Plantlet regeneration enhances solasodine productivity in hairy root cultures of <Emphasis Type="Italic">Solanum khasianum</Emphasis> Clarke

Summary Shoot regeneration in hairy root cultures of Solanum khasianum Clarke influences root growth, solasodine production. and permeabilization of solasodine into the medium. These parameters are dependent on exogenously supplied auxin and cytokinin: the effect being both concentration-and clone-dependent. Hairy root cultures with no shoot regeneration showed high permeabilization of solasodine into the medium by the sixth week of incubation, suggesting the medium acts as a sink for the solasodine synthesized by the roots. Solasodine in the culture medium was toxic to the transformed roots and caused browning of root tips. In a separate set of experiments, the hairy root cultures showed regeneration of approximately 50–70 mm long shoots after treatment with indole-3-acetic acid and kinetin. These hairy root cultures had inereased levels of solasodine production, compared to cultures without shoot regeneration. The plantlets formed in the hairy root cultures accumulated some of the solasodine, thereby reducing its permcabilization into the medium. Transport of solasodine from root to shoot reduced the toxic effect of solasodine in the root zone and extended the exponential growth phase by 8-10d.     

Use of alginate and cryo-protective sugars to improve the viability of lactic acid bacteria after freezing and freeze-drying

Summary In the present paper, the effect of cryo-protective sugars on the survival rate of different strains of Lactic Acid Bacteria (LAB, Lactobacillus acidophilus, Lactobacillus delbrueckii subspbulgaricus, Streptococcus salivarius subsp.thermophilus), after freezing or freeze-drying procedures, was compared. The cells were incubated at 4 °C in 32% final concentration sugar solutions (trehalose, maltose, sucrose, glucose and lactose), and viability was evaluated by the enumeration of colony-forming units. All sugars tested showed a protective effect on cell viability as compared to isotonic solution, especially after freeze-drying procedures (log c.f.u./ml ranging between 1.16 and 2.08, P < 0.001). Furthermore, the resistance to different stress agents (lysozyme, pepsin, bile salts) was estimated. Trehalose was the most effective sugar in preserving bacterial viability [% (log c.f.u. trehalose/log c.f.u. isotonic solution) ranging between 124 and 175, P < 0.001] although each strain showed a different sensitivity. Finally, the protective effect of immobilization of LAB in Ca-alginate beads was compared to that exercised by trehalose. The immobilization induced a good survival rate but lower as compared to the trehalose effect, mainly after freeze-drying in the presence of the selective agents [% (log c.f.u. alginate/log c.f.u. trehalose ranging between 81.1 and 94.5, P < 0.0001]. The protective effect of trehalose was evident in particular for Lactobacillus delbrueckii subsp. bulgaricus in presence of lysozyme. Therefore, because of its chemical inertness and low cost, trehalose could be easily utilized as excellent bacterial preservative, both to improve the viability of starter cultures and to obtain probiotic formulations more resistant to a variety of stressful conditions.     

A Putative LEA Protein,but no Trehalose,is Present in Anhydrobiotic Bdelloid Rotifers

Some eukaryotes, including bdelloid rotifer species, are able to withstand desiccation by entering a state of suspended animation. In this ametabolic condition, known as anhydrobiosis, they can remain viable for extended periods, perhaps decades, but resume normal activities on rehydration. Anhydrobiosis is thought to require accumulation of the non-reducing disaccharides trehalose (in animals and fungi) or sucrose (in plant seeds and resurrection plants), which may protect proteins and membranes by acting as water replacement molecules and vitrifying agents. However, in clone cultures of bdelloid rotifers Philodina roseola and Adineta vaga, we were unable to detect trehalose or other disaccharides in either control or dehydrating animals, as determined by gas chromatography. Indeed, trehalose synthase genes (tps) were not detected in these rotifer genomes, suggesting that bdelloids might not have the capacity to produce trehalose under any circumstances. This is in sharp contrast to other anhydrobiotic animals such as nematodes and brine shrimp cysts, where trehalose is present during desiccation. Instead, we suggest that adaptations involving proteins might be more important than those involving small biochemicals in rotifer anhydrobiosis: on dehydration, P. roseola upregulates a hydrophilic protein related to the late embryogenesis abundant (LEA) proteins associated with desiccation tolerance in plants. Since LEA-like proteins have also been implicated in the desiccation tolerance of nematodes and micro-organisms, it seems that hydrophilic protein biosynthesis represents a common element of anhydrobiosis across several biological kingdoms.     

Carbohydrate utilization in the thoraces of honey bees (Apis mellifera) during early times of flight

Trehalose levels in the thoraces of honey bees did not change significantly during the first 2–4 min of flight. This effect was seen both for bees flown shortly after removal from the hive and for bees which were flown after a 2 hr starvation period. There was also no detectable activation of the trehalose in isolated mitochondria from bees flown for periods of up to 2 min. However, the glucose content of the thoraces of bees flown shortly after removal from the hive dropped dramatically during the first 15 sec of flight. There was no evidence of a transient increase in the glucose content in the thorax at any of the times studied as would be expected if trehalose were hydrolized. The drop in glucose content at early times of flight was not detected if the bees were starved for 2 hr before flight was started. The changes in fructose content of the thoraces with time were similar to those observed for glucose, but were not statistically significant due to variation among individual bees. The sucrose content of bee thoraces varied greatly and no meaningful conclusions could be reached about how it changed as a function of time of flight.     

Fourier transform infared spectroscopy investigation of protein conformation in spray-dried protein/trehalose powders

Spray drying is a way to generate protein solids (powders), which is also true for lyophilization. Sugars are used to protect proteins from conformational changes and chemical degradations arising from drying processes and storage conditions such as the humidity. The influence of trehalose and humidity on the conformation and hydration of spray-dried recombinant human granolucyte colony stimulating factor (rhG-CSF) and recombinant consensus interferon-alpha (rConIFN) was investigated using Fourier transform IR spectroscopy. The spectral analysis of spray-dried powders in the amide I region demonstrated that trehalose stabilized the alpha-helical conformation of both rhG-CSF and rConIFN proteins. Exposure of the pure protein powders to 33% relative humidity (RH) resulted in the formation of beta sheets and loss of turns but no change in alpha-helical structure. Trehalose reduced the magnitude of the changes in beta sheets and turns. Exposure of the pure protein powders to 75% RH resulted in the loss of alpha-helical conformation with a corresponding increase in beta structures (beta sheets and turns). Trehalose did not protect proteins from the loss of alpha-helical structures, but it reduced the formation of antiparallel beta sheets. Hydrogen-deuterium exchange (H-D exchange) was used to further characterize these hydration-induced conformational changes. At 33% RH the percent exchange of the protein decreased with increasing trehalose content, indicating a greater protection of the protein from H-D exchange by a higher concentration of trehalose. Such protection correlates with decreased conformational changes of the protein by trehalose at this humidity. At 75% RH the degree of H-D exchange of the protein was insensitive to the powder composition in all powders. Surprisingly, the H-D exchange of trehalose was low at about 20-25%, which was nearly independent of the protein/trehalose ratio and humidity, indicating that the exchangeable protons on trehalose molecules are highly protected in protein-containing powders. The observed protein hydration is related to the effect of trehalose on the conformational changes of the protein under humidity.     

植物激素对砀山酥梨脱病毒苗增殖生长的影响

砀山酥梨脱病毒苗培养基中添加外源激素能通过调节内源激素的含量,从而控制脱病毒苗的增殖和生长。苄基腺嘌呤(benzyladen ine,BA)处理可提高脱病毒苗内源玉米素核苷(zeatin riboside,ZR)含量,而脱病毒苗的有效增殖芽数与IAA/ZR比值呈显著负相关;1萘-乙酸(1-naphthalene acetic ac id,NAA)处理可显著提高内源吲哚乙酸(indole acetic ac id,IAA)含量,较高的内源IAA含量有利于芽梢的生长;继代组培苗体内含有一定量的内源赤霉素(G iberllic Ac id,GA1 3),适量的外源GA3处理,可提高内源GA1 3含量而显著降低脱落酸(absc isic ac id,ABA)含量,促进芽梢的伸长和叶面积的扩大。     

克隆植物中国沙棘生长对外源植物激素的响应

关于植物克隆生长调节问题,目前集中于外在机制的研究。为了探讨中国沙棘克隆生长调节的内在机制,采用3×3回归设计进行田间试验,期望了解不同生长性状对IAA和CTK用量及其配比的响应规律。结果表明:(1)生长性状对激素用量的响应规律呈典型的钟形曲面模式,即各生长指标均存在一个产量峰值,峰值以前生长指标随IAA、CTK用量的增大而提高,峰值以后生长指标随IAA、CTK用量的增大而下降。(2)不同生长指标对激素用量及其配比的响应规律具有一定差异,较高的IAA比例有利于促进树高生长,较高的CTK比例有利于促进地径和冠幅生长,而几乎相等的IAA和CTK用量有利于种群生物量积累。(3)在激素用量适宜的情况下,中国沙棘生长潜力得到充分发挥,形成高大的个体,较多的子株,有利于提高种群对生境资源的占据和利用,并提高排斥其他植物种类入侵的能力;当激素用量过高或过低时,中国沙棘以降低生长量为代价,形成矮小的个体,减少子株数量,有利于削弱个体之间的竞争。这一结果为了解中国沙棘克隆生长内在调节机制提供了线索。(4)根据不同生长指标的激素效应方程,求出了相应的IAA和CTK的最佳用量和最佳配比,以及合理施激素区域和最低成本线。(5)克隆子株数量增幅与地径和冠幅生长量增幅呈极显著正相关、与种群生物量增幅呈显著正相关,即适宜的IAA和CTK用量既可加速个体生长、也能促进克隆子株的产生。     

Collapse temperature of solutions important for lyopreservation of living cells at ambient temperature

In this study, the collapse temperature was determined using the freeze‐drying microscopy (FDM) method for a variety of cell culture medium‐based solutions (with 0.05–0.8 M trehalose) that are important for long‐term stabilization of living cells in the dry state at ambient temperature (lyopreservation) by freeze‐drying. Being consistent with what has been reported in the literature, the collapse temperature of binary water‐trehalose solutions was found to be similar to the glass transition temperature (T′_g ～ ?30°C) of the maximally freeze‐concentrated trehalose solution (～80 wt% trehalose) during the freezing step of freeze‐drying, regardless of the initial concentration of trehalose. However, the effect of the initial trehalose concentration on the collapse temperature of the cell culture medium‐based trehalose solutions was identified to be much more significant, particularly when the trehalose concentration is less than 0.2 M (the collapse temperature can be as low as ?65°C). We also determined that cell density from 1 to 10 million cells/mL and ice seeding at high subzero temperatures (?4 and ?7°C) have negligible impact on the solution collapse temperature. However, ice seeding does significantly affect the ice crystal morphology formed during the freezing step and therefore the drying rate. Finally, bulking agents (mannitol) could significantly affect the collapse temperature only when trehalose concentration is low (<0.2 M). However, improving the collapse temperature by using a high concentration of trehalose might be preferred to the addition of bulking agents in the solutions for freeze‐drying of living cells. We further confirmed the applicability of the collapse temperature measured with small‐scale (2 µL) samples using the FDM system to freeze‐drying of large‐scale (1 mL) samples using scanning electron microscopy (SEM) data. Taken together, the results reported in this study should provide useful guidance to the development of optimal freeze‐drying protocols for lyopreservation of living cells at ambient temperature for easy maintenance and convenient wide distribution to end users, which is important to the eventual success of modern cell‐based medicine. Biotechnol. Bioeng. 2010;106: 247–259. © 2010 Wiley Periodicals, Inc.     

外源SOD和APX基因在转基因烟草中的表达与遗传

分析转超氧化物歧化酶基因（SOD）或抗坏血酸过氧化物酶基因（APX）烟草及其自交和杂交后代的叶片中超氧化物歧化酶（SOD）和过氧化物酶（POD）活性的结果表明：转基因烟草的SOD和POD活性在终花期最强,不同叶位叶中SOD活性差异不明显,POD活性以下部叶为最高;转基因烟草的SOD或POD活性显著高于近等基因的非转基因品系。杂交后代（F1、F2）的SOD活性能保持稳定,略高于亲本;自交后代（S1～S3）与自交亲本的SOD和POD活性相当。     

D-trehalose/D-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis: the binding of trehalose and maltose results in different protein conformational states

In this work, we used fluorescence spectroscopy, molecular dynamics simulation, and Fourier transform infrared spectroscopy for investigating the effect of trehalose binding and maltose binding on the structural properties and the physical parameters of the recombinant D-trehalose/D-maltose binding protein (TMBP) from the hyperthermophilic archaeon Thermococcus litoralis. The binding of the two sugars to TMBP was studied in the temperature range 20 degrees-100 degrees C. The results show that TMBP possesses remarkable temperature stability and its secondary structure does not melt up to 90 degrees C. Although both the secondary structure itself and the sequence of melting events were not significantly affected by the sugar binding, the protein assumes different conformations with different physical properties depending whether maltose or trehalose is bound to the protein. At low and moderate temperatures, TMBP possesses a structure that is highly compact both in the absence and in the presence of two sugars. At about 90 degrees C, the structure of the unliganded TMBP partially relaxes whereas both the TMBP/maltose and the TMBP/trehalose complexes remain in the compact state. In addition, Fourier transform infrared results show that the population of alpha-helices exposed to the solvent was smaller in the absence than in the presence of the two sugars. The spectroscopic results are supported by molecular dynamics simulations. Our data on dynamics and stability of TMBP can contribute to a better understanding of transport-related functions of TMBP and constitute ground for targeted modifications of this protein for potential biotechnological applications.