Molecularly imprinted polymer-assisted sample clean-up of ochratoxin A from red wine: merits and limitations

A new analytical method for the determination of the carcinogenic mycotoxin ochratoxin A (OTA) in red wines has been developed involving a two-dimensional solid-phase extraction (SPE) clean-up protocol on C18-silica and a target-selective molecularly imprinted polymer (MIP). Prior removal of the interfering acidic matrix compounds by C18 solid-phase extraction was crucial for a successful clean-up as direct sample loading onto the MIP led to poor recoveries. The combined solid-phase extraction protocol afforded extracts suitable for sensitive ochratoxin A quantification by HPLC-fluorescence detection. Preliminary validation of the method performance with spiked (0.033-1.0 ng OTA/ml) and commercial red wines provided recoveries >90% and < 10%, with limit of detection (LOD) and limit of quantification (LOQ) of 0.01 and 0.033 ng/ml. However, a similarly favorable performance characteristics was observed in control experiments in which the MIP was replaced by the corresponding non-imprinted polymer (NIP). These findings provide evidence that under the employed experimental conditions specific analyte binding to imprinted binding sites plays a minor role in selective OTA retention. In the framework of this study, other problems inherent to MIP-based solid-phase extraction have been addressed. These include the reproducible preparation of MIP materials with consistent molecular recognition characteristics, the potential for repeated use of MIP, unfavorable polymer swelling in application-relevant solvents, potential sample contamination by template bleeding, and slow analyte binding kinetics.     

The role of water on the equilibrium of esterification by immobilized lipase packed-bed column reactor

Summary The role of water on the continous synthesis of geranyl esters by immobilized lipase fromHumicola lanuginosa No. 3 was studied in a packed-bed column reactor (PBR) installed with a molecular sieve column for water extraction. The conversion degress by PBR were highly influenced by the water concentration throughout the reaction which acted as a determinant on the reaction equilibrium. Almost 100% conversion of geranyl laurate could be achieved under the optimum water-controlled conditions. The stability of the PBR system was for 35 days with the half-life of 38 days.     

SEASONAL VARIABILITY OF PHYSICOCHEMICAL AND RHEOLOGICAL PROPERTIES OF ULVAN IN TWO ULVA SPECIES (CHLOROPHYTA) FROM THE BRITTANY COAST1

The seasonal variability in the extraction yield, physicochemical characteristics, and rheological properties of ulvan from two Ulva species contributing to Brittany “green tides” has been studied. These seaweeds were collected in the water column for Ulva armoricana Dion, de Reviers et Coat and on hard substrata for Ulva rotundata Bliding. The maximum ulvan extraction efficiency was not related to the maximum ulvan content in the seaweeds, but with the active growth period of the seaweeds. Ulvan chemical structure, macromolecular characteristics, and rheological properties were affected by both species and seasons. The proportion of high‐molecular‐weight ulvan was the major factor positively correlated with the gelling properties. Characteristics of ulvan from U. rotundata subjected to tides were more affected by seasons than ulvan from U. armoricana living in a more constant environment. These results point to several useful recommendations concerning Ulva sp. biomass collected with regard to ulvan characteristics and uses.     

十字花科植物种子低分子RNA提取方法比较

非编码RNA不翻译成蛋白质,它们通过转录、转录后及翻译水平调控靶基因表达,在植物生长发育及逆境胁迫中发挥功能。目前,大量种子萌发期特异表达的非编码RNA (Non-coding RNA)已被发现,高效提取种子低分子RNA是对其进行研究的关键。本研究将介绍一种改良SDS RNA提取方法,并与Trizol、CTAB法、RNA提取试剂盒进行比较。结果表明:这种方法可以高效提取用于Northern blotting、RT-PCR等分子生物学分析的十字花科植物种子低分子RNA。改良SDS RNA提取方法为种子非编码RNA研究、种子萌发生理及分子育种研究提供了帮助。     

Effect of saliva processing on bacterial DNA extraction

More than 700 bacterial species inhabit oral cavity of humans. Various oral diseases are related to changes in the structure of this complex community. Their pathogenesis can, thus, be better understood by study of oral microbial flora. As many bacteria are refractory to cultivation, molecular approaches based on PCR followed by downstream analysis are more suitable for community analysis than culture dependent methods. Effective DNA extraction from the sample matrix is a fundamental part of the pre-analytical phase but it can be influenced by processing of the starting material. The aim of this study was to analyze the effects of saliva processing on DNA extraction using several non-commercial isolation procedures. Bacterial chromosomal DNA was extracted from three different sample matrices: fresh saliva, diluted saliva and pelleted saliva using four different extraction methods: phenol chloroform protocol, benzyl-chloride protocol, extraction with Chelex-100 and extraction with Triton X. Extraction from different saliva samples and the use of different extraction methods significantly affected the effectiveness of DNA extraction. The most suitable material for bacterial DNA extraction for molecular analysis is a fresh saliva sample. The most effective methods for isolating salivary DNA are the benzyl-chloride protocol and Chelex-100 extraction. Our results have implications for studies concentrating on salivary microbiome and its role in the pathogenesis of oral diseases.     

The selective participation of brain-specific non-histone proteins of chromatin Np-3,5 during the reproduction of a defensive habit to food in edible snails]

The role of brain-specific nonhistone proteins of chromatine Np-3.5 in the processes of reproduction of elaborated defensive habit to food was studied in previously learning snails. It was found, that gamma-globulines to Np-3.5 during tens of minutes inhibited behavioural and neuronal reactions elicited by a definite conditioned stimulus--carrot juice, without changing reactions to other conditioned stimulus--apple juice. gamma-globulines to other nonhistone proteins of chromatine did not influence the reproduction of food rejection habits. It was supposed that brain-specific nonhistone proteins of chromatine Np-3.5 were selectively involved in the molecular processes providing for neurophysiological mechanisms of information extraction from the long-term memory.     

Optimization of chitin extraction from shrimp shells

The aim of this paper is to define optimal conditions for the extraction of chitin from shrimp shells. The kinetics of both demineralization and deproteinization with, in the latter case, the role of temperature are studied. The characterization of the residual calcium and protein contents, the molecular weights, and degrees of acetylation (DA) allows us to propose the optimal conditions as follows. The demineralization is completely achieved within 15 min at ambient temperature in an excess of HCl 0.25 M (with a solid-to-liquid ratio of about 1/40 (w/v)). The deproteinization is conveniently obtained in NaOH 1 M within 24 h at a temperature close to 70 degrees C with no incidence on the molecular weight or the DA. In these conditions, the residual content of calcium in chitin is below 0.01%, and the DA is almost 95%.     

A comparison study on microwave-assisted extraction of Artemisia sphaerocephala polysaccharides with conventional method: Molecule structure and antioxidant activities evaluation

The conventional extraction methods for polysaccharides were time-consuming, laborious and energy-consuming. Microwave-assisted extraction (MAE) technique was employed for the extraction of Artemisia sphaerocephala polysaccharides (ASP), which is a traditional Chinese food. The extracting parameters were optimized by Box-Behnken design. In microwave heating process, a decrease in molecular weight (M_w) was detected in SEC-LLS measurement. A d_f value of 2.85 indicated ASP using MAE exhibited as a sphere conformation of branched clusters in aqueous solution. Furthermore, it showed stronger antioxidant activities compared with hot water extraction. The data obtained showed that the molecular weights played a more important role in antioxidant activities.     

一株高产碱性内切聚半乳糖醛酸酶生产菌的筛选与鉴定

从碱性土样中经分离筛选得到1株固态发酵产碱性内切聚半乳糖醛酸酶活力较高的菌株。经提酶条件优化得到较优的酶液提取提条件为30倍Na2CO3/NaHCO3缓冲液提取1 h。从形态特征、培养特征、生理生化特征以及分子生物学鉴定结果来看,该菌株属B.gibsonii。     

Alkaline phosphatase in Hirudo medicinalis L.: partial purification and study of certain molecular and catalytic properties

The authors carried out extraction and purification of an alkaline phosphatase (AP) from Hirudo medicinalis and studied certain characteristics of the enzyme. After homogenizing specimens of H. medicinalis and centrifuging the homogenate at 70,000 g, the enzyme, likely membrane-bound, was obtained in a soluble form by treating the sediment with n-butanol. It was then purified by acetone fractionation and Sephadex G-200 chromatography. A fairly good purification degree was achieved; the enzyme appears to be in a single form and displays a molecular weight of about 268,000. The optimum pH (9.5) is lower than the ones generally observed as regards APs from both Invertebrata and Mammalia. The studied AP displays a strong substrate inhibition, similar to that concerning Metazoa at a higher evolutionary level (Mollusca, Echinodermata). On the contrary, the enzyme-substrate affinity, as shown by Km value (2.447 mM), is lower than as regards APs from more advanced organisms (Echinodermata, Mammalia).     

Changes in the serum protein composition in experimental hypercholesterolemia

Cholesterol accumulation, quantitative changes and composition of lipoproteins, total proteins of blood serum and protein fractions obtained by acid extraction are studied in hypercholesterinemia dynamics in rabbits. It is found that the initial period of cholesterinosis in blood serum is marked by an increased content of total proteins and proteins extracted by acid followed, however, by substantial lowering of the level of these compounds. The proteins obtained by acid extraction are characterized by more explicit changes. This permits assuming their important role in the pathogenesis of hypercholesterinemia. The obtained results make it possible to state that the investigated proteins possess the alkaline properties. The data available in literature on the ability of alkaline polypeptides to bind cholesterol permit assuming that the investigated proteins have the same properties and, hence, can participate in the molecular mechanisms of cholesterol transport as well as in the processes of synthesis and transformations of separate classes of lipoproteins.     

粪便DNA分析技术在分子生态学研究中的机遇与挑战

随着粪便DNA分析技术的不断发展与完善,其越来越多地被应用于分子生态学研究中,特别是野生动物的遗传状况评估研究。粪便DNA的获取可以在不干扰,甚至无需观察到动物本身的情况下展开,因此避免了取样活动可能给野生动物带来的干扰或伤害,极大地促进了野生动物分子生态学的研究。虽然粪便DNA分析技术在其建立伊始因DNA质量问题而受到了一定的挑战,但自其建立至今,研究者发展了多种技术来克服这一问题。现已能获得较高质量的粪便DNA,并将基因分型错误率控制在较低水平。本文将结合我们在粪便DNA分析技术上所积累的经验,从粪便样品采集、保存、DNA提取、PCR扩增以及等位基因分型等各个环节对该技术进行详细探讨,以期阐明该技术在野生动物分子生态学研究中所面临的机遇与挑战,进一步推动其在我国野生动物保护研究中的应用与发展。     

发菜藻蓝蛋白分离纯化的研究

以发菜为材料,比较了提取液类型和饱和硫酸铵浓度对藻蓝蛋白提取的影响,并对藻蓝蛋白的提取程序和部分特性进行了研究。结果表明:50 mmol/L KP缓冲液(pH值7.2)是合适的提取液,体积分数为40%~50%饱和硫酸铵盐析效果优于其它浓度。经过DEAE-Toyopeal 650 S离子交换层析和SuperdexTM200凝胶过滤层析后,藻蓝蛋白纯度达6.2,最大吸收峰位于615 nm,荧光发射峰位于649 nm,由α和β2个亚基组成,其分子质量分别为18 051.17和19 142.27 Da。因此,发菜藻蓝蛋白分离纯化较为理想的程序为:藻粉→50 mmol/L KP缓冲液(pH值7.2)浸泡→French pressure(1 500 kg/cm2)破碎细胞→40%~50%饱和硫酸铵盐析→DEAE-Toyopeal 650 S离子交换层析→SuperdexTM200凝胶过滤层析→较纯的藻蓝蛋白。     

Comparison of different extraction methods of polysaccharides from cup plant (Silphium perfoliatum L.)

The purpose of the present research was to compare the effects of four extraction methods on the extraction yields, physicochemical characteristics, and antioxidant activities of polysaccharides from the cup plant (Silphium perfoliatum L.) (CPPs). The extraction methods included the hot water extraction method (HEM), ultrasonic-assisted extraction method (UEM), enzyme-assisted extraction method (EEM), and the ultrasonic-enzyme-assisted extraction method (UEEM). Four corresponding CPP extracts, HEM-CPPs, UEM-CPPs, EEM-CPPs, and UEEM-CPPs, were obtained. The results indicated that there were notable differences between the extraction methods regarding the yield, average molecular weight, uronic acid content, monosaccharide proportions, and the antioxidant activities of the CPPs. HEM-CPPs were extracted with the lowest yield (6.44%). EEM-CPPs had the largest extraction yield (9.87%) and the most notable antioxidant abilities of ferric reducing power and free radical scavenging activity. According to correlation analysis, the higher antioxidant abilities of EEM-CPPs might be due to their smaller average molecular weight and a higher content of uronic acid than those of the other extracted CPPs. Therefore, EEM could serve as a promising technique for extracting CPPs.     

Comparative analysis of DNA extraction methods to study the body surface microbiota of insects: A case study with ant cuticular bacteria

High‐throughput sequencing of the 16S rRNA gene has considerably helped revealing the essential role of bacteria living on insect cuticles in the ecophysiology and behaviour of their hosts. However, our understanding of host‐cuticular microbiota feedbacks remains hampered by the difficulties of working with low bacterial DNA quantities as with individual insect cuticle samples, which are more prone to molecular biases and contaminations. Herein, we conducted a methodological benchmark on the cuticular bacterial loads retrieved from two Neotropical ant species of different body size and ecology: Atta cephalotes (~15 mm) and Pseudomyrmex penetrator (~5 mm). We evaluated the richness and composition of the cuticular microbiota, as well as the amount of biases and contamination produced by four DNA extraction protocols. We also addressed how bacterial community characteristics would be affected by the number of individuals or individual body size used for DNA extraction. Most extraction methods yielded similar results in terms of bacterial diversity and composition for A. cephalotes (~15 mm). In contrast, greater amounts of artefactual sequences and contaminations, as well as noticeable differences in bacterial community characteristics were observed between extraction methods for P. penetrator (~5 mm). We also found that large (~15 mm) and small (~5 mm) A. cephalotes individuals harbour different bacterial communities. Our benchmark suggests that cuticular microbiota of single individual insects can be reliably retrieved provided that blank controls, appropriate data cleaning, and individual body size and functional role within insect society are considered in the experiment.     

Extraction of DNA from exfoliative cytology specimens and its suitability for analysis by the polymerase chain reaction

The extraction of DNA from archival exfoliative cytology samples would allow the molecular biological analysis of this readily available material using the polymerase chain reaction (PCR). We have quantitatively and qualitatively studied the extraction of DNA from a variety of cytological preparations. For both fresh and archival cervical smears, overnight incubation with proteinase K produces high yields of high molecular weight DNA, but simply boiling the samples produces DNA suitable for PCR amplification of a single copy gene. Increasing the proteinase K incubation to several days allows the extraction of DNA from fixed and stained archival cytology slides from a variety of sites. The extracted DNA was again suitable for PCR analysis. Fresh and archival cytological material can be utilized for molecular biological study of disease processes using PCR. Archival cytological material is probably the best source of DNA and RNA after stored frozen tissue.     

Immunochemical findings about the high molecular weight surface antigens of Shigella sonnei]

The authors studied immunochemical properties of the high molecular fraction of surface soluble antigens obtained by extraction with salt solutions from Sh. sonnei (virulent strain 1041) dried with acetone. The high molecular fraction was isolated by gel-filtration on Sepharose-4B. Along with the O-somatic antigen, this fraction contained thermostable and thermolabile antigens resistant to trypsin and RNA-ase treatment, and also protein-containing antigens disintegrated by trypsin. In difference from the O-somatic antigen, one of the thermostable components was completely precipitated with 50% alcohol.     

用超临界流体技术萃取分离香茅油的研究

作者用超临界流体循环萃取装置对香茅油在超临界CO_2中的溶解度以及香茅油中主要成份香茅醛、香茅醇和香叶醇的分离特性进行研究。分别得出溶解度、分离特性与萃取压力、温度的关系及其相应的数学关联式。用这些关联式计算的结果符合工程需要,为单离香茅油工业化生产提供了理论基础。     

Effect of enzyme inhibitors on profiles of IR-ACTH/beta-endorphin in brain and pituitary

Processing of pro-opiomelanocortin (POMC) in brain and pituitary results in various proportions of multiple peptides appearing as immunoreactive (IR-) ACTH and IR-beta-endorphin. Because it is desirable that molecular profiles of POMC-related peptides reflect in vivo processing and not isolation artifact, inhibition of proteolytic activity during extraction is critical. Although enzyme inhibitors are frequently used during extraction of POMC-related peptides, their benefit or necessity has not been established. To determine the benefit of using enzyme inhibitors for studying molecular profiles of IR-ACTH and IR-beta-endorphin from rat brain and pituitary, chromatographic profiles were compared to assess the effect of each of several enzyme inhibitors. Results suggested that the enzyme inhibitors studied provided no additional benefit in terms of inhibition of extraction proteolysis over that provided by a strong acid and heat inactivation.     

Pectin extraction in the presence of alcohols

A theoretical discussion of the extraction process and especially of the factors influencing extractant penetration in pectin-containing raw materials is presented. The necessity of adding surfactant in the pectin extraction is proved. Experiments were carried out with two types of apple pressings differing in anhydrouronic acid content and quantity of extractable pectin.

The addition of low molecular alcohols in concentrations from 1% to 3% to the acid extragent resulted in an acceleration of extraction and increase in the pectin yield by 55–90%. Ethylene glycol, glycerol and diethylene glycol had a better effect than monohydric alcohols. The effect of the process duration on the pectin yield during acid extraction was studied. A 25-min extraction was sufficient for taking out the extractable pectin. It was shown that the addition of alcohols resulted in a measurable increase in the pectin gel strength.