The inhibition of complement-dependent hemolysis of liposomes containing cerebroside sulfate

Cerebroside sulfate (CGS) was found to be capable of inhibiting complement-dependent hemolysis. The activity dependence of CGS-containing liposomes on their composition was studied. Mixtures of CGS with phosphatidylethanolamine, phosphatidylserine, sphingomyelin from cattle brain, cerebroside from cattle spinal cord (CG), and egg yolk phosphatidylcholine (ePC) were investigated. In the case of binary CGS/ePC mixtures, the antihemolytic activity varied nonlinearly with an increase in the mass part of CGS: it sharply increased with an increase in the CGS part from 0.3 to 0.5 and decreased by 20-30% of the maximum value with an increase in the CGS part from 0.9 to 1. On the basis of these experiments, the optimum distance between the charged groups of CGS was estimated to be 0.92-1.6 nm. In the ternary compositions of 4:3:3 CGS/ePC/polar lipid, only CG increased the activity of liposomes as compared to that of liposomes from the 4:6 CGS/ePC. The preliminary incubation of CGS-containing liposomes with complement decreased hemolysis more effectively than incubation with other components of the hemolytic system. This suggests that the interaction of CGS-containing liposomes with the complement proteins is responsible for their antihemolytic activity.     

The inhibition of complement-dependent hemolysis by liposomes containing cerebroside sulfate

Cerebroside sulfate (CGS) was found to be capable of inhibiting complement-dependent hemolysis. The activity dependence of CGS-containing liposomes on their composition was studied. Mixtures of CGS with phosphatidylethanolamine, phosphatidylserine, sphingomyelin from cattle brain, cerebroside from cattle spinal cord (CG), and egg yolk phosphatidylcholine (ePC) were investigated. In the case of binary CGS/ePC mixtures, the antihemolytic activity varied nonlinearly with an increase in the mass part of CGS: it sharply increased with an increase in the CGS part from 0.3 to 0.5 and decreased by 20–30% of the maximum value with an increase in the CGS part from 0.9 to 1. On the basis of these experiments, the optimum distance between the charged groups of CGS was estimated to be 0.92–1.6 nm. In the ternary compositions of 4:3:3 CGS/ePC/polar lipid, only CG increased the activity of liposomes as compared to that of liposomes from the 4:6 CGS/ePC. The preliminary incubation of CGS-containing liposomes with complement decreased hemolysis more effectively than incubation with other components of the hemolytic system. This suggests that the interaction of CGS-containing liposomes with the complement proteins is responsible for their antihemolytic activity.     

Influence of chemical analogues of microbial autoregulators on the sensitivity of DNA to UV radiation

We established that chemical analogues of alkylhydroxybenzenes (AHB), belonging to alkylresorcinols and functioning as microbial autoregulatory d1 factors, enhance the UV resistance of various DNA molecules of different origin and conformation. These include the linear DNA of the lambda phage, bovine spleen DNA, and the DNA of the pUC19 plasmid that is composed of a number of annular (supercoiled and relaxed) and linearized molecules. Irradiating DNA with UV light (lambda = 254 nm) in the presence of methylresorcinol (MR) or hexylresorcinol (HR) results in comparatively insignificant DNA destruction as evidenced by our data on the electrophoretic mobility pattern in agarose gel. Using the linear Hind III restricts of the lambda phage DNA, we revealed that the protective effect of AHB varies depending on their chemical structure (it is more manifest with HR than MR) and concentration. Importantly, the effect of HR on bovine spleen DNA was based on its protective activity and manifested itself after a long incubation period. Studies using the pUC19 plasmid demonstrated that AHB, apart from increasing the resistance of linearized DNA molecules to UV irradiation, prevented both the supercoiled annular-supercoiled relaxed and the supercoiled relaxed-linearized transitions. The possible mechanisms of the UV-protective effect of AHB on DNA and their contributions to the resistance of dormant microbial forms to environmental factors are discussed.     

Influence of chemical analogues of microbial autoregulators on the sensitivity of DNA to UV radiation

We established that chemical analogues of alkylhydroxybenzenes (AHB), belonging to alkylresorcinols and functioning as microbial autoregulatory d₁ factors, enhance the UV resistance of various DNA molecules of different origin and conformation. These include the linear DNA of the λ phage, bovine spleen DNA, and the DNA of the pUC19 plasmid that is composed of a number of annular (supercoiled and relaxed) and linearized molecules. Irradiating DNA with UV light (λ = 254 nm) in the presence of methylresorcinol (MR) or hexylresorcinol (HR) results in comparatively insignificant DNA destruction as evidenced by our data on the electrophoretic mobility pattern in agarose gel. Using the linear HindIII restricts of the λ phage DNA, we revealed that the protective effect of AHB varies depending on their chemical structure (it is more manifest with HR than MR) and the concentration. Importantly, the effect of HR on bovine spleen DNA was based on its protective activity and manifested itself after a long incubation period. Studies using the pUC19 plasmid demonstrated that AHB, apart from increasing the resistance of linearized DNA molecules to UV irradiation, prevented both the supercoiled annular-supercoiled relaxed and the supercoiled relaxed-linearized transitions. The possible mechanisms of the UV-protective effect of AHB on DNA and their contributions to the resistance of dormant microbial forms to environmental factors are discussed.     

Prolonged circulation time in vivo of large unilamellar liposomes composed of distearoyl phosphatidylcholine and cholesterol containing amphipathic poly(ethylene glycol).

The effect of poly(ethylene glycol) (PEG) on the circulation time of liposomes in mice was examined by employing amphipathic PEGs (phosphatidylethanolamine (PE) derivatives of PEG) with average molecular weights of 1000, 2000, 5000 and 12,000. The activity of dioleoyl phosphatidylethanolamine-PEG (DOPE-PEG) in prolonging the circulation time of egg phosphatidylcholine/cholesterol large unilamellar liposomes (ePC/CH LUVs) (200 nm) was proportional to the molecular weight of PEG, i.e., 12000 = 5000 greater than 2000 greater than 1000. On the other hand, inclusion of distearoylphosphatidylethanolamine-PEG (DSPE-PEG) or dipalmitoyl-phosphatidylethanolamine-PEG (DPPE-PEG) of low molecular weight such as 1000 and 2000 in distearoylphosphatidylcholine (DSPC)/CH LUVs or dipalmitoyl phosphatidylcholine (DPPC)/CH LUVs effectively increased their blood circulation time. At least 3 mol% of amphipathic PEG in liposomes was required for activity. Addition of CH, which has a bilayer-tightening effect, to DSPC/CH/DSPE-PEG2000 LUVs further increased the blood residence time. A size of less than 300 nm was essential for prolonging the residence time of amphipathic PEG-containing liposomes in blood. DSPC/CH/DSPE-PEG2000 LUVs (1:1:0.13, m/m) containing 6 mol% of PEG and 200 nm in diameter remained in the circulation for over 24 h after injection and may be clinically useful for sustained release of an entrapped drug in the bloodstream and for drug accumulation in solid tumors.     

The effect of alkylhydroxybenzenes on functional and operational stability of antibodies

The influence of alkylhydroxybenzene (AHB) homologues of various hydrophobicity on antibody interaction with corresponding antigens under denaturing conditions (functional stability) and suboptimal conditions (operational stability) was revealed. AHBs were found to modulate the antibody sensitivity to heat denaturation and UV irradiation and expand the pH range of activity in different directions. These effects were found to depend upon the specific chemical nature and the AHB concentrations applied. Mechanisms of the AHB effect and possible perspectives of their use as antibody stabilizers are discussed.     

The effect of alkylhydroxybenzenes on the antigen-binding capacity of antibodies

The effect of the chemical analogues of microbial extracellular autoregulators belonging to alkylhydroxybenzenes (AHB), hexylresorcinol (HR), and methylresorcinol (MR), on the interactions between specific antibodies and the corresponding antigens was studied. Nonlinear dependency of the inhibition of binding of AHB-modified antibodies on the AHB chemical structures and concentrations was revealed by enzyme immunoassay. Hexylresorcinol was shown to decrease the antibody affinity and avidity indices and simultaneously increase the indices of nonspecific binding of AHB-modified antibodies to antigens, thereby promoting the formation of “false” antigen-antibody complexes. The nonspecificity of the influence of AHB on the antigenbinding capacity of antibodies is an important characteristic of these effects, which allows us to consider AHB as unique “superhaptenes”.     

Effect of a chemical analogue of autoinducers of microbial anabiosis on the Ca2+ response of mycelial fungi

The microbial alkylhydroxybenzenes (AHB), autoinducers of anabiosis, or d1 factors, participate in stress response of mycelial fungi, as determined from changes in intracellular Ca2+ concentration. By using the genetically modified strain Aspergillus awamori 66A, which produces a recombinant Ca2+-dependent protein aequorin, the dynamics of Ca2+ was studied in the cytosol of cells exposed to mechanical shock in the presence of the protective doses (0.001-0.01% w/vol) of a chemical AHB analogue, 4-n-hexylresorcinol. Like under stressful conditions, Ca2+ concentration increases in the cell cytosol in response to enhanced AHB level in a growing fungal culture; thus, AHB is perceived by cells as a stress signal. The level of cell response, which was determined from the amplitude of luminescence dependent on the Ca2+ concentration in cytosol was related to the physiological age of the cells and AHB concentration. Micromycete preincubation with AHB was found to protect cells from subsequent stress; this was reflected in the Ca2+ response. The protective AHB effect was manifested as (1) a significant decrease in the amplitude of luminescence and, thus, in Ca2+ accumulation in the cytosol during subsequent mechanical stress (as compared to the control--mechanical stress only); (2) development of the secondary Ca2+ response, which was not observed in the control; (3) a high level of Ca2+ retained in the cytosol for a long time in the presence of AHB (as compared to the control without preincubation with AHB). The mechanisms underlying the AHB effect on the Ca2+ transport systems are discussed.     

Regulation of the functional activity of lysozyme by alkylhydroxybenzenes

In our study, we investigated the capacity of alkylhydroxybenzenes (AHB), which are microbial anabiosis autoinducers, for alteration of the enzymatic activity of the hen egg-white lysozyme, as well as the efficiency of hydrolysis of specific (peptidoglycan) and nonspecific (chitin) substrates catalyzed by lysozyme. AHB homologues (C7-AHB and C12-AHB), which differ in their hydrophobicity and effects in their interaction with lysozyme, were used as modifying agents. C7-AHB stimulated enzymatic activity within the whole range of concentrations used (10^?7?10^?3 M). More hydrophobic C12-AHB exhibited this ability only at low concentrations and inhibited fermentative activity at high concentrations, acting as a mixed-type inhibitor. Both AHB homologues caused changes in the hydrophobicity of lysozyme molecules. An increase in the affinity level between the C7-AHB-modified enzyme and the nonspecific substrate (colloidal chitin or cell wall polymers of Saccharomyces sp.) was observed, which manifested itself in the enhancement of the hydrolysis rate by 200–500% (as compared to the native enzyme). A significant effect on the efficiency of the lysozyme-catalyzed modifications of the substrate (peptidoglycan, colloidal chitin) structure as a result of its complexation with AHB was demonstrated. A stabilizing effect of C7-AHB and C12-AHB was revealed, which ensured a high level of activity of the AHB-modified enzyme (as compared to the control) after heat treatment (functional stability), as well as at nonoptimal temperatures of catalysis (operational stability). The biological significance of lysozyme modification with AHB and the practical aspects of its application are discussed.     

Non-species-specific effects of unacylated homoserine lactone and hexylresorcinol, low molecular weight autoregulators, on the growth and development of bacteria

We conducted a comparative study of the effects of alpha-amino-gamma-butyrolactone, the common structural element of extracellular microbial regulators of the homoserine lactone (HSL) group, and of 4-n-hexylresorcinol, an autoregulator of the alkylhydroxybenzene (AHB) group, on the growth and development of gram-positive and gram-negative bacteria. We revealed non-species-specific effects of HSL and AHB and characterized their concentration dependencies. The addition of 10(-5)-10(-3) M HSL or 10(-5)-10(-4) M AHB during the exponential growth phase of the cultures grown on balanced media resulted in cell division arrest and accelerated the transition to the stationary phase that culminated in endospore formation in Bacillus cereus, Alicyclobacillus tolerans, and Sulfobacillus thermosulfidooxidans. When bacilli grew under the cultivation conditions that resulted in a low-zero spore percentage, 10(-4)-10(-3) M HSL cancelled the inhibition of spore formation. In the gram-negative bacteria Pseudomonas aurantiaca and Azotobacter vinelandii, AHB at concentrations of 10(-4) to (1.5-2.5) 10(-4) M induced the formation of dormant cells. Studies with the actinobacterium Streptomyces avermitilis revealed that the HSL effect varied depending on the age of the test cultures. The addition of 10(-4) M HSL during the lag phase of a submerged streptomycete culture accelerated its transition to the stationary phase and induced the formation of endospores, the dormant cells that are regarded as alternatives to exospores (conidia). If HSL (3.64 and 4.55 mg per 1cm2 disc) was locally added to a surface S. avermitilis culture, the growing mycelium formed rings that differed in their density, in the extent of the development of aerial mycelium, and in the presence/absence of exospores. Ring-shaped growth of streptomycete mycelia was also induced by 0.075-0.75 mg of AHB; however, unlike HSL, AHB repressed exospore formation. The data on non-species-specific effects of HSL and AHB suggest that they may perform regulatory functions on the microbial community level.     

A Comparative Study of Varroa Jacobsoni Reproduction in Worker Cells of Honey Bees (Apis Mellifera) in England and Africanized Bees in Yucatan, Mexico

The aim of this study was to investigate an underlying mechanism of the apparent tolerance of Africanized honey bees (AHB) to Varroa jacobsoni mites in Mexico. This was achieved by conducting the first detailed study into the mites' reproductive biology in AHB worker cells. The data was then compared directly with a similar study previously carried out on European honey bees (EHB) in the UK. A total of 1071 singly infested AHB worker cells were analyzed and compared with the data from 908 singly infested EHB worker cells. There was no significant difference between the number of mother mites dying in the cells (AHB = 2.0%, EHB = 1.8%); the mean number of eggs laid per mite (AHB = 4.86, EHB = 4.93); the number of mites producing no offspring (AHB = 12%, EHB = 9%); and developmental times of the offspring in worker cells of AHB and EHB. However, there was a major difference between the percentage of mother mites producing viable adult female offspring (AHB = 40%, EHB = 75%). This was caused by the increased rate of mite offspring mortality suffered by the first (male) and second (female) offspring in AHB worker cells. Therefore, only an average of 0.7 viable adult female offspring are produced per mite in AHB, compared to 1.0 in EHB.     

Expression of synthetic genes coding for completely new, nutritionally rich, artificial proteins

Synthetic genes (A, AB and AHB) constructed and cloned into pKK233-2 vector were recloned from the parent plasmid into the new procaryotic expression vectors pGFY221N and pBI052. Gene AF-B (coding for all amino acids besides phenylalanine) was obtained by 'cassette mutagenesis' from gene AB. The plasmid pGFY221N was constructed from pGFY218L by replacing the PstI by an NcoI site; plasmid pBI052 was derived from pGFY221N through replacing the 221-bp EcoRI/NcoI fragment with a synthetic DNA segment of 52 bp representing the Escherichia coli atpE gene translational initiation region. The genes A, AB, AHB and AF-B in the vector pGFY221N were expressed with a six-amino-acid-long leader sequence; in pBI052 the genes were expressed directly. In vitro expression experiments were successfully with all the genes except with the AHB gene integrated into pGFY221N. In the E. coli minicell system expression was demonstrated with the A gene in pGFY221N and the AF-B and AHB genes in pBI052. Complete translation of the expressed genes AB, AF-B and AHB in either the in vitro or in vivo systems could be shown by using 35S-labelled N-terminal methionine and C-terminal cysteine. Both amino acids occur only once in the peptide sequences.     

6-mercaptopurine and daunorubicin double drug liposomes-preparation, drug-drug interaction and characterization

This article addresses and investigates the dual incorporation of daunorubicin (DR) and 6-mercaptopurine (6-MP) in liposomes for better chemotherapy. These drugs are potential candidates for interaction due to the quinone (H acceptor) and hydroxyl (H donor) groups on DR and 6-MP, respectively. Interactions between the two drugs in solution were monitored by UV/Vis and fluorescence spectroscopy. Interaction between the two drugs inside the liposomes was evaluated by HPLC (for 6-MP) and by fluorescence spectroscopy (for daunorubicin) after phospholipase-mediated liposome lysis. Our results provide evidence for the lack of interaction between the two drugs in solution and in liposomes. The entrapment efficiencies of 6-MP in the neutral Phosphatidyl choline (PC):Cholesterol (Chol):: 2:1 and anionic PC:Chol:Cardiolipin (CL) :: 4:5:1 single and double drug liposomes were found to be 0.4% and 1.5% (on average), respectively. The entrapment efficiencies of DR in the neutral and anionic double drug liposomes were found to be 55% and 31%, respectively. The corresponding entrapment of daunorubicin in the single drug liposomes was found to be 62% on average. Our thin layer chromatography (TLC) and transmission electron microscopy (TEM) results suggest stability of lipid and liposomes, thus pointing plausible existence of double drug liposomes. Cytotoxicity experiments were performed by using both single drug and double drug liposomes. By comparing the results of phase contrast and fluorescence microscopy, it was observed that the double drug liposomes were internalized in the jurkat and Hut78 (highly resistant cell line) leukemia cells as viewed by the fluorescence of daunorubicin. The cytotoxicity was dose dependent and had shown a synergistic effect when double drug liposome was used.     

Monensin intercalation in liposomes: effect on cytotoxicities of ricin, Pseudomonas exotoxin A and diphtheria toxin in CHO cells.

Monensin, a carboxylic ionophore was intercalated in liposomes (liposomal monensin) and its effect on cytotoxicities of ricin, Pseudomonas exotoxin A and diphtheria toxin in CHO cells was studied. Intercalation of monensin in liposomal bilayer is found to have no effect on its stability and interaction with cells. Liposomal monensin (1 nM) substantially enhance the cytotoxicities of ricin (62-fold) and Pseudomonas exotoxin A (11.5-fold) while it has no effect on diphtheria toxin. This observed effect is highly dependent on the liposomal lipid composition. The potentiating ability of monensin (1 nM) in neutral vesicles is significantly higher (2.2-fold) as compared to negatively charges vesicles. This ability is drastically reduced by incorporation of stearylamine in liposomes and is found to be dependent on the density of stearylamine as well as on the concentration of serum in the medium. Monensin in liposomes containing 24 mol% stearylamine has a very marginal effect on the cytotoxicity of ricin (7.5-fold) which is further reduced (1.5-fold) in the presence of 20% serum. The uptake of 125I-gelonin from neutral vesicles is significantly higher (approximately 2.0-fold) than that from the negative vesicles. The uptake from positive vesicles is highly dependent on the concentration of stearylamine. The reduction in the lag period (30 min) of ricin action by monensin in neutral and negative vesicle is comparable with free monensin. However, monensin in positive vesicle has no effect on it. These studies have suggested that liposomes could be used as a delivery vehicle for monensin for selective elimination of tumor cells in combination with hybrid toxins.     

Non-species-specific effects of unacylated homoserine lactone and hexylresorcinol, low molecular weight autoregulators, on the growth and development of bacteria

We conducted a comparative study of the effects of α-amino-γ-butyrolactone, the common structural element of extracellular microbial regulators of the homoserine lactone (HSL) group, and of 4-n-hexylresorcinol, an autoregulator of the alkylhydroxybenzene (AHB) group, on the growth and development of grampositive and gram-negative bacteria. We revealed non-species-specific effects of HSL and AHB and characterized their concentration dependencies. The addition of 10^?5?10^?3 M HSL or 10^?5?10^?4 M AHB during the exponential growth phase of the cultures grown on balanced media resulted in cell division arrest and accelerated the transition to the stationary phase that culminated in endospore formation in Bacillus cereus, Alicyclobacillus tolerans, and Sulfobacillus thermosulfidooxidans. When bacilli grew under the cultivation conditions that resulted in a low-zero spore percentage, 10^?4?10^?3 M HSL cancelled the inhibition of spore formation. In the gram-negative bacteria Pseudomonas aurantiaca and Azotobacter vinelandii, AHB at concentrations of 10^?4 to (1.5?2.5)×10^?4 M induced the formation of dormant cells. Studies with the actinobacterium Streptomyces avermitilis revealed that the HSL effect varied depending on the age of the test cultures. The addition of 10^?4 M HSL during the lag phase of a submerged streptomycete culture accelerated its transition to the stationary phase and induced the formation of endospores, the dormant cells that are regarded as alternatives to exospores (conidia). If HSL (3.64 and 4.55 mg per 1 cm² disc) was locally added to a surface S. avermitilis culture, the growing mycelium formed rings that differed in their density, in the extent of the development of aerial mycelium, and in the presence/absence of exospores. Ring-shaped growth of streptomycete mycelia was also induced by 0.075–0.75 mg of AHB; however, unlike HSL, AHB repressed exospore formation. The data on non-species-specific effects of HSL and AHB suggest that they may perform regulatory functions at the microbial community level.     

Preservation of dried liposomes in the presence of sugar and phosphate

It has been well established that sugars can be used to stabilize liposomes during drying by a mechanism that involves the formation of a glassy state by the sugars as well as by a direct interaction between the sugar and the phospholipid head groups. We have investigated the protective effect of phosphate on solute retention and storage stability of egg phosphatidylcholine (egg PC) liposomes that were dried (air-dried and freeze-dried) in the presence of sugars and phosphate. The protective effect of phosphate was tested using both glucose (low T(g)) and sucrose (high T(g)) by measuring leakage of carboxyfluorescein (CF), which was incorporated inside the vesicles. Liposomes that were dried with glucose or phosphate alone showed complete leakage after rehydration. However, approximately 30% CF-retention was obtained using mixtures of phosphate and glucose. Approximately 75% CF-retention was observed with liposomes that were dried with sucrose. The solute retention further increased to 85% using mixtures of phosphate and sucrose. The pH of the phosphate buffer prior to drying was found to have a strong effect on the solute retention. Fourier transform infrared spectroscopy studies showed that phosphate and sugars form a strong hydrogen bonding network, which dramatically increased the T(g). The HPO(4)(2-) form of phosphate was found to interact stronger with sugars than the H(2)PO(4)(-) form. The increased solute retention of liposomes dried in the sugar phosphate mixtures did not coincide with improved storage stability. At temperatures below 60 degrees C the rate of solute-leakage was found to be strikingly higher in the presence of phosphate, indicating that phosphate impairs storage stability of dried liposomes.     

Effect of a chemical analogue of autoinducers of microbial anabiosis on the Ca<Superscript>2+</Superscript> response of mycelial fungi

The microbial alkylhydroxybenzenes (AHB), which are anabiosis autoinducers also termed d₁ factors, participate in the stress response of mycelial fungi, as determined from changes in intracellular Ca²⁺ concentration. By using the genetically modified strain Aspergillus awamori 66A, which produces the recombinant Ca²⁺-dependent protein aequorin, the dynamics of Ca²⁺ was studied in the cytosol of cells exposed to mechanical shock in the presence of protective doses (0.001–0.01% w/vol) of a chemical AHB analogue, 4-n-hexylresorcinol. As under stressful conditions, Ca²⁺ concentration increases in the cell cytosol in response to an enhanced AHB level in a growing fungal culture; thus, AHB is perceived by cells as a stress signal. The level of cell response, which was determined from the amplitude of luminescence dependent on the Ca²⁺ concentration in the cytosol, was related to the physiological age of the cells and the AHB concentration. Micromycete preincubation with AHB was found to protect cells from subsequent stress; this was reflected in the Ca²⁺ response. The protective AHB effect was manifested as (1) a significant decrease in the amplitude of luminescence and, thus, in Ca²⁺ accumulation in the cytosol during subsequent mechanical stress (as compared to the control—mechanical stress only); (2) development of a secondary Ca²⁺ response, which was not observed in the control; and (3) a high level of Ca²⁺ retained in the cytosol for a long time in the presence of AHB (as compared to the control without preincubation with AHB). The mechanisms underlying the AHB effect on Ca²⁺ transport systems are discussed.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 741–750.Original Russian Text Copyright © 2004 by Kozlova, Kupriyanova-Ashina, Egorov, El-Registan.     

Adaptive functions of extracellular autoregulators of microorganisms

Information about the functions of extracellular autoregulators, which adapt microorganisms to the stresses "scheduled" in the development cycle of microbial cultures (stresses of new medium, starvation, or space exhaustion (high cell density)) is summarized in the review. In a number of bacteria and yeasts, derivatives of alkylhydroxybenzenes (AHB), particularly of the class of alkyl resorcinols, act as autoregulators with adaptogenic functions. The chemical structure of AHB determines their amphiphility; capacity for physical and chemical interaction with membrane lipids, proteins, and DNA; properties as natural modifiers of biological membranes and enzymes; and the expression of antioxidant activity. Increase of AHB concentration up to the critical level (10(-5)-10(-4) M) results in cessation of cell division and in transition of the microbial culture to the stationary phase; further increase to 10(-4)-10(-3) M induces a transition of some of the cells of a post-stationary culture to the anabiotic state with the formation of cystlike resting cells (CRC), even in non-spore-forming bacteria. AHB participate in the regulation of the phenotypic variability of bacteria. The dynamics of extra- and intracellular concentrations of AHB in growing microbial cultures and the polymodality of their effect determine the adaptogenic functions of AHB as autoinhibitors of culture growth, autoinducers of anabiosis, and autoinhibitors of germination of resting forms. Manifestation of any given function depends on the concentration of AHB, the physiological state of the recipient cells, and on environmental factors. The species nonspecificity of AHB effects points to their significant role in the regulation of the development and functioning of microbial communities.     

Determination of products of acetohydroxy acid synthase by the colorimetric method, revisited

The enzyme acetohydroxy acid synthase (AHAS, EC 4.1.3.18) catalyzes two competing reactions of physiological importance: condensation of two molecules of pyruvate to form acetolactate (AL) or condensation of pyruvate and 2-ketobutyrate to form acetohydroxybutyrate (AHB). The activity of AHAS is most frequently analyzed using the Westerfeld method, in which the acetoin formed upon decarboxylation of AL is determined by colorimetric reaction with creatine and alpha-naphthol. However, there has been confusion as to the interpretation of the results of this assay in the presence of both substrates, conditions which lead to formation of both AL and AHB. By applying this assay to enzymatically prepared samples of AL and AHB which have also been analyzed by two other independent methods, we show here that the color yield for AHB in the commonly used assay is 35-40% that for equivalent amounts of acetoin or AL. The relative color yield is not significantly affected by varying the time or temperature of various steps in the color-forming reaction. This information could in principle be used, together with an independent specific assay for AHB, to determine the composition of an AHAS product mixture; it would, however, be less accurate than a simultaneous chromatographic method.