Mechanism of lysis of Escherichia coli by ethanol and other chaotropic agents.

Ethanol has been shown to inhibit the assembly of cross-linked peptidoglycan and to induce cell lysis in Escherichia coli. These effects of ethanol appear to result from the weakening of hydrophobic interactions by ethanol rather than from the intercalation of ethanol into membranes. Other chaotropic agents also inhibited cross-linking and induced lysis. The potency of chaotropic anions with regard to this effect followed the expected chaotropic series. Antichaotropic agents, which strengthened hydrophobic interactions, antagonized ethanol-induced lysis. The weakening of hydrophobic interactions by ethanol is proposed as a general mechanism by which ethanol and other chaotropic agents could affect membrane-associated enzyme activities.     

Membrane protein folding: how important are hydrogen bonds?

Water is an inhospitable environment for protein hydrogen bonds because it is polarizable and capable of forming competitive hydrogen bonds. In contrast, the apolar core of a biological membrane seems like an ideal environment for hydrogen bonds, and it has long been assumed that hydrogen bonding should be a powerful force driving membrane protein folding. Nevertheless, while backbone hydrogen bonds may be much stronger in membrane proteins, experimental measurements indicate that side chain hydrogen bond strengths are not strikingly different in membrane and water soluble proteins. How is this possible? I argue that model compounds in apolar solvents do not adequately describe the system because the protein itself is ignored. The protein chain provides a rich source of competitive hydrogen bonds and a polarizable environment that can weaken hydrogen bonds. Thus, just like water soluble proteins, evolution can drive the creation of potent hydrogen bonds in membrane proteins where necessary, but mitigating forces in their environment must still be overcome.     

直链脂肪醇对豚鼠乳头肌慢动作电位的影响

本工作用相平面法观察和分析了乙醇、丁醇、己醇和辛醇对豚鼠乳头肌慢动作电位(APA,APD50,APD90,(?)_(max),(?)′_(max))及其所对应的钙电流(I_(max))和钾电流(I′_(max))的影响。结果表明:这四种脂肪醇对钙电流均有抑制作用,而对钾电流的影响,随碳原子数的加大从增强(乙醇)逐渐转化为削弱(已醇,辛醇)。并且碳链越长,产生这些效应所需脂肪醇的浓度越低,这提示醇的作用可能与它们的疏水性相关。     

Hydrogen bonding between anhydrous cholesterol and phosphatidylcholines: an infrared spectroscopic study

Fourier transform infrared spectroscopy performed with a high pressure diamond anvil cell was used to study hydrogen bonding between anhydrous phosphatidylcholines and cholesterol at the molar ratio 4:1. The hydroxyl group of cholesterol which acts as a proton donor, engages in strong hydrogen bonding to the sn-2 chain carbonyl group of DMPC, DPPC and HPPC and in weak hydrogen bonding to the phosphate group of all these phospholipids. No evidence of hydrogen bonding between cholesterol and the sn-1 chain carbonyl group of DMPC and DPPC was found. From a comparison of the relative hydrogen-bond strengths between cholesterol or water and the sn-2 chain carbonyl and phosphate groups of all these phospholipids, it is predicted that in aqueous dispersions of cholesterol containing phospholipids, the hydrogen bond of cholesterol to the phosphate group would be replaced by that of water, while the hydrogen bond of cholesterol to the sn-2 chain carbonyl group would remain intact.     

Effects of ethanol on the Escherichia coli plasma membrane.

The effects of ethanol on the fluidity of Escherichia coli plasma membranes were examined by using a variety of fluorescent probes: 1,6-diphenyl-1,3,5-hexatriene, perylene, and a set of n-(9-anthroyloxy) fatty acids. The anthroyloxy fatty acid probes were used to examine the fluidity gradient across the width of the plasma membrane and artificial membranes prepared from lipid extracts of plasma membranes. Ethanol caused a small decrease in the polarization of probes primarily located near the membrane surface. In comparison, hexanol decreased the polarization of probes located more deeply in the membrane. Temperature had a large effect on probes located at all depths. The effects of ethanol on E. coli membranes from cells grown with or without ethanol were also examined. Plasma membranes isolated from cells grown in the presence of ethanol were more rigid than those from control cells. In contrast to plasma membranes, artificial membranes prepared from lipid extracts of ethanol-grown cells were more fluid than those from control cells. These differences are explained by analyses of membrane composition. Membranes from cells grown in the presence of ethanol are more rigid than those from control cells due to a decrease in the lipid-to-protein ratio. This change more than compensates for the fluidizing effect of ethanol and the ethanol-induced increase in membrane C18:1 fatty acid which occurs during growth. Our results suggest that the regulation of the lipid-to-protein ratio of the plasma membrane may be an important adaptive response of E. coli to growth in the presence of ethanol.     

On the dependence of molecular conformation on the type of solvent environment: a molecular dynamics study of cyclosporin A

The dependence of the conformation of cyclosporin A (CPA), a cyclic undecapeptide with potent immunosuppressive activity, on the type of solvent environment is examined using the computer simulation method of molecular dynamics (MD). Conformational and dynamic properties of CPA in aqueous solution are obtained from MD simulations of a CPA molecule dissolved in a box with water molecules. Corresponding properties of CPA in apolar solution are obtained from MD simulations of CPA in a box with carbontetrachloride. The results of these simulations in H2O and in CCl4 are compared to each other and to those of previous simulations of crystalline CPA and of an isolated CPA molecule. The conformation of the backbone of the cyclic polypeptide is basically independent of the type of solvent. In aqueous solution the beta-pleated sheet is slightly weaker and the gamma-turn is a bit less pronounced than in apolar solution. Side chains may adopt different conformations in different solvents. In apolar solution the hydrophobic side chain of the MeBmt residue is in an extended conformation with its hydroxyl group hydrogen bonded to the backbone carbonyl group. In aqueous solution this hydrophobic side chain folds over the core of the molecule and the mentioned hydrogen bond is broken in favor of hydrogen bonding to water molecules. The conformation obtained from the MD simulation in CCl4 nicely agrees with experimental atom-atom distance data as obtained from nmr experiments in chloroform. In aqueous solution the relaxation of atomic motion tends to be slower than in apolar solution.     

Bacterial peptidoglycan (murein) hydrolases

Most bacteria have multiple peptidoglycan hydrolases capable of cleaving covalent bonds in peptidoglycan sacculi or its fragments. An overview of the different classes of peptidoglycan hydrolases and their cleavage sites is provided. The physiological functions of these enzymes include the regulation of cell wall growth, the turnover of peptidoglycan during growth, the separation of daughter cells during cell division and autolysis. Specialized hydrolases enlarge the pores in the peptidoglycan for the assembly of large trans-envelope complexes (pili, flagella, secretion systems), or they specifically cleave peptidoglycan during sporulation or spore germination. Moreover, peptidoglycan hydrolases are involved in lysis phenomena such as fratricide or developmental lysis occurring in bacterial populations. We will also review the current view on the regulation of autolysins and on the role of cytoplasm hydrolases in peptidoglycan recycling and induction of beta-lactamase.     

Development of a method for alkaline extraction of DNA from Brucella for diagnosing brucellosis using the polymerase chain reaction

Several methods of alkaline extraction of chromosome DNA from Brucella in the presence of 50 microliters model diagnostic material blood serum are developed for the diagnosis of brucellosis by the polymerase chain reaction (PCR). These methods are based on the capacity of NaOH to effectively denature proteins and destroy Brucella cell wall, thus isolating the genome DNA without exposure to proteolytic enzymes, detergents, deproteinization, or pH neutralization. The first method consists in alkaline lysis by 0.2-1.0 M NaOH followed by DNA precipitation with two ethanol volumes in the presence of 0.1 M NaCl, washing of the resultant precipitate in 80% ethanol, drying of the precipitate, and dissolving in distilled water. The second method includes alkaline lysis in the presence of 0.3 M NaCl with NaOH concentrations of 0.5-4.3 M and the stages of DNA sedimentation, washing of precipitate, its drying and dissolving similar to those in alkaline lysis. The third method consists in alkaline lysis-precipitation by 0.2-05 M NaOH in the presence of 0.1 M NaCl and 64% ethanol, followed by DNA preparation stages similar to those in alkaline lysis. The best results were achieved by alkaline lysis in the presence of 0.3 M NaCl at NaOH concentrations of 0.7 and 2.1 M, which meant theoretical levels of sensitivity 140 and 86 Brucella cells, respectively.     

The n-alcohol site in the nicotinic receptor pore is a hydrophobic patch

Alcohols and volatile anesthetics inhibit peripheral nicotinic acetylcholine receptors noncompetitively, primarily via an open-channel block mechanism. Analysis of hydrophobic mutations near the middle of the pore-forming M2 domains suggested that alcohols interact with the pore in this vicinity. To establish the extent of this inhibitory site, we created a series of hydrophobicity-altering mutations scanning most of the alpha subunit M2 domain. Using both single-channel and rapid patch perfusion electrophysiology, we measured how these mutations affect nAChR sensitivity to ethanol and hexanol. We find a near-contiguous series of amino acids in alpha-M2, extending from alphaL250 (8') to alphaV255 (13'), where mutagenesis strongly influences inhibition by alcohols. These results support the existence of a large inhibitory patch in the nAChR pore lining where interactions with alcohols are primarily due to hydrophobic forces. Ethanol appears to interact with deeper regions of this site than does hexanol. Because alcohols apparently act as open-channel blockers, we infer from our results that most of the residues between alphaL250 and alphaV255 are exposed to the aqueous environment of the pore when the channel is open. The location and extent of this site can explain why small alcohols occupy the nAChR pore at the same time as larger alcohols or charged blockers, while two large alcohols bind in a mutually exclusive manner.     

Effect of ninhydrin on the biochemical and histopathological changes induced by ethanol in gastric mucosa of rats

Studies on the effect of ninhydrin in the normal gastric mucosa and against the ethanol induced gastric injury were undertaken in rats in view of the presence of a carbonyl function as well as hydroxyl groups in its chemical structure. In spite of its potentials to generate hydroxyl radicals, it is deemed to possess antioxidant property by virtue of its electrophilic nature. Recent studies have shown gastro-protection to mediate through a reaction between the electrophilic compounds and sulfhydryl groups of the mucosa. Hence it was found worthwhile to evaluate the interaction between the oxidant and antioxidant functions in the structure of the same compound. The effects of ninhydrin pretreatment on gastric mucosal injuries caused by 80% ethanol, 25% NaCl and 0.2M NaOH were investigated in rats. The gastric tissue in ethanol-treated rats was analyzed for different histopathological lesions. In addition, the effects on ethanol-induced changes in the gastric levels of proteins, nucleic acids, non-protein sulfhydryl (NP-SH) and malondialdehyde (MDA) were also evaluated. Ninhydrin, as such, failed to induce any significant changes in normal gastric mucosa, while its pretreatment at oral doses of 5, 10 and 20 mg/kg was found to provide a dose-dependent protection against the ulcers induced by ethanol, NaOH and NaCl. The results of histopathological evaluation revealed a protective effect of ninhydrin on congestion, hemorrhage, edema, erosions and necrosis caused by ethanol. Furthermore, the pretreatment afforded a dose-dependent inhibition of the ethanol-induced depletion of proteins, nucleic acids, NP-SH and increase of MDA in the gastric tissue. The results obtained clearly demonstrate the anti-ulcerogenic activity of ninhydrin. The exact mechanism of action is not known. However, the carbonyl function in ninhydrin appears to achieve antioxidant balance and protect the gastric mucosa from the ethanol-induced gastric injury. Further studies are warranted to investigate the toxicity and detailed mechanism of action of this potent compound before any clinical trials, especially at the effective lower doses.     

Roles of histidine 784 and tyrosine 639 in ribose discrimination by T7 RNA polymerase

On the basis of their recently described T7 RNA polymerase-T7 promoter crystal structure, Cheetham et al. [(1999) Nature 399, 80] propose that discrimination of the hydrogen bonding character of the elongating NTP ribose 2'-substituent involves a hydrogen bond to histidine 784. This would contradict a previous conclusion, based on the characterization of mutant RNAPs, that discrimination of the hydrogen bonding character of the ribose 2'-substituent depends solely on the hydroxyl group of tyrosine 639. To resolve this point, we prepared and characterized histidine 784 point mutants. We find that while these mutations reduce the activity of the polymerase, they do not significantly reduce the level of ribose discrimination. Furthermore, a mutant with alanine at position 784 preferentially utilizes NTPs with 2'-substituents capable of acting as hydrogen bond donors or acceptors (2'-OH and 2'-NH(2)) over NTPs with substituents that lack such properties (2'-F and 2'-H). In contrast, mutation of tyrosine 639 to phenylalanine eliminates discrimination of ribose 2'-group hydrogen bonding character. The effects on ribose discrimination of mutating tyrosine 639 to phenylalanine are independent of the side chain at position 784. These results indicate that histidine 784 is not involved in discrimination of the ribose 2'-group of the elongating NTP. The ability of T7RNAP tyrosine 639, which is conserved in both RNA and DNA polymerases, to select for rNTPs appears to be due to the fact that in RNAPs this tyrosine is available to hydrogen bond to the ribose 2'-OH, while in DNAPs it is hydrogen bonded to a glutamic acid.     

Reaction of Vanadyl with Hydrogen Peroxide. An ESR and Spin Trapping Study

Vanadyl reacts with hydrogen peroxide forming hydroxyl radicals in a Fenton-like reaction. The hydroxyl radicals were spin trapped and identified using 5.5-dimethyl-I-pyrroline-N-oxide (DMPO). The quantity of hydroxyl radicals spin trapped during the reaction between vanadyl and hydrogen peroxide are equal to half of the hydroxyl radicals spin trapped during the reaction between ferrous ions and hydrogen peroxide. Experiments in the presence of formate show that this hydroxyl radical scavenger effectively competes with DMPO preventing the formation of the DMPO-OH adduct. However. in experiments using ethanol as the hydroxyl radical scavenger it was not possible to completely prevent the formation of DMPO-OH. The formation of this additional DMPO-OH in the presence of ethanol does not depend on the concentration of dissolved oxygen, but does depend on the concentration of hydrogen peroxide added to the vanadyl solution. The results suggest that the additional DMPO-OH formed in the presence of ethanol originates from a vanadium (V) intermediate. This intermediate may oxidize DMPO leading to the formation of DMPO-0; which rapidly decomposes forming DMPO-OH.     

Reaction of Vanadyl with Hydrogen Peroxide. An ESR and Spin Trapping Study

Vanadyl reacts with hydrogen peroxide forming hydroxyl radicals in a Fenton-like reaction. The hydroxyl radicals were spin trapped and identified using 5.5-dimethyl-I-pyrroline-N-oxide (DMPO). The quantity of hydroxyl radicals spin trapped during the reaction between vanadyl and hydrogen peroxide are equal to half of the hydroxyl radicals spin trapped during the reaction between ferrous ions and hydrogen peroxide. Experiments in the presence of formate show that this hydroxyl radical scavenger effectively competes with DMPO preventing the formation of the DMPO-OH adduct. However. in experiments using ethanol as the hydroxyl radical scavenger it was not possible to completely prevent the formation of DMPO-OH. The formation of this additional DMPO-OH in the presence of ethanol does not depend on the concentration of dissolved oxygen, but does depend on the concentration of hydrogen peroxide added to the vanadyl solution. The results suggest that the additional DMPO-OH formed in the presence of ethanol originates from a vanadium (V) intermediate. This intermediate may oxidize DMPO leading to the formation of DMPO-0; which rapidly decomposes forming DMPO-OH.     

Synthetic co-culture of autotrophic Clostridium carboxidivorans and chain elongating Clostridium kluyveri monitored by flow cytometry

Syngas fermentation with acetogens is known to produce mainly acetate and ethanol efficiently. Co-cultures with chain elongating bacteria making use of these products are a promising approach to produce longer-chain alcohols. Synthetic co-cultures with identical initial cell concentrations of Clostridium carboxidivorans and Clostridium kluyveri were studied in batch-operated stirred-tank bioreactors with continuous CO/CO₂-gassing and monitoring of the cell counts of both clostridia by flow cytometry after fluorescence in situ hybridization (FISH-FC). At 800 mbar CO, chain elongation activity was observed at pH 6.0, although growth of C. kluyveri was restricted. Organic acids produced by C. kluyveri were reduced by C. carboxidivorans to the corresponding alcohols butanol and hexanol. This resulted in a threefold increase in final butanol concentration and enabled hexanol production compared with a mono-culture of C. carboxidivorans. At 100 mbar CO, growth of C. kluyveri was improved; however, the capacity of C. carboxidivorans to form alcohols was reduced. Because of the accumulation of organic acids, a constant decay of C. carboxidivorans was observed. The measurement of individual cell concentrations in co-culture established in this study may serve as an effective tool for knowledge-based identification of optimum process conditions for enhanced formation of longer-chain alcohols by clostridial co-cultures.     

Accumulation of cAMP in the cells of Candida tropicalis at an early stage of ethanol-induced filamentous growth and its prevention by myo-inositol

Phosphatidylinositol metabolism is enhanced in the cells of Candida tropicalis Pk 233 at an early stage of filamentous growth caused by ethanol, and myo-inositol prevents the ethanol-induced changes in the metabolism and morphology [Uejima et al. (1987) FEBS Lett. 214, 127-129]. The accumulation of cAMP and an increase in adenylate cyclase activity were observed in the cells grown with ethanol to the mid-log phase. Myo-inositol abolished these effects of ethanol also. The activity of cAMP phosphodiesterase was affected by neither ethanol nor myo-inositol. These results suggest that the inositol phospholipid-linked and cAMP-linked signaling pathways may be involved in the mechanism of ethanol-induced filamentous growth of this yeast and also that myo-inositol would affect morphogenesis by controlling these pathways.     

Effect of thiourea on microsomal oxidation of alcohols and associated microsomal functions.

Thiourea and diethylthiourea, two compounds which react with hydroxyl radicals, inhibited NADPH-dependent microsomal oxidation of ethanol and 1-butanol. Inhibition by both compounds was more effective in the presence of the catalase inhibitor, azide. Inhibition by thiourea was noncompetitive with respect to ethanol in the absence of azide but was competitive in the presence of azide. Urea, a compound which does not react with hydroxyl radicals or H2O2, was without effect. Thiourea had no effect on NADH- and NADH-cytochrome c reductase, NADPH oxidase, and NADH- and NADPH-dependent oxygen uptake. Thiourea inhibited the activities of aniline hydroxylase and aminopyrine demethylase. Thiourea, but no other hydroxyl radical scavengers, e.g., dimethyl sulfoxide, mannitol, and benzoate, reacted directly with H202 and decreased H2O2 accumulation in the presence of azide. Therefore the actions of thiourea are complex because it can react with both hydroxyl radicals and H2O2. Differences between the actions of thiourea and those previously reported for dimethyl sulfoxide, mannitol, and benzoate, e.g., effects on drug metabolism, effectiveness of inhibition in the absence of azide, or kinetics of the inhibition, probably reflect the fact that thiourea reacts directly with H2O2 whereas the other agents do not. The current results remain consistent with the concept that microsomal oxidation of alcohols involves interactions of the alcohols with hydroxyl radicals generated from microsomal electron transfer.     

Production of flavor esters by lipases of Staphylococcus warneri and Staphylococcus xylosus

The present paper describes the potential of Staphylococcus warneri and Staphylococcus xylosus lipases in the production of a variety of flavor esters. Both immobilized lipases produced ethyl esters from hexanoic to oleic acids with an optimum at decanoic acid. They esterified aliphatic and branched chain primary alcohols from ethanol to hexanol. Under our standard conditions, acetic, butyric, 2-methyl butyric, 3-methyl butyric, and valeric acids underwent slight esterification.     

Steroid structure and function. Molecular conformation of 4-hydroxyestradiol and its relation to other catechol estrogens

Hydroxylation of estrogens at C(2) or C(4) effects differentially their binding affinity to and dissociation rate from the estrogen receptor. The X-ray crystal structure of 4-hydroxyestradiol (4-OH-E2) is reported here and compared with that of 2-hydroxyestradiol (2-OH-E2), the 2- and 4-hydroxylated derivatives of estrone (E1) and with that of the parent estrogens, E1 and E2. The overall molecular shape and hydrogen bonding patterns of each were examined for their possible relevance to their binding to the estrogen receptor and their biological activity. A shift in the B-ring conformation away from the symmetrical 7 alpha,8 beta-half-chair form toward the 8 beta-sofa form is induced by both 2- and 4-hydroxy substitution. This shift appears to be larger in the case of E2 than E1 derivatives and to be correlated with an observed change in the hydrogen bonding potential of the C(3) hydroxyl. In 4-OH-E2, as in E2 and 4-OH-E1, the C(3) hydroxyl functions both as a hydrogen bond donor and acceptor. In contrast in 2-OH-E2 the hydroxyl functions only as a donor. The markedly reduced affinity of 2-hydroxylated estrogens for the estrogen receptor could be due to a combination of steric interactions, competition between O(2) and O(3) for hydrogen bonds for a common site on the receptor, and to general interference with hydrogen bond formation of O(3). The C(4) hydroxyl participates in the formation of a chain of hydrogen bonds in the solid state that is similar to a chain seen in single crystals of E2. The presence of a similar chain of hydrogen bonds involving O(3) in the receptor site could account for the decreased dissociation rate of the 4-OH-E2 receptor complex.     

Effect of ethanol and osmotic stress on receptor conformation. Reduced water activity amplifies the effect of ethanol on metarhodopsin II formation

The combined effects of ethanol and osmolytes on both the extent of formation of metarhodopsin II (MII), which binds and activates transducin, and on acyl chain packing were examined in rod outer segment disc membranes. The ethanol-induced increase in MII formation was amplified by the addition of neutral osmolytes. This enhancement was linear with osmolality. At 360 milliosmolal, the osmolality of human plasma, 50 mM ethanol was 2.7 times more potent than at 0 osmolality, demonstrating the importance of water activity in in vitro experiments dealing with ethanol potency. Ethanol disordered acyl chain packing, and increasing osmolality enhanced this acyl chain disordering. Prior osmotic stress data showed a release of 35 +/- 2 water molecules upon MII formation. Ethanol increases this number to 49 water molecules, suggesting that ethanol replaces 15 additional water molecules upon MII formation. Amplification of ethanol effects on MII formation and acyl chain packing by osmolytes suggests that ethanol increases the equilibrium concentration of MII both by disordering acyl chain packing and by disrupting rhodopsin-water hydrogen bonds, demonstrating a direct effect of ethanol on rhodopsin. At physiologically relevant levels of osmolality and ethanol, about 90% of ethanol's effect is due to disordered acyl chain packing.     

Organic hydroperoxide-dependent oxidation of ethanol by microsomes: lack of a role for free hydroxyl radicals

Organic hydroperoxides can replace NADPH in supporting the oxidation of ethanol by liver microsomes. Experiments were carried out to evaluate the role of hydroxyl radicals in the organic hydroperoxide-catalyzed reaction. Maximum rates of ethanol oxidation occurred in the presence of either 0.5 mM cumene hydroperoxide or 2.5 mM t-butyl hydroperoxide and were linear for 2 to 4 min. The Km for ethanol was about 12 mM and Vmax was about 8 nmol ethanol oxidized/min/mg microsomal protein. Besides ethanol, the organic hydroperoxides supported the oxidation of longer-chain alcohols (1-butanol), and secondary alcohols (isopropanol). The organic hydroperoxide-supported oxidation of alcohols was not affected by several hydroxyl-radical scavengers such as dimethylsulfoxide, mannitol, or 2-keto-4-thiomethylbutyrate which blocked NADPH-dependent oxidation of alcohols by 50% or more. Iron-EDTA, which increases the production of hydroxyl radicals, increased the NADPH-dependent oxidation of ethanol, whereas desferrioxamine, which blocks the production of hydroxyl radicals, inhibited the NADPH-dependent oxidation of ethanol. Neither iron-EDTA nor desferrioxamine had any effect on the organic hydroperoxide-supported oxidation of ethanol. Cumene-and t-butyl hydroperoxide did not support microsomal oxidation of hydroxyl-radical scavengers. These results suggest that, in contrast to the NADPH-dependent oxidation of ethanol, free-hydroxyl radicals do not play a role in the organic hydroperoxide-dependent oxidation of ethanol by microsomes. Ethanol appears to be oxidized by two pathways in microsomes, one which is dependent on hydroxyl radicals, and the other which appears to be independent of these oxygen radicals.