Determination of triterpenic acids in human serum by high-performance liquid chromatography: triterpenoid interaction with serum protein

Terpenic acids are under development as therapeutic agents in numerous treatments. In support of pharmacokinetic and toxicological evaluations, a robust assay based on high-performance liquid chromatography (HPLC) was developed for the analysis of the terpenoids in human serum. For a clear understanding of the differences in biological activity of these compounds, the interactions between oleanolic or betulinic acids and human serum protein have been studied by ultraviolet-visible (UV-vis) absorption under physiological conditions. A combination of liquid/liquid extraction, centrifugation, and consecutive HPLC resulted in simultaneous separation, identification, and quantification of the oleanolic, betulinic, and ursolic acids. The validity of the developed method was established by determining linearity, recovery, precision, accuracy, limit of detection, and quantification. Detection limits were in the range of 3.3-4.3 ng/mL, and linearity values ranged up to 1 μg/mL. The repeatability of the method was good. All compounds can be well-distinguished by order of elution during liquid chromatography. The pentacyclic triterpenoids have been identified by retention time comparison to pure standards and quantified by an internal standard. The results by UV-vis absorption spectra experiments (240-340 nm) indicate that protein structures have been perturbed in the presence of oleanolic and betulinic acids.     

Fractionation-reconstitution experiments provide insight into the role of endoxylanases in bread-making.

The impact mechanism of endoxylanases in straight dough bread-making was investigated in fractionation-reconstitution experiments. To this end, two European flours with different bread-making characteristics were separated in gluten, prime starch, a squeegee fraction (SQF), and a water-extractable fraction. Whereas the former fractions contained negligible levels of arabinoxylan (AX), the latter contained, respectively, most of the water-unextractable AX (WU-AX) and all of the water-extractable AX (WE-AX). In vitro modification with a Bacillus subtilis endoxylanase allowed controlled solubilization of WU-AX from SQF and controlled degradation of solubilized AX and WE-AX from the water-extractables. It followed from bread-making tests with the reconstituted flours that endoxylanases exert positive loaf volume effects in bread-making by lowering the concentration of WU-AX and increasing that of total soluble AX. Limited degradation of WE-AX and significant breakdown of solubilized AX by endoxylanases, on the other hand, resulted in volume losses when compared to their nondegraded counterparts. The volume increasing effects of endoxylanases are therefore related to their ratio of solubilizing to degrading activity and thus to their substrate specificity.     

Further insight into thermally and pH-induced generation of acrylamide from glucose/asparagine model systems

On the basis of numerous studies on the mechanism of formation of acrylamide (AA) from asparagine and reducing sugars, the decarboxylated Schiff base [ N-( d-glucos-1-yl)-3'-aminopropionamide] and its corresponding Amadori product [ N-(1-deoxy- d-fructos-1-yl)-3'-aminopropionamide) are considered to be possible direct precursors in addition to 3-aminopropionamide (AP). Furthermore, the mechanism of decarboxylation of the initially formed N-( d-glucos-1-yl)asparagine to generate the above-mentioned precursors also remains to be confirmed. To identify the relative importance of AA precursors, the decarboxylated Amadori product (AP ARP) and the corresponding Schiff base were synthesized and their relative abilities to generate AA under dry and wet heating conditions were studied. Under both conditions, the N-( d-glucos-1-yl)-3'-aminopropionamide had the highest intrinsic ability to be converted into AA. In the dry model system, the increase was almost 4-fold higher than the corresponding AP ARP or AP; however, in the wet system, the increase was 2-fold higher relative to AP ARP but >20-fold higher relative to AP. In addition, to gain further insight into the decarboxylation step, the amino acid/sugar reactions were analyzed by FTIR to monitor the formation of the previously proposed 5-oxazolidinone intermediate known to exhibit a peak in the range of 1770-1810 cm (-1). Spectroscopic studies clearly indicated the formation of an intense peak in the indicated range, the precise wavelength being dependent on the amino acid and the sugar used. The identity of the peak was verified by observing a 40 cm (-1) shift when [(13)C-1]-labeled amino acid was used.     

Use of psychrophilic xylanases provides insight into the xylanase functionality in bread making

The bread-improving potential of three psychrophilic xylanases from Pseudoalteromonas haloplanktis TAH3A (XPH), Flavobacterium sp. MSY-2 (rXFH), and unknown bacterial origin (rXyn8) was compared to that of the mesophilic xylanases from Bacillus subtilis (XBS) and Aspergillus aculeatus (XAA). XPH, rXFH, and rXyn8 increased specific bread volumes up to 28%, 18%, and 18%, respectively, while XBS and XAA gave increases of 23% and 12%, respectively. This could be related to their substrate hydrolysis behavior. Xylanases with a high capacity to solubilize water-unextractable arabinoxylan (WU-AX) during mixing, such as XBS and XPH, increased bread volume more than xylanases that mainly solubilized WU-AX during fermentation, such as rXFH, rXyn8, and XAA. Irrespective of their intrinsic bread-improving potential, the dosages needed to increase bread volume to a similar extent were much lower for psychrophilic than for mesophilic xylanases. The xylanase efficiency mainly depended on the enzyme's temperature activity profile and its inhibition sensitivity.     

Cold-set globular protein gels: interactions,structure and rheology as a function of protein concentration

We identified the contribution of covalent and noncovalent interactions to the scaling behavior of the structural and rheological properties in a cold gelling protein system. The system we studied consisted of two types of whey protein aggregates, equal in size but different in the amount of accessible thiol groups at the surface of the aggregates. Analysis of the structural characteristics of acid-induced gels of both thiol-blocked and unmodified whey protein aggregates yielded a fractal dimension (2.3 +/- 0.1), which is in line with other comparable protein networks. However, application of known fractal scaling equations to our rheological data yielded ambiguous results. It is suggested that acid-induced cold-gelation probably starts off as a fractal process, but is rapidly taken over by another mechanism at larger length scales (>100 nm). In addition, indications were found for disulfide cross-link-dependent structural rearrangements at smaller length scales (<100 nm).     

Fractionation and reconstitution experiments provide insight into the role of gluten and starch interactions in pasta quality

Commercial durum wheat (Triticum durum desf.) semolina was fractionated into starch, gluten, and water extractables. Starch surface proteins and surface lipids were removed, and two starches with manipulated granule size distributions were produced to influence starch properties, affecting its interaction with other semolina components. Reconstituted spaghetti was made with untreated (control) or treated starches. The pasta made from the starting semolina material had lower cooking time and was of lower quality than the samples made from reconstituted material. This was not due to changes in gluten properties as a result of the first step of the fractionation process. For the reconstituted samples, starch interaction behavior was not changed after surface protein or surface lipid removal. Starch surface properties thus do not influence the starch interaction behavior, indicating that starch-gluten interaction in raw (uncooked) pasta is mainly due to physical inclusion. All reconstituted pasta samples also had generally the same cooking quality. It was concluded that the small changes in starch gelatinization behavior, caused by the above-mentioned starch modifications, are of little importance for pasta quality.     

Chemical structures of corn stover and its residue after dilute acid prehydrolysis and enzymatic hydrolysis: insight into factors limiting enzymatic hydrolysis

Advanced solid-state NMR techniques and wet chemical analyses were applied to investigate untreated corn stover (UCS) and its residues after dilute acid prehydrolysis (DAP) and enzymatic hydrolysis (RES) to provide evidence for the limitations to the effectiveness of enzyme hydrolysis. Advanced solid-state NMR spectral-editing techniques as well as 1H-13C two-dimensional heteronuclear correlation NMR (2D HETCOR) were employed. Our results indicated that dilute acid prehydrolysis selectively removed amorphous carbohydrates, increased aromatic CH/other protonated -C═C- and enriched alkyl CH and CH2 components. Cinnamic acids were increased, and proteinaceous materials and N-containing degradation or condensation compounds were absorbed or coprecipitated in RES. 2D HETCOR experiments indicated a close association between lignin and the residual carbohydrates. Ketones/aldehydes were not detected in the DAP, in contrast to a report in which an appreciable amount of ketones/aldehydes was generated from the acid pretreatment of a purified cellulose in the literature. This suggested that acid pretreatment may modify the structure of purified cellulose more than biomass and that biomass may be a better substrate than model biopolymers and compounds for assessing structural changes that occur with industrial processing. On the basis of NMR and wet chemical analyses, we found the following factors could cause the limitations to the effectiveness of enzymatic hydrolysis: (1) chemical modification of carbohydrates limited the biologically degradable carbohydrates available; (2) cinnamic acids in the residue accumulated; (3) accessibility was potentially limited due to the close association of carbohydrates with lignin; and (4) proteinaceous materials and N-containing degradation or condensation compounds were absorbed or coprecipitated.     

Fractionation and reconstitution experiments provide insight into the role of starch gelatinization and pasting properties in pasta quality

Commercial durum wheat semolina was fractionated into protein, starch, water-extractable, and sludge fractions. The starch fraction was hydroxypropylated, annealed, or cross-linked to change its gelatinization and pasting properties. Spaghettis were made by reconstitution of the fractions, and their quality was assessed. Hydroxypropylated starches were detrimental for cooked pasta quality. Cross-linked starches made the reconstituted pasta firmer and even brittle when the degree of cross-linking was too high. These results indicate that starch properties play a role in pasta quality, although the gluten remains very important as an ultrastructure agent. It was concluded that, given a certain gluten ultrastructure, starch water uptake and gel properties and/or its interference with or breakdown of the continuous gluten network during cooking determine pasta quality.     

Effects of moisture-induced whey protein aggregation on protein conformation, the state of water molecules, and the microstructure and texture of high-protein-containing matrix

Moisture-induced protein aggregation through intermolecular interactions such as disulfide bonding can occur in a high-protein-containing food matrix during nonthermal processing and storage. The present study investigated the effect of moisture-induced whey protein aggregation on the structure and texture of such high-protein-containing matrices using a protein/buffer model system. Whey proteins in the protein/buffer model systems formed insoluble aggregates during 3 months' storage at temperatures varying from 4 to 45 degrees C, resulting in changes in microstructure and texture. The level of aggregation that began to cause significant texture change was an inverse function of storage temperature. The protein conformation and the state of water molecules in the model system also changed during storage, as measured by differential scanning calorimetry and Fourier transform infrared spectroscopy. During storage, the model system that had an initially smooth structure formed aggregated particles (100-200 nm) as measured by scanning electron microscopy, which lead to an aggregation network in the high-protein-containing matrix and caused a harder texture.     

植物多酚-牛血清白蛋白相互作用及对蛋白质结构的影响

多酚与蛋白质的相互作用取决于蛋白质和多酚的结构与类型,为研究不同植物多酚与牛血清白蛋白（bovine serum albumin,BSA）之间的相互作用,探明不同植物多酚对BSA结构的影响,进而筛选出稳定的植物多酚-BSA复合物。该研究采用原花青素（proanthocyanidins,PC）、儿茶素（catechin,C）、表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)、茶多酚（tea polyphenol,TP）,通过紫外光谱法、荧光光谱法、红外光谱法和分子对接研究植物多酚与BSA相互作用及其对蛋白质结构的影响。结果表明：4种植物多酚-BSA复合物的紫外最大波长红移由大到小为EGCG（365 nm）＞PC（364 nm）、C（364 nm）＞TP（363 nm）;红外光谱发现,酰胺Ⅰ带的吸收峰（1 657.44 cm^-1）发生蓝移由大到小为PC（1 656.15 cm^-1）＞TP（1 632.07 cm^-1）＞EGCG（1 631.49 cm^-1）＞C（1 631.44 cm^-1）,烟酰胺II带峰形变宽;BSA二级结构由β-转角、无规卷曲转变为β-折叠、ɑ-螺旋,β-折叠含量增加C-BSA（40.20%）>PC-BSA（39.50%）>EGCG-BSA（39.32%）>TP-BSA（34.04%）,表明C更能促进BSA的二级结构稳定;荧光光谱表明4种多酚对BSA的猝灭类型均为静态猝灭,且结合位点约为1,表明存在一个结合位点;分子对接结果表明该结合位点位于Sub-domain IIA的疏水腔中,分子间相互作用力主要是氢键、疏水作用力,结合能由小到大为C（-3.72 kJ/mol）＜EGCG（-1.77 kJl/mol）＜PC（-1.02 kJ/mol）＜TP（-0.38 kJ/mol）;4种多酚中C和EGCG与BSA相互作用程度较大,C与BSA的结合能最小,形成的复合物最稳定。通过多酚与蛋白相互作用研究,更好地发挥多酚和蛋白两种组分的功能作用,可为开发功能性多酚-蛋白质复合物产品提供参考。     

Regulation and function of AtNRAMP4 metal transporter protein

The NRAMP gene family encodes integral membrane proteins mediating the transport of a broad range of transition metals in bacteria, fungi, plants, and animals. We studied the regulation of AtNRAMP4 in Arabidopsis. In a previous study, we showed that AtNRAMP3 and AtNRAMP4 transport manganese (Mn), iron (Fe), and cadmium (Cd). In this study, we show that, in contrast to AtNRAMP3, AtNRAMP4 complements the growth phenotype of the zrt1zrt2 Zn uptake deficient yeast mutant. In a previous study, we have shown that, under Fe starvation, AtNRAMP4 mRNA levels are up-regulated in Arabidopsis. To analyze the regulation of AtNRAMP4 at the protein level, we generated specific antibodies against AtNRAMP4 protein. The antiserum was able to recognize a tagged version of AtNRAMP4 expressed in yeast. The antibody did not reveal any change in AtNRAMP4 protein level upon Fe starvation in Arabidopsis thaliana ecotype Columbia plants. In AtNRAMP4 overexpressing plants, high levels of AtNRAMP4 protein could be detected. AtNRAMP4 overexpressing plants display cadmium hypersensitivity in a medium containing 50 μm FeEDTA as Fe source. However, despite the constitutive accumulation of AtNRAMP4 protein, AtNRAMP4 over-expressing plants did not display Cd hypersensitivity under high Fe supply (100 μm FeHBED). AtNRAMP4 over-expressing lines displayed the same sensitivity to Zn as controls under all conditions tested. Our results suggest a translational level for the regulation of AtNRAMP4. Over-expression of AtNRAMP4 in Arabidopsis thaliana confers a slight hypersensitivity to Cd but not to Zn.     

红色荧光标记载体pBacA4DsRed1的构建及其表达

以首次克隆的家蚕Bombyx mori 肌动蛋白（actin4, A4）启动子、红色荧光蛋白基因DsRed1和多聚腺苷酸识别序列SV40为元件，经多次克隆将其插入到 piggyBac转座载体中，构建成pBacA4DsRed1转基因表达载体。将该载体显微注射到胚盘形成前期的蚕卵中，在荧光显微镜下观察其发光情况，结果发现：在胚胎早期发育的第三天，检测到蚕卵内发出较强的红色荧光。由于蚕卵没有红色自发荧光，用DsRed1标记背景干扰小，对注射蚕种无特别选择，在荧光显微镜下观察到明显的红色荧光，表明pBacA4DsRed1载体构建正确且能在蚕卵中表达。红色荧光蛋白(RFP)基因丰富了报告基因的种类，并且与绿色荧光蛋白(GFP)基因一样，可以作为理想的报告基因应用于家蚕的功能基因研究。     

Adsorption behavior of tetracycline on the soil and molecular insight into the effect of dissolved organic matter on the adsorption

Journal of Soils and Sediments - Tetracycline (TC) is one of the most used antibiotics and has accumulated in soil. Its adsorption behavior was studied, and the effect of dissolved organic matter...     

Modification of bovine beta-lactoglobulin by glycation in a powdered state or in an aqueous solution: effect on association behavior and protein conformation

The effect of glycation with lactose on the association behavior and conformational state of bovine beta-lactoglobulin (beta-LG) was studied, using size exclusion chromatography, polyacrylamide gel electrophoresis, proteolytic susceptibility, and binding of a fluorescent probe. Two modification treatments were used, i.e., aqueous solution glycation and dry-way glycation. The results showed that the latter treatment did not significantly alter the nativelike behavior of the protein while the former treatment led to important structural changes. These changes resulted in a specific denatured beta-LG monomer, which covalently associated via the free thiol group. The homodimers thus formed and the expanded monomers underwent subsequent aggregation into a high molecular weight species, via noncovalent interactions. The association behavior of glycated beta-LG is discussed with respect to the known multistep denaturation/aggregation process of nonmodified beta-LG.     

Beer consumption and changes in stability of human serum proteins

The aim of this study was to evaluate the influence of beer consumption (BC) on the functional and structural properties of human serum proteins (HSP). Thirty-eight volunteers (after coronary bypass) were divided into two groups: experimental (EG) and control (CG). Nineteen volunteers of the EG consumed 330 mL per day of beer (about 20 g of alcohol) for 30 consecutive days. The CG volunteers consumed mineral water instead of beer. Blood samples were collected from EG and CG patients before and after the experiment. Albumin (Alb), globulin (Glo), and methanol-precipitable proteins (MPP) from human serum were denatured with 8 M urea. Fluorescence and electrophoresis were employed in order to elucidate urea-induced conformational changes and structural behavior of proteins. The measured fluorescence emission spectra were used to estimate the stability of native and denatured protein fractions before and after BC. It was found that before BC the fractions most stable to urea denaturation were Glo, Alb, and MPP fractions. After BC in most of the beer-consuming patients (EG) some changes in native and denatured protein fractions were detected: a tendency to lower stability and minor structural deviations. These qualitative changes were more profound in MPP than in Alb and Glo. Thus, Glo is more resistible to alcohol influence than Alb, which in turn is more resistible than MPP. No serum protein changes were detected in patients of CG.     

A new insight into the formation of odor active carbonyls by thermally-induced degradation of phospholipids in self-assembly structures

The role of molecular organization in heated aqueous dispersions of egg phosphatidylcholine (PC) and egg phosphatidylethanolamine (PE) was studied with respect to the formation of key odorants. Evidence was found for the crucial role of self-assembly structures adopted by phospholipid molecules on the quantitative composition of volatile constituents. The concentrations of seven aldehydes and one vinyl ketone were determined by isotope dilution assay in heated aqueous dispersions of PC and PE present in various ratios. Addition of PE to PC drastically decreased the amount of (E,E)-2,4-decadienal formed, which cannot be explained by the differences in the fatty acid composition of PC and PE. The free amino group in PE does not explain this phenomenon either, as replacing PE by phosphatidic acid distearylester also reduced the amounts of (E,E)-2,4-decadienal. We suggest that the type of self-assembly structure adopted by phospholipids in water significantly influences the reaction yields. However, the mechanisms leading to the preferred formation of phospholipid-derived odorants in a lamellar phase, as compared to the reversed hexagonal phase, remain unknown.     

New insight into the genetic structure of the Allolobophora chlorotica aggregate in Europe using microsatellite and mitochondrial data

The taxonomic status of Allolobophora chlorotica is still the subject of considerable discussion. After the recent experimental demonstration that the two regularly observed colour morphs (pink and green) were not fully interfertile and likely represent two distinct species, molecular analyses revealed a more complex picture with the occurrence of five mitochondrial lineages (named L1 to L5) in A. chlorotica, two within the green morph and three within the pink morph. Although nuclear markers (AFLPs) confirmed that the pink morph might consist of three different taxa, AFLPs data suggested that the green morph may be a single taxon. In order to further characterize the population genetic structure within A. chlorotica in addition to test for reproductive isolation between lineages, eight polymorphic microsatellite loci were developed using a microsatellite-enriched genomic library from an individual belonging to lineage L2. The number of alleles per locus varied from 9 to 29 in a set of individuals belonging to L2. Considerable sub-structure was observed within L2 populations suggesting a low dispersal capability in this species and a distribution of the individuals in patches that could function as panmictic units. These markers were transferable to the other lineages but only five loci could be amplified consistently in four of the lineages (L1, L2, L3 and L4). Microsatellite data confirmed that the green morph represents a single taxon. Although the integrity of the three previously documented lineages within the pink morph was also generally supported by the data, microsatellites provided evidence for hybridization between lineages and between morphs in the field. Moreover, mitochondrial data revealed the existence of two additional mitochondrial lineages within the pink morph. The taxonomic status of the pink morph remains thus unclear, requiring a thorough and comprehensive study.     

Crocetin, dimethylcrocetin, and safranal bind human serum albumin: stability and antioxidative properties

Crocetin (CRT) and dimethylcrocetin (DMCRT) are derived from crocins which are found in the stigmas of saffron (Crocus sativus L.), while safranal is the main component of saffron's essential oil. The aim of the present study was to examine their interaction with human serum albumin in aqueous solution at physiological conditions using constant protein concentration and various ligand contents. FT-IR and UV-visible spectroscopic methods were used to determine the ligands' binding mode, the binding constant, and the effects of ligand complexation on protein secondary structure. Structural analysis showed that crocetin, dimethylcrocetin, and safranal bind nonspecifically (H-bonding) via protein polar groups with binding constants of Kcrt =2.05 (+/-0.30) x 103 M-1, Kdmcrt = 9.60 (+/-0.35) x 104 M-1, and Ksaf = 2.11 (+/-0.35) x 103 M-1. The protein secondary structure showed no major alterations at low ligand concentrations (1 microM), whereas at higher content (1 mM), decrease of alpha-helix from 55% (free HSA) to 43-45% and increase of beta-sheet from 17% (free HSA) to 18-22% and random coil 7% (free HSA) to 10-14% occurred in the ligand-HSA complexes. The results point to a partial unfolding of protein secondary structure at high ligand content. The antioxidant activity of CRT, DMCRT, and safranal was also tested by the DPPH* antioxidant activity assay, and their IC50 values were compared to that of well-known antioxidants such as Trolox and butylated hydroxy toluene (BHT). The IC50 values of CRT and safranal were 17.8 +/- 1 microg/mL and 95 +/- 1 microg/mL, respectively, while the inhibition of DMCRT reached a point of 38.8%, which corresponds to a concentration of 40 microg/mL, and then started to decrease. The IC50 values of Trolox and BHT were 5.2 +/- 1 microg/mL and 5.3 +/- 1 microg/mL, respectively.     

蛋白质二级结构预测的支持向量机模型研究

蛋白质序列种类繁多，形态、结构和功能极其丰富多样，而已知三维结构的蛋白质数量却非常有限。为了能够利用有限的已知结构模拟和预测未知蛋白的结构，小样本统计学技术非常重要。本文主要探讨统计学习理论应用于蛋白质二级结构预测的模型。说明支持向量机(SVMs)进行蛋白质二级结构模式识别的主要步骤，讨论利用SVMs预测蛋白质二级结构优化过程中样本空间的确定方法。研究氨基酸序列向量化途径以及分析如何选择映射输入空间到特征空间的核函数等问题。