Maillard reaction and enzymatic browning affect the allergenicity of Pru av 1, the major allergen from cherry (Prunus avium)

The influence of thermal processing and nonenymatic as well as polyphenoloxidase-catalyzed browning reaction on the allergenicity of the major cherry allergen Pru av 1 was investigated. After thermal treatment of the recombinant protein rPru av 1 in the absence or presence of carbohydrates, SDS-PAGE, enzyme allergosorbent tests, and inhibition assays revealed that thermal treatment of rPru av 1 alone did not show any influence on the IgE-binding activity of the protein at least for 30 min, thus correlating well with the refolding of the allergen in buffer solution as demonstrated by CD spectroscopic experiments. Incubation of the protein with starch and maltose also showed no effect on IgE-binding activity, whereas reaction with glucose and ribose and, even more pronounced, with the carbohydrate breakdown products glyceraldehyde and glyoxal induced a strong decrease of the IgE-binding capacity of rPru av 1. In the second part of the study, the effect of polyphenoloxidase-catalyzed oxidation of polyphenols on food allergen activity was investigated. Incubation of rPru av 1 with epicatechin in the presence of tyrosinase led to a drastic decrease in IgE-binding activity of the protein. Variations of the phenolic compound revealed caffeic acid and epicatechin as the most active inhibitors of the IgE-binding activity of rPru av 1, followed by catechin and gallic acid, and, finally, by quercetin and rutin, showing significantly lower activity. On the basis of these data, reactive intermediates formed during thermal carbohydrate degradation as well as during enzymatic polyphenol oxidation are suggested as the active chemical species responsible for modifying nucleophilic amino acid side chains of proteins, thus inducing an irreversible change in the tertiary structure of the protein and resulting in a loss of conformational epitopes of the allergen.     

Expression, cloning, and immunological analysis of buckwheat (Fagopyrum esculentum Moench) seed storage proteins

cDNA of buckwheat (Fagopyrum esculentum Moench) was isolated from immature seeds harvested 14 days after pollination. Two genes, designated FA02 and FA18, were found to encode legumin-like proteins and were expressed during seed development. The deduced amino acid sequence of FA02 was identical to the N-terminal amino acid domain of BW24KD, which was believed to be a major buckwheat allergen (Urisu, A.; Kondo, Y.; Morita, Y.; Yagi, E.; Tsuruta, M.; Yasaki, T.; Yamada, K.; Kuzuya, H.; Suzuki, M.; Titani, K.; Kurosawa, K. Isolation and characterization of a major allergen in buckwheat seeds. In Current Advances in Buckwheat Research; Shinshu University Press: Matsumoto, Japan, 1995; pp 965--974). It was predicted that FA02 would be cleaved to generate two separate components, a 41.3 kDa alpha-subunit and a 21 kDa beta-subunit. Antiserum was raised against the deduced FA02 beta-subunit, and immunoblotting of total protein from buckwheat seeds (F. esculentum M. and Fagopyrum tartaricum Gaertn.) revealed that several groups of proteins reacted with the antiserum. Polypeptides in the 23--25 kDa range displayed the greatest reactivity.     

Purification and characterization of an antifungal protein, C-FKBP, from Chinese cabbage

An antifungal protein was isolated from Chinese cabbage (Brassica campestris L. ssp. pekinensis) by buffer-soluble extraction and two chromatographic procedures. The results of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that the isolated Chinese cabbage protein was identical to human FK506-binding protein (FKBP). A cDNA encoding FKBP was isolated from a Chinese cabbage leaf cDNA library and named C-FKBP. The open reading frame of the gene encoded a 154-amino acid polypeptide. The amino acid sequence of C-FKBP exhibits striking degrees of identity with the corresponding mouse (61%), human (60%), and yeast (56%) proteins. Genomic Southern blot analyses using the full-length C-FKBP cDNA probe revealed a multigene family in the Chinese cabbage genome. The C-FKBP mRNA was highly expressed in vegetative tissues. We also analyzed the antifungal and peptidyl-prolyl cis-trans isomerase activity of recombinant C-FKBP protein expressed in Escherichia coli. This protein inhibited pathogenic fungal strains, including Candida albicans, Botrytis cinerea, Rhizoctonia solani, and Trichoderma viride, whereas it exhibited no activity against E. coli and Staphylococcus aureus. These results suggest that recombinant C-FKBP is an excellent candidate as a lead compound for the development of antifungal agents.     

Jug r 4, a legumin group food allergen from walnut (Juglans regia Cv. Chandler)

Allergy to walnut is the most frequently reported tree nut allergy in the United States. Walnut 2S albumin, a vicilin-like protein, and a lipid transfer protein allergen have previously been described. Our objective was to clone and express a cDNA encoding a legumin group protein, assess IgE-binding with sera from walnut allergic patients, and investigate cross-reactivity with selected nuts. Primers were used to obtain the cDNA by 5' and 3' rapid amplification of cDNA ends from walnut mRNA. The cDNA was subcloned into the pMAL-c2X vector and the recombinant fusion protein, named rJug r 4, was expressed in Escherichia coli. The obtained cDNA encoded a precursor protein with a predicted molecular weight of 58.1 kD, which showed significant sequence homology to hazelnut and cashew legumin allergens. Serum IgE from 21 of 37 (57%) patients bound the rJug r 4 fusion protein. In vitro cross-reactivity was demonstrated with hazelnut, cashew, and peanut protein extracts.     

Purification, cloning, and immunological characterization of arginine kinase, a novel allergen of Octopus fangsiao

Arginine kinase (AK) is an important enzyme participating in energy metabolism in invertebrates, but, to date, there have been no reports that AK from octopus is an allergen. In this study, octopus AK was purified, and its molecular biological, immunological, and physicochemical characterizations were analyzed. The results showed that octopus AK was purified and confirmed by mass spectrometry for the first time, and its molecular mass was 38 kDa. The full-length gene sequence of octopus AK encompassed 1209 bp and was predicted to encode a protein with 348 amino acid residues. The homology of octopus AK and crustacean AK was about 54%, but the similarity between their three-dimensional structures was high. Octopus AK could react with mouse anti-shrimp AK and rabbit anti-crab AK polyclonal antibody singly. Octopus AK could also react with specific IgE of the sera from octopus-allergic patients effectively, whereas crab AK could inhibit the reaction between them. Finally, the IgE-binding activity of octopus AK could be reduced in the processes of thermal or acid-alkali treatment. In summary, AK was identified as a novel allergen in octopus, which had a sensitizing ability similar to that of crustacean AK. This is significant in allergy diagnosis and the treatment of octopus-allergic disorders.     

cDNA clone of a putative peanut (Arachis hypogaea L.) trypsin inhibitor has homology with peanut allergens Ara h 3 and Ara h 4

Trypsin inhibitors are pathogenesis-related (PR) proteins, which play an important role in the plant defense mechanism against insects and pathogens. Peanut trypsin inhibitors are low molecular mass seed storage proteins. Like peanut allergens, they are stable to acid and heat, resistant to digestion, and can have a negative impact on human health. In peanut, five Bowman-Birk trypsin inhibitors (BBTI) have been isolated and amino acid sequences published. However, to date, no peanut BBTI sequence is available at both the cDNA and the genomic levels. The objectives of this investigation were (i) to synthesize degenerate oligonucleotides based on conserved regions of published amino acid sequences of BBTI, BII, and BIII; (ii) to isolate, sequence, and analyze at least one positive peanut trypsin inhibitor cDNA clone using the synthesized (32)P-labeled oligonucleotides as probes; and (iii) to determine its trypsin inhibitory activity. Thirty-two degenerate oligonucleotides DNA primers of 24 nucleotides each were synthesized based on the published amino acid sequences of peanut BBTI, and two were selected as probes to screen a peanut Lambda gt 11 cDNA library. Three putative positive clones were isolated, purified, and subcloned, and one was sequenced. Sequence analysis revealed a partial cDNA clone of 643 bp with a start codon. This clone shares 93 and 96% nucleotide sequence homology with peanut allergens Ara h 3 and Ara h 4 cDNA clones, respectively. A trypsin inhibitor assay revealed that peanut allergen Ara h 3 has a trypsin inhibitory activity of 11 238 TIA/mg protein. We concluded that peanut allergen Ara h 3 may also function as a trypsin inhibitor.     

黑苦荞米黄酮提取工艺优化及其降血糖活性研究

为了获得合适的黑苦荞米黄酮提取工艺,该文采用单因素试验和响应面设计,选择乙醇体积分数、料液比、提取时间、提取温度4个因素,优化黑苦荞米黄酮提取工艺。试验结果表明,黑苦荞米黄酮的最佳提取条件是:乙醇体积分数54%,料液比1:24 g/m L,提取时间62 min,提取温度71℃。在此条件下,理论黄酮得率为2.21%,实际黄酮得率为2.20%,相对误差为0.45%。在此基础上进一步研究了黑苦荞米黄酮的α-淀粉酶抑制活性,结果表明7.5 mg/m L的黑苦荞米黄酮对α-淀粉酶活性的抑制率为54.05%,与二甲双胍(5 mg/m L)效果相当;此外,与空白对照相比,50μg/m L黑苦荞米黄酮能显著(P0.05)提高肝脏细胞Hep G2的葡萄糖消耗量(48.73%),并促进肝脏细胞糖原的合成。研究结果表明,黑苦荞米黄酮具有较好的辅助降血糖功效。     

甘薯茎线虫乙酰胆碱酯酶基因ace-3全长cDNA的克隆和序列分析

利用RT- PCR结合RACE技术克隆了甘薯茎线虫（Ditylenchus destructor）乙酰胆碱酯酶基因cDNA,Dd-ace-3,提交GenBank登录号EF583057。该cDNA序列5’端存在反式剪接引导序列SL1,全长2517bp ,包含一个1836bp的开放阅读框;在推导出的611个氨基酸残基的前体蛋白中, N 端的前21个氨基酸残基为信号肽, C-28的位置存在糖基磷脂酰肌醇(GPI)锚定位点,除此之外的562个氨基酸残基是成熟的乙酰胆碱酯酶序列,其预测的分子量为64396.81D。在一级结构中,形成催化活性中心的3个氨基酸残基 (Ser232 ,Glu360 和His479) ,以及在亚基内形成二硫键的6个半胱氨酸完全保守;在电鳐乙酰胆碱酯酶分子的催化功能域中存在14个保守的芳香族氨基酸残基,其中11个在甘薯茎线虫乙酰胆碱酯酶中完全保守。该酶的氨基酸序列与秀丽小杆线虫（Caenorhabditis elegans）ACE-3和ACE-4的同源性达56.1%和50.0%。与其它线虫和物种乙酰胆碱酯酶的聚类分析显示,甘薯茎线虫的乙酰胆碱酯酶与Ш型乙酰胆碱酯酶ACE-3同属一个支系。     

Molecular cloning, characterization, and expression of a cDNA coding Copper/Zinc superoxide dismutase from black porgy

A full-length complementary DNA (cDNA) clone encoding a putative copper/zinc superoxide dismutase (Cu/Zn-SOD) was amplified by a Polymerase Chain Reaction (PCR) based technique from cDNA synthesized from black porgy, Acanthopagrus schlegeli, mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 154 amino acid residues. The deduced amino acid sequence showed slightly higher identity (72.8-78.1%) with shark and swordfish Cu/Zn-SOD than with Cu/Zn-SOD from mammalian (68.1-70.7%) and plant (55.5-56.5%) sources. The residues required for coordinating copper and zinc are conserved as they are among all reported Cu/Zn-SOD sequences. The deduced amino acid sequence lacks mitochondria targeting sequence, which suggests that the black porgy cDNA clone encodes a cytosolic Cu/Zn-SOD. The coding region of Cu/Zn-SOD from black porgy was introduced into an expression vector, pET-20b(+), and transformed into Escherichia coli AD494(DE3)pLysS. A predominant achromatic zone was detected by activity staining of native PAGE. This indicates that the Cu/Zn-SOD cDNA clone can express active Cu/Zn-SOD enzyme in E. coli.     

Cloning and expression of a cDNA coding for catalase from zebrafish (Danio rerio)

A full-length complementary DNA (cDNA) clone encoding a catalase was amplified by the rapid amplication of cDNA ends-polymerase chain reaction (RACE-PCR) technique from zebrafish (Danio rerio) mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 526 amino acid residues and that it had a molecular mass of 59 654 Da. The deduced amino acid sequence showed high similarity with the sequences of catalase from swine (86.9%), mouse (85.8%), rat (85%), human (83.7%), fruit fly (75.6%), nematode (71.1%), and yeast (58.6%). The amino acid residues for secondary structures are apparently conserved as they are present in other mammal species. Furthermore, the coding region of zebrafish catalase was introduced into an expression vector, pET-20b(+), and transformed into Escherichia coli expression host BL21(DE3)pLysS. A 60-kDa active catalase protein was expressed and detected by Coomassie blue staining as well as activity staining on polyacrylamide gel followed electrophoresis.     

RT-PCR克隆籼稻叶绿素a/b结合蛋白基因全长cDNA及序列的in silico分析

根据日本晴cab4基因序列（GenBank：AK104499.1）设计引物,用RT-PCR的方法从籼稻9311中克隆了叶绿素a/b结合蛋白基因的全长cDNA,命名为cab-9311（cab gene from 9311）。insilico分析表明：cab-9311与cab4基因同源性为99%,编码的蛋白含有244个氨基酸,与cab4基因编码的蛋白同源性为98%。蛋白分子质量为26.9kD,理论等电点为6.52。第54位～第216位氨基酸是一个典型的叶绿素a/b结合蛋白功能域（chlorophyll a/bbinding domain）。跨膜分析和蛋白质三级预测显示,该蛋白在C端有一个典型的跨膜区。亚细胞定位分析表明该蛋白定位于叶绿体,是一个叶绿体内囊体膜上的锚定蛋白。     

Gene cloning and characterization of a novel recombinant antifungal chitinase from papaya (Carica papaya)

A chitinase cDNA clone (CpCHI, 1002 bp) was isolated from papaya fruit, which encoded a 275 amino acid protein containing a 28 amino acid signal peptide in the N-terminal end. The predicted molecular mass of the mature protein was 26.2 kDa, and its pI value was 6.32. On the basis of its amino acid sequence homology with other plant chitinases, it was classified as a class IV chitinase. An active recombinant CpCHI enzyme was overexpressed in Escherichia coli. The purified recombinant papaya chitinase showed an optimal reaction temperature at 30 degrees C and a broad optimal pH ranging from 5.0 to 9.0. The recombinant enzyme was quite stable, retaining >64% activity for 3 weeks at 30 degrees C. The spore germination of Alternaria brassicicola could be completely inhibited by a 76 nM level of recombinant CpCHI. Recombinant CpCHI also showed antibacterial activity in which 50% of E. coli was inhibited by a 2.5 microM concentration of the enzyme.     

Detecting potential IgE-reactive sites on food proteins using a sequence and structure database, SDAP-food

The high incidence of food allergies, including oral allergy syndrome, represent major considerations when introducing new crops and foods. A new structural database of allergenic proteins, SDAP-Food, http://fermi.utmb.edu/SDAP/, has been developed to aid in predicting the IgE-binding potential of novel food proteins and cross-reactivities among known allergens. The site is designed to facilitate the first steps of a decision tree approach to determine the allergenicity of a given protein, based on the sequence and structural similarity to known allergens and their IgE binding sites. Immunological tests can then be used to confirm the predictions. A hierarchical procedure for identifying potential allergens, using a physical property-based sequence similarity index, has been designed to identify regions that resemble known IgE binding sites. As an example, SDAP tools were used to find food allergen sequences similar to an IgE binding site of the Jun a 3 allergen from mountain cedar pollen. The SDAP sequence similarity search matched the Jun a 3 epitope to regions in several food allergens, including cherry (Pru av 2), apple (Mal d 2) and pepper (Cap a 1), which are, like Jun a 3, members of the plant pathogenesis-related (PR-5) protein family. Homology modeling, using our EXDIS/DIAMOD/FANTOM program suite, indicated a similar surface location and structure for the potential epitope region on all of these allergens. The quantitative approach presented here can be used as part of a screening process for potential allergenicity of recombinant food products.     

硒对苦荞硒、总黄酮和芦丁含量、分布与累积的影响

采用盆栽试验探讨土壤施硒对苦荞硒、总黄酮和芦丁含量、分布与累积的影响。结果看出,在土壤施硒0.5～2.0 mg/kg范围,苦荞根在苗期（40 d）大量吸收并累积硒,全生育期各器官硒含量极显著提高;在生长中后期(60～80 d) 硒的累积最快,累积量最多。硒在苦荞各器官中的分布为:在40 d时,根＞叶＞茎;60 d时Se0.0处理为叶＞根＞花＞茎,施硒各处理则为花＞根＞叶＞茎;80 d时Se0.0处理以花＞根＞叶＞茎＞子粒,Se0.5处理以花＞叶＞子粒＞茎＞根,施硒≥1.0 mg/kg的处理则为花＞叶＞茎＞根＞子粒。土壤施硒≤1.0 mg/kg促进苦荞生长,提高地上部各器官干重和植株总干重以及各器官总黄酮和芦丁含量与累积量,不改变总黄酮和芦丁的器官分布,增加苦荞中后期对总黄酮的累积;以Se0.5处理效应最佳,各差异达显著水平。过量的硒（Se1.5～2.0 mg/kg）显著抑制苦荞生长,降低各器官干重、总黄酮和芦丁含量与累积,不利于硒在子粒中富集和总黄酮在子粒中分布。表明在低硒土壤上栽培苦荞,土壤施硒以不超过1.0 mg/kg为宜,既能最大限度的提高苦荞各器官硒、总黄酮和芦丁含量和累积量,又可降低施用硒肥的成本和减少硒肥对环境的影响。     

A pectinmethylesterase gene associated with a heat-stable extract from citrus

A putative thermostable pectinmethylesterase (TSPME) protein of 36 kDa was isolated from heat-treated citrus finisher pulp. After purification and partial sequencing of the protein, a reverse genetic approach was used to obtain the complete genomic sequence of a new pectinmethylesterase (PME) gene, CsPME4, from Citrus sinensis (L.) Osb. cv. Valencia. The CsPME4 gene contained two exons of 1203 and 690 bp interrupted by a single positionally conserved intron of 1230 bp. A full-length CsPME4 cDNA clone amplified from Valencia orange juice vesicles shared 98% identity with the genomic clone. The encoded protein of the full-length CsPME4 cDNA shared 66 and 39% amino acid identity with the full-length encoded proteins of the citrus PME, CsPME1, and CsPME3, respectively, whereas the predicted mature protein of CsPME4 shared 80 and 61% identity with the predicted mature proteins of CsPME1 and CsPME3, respectively. Southern analysis demonstrated that CsPME4 was present in at least two copies in the Valencia orange genome. Northern analysis revealed that CsPME4 mRNA was accumulated mainly in young and developing tissues of Valencia orange. Several approaches to express recombinant CsPME4 in different systems failed to obtain active protein. Further research will be necessary to successfully express the putative TSPME gene CsPME4 for biochemical characterization.     

超高压处理改善苦荞淀粉理化性质及益生菌群落

为研究超高压(ultra-high pressure,HHP)处理对苦荞淀粉理化性质及功能性的影响,该文以6个不同压力处理后的苦荞淀粉为材料,利用扫描电镜、X射线衍射仪等分析了其淀粉颗粒大小、晶体结构、溶解度、透光率、冻融稳定性等特性,并通过建立体外模型模拟人体消化以及肠道发酵过程,利用气相色谱质谱联用技术和平板计数法,对小鼠粪便发酵液短链脂肪酸(short chain fatty acid、SCFAs)含量及主要菌群进行了测定。试验结果表明:超高压处理后苦荞淀粉颗粒出现凹陷与粘连状态,并逐渐失去原有形态,但其结晶类型仍为典型的A型,其结晶度在200 MPa时达到最大值41.8%;溶解度和膨胀度均随着压力的增加呈先减小后增大的趋势,并且其透光率会下降,其中在200 MPa时,溶解度和膨胀度都达到了最低0.83%和171%,而冻融稳定性会有一定的改善。此外,超高压处理后苦荞淀粉使肠道中双歧杆菌、乳酸杆菌的数量显著增加(P0.05),而大肠杆菌、肠球菌的生长受到显著抑制(P0.05)。同时,肠道内的pH值显著下降(P0.05),SCFAs中乙酸、丙酸、丁酸显著增加(P0.05),综上所述,超高压处理后苦荞可作为一种良好的天然改善肠道菌群的食物来源。     

嗜热子囊菌光孢变种cbh1基因的cDNA克隆及在毕赤酵母的高效表达

以嗜热子囊菌光孢变种(Thermoascus aurantiacus var. levisporus)总RNA为模板，通过RT-PCR克隆出外切纤维二糖水解酶基因cbh1片段，采用RACE方法获得全长cDNA克隆，其全长为1 710 bp，编码一种由457个氨基酸组成的单肽，推导的氨基酸序列中1～19位为信号肽序列，GenBank的登录号为AY840982。将该片段克隆到毕赤酵母(Pichia pastoris)分泌型表达载体pPIC9K上，获得表达重组质粒pPIC9K/cbh1，转化毕赤酵母GS115，所得重组子经PCR验证后进行诱导表达，筛选出一重组子GSp-15,经144 h诱导后，外切纤维二糖水解酶表达量为1.17 mg/mL，产酶活力为20.3 U/mL。     

Cloning and characterization of a plant defensin VaD1 from azuki bean

A recombinant mungbean defensin VrD1 was previously shown to exhibit antifungal and bruchid-resistant activity. To study the function and regulation of VrD1, genomic DNAs of plant defensins were isolated from Vigna radiata VC6089A and azuki bean Vigna angularis Kao Hsiung No. 6. The azuki bean defensin genomic DNA VaD1 was sequenced and converted to VaD1 cDNA. VaD1 defensin was purified from Vigna angularis Kao Hsiung No. 6 to apparent homogeneity. The complete amino acid sequence of the purified VaD1 was determined and was found to be exactly the same as the sequence deduced from VaD1 cDNA. VaD1 is a basic protein containing 46 amino acids with four conserved disulfide bonds and shares high sequence homology (78.3%) with VrD1. VaD1 inhibited the growth of Fusarium oxysporum, Fusarium oxysporum f. sp. pisi, Staphylococcus epidermidis, and Salmonella typhimurium. VaD1 also inhibited in vitro protein synthesis and bruchid larval development, but was less active than the recombinant VrD1.     

Biochemical characterization of a cambialistic superoxide dismutase isozyme from diatom Thallassiosira weissflogii: cloning, expression, and enzyme stability

A cDNA clone of 1081 bp encoding a second putative superoxide dismutase (SOD) from diatom Thallassiosira weissflogii was cloned by the polymerase chain reaction technique. The cDNA encodes a protein of 286 amino acid residues. Alignment of the truncated SOD sequence containing 217 amino acid residues with Mn-SODs from Vibrio mimicus and Escherichia coli, as well as two Fe-SODs from E. coli and Photobacterium leiognathi, this SOD showed greater homology to Mn-SOD. The residues required to coordinate the manganese ion were conserved in all reported Mn-SOD. The recombinant SOD has a half life of deactivation of 14.7 min at 65 degrees C. Its thermal inactivation rate constant Kd was 3.21 x 10(-2) min(-1). The enzyme was stable in a broad pH range from 4 to 12. The presence of imidazole (up to 0.8 M) and sodium dodecylsulfate (up to 4%) had little effect on the enzyme's activity. The atomic absorption spectrometric assay showed the presence of 0.3 atom of iron/manganese (2:1) in each SOD subunit. Reconstituted activity suggested that diatom SOD was cambialistic Fe/Mn-SOD.