A reevaluation of the chemotactic potency of leukotriene B4 (LTB4)

The chemotactic potency of leukotriene B4 (LTB4) was reevaluated based on an improved purification procedure which combines reversed phase and straight phase high pressure liquid chromatography (RP-HPLC and SP-HPLC). Socalled LTB4 isomer III prepared by RP-HPLC contains two double oxygenated 5,12-dihydroxy acids in addition to LTB4. On a molar basis, the chemotactic activity of LTB4 repurified by SP-HPLC was far greater than that of the other two 5,12-dihydroxy acids and comparable to that of formyl-methionyl-leucyl-phenylalanine (fMLP). The chemotactic activity of LTB4 isomer III is dependent upon the relative concentrations of the double oxygenated 5,12-dihydroxy acids and LTB4. Further purification of peak III by SP-HPLC is required before assessing the biologic activity of LTB4.     

Separation by high-performance liquid chromatography of alpha- and beta-amino acids: application to assays of lysine 2,3-aminomutase and leucine 2,3-aminomutase

Several pairs of alpha- and beta-amino acids were well separated as the ortho-phthalaldehyde derivatives by reversed phase HPLC. These included alpha- and beta-lysine, leucine, tyrosine, serine, aminoisobutyric acid, and beta-hydroxyvaline and its positional isomer. The separation was applicable to assays of lysine 2,3-aminomutase and leucine 2,3-aminomutase. However, for unknown reasons, no leucine 2,3-aminomutase activity could be found in rat liver homogenate.     

Binding of cytochrome P450 monooxygenase and lipoxygenase pathway products by heart fatty acid-binding protein

Arachidonic acid metabolism by lipoxygenases and cytochrome P450 monooxygenases produces regioisomeric hydroperoxyeicosatetraenoic acids (HPETEs), hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs), which serve as components of cell signaling cascades. Intracellular fatty acid-binding proteins (FABPs) may differentially bind these nonprostanoid oxygenated fatty acids, thus modulating their metabolism and activities. Vascular cells, which express heart FABP (H-FABP), utilize oxygenated fatty acids for regulation of vascular tone. Therefore, the relative affinities of H-FABP for several isomeric series of these compounds were measured by fluorescent displacement of 1-anilinonaphthalene-8-sulfonic acid (ANS). In general, H-FABP rank order affinities (arachidonic acid > EETs > HETEs > DHETs) paralleled reversed-phase high-performance liquid chromatography retention times, indicating that the differences in H-FABP affinity were determined largely by polarity. H-FABP displayed a similar rank order of affinity for compounds derived from linoleic acid. H-FABP affinity for 20-HETE [apparent dissociation constant (K(d)') of 0.44 microM] was much greater than expected from its polarity, indicating unique binding interactions for this HETE. H-FABP affinity for 5,6-EET and 11,12-EET (K(d)' of approximately 0.4 microM) was approximately 20-fold greater than for DHETs (K(d)' of approximately 8 microM). The homologous proteins, liver FABP and intestinal FABP, also displayed selective affinity for EET versus DHET. Thus, FABP binding of EETs may facilitate their intracellular retention whereas the lack of FABP affinity for DHETs may partially explain their release from cells. The affinity of H-FABP for EETs suggests that this family of intracellular proteins may modulate the metabolism, activities, and targeting of these potent eicosanoid biomediators.     

Partial purification of hatching activity for Globodera rostochiensis from potato root diffusate

The potential of several chromatographic methods for isolating hatching factors for potato cyst nematodes from potato root diffusate was investigated using a bioassay based on emergence of juveniles from cysts. Gel filtration provided an overall estimate of molecular weight of 437 Da for the hatching activity and ion exchange chromatography indicated that at least 60% of the recovered activity was anionic in nature. Material less polar than the hatching activity could be removed by passing potato root diffusate through a reversed-phase Sep-Pak C₁₈ cartridge and the elutant showed 83.3 ± 4.4% (mean of 32 cysts) of the initial hatching activity. High performance liquid chromatography (HPLC) with a reversed phase, C₁₈ column and gradient elution (0–80% CH₃OH in water) confirmed that much of the hatching activity was polar and that it was not retained by this method of separation. A weak anion exchange resin achieved slight retention of much of the hatching activity and an ion pairing reagent lowered the polarity sufficiently to allow some retention in subsequent reversed phase HPLC on a C_IS column. Both ion exchange and ion pairing HPLC suggested that hatching activity was not chromatographed as a single compound and indicated that fractions able to influence the nucleolus of the nucleus within the dorsal pharyngeal gland cell did not always show hatching activity.     

高效液相荧光法测定大鼠脑内氨基酸类神经递质方法的改良

目的 改良测定大鼠脑组织氨基酸类神经递质的反相高效液相色谱荧光法.方法 改良使用磷酸盐-甲醇-乙腈作为流动相,反相高效液相色谱洗脱,高丝氨酸作为内标,邻苯二甲醛柱前衍生和荧光检测器,检测大鼠大脑皮质、海马、纹状体、中脑、小脑和下丘脑6个脑区中天冬氨酸(Asp)、谷氨酸(Glu)、谷氨酰胺(G1n)、甘氨酸(Gly)、γ-氨基丁酸(GABA)和牛磺酸(Tau)6种氨基酸类神经递质含量.结果 6种氨基酸在20 min内洗脱完全,分离效果良好;在6.25～ 400 μmol/L浓度范围有较好的线性关系,其相关系数不低于0.99;6种氨基酸日内试验精密度范围为1.38％ ～7.59％;日间试验精密度为2.7％～8.68％;6种氨基酸回收率不低于80％.结论 改良后的反相高效液相色谱荧光法灵敏度较高、重复性好,能有效分离检测大鼠脑组织分区中氨基酸类神经递质含量.     

Detecting groups of coevolving positions in a molecule: a clustering approach

Background

Although the patterns of co-substitutions in RNA is now well characterized, detection of coevolving positions in proteins remains a difficult task. It has been recognized that the signal is typically weak, due to the fact that (i) amino-acid are characterized by various biochemical properties, so that distinct amino acids changes are not functionally equivalent, and (ii) a given mutation can be compensated by more than one mutation, at more than one position.

Results

We present a new method based on phylogenetic substitution mapping. The two above-mentioned problems are addressed by (i) the introduction of a weighted mapping, which accounts for the biochemical effects (volume, polarity, charge) of amino-acid changes, (ii) the use of a clustering approach to detect groups of coevolving sites of virtually any size, and (iii) the distinction between biochemical compensation and other coevolutionary mechanisms. We apply this methodology to a previously studied data set of bacterial ribosomal RNA, and to three protein data sets (myoglobin of vertebrates, S-locus Receptor Kinase and Methionine Amino-Peptidase).

Conclusion

We succeed in detecting groups of sites which significantly depart the null hypothesis of independence. Group sizes range from pairs to groups of size ? 10, depending on the substitution weights used. The structural and functional relevance of these groups of sites are assessed, and the various evolutionary processes potentially generating correlated substitution patterns are discussed.     

Identification of very long chain fatty acids from sugar cane wax by atmospheric pressure chemical ionization liquid chromatography-mass spectroscopy

A method is described for the enrichment of very long chain fatty acids (VLCFAs) from total fatty acids of sugar cane wax and their identification as picolinyl esters by means of liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-MS/APCI). The method is based on the use of preparative reversed phase HPLC of 100 mg amounts and their subsequent identification by microbore APCI LC-MS. The combination of these two techniques was used to identify unusual saturated VLCFAs up to C(50).     

Regulation of hepatic cholesterol biosynthesis. Effects of a cytochrome P-450 inhibitor on the formation and metabolism of oxygenated sterol products of lanosterol.

The involvement of oxygenated cholesterol precursors in the regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was studied by examining the effect of ketoconazole on the metabolism of mevalonic acid, lanosterol and the lanosterol metabolites, lanost-8-ene-3 beta,32-diol,3 beta-hydroxylanost-8-en-32-al and 4,4-dimethylcholesta-8,14-dien-3 beta-ol, in liver subcellular fractions and hepatocyte cultures. Inhibition of cholesterol synthesis from mevalonate by ketoconazole at concentrations up to 30 microM was due exclusively to a suppression of cytochrome P-450LDM (LDM = lanosterol demethylase) activity, resulting in a decreased rate of lanosterol 14 alpha-demethylation. No enzyme after the 14 alpha-demethylase step was affected. When [14C]mevalonate was the cholesterol precursor, inhibition of cytochrome P450LDM was accompanied by the accumulation of several labelled oxygenated sterols, quantitatively the most important of which was the C-32 aldehyde derivative of lanosterol. There was no accumulation of the 24,25-oxide derivative of lanosterol, nor of the C-32 alcohol. Under these conditions the activity of HMG-CoA reductase declined. The C-32 aldehyde accumulated to a far greater extent when lanost-8-ene-3 beta,32-diol rather than mevalonate was used as the cholesterol precursor in the presence of ketoconazole. With both precursors, this accumulation was reversed at higher concentrations of ketoconazole in liver subcellular fractions. A similar reversal was not observed in hepatocyte cultures.     

Motor neuron pathfinding following rhombomere reversals in the chick embryo hindbrain.

Motor neurons are segmentally organised in the developing chick hindbrain, with groups of neurons occupying pairs of hindbrain segments or rhombomeres. The branchiomotor nucleus of the trigeminal nerve occupies rhombomeres 2 and 3 (r2 and r3), that of the facial nerve r4 and r5, and that of the glossopharyngeal nerve r6 and r7. Branchiomotor neuron cell bodies lie within the basal plate, forming columns on either side of the ventral midline floor plate. Axons originating in rhombomeres 2, 4 and 6 grow laterally (dorsally) towards the exit points located in the alar plates of these rhombomeres, while axons originating in odd-numbered rhombomeres 3 and 5 grow laterally and then rostrally, crossing a rhombomere boundary to reach their exit point. Examination of the trajectories of motor axons in odd-numbered segments at late stages of development (19-25) showed stereotyped pathways, in which axons grew laterally before making a sharp turn rostrally. During the initial phase of outgrowth (stage 14-15), however, axons had meandering courses and did not grow in a directed fashion towards their exit point. When r3 or r5 was transplanted with reversed rostrocaudal polarity prior to motor axon outgrowth, the majority of axons grew to their appropriate, rostral exit point, despite the inverted neuroepithelial polarity. In r3 reversals, however, there was a considerable increase in the normally small number of axons that grew out via the caudal, r4 exit point. These findings are discussed with relevance to the factors involved in motor neuron specification and axon outgrowth in the developing hindbrain.     

Identification of very long chain unsaturated fatty acids from Ximenia oil by atmospheric pressure chemical ionization liquid chromatography-mass spectroscopy

A method is described for the enrichment of very long chain unsaturated fatty acids from total fatty acids of Ximenia oil and their identification as picolinyl esters by means of liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-MS/APCI). The method is based on the use of preparative reversed phase HPLC and their subsequent identification by microbore LC-MS/APCI. The combination of these two techniques was used to identify unusual unsaturated VLCFAs up to tetracontenoic acid. All four positional isomers of tetratriacontenoic acid were also synthesized to unambiguously confirm their structure.     

Inhibitors of sterol synthesis. Chromatography of acetate derivatives of oxygenated sterols

The separation of the acetate derivatives of a number of oxygenated sterols was achieved by medium pressure liquid chromatography on silica gel columns and by normal and reversed phase high performance liquid chromatography. We have explored the application of these chromatographic systems for the analysis of oxygenated sterols of plasma samples from two normal human subjects. The addition of highly purified [14C]cholesterol to plasma permitted the detection and quantitation of oxygenated sterols formed by autoxidation of cholesterol during processing of the samples. Special attempts to suppress autoxidation of cholesterol included the use of an all-glass closed system for saponification and extraction under argon followed by rapid removal of cholesterol from the polar sterols by reversed phase medium pressure liquid chromatography. Chromatographic analyses of the [3H]acetate derivatives of the polar sterols provided a sensitive approach for the detection and quantitation of the individual oxygenated sterols. Oxygenated sterols detected in plasma included cholest-5-ene-3 beta,26-diol, (24S)-cholest-5-ene-3 beta,24-diol, and cholest-5-ene-3 beta,7 alpha-diol. After correction for their formation by autoxidation of cholesterol during processing of the samples, very little or none of the following sterols were observed: cholest-5-ene-3 beta,7 beta-diol, 5 alpha,6 alpha-epoxy-cholestan-3 beta-ol, 5 beta,6 beta-epoxy-cholestan-3 beta-ol, and cholestane- 3 beta, 5 alpha,6 beta-triol, and the 25-hydroxy, 22R-hydroxy, 21-hydroxy, 20 alpha-hydroxy, and 19-hydroxy derivatives of cholesterol.     

Potential correlation between carboxylic acid metabolites in Biomphalaria alexandrina snails after exposure to Schistosoma mansoni infection

Carboxylic acids play an important role in both aerobic and anaerobic metabolic pathways of both the snail and the parasite. Monitoring the effects of infection by schistosome on Biomphalaria alexandrina carboxylic acids metabolic profiles represents a promising additional source of information about the state of metabolic system. We separated and quantified pyruvic, fumaric, malic, oxalic, and acetic acids using ion-suppression reversed-phase high performance liquid chromatography (HPLC) to detect correlations between these acids in both hemolymph and digestive gland gonad complex (DGG's) samples in a total of 300 B. alexandrina snails (150 infected and 150 controls) at different stages of infection. The results showed that the majority of metabolite pairs did not show significant correlations. However, some high correlations were found between the studied acids within the control group but not in other groups. More striking was the existence of reversed correlations between the same acids at different stages of infection. Some possible explanations of the underlying mechanisms were discussed. Ultimately, however, further data are required for resolving the responsible regulatory events. These findings highlight the potential of metabolomics as a novel approach for fundamental investigations of host-pathogen interactions as well as disease surveillance and control.     

Applanoxidic acids A, B, C and D, biologically active tetracyclic triterpenes from Ganoderma applanatum

Four novel polyoxygenated lanostanoid triterpenes, named applanoxidic acids A, B, C, and D, have been isolated from an Indonesian tropical fungus, Ganoderma applanatum. Their structures were determined by spectrocopic and chemical methods. Biological activity as an anti-tumour promoter was observed for these components.     

Rapid analysis of O-acetylated neuraminic acids by matrix assisted laser desorption/ionization time-of-flight mass spectrometry

N-Acetylneuraminic acid (a sialic acid) occurs mainly as a terminal substituent of oligosaccharides of glycoconjugates. Derivatives of neuraminic acid occur widely, substituted in the amino and hydroxy side chains, as well in the C-9 carbon skeleton. These derivatives are responsible for specific functions of sialic acids during cell-cell, cell-substrate, or cell-virus interactions. The study of O-acetylated neuraminic acids is difficult, because only small amounts are extractable from natural sources and they are generally unstable to acids and bases. We report a new method for the rapid analysis of O-acetylated neuraminic acids, using a combination of reversed phase HPLC and MALDI-TOF mass spectrometry. A mixture of neuraminic acids from bovine submaxillary gland mucins was analysed, as well as neuraminic acids variously substituted in the amino and hydroxy side chains with acetyl and glycolyl groups, respectively. © 1998 Rapid Science Ltd     

High pressure liquid chromatographic analysis of conjugated bile acids in human bile: simultaneous resolution of sulfated and unsulfated lithocholyl amidates and the common conjugated bile acids

A reversed phase high pressure liquid chromatography (HPLC) system capable of simultaneously separating four lithocholyl species (sulfated and unsulfated forms of lithocholylglycine and lithocholyltaurine) as well as the eight other major conjugated bile acids present in human bile is described. The system uses a C18 octadecylsilane column and isocratic elution with methanol phosphate buffer, pH 5.35. Relative bile acid concentration is determined by absorbance at 200 nm. Retention times relative to chenodeoxycholylglycine are reported for the four lithocholic acid forms, the glycine and taurine amidate of the four major bile acids present in human bile (cholic, chenodeoxycholic, ursodeoxycholic, and deoxycholic), and for their corresponding unconjugated forms. Retention times are also reported for the glycine and taurine amidates as well as the unconjugated form of the C23 norderivatives of these bile acids. Maximal absorbance of bile acid amidates is at 200 nm and is very similar for the (unsulfated) glycine and taurine amidates. Sulfated lithocholyl amidates exhibit molar absorptivities at 200 nm which are 1.4 times greater than that of non-sulfated lithocholyl amidates. Unconjugated bile acid absorbance at 200 nm or 210 nm is 20 to 30 times less than that of corresponding peptide conjugates. The method has been applied to samples of gallbladder bile obtained from 14 healthy subjects to define the pattern of conjugated bile acids present in human bile.     

Oxygenation of arachidonic acid by hepatic microsomes of the rabbit. Mechanism of biosynthesis of two vicinal dihydroxyeicosatrienoic acids

[1-14C] Arachidonic (eicosatetraenoic) acid was incubated at 37 degrees C for 15 min with rabbit liver microsomes fortified with NADPH (1 mM). The products were purified by high-pressure liquid chromatography (HPLC) and analyzed by gas chromatography-mass spectrometry. Based on polarity on reversed phase HPLC, the metabolites could be divided into three groups. The major metabolites of lowest polarity were 19- and 20-hydroxyarachidonic acid and 19-oxoarachidonic acid. The major metabolites of medium polarity were two diols, 14,15-dihydroxy-5,-8,11-eicosatrienoic acid and 11,12-dihydroxy-5,8,14-eicosatrienoic acid. Microsomal incubation under atmospheric isotopic oxygen led to incorporation of only one 18O molecule in each diol, indicating that the diols could originate from breakdown of 14(15)-oxido-5,8,11-eicosatrienoic acid and 11(12)-oxido-5,8,14-eicosatrienoic acid, respectively. Major metabolites in the most polar group were 14,15,19- and 14,15,20-trihydroxy-5,8,11-eicosatrienoic acid. 11,12,19- and 11,12,20-trihydroxy-5,8,14-eicosatrienoic acid and 11,12-dihydroxy-19-oxo-5,8,-14-eicosatrienonic acid. About 0.5% of exogenous radioactively labelled arachidonic was covalently bound to microsomal proteins. The metabolites and the protein-bound products were formed in considerably smaller amounts by non-fortified microsomes. Carbon monoxide inhibited this pathway of arachidonic acid metabolism, indicating that these reactions might be catalyzed by the cytochrome P-450-linked monooxygenase systems.     

Use of reversed phase HP liquid chromatography to assay conversion of N-acylglycines to primary fatty acid amides by peptidylglycine-alpha-amidating monooxygenase

Primary fatty acid amides (R-CO-NH2) and N-acylglycines (R-CO-NH-CH2-COOH) are classes of compounds that have only recently been isolated and characterized from biological sources. Key questions remain regarding how these lipid amides are produced and degraded in biological systems. Relative to the fatty acids, little has been done to develop methods to separate and quantify the fatty acid amides and N-acylglycines. We describe reversed phase HPLC methods for the separation of C2-C12 primary fatty acid amides and N-acylglycines and also C12-C22 fatty acid amides. Separation within each class occurs primarily on the basis of simple interactions between the acyl chain and the chromatographic stationary phase, but the polar headgroups on these and related fatty acids and N-acylethanolamides modulate the absolute retention in reversed phase mode. We use these methods to measure the enzyme-mediated, two-step conversion of N-octanoylglycine to octanoamide.     

Analysis of the stereochemistry of lipoxygenase-derived hydroxypolyenoic fatty acids by means of chiral phase high-pressure liquid chromatography

A chiral phase HPLC method was developed for the simultaneous determination of the positional and optical isomers of the lipoxygenase-derived hydroxypolyenoic fatty acids. With a Bakerbond chiral phase HPLC column (dinitrobenzoyl phenylglycine as chiral phase) the positional and optical isomers of the reduced dioxygenation products (by triphenylphosphine or borohydride) of linoleic acid and arachidonic acid were separated after methylation of the carboxylic groups. No cumbersome chemical derivatization such as conversion to a diastereomer was necessary. As compared with the methods used up till now chiral phase HPLC proved to be simpler and more sensitive. About 10 pmol of hydroxy fatty acids suffice for an analysis. The chiral phase HPLC can be used for the preparative separation of the optical antipodes of the lipoxygenase products. An optical purity of more than 90% can be reached in one preparative run. The method was applied to the determination of the stereochemistry of the dioxygenation products of polyenoic fatty acids formed by the lipoxygenases from soybeans, reticulocytes, pea seeds (isoenzyme I and II), tomato fruits, by the quasilipoxygenase activity of hemoglobin, and by the methylene blue-mediated photooxidation of arachidonic acid.     

Prediction of peptide retention volumes in gradient reversed phase HPLC

A method of computation of retention volumes of linear peptides of known composition that contain no more than 25 amino acids in gradient reversed phase HPLC was developed. Tha method is suitable for various acetonitrile gradient profiles. The calculations were carried out on the basis of a statistical model, the parameters of which were experimental dependences of the retention of individual amino acids on acetonitrile concentration. The method developed was used to predict the chromatographic behavior of 34 peptides in four different acetonitrile gradients. The correlation coefficients between the predicted and experimental retention volumes were more than 0.9, and the average relative error of prediction was less than 15%.     

Beta-substituted ethylsulfonyl chlorides as 5'-OH protecting groups in nucleoside and nucleotide chemistry

Three beta-substituted ethylsulfonyl chlorides 1-3 were synthesized and their usefulness as 5'-OH protecting groups for oligonucleotide synthesis studied. The formation of the corresponding 5'-beta-R-ethyl-sulfonic acid esters was investigated in respect to fast and complete capping. The 5'-sulfonates 4-9 were prepared and the deprotection was studied using reversed phase HPLC as the analytical tool.