Oxidation of dimethyl sulfide by various aromatic compound oxygenases from bacteria

As a result of the determination of dimethyl sulfide (DMS) oxidizing activity of bacterial aromatic compound oxygenases, multicomponent monooxygenases (DmpKLMNOP from Pseudomonas sp. CF600, AphKLMNOP from Comamonas testosteroni TA441, and TodABCDEF from Pseudomonas sp. JS150), single component monooxygenases (TfdB from Pseudomonas putida EST4011 and XylMA from Pseudomonas putida mt-2), and dioxygenases (CumA1A2A3A4 from Pseudomonas fluorescens IP01 and PahAaAbAcAd from Pseudomonas putida OUS82) showed DMS-oxidizing activity, while CarAaAcAd from Pseudomonas sp. CA10 and SoxC from Rhodococcus sp. IGTS8 did not. These results indicate the possibilities that these oxygenases might oxidize DMS to DMSO under the natural condition in the environment.Present address: Laboratory of Microbiology, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan     

Growth inhibitory effects of dimethyl sulfoxide and dimethyl sulfone on vascular smooth muscle and endothelial cells in vitro

Summary The growth of bovine aortic smooth muscle and endothelial cells was studied after exposure to dimethyl sulfoxide (DMSO) or its major metabolite, dimethyl sulfone (DMSO₂). Both compounds caused a dose-dependent inhibition of cell growth as determined by [³H]thymidine incorporation and by counting the number of cells with time of exposure in culture. The IC₅₀ of DMSO (concentration which produces 50% inhibition of growth) was 1% for smooth muscle cells and 2.9% for endothelial cells. Similarly, the IC₅₀ of DMSO₂ was also 1% for smooth muscle cells, but was 1.8% for endothelial cells. After a 4-d exposure to either compound, the growth inhibition of smooth muscle cells was completely reversible at 1%, partially reversible at 2 to 3% and completely irreversible at 4%. By comparison, inhibition of endothelial cell growth was completely reversible up to 4% of either compound. It is concluded that the growth of smooth muscle cells was similarly inhibited by DMSO, and DMSO₂, but that smooth muscle cells were more susceptible than endothelial cells to the growth inhibitory effects of these compounds. In addition, DMSO₂ was a more potent inhibitor of cell growth than DMSO and its growth inhibition was less reversible than that produced by DMSO.     

Breakdown of dimethyl sulphide by mixed cultures and by Thiobacillus thioparus

Abstract Dimethyl sulphide (DMS) was degraded by acclimatized activated sludge and by a mixed culture of Thiobacillus thioparus TK-1 and Pseudomonas sp. AK-2. While both these organisms persisted in stable co-culture on DMS, it was found that T. thioparus TK-1 and the derived strain TK-m grew in pure culture on DMS, and oxidized DMS with an apparent K _m of 4.5 × 10⁻⁵ M. During growth, all the DMS-sulphur was oxidized stoichiometrically to sulphate but no methanol was detected in pure cultures of TK-m. DMS-carbon was probably converted to CO₂, since the fixation of ¹⁴CO₂ was progressively diluted during growth of a culture on ¹⁴CO₂ and DMS. Growth yields were consistent with autotrophic growth, dependent on the oxidation of the methyl residues to CO₂ (probably with formaldehyde as a first intermediate) and the sulphide to sulphate. The organism thus appears to exhibit a mixture, from the one substrate, of chemolithotrophic and methylotrophic energy generation supporting autotrophic growth with CO₂ fixation.     

草酸二甲酯对小鼠亚急性吸入毒性的实验研究

为了研究草酸二甲酯吸入染毒对小鼠的急性毒性,观察吸入染毒后小鼠活动状况、记录心电图、计算LD50,实验结束后取血、检测血液学、血液cTnI和生化指标;取脏器,计算脑、心、肝、脾、肺、肾、卵巢、睾丸、附睾脏器系数,观察其病理组织学改变。结果显示,小鼠吸入草酸二甲酯后出现抽搐、烦躁不安、心电图异常等中毒反应,LD50为2.065 4×10-4g/cm3,脑、心、肝、脾、肺、肾、睾丸、卵巢脏器系数明显升高,血液学、血生化指标和cTnI水平出现了异常变化,与对照组比较具有显著性差异(P0.05或P0.01);心、肺、肝、小肠和肾组织出现了严重受损。由此可见,草酸二甲酯吸入染毒具有较强的毒性,可引起小鼠心电图、血液中cTnI水平、血液学和血生化指标异常改变,可导致心、肺、肝、小肠和肾脏出现病变。本实验为乙二醇合成过程中产生的碳酸二甲酯职业危害防治提供实验依据。     

Electronic and spatial structure of dimethyl orthophosphate and orthophosphate

The applicability of different variants of the semiempirical CNDO method to calculate the electronic structure of different conformations of all possible ionic forms of dimethyl orthophosphate and orthophosphate is considered. The CNDO/BW (Boyd-Whitehead parameterization) approximation with selected parameters for the P-O bond is shown to provide the best qualitative and sometimes quantitative agreement with ab initio methods. The dependences of energy and P-O bond strengths in P-O-CH₃ (P-O-H) groups on torsion angles at P-O bonds are obtained in this approximation for dimethyl orthophosphate and orthophosphate. The rotation of these groups is found to stremgthen one P-O bond stronger and labilize another. The energy minima of dimethyl orthophosphate and orthophosphate anions are shown to correspond to conformations where the strengths of the studied P-O bonds are almost the same, i.e., none of the bonds is weakened to a minimum. Protonation of these compounds increases the strength of P-O bonds and decreases the dependence of bond strength on torsion angles. The di-and trianions of dimethyl orthophosphate and orthophosphate are also studied. The growth of negative charge is shown to progressively weaken the P-O bond. The dependence of bond strength on torsion angle for the dianion is less pronounced than that for the monoanion. Calculation results are compared with experimental data known from literature. The significance of the data obtained for revealing essential features of the enzyme cleaving and forming the P-O bond is discussed.     

Inactivation kinetics of mushroom tyrosinase in the dimethyl sulfoxide solution

Mushroom tyrosinase (EC 1.14.18.1) is a kind of copper-containing oxidase that catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones and then forms brown or black pigments. In the present paper, the effects of dimethyl sulfoxide on the enzyme activity for the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) have been studied. The results show that low concentrations of dimethyl sulfoxide (DMSO) can lead to reversible inactivation of the enzyme, and the IC ₅₀ is estimated to be 2.45 M. Inactivation of the enzyme by DMSO is classified as mixed type. The kinetics of inactivation of mushroom tyrosinase at low concentrations of DMSO solution has been studied using the kinetic method of the substrate reaction. The rate constants of inactivation have been determined. The results show the free enzyme molecule is more fragile than the enzyme–substrate complex in the DMSO solution. It is suggested that the presence of the substrate offers marked protection of this enzyme against inactivation by DMSO.     

Dolomite limits acidification of a biofilter degrading dimethyl sulphide

The applicability of dolomite particlesto control acidificationin a Hyphomicrobium MS3inoculated biofilter removingdimethyl sulphide (Me₂S) wasstudied. While direct inoculationof the dolomite particles with theliquid microbial culture was notsuccessful, start-up ofMe₂S-degradation in thebiofilter was observed when thedolomite particles were mixed with33% (wt/wt) of Hyphomicrobium MS3-inoculatedcompost or wood bark material.Under optimal conditions, anelimination capacity (EC) of 1680~g Me₂S m^-3 d^-1 wasobtained for the compost/dolomitebiofilter. Contrary to a wood barkor compost biofilter, no reductionin activity due to acidificationwas observed in these biofiltersover a 235 day period because ofthe micro environmentneutralisation of the microbialmetabolite H₂SO₄ with thecarbonate in the dolomite material.However, performance of thebiofilter decreased when themoisture content of the mixedcompost/dolomite material droppedbelow 15%. Next to this, nutrientlimitation resulted in a gradualdecrease of the EC andsupplementation of a nitrogensource was a prerequisite to obtaina long-term high EC (> 250 gMe₂S m^-3 d^-1) forMe₂S. In relation to thisnitrogen supplementation, it wasobserved that stable ECs forMe₂S were obtained when thisnutrient was dosed to the biofilterat a Me₂S-C/NH₄Cl-Nratio of about 10.Abbreviations:DW – dry weight,EC – elimination capacity,Me₂S – dimethyl sulphide,OL – organic loading rate,VS - volatile solids     

纤维素酶在二甲基亚砜微扰条件下的动力学行为和光谱学变化

为了探索二甲基亚砜对纤维素酶催化活性的影响,以羧甲基纤维素钠(CMC)为底物来研究纤维素酶纯酶在二甲基亚砜中的动力学变化、紫外吸收光谱、紫外差示光谱和荧光发射光谱。实验表明:在3%的二甲基亚砜中,纤维素酶的催化活性下降了46.78%;其Km值从缓冲液中的2.500 mg/mL上升到二甲基亚砜中的3.922 mg/mL;在二甲基亚砜中,酶分子的肽键紫外吸收稍有改变,但其氨基酸基团的紫外吸收没有改变;其紫外差示光谱出现明显的正峰和负峰;其荧光发射光谱没有改变。研究结果证明:二甲基亚砜通过轻微改变酶分子的肽链结构,使分子构象改变,导致酶分子对底物的亲和力下降,从而降低其催化活性。     

Conformational studies of vasopressin analogues modified with N-methylphenylalanine enantiomers in dimethyl sulfoxide solution

The conformations of [Arg8]vasopressin (AVP) analogues substituted at positions 2 and 3 with N-methylphenylalanine (MePhe) enantiomers were earlier investigated by using nuclear magnetic resonance (NMR) spectroscopy in aqueous solution. A comparison of the results obtained in H2O/D2O (9:1) and DMSO-d6 has shown the structures in the first solution to be more flexible than those in DMSO-d6. This is manifested by a higher percentage of minor conformations in H2O/D2O. The largest differences between the NMR spectra in both solvents were noticed for [MePhe2, D-MePhe3]AVP (II) and [D-Cys1,MePhe2,D-MePhe3]AVP (III). Namely, in the ROESY spectra in aqueous solution, the cis/trans isomerization between MePhe2-DMePhe3 and D-Cys1-MePhe2 for II and III, respectively, is observed, while in DMSO-d6, the appropriate cross peaks indicate isomerization across the Cys6-Pro7 peptide bond. In the case of the remaining peptides, the position of cis/trans isomerization is the same in aqueous solution and in dimethyl sulfoxide. [D-MePhe2,MePhe3]AVP (V) displays low antiuterotonic and antipressor activities, while [D-MePhe2,)]AVP (IV) is a weak but selective blocker of oxytocin (OT) receptors in the uterus. The former shows similar conformational preferences as another antagonist of V1a and OT receptors-namely, [Acc2,D-Arg8]VP (Acc: 1-aminocyclohexane-1-carboxylic acid)-investigated by us. In the case of IV, the cis peptide bond between residues at positions 2 and 3 might be the reason for selectivity.     

Aerosol generation by metered-dose inhalers containing dimethyl ether/propane inverse microemulsions

Water soluble compounds were incorporated into metered-dose inhalers (MDIs) by using water-in-propellant lecithin microemulsions, in which dimethyl ether (DME) and propane acted as both continuous phase and propellant. Lecithin, water, and water soluble compounds were added to glass MDI containers, valves were crimped on, and propellants were added using a pressure burette. Aerosols were produced using commercially available actuators, and inertial impaction was used to determine the mass median aerodynamic diameter (MMAD), geometric standard deviation (GSD), and fine particle fraction (FPF) of the resulting aerosols. The DME/propane/lecithin, microemulsion MDIs generated aerosols with particle size distributions suitable for pulmonary delivery (eg, MMAD 3.1 μm, FPF 59% for DME with lecithin content 3%, water content 2.5% [wt/wt]). Increasing water concentration (up to 8% wt/wt) was correlated with a reduction in FPF. Freezing and rewarming had no adverse effect on MMAD, GSD, or FPF. Storage of microemulsion samples for up to 3 weeks did not adversely affect the MMAD, GSD, or FPF. This approach may enable the pulmonary delivery of water soluble therapeutic agents via MDIs.     

三种典型赤潮藻产生与消耗二甲基硫化物的速率估算

采用抑制剂加入法估算了中肋骨条藻、棕囊藻和东海原甲藻在不同生长期内二甲基硫化物的产生与消耗速率.结果表明:颗粒态二甲基巯基丙酸(DMSPp)和颗粒态二甲亚砜(DMSOp)在3种藻类的不同生长期内均为净消耗,溶解态二甲基巯基丙酸(DMSPd)和溶解态二甲亚砜(DMSOd)的含量受藻类产生与细菌病毒消耗控制,在藻类不同生长期内存在不同的产生与消耗速率,而二甲基硫(DMS)在3种藻不同生长期内均为净产生.同一种藻在不同生长期内以及不同藻在相同生长期内二甲基硫化物的产生与消耗速率均存在较大差异,表明藻类的生理状态和种间差异均对二甲基硫化物的产生与消耗速率产生影响.     

Biodegradation of dimethyl phthalate with high removal rates in a packed-bed reactor

Biological treatment of a dimethyl phthalate (DMP)-containing waste stream was evaluated in packed-bed bioreactors using an acclimated mixed bacterial culture. The passive immobilization start-up strategy was successful in the development of a stable biofilm on the packing material in the reactor. Nutrient supplementation significantly improved the removal efficiency. High removal rates with 100% efficiencies of DMP removal were achieved up to the phthalate-loading rate of 560 g/m³ h.     

The modulation of membrane structure and stability by dimethyl sulphoxide (Review)

Dimethyl sulphoxide is a widely used agent in cell biology. It is well known as a cryoprotectant, cell fusogen and a permeability enhancing agent. These applications depend, to a greater or lesser extent, on the effect of dimethyl sulphoxide on the stability and dynamics of biomembranes. The aim of this review is to examine progress of the research which has been directed towards studies of the interactions between dimethyl sulphoxide and membranes, particularly that with the lipid components of cell membranes, as seen in its effects on model membrane systems. Models are proposed to explain the mechanism whereby dimethyl sulphoxide may mediate its effects on biological functions by its effects on the stability and properties of the membrane lipid matrix.     

Formation of 5-carbomethoxyvaleramidine during hydrolysis of the protein cross-linking agent dimethyl adipimidate

Imidoesters have been used in biological studies to measure interresidue distances of proteins and macromolecular complexes, and in hematology as antisickling agents. Treatment of human red blood cells with¹⁴C-labeled dimethyl adipimidate (DMA), a bifunctional imidoester with antisickling properties, was followed by gradual loss of radioactivity from the treated cells. The radioactive compound released was isolated by thin-layer chromatography and identified by high-resolution mass spectrometry and by carbon-13 nuclear magnetic resonance, ultraviolet, and infrared spectroscopy as 5-carbomethyoxyvaleramidine, which was also shown to be the major product of DMA hydrolysis in vitro at physiologic pH in phosphate buffer. High-resolution mass spectrometry studies indicated that this product is formed via cyclization to a reactive intermediate (7-methoxy-2-imino-3,4,5,6-tetrahydro-2H-azepine) followed by hydrolysis. The intermediate exhibited strong UV absorbance, maximal at 232 nm. Such an intermediate would be capable of participating in cross-linking reactions which would have smaller dimensions than those observed with the imidoester in its extended form. The hydrolysis product, an unreactive species, should have no toxic effects on individuals receiving infusions of DMA-treated red cells.     

Isobutyraldehyde as a competitor of the dimethyl sulfide degrading activity in biofilters

Upon inoculation with Hyphomicrobium MS3, theelimination capacity of a lab-scale biofilter for theodorant dimethyl sulfide (Me₂S) can be stronglyincreased from less than 10 to more than 35 and 1000 gm^-3d^-1 using wood bark and compost as acarrier material, respectively. However, uponsupplementation of isobutyraldehyde (IBA) as a secondgaseous substrate, sequential degradation profiles of IBAand Me₂S in physically separated sections wereobserved in the Hyphomicrobium MS3-inoculatedwood bark and compost biofilters. Contrary to this, thebiofiltration efficiency for Me₂S remainedunaffected upon the supplementation of toluene as asecond gaseous substrate. Batch experiments with theliquid Hyphomicrobium MS3 culture confirmed thecompetitive effect of IBA on the Me₂S degradingactivity: in the presence of both compounds, Hyphomicrobium MS3 preferred degradation of thecarbonyl compound. In technical terms, this means thatthe complete purification of a waste gas stream containingboth IBA and Me₂S should be performed usingsufficiently high or bistage HyphomicrobiumMS3-inoculated biofilters. Design criteria have to beconceived in this respect.     

Microbial characterization and quantification of an anaerobic sludge degrading dimethyl phthalate

Aims:  Characterization and quantification of microbial community in dimethyl phthalate (DMP)-degrading anaerobic sludge using molecular techniques.
Methods and Results:  An enriched anaerobic sludge effectively degrading over 99% of dimethyl phthalate in an upflow anaerobic sludge blanket (UASB) reactor for 530 days was characterized and quantified by 16S rRNA-based molecular methods. A total of 78 Bacteria clones were classified into 22 operational taxonomic units (OTUs) in nine divisions, including Firmicutes , Proteobacteria, Chloroflexi, Thermotogae , Bacteroidetes/Chlorobi , Spirochaetes , Acidobacteria and two candidate divisions. The two most abundant OTUs were likely responsible, respectively, for the de-esterification of DMP and the subsequent phthalate degradation. The outer layer of the granule was dominated by Bacteria; whereas the interior was by Archaea , of which 89 ± 5% were acetoclastic Methanosaetaceae and 11 ± 5% hydrogenotrophic Methanomicrobiales .
Conclusions:  Twenty-two Bacteria OTUs in DMP-degrading anaerobic sludge distributed in nine divisions. The two most abundant OTUs were likely responsible respectively for the de-esterification of DMP and the subsequent phthalate degradation. Layered granular microstructure of DMP-degrading anaerobic sludge suggested that the rate of DMP de-esterification is faster than its inward diffusion rate.
Significance and Impact of the Study:  This work is the first study to characterize and quantify the microbial community in the anaerobic phthalic ester degrading sludge from the anaerobic reactor.     

Enhanced expression of various exogenous genes in recombinant Chinese hamster ovary cells in presence of dimethyl sulfoxide

Dimethyl sulfoxide (DMSO) (1% v/v) stimulated stable transformed Chinese hamster ovary (CHO) cells to synthesize different recombinant proteins and repress their proliferation rate. The expressions of a fusion protein and -galactosidase were increased 1.6- and 1.4-fold after adding DMSO. The expression of fusion protein was increased by up to 2.8-fold that of uninduced control by the simultaneous addition of DMSO and pentanoic acid. However, DMSO did not increase the production of the monoclonal antibody (immunoglobulin G) of three hybridoma cell lines (OKM1, OKT4 and HyGPD-YK-1-1), although it could inhibit the growth rates of the hybridoma.     

Metabolism of the dimethyl ester of [2,3-13C]succinic acid in rat hepatocytes

Hepatocytes prepared from overnight fasted rats were incubated for 120 min in the presence of the dimethyl ester of [2,3-13C]succinic acid (10 mM). The identification and quantification of 13C-enriched metabolites in the incubation medium were performed by a novel computational strategy for the deconvolution of NMR spectra with multiplet structures and constraints. The generation of 13C-labelled metabolites, including succinate, fumarate, malate, lactate, alanine, aspartate and glucose accounted for about half of the initial amount of the ester present in the incubation medium. A fair correlation was observed between the experimental abundance of each 13C-labelled glucose isotopomer and the corresponding values derived from a model for the metabolism of [2,3-13C]succinate. Newly formed glucose was more efficiently labelled in the carbon C5 than C2, as well as the carbon C6 than C1, supporting the concept that D-glyceraldehyde-3-phosphate may undergo enzyme-to-enzyme channelling between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase.