Novel Long‐Chain Diol Phospholipids from Some Bacteria Belonging to the Class Thermomicrobia

Polar lipids of bacteria from the class Thermomicrobia are known to contain long‐chain 1,2‐diols instead of glycerol, although the nature of polar head groups has not been investigated. We have studied phospholipid classes of two species from the class Thermomicrobia—Sphaerobacter thermophilus and Thermomicrobia sp. WKT50.2. TLC and ³¹P‐NMR analysis of polar lipids revealed that both S. thermophilus and Thermomicrobia sp. WKT50.2 contain the same set of four major phospholipid classes. Structures of the novel phospholipids in S.thermophilus were established as 2‐acylalkyldiol‐1‐O‐phosphoinositol, 2‐acylalkyldiol‐1‐O‐phosphoinositol mannoside, 2‐acylalkyldiol‐1‐O‐phospho‐acylmannoside, and 2‐acylalkyldiol‐1‐O‐phosphoinositol acylmannoside. To our knowledge, this is the first report of a phospholipid with a mannose directly bound to the phosphate. We also analyzed fatty acids and long‐chain 1,2‐diols of S. thermophilus and Thermomicrobia sp. WKT50.2 and compared our data with available information for T. roseum. All species share a similar set of fatty acids, with 12‐Me 18:0 being the major fatty acid. The major diol in S. thermophilus was identified as 13‐Me 19:0 (66.2 %). The 21:0 diol was the major component both in Thermomicrobia sp. WKT50.2 (50.6 %) and in T. roseum (56.6 %).     

云南热泉亚栖热菌质粒与其部分表型关系的初步研究

从云南洱源、腾冲热泉分离到108株专性嗜热非芽孢菌,经形态、革兰氏染色及生理生化实验初步鉴定为亚栖热菌。通过碱裂解及试剂盒抽提质粒,琼脂糖凝胶电泳检测,13株菌含有质粒。用AO、SDS对RH17-H进行化学消除后对比消除前后色素和耐高温性变化,显示与质粒无决定性关系。     

Phospholipids of New Zealand Edible Brown Algae

Edible brown algae have attracted interest as a source of beneficial allenic carotenoid fucoxanthin, and glyco‐ and phospholipids enriched in polyunsaturated fatty acids. Unlike green algae, brown algae contain no or little phosphatidylserine, possessing an unusual aminophospholipid, phosphatidyl‐O‐[N‐(2‐hydroxyethyl) glycine], PHEG, instead. When our routinely used technique of ³¹P‐NMR analysis of phospholipids was applied to the samples of edible New Zealand brown algae, a number of signals corresponding to unidentified phosphorus‐containing compounds were observed in total lipids. NI (negative ion) ESI QToF MS spectra confirmed the presence of more familiar phospholipids, and also suggested the presence of PHEG or its isomers. The structure of PHEG was confirmed by comparison with a synthetic standard. An unusual MS fragmentation pattern that was also observed prompted us to synthesise a number of possible candidates, and was found to follow that of phosphatidylhydroxyethyl methylcarbamate, likely an extraction artefact. An unexpected outcome was the finding of ceramidephosphoinositol that has not been reported previously as occurring in brown algae. An uncommon arsenic‐containing phospholipid has also been observed and quantified, and its TLC behaviour studied, along with that of the newly synthesised lipids.     

Inactivation and Unfolding of the Hyperthermophilic Inorganic Pyrophosphatase from Thermus thermophilus by Sodium Dodecyl Sulfate

Inorganic pyrophosphatase (PPase, EC 3.6.1.1) is an essential constitutive enzyme for energy metabolism and clearance of excess pyrophosphate. In this research, we investigated the sodium dodecyl sulfate (SDS)-induced inactivation and unfolding of PPase from Thermus thermophilus (T-PPase), a hyperthermophilic enzyme. The results indicated that like many other mesophilic enzymes, T-PPase could be fully inactivated at a low SDS concentration of 2 mM. Using an enzyme activity assay, SDS was shown to act as a mixed type reversible inhibitor, suggesting T-PPase contained specific SDS binding sites. At high SDS concentrations, T-PPase was denatured via a two-state process without the accumulation of any intermediate, as revealed by far-UV CD and intrinsic fluorescence. A comparison of the inactivation and unfolding data suggested that the inhibition might be caused by the specific binding of the SDS molecules to the enzyme, while the unfolding might be caused by the cooperative non-specific binding of SDS to T-PPase. The possible molecular mechanisms underlying the mixed type inhibition by SDS was proposed to be caused by the local conformational changes or altered charge distributions.     

Rapid Identification and Relative Quantification of the Phospholipid Composition in Commercial Lecithins by <Superscript>31</Superscript>P NMR

³¹P NMR analysis of samples prepared in a sodium cholate detergent system was used as a method for the identification and quantification of enzymatic hydrolysis products of lecithin. To precisely characterize all of the hydrolysis products from commercial lecithin, a series of enzymatic reactions of each phospholipid with phospholipase PLA1 were conducted and monitored by ³¹P NMR at different times. Twenty-six phosphorus-containing hydrolysis products from six classes of phospholipids (PC, PI, PS, PE, PG, PA) were found and determined by ³¹P NMR measurement. The impact of pH on the chemical shift values for these hydrolysis products was observed and reported. To the best of our knowledge, this is the first report of ³¹P-NMR chemical shift values for entire lyso-phospholipids hydrolyzed from 6 classes of phospholipids. Rapid and routine analysis of phospholipid composition in commercial lecithins by ³¹P NMR was achieved without the need of phospholipid standards.     

The Identification and Quantification of Steryl Glucosides in Precipitates from Commercial Biodiesel

There have been several discoveries of unexpected precipitates in manufacturing facilities, transport vessels, and storage tanks containing commercial biodiesel. In some cases these have been formed during storage at temperatures above the cloud point of the fuel. High performance liquid chromatography (HPLC) and mass spectrometry (MS) methods were applied to 24 field receipt samples of solids from such biodiesels. The analyses revealed the presence of steryl glucosides (SG), common phytosterols (plant sterol) found in crude soybean oil and many other plant materials, in these biodiesel precipitates. Quantitative analysis of the solids revealed SG concentrations as high as 68 wt% of the provided material (which had not been previously washed with solvent to remove entrained biodiesel). In some samples no SG were present. In others they constituted all of the non-biodiesel material present. Monacylglycerols and diacylglycerols, the products of incomplete transesterification of triacylglycerols, were also present in some samples. The normal phase and reverse phase methods described in this report could be used to analyze SG quantitatively from biodiesel precipitates with an HPLC instrument equipped with either an evaporative light-scattering detector (ELSD) or a more common UV detector operating at 205 nm. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Rapid and Simple Identification and Quantification of Components in Detergent Formulations by Nuclear Magnetic Resonance Spectroscopy

A rapid, inexpensive, and simple high-resolution NMR spectroscopic method is outlined for determining the content of surfactants and other low-molecular-weight organic compounds in detergent formulations. With simple sample preparation, quantitative results can be obtained from an internal standard and/or the method can be used as a fingerprint analysis of the surfactant composition. The NMR sample is prepared by suspending the detergent sample in deuterated acetic acid and thus dissolving surfactants and other organic compounds. Any content of carbonate will be liberated as CO₂, whereas other inorganic materials are removed by centrifugation. From one-dimensional ¹H and two-dimensional HSQC NMR spectra, the surfactant components and low-molecular-weight organic compounds can be identified from reference spectra. Intensities of signature signals in the one-dimensional ¹H NMR spectrum are used for quantification by comparing with an internal standard. Furthermore, it is demonstrated how ³¹P NMR can be used to identify and quantify phosphonate-type chelators.     

Fatty Acid and Lipid Profiles with Emphasis on n-3 Fatty Acids and Phospholipids from Ciona intestinalis

In order to establish Ciona intestinalis as a new bioresource for n‐3 fatty acids‐rich marine lipids, the animal was fractionated into tunic and inner body tissues prior to lipid extraction. The lipids obtained were further classified into neutral lipids (NL), glycolipids (GL) and phospholipids (PL) followed by qualitative and quantitative analysis using GC‐FID, GC–MS, ¹H NMR, 2D NMR, MALDI‐TOF‐MS and LC–ESI–MS methods. It was found that the tunic and inner body tissues contained 3.42–4.08 % and 15.9–23.4 % of lipids respectively. PL was the dominant lipid class (42–60 %) irrespective of the anatomic fractions. From all lipid fractions and classes, the major fatty acids were 16:0, 18:1n‐9, C20:1n‐9, C20:5n‐3 (EPA) and C22:6n‐3 (DHA). The highest amounts of long chain n‐3 fatty acids, mainly EPA and DHA, were located in PL from both body fractions. Cholestanol and cholesterol were the dominant sterols together with noticeable amounts of stellasterol, 22 (Z)‐dehydrocholesterol and lathosterol. Several other identified and two yet unidentified sterols were observed for the first time from C. intestinalis. Different molecular species of phosphatidylcholine (34 species), sphingomyelin (2 species), phosphatidylethanolamine (2 species), phosphatidylserine (10 species), phosphatidylglycerol (9 species), ceramide (38 species) and lysophospholipid (5 species) were identified, representing the most systematic PL profiling knowledge so far for the animal. It could be concluded that C. intestinalis lipids should be a good alternative for fish oil with high contents of n‐3 fatty acids. The lipids would be more bioavailable due to the presence of the fatty acids being mainly in the form of PL.     

Profiling and Imaging of Phospholipids in Brains of Abcd1‐Deficient Mice

ABCD1 is a gene responsible for X‐linked adrenoleukodystrophy (X‐ALD), and is critical for the transport of very long‐chain fatty acids (VLCFA) into peroxisomes and subsequent β‐oxidation. VLCFA‐containing lipids accumulate in X‐ALD patients, although the effect of ABCD1‐deficiency on each lipid species in the central nervous system has not been fully characterized. In this study, each phospholipid and lysophospholipid species in Abcd1‐deficient mice brains were profiled by liquid chromatography‐mass spectrometry. Among the phospholipid and lysophospholipid species that are significantly more enriched in Abcd1‐deficient mice brains, VLCFA were present in 75, 15, 5, 4, and 1 species of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, and lysophosphatidylethanolamine, respectively. Most VLCFA were incorporated at the sn‐1 position of phosphatidylcholine and phosphatidylethanolamine. Among the phospholipid species that are significantly less enriched in Abcd1‐deficient mice brains, odd‐numbered saturated or mono‐unsaturated fatty acyl moieties are contained in all phosphatidylcholine species. In addition, a number of phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine species contained highly unsaturated fatty acyl moieties. Intriguingly, 44:1 phosphatidylcholine with VLCFA was mainly distributed in the gray matter, such as the cortex, but not in the white matter in the cerebrum and cerebellum. These results show that ABCD1‐deficiency causes metabolic alternation of long‐chain fatty acids and VLCFA. Moreover, our results imply a molecular mechanism for the incorporation of saturated or monounsaturated VLCFA into the sn‐1 position of phospholipids, and also indicate that the distribution of phospholipids with VLCFA may correlate with the development of X‐ALD.     

The effect of different structure parameters on the crosslinking behaviour and network performance of LDPE

In the present study, we investigate the crosslinking behaviour of 6 ordinary low density polyethylenes, and the effect of relatively modest changes in the polyethylene structure on the properties of the resulting network. The observed variations in gel formation are mainly explained by differences in molecular weight, where both and affect the results. The presence of long chain branches (LCB) present on the polymer main chain is shown to have a significant effect on the network quality. The occurrence of LCB affects the ability in making effective entanglements, a behaviour which is largely dependent on the length of the branches. At high LCB frequencies, the long chain branches are relatively short and therefore more prone to disentangle. The frequency and the amount of LCB, together with the molecular weight, have a large impact on the coil size. The coil size is believed to be an important parameter for the crosslinking behaviour, as a large coil size facilitates interconnections between adjacent polymer coils. In addition, LCB increases the probability of creating intramolecular crosslinks. However, this study shows that a very high amount of LCB is needed in order to obtain any significant effect on the network quality originating from intramolecular crosslinks.     

长链α-烯烃的生产与应用前景

简要地阐述了国内外长链α 烯烃的生产现状及国内市场情况 ,并根据我国国情 ,提出了蜡裂解制取长链α 烯烃的工艺路线。并以 5 4 # 半炼蜡为原料进行蜡裂解试验 ,通过精密分馏将目的烯烃切割成不同馏分 ,进行下游产品的评定试验。试验结果表明 ,以半炼蜡为原料 ,通过裂解工艺 ,可以生产出质量合格的符合下游产品应用要求的长链α 烯烃产品 ,解决了国内部分行业的急需 ,具有很好的社会效益和经济效益     

Fibroblast Growth Factor-21 and the Beneficial Effects of Long-Chain n-3 Polyunsaturated Fatty Acids

Long‐chain n‐3 polyunsaturated fatty acids (LC n‐3 PUFA) in the diet protect against insulin resistance and obesity. Fibroblast growth factor‐21 (Fgf21) is a hormonal factor released mainly by the liver that has powerful anti‐diabetic effects. Here, we tested whether the beneficial metabolic effects of LC n‐3 PUFA involve the induction of Fgf21. C57BL/6 J mice were exposed to an obesogenic, corn‐oil‐based, high‐fat diet (cHF), or a diet in which corn oil was replaced with a fish‐derived LC n‐3 PUFA concentrate (cHF + F) using two experimental settings: short‐term (3 weeks) and long‐term treatment (8 weeks). CHF + F reduced body weight gain, insulinemia, and triglyceridemia compared to cHF. cHF increased plasma Fgf21 levels and hepatic Fgf21 gene expression compared with controls, but these effects were less pronounced or absent in cHF + F‐fed mice. In contrast, hepatic expression of peroxisome proliferator‐activated receptor (PPAR)‐α target genes were more strongly induced by cHF + F than cHF, especially in the short‐term treatment setting. The expression of genes encoding Fgf21, its receptors, and Fgf21 targets was unaltered by short‐term LC n‐3 PUFA treatment, with the exception of Ucp1 (uncoupling protein 1) and adiponectin genes, which were specifically up‐regulated in white fat. In the long‐term treatment setting, the expression of Fgf21 target genes and receptors was not differentially affected by LC n‐3 PUFA. Collectively, our findings indicate that increased Fgf21 levels do not appear to be a major mechanism through which LC n‐3 PUFA ameliorates high‐fat‐diet‐associated metabolic disorders.     

Identification of a Δ4‐desaturase from the microalga Ostreococcus lucimarinus

Very long chain PUFA (VLCPUFA) like DHA are essential and health‐beneficial components of the human diet. Due to a shortfall in VLCPUFA supply, research is ongoing to establish VLCPUFA production in heterologous systems like for example oilseed plants. For this purpose it is crucial to identify the required enzymes from primary producers of VLCPUFA. Here, we describe a cDNA from the microalga Ostreococcus lucimarinus coding for a Δ4‐fatty acid desaturase. It exhibits a cytochrome b₅ domain fused to its amino terminus and three histidine boxes that are typically found in front‐end desaturases. Heterologous expression of the partly codon‐optimized version of the cDNA in yeast revealed that the encoded protein catalyzes the desaturation of (n‐3)‐ as well as (n‐6)‐substrates with a preference for VLCPUFA. In yeast it localized at the endoplasmic reticulum (ER) membrane and analysis of the product distribution into different lipid classes suggested that the enzyme most likely acts in a lipid‐dependent manner. Practical applications : The identified Δ4‐desaturase may be useful for the production of DHA in transgenic oleaginous organisms like annual oilseed crops.     

The effect of chain packing on the thermal and dynamic mechanical behaviour of liquid‐crystalline epoxy thermosets

Liquid‐crystalline thermosets (LCTs) have received considerable attention since they exhibit many interesting properties. However, some aspects concerning LCTs are still unexplored. In particular, the structure–property relationships, as far as the organization of liquid‐crystalline (LC) domains in cured thermosets is concerned, have not been fully elucidated yet. We investigated the effect of the presence of a nematic mesophase on the thermal and dynamic mechanical behaviour of p‐(2,3‐epoxypropoxy)phenyl‐p‐(2,3‐epoxypropoxy)benzoate cured with 2,4‐diaminotoluene. Fourier transform infrared spectral analysis showed that epoxy group conversion was complete in both LC and isotropic (ISO) systems; moreover, a greater amount of intermolecular hydrogen bonding in the LC material was found. Thermogravimetric analysis evidenced similar thermal stability for the two systems, but a kinetic analysis of the data showed that the degradation process is more complex for the LC sample, and is characterized by higher activation energy. Dynamic mechanical thermal analysis (DMTA) showed lower glass transition temperature values for the LC system. Solid‐state NMR analysis evidenced lower paramagnetic oxygen absorption for this system. DMTA results show that the ISO material possesses a larger number of physical crosslinks, which act as extra constraints on the molecular motions. In the case of the LCT, the formation of a locally more dense structure is postulated, where the presence of more extended macromolecular segments leads to a smaller number of physical crosslinks, as also confirmed by solid‐state NMR analysis. A more compact molecular packing also leads to an increase of the activation energies of the thermal degradation process of the LC system. Copyright © 2010 Society of Chemical Industry     

Comparison of the Activity of a Rhodium‐Biphephos Catalyst in Thermomorphic Solvent Mixtures and Microemulsions

Tunable solvent systems provide optimal reaction conditions for homogeneous catalysis as well as appropriate conditions for separating the product from the catalyst via temperature switches. Two tunable solvent systems are presented based on different thermodynamic concepts, namely thermomorphic multicomponent solvent systems and dispersions of immiscible liquids, stabilized by nonionic surfactants as microemulsions. Both systems were applied for the regioselective hydroformylation of the model substrate 1‐dodecene with a Rhodium/ligand catalyst system. The accessibility of the catalyst for the reactants was compared between the molecularly dispersed mixtures and the colloidally dispersed microemulsion systems which are heterogeneous on the microscopic level.     

Identification of a potent inhibitor of human dual-specific phosphatase, VHR, from computer-aided and NMR-based screening to cellular effects

Human vaccinia H1-related phosphatase (VHR) is a dual-specific phosphatase (DSPs) that plays an important role in the mitogen-activated protein (MAP) kinase cascade regulation. It is also a potential drug target for diseases that are related to immune response. By combining a virtual and NMR-based ligand-screening strategy, we successfully identified four VHR inhibitors, of which GATPT ((glucosamine-aminoethoxy)triphenyltin) can bind to VHR with a K(i) value of 2.54 muM. The putative binding mode of GATPT was constructed by a molecular docking simulation to provide structural insights into the ligand-binding mechanism. Furthermore, we found that this compound can significantly inhibit the dephosphorylation of the extracellular regulated kinases (ERKs), and c-Jun N-terminal kinases (JNKs) and block the G(1)-S phase transition in the cell cycle. Therefore, GATPT is a promising lead structure for designing more effective inhibitors of VHR.     

Rapid Biophysical Characterization and NMR Spectroscopy Structural Analysis of Small Proteins from Bacteria and Archaea

Proteins encoded by small open reading frames (sORFs) have a widespread occurrence in diverse microorganisms and can be of high functional importance. However, due to annotation biases and their technically challenging direct detection, these small proteins have been overlooked for a long time and were only recently rediscovered. The currently rapidly growing number of such proteins requires efficient methods to investigate their structure–function relationship. Herein, a method is presented for fast determination of the conformational properties of small proteins. Their small size makes them perfectly amenable for solution-state NMR spectroscopy. NMR spectroscopy can provide detailed information about their conformational states (folded, partially folded, and unstructured). In the context of the priority program on small proteins funded by the German research foundation (SPP2002), 27 small proteins from 9 different bacterial and archaeal organisms have been investigated. It is found that most of these small proteins are unstructured or partially folded. Bioinformatics tools predict that some of these unstructured proteins can potentially fold upon complex formation. A protocol for fast NMR spectroscopy structure elucidation is described for the small proteins that adopt a persistently folded structure by implementation of new NMR technologies, including automated resonance assignment and nonuniform sampling in combination with targeted acquisition.     

Absolute Stereochemistry of 1,2-Diols from Lipids of Thermomicrobia

1,2‐Diol based phospholipids are a well‐known feature of bacteria from the class Thermomicrobia. Since these bacteria contain only lipids with an alkyldiol‐1 ‐phosphate backbone instead of sn‐glycero‐3‐phosphate, it is important to elucidate the stereochemistry of the 1,2‐diols. We have studied the absolute stereochemistry of long‐chain 1,2‐diols isolated from Thermorudis pharmacophila (formerly known as Thermomicrobia sp. WKT50.2) by nuclear magnetic resonance (NMR) using α‐methoxyphenylacetic acid (MPA). Low‐temperature (?60 °C) NMR of bis‐(R)‐MPA ester showed (R) stereochemistry of the 1,2‐diols. This is the first report concerning the stereochemistry of natural 1,2‐diols, which replace the glyceride moiety in phospholipids. The (R) stereochemistry of the diols is expected as it is the same configuration as for the common bacterial lipid backbone—sn‐glycero‐3‐phosphate. This is the first application of low‐temperature NMR of a single MPA derivative for assignment of stereochemistry of natural 1,2‐diols. The results were confirmed by the comparison of NMR data with bis‐(R)‐MPA ester of (R) and rac‐1,2‐octanediol.