aggregates (2 to 10/clump) exhibit a constantly decreasing disinfection rate with bromine. At a survival level of 10(-3) for single virions, the aggregated preparations have lost only 99% of their plaque titer and 10(-4) is reached only after 4 s of exposure. The disinfection rate does not appear to be a simple function of the size and frequency of aggregates in the virus suspension even when the aggregates contain no foreign material. Unpurified virus preparations (crude freeze-thaw lysates of infected cells) are shown, by zonal centrifugation, to contain 50% to over 90% of the infectivity in large, fast sedimenting aggregates. Such aggregates would strongly influence the bromine resistance of virus in polluted water.     

Initial fast reaction of bromine on reovirus in turbulent flowing water.

An apparatus is described for precise observation of the kinetics of the initial fast reaction of bromine with reovirus in turbulent flowing water. When quantitative electron microscopy shows that virus suspensions are essentially all single particles, the loss of infectivity follows first-order kinetics, the plaque titer falling at the rate of 3 log10 units/s at pH 7, 2 C, and at a 3-muM bromine concentration. Virus suspensions containing small aggregates (2 to 10/clump) exhibit a constantly decreasing disinfection rate with bromine. At a survival level of 10(-3) for single virions, the aggregated preparations have lost only 99% of their plaque titer and 10(-4) is reached only after 4 s of exposure. The disinfection rate does not appear to be a simple function of the size and frequency of aggregates in the virus suspension even when the aggregates contain no foreign material. Unpurified virus preparations (crude freeze-thaw lysates of infected cells) are shown, by zonal centrifugation, to contain 50% to over 90% of the infectivity in large, fast sedimenting aggregates. Such aggregates would strongly influence the bromine resistance of virus in polluted water.     

Changes in pH associated with clotting of fibrinogen. Kinetic studies of the pH shift and correlation of the pH change with the release of fibrinopeptides and the ensuing polymerization

The effect of the initial pH and the concentrations of thrombin, fibrinogen, and Ca2+ upon the rate of pH change associated with clotting of bovine fibrinogen by human thrombin was investigated at pH 6.80, 7.80, and 8.80, 0.3 ionic strength, 25 degrees C, and 19.5 mg/mL final fibrinogen concentration. At pH 6.80 and 7.80, the reaction was first order, with rate constant k1. At pH 8.80, a first-order reaction of the release of H+ (k1) was followed by a partial rebinding of these in a reaction consecutive to the first one (k2). At each of the above pH values, k1 was proportional to thrombin concentration in the 0.05-3.0 min-1 range investigated. The k1 constants were 0.111 +/- 0.001, 0.250 +/- 0.005, and 0.190 +/- 0.002 min-1 (NIH thrombin units)-1 mL-1 at pH 6.80, 7.80, and 8.80, respectively. Plots of log rate vs log thrombin concentration of these data were linear with slopes close to 1 at all three pH values. The rate of the second reaction (k2) was independent of both the thrombin and the initial fibrinogen concentration. The pH dependence of k1 exhibited a bell-shaped curve that could be resolved into the effect of one group with a pK of 7.27 that increased the rate and another with a pK of 9.22 that decreased the rate. With constant thrombin concentration but varying fibrinogen concentration, plots of 1/k1 vs [fibrinogen] were linear, but the lines did not pass through the origin. From the slope and intercept, kcat and KM of the Michaelis-Menten equation could be calculated. The same parameters were obtained also from initial velocity vs [fibrinogen] plots. Values of kcat were consistent and accurate; those of KM were more scattered. KM was (22.4-34.2) X 10(-6) M at pH 6.80 and approximately 7 X 10(-6) M in the pH 7.26-8.80 range. The latter value, pertaining to the release of H+ ions, is in agreement with values in the literature for KM of the release of fibrinopeptide A by thrombin in the 7.4-8.0 pH range. The value of kcat s-1 (unit of thrombin)-1 mL-1 increases from 1.2 X 10(-10) s-1 unit of thrombin-1 mL-1 at pH 6.80 to 2.46 X 10(-10) at pH 7.80 and then decreases to 2.01 X 10(-10) 10(-1) (units of thrombin)-1 mL-1 at pH 8.80. The kcat values are significantly lower than those in the literature for the release of fibrinopeptide A.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of allosteric effectors and temperature on oxygen binding properties and the Bohr effect of bovine hemoglobin

The O2 binding properties of bovine Hb were examined. The increase in Cl- and DPG concentration enhanced P50. A reduction in n(max) was observed at high Cl- concentration, while DPG had little effect on n(max). An increase in Cl- concentration enhanced the Bohr effect, the magnitude of which reached a maximum at 0.1 M Cl- and 20 degrees C. This concentration is nearly equal to that at the highest slope of the log P50 vs. log [Cl-] plot, and also equal to the physiological Cl- concentration (0.1 M) of bovine blood. Furthermore, the influence of Cl- concentration on the Bohr effect is independent of temperature. On the other hand, in the absence of Cl-, bovine Hb is sensitive to DPG; an increase in DPG concentration enhanced the Bohr effect, which reached a maximum at 3 mM DPG and 20 degrees C. This concentration is nearly equal to that at the highest slope of the log P50 vs. log [DPG] plot. At low DPG concentrations, the DPG effect on the Bohr effect became small with increasing temperature, whereas at high DPG concentrations, the DPG effect was insensitive to temperature changes. At the physiological concentration of DPG (0.5 mM), increases in both Cl- concentration and temperature diminished the DPG effect. At the physiological concentrations of Cl- and DPG, the Bohr effect was -0.36 at 37 degrees C. The deltaH value at the physiological concentrations of Cl- and DPG was approximately -5.8 kcal/mol at pH 7.4. These results indicate that Cl- and temperature are important determinants of the O2 binding properties of bovine Hb.     

The effects of temperature on the respiration and production of the freshwater nematode Anonchus sp.

Summary Growth and respiration were measured in a species of Anonchus (Nematoda: Plectidae) at 5°C, 10°C, 15°C, 20°C and 25°C. At 5°C no growth was measurable but the organisms remained active. Maximum production occurred at 15°C, but the highest rate of growth occurred at 20°C. Thus, adult size attained is dependent on the temperature of growth. Respiratory energy losses derived from Cartesian diver microrespirometry, increased with temperature up to 25°C. Regression coefficients (b values) derived from a log log linear regression of weight against oxygen consumption varied between 0.574–1.793, the lowest value being attained at 5°C, the highest at 20°C. Based on Q₁₀, production and respiratory energy losses the optimum temperatures for Anonchus appears to lie between 10°C–15°C.     

A laboratory hot tub model for disinfectant efficacy evaluation

This paper describes a novel laboratory hot tub (LHT) apparatus and associated standard operating procedure (SOP) designed to reproduce the key biological, chemical, and engineering parameters associated with recreational and therapeutic hot tubs. Efficacy, as measured quantitatively by log reduction values, was determined against both biofilm and planktonic bacteria. When the LHT was run according to the SOP, with no antimicrobial treatment, a consistent level of bacterial contamination occurred. The means of log10 viable cell densities (+/- the repeatability standard deviation of log densities) were 7.2 (+/-0.31) for the bulk water (density in units of cfu ml-1), 5.3 (+/-0.56) for the coupons (density in units of cfu cm-2), and 6.6 (+/-0.50) for the filters (density in units of cfu cm-2). When control and chlorine treated LHTs were run in parallel, the log reduction increased significantly with chlorine concentration for samples of planktonic bacteria in the bulk water (p=0.016), biofilm bacteria on the coupons (p=0.09) and biofilm bacteria on the filter (p=0.005), indicating that the method was sensitive to chlorine concentration. The method also displayed sensitivity by differentiating between chlorine and bromine treatments; in every case, chlorine produced a greater log reduction than did the same concentration of bromine. The model and SOP were shown to be rugged with respect to slight changes in fluid mixing intensity, water chemistry (saturation index), inoculum size, and organic loading. The LHT and associated SOP provide a reliable second tier in a three-tiered testing process, in which the first tier is a suspension test and the final tier is a field test.     

Factors affecting the kinetics of DNA reassociation in phenol–water emulsion at high DNA concentrations

DNA reassociation kinetics using the phenol emulsion reassociation technique (PERT) [Kohne, D. E., Levison, S. A. & Byers, M. J. (1977) Biochemistry 16 , 5329–5341] has been investigated at high DNA concentrations using an endonuclease S1 assay of reaction progress. Apparent second-order rate constants fall on two intersecting straight lines when presented as a function of DNA concentrations on a log–log plot. In the low DNA concentration range, the rate constants drop about 10-fold when concentration increases 1000-fold. In the high DNA concentration range, the rate constants drop more than 10-fold when concentration increases 10-fold. The slopes of these lines are the same in different solvents and at different temperatures. The intersection between the lines occurs when the available catalytic surface is saturated. At high DNA concentrations, high-complexity heterologous denatured DNA apparently competes 2–4 times better for the surface than homologous DNA because it does not participate in a reassociation reaction. Native and partially native DNA molecules cannot compete with single-stranded DNA for a saturated surface. At high DNA concentrations, reactions using PERT become dependent on the single-strand DNA length. Increasing length lowers reassociation rates.     

Inactivation of Bacillus anthracis spores by liquid biocides in the presence of food residue

Biocide inactivation of Bacillus anthracis spores in the presence of food residues after a 10-min treatment time was investigated. Spores of nonvirulent Bacillus anthracis strains 7702, ANR-1, and 9131 were mixed with water, flour paste, whole milk, or egg yolk emulsion and dried onto stainless-steel carriers. The carriers were exposed to various concentrations of peroxyacetic acid, sodium hypochlorite (NaOCl), or hydrogen peroxide (H(2)O(2)) for 10 min at 10, 20, or 30 degrees C, after which time the survivors were quantified. The relationship between peroxyacetic acid concentration, H(2)O(2) concentration, and spore inactivation followed a sigmoid curve that was accurately described using a four-parameter logistic model. At 20 degrees C, the minimum concentrations of peroxyacetic acid, H(2)O(2), and NaOCl (as total available chlorine) predicted to inactivate 6 log(10) CFU of B. anthracis spores with no food residue present were 1.05, 23.0, and 0.78%, respectively. At 10 degrees C, sodium hypochlorite at 5% total available chlorine did not inactivate more than 4 log(10) CFU. The presence of the food residues had only a minimal effect on peroxyacetic acid and H(2)O(2) sporicidal efficacy, but the efficacy of sodium hypochlorite was markedly inhibited by whole-milk and egg yolk residues. Sodium hypochlorite at 5% total available chlorine provided no greater than a 2-log(10) CFU reduction when spores were in the presence of egg yolk residue. This research provides new information regarding the usefulness of peroxygen biocides for B. anthracis spore inactivation when food residue is present. This work also provides guidance for adjusting decontamination procedures for food-soiled and cold surfaces.     

Response of Campylobacter jejuni to sodium chloride.

Studies were done to provide more comprehensive information on the response of Campylobacter jejuni and nalidixic acid-resistant, thermophilic Campylobacter (NARTC) to sodium chloride at 4, 25, and 42 degrees C. Three strains of C. jejuni were studies, and all could grow at 42 degrees C in the presence of 1.5% NaCl, but not 2.0% NaCl. At the same temperature, NARTC could grow in 2.0% NaCl and was substantially more tolerant to 2.5 and 4.5% NaCl than was C. jejuni. Both C. jejuni and NARTC grew poorly in the absence of added NaCl and grew best in the presence of 0.5% NaCl at 42 degrees C. At 25 degrees C, NaCl concentrations of 1.0 to 2.5% were protective to NARTC, but the same concentrations of salt generally enhanced the rate of death of C. jejuni. At 4 degrees C, both C. jejuni and NARTC were sensitive to 1.0% or more NaCl; however, the rate of death at this temperature was substantially less than that which occurred at 25 degrees C. A 3 log10 decrease of cells occurred in 4.5% NaCl after 1.2 to 2.1 days at 25 degrees C, and a similar reduction in cells took approximately 2 weeks at the same salt concentration and 4 degrees C. Although C. jejuni grows best in the presence of 0.5% NaCl, the presence of NaCl at concentrations as low as 1.0% may retard growth or increase rate of death; hence, it is advisable that growth media used for recovering or enumerating this organism contain 0.5% NaCl, but not 1.0% or more NaCl.     

Efficacy of copper and silver ions and reduced levels of free chlorine in inactivation of Legionella pneumophila.

Water disinfection systems utilizing electrolytically generated copper and silver ions (200 and 20, 400 and 40, or 800 and 80 micrograms/liter) and low levels of free chlorine (0.1 to 0.4 mg/liter) were evaluated at room (21 to 23 degrees C) and elevated (39 to 40 degrees C) temperatures in filtered well water (pH 7.3) for their efficacy in inactivating Legionella pneumophila (ATCC 33155). At room temperature, a contact time of at least 24 h was necessary for copper and silver (400 and 40 micrograms/liter) to achieve a 3-log10 reduction in bacterial numbers. As the copper and silver concentration increased to 800 and 80 micrograms/liter, the inactivation rate significantly (P less than or equal to 0.05) increased from K = 2.87 x 10(-3) to K = 7.50 x 10(-3) (log10 reduction per minute). In water systems with and without copper and silver (400 and 40 micrograms/liter), the inactivation rates significantly increased as the free chlorine concentration increased from 0.1 mg/liter (K = 0.397 log10 reduction per min) to 0.4 mg/liter (K = 1.047 log10 reduction per min). Compared to room temperature, no significant differences were observed when 0.2 mg of free chlorine per liter with and without 400 and 40 micrograms of copper and silver per liter was tested at 39 to 40 degrees C. All disinfection systems, regardless of temperature or free chlorine concentration, showed increase inactivation rates when 400 and 40 micrograms of copper and silver per liter was added; however, this trend was significant only at 0.4 mg of free chlorine per liter.     

Reactivity and pH dependence of thiol conjugation to N-ethylmaleimide: detection of a conformational change in chalcone isomerase

The reactivity of simple alkyl thiolates with N-ethylmaleimide (NEM) follows the Br?nsted equation, log kS- = log G + beta pK, with G = 790 M-1 min-1 and beta = 0.43. The rate constant for the reaction of the thiolate of 2-mercaptoethanol with NEM is 10(7) M-1 min-1, whereas the rate constant for the reaction of the protonated thiol is less than 0.0002 M-1 min-1. The intrinsic reactivity of the protonated thiol (SH) is over (5 X 10(10]-fold less than the thiolate (S-) and makes a negligible contribution to the reactivity of thiols toward NEM. The rate of NEM modification of chalcone isomerase was conveniently measured by following the concomitant loss in enzymatic activity. The pseudo-first-order rate constants for inactivation show a linear dependence on the concentration of NEM up to 200 mM and yield no evidence for noncovalent binding of NEM to the enzyme. Evidence is presented demonstrating that the modification of chalcone isomerase by NEM is limited to a single cysteine residue over a wide range of pH. Kinetic protection against inactivation and modification by NEM is provided by competitive inhibitors and supports the assignment of this cysteine residue to be at or near the active site of chalcone isomerase. The pH dependence of inactivation of the enzyme by NEM indicates a pK of 9.2 for the cysteine residue in chalcone isomerase. At high pH, the enzymatic thiolate is only (3 X 10(-5))-fold as reactive as a low molecular weight alkyl thiolate of the same pK, suggesting a large steric inhibition of reaction on the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

APS kinase from Penicillium chrysogenum. Dissociation and reassociation of subunits as the basis of the reversible heat inactivation

Adenosine-5'-phosphosulfate (APS) kinase from Penicillium chrysogenum, loses catalytic activity at temperatures greater than approximately 40 degrees C. When the heat-inactivated enzyme is cooled to 30 degrees C or lower, activity is regained in a time-dependent process. At an intermediary temperature (e.g. 36 degrees C) an equilibrium between active and inactive forms can be demonstrated. APS kinase from P. chrysogenum is a dimer (Mr = 57,000-60,000) composed of two apparently identical subunits. Three lines of evidence suggest that the reversible inactivation is a result of subunit dissociation and reassociation. (a) Inactivation is a first-order process. The half-time for inactivation at a given temperature is independent of the original enzyme concentration. Reactivation follows second-order kinetics. The half-time for reactivation is inversely proportional to the original enzyme concentration. (b) The equilibrium active/inactive ratio at 36 degrees C increases as the total initial enzyme concentration is increased. However, Keq,app at 5 mM MgATP and 36 degrees C calculated as [inactive sites]2/0.5 [active sites] is near-constant at about 1.7 X 10(-8) M over a 10-fold concentration range of enzyme. (c) At 46 degrees C, the inactive P. chrysogenum enzyme (assayed after reactivation) elutes from a calibrated gel filtration column at a position corresponding to Mr = 33,000. Substrates and products of the APS kinase reaction had no detectable effect on the rate of inactivation. However, MgATP and MgADP markedly stimulated the reactivation process (kapp = 3 X 10(5) M-1 X s-1 at 30 degrees C and 10 mM MgATP). The kapp for reactivation was a nearly linear function of MgATP up to about 20 mM suggesting that the monomer has a very low affinity for the nucleotide compared to that of the native dimer. Keq,app at 36 degrees C increases as the MgATP concentration is increased. The inactivation rate constant increased as the pH was decreased but no pK alpha could be determined. The reactivation rate constant increased as the pH was increased. An apparent pK alpha of 6.4 was estimated.     

Kinetics and mechanism of the oxidation of ammineruthenium(II) complexes by bromine

The reactions of Ru(NH₃)₅py²⁺, Ru(NH₃)₄bpy²⁺, Ru₂(NH₃)₁₀pz⁵⁺, RuRh(NH₃)₁₀pz⁵⁺ and Ru(NH₃)₅pz²⁺ with bromine are first-order in ruthenium and first-order in bromine. The rates decrease with increasing bromide ion concentration and, except for Ru(NH₃)₅pz²⁺, are independent of hydrogen ion concentration. The reactions are postulated to proceed via outer-sphere, one-electron transfer from Ru(II) to Br₂ with the formation of Br₂⁻ as a reactive intermediate. The bromide inhibition is ascribed to the formation of Br₃⁻ which is unreactive in outer-sphere reactions because of the barrier imposed by the need to undergo reductive cleavage. The reaction of Ru(NH₃)₅pz²⁺ is inhibited by hydrogen ions. The hydrogen ion dependence shows that Ru(NH₃)₅pzH³⁺ has a pK_a of 2.49 and is at least 500 times less reactive than Ru(NH₃)₅pz²⁺. The reaction of Ru₂(NH₃)₁₀pz⁴⁺ with bromine is biphasic. The second phase has a rate identical to that of the Ru₂(NH₃)₁₀pz⁵⁺-Br₂ reaction. A detailed analysis shows that the reaction of Ru₂(NH₃)₁₀pz⁴⁺ with bromine proceeds by a sequence of one-electron steps, Br₂⁻ being produced as an intermediate. A linear free energy relationship between rate constants and equilibrium constants, obeyed for all the reactions studied, provides an estimate of 1.5 × 10² M⁻¹ s⁻¹ for the self-exchange rate constant of the Br₂/Br₂⁻ couple.     

Determination of log P for pressurized carbon dioxide and its characterization as a medium for enzyme reaction

The log P value of pressurized CO(2) at 50 degrees C was determined from the solubility of 1-octanol in CO(2) and compared with other solvent parameters such as permittivity, epsilon, and polarity parameter, E(T)(30). The log P indicated that pressurized CO(2) is rather hydrophilic although it seems hydrophobic being judged from epsilon(r) and E(T)(30). With a change in pressure from 3 to 11.8 MPa, the log P changed from 0.9 to 2.0 while epsilon(r) and E(T)(30) changed only slightly. The log P was linearly correlated to the logarithm of the solubility of water among organic solvents. Pressurized CO(2) was located close to the linear correlation line among the solvents at high pressure (>11 MPa) but its location deviated to the hydrophilic side with a decrease in pressure. Lipase-catalyzed esterification of stearic acid with ethanol and hydrolysis of ethyl stearate were carried out in pressurized CO(2), benzene (log P = 2.0), and n-hexane (log P = 3.5). In spite of the lowest log P value for CO(2), the reaction rate in CO(2) was the highest among solvents tested in pressure range over 10 MPa. The reaction rate was strongly dependent on pressure of CO(2).     

Oxygen consumption by ammocoetes of the lampreyGeotria australis in air

 When covered by moistened lint-free gauze, the larvae (ammocoetes) of the lamprey Geotria australis survived, without apparent discomfort, for 4 days in water-saturated air at 10, 15 and 20 °C. In air, the mean standard rates of O₂ consumption of medium to large ammocoetes of G. australis (xˉ=0.52 g) at 10, 15 and 20 °C were 14.5, 35.7 and 52.1 μl⋅g^-1⋅h^-1, respectively. At 15 °C, the slope of the relationship between log O₂ consumption (μl O₂⋅h^-1) and log body weight for ammocoetes over a wide range in body weight was 0.987. The Q ₁₀s for rate of O₂ consumption between 10 and 15 °C, 15 and 20 °C and 10 and 20 °C were 4.9, 2.9 and 3.6, respectively. Our results and observations of the ammocoetes suggest that, when out of water, larval G. australis derives most of its O₂ requirements from cutaneous respiration, particularly at lower temperatures. This would be facilitated by the small size and elongate shape (and thus a relatively high surface-to-volume ratio), low metabolic rate, thin dermis, extensive subdermal capillary network and high haemoglobin concentration of larval G. australis. Accepted: 28 March 1996     

Survival of Escherichia coli O157 in abattoir waste products

AIMS: This study monitored survival and growth of Escherichia coli O157 in ovine and bovine abattoir waste. METHODS: Blood and gut contents were inoculated separately with cocktails of E. coli O157. Samples were stored aerophilically and microaerophilically at 5 degrees C, 15 degrees C and 30 degrees C to represent storage at different container depths and at extremes of UK ambient temperature. CONCLUSIONS: Results showed survival of E. coli O157 was irrespective of oxygen content with no significant differences observed between aerophilic and microaerophilic environments. Numbers of E. coli O157 in ovine and bovine gut contents showed no change when stored at 5 degrees C and increased 1-2 log(10) at 15 degrees C and 30 degrees C in 28 h. In ovine and bovine blood, irrespective of storage temperature, there was a 0.5-2 log(10) reduction or no change in numbers except in ovine blood stored at 30 degrees C where the fall in numbers was followed by a 3 log(10) increase. In aged (stored at 4 degrees C for 18 h before spiking) bovine blood there was no significant change in numbers at 5 degrees C while at 15 degrees C there was 2 log(10) rise after 48 h. At 30 degrees C there was an initial 1 log(10) decrease in numbers followed by a 1 log(10) rise over the following 40 h. SIGNIFICANCE AND IMPACT OF STUDY: Abattoir wastes may become contaminated from animals infected with Verocytotoxigenic E. coli O157 and in certain storage conditions these pathogens could significantly increase in numbers. There is need for care in abattoir waste disposal, not only for personnel subject to direct contact, but also in the prevention of cross contamination to adjacent land and water courses which could indirectly infect humans.     

Fission-spectrum neutrons at a low dose rate enhance neoplastic transformation in the linear, low dose region (0-10 cGy)

The neoplastic transformation of C3H 10T1/2 cells induced by fission-spectrum neutrons delivered at a high dose rate is linear up to 40 cGy. Reducing the dose rate increases the frequency of transformation in the low dose region. At a dose rate of 0.086 cGy min-1, the initial part of the induction curve remains linear but it has a slope 9-fold greater than the initial part of the curve at a high dose rate.     

Spontaneous transfer of retinoic acid, retinyl acetate, and retinyl palmitate between single unilamellar vesicles

The transfer of retinoic acid, retinyl acetate, and retinyl palmitate between single unilamellar vesicles was studied by resonance energy transfer. The retinoic acid transfers spontaneously between single unilamellar vesicles with a first order rate constant of 9.6 s-1 at 15 degrees C and pH 7.4. At 30 degrees C, the transfer rate was 3.5 times faster than that at 10 degrees C. At pH 7.4, the transfer rate was almost 2 orders of magnitude faster than that observed at pH 1.6. Increasing the concentration of NaCl decreased the retinoic acid transfer rate significantly. Retinyl acetate transfers with a rate constant of 0.15 s-1, but no spontaneous transfer of retinyl palmitate was observed over 60 min. The evidence supports the proposal that retinoic acid and retinyl acetate transfer between single unilamellar vesicles occur via the aqueous phase. In contrast, no spontaneous transfer of retinyl palmitate was observed. However, linear free energy relationships and the thermodynamic parameters for retinyl acetate transfer permit the calculation of rate constant for retinyl palmitate transfer.     

Monohalogenated maleimides as potential agents for the inhibition of Pseudomonas aeruginosa biofilm

New monohalogenated maleimide derivatives (with bromine, chlorine or iodine) were synthesized to test the effect of halogen atoms in inhibiting the formation of Pseudomonas aeruginosa biofilm. The evaluation of their biological activities clearly defines a structure–activity relationship. In this study, the bactericidal action of the three compounds was observed at the concentration range 0.3–5.0 mM on Luria-Bertani agar plates. The halogen atom of these molecules was critical in modulating the antibacterial activity, with a slightly higher effectiveness for chlorine. Confocal laser scanning microscopy was used to examine P. aeruginosa biofilms cultivated in flow cells. At concentration as low as 40 μM, the bromine and iodine compounds displayed a total inhibition towards the formation of bacterial biofilm. At this concentration, the bacterial attachment to glass surfaces was strongly affected by the presence of bromine and iodine whereas the chlorine derivative behaved as a bactericidal compound. A bioluminescent reporter strain was then used to detect the effect of the chemically synthesized maleimides on quorum sensing (QS) in P. aeruginosa. At the concentration range 10–100 μM, bioluminescence assays reveal that halogenated maleimides were able to interfere with the QS of the bacterium. Although the relationship between the weak inhibition of cell-to-cell communication (15–55% of the signal) and the high inhibition of biofilm formation has not been elucidated clearly, the results demonstrate that bromo- and iodo-N-substituted maleimides bromine and iodine may be used as new potent inhibitors that control bacterial biofilms.