Isolation of a napin-like polypeptide with potent translation-inhibitory activity from Chinese cabbage (Brassica parachinensis cv green-stalked) seeds.

A heterodimeric napin-like polypeptide was isolated from Brassica parachinensis seeds with a procedure involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, FPLC-ion exchange chromatography on Mono S and FPLC-gel filtration on Superdex 75. The N-terminal sequence of the 5 kDa subunit of the polypeptide (PAGPFRIPKKRKKEE) showed high homology with other 2S storage proteins like napins and albumins. The polypeptide potently inhibited translation in a cell free system with an IC50 of 6.2 nM. The translation-inhibiting activity of the polypeptide was relatively stable in the pH range 6-11 and in the temperature range 10-50 degrees C.     

A napin-like polypeptide from dwarf Chinese white cabbage seeds with translation-inhibitory, trypsin-inhibitory, and antibacterial activities

Napins are 1:1 disulfide-linked complexes of a smaller (ca. 4kDa) subunit and a larger (ca. 10kDa) subunit. The intent of the present study was to ascertain the production of napin by the seeds of a Brassica species that has not been examined previously, and also to explore new biological activities of the napin. A heterodimeric 11-kDa napin-like polypeptide has been isolated from Chinese white cabbage (Brassica chinensis cv dwarf) seeds with a protocol comprising ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S and FPLC-gel filtration on Superdex 75. The N-terminal sequence of the 7-kDa subunit manifests striking similarity to napin large chain, albumin and trypsin inhibitor. The N-terminal sequence of the 4-kDa subunit is homologous to napin large chain and an antimicrobial peptide. The napin-like polypeptide inhibited translation in the rabbit reticulocyte system with an IC50 of 18.5nM. This translation-inhibitory activity was stable between pH 4 and 11, and between 10 and 40 degrees C. The polypeptide inhibited trypsin with a higher potency ( IC50 = 8.5 microM) than it inhibited chymotrypsin (IC50 = 220 microM), but was devoid of ribonuclease and antifungal activities. It manifested antibacterial activity against Pseudomonas aeruginosia, Bacillus subtilis, Bacillus cereus, and Bacillus megaterium. The results revealed that the napin-like polypeptide from Chinese white cabbage seeds exhibited some potentially exploitable activities.     

A napin-like polypeptide with translation-inhibitory, trypsin-inhibitory, antiproliferative and antibacterial activities from kale seeds.

A heterodimeric napin-like polypeptide with translation-inhibiting and antibacterial activities has been isolated from kale seeds. The purification procedure entailed ion-exchange chromatography on dielthylaminoethyl (DEAE)-cellulose, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S, and gel filtration by FPLC on Superdex 75. The napin-like polypeptide was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and Mono S. Its 7-kDa large subunit differs in N-terminal amino acid sequence from the 4-kDa small subunit. The polypeptide inhibited translation in the rabbit reticulocyte lysate system with an IC50 of 37.5 nM. This activity was preserved between pH 5 and pH 11, and between 10 and 40 degrees C. It fell to a low level at pH 3 and pH 13 and at 70 degrees C. Antibacterial activity against Bacillus, Megabacterium, and Pseudomonas species and antiproliferative activity against leukemia L1210 cells were observed. However, the polypeptide did not exert antifungal, ribonuclease, or protease activity.     

秋季菜心田主要害虫生态位

对福州郊区菜心田害虫种群的生态位进行了初步研究。结果表明,黄曲条跳甲、小菜蛾和蚜虫为秋季菜心田最主要的害虫。不同害虫的生态位上存在明显分化,黄曲条跳甲在时间生态位上占有较多资源,小菜蛾则在空间生态位上占有相当多资源。并分析探讨了田间不同害虫种群混合发生的竞争机制及相应的害虫控制策略。     

Peroxidase and chitinase isoenzyme activities during root infection of Chinese cabbage with Plasmodiophora brassicae

Roots of two Chinese cabbage (Brassica campestris L. ssp. pekinensis) varieties, one tolerant and one susceptible, were inoculated with Plasmodiophora brassicae in liquid medium and in soil. Chitinase and peroxidase activities were determined in roots and shoots 1–21 days after inoculation with resting spores of Plasmodiophora and the enzyme activities compared with healthy tissue of the same age. In infected roots of the susceptible variety ‘Granat’ chitinase activity was higher than in the control 10 days after inoculation with spores. In the tolerant variety ‘Parkin’ we detected an increase in chitinase activity at the same time, which was about twice that of ‘Granat’. Chitinase activity in ‘Granat’ was also enhanced on day 13, 14 and 17 after inoculation, whereas chitinase activity in ‘Parkin’ was lower in the infected roots than in the controls during that period. In the shoots no correlation between chitinase activity and infection in the two varieties was observed. Chitinase from Chinese cabbage was further characterized and showed a pH optimum at pH 4.5–5.5 and a temperature optimum at 35–45°C. After isoelectric focusing 7 isoenzymes were discovered, but there were almost no differences between infected and healthy root extracts. Two isoenzymes with pI 8.7 and 8.8 showed cross-reactivity with an antiserum against bean chitinases. The molecular mass of these isoenzymes was determined as 33 kDa. Total peroxidase activity was generally higher in root tissue of both varieties than in the shoots. Peroxidase activity was increased most prominently in infected ‘Granat’ roots on day 13 after inoculation and of both varieties on day 17 compared to the controls. In clubbed tissue of ‘Granat’ a specific peroxidase isoenzyme appeared the first time 21 days after inoculation and was most prominent 28–30 days after inoculation. This isoenzyme had a molecular mass of ca 24 kDa and a pI of ca 8.8. With respect to our results the strategy of the Plasmodiophorales for plant attack is discussed.     

A novel peptide with ribonuclease and translation-inhibitory activities from fruiting bodies of the oyster mushroom Pleurotus ostreatus.

From the fresh fruiting bodies of the oyster mushroom a peptide with a molecular weight of 9 kDa and demonstrating a novel N-terminal sequence GPCYLVAFYESSGRR was isolated. The isolation procedure involved ion exchange chromatography on CM-Sepharose and Mono S. The peptide was adsorbed on both types of chromatographic media. The peptide demonstrated a ribonuclease activity of 650 U/mg toward yeast transfer RNA. It inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of 15 nM.     

小菜蛾取食后上海青的保护酶活力变化

上海青遭受小菜蛾取食为害后,0.5小时内叶片的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)、多酚氧化酶(PPO)和总酚含量发生变化。就一天内的变化趋势而言,受害叶片的SOD、CAT、POD、PPO活力和总酚含量总体上比正常叶片低,但在15：00时,正常叶片的SOD、CAT和POD活力最低时,受害叶片的三种酶活力却明显上升,在11：00时,正常叶片PPO活力为最低值时,受害叶片的PPO活力达最高,这可能是过度胁迫造成的,正常叶片的PPO活力与总酚量负相关,但受害叶片不呈现这种关系,此外,受害植株的未受害叶的保护酶活力也不同程度地发生变化,可见上海青对小菜蛾的取食胁迫后保护酶的反应是系统性的。     

Construction of random sheared fosmid library from Chinese cabbage and its use for Brassica rapa genome sequencing project

As a part of the Multinational Genome Sequencing Project of Brassica rapa, linkage group R9 and R3 were sequenced using a bacterial artificial chromosome (BAC) by BAC strategy. The current physical contigs are expected to cover approximately 90% euchromatins of both chromosomes. As the project progresses, BAC selection for sequence extension becomes more limited because BAC libraries are restriction enzyme-specific. To support the project, a random sheared fosmid library was constructed. The library consists of 97536 clones with average insert size of approximately 40 kb corresponding to seven genome equivalents, assuming a Chinese cabbage genome size of 550 Mb. The library was screened with primers designed at the end of sequences of nine points of scaffold gaps where BAC clones cannot be selected to extend the physical contigs. The selected positive clones were end-sequenced to check the overlap between the fosmid clones and the adjacent BAC clones.Nine fosmid clones were selected and fully sequenced. The sequences revealed two completed gap filling and seven sequence extensions, which can be used for further selection of BAC clones confirming that the fosmid library will facilitate the sequence completion of B. rapa.     

大白菜细胞质雄性不育保持系3411-7--RAPD特异扩增片段的克隆测序

利用RAPD技术从大白菜细胞质雄性不育保持系3411-7的DNA中得到一个特异扩增片段MOPB04600。回收该特异扩增片段并将其克隆到pGEM-TEasy载体上进行序列测定。结果表明该片段全长600bp,其碱基组成为A+T=72.33%。通过与GenBank+EMBL+DDBJ+PDB中的455,972个序列进行同源性比较,同源性均小于30%,表明该片段为一新发现的序列。并对该特异片段的来源及其可能的作用进行探讨。     

Construction of random sheared fosmid library from Chinese cabbage and its use for Brassica rapa genome sequencing project

As a part of the Multinational Genome Sequencing Project of Brassica rapa,linkage group R9 and R3 were sequenced using a bacterial artificial chromosome(BAC) by BAC strategy.The current physical contigs are expected to cover approximately 90%euchromatins of both chromosomes.As the project progresses,BAC selection for sequence extension becomes more limited because BAC libraries are restriction enzyme-specific.To support the project,a random sheared fosmid library was constructed.The library consists of 9...     

干旱胁迫对菜苔叶片保护酶活性和膜脂过氧化的影响

以3个不同耐旱性的菜苔（Brassica parachinensis L.H.Bailey）品种为试材,研究了干旱胁迫对叶片保护酶活性和膜脂过氧化的影响及其与抗旱性的关系。干旱胁迫条件下,菜苔叶片的电解质外渗率和MDA含量呈上升趋势,叶绿素含量、抗坏血酸含量和SOD活性呈下降趋势,CAT活性表现为先上升后下降。耐旱品种比不耐旱品种具有较高的叶绿素含量和抗坏血酸含量,具有较低的电解质外渗率和MDA含量;耐旱品种的SOD活性比不耐旱品种下降幅度小。轻度干旱胁迫下,耐旱品种的CAT活性上升幅度比不耐旱品种高;重度干旱胁迫下耐旱品种的CAT活性下降程度比不耐旱品种低。耐旱品种的POD活性在干旱条件下先上升而后降低,不耐品种的POD活性处于下降趋势。干旱6d后,耐旱品种的SOD、CAT和POD活性显著高于不耐旱品种。     

Medium and genotype factors influencing shoot regeneration from cotyledonary explants of Chinese cabbage (Brassica campestris L. ssp. pekinensis)

Medium conditions for reliable shoot regeneration from cotyledonary explants of Chinese cabbage were examined. Maximum shoot regeneration was obtained in the presence of 5 mg/l BA and 0.5 mg/l NAA. Shoot induction was further improved by the addition of AgNO₃ as well as higher concentrations (1.2–1.6%) of agar in the regeneration medium. When 123 genotypes were tested, a large variation in regeneration frequency was observed, ranging from 95% to 0%. Shoot regeneration frequency was not related to origin and days to maturity of the genotypes. Ethylene production from cultured explants seemed to play an important role in shoot regeneration. Explants of highly responsive genotypes or if cultured on the medium solidified with a higher concentration of agar generally showed low levels of ethylene production. However, AgNO₃, which also enhanced shoot induction, resulted in an increase in ethylene production. The possible interaction between ethylene and shoot regeneration is discussed. Received: 26 September 1997 / Revision received: 6 March 1998 / Accepted: 20 March 1998     

Molecular and functional characterization of a cyclophilin with antifungal activity from Chinese cabbage

An antifungal protein that inhibits the growth of filamentous fungal pathogens was isolated from Chinese cabbage (Brassica campestris L. ssp. pekinensis) by affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose. The N-terminal amino acid sequence of the protein was highly homologous to that of plant cyclophilins and consequently the protein was denoted as C-CyP. To understand the antifungal activity of C-CyP, we isolated a cDNA encoding its gene from a Chinese cabbage leaf cDNA library. The Chinese cabbage genome bears more than one C-CyP gene copy and C-CyP mRNA is highly expressed in all tissues except the seeds. Recombinant C-CyP catalyzed the cis-trans inter-conversion of the Ala-Pro bond of the substrate, which indicates this protein has peptidyl-prolyl cis-trans isomerase activity. It also inhibited the growth of several fungal pathogens.     

Total extract of Beta vulgaris var. cicla seeds versus its purified phenolic components: antioxidant activities and antiproliferative effects against colon cancer cells

Introduction – Beta vulgaris var. cicla (BV) leaves contain chemopreventive compounds that have been investigated for new drug discovery. These compounds belong to the family of the apigenin‐glycosides. Since the leaves are seasonal products containing high percentages of water, they are easily degradable during storage in fresh conditions. To be stored they require a drying process, consuming time and a large amount of energy. The extraction of apigenin‐glycosides may also be conveniently performed from BV seeds, which represent a stable and year‐long available biomass. Objectives – The present report was undertaken to find a strategy of purification of bioactive flavonoids from BV seeds and test their ability to inhibit proliferation both on human colon cancer (RKO) cells and normal human fibroblasts (HF). Materials and methods – The ethyl‐acetate extract of BV seeds was fractionated on a Sephadex LH 20 column. A fraction of this extract, labeled as P4, exploited a marked antiproliferative activity on RKO cells. The components of P4 were purified on an RP₁₈ column chromatography and identified by HPLC‐ESI‐MS as 2,4,5‐trihydroxybenzaldehyde, 2,5‐dihydroxybenzaldehyde, vanillic acid, xylosylvitexin, glucopyranosyl‐glucopyrasyl‐rhamnetin and glucopyranosyl‐xylosyl‐rhamnetin. All of them were tested for cytostatic and cytotoxic activity on RKO and HF cells. Results – Xylosylvitexin exhibited the strongest antiproliferative activity on RKO cells, together with an enhancement of the apoptosis, an increase of cells in the G₁ phase and a reduction of cells in the S phase; on the contrary, the proliferation of HF was significantly stimulated. Conclusion – Xylosylvitexin is the main and more efficient chemopreventive compound in BV seeds, but the natural cocktail of molecules, represented by P4 fraction, showed a better compromise between the antiproliferative activity on RKO cells and the enhancement of HF proliferation. Copyright © 2011 John Wiley & Sons, Ltd.     

丝瓜籽中一种具有翻译抑制活性和胰蛋白酶抑制剂活性的多肽——Luffin P1的纯化和性质

通过硫酸铵分级沉淀、CM 5 2阳离子交换层析、蓝胶亲和层析和FPLCMonoS阳离子交换层析 ,从丝瓜籽抽提液中分离到一种多肽luffinP1。经MALDI TOFMS测得其分子量为 5 2 2 6 .5。氨基酸序列测定及同源性分析发现 ,luffinP1的N端 11个氨基酸序列与丝瓜籽中的一种 6 .5K富含Arg Glu的多肽AGRP的部分序列相同 ,并与南瓜籽中一种胰蛋白酶抑制剂C2肽具有很高的同源性。体外分析表明 ,luffinP1同时具有两种生物活性 :(1)对兔网织红细胞裂解液系统蛋白质生物合成有较强的抑制作用 ,IC50 为 0 .6nmol L ;(2 )具有明显的胰蛋白酶抑制活性 ,IC50 为 2 2 μmol L。     

Purification and characterization of charantin, a napin-like ribosome-inactivating peptide from bitter gourd (Momordica charantia) seeds.

A peptide designated charantin, with a molecular mass of 9.7 kDa, was isolated from bitter gourd seeds. The procedure comprised affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on Mono S and gel filtration on Superdex 75. The N-terminal sequence of charantin exhibited marked similarity to that of the 7.8-kDa napin-like peptide previously isolated from bitter gourd seeds. Charantin inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of 400 nm, a potency lower than that of the previously reported small ribosome-inactivating protein gamma-momorcharin (IC50 = 55 nm) which also exhibited an abundance of arginine and glutamate/glutamine residues. Charantin reacted positively in the N-glycosidase assay, yielding a band similar to that formed by the small ribosome-inactivating proteins gamma-momorcharin and luffin S.     

Identification of indole-3-acetaldehyde and indole-3-acetaldehyde reductase in Chinese cabbage

Indole-3-acetaldehyde (IAAId) was identified as a natural compound in Chinese cabbage ( Brassica campestris L. ssp. pekinensis cv. Granat) seedlings by chemical conversion to indole-3-acetaldoxime (1AOX) followed by mass spectroscopy. The lAAId reductase (EC 1.2. 3.1), an enzyme with a molecular mass of 32 kDa, was extracted, purified 5-fold and characterized. The enzymatic IAAld reduction showed a pH optimum at 6–7 and a marked preference for NADPH as cofactor The K_m value for IAAld was 125 μ M , for NADPH 36 μ M . The enzyme reaction was inhibited at high NADPH concentrations (>200 μ M ) and modulated by IAA and indole-3-ethanol (IEt). Sulfhydryl reagents inhibited IEt formation, suggesting the participation of SH-groups in the reaction. Phenylacetaldehyde and benzaldehyde were competitive substrates, while acetaldehyde acted partly as an inhibitor, and partly as an activator on the IAAld reduction. IAAld reductase activity was also detected in other Brassica species. The importance of this enzyme is discussed with respect to the possibilities of IAA biosynthesis in the Brassicaceae.