Further analogues of plant peptide hormone phytosulfokine-alpha (PSK-alpha) and their biological evaluation.

Phytosulfokine-alpha (PSK-alpha), a sulfated growth factor of structure H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH universally found in both monocotyledons and dicotyledons, strongly promotes proliferation of plant cells in culture. In studies on the structure/activity relationship of PSK-alpha the synthesis was performed of a series of a further 23 analogues modified in position 1, 3 or 4 as well as simultaneously in positions 1 and 3 of the peptide chain. Peptides were synthesized by the solid phase method according to the Fmoc procedure on a Wang-resin. Free peptides were released from the resin by 95% TFA in the presence of EDT. All peptides were tested by competitive binding assay to the carrot membrane using 3H-labelled PSK-alpha according to the test of Matsubayashi et al. Among these peptide analogues, [H-Phe(4-Cl)1]-PSK-alpha (IV), [H-Phe(4-I)1]-PSK-alpha (VII), and [Phe(4-Cl)3]-PSK-alpha (XI) retained 30% PSK-alpha activity. Analogue [Tyr(PO3H2)3]-PSK-alpha (IX) showed 10% of PSK-alpha activity.     

Plant peptide hormone phytosulfokine (PSK-alpha): synthesis of new analogues and their biological evaluation.

Phytosulfokine-alpha (PSK-alpha), a sulfated growth factor (H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH) universally found in both monocotyledons and dicotyledons, strongly promotes proliferation of plant cells in culture. In our studies on structure/activity relationship in PSK-alpha the synthesis of a series of analogues was performed: [H-D-Tyr(SO3H)1]- (9), [H-Phe(4-SO3H)1]- (10), [H-D-Phe(4-SO3H)1]- (11), [H-Phg(4-SO3H)1]- (12), [H-D-Phg(4-SO3H)1]- (13), H-Phe(4-NHSO2CH3)1]- (14), [H-D-Phe(4-NHSO2CH3)1]- (15), [H-Phe(4-NO2)1]- (16), [H-D-Phe(4-NO2)1]- (17), [H-Phg(4-NO2)1]- (18), [H-D-Phg(4-NO2)1]- (19), [H-Hph(4-NO2)1]- (20), [H-Phg(4-OSO3H)1]- (21), [Phe(4-NO2)3]- (22), [Phg(4-NO2)3]- (23), [Hph(4-NO2)3]- (24), [H-Phe(4-SO3H)1, Phe(4-SO3H)3]- (25) [H-Phe(4-NO2)1, Phe(4-NO2)3]- (26), [H-Phg(4-NO2)1, Phg(4-NO2)3]- (27), [H-Hph(4-NO2)1, Hph(4-NO2)3]- (28) and [Val3]- PSK-alpha (29). For modification of the PSK-alpha peptide chain the novel amino acids and their derivatives were synthesized, such as: H-L-Phg(4-SO3H)-OH (1), H-D-Phg(4-SO3H)-OH (2), Fmoc-Phg(4-SO3H)-OH (3), Fmoc-D-Phg(4-SO3H)-OH (4), Boc-Phg(4-NHSO2CH3)-OH (5), Boc-D-Phg(4-NHSO2CH3)-OH (6) Boc-Phe(4-NHSO2CH3)-OH (7), and Boc-D-Phe(4-NHSO2CH3)-OH (8). Peptides were synthesized by a solid phase method according to the Fmoc procedure on a Wang-resin. Free peptides were released from the resin by 95% TFA in the presence of EDT. All peptides were tested by competitive binding assay to the carrot membrane using 3H-labelled PSK according to the Matsubayashi et al. test.     

Alkaloid production by plant cell suspension cultures of Holarrhena antidysenterica: I. Effect of major nutrients

The effect of major nutrients on growth and alkaloid production by plant cell culture of Holarrhena antidysenterica was studied with a view to increasing the yield of the alkaloid conessine, a therapeutic drug used for treatment of dysentery and helminthic disorders. The studies resulted in development of a modified Murashige and Skoog (MS) medium that contained 60 mM total nitrogen with a NH(4) (+)-to-NO(3) (-) ratio of 5:1, 0.25 mM phosphate, and 40 g/L sucrose. The growth regulators 2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin (Kn) were also found to affect the synthesis of alkaloid. Using an optimal level of inoculum (3 g/L), the modified medium resulted in alkaloid synthesis of 0.66 g/100 g dry cell weight, which represented a 4.25-fold increase over that obtained in standard MS medium.     

Effect of carbon dioxide on cell growth and saponin production in suspension cultures of Panax ginseng

The effects of carbon dioxide supply within the range of 1–5 % (along with purified air), on cell culture of Panax ginseng were investigated in a balloon type bubble bioreactor containing 4 dm³ of Murashige and Skoog (MS) medium supplemented with 7.0 mg dm⁻³ indolebutyric acid, 0.5 mg dm⁻³ kinetin and 30 g dm⁻³ sucrose. A 1 % CO₂ supply was found beneficial for the production of cell mass; however, increasing CO₂ concentration to 2.5 and 5 % decreased the biomass accumulation. CO₂ enrichment was not beneficial for saponin production and 1, 2.5, and 5 % CO₂ supply resulted in decrease in saponin accumulation up to 11.6, 19.5, and 50.6 %, respectively.     

Effect of cyclophosphamide and 61.22 GHz millimeter waves on T-cell, B-cell, and macrophage functions

The present study was undertaken to investigate whether millimeter waves (MMWs) at 61.22 GHz can modulate the effect of cyclophosphamide (CPA), an anti-cancer drug, on the immune functions of mice. During the exposure each mouse's nose was placed in front of the center of the antenna aperture (1.5 x 1.5 cm) of MMW generator. The device produced 61.22 +/- 0.2 GHz wave radiation. Spatial peak Specific Absorption Rate (SAR) at the skin surface and spatial peak incident power density were measured as 885 +/- 100 W/kg and 31 +/- 5 mW/cm(2), respectively. Duration of the exposure was 30 min each day for 3 consecutive days. The maximum temperature elevation at the tip of the nose, measured at the end of 30 min, was 1 degrees C. CPA injection (100 mg/kg) was given intraperitoneally on the second day of exposure to MMWs. The animals were sacrificed 2, 5, and 7 days after CPA administration. MMW exposure caused upregulation in tumor necrosis factor-alpha (TNF-alpha) production in peritoneal macrophages suppressed by CPA administration. MMWs also caused a significant increase in interferon-gamma (IFN-gamma) production by splenocytes and enhanced proliferative activity of T-cells. Conversely, no changes were observed in interleukin-10 (IL-10) level and B-cell proliferation. These results suggest that MMWs accelerate the recovery process selectively through a T-cell-mediated immune response.     

Semicontinuous bioreactor production of a recombinant human therapeutic protein using a chemically inducible viral amplicon expression system in transgenic plant cell suspension cultures

Plant cell culture is an alternative for the production of recombinant human therapeutic proteins because of improved product safety, lower production cost, and capability for eukaryotic post‐translational modification. In this study, bioreactor production of recombinant human alpha‐1‐antitrypsin (rAAT) glycoprotein using a chemically inducible Cucumber mosaic virus (CMV) viral amplicon expression system in transgenic Nicotiana benthamiana cell culture is presented. Optimization of a chemically inducible plant cell culture requires evaluation of effects of timing of induction (TOI) and concentration of inducer (COI) on protein productivity and protein quality (biological functionality). To determine the optimal TOI, the oxygen uptake rate (OUR) of the plant cell culture was chosen as a physiological indicator for inducing maximum rAAT expression. Effects of COI on rAAT production were investigated using a semicontinuous culture, which enables the distinction between effects of growth rate and effects of inducer concentration. An optimized semicontinuous bioreactor operation was further proposed to maximize the recombinant protein production. The results demonstrated that the transgenic plant cells, transformed with the inducible viral amplicon expression system, maintain higher OUR and exhibit lower extracellular protease activity and lower total phenolics concentration in the optimized semicontinuous bioreactor process than in a traditional batch bioreactor operation, resulting in a 25‐fold increase in extracellular functional rAAT (603 µg/L) and a higher ratio of functional rAAT to total rAAT (85–90%). Surprisingly, sustained rAAT production and steady state, long‐term bioreactor operation is possible following chemical induction and establishment of the viral amplicons. Biotechnol. Bioeng. 2010; 106: 408–421. © 2010 Wiley Periodicals, Inc.     

红豆杉细胞悬浮培养中不同添加物对细胞生长和紫杉醇合成的影响

采用正交实验检测红豆杉（Ｔａｘｕｓ ｃｈｉｎｅｎｓｉｓ（Ｐｉｌｇｅｒ）Ｒｅｈｄ．）细胞悬浮培养中水杨酸、Ｄ－果糖、甘露醇和硫酸镧对细胞生长和紫杉醇（ｔａｘｏｌ）积累的影响。添加１０ｇ／ＬＤ－果糖,可使细胞的鲜重和干重明显增加;添加６０ｇ／Ｌ甘露醇使细胞的鲜重和干重明显减少;１ｍｇ／Ｌ水杨酸仅使细胞鲜重增加,对干重影响不明显;硫酸镧对细胞生长无明显影响。单独添加这４种物质,紫杉醇含量均下降,同时添加     

Effect of norleucine on growth,protein and catechol oxidase synthesis in tobacco suspension cultures

Tobacco cells were grown in artificial media with defined amino acid composition. In such media, the addition of methionine or norleucine caused increases in the specific activity of the catechol oxidase, while in the normal medium norleucine depressed it. The differences of the effect of norleucine on synthesis of catechol oxidase and on cell growth is demonstrated, as is the reversibility of the norleucine effect by methionine. The incorporation of norleucine into a purified enzyme fraction is shown. The change in the electrophoretic patterns of the enzyme during growth in the absence and presence of norleucine was followed. [¹⁴C]-Leucine incorporation by control and norleucine treated cells was examined and it was shown that protein synthesis in the norleucine treated cells was markedly changed and total incorporation reduced. Incorporation into soluble protein was reduced, but increased in the 20 000 g precipitate fraction. Nevertheless use of autoradiography indicates that some catechol oxidase is apparently synthesised in the presence of norleucine.     

Effect of auxins on the formation of ubiquinone by tobacco plant cells in suspension culture

Ubiquinone (UQ) formation in BY-2 tobacco cells was especially promoted by a high concentration of 2,4-D. 2,4,5-T, MCP and NAA also promoted UQ formation in these cells. The UQ content in the cells cultured at high concentrations of 2,4-D was higher than that of controls throughout the culture period. The addition of 2,4-D at an early period in cell growth was very effective in promoting UQ formation, but addition at the stationary phase was ineffective. Cell growth was improved by adding phosphate to the medium but UQ content was decreased. UQ content decreased slowly during subculturing, whereas cell growth recovered gradually.     

植物生长调节物质IP－1号对木薯产量及其生物性状的影响

１９９０和１９９１年在木薯（ＭａｎｉｈｏｔｅｓｃｕｌｅｎｔａＣｒａｎｔｚ）生长期以植物生长调节物质ＩＰ－１号０,２０,３０和４０ｐｐｍ进行叶面喷洒,结果表明：３０ｐｐｍ处理可使木薯块根产量平均增加５４．４４％,块根淀粉含量平均提高２０．８１％。单株最大薯重提高３１．５５％,块根数增加２１．１７％,块根长度增长１７．６２％,地上部鲜重增加３４．３６％,植株高度增加４．３６％,植株收获期保留青叶数增加１９．４２％,主茎直径增加６．２６％,块根直径增加２．５８％,叶片的叶绿素和蛋白质含量分别提高５．５７％和２５．９６％,叶片光合作用强度提高１５．８６％,而对主茎高度、主茎节数没有明显影响。     

岩黄连细胞生长与营养物质消耗的动态学研究

在岩黄连细胞悬浮培养过程中,对培养液pH值,碳源、氮源和磷酸盐含量,以及细胞生物量和生物碱含量进行测定,分析其动态变化过程。结果显示培养液pH值在培养初期降低,后逐渐升高;碳源在培养过程中逐渐被利用,磷酸盐和氮源在培养中期几乎耗尽,其中磷酸盐的消耗速率最快;悬浮细胞的生长周期为20 d左右,第18天细胞鲜重和干重达最大,而第21天脱氢卡维丁和小檗碱的含量最高,分别为8.22mg/L和4.31mg/L。结果表明营养物质(碳、氮和磷)的吸收与细胞生长以及生物碱的合成密切相关,营养元素的相对消耗速率为磷>氮>碳,推测氮和磷是影响岩黄连细胞培养的主要因素。     

Solution structure of the plant defensin VrD1 from mung bean and its possible role in insecticidal activity against bruchids

Vigna radiata plant defensin 1 (VrD1) is the first reported plant defensin exhibiting insecticidal activity. We report herein the nuclear magnetic resonance solution structure of VrD1 and the implication on its insecticidal activity. The root-mean-square deviation values are 0.51 +/- 0.35 and 1.23 +/- 0.29 A for backbone and all heavy atoms, respectively. The VrD1 structure comprises a triple-stranded antiparallel beta-sheet, an alpha-helix, and a 3(10) helix stabilized by four disulfide bonds, forming a typical cysteine-stabilized alphabeta motif. Among plant defensins of known structure, VrD1 is the first to contain a 3(10) helix. Glu26 is highly conserved among defensins; VrD1 contains an arginine at this position, which may induce a shift in the orientation of Trp10, thereby promoting the formation of this 3(10) helix. Moreover, VrD1 inhibits Tenebrio molitor alpha-amylase. Alpha-amylase has an essential role in the digestion of plant starch in the insect gut, and expression of the common bean alpha-amylase inhibitor 1 in transgenic pea imparts complete resistance against bruchids. These results imply that VrD1 insecticidal activity has its basis in the inhibition of a polysaccharide hydrolase. Sequence and structural comparisons between two groups of plant defensins having different specificity toward insect alpha-amylase reveal that the loop between beta2 and beta3 is the probable binding site for the alpha-amylase. Computational docking experiments were used to study VrD1-alpha-amylase interactions, and these results provide information that may be used to improve the insecticidal activity of VrD1.     

Effect of plant growth regulators on in vitro fiber development from unfertilized and fertilized Egyptian cotton ovules

Unfertilized and fertilized ovules of Gossypium barbadense Giza 45 (extra long staple variety) were used to study the effect of plant growth substances (auxins, gibberellins and cytokinins) on in vitro fiber initiation and development. Kinetin, alone did not increase total fiber unit (TFU) of unfertilized ovules, while an increase in TFU value occurred when a constant level of IAA and GA₃ were used either separately or in combination in the liquid medium. GA₃ used alone, produced a higher TFU value than that produced by IAA, whilst, IAA with a constant level of GA₃ (5 M) produced the highest value of TFU. GA₃ with a constant level of IAA (5 M) produced a lower TFU value. Kinetin reduced the stimulatory effect of IAA and GA₃ on TFU value when used in combination with either substance. In fertilized ovules, the highest level of TFU was reached when IAA, with a constant level of GA₃, was added to the medium, whilst its lowest level was obtained when IAA was used alone. Estimation of in vitro fiber production, as well as the effect of growth substances used in different concentrations on in vitro fiber initiation and development from unfertilized and fertilized ovules of Egyptian cotton varieties Gossypium barbadense Giza 45 are discussed.     

水杨酸、茉莉酸甲酯和乙烯利对野葛细胞悬浮培养生产葛根素的影响

野葛〔Ｐｕｅｒａｒｉａｌｏｂａｔａ(Ｗｉｌｌｄ.)Ｏｈｗｉ〕是我国及其他东亚国家传统的药用资源,其主要药用成分为葛根素,广泛用于治疗多种心血管疾病[1]。由于近年来生态环境的人为破坏,野生野葛资源已不能满足人们对葛根素药物的需求,通过细胞工程可以实现野葛资源的可持续性开发。本项目组已经建立了野葛组织培养和植株再生[2]及细胞悬浮培养体系[3],研究结果表明,悬浮细胞培养物能够生产葛根素,但产量仍有待于提高[3]。已知水杨酸(ｓａｌｉｃｙｌｉｃａｃｉｄ,ＳＡ)、乙烯和茉莉酸类化合物(ｊａｓｍｏｎａｔｅｓ,ＪＡｓ)能够在高…     

Generation and maintenance of suspension cultures from cotyledons and their organogenic potential of two mangrove species, Sonneratia alba and S. caseolaris

Liquid cultures were successfully generated from cotyledons of two Sonneratia species, S. alba and S. caseolaris in Murashige and Skoog (MS) medium containing 0.1 μmol L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Adventitious roots differentiated from cotyledons of S. alba. Proliferated cells were subcultured and a large volume of suspension cells was subsequently established in 100-mL flasks. All the cytokinins tested inhibited cell proliferation. After three years of culture, the potential to differentiate was tested as indicated by greening of the cells. Greening occurred when suspension cells were transferred to solid MS medium with and without 0.1 μmol L⁻¹ 2,4-D. Greening was stimulated by low concentrations of the weak auxins indolebutyric acid (IBA) and naphthaleneacetic acid (NAA) while 2,4-D stimulated late-stage greening. Abscisic acid (ABA) inhibited greening. Gibberellic acid (GA₃) at 1.0 μmol L⁻¹ stimulated callus greening and was not inhibitory even when tested at high concentrations. Cytokinins were inhibitory in combination with 0.1 μmol L⁻¹ of either IBA or NAA. The cause of different effects of plant hormones on growth and differentiation was discussed. Small-scale liquid media and 24-well culture plates of solid media methods developed in this paper are suitable for the optimization of hormonal conditions for cell proliferation and differentiation.     

Effect of oxygen supply on cell growth and saponin production in bioreactor cultures of Panax ginseng

The effects of oxygen supply within the range 20.8–50% (using pure oxygen and air), on cell cultures of Panax ginseng were investigated in a balloon-type bubble bioreactor (5 L capacity, containing 4 L Murashige and Skoog medium, supplemented with 7.0 mg L⁻¹ indolebutyric acid, 0.5 mg L⁻¹ kinetin and 30 g L⁻¹ sucrose). A 40% oxygen supply was found to be optimal for the production of both cell mass and saponin yielding values of 12.8 g (DW) L⁻¹, 4.5 mg (g DW)⁻¹ on day 25, respectively. Low (20.8%, 30%) and high (50%) oxygen concentration supplies were unfavorable to cell growth and saponin accumulation. The results indicate that oxygen supplementation to bioreactor-based ginseng cultures was beneficial for biomass accumulation and saponin production.     

Physico-chemical factors influencing the shikonin derivatives production in cell suspension cultures of Arnebia euchroma (Royle) Johnston, a medicinally important plant species

Cell suspension cultures of Arnebia euchroma were raised from in vitro leaf-derived friable callus on liquid MS [Murashige and Skoog] medium supplemented with BAP (6-benzylaminopurine) (10.0 μM) and IBA (indole-3-butyric acid) (5.0 μM). A two-stage culture system was employed using growth and production medium for cell biomass and shikonin derivatives, respectively. Factors such as light, temperature, sucrose and pH (hydrogen ion concentration) were studied to observe their effect on the shikonin derivative production. Light conditions completely inhibited shikonin derivative production. Out of different temperature regimes tested, the highest yield (586.17 μg/g FW) was found at 25°C. Maximum production (656.14 μg/g FW) was observed in 6% sucrose. An alkaline pH (7.25-9.50) favoured shikonin derivative production. The results showed that physical and chemical factors greatly influence the production of shikonin derivatives in cell suspension cultures of A. euchroma. Therefore, by employing optimum culture conditions, it is possible to enhance the production of secondary compounds from the cells. The factors optimized for in vitro production of shikonin derivatives during the present study can successfully be employed for their large-scale production in bioreactors.     

Trifluoroethanol effects on helix propensity and electrostatic interactions in the helical peptide from ribonuclease T1.

Trifluoroethanol (TFE) is often used to increase the helicity of peptides to make them usable as models of helices in proteins. We have measured helix propensities for all 20 amino acids in water and two concentrations of trifluoroethanol, 15 and 40% (v/v) using, as a model system, a peptide derived from the sequence of the alpha-helix of ribonuclease T1. There are three main conclusions from our studies. (1) TFE alters electrostatic interactions in the ribonuclease T1 helical peptide such that the dependence of the helical content on pH is lost in 40% TFE. (2) Helix propensities measured in 15% TFE correlate well with propensities measured in water, however, the correlation with propensities measured in 40% TFE is significantly worse. (3) Propensities measured in alanine-based peptides and the ribonuclease T1 peptide in TFE show very poor agreement, revealing that TFE greatly increases the effect of sequence context.