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1.
Zymomonas mobilis (ATCC 29191) was grown either aerobically or anaerobically in the presence of 2% (wt/vol) glucose and 0, 3, or 6% (vol/vol) ethanol. The rates of growth and the composition of hopanoids, cellular fatty acids, and other lipids in the bacterial membranes were quantitatively analyzed. The bacterium grew in the presence of 3% and 6% ethanol and was more ethanol tolerant when grown anaerobically. In the absence of ethanol, hopanoids comprised about 30% (by mass) of the total cellular lipids. Addition of ethanol to the media caused complex changes in the levels of hopanoids and other lipids. However, there was not a significant increase in any of the hopanoid lipid classes as ethanol concentration was increased. As previously reported, vaccenic acid was the most abundant fatty acid in the lipids of Z. mobilis, and its high constitutive levels were unaffected by the variations in ethanol and oxygen concentrations. A cyclopropane fatty acid accounted for 2.6–6.4 wt % of the total fatty acids in all treatments. Received: 12 November 1996 / Accepted: 25 February 1997  相似文献   

2.
Hopanoids are steroid‐like lipids from the isoprenoid family that are produced primarily by bacteria. Hopanes, molecular fossils of hopanoids, offer the potential to provide insight into environmental transitions on the early Earth, if their sources and biological functions can be constrained. Semiquantitative methods for mass spectrometric analysis of hopanoids from cultures and environmental samples have been developed in the last two decades. However, the structural diversity of hopanoids, and possible variability in their ionization efficiencies on different instruments, have thus far precluded robust quantification and hindered comparison of results between laboratories. These ionization inconsistencies give rise to the need to calibrate individual instruments with purified hopanoids to reliably quantify hopanoids. Here, we present new approaches to obtain both purified and synthetic quantification standards. We optimized 2‐methylhopanoid production in Rhodopseudomonas palustris TIE‐1 and purified 2Me‐diplopterol, 2Me‐bacteriohopanetetrol (2Me‐BHT), and their unmethylated species (diplopterol and BHT). We found that 2‐methylation decreases the signal intensity of diplopterol between 2 and 34% depending on the instrument used to detect it, but decreases the BHT signal less than 5%. In addition, 2Me‐diplopterol produces 10× higher ion counts than equivalent quantities of 2Me‐BHT. Similar deviations were also observed using a flame ionization detector for signal quantification in GC. In LC‐MS, however, 2Me‐BHT produces 11× higher ion counts than 2Me‐diplopterol but only 1.2× higher ion counts than the sterol standard pregnane acetate. To further improve quantification, we synthesized tetradeuterated (D4) diplopterol, a precursor for a variety of hopanoids. LC‐MS analysis on a mixture of (D4)‐diplopterol and phospholipids showed that under the influence of co‐eluted phospholipids, the D4‐diplopterol internal standard quantifies diplopterol more accurately than external diplopterol standards. These new quantitative approaches permit meaningful comparisons between studies, allowing more accurate hopanoid pattern detection in both laboratory and environmental samples.  相似文献   

3.
Novel hopanoid cyclases from the environment   总被引:1,自引:0,他引:1  
Hopanoids are ubiquitous isoprenoid lipids found in modern biota, in recent sediments and in low-maturity sedimentary rocks. Because these lipids primarily are derived from bacteria, they are used as proxies to help decipher geobiological communities. To date, much of the information about sources of hopanoids has come from surveys of culture collections, an approach that does not address the vast fraction of prokaryotic communities that remains uncharacterized. Here we investigated the phylogeny of hopanoid producers using culture-independent methods. We obtained 79 new sequences of squalene-hopene cyclase genes (sqhC) from marine and lacustrine bacterioplankton and analysed them along with all 31 sqhC fragments available from existing metagenomics libraries. The environmental sqhCs average only 60% translated amino acid identity to their closest relatives in public databases. The data imply that the sources of these important geologic biomarkers remain largely unknown. In particular, genes affiliated with known cyanobacterial sequences were not detected in the contemporary environments analysed here, yet the geologic record contains abundant hopanoids apparently of cyanobacterial origin. The data also suggest that hopanoid biosynthesis is uncommon: < 10% of bacterial species may be capable of producing hopanoids. A better understanding of the contemporary distribution of hopanoid biosynthesis may reveal fundamental insight about the function of these compounds, the organisms in which they are found, and the environmental signals preserved in the sedimentary record.  相似文献   

4.
HPLC fluorometric methods have been used to analyze trace amounts of d-amino acids in biological samples. In this study, we established an expedient column-switching fluorometric HPLC system that would improve the analysis of d-amino acids, in particular d-aspartate (Asp). Our system consists of the fluorogenic derivatization of amino acids with NBD-F and two chromatographic steps, one that separates individual amino acids in reverse phase mode and another that separates the chiral forms of each amino acid in normal-phase mode. The two separation steps are linked through a trapping column by an automated column-switching system. In addition, sample preparation is simplified and improved, where trichloroacetic acid is used for deproteinization, and borate buffer, pH 9.5 is employed for the fluorescent derivatization. The detection limit for d-Asp in culturing medium is 5 nM. The resulting peak heights correlated well with concentrations that ranged from 12.5 to 250 nM for both d- and l-Asp. The present method was applied to determine d- and l-Asp levels in cell culturing medium, and within cells of MPT1 cell line. The detected cellular levels of d- and l-Asp agree with those detected by our previous method. In addition, this method was used to measure d- and l-Asp levels in rat blood samples, and the results are consistent with the reported values.  相似文献   

5.
Cyanobacteria are key players in the global carbon and nitrogen cycles and are thought to have been responsible for the initial rise of atmospheric oxygen during the Neoarchean. There is evidence that a class of membrane lipids known as hopanoids serve as biomarkers for bacteria, including many cyanobacteria, in the environment and in the geologic record. However, the taxonomic distributions and physiological roles of hopanoids in marine cyanobacteria remain unclear. We examined the distribution of bacteriohopanepolyols (BHPs) in a collection of marine cyanobacterial enrichment and pure cultures and investigated the relationship between the cellular abundance of BHPs and nitrogen limitation in Crocosphaera watsonii, a globally significant nitrogen‐fixing cyanobacterium. In pure culture, BHPs were only detected in species capable of nitrogen fixation, implicating hopanoids as potential markers for diazotrophy in the oceans. The enrichment cultures we examined exhibited a higher degree of BHP diversity, demonstrating that there are presently unaccounted for marine bacteria, possibly cyanobacteria, associated with the production of a range of BHP structures. Crocosphaera watsonii exhibited high membrane hopanoid content consistent with the idea that hopanoids have an important effect on the bulk physical properties of the membrane. However, the abundance of BHPs in C. watsonii did not vary considerably when grown under nitrogen‐limiting and nitrogen‐replete conditions, suggesting that the role of hopanoids in this organism is not directly related to the physiology of nitrogen fixation. Alternatively, we propose that high hopanoid content in C. watsonii may serve to reduce membrane permeability to antimicrobial toxins in the environment.  相似文献   

6.
Sphingolipids are an important class of lipids due to their role as biologically active molecules and as intracellular second messengers. Sphingolipid metabolites are involved in a wide variety of important biological processes including signal transduction and growth regulation. Simple, quantitative analytical methods are needed to assay these complex lipids, in order to study their biological functions. The current methods used to quantify ceramides and long-chain sphingoid bases are primarily based on derivatization with uv or fluorescent tags and with radioactive-based enzymatic assays. A method was developed to separate ceramides and sphingoid bases by normal-phase high-performance liquid chromatography and detect them directly with evaporative light-scattering detection. Ceramides and the sphingoid bases phytosphingosine, dihydrosphingosine, sphingosine, and sphingosine 1-phosphate were resolved with a rapid and quantitative assay in the nanomole range. Yeast extracts grown to various time points were assayed for ceramide and sphingoid bases using a simple, isocratic HPLC system. Both ceramide and phytosphingosine, the primary sphingoid base present in yeast cell extracts, were detected in yeast cell extracts. Phytosphingosine was resolved as a sharp peak with the addition of triethylamine and formic acid modifiers to a chloroform/ethanol mobile phase. This method demonstrates the first direct assay of both ceramides and sphingoid bases.  相似文献   

7.
Lipid bilayer membranes were made from hopanoid phosphatidylcholine mixtures dissolved in decane. The specific capacity of the mixed membranes was found to increase with increasing hopanoid content. This indicates an interaction between hopanoids and lipids which leads to a reduction of the chemical potential of the solvent in the membranes.The structural properties of mixtures of hopanoids and phosphatidylcholines were investigated using charged probe molecules, the negatively charged lipophilic ions dipicrylamine (DPA) and tetraphenylborate (TØB) and the positively charged potassium complex PV-K+ (PV, cyclo (D-Val-L-Pro-L-Val-D-Pro)3). The transport properties of the lipophilic ions in the mixed membranes indicate that the electrical properties like dipolar potential and surface potentials of phosphatidylcholine membranes are not changed by the insertion of the hopanoids. The translocation rate constant K of the PV-K+ complex is drastically reduced in the hopanoid phosphatidylcholine membranes with increasing hopanoid content. This effect is discussed on the basis of an alteration of the microviscosity in the mixed membranes. There exists a close analogy between the action of cholesterol and hopanoids in bilayer membranes from phosphatidylcholines.A bilayer membrane composed of di-ω-cyclohexyldodecanoyl-phosphatidylcholine (DCPC) was found to possess a higher specific capacity as compared to other phosphatidylcholines. Also a lower translocation rate constant for PV-K+ was observed which may be caused by the relative high microviscosity of this lipid even above the phase transition temperature.  相似文献   

8.
Free amino acid concentrations in needles of drought-stressed and shaded seedlings of ponderosa pine (Pinus ponderosa Dougl. ex Laws.) were analyzed to determine the influence of low irradiance on a biochemical response to drought stress. A convenient method was developed for separating and quantitating amino acids in needle extracts using derivatization with dansyl chloride and reversed-phase high-performance liquid chromatography (HPLC). Twenty-one amino acids were separated on an Ultrasphere ODS CIS column and detected with a fluorescence spectrophotometer. As little as 10 pmol of each were detected with good peak separation and reproducible retention times. The results of HPLC analysis showed that drought and shading induced an increase in total amino acid concentrations in needles; shading had the greater effect. Arginine and proline concentrations increased most in needles of drought-stressed seedlings and remained high in unshaded seedlings recovering from drought 48 h after rewatering. Arginine and glutamine increased most in the shaded seedlings, which did not survive severe drought. The large increase in arginine in both drought-stressed and severely shaded seedlings suggests that sequestering and storage of ammonia are important when stress reduces carbon fixation.  相似文献   

9.
Procedures are described for the rapid and quantitative analysis of cyanobacterial heterocyst-type glycolipids (HGs) by normal-phase HPLC of their per-O-benzoylated derivatives. Total lipids are obtained from 1 ml of nitrogen-fixing cyanobacterial culture by triplicate extraction with chloroform/methanol, 1/1 (v/v), and the HGs are isolated from other complex lipids by preparative silica gel TLC. A C18 solid-phase extraction cartridge is used to ensure quantitative salt-free recovery of the HGs, and the purified glycolipids are then rendered uv-absorbing by a per-O-benzoylation derivatization reaction for which optimal conditions have been established. Derivatives are analyzed within 12 min on a 3-microns silica HPLC column using a linear gradient of 2-propanol in n-hexane and uv monitoring at 230 nm. The reaction product was also used to determine the relative proportions of the glucosyl and galactosyl epimers of individual members of this class of glycolipid.  相似文献   

10.
A method for the determination of rofecoxib in human plasma is described. After the addition of an internal standard, buffered (pH 5) plasma samples are extracted with hexane–methylene chloride (50:50, v/v). The extracts are evaporated to dryness and reconstituted in mobile phase. Upon exposure to UV light, the analyte was found to undergo a stilbene–phenanthrene-like photocyclization reaction with the resulting formation of a highly fluorescent species. Thus, the plasma extracts were analyzed via HPLC with post-column photochemical derivatization and fluorescence detection. The assay has been validated in the concentration range of 0.5–100 ng/ml using 1-ml samples. The method has been successfully utilized to support human clinical pharmacokinetic studies.  相似文献   

11.
A highly sensitive HPLC method for the determination of decarboxylated S-adenosylmethionine (dc-SAM) by fluorometric detection was developed. The reaction of dc-SAM and its analogs with chloroacetaldehyde leads to the corresponding 1,N6-etheno derivatives. These highly fluorescent derivatives were fully characterized through their proton nuclear magnetic resonance spectra and/or mass spectra. This derivatization procedure has been applied to the analysis of dc-SAM in rat and human urine. After a simple cation exchange column prepurification, the urine extracts were derivatized with chloroacetaldehyde and analyzed by reversed-phase HPLC with fluorometric detection. The method allowed the determination of subpicomole amounts of dc-SAM and was shown to be highly reproducible with the use of decarboxylated S-adenosylethionine as internal standard. The application of the method to the analysis of urine of rats treated with MDL 72175, a potent ornithine decarboxylase inhibitor, showed that the dc-SAM levels increased in a dose-related fashion.  相似文献   

12.
A novel procedure to specifically quantify low amounts of pipecolic acid and structurally related compounds in several types of biological materials has been characterized. From crude extracts of various types of biological material, the first step was to clear all low-molecular-weight compounds containing primary amino groups by a treatment of nitrous acid. Using a microwave-assisted reaction, the remaining substances containing secondary amino groups were then derivatized with ninhydrin and made soluble in glacial acetic acid. The derivatives produced were resolved by reverse-phase HPLC and detected by spectrophotometry at 570nm. This procedure allowed more rapid determination of pipecolic acid since microwave heating shortened the time needed for derivatization compared with heating at 95 degrees C in a water bath. The complete analysis of the chromogens for pipecolic acid and related substances was achieved in 20min. Under such conditions, the detection threshold for pipecolic acid was about 20pmol. The suitability of the technique was assessed in various biological matrices known to contain significant amounts of this amino acid. The data obtained are in accordance with those available in the literature. To our knowledge, this is the first method using the ninhydrin reaction in a precolumn, microwave-assisted derivatization procedure for detection and determination of heterocyclic alpha-amino acids.  相似文献   

13.
Melatonin, the chief secretory product of the vertebrate pineal gland is suspected to be a ubiquitous molecule principally involved in the transduction of photoperiodic information. Besides vertebrates, melatonin has been detected throughout phylogeny in numerous non-vertebrate taxa. In the present study, the occurrence of melatonin in Antarctic krill Euphausia superba and its possible role in mediating seasonal metabolic changes was evaluated. Melatonin was quantified by enzyme linked immunosorbent assay (ELISA) in high performance liquid chromatography (HPLC) purified extracts of eyestalks and hemolymph of krill sampled in the Lazarev Sea during the Antarctic winter and summer. In addition, oxygen uptake rates and the activities of the metabolic enzyme malate dehydrogenase (MDH) were recorded to assess the metabolic status of krill. Validation of melatonin measurements was carried out on the basis of three different extraction methods with parallel determination of melatonin by ELISA in crude extracts and in HPLC purified extracts, and after derivatization of melatonin under alkaline conditions in the presence of hydrogen peroxide. A significantly higher respiration rate and MDH activity was found in summer krill than in winter krill indicating that krill was in a state of reduced metabolic activity during winter. However, neither during winter nor during summer there were detectable melatonin concentrations in the visual system or hemolymph of krill. Based on these results, we question a mediating role of melatonin in the control of seasonal metabolic changes in Antarctic krill.  相似文献   

14.
Hopanoid lipids have been discovered recently in a number of nitrogen-fixing soil bacteria and in Bradyrhizobium bacteria which fix nitrogen in association with legume plants. We report here an investigation of the hopanoid content in an additional number of soil bacteria capable of living in close association with plants. Of the strains investigated, hopanoids were discovered in phototrophic, nitrogen-fixing bacteria and in an extended number of Bradyrhizobium strains. Strains in which hopanoids so far have not been found belong to the following genera: Rhizobium, Sinorhizobium, Phyllobacterium, Agrobacterium, and Azoarcus. To address the function of hopanoids in Bradyrhizobium, we cloned the gene coding for a key enzyme of hopanoid biosynthesis, the squalene-hopene cyclase, and expressed the gene in E. coli. The recombinant enzyme catalyzed in vitro the cyclization of squalene to hopanoid derivatives.Abbreviations SHC squalene-hopene cyclase - shc squalene-hopene cyclase gene  相似文献   

15.
A method for the separation and quantification of the levels of alanopine and strombine in neutralized, perchloric acid extracts of tissues of marine invertebrates is presented. The method is based on high-performance liquid chromatographic (HPLC) separation, postcolumn derivatization using o-phthaldialdehyde and sodium hypochlorite, and subsequent fluorometric detection. Isocratic separation results in the rapid elution of alanopine and strombine, with elution times of 4.7 and 5.4 min, respectively. The sensitivity of this method is in the range 50-250 pmol. However, the fluorometric detection approach provides the capability for even greater sensitivity.  相似文献   

16.
Gibberellin (GA)-like substances were analyzed in extracts from cotyledons and phloem exudate collected from cotyledons in photoinduced and vegetative seedlings of the short-day plant Pharbitis nil Chois. var. Violet, using high performance liquid chromatography (HPLC) and the dwarf rice bioassay, to see whether any specific GA-like substances were transported from the photoinduced cotyledons via phloem. Cotyledon extracts exhibited five peaks of free GA-like activity in HPLC, whereas only one or two active peaks were detected in phloem exudate extracts. The level of free GA-like activity was considerably lower in phloem exudate than in the cotyledons. In five out of six analyses of cotyledons and phloem exudate, there were substantially higher levels of free GA-like substances in photoinduced plants. Conjugated GA-like substances were present in much higher levels than free GA-like substances in the cotyledon extracts but the levels were not influenced by daylength. In phloem exudate extracts there was no conjugated GA-like substances. The free GA-like substances that are transported via phloem cochromatographed with GA5/20 and GA19 on HPLC. These were significantly higher in photoinduced plants and thus could have some influence on the photoperiodically-induced flowering in P. nil.  相似文献   

17.
1. Circumoesophageal ganglia and foot muscle of the garden snail. Helix aspersa, were subjected to immunocytochemistry using antisera to the tachykinins, substance P (SP), neurokinin A (NKA), kassinin (KAS) and eledoisin (ELE).2. Immunoreactivity in neuronal somata and fibres was detected only with the SP antiserum.3. SP and NKA radioimmunoassays were performed on extracts of Circumoesophageal ganglia. In common with immunocytochemistry, immunoreactivity was only detected with the SP antiserum.4. Gel permeation chromatography of extracts resolved a single peak of immunoreactivity eluting slightly later than synthetic mammalian SP. Reverse-phase HPLC of immunoreactive fractions resolved two immunoreactive peptides representing oxidised and reduced forms of a single peptide.5. These data suggest that the nervous system of H. aspersa contains a single tachykinin with C-tenninal structural characteristics similar to mammalian SP.  相似文献   

18.
Abstract A gas-liquid chromatographic method for the quantitative determination of hopanoids with an elongated side chain was developed. After extraction of lipids from Bacillus acidocaldarius , periodate oxidation, reduction and acetylation the 1-hydroxyethane-29-hopane acetate was quantitated by comparison with 1-octadecanyl-glycerol ether. The contents of extended hopanoids increased strongly with increasing temperature and moderately with decreasing pH. Hopene showed no significant alteration with temperature and pH.  相似文献   

19.
Geobacter metallireducens and G. sulfurreducens have been classified as strictly anaerobic bacteria which grow and thrive in subsurface and sediment environments. Hopanoids are pentacyclic triterpenoid lipids and are important for bacterial membrane stability and functioning. Hopanoids predominantly occur in aerobically growing bacteria of oxic environments. They rarely have been found in facultatively anaerobic bacteria and, to date, not at all in strict anaerobes. Our research shows that anaerobically grown G. metallireducens and G. sulfurreducens bacteria contain a range of hopanoid lipids, such as diploptene (i.e. hop-22(29)-ene) and hop-21-ene, and more complex, elongated hopanoids. In geological formations, diagenetic derivatives of hopanoids are widely used as biomarkers and are recognized as molecular fossils of bacterial origin. To date, these biomarkers have largely been interpreted as those derived from ancient oxic environments. Our findings presented here suggest that this interpretation needs to be re-evaluated. In addition to the origin in oxic environments, 'geohopanoids' may originate from ancient anaerobic environments as well.  相似文献   

20.
Analysis of the branched, medium-chain fatty acid anticonvulsant, valproic acid, and its unsaturated metabolites by gas chromatography with electron-capture detection suffered from background interference caused by the derivatizing reagent pentafluorobenzyl bromide. Background was reduced by keeping the derivatization anhydrous, using an inert solvent, minimizing the amount of pentafluorobenzyl bromide, using hypernucleophilic bases and displacing the derivatization solvent with isooctane. However, these strategies proved difficult to reproduce. Post-derivatization clean-up with HPLC was much more reliable and provided sufficient sensitivity for the analysis of extracts of plasma and brain homogenate. The assay was validated for plasma and brain samples from humans, rats and mice.  相似文献   

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