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.     

Long-term preservation of DNA in aqueous solutions in the presence of the chemical analogues of microbial autoregulators

The fact of long-term preservation of the physicochemical properties of DNA molecules in aqueous solutions in complexes with methylresorcinol, hexylresorcinol, and tyrosol, the chemical analogues of microbial autoregulators (d1 factors) from the group of alkylhydroxybenzenes (AOB), was established. Compared to the control variants of storage of aqueous DNA solutions, the AOB influence consisted in the sum of correlating effects: the prevention of DNA degradation (according to spectrophotometric parameters) and the preservation of its viscous characteristics and electrophoretic mobility. The initial DNA properties were preserved to the greatest degree in the presence of hexylresorcinol, the compound with the longest alkyl radical. Possible mechanisms of the protective action of alkylhydroxybenzenes in relation to DNA are discussed, namely, the prevention of its hydrolysis due to isolation from the aqueous environment and maintaining DNA stability in the dormant forms of microorganisms.     

IR spectroscopic research on the impact of chemical analogues of autoregulatory d<Subscript>1</Subscript> factors of microorganisms on structural changes in DNA

Using IR spectroscopy, we investigated the impact of chemical analogues of autoregulatory d₁ factors of microorganisms (methylresorcinol, hexylresorcinol, and tyrosol) on the conformational changes in DNA in films upon altering (decreasing) the relative humidity. We analyzed the appearance/disappearance of characteristic absorption bands of A and B DNA forms and determined D ₁₀₈₈/D ₁₂₂₄, the ratio between the band intensities of symmetrical and asymmetrical oscillations in their phosphate groups. The data obtained suggest the slowing down of the B → A structural transition in DNA in the presence of methylresorcinol and its speeding up in the presence of tyrosol. We discuss the mechanisms of this phenomenon in relation to the chemical composition of d 1 factors and their biological function.     

Mechanisms of Interaction between DNA and Chemical Analogues of Microbial Anabiosis Autoinducers

The alkylhydroxybenzene (AHB) autoregulatory factors d₁ (fd₁) of microorganisms have been found to directly interact with highly polymeric DNA. This circumstance results in changes, related to alterations in the topology of this macromolecule, in DNA physicochemical properties. The physicochemical properties of DNA in the presence of chemical analogues of microbial AHBs (methylresorcinol; hexylresorcinol; and 2-(4-hydroxyphenyl)ethane-1-ol, also known as tyrosol) were investigated using adsorption spectrophotometry, fluorometry, heat denaturation, viscosimetry, and electrophoresis in agarose gel. A number of concordant effects pointing to DNA-AHB interactions were revealed that manifest themselves in the hypochromic properties of the resulting complexes, an increase in their melting temperature and viscosity, a decrease in their electrophoretic mobility, and a change in the fluorescent properties of AHBs upon complexation with DNA. Such alterations were particularly significant in the presence of hexylresorcinol, which possessed the maximum alkyl radical length among the fd₁ analogues tested. Using atomic force microscopy, we visualized the micellelike DNA structures forming in the presence of AHBs. The results obtained provided the basis for developing a hypothetical model of the interaction between the biopolymer macromolecule and low-molecular-weight AHBs that takes into account the differences in the hydrophobicity of individual AHB homologues functioning as ligands. In terms of our model, we discuss AHB involvement in the stabilization of DNA and alteration of its topology, i.e., in the process related to intragenomic rearrangements, which account for the intrapopulational variability of bacteria, including dissociation processes.     

Stabilization of enzymes by dormancy autoinducers as a possible mechanism of resistance of resting microbial forms

Alkyl-substituted hydroxybenzenes (AHBs), autoinducers of microbial dormancy (ord ₁ factors), were found to stabilize the structure of protein macromolecules, making them metabolically less active and more resistant to stresses. In vitro experiments with theBacillus intermedius ribonuclease and chymotrypsin showed that the degree of the physical and chemical stability of these enzymes treated with AHBs depends on their concentration and incubation time. Experiments with RNase, which is capable of refolding, i.e., renaturation after heat denaturation, revealed that AHBs efficiently interact with both intact and denatured proteins. The data obtained allow the inference to be made thatd ₁ factors may play the role of natural chemical chaperons, blocking metabolism in dormant cells through the formation of catalytically inactive thermostable complexes with enzymes.     

The role of microbial dormancy autoinducers in metabolism blockade

Alkyl-substituted hydroxybenzenes (AHBs), which are autoinducers of microbial dormancy (d ₁ factors), were found to stabilize the structure of protein macromolecules and modify the catalytic activity of enzymes. In vitro experiments showed that C₆-AHB at concentrations from 10⁻⁴ to 10⁻² M, at which it occurs in the medium as a true solution and a micellar colloid, respectively, nonspecifically inhibited the activity of chymotrypsin, RNase, invertase, and glucose oxidase. C⁶-AHB-induced conformational alterations in protein macromolecules were due to the formation of complexes, as evidenced by differences in the fluorescence spectra of individual RNase and C₆-AHB and their mixtures and in the surface tension isotherms of C₆-AHB and trypsin solutions. Data on the involvement of dormancy autoinducers in the posttranslational modification of enzymes and their inhibition will provide further insight into the mechanisms of development and maintenance of dormant microbial forms.     

贺兰山不同海拔植被下土壤微生物群落结构特征

为明确海拔变化对干旱区山地森林土壤微生物群落的影响,揭示环境因子改变后土壤微生物群落结构特征及影响因素。对贺兰山5个海拔梯度土壤理化性质进行测定,同时采用磷酸脂肪酸(PLFA)图谱法分析土壤微生物群落组成,通过主成分分析、冗余分析(RDA)探究土壤理化性质与土壤微生物群落相对丰度之间的相关关系。结果表明:土壤养分含量在不同海拔之间差异性显著(P<0.05),土壤有机碳和全氮含量随海拔的升高而升高,全磷含量随海拔升高先升高再降低再升高;土壤微生物量随海拔升高先升高后降低,土壤微生物的相对丰度在不同海拔之间存在差异(P<0.05);主成分分析表明,与第1主成分相关性较强的微生物类群为革兰氏阳性细菌(G~+)、革兰氏阴性细菌(G~-)和真菌;与第2主成分相关性较强的微生物类群为放线菌、原生动物和非特异性细菌。非特异性细菌和真菌与各土壤因子之间均有显著相关关系,而放线菌、G~+和G~-与各土壤因子相关性较弱,原生动物与土壤全磷含量的关系密切。海拔是影响特征微生物分布的重要因素,特征微生物的含量和相对丰度随海拔的升高先升高后降低,符合山地生态学中的"中部膨胀"理论。探明了贺兰山不同海...     

Stabilization of yeast hexokinase A by polyol osmolytes: correlation with the physicochemical properties of aqueous solutions

Osmolytes of the polyol series are known to accumulate in biological systems under stress and stabilize the structures of a wide variety of proteins. While increased surface tension of aqueous solutions has been considered an important factor in protein stabilization effect, glycerol is an exception, lowering the surface tension of water. To clarify this anomalous effect, the effect of a series of polyols on the thermal stability of a highly thermolabile two domain protein yeast hexokinase A has been investigated by differential scanning calorimetry and by monitoring loss in the biological activity of the enzyme as a function of time. A larger increase in the T(m) of domain 1 compared with that of domain 2, varying linearly with the number of hydroxyl groups in polyols, has been observed, sorbitol being the best stabilizer against both thermal as well as urea denaturation. Polyols help retain the activity of the enzyme considerably and a good correlation of the increase in T(m) (DeltaT(m)) and the retention of activity with the increase in the surface tension of polyol solutions, except glycerol, which breaks this trend, has been observed. However, the DeltaT(m) values show a linear correlation with apparent molal heat capacity and volume of aqueous polyol solutions including glycerol. These results suggest that while bulk solution properties contribute significantly to protein stabilization, interfacial properties are not always a good indicator of the stabilizing effect. A subtle balance of various weak binding and exclusion effects of the osmolytes mediated by water further regulates the stabilizing effect. Understanding these aspects is critical in the rational design of stable protein formulations.     

A kinetic study of D-glucose oxidation by bromine in aqueous solutions

The kinetics of the oxidation of D-glucose to D-gluconic acid by bromine in aqueous solution were studied using potentiometric techniques and theoretical considerations of complex bromine-bromide-pH equilibria. The pH has a strong influence on reaction rate. At pH < 8 the reaction is very slow, while in the pH range pH 8-9.5 the reaction is sufficiently fast and seems optimal for the reaction. The proposed active species at that pH region is hypobromous acid. At pH > 9.5, the reaction is further accelerated due to the formation of hypobromite. The proposed kinetics expression for gluconic acid formation, based on the determined kinetic parameters at pH 9.24, is of the form dc(GA)/dt = 160c(2)(G)c(o)(HOBr)c(o)(H(+)c(o)(Br)     

Effect of alkylation with streptozotocin on the secondary structure of DNA

S₁ nuclease hydrolysis and hydroxyapatite chromatography were used to study the effect of the alkylating antibiotic, streptozotocin, on the secondary structure of DNA. Native calf thymus DNA was alkylatedin vitro with increasing concentrations of streptozotocin and subjected to S 1 nuclease hydrolysis. An increasing degree of DNA degradation was seen, suggesting a destabilization of the secondary structure. Indirect evidence, deduced from alkaline hydrolysis, effect of NaCl on S₁ nuclease hydrolysis, and hydroxyapatite chromatographic analysis of alkylated DNA, suggested a significant alkylation of DNA phosphates in addition to DNA bases. Nictotinamide has been reported to alter the cytotoxic and carcinogenic effects of streptozotocin. Our experiments indicate that in the presence of nicotinamide, streptozotocin causes the formation of a greater proportion of alkylated bases in relation to alkyl phosphotriesters. This may have significance in relation to the differential cytotoxicity of streptozotocin in the absence and presence of nicotinamide.     

Influence of formamide on the thermal stability of DNA

The utility of formamide in the denaturation and renaturation of DNA has been examined. The melting temperature of duplex DNA is lowered by 0·6°C per per cent formamide. The depression of melting temperature is independent of the GC content. Formamide also increases the width of the thermal transition. Upto 30%, it does not affect the rate of DNA reassociation     

Effect of alkylhydroxybenzenes,microbial anabiosis inducers,on the structural organization of <Emphasis Type="Italic">Pseudomonas aurantiaca</Emphasis> DNA and on the induction of phenotypic dissociation

We revealed a relationship between alkylhydroxybenzene (AHB)-induced changes in the structural organization of supramolecular complexes (SC) of the DNA of Pseudomonas aurantiaca and the phenotypic dissociation of this bacterium. The addition of 0.1–0.3 mM hexylresorcinol (C₆-AHB), a chemical analogue of microbial anabiosis autoinducers, caused the formation of cystlike refractile cells (CRC) in these gram-negative, nonsporulating bacteria. Inoculating pseudomonad CRC on solid nutrient media resulted in phenotypic dissociation of the microbial population that yielded several variants with different colony structure and morphology. This manifested itself in the conversion of the original S-colony-forming phenotype into the R form and in the formation of less pigmented colonies. These transitions were possibly linked to AHB-induced structural changes in the DNA. In vitro studies revealed that AHB could interact with DNA SC, resulting in their structural modification that manifested itself in changes in their viscoelasticity. DNA supramolecular complexes isolated from proliferating, stationary-phase, and anabiotic P. aurantiaca cells differed in their viscoelasticity and capacity to interact with AHB homologues with different hydrophobicity, such as hexylresorcinol and methylresorcinol (C₁-AHB). The DNA SC from actively proliferating cells were characterized by smaller viscoelasticity compared with those from stationary-phase and anabiotic cells, due to the difference in the DNA superspiralization degree and the physiological age of the bacteria involved. C₆-AHB produced a pronounced relaxing effect on the DNA SC from exponential-phase P. aurantiaca cells. The less hydrophobic C₁-AHB produced a similar relaxing effect on the DNA SC from stationary-phase cells. The curve of the dose-effect dependence of C₆-AHB had a breaking point within the submillimolar (10^–4 M) concentration range. These concentrations induce the formation of cystlike anabiotic pseudomonad cells that are characterized by an unstable phenotype and dissociate into distinct variants upon inoculation on solid media.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 157–165.Original Russian Text Copyright © 2005 by Mulyukin, Vakhrushev, Strazhevskaya, Shmyrina, Zhdanov, Suzina, Duda, Kozlova, El-Registan.     

湖泊微生物硝化过程研究进展

湖泊中微生物介导的硝化作用在生境内部氮周转和温室气体N_2O释放方面扮演着关键的角色。因此,研究湖泊微生物硝化过程及速率有助于我们整体评估湖泊生境内部的氮循环状态,全面认识湖泊响应区域乃至全球气候变化的规律。本文综述了湖泊生境中硝化过程及其驱动微生物和影响因素,包括氨氧化过程、亚硝酸盐氧化过程和完全氨氧化过程,同时聚焦前沿,归纳了氨氧化古菌、氨氧化细菌和完全氨氧化菌产生N_2O的机制和相对贡献。最后对湖泊硝化过程研究现状和未来发展方向提出总结和展望。     

Multinuclear NMR and molecular modelling investigations on the structure and equilibria of complexes that form in aqueous solutions of Ca and gluconate

Complexation of d-gluconate (Gluc⁻) with Ca²⁺ has been investigated via ¹H, ¹³C and ⁴³Ca NMR spectroscopy in aqueous solutions in the presence of high concentration background electrolytes (1 M ? I ? 4 M (NaCl) ionic strength). From the ionic strength dependence of its formation constant, the stability constant at 6 ? pH ? 11 and at I → 0 M has been derived (). The protonation constant of Gluc⁻ at I = 1 M (NaCl) ionic strength was also determined and was found to be log K_a = 3.24 ± 0.01 (¹³C NMR) and log K_a = 3.23 ± 0.01 (¹H NMR). It was found that ¹H and ¹³C NMR chemical shifts upon complexation (both with H⁺ and with Ca²⁺) do not vary in an unchanging way with the distance from the Ca²⁺/H⁺ binding site. From 2D ¹H-⁴³Ca NMR spectra, simultaneous binding of Ca²⁺ to the alcoholic OH on C2 and C3 was deduced. Molecular modelling results modulated this picture by revealing structures in which the Gluc⁻ behaves as a multidentate ligand. The five-membered chelated initial structure was found to be thermodynamically more stable than that derived from a six-membered chelated initial structure.     

Isolation and site-directed mutagenesis of DNA methyltransferase SssI

Prokaryotic DNA methyltransferase SssI (M.SssI) methylates cytosines at C5 in CpG sequences. Bacterial strains that produced M.SssI and its mutants as His₆-tagged proteins were constructed. To verify the role of Ser300 in recognizing the CpG sequence by the enzyme, Ser300 was replaced by Gly or Pro. The substitutions had virtually no effect on DNA binding and methylation by M.SssI apart from a slight decrease in binding in the case of S300P. It was assumed that no contact with DNA is formed by the side chain of Ser300 and the carbonyl oxygen and amide nitrogen of its peptide bonds. In addition, Ala was substituted for highly conserved Val188, presumably involved in stabilization of the flipped-out cytosine during the reaction. The substitution decreased fivefold the dissociation constant of the enzyme-substrate complex and halved the initial rate of DNA methylation. Despite the lack of a considerable effect of V188A, it was assumed that Val188 does form a contact with the target cytosine, but such a contact is formed with Ala in the case of the V188A mutant.     

DNA Damage in Astrocytes Exposed to Fumonisin B1

Fumonisins are a group of toxic metabolites mainly produced by Fusarium moniliforme and Fusarium proliferatum, fungi that commonly occur on corn throughout the world. Fumonisin B₁ (FB₁), structurally resembling sphingoid bases, is an inhibitor of ceramide synthase, a key enzyme involved in de novo sphingolipid biosynthesis and in the reacylation of free sphingoid bases derived from sphingolipid turnover. This inhibitory effect leads to accumulation of free sphinganine (SA) and sphingosine (SO), inducing cell death. However, little is known on the down stream effectors activated by these sphingolipids in the cell death signaling pathway. We exposed rat astrocytes to FB₁ with the aim of evaluating the involvement of oxygen free radicals and of some other biochemical pathways such as caspase-3 activity and DNA damage. Our results indicate that FB₁ treatment (48, 72 h and 6 days in vitro, DIV, and 10, 50, 100 M) does not affect cell viability. Conversely, after 72 h of treatment, FB₁ (50 and 100 M) induced DNA damage and an enhancement of caspase-3 activity compared to controls. In addition, FB₁ increased the expression of HSP70 at 10 and 50 M at 48, 72 h, and 6 DIV of treatment. We conclude that DNA damage of apoptotic type in rat astrocytes is caused by FB₁ and that the genotoxic potential of FB₁ has probably been underestimated and should be reconsidered.     

Global DNA methylation in a population with aflatoxin B1 exposure

We previously reported that global DNA hypomethylation, measured as Sat2 methylation in white blood cells (WBC), and aflatoxin B₁ (AFB₁) exposure were associated with increased hepatocellular carcinoma risk. In this study, we assessed the association between AFB₁ exposure and global DNA methylation. We measured LINE-1 and Sat2 methylation in WBC DNA samples from 1140 cancer free participants of the Cancer Screening Program (CSP) cohort. Blood and urine samples were used to determine the level of AFB₁-albumin (AFB₁-Alb) adducts and urinary AFB₁ metabolites. In continuous models, we found reverse associations of urinary AFB₁ with LINE-1 and Sat2 methylation. The odds ratio (OR) per 1 unit decrease were 1.12 (95%CI = 1.03–1.22) for LINE-1 and 1.48 (95%CI = 1.10–2.00) for Sat2 methylation. When compared with subjects in the highest quartile of LINE-1, we found that individuals in the 2nd and 3rd quartiles were less likely to have detectable AFB₁-Alb adducts, with ORs (95%CI) of 0.61 (0.40–0.93), 0.61 (0.40-.94), and 1.09 (0.69–1.72), respectively. The OR for detectable AFB₁-Alb was 1.81 (95%CI = 1.15–2.85) for subjects in the lowest quartile of Sat2 methylation. The OR for detection of urinary AFB₁ for those with LINE-1 methylation in the lowest quartile compared with those in the highest quartile was 1.87 (95%CI = 1.15–3.04). The corresponding OR was 1.75 (95%CI = 1.08–2.82) for subjects in the lowest quartile of Sat2 methylation. The association between AFB₁ exposure and global DNA methylation may have implications for the epigenetic effect of AFB₁ on hepatocellular carcinoma development and also suggests that changes in DNA methylation may represent an epigenetic biomarker of dietary AFB₁ exposure.     

Oxidation-Reduction Reactions of Iron Bleomycin in the Absence and Presence of DNA

Using the pulse radiolysis technique, we have demonstrated that bleomycin-Fe(III) is stoichiometrically reduced by CO₂^? to bleomycin-Fe(II) with a rate of (1.9 ± 0.2) × 10⁸M^?1s^?1. In the presence of calf thymus DNA, the reduction proceeds through free bleomycin-Fe(III) and the binding constant of bleomycin-Fe(III) to DNA has been determined to be (3.8 ± 0.5) x 10⁴ M^?1. It has also been demonstrated that in the absence of DNA O₂^?1 reacts with bleomycin-Fe(III) to yield bleomycin-Fe(II)O₂, which is in rapid equilibrium with molecular oxygen, and decomposes at room temperature with a rate of (700 ± 200) s^?1. The resulting product of the decomposition reaction is Fe(III) which is bound to a modified bleomycin molecule. We have demonstrated that during the reaction of bleomycin-Fe(II) with O₂, modification or self-destruction of the drug occurs, while in the presence of DNA no destruction occurs, possibly because the reaction causes degradation of DNA.     

DNA isolation by a rapid method from human blood samples: Effects of MgCl2, EDTA,storage time,and temperature on DNA yield and quality

The isolation of DNA from whole blood by a modified rapid method (RM) was tested using various detergents and buffer conditions. Extraction of DNA with either NP-40 or Triton X-100 gave a high yield of undegraded DNA in less than an hour. The concentration of magnesium ion in the buffers was critical to obtaining intact, high molecular weight (HMW) DNA. Greater than 10 mM MgCl₂ led to degradation. Addition of EDTA to the buffer inhibits this degradation. Preparation of DNA from blood stored at room temperature or incubated at 37°C for 24 hr resulted in the same amount and quality of DNA as from samples frozen at −70°C. DNA from blood samples that had undergone more than four freeze-thaw cycles was found to be partially degraded. The modified RM can be applied to extract DNA from as little as 10 μl of blood (340 ng of DNA) and from dried blood samples. DNA samples remained intact and undegraded for longer times when DNA was dissolved in higher concentrations of EDTA. This work was supported by grants from the Indiana Department of Mental Health and PHS RO1 AG10297.