Characterization of sulfate-reducing bacteria in yeast industry waste by microcalorimetry and PCR amplification

Sulfate-reducing bacteria (SRB) were isolated from anaerobic sludge of yeast factory wastewater treatment plant by cultivation on Postgate C medium. Microcalorimetry was used to monitor the anaerobic digestion processes and to measure the growth rates of sulfate-reducing bacteria. The maximum growth rates determined by microcalorimetry and ATP analysis were different—μ_max(dQ/dt) = 0.165 ± 0.008 h⁻¹ and μ_max(N_ATP) = 0.207 ± 0.013 h⁻¹. Experiments on the cultivation of SRB from yeast industry wastewater treatment plant in batch culture showed that during the first 20 h the concentration of sulfate decreased from 78.3 mM down to 62.2 mM while the increase of sulfide production was negligible. Perceptible amount of sulfide (7.82 mM) appeared on the 33.5 h of fermentation together with a peak on the power–time curve and considerable increase in the cell count (1.26 × 10⁹). First steps of sulfate metabolism (activation of sulfate by ATP sulfurylase, production of H₂) are accompanied by endothermic heat effects, therefore the values of thermal power remain moderate until the evolution of sulfide starts. The influence of green microalgae Chlorococcum sp. (preparation Biotreat 100) on the growth characteristics of microorganisms was also studied. Identification of one SRB strain was started by sequencing of PCR-amplified 16S rRNA gene. Two sets of primers were used for PCR amplification, both specific for domain Bacteria but giving different gene fragments. PCR-products were purified with JETQUICK kit according to the manufacturer instructions.     

Dibutyltin-3-hydroxyflavone titrates a dissociable component (cofactor) of mitochondrial ATP synthase: An energy-transfer component linked to the ubiquinone pool

Dibutyltin-3-hydroxyflavone, Bu₂Sn(of), is a new fluorescence probe inhibitor of F₁F₀-ATPase and oxidative phosphorylation which inhibits by titration of an unidentified component of F₀. Its site of action is closely related to that of the trialkyltins and of venturicidin. This F₀ component is part of a pool of this component which is present in the heart mitochondrial inner membrane at levels of 5–7 nmol (mg protein)^?1 [18 ± 3 Bu₂Sn(of) sites per mol F₁F₀-ATPase]. However, ATPase activity in submitochondrial particles is near maximally inhibited by titration of approx. three Bu₂Sn(of) sites per mol F₁F₀-ATPase. Over 60% (60–80%) of the Bu₂Sn(of) interaction sites can be lost during the purification of F₁F₀-ATPase from submitochondrial particles. The number of Bu₂Sn(of) interaction sites in various F₁F₀-ATPase preparations is variable. The high numbers of Bu₂Sn(of) sites per mol F₁F₀-ATPase for heart mitochondria (18–21) and submitochondrial particles (15–19.5) decline in ATP synthase (11–15) to the low values obtained in Complex V (7–10.5) and the minimal values observed in highly purified F₁F₀?ATPase (3.5–5.6), thus indicating a variable dissociable component or cofactor of ATP synthase. The Bu₂Sn(of) interaction site, a component of ATP synthase, is responsive to the redox status of the respiratory chain and the interaction with Bu₂Sn(of) is with the reduced form of this component. Fluorescence titration studies show that this component is in redox equilibrium with the ubiquinone pool of the respiratory chain. It is proposed that this redox component serves as an inhibitor titratable cofactor pool which cycles through an F₀ interaction site (or sites) via a system which serves as an energy-transfer link between the respiratory chain and ATP synthase.     

The origin of enhanced photocatalytic decomposition of ATP on TiO2 powders as induced by surface modification with lanthanide ions: important roles of ATP adsorption to the modified surface

Surface modification of TiO₂ powders with lanthanide salts (EuCl₃ or YbCl₃) enhanced photocatalytic decomposition of ATP. In comparison with the unmodified catalyst, a 3-fold increase in the ATP decay rate was observed with TiO₂ powders as prepared in 5 mM EuCl₃ (or YbCl₃) solution. The reason was ascribed to enrichment of ATP to TiO₂ powder with the lanthanide ions. Evidence was obtained by adsorption experiments where ATP concentration was monitored after dispersing TiO₂ powders into the solution. The ATP molecules adsorbed faster to TiO₂ powder and strongly stabilized there in the presence of Yb³⁺ than otherwise.     

Metabolomic-based investigation of Yinlan alleviating hyperlipidemia by inhibiting blood stasis and phlegm turbidity through the PXR-CYP3A4-ABCB1-FXR pathway

Yinlan lipid regulatory capsule (YL) is a composite traditional Chinese medicine (TCM) new drug to alleviate hyperlipidemia, while its therapeutic mechanism in vivo was not clarified with nontargeted metabolomics investigation. An animal model was established in rats fed a high-fat diet, and their body weights, body mass index (BMI) and blood cholesterol levels were measured. Serum, liver and kidney tissue samples were also extracted for PXR-CYP3A4-ABCB1-FXR signaling pathway research using PCR and UHPLC–MS. The obtained plasma samples were analyzed by UHPLC-Q-TOF-MS metabolomic investigation, which revealed PXR-CYP3A4-related metabolites and changes induced by YL. Finally, the key metabolites were chosen as index components, and their levels in the serum, liver, small intestine and bile were used for simultaneous UHPLC–MS-MS determination. The results indicated that YL was effective in rebalancing blood TG and TC levels (compared to controls). With respect to the PXR-CYP3A4-ABCB1 pathway, as a result of YL’s effect, gene expression or activity of the two targets decreased significantly in both the liver and kidney. The same trend was observed in the serum samples mentioned above. Metabolomics screening and data revealed that 44 metabolites can be regarded as biomarkers related to hyperlipidemia, fatty acids synthesis, and body energy consumption, as well as synthesis, transportation and exertion of cholesterol. YL’s treatment focused on 26 of them, primarily bile acids, indicating that the antihyperlipidemic effect of this drug lies in its inhibitory activity of cholesterol metabolism. Subsequent analysis of those in vivo components revealed that significant increases (compared to the model group) occurred in the blood, liver, small intestine and bile in groups that received medium and high doses of YL (while the low dose was relatively unchanged). Those target components exhibit a close relationship with PXR and/or CYP3A4. The use of YL repressed PXR expression and subsequently decreased CYP3A4 activity. As a result, synthesis of related bile acids increased, while cholesterol levels decreased, consequently leading to the attenuation of hyperlipidemia. This study comprehensively investigated the antihyperlipidemia mechanism of YL based on its repression of PXR-CYP3A4 activity and related metabolite yield, establishing an accurate method for evaluating the therapeutic effect of YL.     

土壤微生物ATP、脱氢酶和尿素酶活性的ED50值评价外源镧的生物毒性

０IntroductionRareearthsarenowappliedwidelyinChina，whichcanimprovecropsyieldsandthetheirqualit－ies犤１犦．Thebeneficialeffectsmaybeduetothestimu－latoryeffectsoftheseelementsonthenutrientuptakebyplantsorontheincreasingofchlorophyllsynthesisintheplants犤２犦．Whilealotofresearcheshavebeendoneontheimprovednutritionofcropsafterapplica－tionofrareearths，muchlessattentionhasbeenpaidtothedeteriorationofsoilqualityduetotheapplicationofrareearthsforyears犤３犦．Scientistshavediscoveredthataccumula…     

Enzyme inhibitors as chemical tools to study enzyme catalysis: rational design, synthesis, and applications

Carefully designed molecules that are intimately related to the reaction mechanism of enzymes are often highly selective and potent inhibitors that serve as extremely useful chemical probes for understanding the reaction mechanism and structure of enzymes. This article describes the design, synthesis, and applications of specific inhibitors of two mechanistically distinct groups of enzymes, ATP-dependent amide ligases and Ser- and Thr-hydrolases. Our strategy is based on the premise that stable analogues of the transition state (transition-state analogues) are highly potent inhibitors that serve as good mechanistic probes, and that a key structure of a good inhibitor of one enzyme is also utilized for the inhibitors of other enzymes that share the same chemistry in their catalyzed reactions, irrespective of the degree of structural similarity and evolutionary link between the enzymes. According to these principles, we designed and synthesized a series of phosphinate- and sulfoximine-based transition-state analogue inhibitors of glutathione synthetase, gamma-glutamylcysteine synthetase and asparagine synthetase. For the second group of enzymes, we synthesized a gamma-monofluorophosphono glutamate analogue for mechanism-based affinity labeling of gamma-glutamyltranspeptidase and fluorescent phosphonic acid esters for the active-site titration of lipase. These inhibitors were used successfully as ligands for detailed kinetic analyses, X-ray crystallography, and mass analysis of the enzymes to identify the key amino acid residues responsible for catalysis and substrate recognition in the transition state.     

Nucleotide/Protein Interaction: Energetic and Structural Features of Na,K-ATPase

Microcalorimetric titrations allow to recognize and investigate high-affinity ligand binding to Na,K-ATPase. Titrations with the cardiac glycoside Ouabain, which acts as a specific inhibitor of the enzyme, have provided not only the thermodynamic parameters of high-affinity binding with a stoichiometric coefficient of about 0.6 but also evidence for low-affinity binding to the lipid. The marked enthalpic contribution of -95 kJ mol^-1 at 298.2 K is partially compensated by a large negative entropy change, attributed to an increased interaction between water and the protein. The calorimetric ADP and ATP titrations at 298.2 K are indicative of high-affinity nucleotide binding either in 3 mM NaCl, 3 mM MgCl₂ or at high ionic strength such as 120 mM choline chloride. However, no binding is detected in the buffer solution alone at low ionic strength. The affinities for ADP and ATP are similar, around 10⁶ M^-1 and the stoichiometric coefficients are close to that of Ouabain binding. The exothermic binding of ADP is characterized by a ΔH and ΔS value of -65 kJ mol^-1 and -100 J mol^-1 K^-1, respectively. TheΔH value for ATP binding is larger than for ADP and is compensated by a larger, unfavorable ΔS value. This leads to an enthalpy/entropy compensation, which could express that H-bond formation represents the major type of interaction. As for Ouabain, the negative ΔS values that are also characteristic of nucleotide binding can indicate an increase of solvate interaction with the protein due to a conformational transition occurring subsequent to the binding process. The resulting binding constants are discussed with regard to the results of other studies employing different techniques. A molecular interaction model for nucleotide binding is suggested. This revised version was published online in July 2006 with corrections to the Cover Date.     

紫色酸性磷酸酯酶模型化合物水解ATP的研究

紫色酸性磷酸酯酶（PAPS）是含铁的糖蛋白，能够从植物和动物体内分离出来，在微酸性条件下，能催化水解磷酸酯阻］，该类酶具双核铁中心活性部位，每个蛋白有两个铁原子，两个铁原子以ellelll形式存在时，具有催化活性·近年来，人们对研究磷酸酯水解酶感兴趣，是因为这个课题     

Simulations of the active transport of a neutral solute based on a kinase-channel-phosphatase topology

Simulations of coupled interactions involving two opposite enzymatic reactions, solute diffusions, and electrostatic interactions between membrane charges and charged solutes were conducted under a fixed kinase-channel-phosphatase (KCP) topology oriented from the outside to the inside of a porous membrane structure. Depending on the kinase and phosphatase locations, we recently demonstrated that an active transport of a phosphorylated substrate may occur via the opposite topology, that is, a PCK topology. The present analysis demonstrates that, under a KCP membrane topology, which also behaves as a specific ATP-dependent transporter, the active transport of a neutral substrate may occur. This analogous active transport appears to be dependent on the phosphatase location and on the membrane surface potentials. A broad analysis of the role played by the main parameters taken into account in the model was conducted in order to define precisely the physico-chemical conditions and the membrane topology needed for the highest active transports within the shortest time.