Diamine Oxidase from Cultured Roots of Hyoscyamus niger: Its Function in Tropane Alkaloid Biosynthesis

Diamine oxidase was partially purified from cultured roots of Hyoscyamus niger L. that produce considerable amounts of tropane alkaloids, and then characterized. N-Methylated amines inhibited the activity of the enzyme more strongly than the corresponding primary amines. N-Methylputrescine was the best substrate of those studied, the respective K_m values for it and for putrescine and cadaverine being 0.33, 2.85, and 6.25 millimolar. The specificity constants V_max/K_m for putrescine and cadaverine were 11 and 1% of the constant for N-methylputrescine. Marked specificity for the N-methylated diamine would enable the Hyoscyamus enzyme to function specifically in tropane alkaloid biosynthesis.     

Organogenesis, Differentiation and Histolocalization of Alkaloids in Cultured Tissues and Organs of Duboisia myoporoides R. Br.

Duboisia myoporoides R. Br. shoots were regenerated from non-organogenicand organogenic calli induced with nine different cytokinin/auxincombinations. Alkaloid colour reagents localized tropane alkaloidsin the vascular regions which had large cells in the secondaryxylem of the basal stem sections of the non-rooted shoots. Tropanealkaloids were localized in shoots regenerated from calli inducedwith two different cytokinin/auxin combinations. No alkaloidswere localized in shoots regenerated from calli induced withother cytokinin/auxin combinations. However, only nicotine wasdetected by GC-MS in the non-rooted shoots regenerated fromcalli induced with three different cytokinin/auxin combinations.Tropane alkaloids were also localized in xylem cells of rootsregenerated from calli induced with two different cytokininand auxin combinations independently. The presence or absenceof nicotine, hyoscyamine and scopolamine in different culturedplant materials was confirmed by GC-MS, indicating that althoughthe root is the main site for alkaloid biosynthesis, with suitablecell differentiation, alkaloid biosynthesis may take place incultured shoots without root initiation. Copyright 2000 Annalsof Botany Company Duboisia myoporoides, Corkwood tree, Solanaceae, tropane alkaloid, alkaloid localization, shoot culture, root culture, iodoplatinate     

一种紫菜多糖的制备及对淋巴细胞生长的影响

用DEAE－纤维素和SephadexG-200柱层析法分离纯化条斑紫菜的热水提取物,从中得到多糖PY2,并测出其分子量为2．0％10^4。用紫外和红外光谱对PY2的性质进行了鉴定。进一步测定了PY2对体外培养的小鼠骨髓细胞及淋巴细胞生长的影响。结果表明,PY2是一种少见的紫菜多糖,它不含有大多数紫菜侈糖具有的3,6－内醚－兰乳糖和硫酸基,它对小鼠脾脏淋巴细胞、胸腺淋巴细胞以及混合淋巴细胞的增殖有一定的抑制作用,而对骨髓细胞的增殖没有明显的影响。     

Red rot resistance in interspecific protoplast fusion product progeny of Porphyra yezoensis and P. tenuipedalis (Bangiales, Rhodophyta)

Nine primary regenerants were recovered by interspecific protoplast fusion of Porphyra yezoensis Ueda T‐14 (Py) (cultivated Porphyra) and Porphyra tenuipedalis Miura (Pt). This combination is difficult to achieve with conventional sexual hybridization, yet is important in that non‐cultivated P. tenuipedalis is partially resistant (PR) to red rot disease, caused by the microbial pathogen, Pythium porphyrae Takahashi et Sasaki. Out of the nine primary regenerants, two strains (Py‐Pt‐4 and Py‐Pt‐7) were like the parent, P. tenuipedalis, while the rest were like the other cultivated parent P. yezoensis T‐14 in their life cycle. Red rot resistance was assessed in parents and interspecific fusion product progeny (FPP) by exposing the foliose thalli to equivalent infection and measuring two parameters of the host‐pathogen interactions: supported fungal biomass and amount of disease produced. Intermediate resistance between P. yezoensis T‐14 (1.00) and P. tenuipedalis (0.13) was observed in two of the Py‐type FPP, Py‐Pt‐2F₂ (0.25) and Py‐Pt‐5F₂ (0.23). Stable inheritance of resistance was observed through two subsequent generations. The morphologic and reproductive characteristics of the regenerated foliose thalli, and nature of host‐pathogen interactions were used to further verify the hybrid origin of the FPP. Host‐pathogen interactions were followed using epi‐fluorescence and scanning electron microscopy (SEM). The zoospores encysted at higher rates on the susceptible cultivated parent (P. yezoensis T‐14) germinated immediately and the short germ tubes formed appres‐soria and penetrated the algal cells near the site of encystment. While on the PR parental (P. tenuipedalis) and partially resistant FPP (PRFPP) progeny (Py‐Pt‐2F₂ and Py‐Pt‐5F₂) the low rate of zoospore encystment was followed by cyst germination, but only a few of the germ tubes formed appressoria and penetrated the thallus surface. Long germ tubes (with no appressoria) were seen growing on the thallus surface without host penetration. The minimal rate of encystment concomitant with low rate of appressorium formation on the PR parent and PRFPP was observed as the major factor responsible for the partial resistance in these thalli.     

Research Note: Simple molecular discrimination of cultivated Porphyra species (Porphyra yezoensis and Porphyra tenera) and related wild species (Bangiales,Rhodophyta)

To discriminate between cultivated Porphyra species (Porphyra yezoensis and Porphyra tenera) and closely related wild Porphyra species, we developed a polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) analysis of the rbcL gene using five restriction enzymes. Although our previous PCR‐RFLP analyses of internal transcribed spacer (ITS) rDNA and plastid RuBisCO spacer regions could not always discriminate wild P. yezoensis, wild P. tenera, and closely related wild species, the PCR‐RFLP profiles of the rbcL gene were useful in discriminating samples collected from natural habitats. Therefore, PCR‐RFLP analysis of the rbcL gene will help in the simple identification of a large number of samples, not only for the establishment of reliable cultures as breeding material, but also for the taxonomic investigations of species that are closely related to cultivated Porphyra.     

紫菜多糖对免疫细胞及肿瘤细胞生长的影响（英文）

采用细胞培养技术测定从条斑紫菜中得到的多糖PY3对小鼠免疫细胞及人肿瘤细胞K562生长的影响。结果表明,PY3对小鼠骨髓细胞和脾脏淋巴细胞的增殖以及对混合淋巴细胞反应均有一定的促进作用。PY3对血癌细胞K562的生长有一定的抑制作用,研究表明多糖PY3不仅能够提高小鼠免疫细胞的功能,而且有一定的抗肿瘤作用。     

Effects of Culture Conditions on Tropane Alkaloid Formation in Hyoscyamus niger Suspension Cultures

Tropane alkaloid formation was studied under various culture conditions in suspension cultures of Hyoscyamus niger L. High aeration increased cell growth and the contents of hyoscyamine and scopolamine. White’s medium inhibited cell growth but increased the hyoscyamine content. The low concentrations of nitrogen and phosphate in White’s medium were responsible for such effects. NAA promoted cell growth but inhibited hyoscyamine formation. A hyoscyamine content of 0.05% dry weight (DW) (0.14 mg in 25 ml of culture medium) was achieved in modified LS medium in which the phosphate concentration was decreased to 0.2 mm and the auxin omitted.

Effects of precursor feeding on alkaloid formation were also studied with the putative precursors of hyoscyamine. N-Methylputrescine, tropine, phenylalanine, and tropic acid increased alkaloid formation, but the promotive effect of these precursors varied from one experiment to another.     

条斑紫菜R-藻红蛋白提纯工艺研究

对条斑紫菜叶状体R-藻红蛋白提纯方法进行了研究和分析。首先分别采用冻融法、化学试剂法、溶胀法等单一及组合细胞破碎方法对条斑紫菜细胞进行破碎,结果表明溶胀 组织捣碎法效果最佳。其次用25%～60%饱和度范围内的硫酸铵沉淀法粗提藻红蛋白,发现25%～45%硫酸铵分步沉淀法效果最好,再经结晶法盐析纯度(D565nm/D280nm)达到2.088。盐析液用SephadexG-25层析柱脱盐,再经羟基磷灰石(HA)柱层析,纯度可达到4.98。R-藻红蛋白的最大吸收峰在565nm,其室温荧光发射峰为578nm。SDS-PAGE结果显示,R-藻红蛋白可分为α、β两个亚基,Mr分别为17.0×103和19.0×103。     

Confirmation of cultivated Porphyra tenera (Bangiales, Rhodophyta) by polymerase chain reaction restriction fragment length polymorphism analyses of the plastid and nuclear DNA

Polymerase chain reaction restriction fragment length polymorphism (PCR‐RFLP) analysis of the plastid ribulose‐1,5‐bisphosphate carboxylase (RuBisCo) spacer region was developed for a more reliable and rapid species identification of cultivated Porphyra in combination with PCR‐RFLP analysis of the nuclear internal transcribed spacer (ITS) region. From the PCR‐RFLP analyses of the plastid and nuclear DNA, we examined seven strains of conchocelis that were used for cultivation as Porphyra tenera Kjellman but without strict species identification. The PCR‐RFLP analyses suggested that two strains, C‐32 and 90‐02, were cultivated P. tenera and that the other five strains, C‐24, C‐28, C‐29, C‐30 and M‐1, were Porphyra yezoensis f. narawaensis Miura. To identify species more accurately and to reveal additional genetic variation, the two strains C‐32 and 90‐02 were further studied by sequencing their RuBisCo spacer and ITS‐1 regions. Although RuBisCo spacer sequences of the two strains were identical to each other, each of their ITS‐1 sequences showed a single substitution. The sequence data again confirmed that the two strains (C‐32 and 90‐02) were cultivated P. tenera, and suggested that the two strains showed some genetic variation. We concluded that PCR‐RFLP analysis of the plastid and nuclear DNA is a powerful tool for reliable and rapid species identification of many strains of cultivated Porphyra in Japan and for the collection of genetically variable breeding material of Porphyra.     

条斑紫菜粗多糖对淋巴细胞和支持细胞的增殖作用

目的:探讨条斑紫菜粗多糖的一些生物学功能,包括促进淋巴细胞增殖以及促进支持细胞生长的功能。方法:将原代培养的大鼠和小鼠脾淋巴细胞以及大鼠睾丸支持细胞分别接种于96孔板中,将不同浓度的条斑紫菜粗多糖(0.05、0.5、5、10mg/mL)加入培养,每种浓度设6个复孔,并设对照组孔。采用四氮甲唑蓝(MTT)法检测细胞的增殖率。结果:条斑紫菜粗多糖能够显著地促进大鼠、小鼠脾淋巴细胞和大鼠睾丸支持细胞的增殖,增殖率随多糖浓度的提高而升高;当多糖浓度为10mg/mL时,增殖率分别为137.3%、149%和454.5%。结论:条斑紫菜粗多糖具有提高免疫力及促进生殖功能的作用,其对睾丸支持细胞的增殖作用目前还未见报道。     

悬浮培养中不同理化因子对烟草发状根生长及烟碱合成的影响

悬浮培养中烟草(Nicotiana tabacum)发状根的生长及烟碱合成受到基本培养基浓度、初始pH值、激素种类和浓度等因素的影响.一个5 cm长的根尖,悬浮在40 mL、pH值为6.0、附加1.0 mg·L-1IAA的1/2 MS液体培养基中,28℃、散光条件下培养30天,烟碱产量可达0.241 mg·mL-1.     

悬浮培养中不同理化因子对烟草发状根生长及烟碱合成的影响

悬浮培养中烟草(Nicotiana tabacum)发状根的生长及烟碱合成受到基本培养基浓度、初始pH值、激素种类和浓度等因素的影响。一个5 cm长的根尖, 悬浮在40 mL、pH值为6.0、附加1.0 mg.L－1IAA的1/2 MS液体培养基中, 28 ℃、散光条件下培养30天, 烟碱产量可达0.241 mg.mL－1。     

Effects of light photon flux density and spectral quality on photosynthesis and respiration in Porphyra yezoensis (Bangiales, Rhodophyta)

A comparative study on the effects of photon flux density and spectral quality on photosynthesis and respiration in the marine red alga Porphyra yezoensis Ueda was conducted using a light dispenser. Results showed that the photosynthetic response, expressed in light utilization efficiency (LUE) for preselected wave bands of photosynthetically active radiation, could be ranked as follows: white > green > red > blue. Differences in LUE were also found between conchocelis and gametophyte stages and different strains of the alga. Pre-illumination light compensation, post-illumination light compensation, light saturation, respiration and photorespiration were also measured and compared. The relationships between light exposure, photosynthetic capacity, and the natural environmental conditions are discussed.     

Effect of Yeast Extract on h6h Gene Expression and Tropane Alkaloids Production in Atropa belladonna L. Hairy Roots

Russian Journal of Plant Physiology - Plant alkaloids, are one of the largest groups of pharmaceutical metabolites. Scopolamine and atropine are most common tropane alkaloids in Atropa belladonna...     

Comparison of Cell Growth and Alkaloid Production of Catharanthus roseus Cells Cultured in Shake Flask and in Bioreactor

The cell growth and alkaloid production of Catharanthus roseus (L.) G. Don cells cultured in the shake flasks with different volumes and in the stirred tank bioreactor (10 L) were compared. Cell dry weight and alkaloid production showed no significant difference in the small volume scale-up shake flasks. When more broths were added to a certain volume in the shake flask, both cell weight and alkaloid production were decreased. The maximum cell dry weight was similar between the cell cultures in the shake flask and the bioreactor, but the alkaloid production of cells was much less in the bioreactor. Gas regime and shear stress were recognized to be the main factors contributing the important effect on alkaloid production during the scale-up processes.     

Alkaloid biosynthesis in somatic hybrids of Duboisia leichhardtii F. Muell. and Nicotiana tabacum L.

Somatic hybrids of Duboisia leichhardtii and Nicotiana tabacum were obtained by electrofusion followed by individual cloning. The hybrid nature of the cloned cells and regenerated shoots was confirmed by cytological investigation and ribosomal-DNA analysis, respectively. The hybrid plantlets predominantly produced nicotine, while Duboisia plantlets produced both tropane and nicotine alkaloids. Activities involved in tropane-alkaloid biosynthesis were examined in a series of precursor-feeding experiments. The presence in the hybrid plants of activities responsible for the reduction of tropinone, the hydroxylation and epoxidation of hyoscyamine, and the conversion of nicotine to nornicotine demonstrated the presence of the Duboisia genes for these enzyme activities.We thank Mr. T. Shikanai, Kyoto University, for the preparation of rice rRNA. We also appreciate Dr. J.H. Fitchen, Kyoto University, for critical discussion and English correction.     

Effects of phthalate esters on dendritic cell subsets and interleukin-4 production in fluorescein isothiocyanate-induced contact hypersensitivity

Phthalate esters with short alkyl chains, such as di-ethyl (DEP), di-n-propyl (DPP), and di-butyl phthalate (DBP), have adjuvant effects on an FITC-induced contact hypersensitivity mouse model. The adjuvant effects of DPP and DBP are associated with enhanced trafficking of FITC-presenting CD11b(+) dendritic cells (DC). DEP has relatively weak activity as to FITC-positive cell migration. Here we demonstrated that DBP and DPP also increased the number of FITC-positive CD8alpha(+) DC in draining lymph nodes. We also found enhanced production of interleukin-4 in draining lymph nodes after FITC sensitization with DEP, DPP, or DBP, suggesting an additional adjuvant mechanism of phthalate esters.     

外源植物生长调节物质对烟草根中烟碱含量和烟碱合成酶活性变化的生理效应

水培法研究烟草打顶和喷施外源生长调节物质的结果表明:打顶的比不打顶的烟草根中鸟氨酸脱羧酶(ODC)、腐胺N-甲基转移酶(PMT)和N-甲基腐胺氧化酶(MPO)活性升高,烟叶中烟碱含量剧增;打顶喷施ABA和6-BA烟叶中烟碱含量升高,喷施IAA和GA3的下降,IAA的效果更明显.