Purification and characterization of a lectin from wild sunflower (Helianthus tuberosus L.) tubers

A lectin (HTTL) was isolated from Helianthus tuberosus L. (wild sunflower) tubers using ion-exchange chromatography, gel filtration, and affinity chromatography. The lectin agglutinated both untreated and trypsin-treated rabbit erythrocytes and did not agglutinate human blood cells of groups A, B, and O. The gel filtration showed the native molecular mass of 72 kDa and subunit molecular masses of 17 and 18.5 kDa on 12% SDS-PAGE. The lectin activity was inhibited by D-mannose. The tetrameric protein revealed a unique characteristic by forming a broad zone of protein in native PAGE at pH 8.3, which dissociated into seven subunits of varying e/m ratios on acid gel at pH 4.3. These seven bands revealed two polypeptide species of molecular masses 17 and 18.5 kDa on 12% SDS-PAGE, as in the case of the native protein. The result indicated that of the seven subunits, three were homotetramers of 17 kDa, one was a homotetramer of 18.5 kDa, and three were heterotetramers of 17 and 18.5 kDa. The lectin was thermostable with broad pH optima (pH 4-8) and had no requirement for divalent metal cations for its activity. The amino acid composition showed that the lectin contained higher amounts of glycine, alanine, and lysine, but no methionine. The sugar content was estimated to be 5.3% mannose equivalent. The HTTL was mitogenic to mouse spleen (total) cells at 25 microg/ml concentration. The lectin showed characteristics different from those of the earlier reported H. tuberosus tuber lectins and hence opens up a new avenue to investigate the structure-function relationship of lectin in Helianthus species.     

Ethanol production from Jerusalem artichoke tubers (Helianthus tuberosus) using Kluyveromyces marxianus and Saccharomyces rosei

This article examines the potential of Jerusalem artichoke as a source for ethanol and single-cell protein SCP. In addition, experimental results are presented on batch fermentation kinetics employing two strains of Kluyveromyces marxianus and one strain of Saccharomyces rosei grown on the extract derived from the tubers of Jerusalem artichoke. Of the three cultures examined, Kluyveromyces marxianus UCD (FST) 55-82 was found to be the best producer of ethanol grown in a simple medium at 35 degrees C. The ethanol production was found to be growth-associated having a mu(max) = 0.41. h(-1) and the ethanol and biomass yields were determined to be Y(p/s) = 0.45 (88% of the theoretical) and Y(x/s) = 0.04 with 92% of the original sugars utilized. On the basis of carbohydrate yields of Jerusalem artichoke reported in the literature and these batch kinetic studies with K. maxxianus, the calculated ethanol yields were found to range from 1400 kg ethanol acre (-1) yr(-1)to a maximum of 2700 kg ethanol acre (-1) yr(-1). The SCP yields for K. marxianus were calculated to range between 130 to 250 kg dry wt cell acre (-1) yr(-1). The potential for developing an integrated process to produce ethanol and SCP is also discussed.     

Molecular characterization of a novel peroxidase isolated from the ligninolytic fungus Pleurotus eryngii

A haem peroxidase different from other microbial, plant and animal peroxidases is described. The enzyme is secreted as two isoforms by dikaryotic Pleurotus eryngii in peptone-containing liquid medium. The corresponding gene, which presents 15 introns and encodes a 361-amino-acid protein with a 30-amino-acid signal peptide, was isolated as two alleles corresponding to the two isoforms. The alleles differ in three amino acid residues and in a seven nucleotide deletion affecting a single metal response element in the promoter. When compared with Phanerochaete chrysosporium peroxidases, the new enzyme appears closer to lignin peroxidase (LiP) than to Mn-dependent peroxidase (MnP) isoenzymes (58–60% and 55% identity respectively). The molecular model built using crystal structures of three fungal peroxidases as templates, also showed high structural affinity with LiP (C_α-distance 1.2 Å). However, this peroxidase includes a Mn²⁺ binding site formed by three acidic residues (E36, E40 and D175) near the haem internal propionate, which accounts for the ability to oxidize Mn²⁺. Its capability to oxidize aromatic substrates could involve interactions with aromatic residues at the edge of the haem channel. Another possibility is long-range electron transfer, e.g. from W164, which occupies the same position of LiP W171 recently reported as involved in the catalytic cycle of LiP.     

Growth and Differentiation of Plant Tissue Cultures: I. Changes Accompanying the Growth of Explants from Helianthus tuberosus tubers

Explants derived from artichoke tubers proliferate rapidly whencultured aseptically on a nutrient medium containing sugar,mineral salts, coconut milk, and 2, 4 dichlorophenoxyaceticacid. This rapid growth, which occurs in three distinct stages,is accompanied by massive changes in numbers of cells, freshand dry weight, and pronounced changes in the rates of gaseousexchange. The very short lag phase is followed by a period ofexponential cell division during which the original cellularpattern is partially obliterated. At the end of this growthphase redifferentiation occurs, tracheidea are formed in largenumbers, and distinctive nodular bodies appear which resemblemeristems. Under the conditions of these experiments they donot develop further.     

Plasmalemma fluidity in parenchyma cells from Jerusalem artichoke (Helianthus tuberosus L.) tubers during the break of dormancy

Plasmalemma-enriched fractions were isolated from Jerusalem artichoke tubers along the time course of dormancy break produced by cold treatment. A decrease of membrane fluidity was noted from the 3^rd to the 8^th week of this treatment, as well as a decrease of plasmalemma NADH dehydrogenase activity from the 5^th to the 8^th week. The plasmalemma lipid extracts studied revealed two major phospholipidic components: phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Their respective quantities decreased until the 12^th week, where the phosphatidylcholine level is lower than the phosphatidylethanolamine one. The observed changes are discused in relation to dormant and non-dormant states of tubers and the breaking of dormancy.     

Purification and characterization of the reconstitutively active adenine nucleotide carrier from mitochondria of Jerusalem artichoke (Helianthus tuberosus L.) tubers

The adenine nucleotide carrier from Jerusalem artichoke (Helianthus Tuberosus L.) tubers mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxapatite and Matrex Gel Blue B in the presence of cardiolipin and asolectin. SDS gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 33 kDa. When reconstituted in liposomes, the adenine nucleotide carrier catalyzed a pyridoxal 5-phosphate-sensitive ATP/ATP exchange. It was purified 75-fold with a recovery of 15% and a protein yield of 0.18% with respect to the mitochondrial extract. Among the various substrates and inhibitors tested, the reconstituted protein transported only ATP, ADP, and GTP and was inhibited by bongkrekate, phenylisothiocyanate, pyridoxal 5-phosphate, mersalyl and p-hydroxymercuribenzoate (but not N-ethylmaleimide). Atractyloside and carboxyatractyloside (at concentrations normally inhibitory in animal and plant mitochondria) were without effect in Jerusalem artichoke tubers mitochondria. V _max of the reconstituted ATP/ATP exchange was determined to be 0.53 mol/min per mg protein at 25°C. The half-saturation constant K _m and the corresponding inhibition constant K _i were 20.4 M for ATP and 45 M for ADP. The activation energy of the ATP/ATP exchange was 28 KJ/mol between 5 and 30°C. The N-terminal amino acid partial sequence of the purified protein showed a partial homology with the ANT protein purified from mitochondria of maize shoots.     

Stressed Jerusalem artichoke tubers (Helianthus tuberosus L.) excrete a protein fraction with specific cytotoxicity on plant and animal tumour cell

Wounds from Jerusalem artichoke (Helianthus tuberosus L.) tubers excrete bioactive metabolites from a variety of structural classes, including proteins. Here we describe a protein specifically active against tumour cells arising either from human, animal or plant tissues. The non-tumour animal cells or the plant callus cells are not sensitive to these excreta. The active product was only obtained after a wound-drought stress of plant tubers. The cytotoxicity varies according to the tumour cell type. For instance, some human tumour cell lines and especially the human mammary tumour cells MDA-MB-231 were shown to be very susceptible to the active product. The active agent is shown to contain an 18-kDa polypeptide with homology to a superoxide dismutase (SOD). A 28-kDa polypeptide, related to an alkaline phosphatase (AP), was shown to be tightly linked to this 18-kDa polypeptide. The excreted 28-kDa polypeptide also displayed a consensus sequence similar to the group of DING proteins, but with a smaller molecular weight. The superoxide dismutase polypeptide was shown to be involved in the antitumour activity, but the presence of smaller factors (MW<10 kDa), such as salicylic acid, can enhance this activity.     

Protein and mineral concentrations in tubers of selected genotypes of wild and cultivated Jerusalem-artichoke (Helianthus tuberosus,asteraceae)

Nineteen wild and cultivated genotypes of Jerusalem-artichoke (Helianthus tuberosus) whose tubers are used as food were analyzed for protein and mineral content at three stages of growth. The protein content of the tubers is comparable to or higher than that of other common root-type crops. Adequate macrominerals of calcium, magnesium, and phosphorus were found in Jerusalem-artichoke. Potassium and sodium concentrations were higher than other root crops. Trace elements (manganes, zinc, and copper) were present in adequate amounts, with iron content higher than several other root crops. Wild and cultivated Jerusalem-artichoke genotypes appear to contain adequate protein and minerals to contribute significantly toward a nutritionally balanced diet.     

菊芋类金属硫蛋白基因htMT2的克隆及其表达特征分析

从菊芋 (HelianthustuberosusL .)块茎cDNA文库中得到了一个新的植物类金属硫蛋白基因htMT2的cDNA序列 ,全长 5 0 9bp ,包括 2 4 0bp的开放阅读框、6 2bp的 5′端非翻译区、2 0 7bp的 3′端非翻译区。通过PCR获得了 2个htMT2编码区的部分基因组片段htMTG_1及htMTG_2 ,长度分别为 986bp和 982bp。分析表明两个基因组片段均包含 3个外显子及 2个内含子 ,编码一个由 79个氨基酸残基组成的多肽 ,与从htMT2推测的多肽完全一致 ,该多肽具有植物类金属硫蛋白的典型结构特征 ,N端及C端结构域富含Cys ,分别具有 8个和 7个Cys残基 ,上述两个结构域被一个无Cys的中间区分开。Southern杂交结果表明 ,htMT2在菊芋基因组中以小基因家族的形式存在。Northern杂交结果表明htMT2在叶片、叶柄、茎及块茎中均有表达 ,在茎中有较高水平的表达 ,但在根中未检测到杂交信号。经Cu2 处理后 ,htMT2在茎中的表达量显著降低。与其他 2型金属硫蛋白的序列同源性比较及htMT2对金属离子处理的反应均表明 ,htMT2是一种新的植物类金属硫蛋白基因。     

菊芋试管苗的初代培养及愈伤组织不定芽诱导

菊芋（Helianthus tuberosus Linn.）为菊科（Asteraceae）向日葵属（Helianthus Linn.）多年生草本植物,耐寒、耐旱、耐贫瘠、耐盐碱[1];其地下块茎富含菊糖,还可通过发酵生产乙醇,在功能性食用多糖及生物能源方面的开发潜力巨大。菊芋主要通过块茎进行无性繁殖,其种子成活率和发芽率均很低[2],严重阻碍了菊芋的杂交育种。近年来以植物组织培养为基础的一系列现代育种技术为菊芋的种质改良提供了新途径,但由于菊芋的愈伤组织难以诱导不定芽或体胚发生,导致以农杆菌转化为主的转基因育种技术的应用受到限制。     

A micro-batchwise technique method for rapid reconstitution of functionally active mitochondrial ADP/ATP carrier from Jerusalem artichoke (Helianthus tuberosus L.) tubers

A method for rapid reconstitution of ADP/ATP carrier from Jerusalem artichoke (Helianthus tuberosus L.) tubers mitochondria in proteoliposomes is described. The method is based on the well known property of the Amberlite resin to absorb the detergent allowing proteoliposome formation. This has been achieved by a micro-batchwise technique, using a rotating plate stirrer. An evaluation of the optimal conditions, in comparison with the more usual column method is presented. The purified ADP/ATP carrier, incorporated in proteoliposomes by this method, shows a high transport activity and a higher specific activity with respect to proteoliposomes obtained by the column procedure. Furthermore the proteoliposomal preparations are more homogeneous in size, with a diameter ranging from 300 to 350 nm. The method is suitable for the reconstitution of other membrane transport proteins.     

Production and Characterization of Monoclonal Antibodies against NADPH-Cytochrome P-450 Reductases from Helianthus tuberosus

Monoclonal antibodies (mAbs) against a plant NADPH-cytochrome P-450 (Cyt P-450) reductase from Jerusalem artichoke (Helianthus tuberosus) tuber were prepared. These antibodies were produced by hybridoma resulting from the fusion of spleen cells from a rat immunized with a purified preparation of the reductase and mouse myeloma cells. The mAbs thus obtained were screened for their interaction with the reductases, first in western dots and then in blots, and for their ability to inhibit the NADPH-cytochrome c (Cyt c) reductase activity from Jerusalem artichoke microsomes. Among the 11 clones giving a positive response on western blots, only 6 were also able to inhibit microsomal NADPH-Cyt c reductase activity, and the microsomal Cyt P-450 monooxygenase activities dependent upon electrons transferred by the reductase. Thus, two families of mAbs were characterized: a family of mAbs that interact with epitopes of the reductase implicated in the reduction of Cyt P-450 by NADPH (binding sites for NADPH, flavin mononucleotide, flavin adenine dinucleotide, and Cyt P-450), and a structural family, whose members recognize epitopes outside the active site of the reductases. These mAbs specifically recognize the reductase, and all of them interact with all of the isoforms, indicating that important primary or secondary structural analogies exist between the isoforms, not only at the active site, but also at the level of epitopes not directly associated with catalytic activity.     

高效液相色谱-电化学(库仑电极)阵列检测海水处理对菊芋幼苗体内氯原酸及小分子物质的影响

以不同浓度海水处理菊芋幼苗体,利用高效液相色谱-电化学(库仑电极)阵列检测技术检测不同处理时间植物体内氯原酸及小分子物质的差异.结果表明:0%海水处理下氯原酸含量变化不显著,而15%和30%海水处理下氯原酸含量变化显著,15%海水处理下在1 h时较2 h和3 h时高,而30%海水处理下在3 h时较1 h和2 h时高.处理后菊芋幼苗体植株产生的其他一些未知的小分子物质尚有待定性和进一步考察其变化规律.     

Cloning and sequence analysis of cDNA coding for a lectin from Helianthus tuberosus callus and its jasmonate-induced expression

Two lectins (designated as HTA I and HTA II) that seemed to be isolectins were found in Helianthus tuberosus callus. cDNA encoding HTA I was isolated from a ZAP Express expression library by immunoselection by using the anti-HTA antiserum. The sequence of this cDNA consisted of 432 bp nucleotides coding for a polypeptide of 143 amino acid residues (Mr, 15,314). When introduced into E. coli, the cDNA directed the synthesis of active HTA I as indicated by the hemagglutination activity. The deduced amino acid sequence showed homology with some lectins and jasmonate-induced proteins. When callus was cultured in the presence of methyl jasmonate (MeJA), the hemagglutination activity increased in a dose-dependent manner. The levels of expression of the HTA protein and of the corresponding mRNA also increased in the treated callus. In view of these results, HTA I is considered to be a jasmonate-induced protein.     

Molecular identification of vaginal lactobacilli isolated from Bulgarian women

Lactobacilli play an important role in maintaining the vaginal health of women. The development of suitable bacterial replacement therapies for the treatment of vaginosis requires knowledge of the vaginal lactobacilli species representation. The aim of this study was to identify at the species level vaginal Lactobacillus isolates obtained from Bulgarian women in childbearing age by using different molecular methods. Twenty-two strains of lactobacilli isolated from vaginal samples were identified and grouped according to their genetic relatedness. A combined approach, which included amplified ribosomal DNA restriction analysis (ARDRA), ribotyping and polymerase chain reaction (PCR) with species-specific oligonucleotide primers was applied. All vaginal isolates were grouped into 5 clusters in␣comparison with a set of 21 reference strains based␣on the initial ARDRA results, which was then confirmed by ribotyping. Finally, the strains were subjected to PCR analysis with eight different species-specific primer pairs, which allowed most of␣them to be classified as belonging to one of␣the␣following species: Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus and Lactobacillus plantarum. In conclusion, this study suggests that the most straightforward identification strategy for vaginal lactobacilli would be grouping by ARDRA or ribotyping, followed by PCR specific primers identification at species level.     

Purification and characterization of the enzymes of fructan biosynthesis in tubers of Helianthus tuberosus 'Colombia': I. Fructan: fructan fructosyl transferase

Fructan: fructan fructosyl transferase (FFT), one of the enzymesinvolved in the synthesis of ß-2,1 linked fructosepolymers has been purified 205-fold from tubers of Helianthustuberosus harvested in the accumulation phase. The molecularweight of the native as well as the SDS-denatured protein isapproximately 70 kDa. On IEF, the protein was separated intofive molecular species with pl values between pH 4.5–5.0.The optimum pH for fructosyl transfer activity was between 5.5–7.0.Temperature optimum was in the range of 25-35° C; the Q10value between 25 and 5° C was 1.14. FTT catalysed the self-transferof fructosyl groups with GF₂, GF₃, GF₄ or GF₅ as substrate andacceptor. The rate of elf-transfer with both GF₂ and GF₃ increasedlinearly with substrate concentration up to 100 mol m^–3and was still not saturated at 600 and 300 mol m^–3, respectively.FFT was unable to hydrolyse GF or to catalyse the self-transferwith GF but could mediate the transfer of fructosyl units frominulin on to GF. Key words: Fructan: fructan fructosyl transferase, Helianthus tuberosus, Jerusalem artichoke, purification, kinetics     

蜈蚣肠道共生菌来源的吩嗪类化合物分离与鉴定

动植物共生菌是药物发现的重要来源之一。为了获取结构新颖且具有良好生物活性的微生物次生代谢产物,本文利用划线分离方法从蜈蚣肠道中分离获得一株共生菌WG4(未鉴定),经土豆液体发酵培养,发酵液经乙酸乙酯萃取、浓缩,获得次级代谢产物浸膏。浸膏经硅胶柱层析、高效液相色谱等分离方法反复纯化,获得3个化合物。通过核磁共振谱和低分辨质谱测试,3个化合物分别鉴定为1-羟基吩嗪(1)、吩嗪-1-甲酰胺(2)和吩嗪-1-羧酸(3)。据文献报道,吩嗪化合物具有抗菌、抗虫、抗病毒、抗肿瘤等生物活性,本实验证实了动植物来源的共生菌具有生产药源化合物的潜力。