Occurrence, identification, and bacterial mutagenicity of heterocyclic amines in cooked food

Potent mutagenic activity in Salmonella bacteria has been reported in cooked foods in numerous laboratories worldwide. Determining the human risk from exposure to these biologically active compounds in our diet requires genotoxic and carcinogenic evaluation of the chemicals coupled with determination of the dose consumed. Thus, knowledge of the exact structure of the mutagens present in the food and enough synthesized material for biological assessment are essential for this evaluation. To reach this goal, isolation of these compounds requires the Ames/Salmonella assay to guide the purification and identification process. Mass and NMR spectrometry are used to identify the isolated compounds. Finally, these findings are followed by synthesis of the exact isomer. The predominant class of mutagens found in cooked foods of the western diet are amino-imidazo-quinoxalines, amino-imidazo-pyridines and amino-imidazo-quinolines, collectively called amino-imidazoazaarenes (AIAs). Mass amounts of these specific compounds range from less than 1 to 70 ng/g of meat. The mutagens are formed from the heating of natural precursors (creatinine, amino acids, and possibly sugars) in the food. These AIAs are some of the most potent mutagens ever tested in Salmonella bacteria with the number and position of methyl groups having an important influence on the mutagenic activity.     

Identification and field optimisation of the female sex pheromone of the rice leaffolder,Cnaphalocrocis medinalis in India

Analysis of ovipositor washings from virgin femaleCnaphalocrocis medinalis Guenée (Lepidoptera: Pyralidae) of Indian origin by linked gas chromatography and electroantennography indicated the presence of three electrophysiologically-active compounds. These were identified on the basis of their gas chromatographic retention times and mass spectra as (Z)-11-hexadecenyl acetate, (Z)-13-octadecenyl acetate and (Z)-13-octadecen-1-ol with (Z)-13-octadecenyl acetate present in amounts of between 0.25 and 1.5 ng per ovipositor and the other two components at less than 10% of this. Trace quantities of octadecyl acetate were identified by mass spectrometry but no electroantennographic responses were observed to this compound. Field trials conducted with a range of blends of the three electrophysiologically-active compounds showed that blends containing between 5% and 30% (Z)-11-hexadecenyl acetate in (Z)-13-octadecenyl acetate dispensed from either white rubber septa or polythene vials were more attractive to male moths than a virgin female moth. Addition of (Z)-13-octadecen-1-ol reduced attractiveness to male moths in the blends and concentrations tested.     

Uniform 13C isotope labeling of proteins with sodium acetate for NMR studies: application to human carbonic anhydrase II

Uniform double labeling of proteins for NMR studies can be prohibitively expensive, even with an efficient expression and purification scheme, due largely to the high cost of [13C6, 99%]glucose. We demonstrate here that uniformly (greater than 95%) 13C and 15N double-labeled proteins can be prepared for NMR structure/function studies by growing cells in defined media containing sodium [1,2-13C2, 99%]acetate as the sole carbon source and [15N, 99%]ammonium chloride as the sole nitrogen source. In addition, we demonstrate that this labeling scheme can be extended to include uniform carbon isotope labeling to any desired level (below 50%) by utilizing media containing equal amounts of sodium [1-13C, 99%]acetate and sodium [2-13C, 99%]acetate in conjunction with unlabeled sodium acetate. This technique is less labor intensive and more straightforward than labeling using isotope-enriched algal hydrolysates. These labeling schemes have been used to successfully prepare NMR quantities of isotopically enriched human carbonic anhydrase II. The activity and the 1H NMR spectra of the protein labeled by this technique are the same as those obtained from the protein produced from media containing labeled glucose; however, the cost of the sodium [1,2-13C2, 99%]acetate growth media is considerably less than the cost of the [13C6, 99%]glucose growth media. We report here the first published 13C and 15N NMR spectra of human carbonic anhydrase II as an important step leading to the assignment of this 29-kDa zinc metalloenzyme.     

Volatiles from rhizomes of Rhodiola rosea L

Terpenes and aroma volatiles from rhizomes of Rhodiola rosea L. from Norway have been isolated by both steam distillation and headspace solid-phase micro-extraction coupled with gas chromatography and mass spectrometry analysis. The dried rhizomes contained 0.05% essential oil with the main chemical classes: monoterpene hydrocarbons (25.40%), monoterpene alcohols (23.61%) and straight chain aliphatic alcohols (37.54%). n-Decanol (30.38%), geraniol (12.49%) and 1,4-p-menthadien-7-ol (5.10%) were the most abundant volatiles detected in the essential oil, and a total of 86 compounds were identified in both the SD and HS-SPME samples. Geraniol was identified as the most important rose-like odour compound besides geranyl formate, geranyl acetate, benzyl alcohol and phenylethyl alcohol. Floral notes such as linalool and its oxides, nonanal, decanal, nerol and cinnamyl alcohol highlight the flowery scent of rose root rhizomes.     

New principles of ion exchange preparative chromatography and their applications for isolation, purification and super purification of antibiotics]

New types of structurally segregated heteronetwork biosorbents with given parameters of heterogeneity and porosity have been developed. Physico-chemical characteristics of the biosorbents on the basis of which one can predict optimal structures of ion exchangers to be used in preparative chromatography of biologically active compounds were studied. A new principle of sequential displacement of ions of organic compounds, in particular antibiotics, adsorbed on selective biosorbents with a high adsorption capacity was developed, which enables purification and superpurification of the desired compound. The method is based on the effect of small thermodynamic shifts in physico-chemical parameters of the elution system, which results in preparative separation of substances with close properties and in purification of the desired compound from microadmixtures. The "small shift effect" is realized in the case of limiting thermodynamic and kinetic parameters of heterogeneous mass exchange and with biosorbents possessing a highly selective adsoprtion capacity.     

epi-Cubebanes from Solidago canadensis

GC-MS of the essential oil prepared by hydrodistillation of the green parts of a specimen of Solidago canadensis collected near Katowice, Poland, revealed two new sesquiterpene hydrocarbons. Their EI mass spectra resembled the mass spectrum of beta-ylangene (1) but the retention indices of the new compounds differed markedly from this known compound. After isolation of the new compounds by preparative GC their investigation by one- and two-dimensional NMR techniques resulted in the identification of 6-epi-alpha-cubebene (2) (minor constituent, 1.5%) and 6-epi-beta-cubebene (3) (major constituent, 20.5%).     

Application of LC/MS systems to structural characterization of flavonoid glycoconjugates

Mass spectrometry is currently one of the most versatile and sensitive instrumental methods applied to structural characterization of plant secondary metabolite mixtures isolated from biological material. Plant tissues contain thousands of natural products fulfilling different roles in plant physiology and biochemistry. These natural products have various biological activities in respect to plants synthesizing them, in their responses to different environmental stresses and are also active principles of food supplements and pharmaceuticals of plant origin. Flavonoids constitute a large group of phenolic secondary metabolites and are probably produced by all terrestrial plant species. More than 9000 glycoconjugates of flavonoids are presently known in the plant kingdom and more than 50 of them may be present in a single plant. For this reason methods of identification and analysis of this group of compounds are particularly demanded. Due to a high number of metabolites present in plant extracts, the isolation and purification of most compounds in amounts suitable for unambiguous characterization with NMR methods is often impossible. For these reasons elaboration of strategies for sufficiently precise structural characterization of compounds present in mixture samples is currently a primary task. Mass spectrometry, thanks to application of different physical phenomena for ionization, separation and detection of analyzed molecules, became the method of choice among analytical methods applied for identification, structural characterization and quantitative analysis of the natural products. Methods of analysis of differently substituted flavonoids (O- and C-glycosides, differentiation of various oligosaccharidic substituents, detection of acylated compounds) are presented in the paper. A proper application of mass spectrometric methods in well-defined and strictly controlled technical parameters of analysis permits obtaining important structural information. Among others, recording collision induced dissociation mass spectra allows identification of compounds after comparison of the registered MS spectra with these present in the existing databases.     

Pathway for Ubiquinone Biosynthesis in Escherichia coli K-12: Gene-Enzyme Relationships and Intermediates

Seven ubiquinone-deficient mutants of Escherichia coli, each of which accumulates two phenolic precursors of ubiquinone, have been characterized, and the accumulated compounds have been identified. The mutants accumulate small quantities of 2-octaprenyl-6-methoxyphenol, which was isolated and characterized by nuclear magnetic resonance and mass spectrometry, and relatively large amounts of 2-octaprenylphenol, a compound previously identified from E. coli. They also accumulate small quantities of a compound identified as 2-(hydroxyoctaprenyl)phenol although the relevance of this compound to the biosynthesis of ubiquinone is not clear. The results of genetic analysis suggest that each of the mutants carries a mutation in a gene (designated ubiH) which is located at about min 56 on the E. coli chromosome and is co-transducible with the serA and lysB genes. Based on information obtained from this and previous studies with ubiquinone-deficient mutants, a pathway is proposed for ubiquinone biosynthesis in E. coli, and a summary of the known gene-enzyme relationships is given.     

珠子草化学成分的研究

利用大孔树脂吸附和多种柱层析方法,从珠子草中分离得到5个化合物,根据理化数据和波谱学等方法鉴定为柯里拉京(1)、芦丁(2)、isobubbialine(3)、丁二酸(4)和没食子酸(5)。根据2D-NMR修正了化合物3的部分碳信号归属,归属了化合物1的碳氢谱数据。     

Diurnal regulation of scent emission in rose flowers

Previous studies have shown diurnal oscillation of scent emission in rose flowers with a peak during the day (Helsper in Planta 207:88–95, 1998; Picone in Planta 219:468–478, 2004). Here, we studied the regulation of scent production and emission in Rosa hybrida cv. Fragrant Cloud during the daily cycle and focused on two terpenoid compounds, germacrene D and geranyl acetate, whose biosynthetic genes have been characterized by us previously. The emission of geranyl acetate oscillated during the daily light/dark cycle with a peak early in the light period. A similar daily fluctuation was found in the endogenous level of this compound and in the expression of its biosynthetic gene, alcohol acetyl transferase (RhAAT). The rhythmic expression of RhAAT continued under conditions of constant light or darkness, indicating regulation by the endogenous circadian clock. However, the accumulation and emission of geranyl acetate ceased under continuous light. Our results suggest that geranyl acetate production is limited by the level of its substrate geraniol, which is suppressed under constant light conditions. The emission of germacrene D also oscillated during the daily cycle with a peak early in the light period. However, the endogenous level of this compound and the expression of its biosynthetic gene germacrene D synthase (RhGDS) were constant throughout the day. The diurnal oscillation of germacrene D emission ceased under continuous light, suggesting direct regulation by light. Our results demonstrate the complexity of the diurnal regulation of scent emission: although the daily emission of most scent compounds is synchronized, various independently evolved mechanisms control the production, accumulation and release of different volatiles. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chemical composition of volatiles from cortical oleoresin of Pseudotsuga menziesii

Pseudotsuga menziesii cortical oleoresin was found to contain 1·7% of oxygenated terpenoids and compounds of similar volatility composed of linalool, methylsalicylate, bornyl acetate, citronellol, geranyl acetate, methylthymol, citronellyl acetate, terpinen-4-ol, borneol, isopulegol, anethole, terpinen-4-ol acetate, camphor, geraniol, neryl acetate, and nerol. Sesquiterpenoid hydrocarbons were low (only 0·07%) and contained sibirene and longifolene as main constituents, with β-caryophyllene, γ-muurolene, γ-cadinene (identified by IR), and 20 additional compounds in small amounts. p-Cymen-8-ene was identified in monoterpene hydrocarbon fraction.     

Development of a protocol for the semi-synthesis and purification of betaxanthins

A method for the analytical and semi-preparative chromatographic purification of betaxanthins is described together with an improved procedure for the semi-synthesis of these compounds from betalamic acid. Standard conditions for obtaining preparative amounts of betaxanthins free of the precursor amino acids are provided. Following this procedure, 14 pure betaxanthins were obtained with yields of up to 100%. A simple reversed-phase HPLC protocol for pigment identification and quantification is also provided. Calibration for betaxanthins is reported for the first time using the synthesised and purified pigments as standards. Structures were confirmed by UV-vis spectroscopy, HPLC retention times and electrospray ionization mass spectrometry. Betaxanthins can be obtained pure, and in sufficient amounts for further studies, which opens up new perspectives in the research and applications of these pigments.     

Preparative purification of pig red blood cell hexokinase type III using a new efficient chromatographic support.

In this paper we report the purification of pig erythrocyte hexokinase type III, at preparative level, using 52 liters of starting material (hemolysate). This was possible using a new efficient anion exchanger support, the Toyopearl DEAE 650 M which allows completely to change the strategy of removing hemoglobin from hemolysates, permitting to handle large amounts of starting material and reducing work would have required months using conventional anion exchanger supports, to only 2-3 days. Furthermore, we have tested the binding of other red blood cell enzymes to the Toyopearl DEAE 650 M, showing a wider potential use of this chromatographic support for their purification at a preparative level.     

Enzymatic hydrolysis of (chloromethyldimethylsilyl)-2-propenyl acetate isomers: atypic specificity of Candida antarctica lipase

Enzymatic hydrolysis of a mixture of (chloromethyldimethylsilyl)-2-propenyl acetate isomers was investigated by using immobilized Candida antarctica lipase as biocatalyst. TLC analysis and 1H NMR spectroscopy were used to monitor the extent of the reaction. At 60 degrees C, the enzyme exhibited a high selectivity towards 3-(chloromethyldimethylsilyl)-2-propenyl acetate which was almost quantitatively hydrolyzed, whereas, only 11% of 2-(chloromethyldimethylsilyl)-2-propenyl acetate reacted with the lipase. Consequently, the unreacted acetate was readily purified from the reaction medium by flash column chromatography and deacetoxylated in acidic methanol to give the corresponding hydroxy compound in a 71% global yield. On the other hand, without lipase, chemical treatment of the acetate mixture resulted in much lower yields in hydroxy compounds followed by a tedious purification process.     

Volatile organic compounds released by Rumex confertus following Hypera rumicis herbivory and weevil responses to volatiles

We report in this study large induction of volatile organic compounds (VOCs) from a single inflorescence of mossy sorrel (Rumex confertus Willd., Polygonaceae), by herbivory of the weevil (Hypera rumicis L., Coleoptera: Curculionidae). VOCs blend induced by the weevil herbivory included 1 green leaf volatiles (GLVs) ((Z)‐3‐hexen‐1‐yl acetate), five terpenes ((Z)‐β‐ocimene, linalool, geranyl acetate, β‐caryophyllene and (E)‐β‐farnesene), three esters (benzyl acetate, methyl salicylate and methyl anthranilate) and one aromatic heterocyclic organic compound (indole). Uninjured plants produced only detectable amounts of VOCs. A Y‐tube experiment revealed that both females and males of H. rumicis were not attracted to any of tested concentrations (1, 5, 25, 125 ng/min). Also both females and males were significantly repelled by the highest concentrations (25 and 125 ng/min). Additionally, concentration of 5 ng/min proved to be repellent for females of H. rumicis.     

Optimized bacterial expression and purification of the c-Src catalytic domain for solution NMR studies

Progression of a host of human cancers is associated with elevated levels of expression and catalytic activity of the Src family of tyrosine kinases (SFKs), making them key therapeutic targets. Even with the availability of multiple crystal structures of active and inactive forms of the SFK catalytic domain (CD), a complete understanding of its catalytic regulation is unavailable. Also unavailable are atomic or near-atomic resolution information about their interactions, often weak or transient, with regulating phosphatases and downstream targets. Solution NMR, the biophysical method best suited to tackle this problem, was previously hindered by difficulties in bacterial expression and purification of sufficient quantities of soluble, properly folded protein for economically viable labeling with NMR-active isotopes. Through a choice of optimal constructs, co-expression with chaperones and optimization of the purification protocol, we have achieved the ability to bacterially produce large quantities of the isotopically-labeled CD of c-Src, the prototypical SFK, and of its activating Tyr-phosphorylated form. All constructs produce excellent spectra allowing solution NMR studies of this family in an efficient manner. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation and characterization of naturally occurring cyanogenic compounds

The literature dealing with the detection, isolation, purification and characterization of cyanogenic glycosides has been integrated with spectral and chemical data as well as other techniques from our laboratory to establish a method for the positive identification of glycosides of this type. The compounds are arranged into biosynthetically related groups (those derived from l-phenylalanine; l-tyrosine; l-leucine, l-valine; l-isoleucine; those with cyclopentene rings and pseudocyanogenic glycosides) and features of each of the above procedures are critically reviewed and spectral data for each group presented (IR, MS, UV and NMR). The NMR spectra of TMS ethers of cyanogenic glycosides have proven especially useful in chemical structure determination. This information is sufficient to permit identification of any of the 26 known glycosides as well as certain uncharacterized ones.     

Enzymatic synthesis of dTDP-4-amino-4,6-dideoxy-D-glucose using GerB (dTDP-4-keto-6-deoxy-D-glucose aminotransferase)

Over-expressed GerB (dTDP-4-keto-6-deoxy-d-glucose aminotransferase) of Streptomyces sp. GERI-155 was used in the enzymatic synthesis of dTDP-4-amino-4,6-dideoxy-D-glucose (2) from dTDP-4-keto-6-deoxy-D-glucose (1). [Carbohydrate structure: see text]. Five enzymes including dTMP kinase (TMK), acetate kinase (ACK), dTDP-glucose synthase (TGS), dTDP-glucose 4,6-dehydratase (DH), and dTDP-4-keto-6-deoxy-d-glucose aminotransferase (GerB) were used to synthesize 2 on a large scale from glucose-1-phosphate and TMP. A conversion yield of up to 57% was obtained by HPLC peak integration given a reaction time of 270min. After purification by two successive preparative HPLC systems, the final product was identified by HPLC and then analyzed by (1)H, (13)C, (1)H-(1)H COSY NMR spectrometry.     

Expression, refolding, and isotopic labeling of human serum albumin domains for NMR spectroscopy

Many different compounds bind to human serum albumin (HSA), which can be a significant problem in the drug discovery process. To aid in the design of drug molecules that do not bind to HSA, the structures of HSA/ligand complexes would be very useful. However, little information has been reported on the structures of small molecules complexed to HSA. In this paper, we describe a procedure for preparing isotopically labeled domains of HSA for nuclear magnetic resonance (NMR) studies. The procedure involves the expression in Escherichia coli, refolding, and a multistep purification. Domains I and III are capable of folding into stable structural units and producing well resolved (15)N/(1)H correlation spectra, whereas domain II forms significant aggregates at sub-millimolar concentration. Using our protocols, isotopically labeled and properly folded domains I and III can be effectively produced in large quantities for NMR-based structural studies and NMR-based screening. This provides a valuable tool for obtaining structural information on HSA/ligand complexes by NMR which will be useful in drug discovery.     

龙胆草地上部分的化学成分

为研究龙胆草（Gentiana scabra Bunge）地上部分的化学成分,利用硅胶柱色谱对其乙醇提取物进行分离纯化,根据理化性质和波谱方法鉴定了化合物的结构。分离得到β-香树脂醇（1）、β-谷甾醇（2）、β-香树脂醇乙酸酯（3）、乌苏醇（4）、齐墩果酸（5）、6-去甲氧基-7-甲基茵陈色原酮（6）。其中化合物6为首次从龙胆科植物中分离得到的具有2-苯氧基色原酮骨架的天然酚类成分,本文对其1D和2DNMR谱的特征及EI质谱主要碎片的可能裂解方式进行了讨论,利用单晶X-射线衍射方法对其晶体结构进行了分析,为该类天然产物的结构表征提供了依据。