Inhibitory Effect of Copper Complex of Indomethacin on Bacteria Studied by Microcalorimetry

A microcalorimetric technique based on the bacterial heat output was explored to evaluate the effect of copper–indomethacin complex on Staphylococcus aureus and Escherichia coli. The extent and duration of the inhibitory effect on the metabolism as judged from the rate constant (k) in log phase, half inhibitory ratio (IC₅₀). The rate constant of bacteria in the presence of the drugs decreased with increasing concentrations of the drugs. The copper complex exhibited higher antibacterial activity than the parent drug whose IC₅₀ value was 1.5 and 2.3 times lower than that of indomethacin to S. aureus and E. coli, respectively. It was indicated that when the copper ion is coupled with indomethacin, the drug is more potent as a bacteriostatic.     

Antibacterial Activities of Novel Diselenide-bridged <Emphasis Type="Italic">bis</Emphasis>(Porphyrin)s on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> Investigated by Microcalorimetry

The actions of two novel diselenide-bridged bis(porphyrin)s (1 and 2) on Staphylococcus aureus growth was investigated by microcalorimetry at 37.00°C, compared with that of Na₂SeO₃. Differences in their capacities to inhibit the growth metabolism of S. aureus were observed. By analyzing the power–time curves, crucial parameters such as the rate constant of bacterial growth (k), inhibitory rate (I), and generation time (t _G) were determined. The growth rate constant (k) of S. aureus (in the log phase) in the presence of the drugs decreased with increasing concentrations of the drugs regularly. The relationship of k and c is nearly linear for diselenide-bridged bis(porphyrin) 2. The sequence of the antibacterial activities of these selenium compounds tested was 2 > 1 > Na₂SeO₃.     

Kinetics of the action of three selenides on Staphylococcus aureus growth as studied by microcalorimetry

The effect of three kinds of selenide on Staphylococcus aureus growth was studied by means of microcalorimetry. Differences in their capacities to inhibit the metabolism of this bacterium were observed. The rate constant k (in the log phase) in the presence of the compounds decreased with increasing concentrations of the compounds. The relationship of k and c is nearly linear for the selenium compounds. Judged from the rate constant, k, and the half-inhibitory concentration IC₅₀, the experimental results reveal that the sequence of antibiotic activity of the three tested selenides compounds is (2-hydroxy benzyl imino)ethyl n-hexyl selenide> n-butyl(2-hydroxy benzyl imino)ethyl selenide>bis[(2,4-dihydroxy benzyl imino)ethyl] selenide.     

Study of Antibacterial Activity of ZnZnbisporphyrin Complexes and its Free Components on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> by Microcalorimetry

The antibacterial effect of Zn(II), tetraphenyl porphyrin (TPP), propdioxyl bridged tetraphenyl bisporphyrin 1, and its metallobisporphyrin complexes (ZnMnbisporphyrin 2 and ZnZnbisporphyrin 3) towards Staphylococcus aureus growth was investigated by microcalorimetry at 37°C. Differences in their capacities to inhibit the growth metabolism of S. aureus were observed. By analyzing the power–time curves, crucial parameters such as the rate constant of bacterial growth (k), inhibitory ratio (I), and generation time (t _G) were determined. The growth rate constant (k) of S. aureus (in the log phase) in the presence of the drugs decreased linearly with increasing concentrations of the complexes. The sequence of the antibacterial activities of these compounds tested was 3 > 2 > 1 > Zn(II) > TPP. ZnZnbisporphyrin 3 is proposed to benefit from the synergetic effects of Zn(II) and 1.     

Study of the thermokinetic properties of copper(II) on Escherichia coli growth

By using an LKB2277 Bioactivity Monitor, stop-flow mode, the power-time curves of Escherichia coli at 37°C affected by Cu(II) were determined. Some parameters, such as growth rate constants k, inhibitory ratio I, the heat output Q_log in the log phase, and the generation times G were obtained. According to these parameters, we found that a low concentration of Cu(II) (0–20 μg/mL) had an promoting action on the growth of E. coli, but a high concentration of Cu(II) (40–100 μg/mL) had an inhibitory action. The toxicity of Cu(II) can also be expressed as the half-inhibitory concentration IC₅₀; the value is 69.7 μg/mL. The assay is quantitative, inexpensive, and versatile.     

光枝勾儿茶内生真菌多样性及抑菌活性研究

为探索贵州苗药光枝勾儿茶内生真菌类群特征、分布部位及其抑菌活性,该研究采用传统方法对贵州省贵阳市和黔西市光枝勾儿茶内生真菌进行分离,并基于分子生物学及统计学对其分类地位进行鉴定及多样性评价,最后通过微量肉汤倍比稀释法筛选具有抑菌活性的菌株。结果表明:(1)从光枝勾儿茶中分离到191 株内生真菌,隶属于3 个门5 个纲10 个目15 个科19 个属,优势属为叶点霉属(Phyllosticta)、间座壳属(Diaporthe)、葡萄座腔菌属(Botryosphaeria)和刺盘孢属(Colletotrichum)。(2)黔西光枝勾儿茶内生真菌香农-维纳多样性指数(H''_Q=2.112)较贵阳(H''_G=1.801)高,索伦森相似性指数Cs_G-Q为0.923,不同组织香农-维纳多样性指数为茎(H''_S=2.004)>根(H''_R=1.764)>叶(H''_L=1.654)>果实(H''_F=1.473),茎和叶内生真菌的索伦森相似性最高(Cs_S-L=0.667)。(3)筛选出的21株内生真菌对供试菌大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)和沙门氏菌(Salmonella enterica)具有抑菌效果,其中Diaporthe sp. QX4G6对大肠杆菌、金黄色葡萄球菌和沙门氏菌的最小抑菌浓度分别为12.5、6.25、12.5 mg·mL^-1,最小杀菌浓度分别为12.5、6.25、12.5 mg·mL^-1。以上研究结果揭示了光枝勾儿茶蕴藏丰富的内生真菌资源,不同地区及组织内生真菌类群组成有差异,多个分离菌株具有抗菌活性,为光枝勾儿茶内生真菌天然抗菌药物或药源研发奠定了基础。     

Microcalorimetric Study of <Emphasis Type="Italic">Tetrahymena</Emphasis> Growth Affected by Copper(II) Complexes

By means of microcalorimetry, the effect of four copper(II) complexes on Tetrahymena growth was investigated. The extent and duration of the inhibitory effect on the metabolism, judged by the rate constant, k, and the half inhibition concentration, IC₅₀, varied with the different complexes. The results showed that the half inhibition concentrations IC₅₀ of CuCl₂, (C₉H₆NO)₂Cu and [Cu(phen)₂]Cl₂⋅6H₂O were 9.9 × 10⁻⁴, 2.0 × 10⁻⁴, and 2.6 × 10⁻⁴ mol/L, respectively. The sequence of antibiotic activity of these three complexes was: (C₉H₆NO)₂Cu > [Cu(phen)₂]Cl₂⋅6H₂O > CuCl₂. The growth rate constants of [Cu(phen)₃]Cl₂⋅6H₂O did not change obviously with the increase of concentrations, but [Cu(phen)₃]Cl₂⋅6H₂O also can prolong the time of Tetrahymena growth.     

怀槐细胞悬浮培养的结构化动力学模型

为探明怀槐细胞生长、异黄酮染料木素合成与底物消耗间的关系,建立了怀槐细胞悬浮培养的结构化动力学模型。模型预测分析了胞内外的蔗糖代谢、胞内结构组分变化、胞内中间组分的变化、细胞呼吸损失以及胞内外异黄酮染料木素的合成情况。模型各参数灵敏度的分析表明kM_b1、k_b2 和k_p是最为灵敏的参数,其调节10%时,目标函数变化的最大比例分别达12.8%、4.61%和2.54%,其它参数对目标函数变化的影响均小于0.5%。该模型预测值与实验值具有较好的吻合性。     

Effect of <Emphasis Type="Italic">poxB</Emphasis> gene knockout on metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis> based on growth characteristics and enzyme activities

The effect of poxB gene knockout on metabolism in Escherichia coli was investigated in the present paper based on the growth characteristics and the activities of the enzymes involved in the central metabolic pathways. The absence of pyruvate oxidase reduced the glucose uptake rate and cell growth rate, and increased O₂ consumption and CO₂ evolution. The enzyme assay results showed that although glucokinase activity increased, the flux through glycolysis was reduced due to the down-regulation of the other glycolytic enzymes such as 6-phosphofructosekinase and fructose bisphosphate aldolase in the poxB mutant. TCA cycle enzymes such as citrate synthase and malate dehydrogenase were repressed in the poxB mutant when the cells were cultivated in LB medium. The pyruvate oxidase mutation also resulted in the activation of glucose-6-phosphate dehydrogenase and acetyl-CoA synthetase. All these results suggest that pyruvate oxidase is not only a stationary-phase enzyme as previously known, and that the removal of the poxB gene affects the central metabolism at the enzyme level in E. coli.     

A new esterase showing similarity to putative dienelactone hydrolase from a strict marine bacterium, <Emphasis Type="Italic">Vibrio</Emphasis> sp. GMD509

Vibrio sp. GMD509, a marine bacterium isolated from eggs of the sea hare, exhibited lipolytic activity on tributyrin (TBN) plate, and the gene representing lipolytic activity was cloned. As a result, an open reading frame (ORF) consisting of 1,017 bp (338 aa) was found, and the deduced amino acid sequence of the ORF showed low similarity (<20%) to α/β hydrolases such as dienelactone hydrolases and esterase/lipase with G–X₁–S–X₂–G sequence conserved. Phylogenetic analysis suggested that the protein belonged to a new family of esterase/lipase together with various hypothetical proteins. The enzyme was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme (Vlip509) showed the best hydrolyzing activity toward p-nitrophenyl butyrate (C₄) among various p-nitrophenyl esters (C₂ to C₁₈), and optimal activity of Vlip509 occurred at 30°C and pH 8.5, respectively. Kinetic parameters toward p-nitrophenyl butyrate were determined as K _m (307 μM), k _cat (5.72 s⁻¹), and k _cat/K _m (18.61 s⁻¹ mM⁻¹). Furthermore, Vlip509 preferentially hydrolyzed the S-enantiomer of racemic ofloxacin ester. Despite its sequence homology to dienelactone hydrolase, Vlip509 showed no dienelactone hydrolase activity. This study represents the identification of a novel lipolytic enzyme from marine environment.     

Antioxidant Activity of Supramolecular Water-Soluble Fullerenes Evaluated by β-Carotene Bleaching Assay

We investigated the antioxidant activity of supramolecular water-soluble fullerenes, polyvinylpyrrolidone (PVP)-entrapped C₆₀, and γ-cyclodextrin (CD)-bicapped C₆₀, based on comparable β-carotene bleaching assay. Antioxidant activity against reactive oxygen species (ROS) generated by three different methods, (i) autoxidation of linoleic acid, (ii) hydrogen peroxide promoter, and (iii) photoirradiation, was evaluated as percent of inhibition relative to a control experiment in view of the bleaching rate constant (k _obs) as well as the persistent absorbancy of β-carotene. Water-soluble fullerenes exhibit significant inhibitory effects on the oxidative discoloration of β-carotene in any system.     

A mathematical model for the vertical distribution of chlorophyllA in estuarine intertidal sediments

Coastal and estuarine intertidal sediments are commonly colonized by dense populations of microphytobenthos. Due to wind and tides, important fractions of microphytobenthic populations may be buried. A mathematical model describing the depth variation of chlorophyll a in intertidal sediments was developed and experimentally tested. The model assumed first-order chlorophylla degradation and a constant mean burial velocity which resulted in a negative exponential variationC _Z =C _O exp{-k/vz} (C _Z andC _O =chlorophylla concentration at depth zand at the surface;k=specific degradation rate of chlorophyll a to pheopigments;V=mean burial velocity). Chlorophylla concentration depth profiles in different sediment types measured at the Tagus estuary and Ria Formosa (Portugal) were used to validate the model. The model was adjusted to field data. The chlorophyll a degradation rate was measured in a microcosm experiment under total darkness and no tidal action, and sampled during three months. This rate was shown to be independent of time and depth for the upper 0–15 mm depth interval. This result allowed the estimation ofV for each sampling site. Comparison of predicted and observed temporal data further confirmed the validity of the model andk andV values. Despite its simplicity, the proposed model adequately described the depth distribution of chlorophylla in different types of intertidal sediments. The model allowed the quantitative characterization of the buried microphytobenthic biomass (depth-integrated biomass) and the assessment of its importance as potentially productive stock of cells.     

Decolorization kinetics of a recombinant Escherichia coli strain harboring azo-dye-decolorizing determinants from Rhodococcus sp.

A 6.3 kb DNA fragment containing genes responsible for azo-dye decolorization was cloned and expressed in Escherichia coli. The resulting recombinant strain E. coli CY1 decolorized 200 mg azo dye (C.I. Reactive Red 22) l^–1 at 28 °C at 8.2 mg g cell^–1 h^–1, while the host (E. coli DH5) had no color-removal activity. Addition of 0.5 mM isopropyl--d-thiogalacto-pyranoside (IPTG) increased the decolorization rate 3.4-fold. The dependence of the decolorization rate on initial dye concentration essentially followed Monod-type kinetics and the maximal rate occurred with the dye at 600 mg l^–1. The decolorization rate of E. coli CY1 was optimal at 40 °C and pH 11. Aeration (increased dissolved O₂ level) strongly inhibited the decolorization, but decolorization occurred effectively under static incubation conditions (no agitation was employed). The CY1 strain also exhibited excellent stability during repeated-batch operations.     

Sensitivity to detergents and plasmid curing in Enterococcus faecalis

This research reports the sensitivity of a clinical isolate of Enterococcus faecalis to sodium N-lauroylsarcosinate (sarkosyl) and sodium dodecyl sulfate (SDS), as well as the efficiency of these detergents in curing the strain. Compared to Escherichia coli, Enterococcus faecalis was very sensitive to both detergents, with minimum inhibitory concentrations (MIC) for the latter being 100 times lower than for Escherichia coli. The clinical isolate of Enterococcus faecalis used in this study exhibited plasmid-borne resistance to kanamycin (MIC 2 mg/ml) and tetracycline (MIC 50 μg/ml); 3% curing was observed after growth in the presence of sarkosyl but no curing was observed after growth in the presence of either SDS or acridine orange. In contrast, 35% curing of plasmid-bearing Escherichia coli was observed after growth in the presence of either SDS or acridine orange, but none was observed after growth in the presence of sarkosyl.     

Cloning, expression, and characterization of a new deoxyribose 5-phosphate aldolase from Yersinia sp. EA015

A new deoC gene encoding deoxyribose 5-phosphate aldolase (DERA) was identified in Yersinia sp. EA015 isolated from soil. The DERA gene had an open reading frame (ORF) of 672 base pairs encoding 223 amino acids to yield a protein of molecular mass 24.8 kDa. The amino acid sequence was 94% identical to that of DERA from Yersinia intermedia ATCC 29909. DERA was over-expressed in Escherichia coli and purified using Ni–NTA affinity chromatography. The specific activity was 137 μmol/min/mg. The Michaelis constant (k_m value) of DERA was 9.1 mM. DERA was optimally active at pH 6.0 and 50 °C. DERA was tolerant to a high concentration (300 mM) of acetaldehyde.     

Cold adapted features of Vibrio salmonicida catalase: characterisation and comparison to the mesophilic counterpart from Proteus mirabilis

The gene encoding catalase from the psychrophilic marine bacterium Vibrio salmonicida LFI1238 was identified, cloned and expressed in the catalase-deficient Escherichia coli UM2. Recombinant catalase from V. salmonicida (VSC) was purified to apparent homogeneity as a tetramer with a molecular mass of 235 kDa. VSC contained 67% heme b and 25% protoporphyrin IX. VSC was able to bind NADPH, react with cyanide and form compounds I and II as other monofunctional small subunit heme catalases. Amino acid sequence alignment of VSC and catalase from the mesophilic Proteus mirabilis (PMC) revealed 71% identity. As for cold adapted enzymes in general, VSC possessed a lower temperature optimum and higher catalytic efficiency (k _cat/K _m) compared to PMC. VSC have higher affinity for hydrogen peroxide (apparent K _m) at all temperatures. For VSC the turnover rate (k _cat) is slightly lower while the catalytic efficiency is slightly higher compared to PMC over the temperature range measured, except at 4°C. Moreover, the catalytic efficiency of VSC and PMC is almost temperature independent, except at 4°C where PMC has a twofold lower efficiency compared to VSC. This may indicate that VSC has evolved to maintain a high efficiency at low temperatures.     

Factors affecting conjugative transfer rates of plasmids in batch and continuous cultures

Factors affecting the rates of plasmid transfer were investigated using Escherichia coli LC102 bearing a conjugative plasmid R100-1 and E. coli DH1. The rate constant of transconjugant increase, k_ti, was used for presenting the degree of plasmid transmissibility instead of the plasmid transfer efficiency (pte). The rate constant was defined as the specific rate of transconjugant increase (srti, the number of transconjugants per donor per h) divided by the recipient cell concentration. The k_ti values ranged between 10⁻¹⁰ and 10⁻¹⁵ ml cells⁻¹ h⁻¹, when estimated under various conditions. Moderate liquid agitation had a favorable effect on k_tf but agitation rates higher than 33 s⁻¹ (intergrated shear force) greatly decreased the value of k_ti. The transconjugant-forming activity of the cells growing in continuous culture did not significantly change with the dilution rate, except those growing at dilution rates less than 0.1 h⁻¹. The rate constant k_ti at temperatures of 10–15°C was as low as the detection limit (10⁻¹⁵ ml cells⁻¹ h⁻¹).     

A novel site for streptomycin resistance in the “530 loop” of chloroplast 16S ribosomal RNA

The chloroplast gene for 16S rRNA was cloned from two maternally inherited streptomycin-resistant mutants ofNicotiana differing in degree of resistance at the whole plant and isolated chloroplast level. A single-nucleotide change in the 16S rRNA gene was detected for each mutant: a C to T transition at nucleotide 860 (Escherichia coli coordinate C₉₁₂) which is an often mutated site, and a novel transition of C to T at nucleotide 472 (E. coli coordinate C₅₂₅). The novel mutation is located in the phylogenetically conserved 530 loop.     

On the relationships between absolute and allometric shell growth in unionid mussels (Bivalvia,Unionidae) from European Russia

The heuristic analysis of the relationship between the parameters of equations of linear (L. von Bertalanffy) and allometric shell growth was carried out on Unio pictorum (Linnaeus, 1758), U. tumidus Retzius, 1778, and U. crassus Retzius 1778 (Unionidae) from European Russia. Growth constants of the shell, k, were studied for length (k _L ), height (k _h ), and convexity (k _C ). It was shown that the ratios of growth constants (k _h /k _L , k _C /k _h , and k _C/k _L ) and parameter bfrom the allometric equations (h=aL ^b ;B=ah ^b ; C=aL ^b ) are connected by a strong negative correlation. If the b parameter is more than 1 (positive allometry), the growth constant relations are less than one, if b < 1 (negative allometry), the growth constant proportions are more than 1, and, in case when b is close to 1, (isometry), the growth constant proportions are also close to 1. All three types of allometric growth (isometry and both positive and negative allometry) are observed in the studied mussels.     

Kinetics and Mechanism of the Peptide Synthesis in Solution

The kinetics of the reaction of Boc-Xaa fluorophenyl esters (where Xaa = Ala, Val, Phe, Ser, Leu, Gly, Met, Pro, or Ile) with leucinamide was studied in order to measure changes in fluorescence emission at 375 nm of the fluorophenyl chromophore accompanying the reaction. It was found that the experimental kinetic data could not be described by a simple scheme of the second order reaction. Measurements of the kinetic parameters of the reaction at various initial concentrations of reagents indicated that the reaction rate can be expressed as: = kC _N ^a C _AE ^b , where k is the reaction rate constant, C _N is the concentration of leucinamide, and C _AE is the concentration of fluorophenyl ester. The a and b reaction orders were close to 1/2 and 3/2 for Xaa = Ala, Val, Phe, Ser, or Leu, 1/2 and 1 for Gly, Met, or Pro, and 1 and 2 for Ile. The experimental equations for the reaction rate can theoretically be derived from a single scheme of chain reactions with various deactivation ways for active intermediates.