一种快速提取葱属植物 DNA 的方法

目的：针对分子育种工作的繁琐性及葱属植物次生代谢物较多的特点,研究一种利用普通试剂及简单仪器高效快速提取葱属植物DNA的方法。方法：对碱处理法稍加改进,在碱性环境中高温裂解细胞,将释放的DNA保存在Tris缓冲液中,用PCR检测提取质量并分析DNA的保存时间。结果：与用试剂盒提取的DNA相比,扩增产物无显著差异,利用此方法提取DNA并分析了13个洋葱品种的细胞质雄性不育类型;保存时间分析显示,DNA在常温下只能保存5d,在4℃或-20℃可至少保存2个月。结论：该方法方便快速、成本低、适于田间操作,得到的DNA可满足分子生物学实验的基本要求,可用于以PCR为基础的分子标记辅助育种。     

An improved method for preparing refrozen rethawed human lymphocytes on plates for microcytotoxicity studies.

This report describes simplified methods for the initial freezing and thawing of human lymphocytes and the subsequent use of these cells after refreezing on cytotoxicity plates, storage, and a second thaw. The proposed initial freeze method eliminates some technical inconveniences required previously such as chilling of cells prior to addition of DMSO, preparing cryoprotective mixtures just prior to freezing, controlled rate of freezing and thawing and the washing of cells after thawing. However, pH of the media, blood freshness, type of storage tube used, and constant storage temperature were found to be very important to maintain good cell viability. Most lymphocytes maintain an average viability of 85 to 95% for at least a year when prepared according to the present freezing and thawing technique.When panels of lymphocytes are prepared for refrozen rethawed cytotoxicity test plates, the thaw time between freezes must be brief. Production of test plates on ice, however, was not found to be necessary. As the period of storage of refrozen cells on plates increases, viability of the cells after a second thaw decreases and treatment with DNase to enzymatically remove the dead cells is useful. With this procedure, refrozen rethawed lymphocytes up to a year old can be prepared on microcytotoxicity test plates with average viabilities of 90 ± 1%.     

Induction of chromosomal aberrations in CHO cells by restriction endonucleases: effects of blunt- and cohesive-ended double-strand breaks in cells treated by 'pellet' methods

In recent reports it has been suggested that restriction endonucleases (RE) producing cohesive-ended double-strand breaks (dsb), are of comparable effectiveness to those producing blunt-ended dsb in causing chromosomal aberrations (CA) in mammalian cells. In several of these reports, trypsinized cells or suspension cultures were treated as cell 'pellets' in small volumes containing RE and storage buffers. In this study we have examined this by comparing 2 'pellet' methods in which trypsinized Chinese hamster cells were treated with RE in small volumes, after cells were centrifuged to a pellet. In the first method, cells were treated with RE in storage buffer as previously reported (e.g. Obe et al., 1985). In the second method, cells were treated as pellets with Sendai virus and purified RE. For both methods we show that the frequency of chromosomal aberrations was higher in cells treated with RE causing blunt-ended dsb than those causing cohesive-ended dsb. The first method however was found to lead to substantial loss in cell viability. The results strengthen the conclusion drawn from our earlier work, using treatment of attached V79 or CHO-K1 cells with Sendai virus, that cohesive-ended dsb are less effective than blunt-ended dsb in causing chromosomal aberrations.     

Recovery of carotenoids from ensilaged shrimp waste

A method for fermentation of shrimp waste was standardized using a statistically designed experiment, with respect to three variables namely, levels of glucose and starter culture and time of fermentation. The optimized levels for achieving the desired pH was 20.5% glucose, 19.5x10(4)cells/g of starter culture and fermentation time of 70h. Recovery of carotenoids from fermented and acid ensiled shrimp waste was assessed during 75 days of storage. Acid ensilaging resulted in the reduction of solvent extraction yield of carotenoids from 43.09 to 26.76 microg/g by the end of 75 days of storage. The yield of oil extracted carotenoids was higher in both types of silage at the end of 75 days storage compared to the initial yield, being 31.30 microg/g in fermented silage and 26.18 microg/g in acid silage. The results indicated the usefulness of fermentation as a method for stabilization and recovery of carotenoids in the shrimp waste.     

Development of an attached strain from a continuous insect cell line

Summary A continuous attached cell strain has been developed from the IPRI-CF-124 line of the spruce budworm,Choristoneura fumiferana. This was done by discarding suspended cells at each passage, rinsing attached cells with 0.05% trypsin and using only the strongly attached cells for subculturing. The method is very effective in that the proportion of attached cells increased from 6% in the parent cell line to 97% in the new cell strain after 20 passages. The attachment and growth properties are stable after storage of cells in liquid nitrogen. The new cell strain is designated IPRI-CF-124T and has a population doubling time comparable to that of the parent cell line. Contribution No.: 329.     

A liver micronucleus assay using young rats exposed to diethylnitrosamine: methodological establishment and evaluation

We have developed a simple liver micronucleus assay using young rats (up to 4 weeks old) to assess cytogenetic damage of chemicals in liver cells. Diethylnitrosamine (DEN) was used as a model rodent hepatocarcinogen in this study. Compared to the partial hepatectomy method most commonly used for the liver micronucleus assay, the present assay method showed equal or even higher practicability. The young rat liver micronucleus assay was also characterized for rat strain differences, sampling time after treatment, single treatment vs. split treatment, age of animals, administration route, and staining method. Although based on one model chemical, we propose an acceptable protocol for the micronucleus assay using young rat liver as follows: Up to 4-week-old rats should be used; oral or intraperitoneal administration can be used; single or repeated treatment protocols can be applied; sampling time is 3-5 days after the last treatment; hepatocytes are prepared by the collagenase perfusion method; and cells are stained with the AO-DAPI double staining method.     

Studies of the deformability of preserved red blood cells using a simple filtration method]

The applied method to measure the filtrability of red blood cells is accurate enough for the study of the effects of the conditions of storage on their deformability. In this work the standard deviation of the method, the mean biological deviation of the deformability of human red blood cells and their deformability in the course of storage in the ACD-AG-medium have been investigated. Considerable loss of deformability occurs towards the end of storage time of the cells, which is not reversible be restitution of ATP alone. The applicability of this method as a simple test of the vitatity of preserved erythrocytes if to be tested.     

Fuzzy ART-based place recognition for visual loop closure detection

The automatic place recognition problem is one of the key challenges in SLAM approaches for loop closure detection. Most of the appearance-based solutions to this problem share the idea of image feature extraction, memorization, and matching search. The weakness of these solutions is the storage and computational costs which increase drastically with the environment size. In this regard, the major constraints to overcome are the required visual information storage and the complexity of similarity computation. In this paper, a novel formulation is proposed that allows the computation time reduction while no visual information are stored and matched explicitly. The proposed solution relies on the incremental building of a bio-inspired visual memory using a Fuzzy ART network. This network considers the properties discovered in primate brain. The performance evaluation of the proposed method has been conducted using two datasets representing different large scale outdoor environments. The method has been compared with RatSLAM and FAB-MAP approaches and has demonstrated a decreased time and storage costs with broadly comparable precision recall performance.     

Carboxylic ester hydrolases in the thyroid gland of the guinea-pig. A light microscopic study

Synopsis The location of cholinesterase and non-specific esterase in the thyroid gland of the guinea-pig was studied with the light microscope. It was found that the inxodyl method for non-specific esterase activity under special conditions is superior to the cholinesterase method in a number of respects for the demonstration of the intra-, inter-and parafollicular cells. When using the indoxyl method the incubation period can be reduced from 2.5–3 hr to 40–50 min. Further, the reaction can be followed during the incubation. False localization of the reaction products is avoided, and nerves and erythrocytes are not stained.By varying the fixation time and the time of storage in gum arabic-sucrose, it was found that the miscellaneous activity of non-specific esterase in APUD cells (C-cells) and follicle cells may be due to both factors. In fresh tissue the activity of the enzyme was equal in follicle and C cells.Special cyst-like structures containing an esterase which is NaF-resistant whenn-naphthyl acetate is employed as a substrate and which gives a strong reaction at low pH values when 5-bromo indoxyl acetate is the substrate, are described, and their nature and possible origin are discussed.     

Prolactin and insulin are targeted to the regulated pathway in GH4C1 cells, but their storage is differentially regulated

PRL storage in GH4C1 cells, rat pituitary tumor cells, can be induced by treatment with a combination of estradiol, epidermal growth factor, and bovine insulin. This increase in storage is characterized by a preferential increase in intracellular PRL compared with secreted PRL and a 50-fold increase in the number of secretory granules. Treatment with the combined hormones stimulates PRL synthesis approximately 6-fold, but this effect is not sufficient to increase PRL storage, because epidermal growth factor alone increases PRL synthesis to the same extent without affecting storage. The cDNA for human proinsulin down-stream of the RSV-LTR promoter was transfected into GH4C1 cells to determine whether storage of another protein known to be targeted to the regulated pathway would also increase with hormone treatment. Proinsulin and PRL release were stimulated over the same time course and to the same peak height, compared to basal release, by both KCl and TRH, indicating that proinsulin is targeted to the regulated pathway in GH4C1 cells. There was little intracellular processing of proinsulin to insulin. Proinsulin synthesis increased 3.9-fold with the combined treatment, assessed by accumulation of proinsulin immunoreactivity in the medium and increases at the mRNA level. Treatment with the combined hormones did not cause the preferential increase in intracellular proinsulin that occurred with PRL; the increase in intracellular proinsulin could be accounted for primarily by effects on synthesis. These results suggest that storage of the two hormones can be differentially regulated.     

Hormonal regulation of prolactin storage in a clonal strain of rat pituitary tumor cells

GH4C1 cells (GH cells) are a clonal strain of rat pituitary tumor cells which secrete prolactin. GH cells have been used to study hormone secretion, but they store relatively little prolactin compared to normal prolactin-secreting cells. They are not suitable, therefore, for studying some aspects of pituitary function. We have found that the amount of prolactin GH cells store can be regulated. When GH cells were plated at 10(6) cells/well and treated for six days with 180 nM insulin or 1 nM estradiol, there was a 60 percent increase in prolactin storage compared to control cells. Insulin and estradiol in combination acted synergistically to cause a 190 percent increase in prolactin storage. In contrast, they were additive in increasing extracellular prolactin; there was a 40 percent increase in extracellular prolactin after insulin, a 20 percent increase after estradiol, and a 50 percent increase after insulin plus estradiol. The increases in prolactin storage were always greater than the increases in extracellular prolactin. The increases in prolactin storage were dose-dependent and reached maximal levels after four days of treatment with 180 nM insulin plus 1 nM estradiol. Reducing the plating density to 10(3) cells/well increased the response to insulin and estradiol to nineteenfold. Epidermal growth factor (10 nM) acted synergistically with estradiol and insulin in combination to increase prolactin storage 27-fold. The insulin- and estradiol-induced increase in extracellular prolactin was caused by a specific increase in the rate of prolactin synthesis. The fractional increase in prolactin storage above the increase in prolactin production could not be explained by an increase in prolactin synthesis, an increase in intracellular transit time, or a change in the cell-cycle distribution of the population. Hormone storage can, therefore, be regulated independently from other processes which control hormone production. The prolactin stored in response to insulin and estradiol was releasable by potassium depolarization. Following depletion of intracellular prolactin by depolarization, the cells retained their increased capacity for prolactin storage. The ability to increase prolactin storage will make GH cells a more useful system in which to study pituitary function.     

弗里熊蜂蜜罐中糖液成分分析

【目的】熊蜂是众多植物的重要传粉昆虫,以采集并贮藏花蜜和花粉为主要食物。本研究旨在探究熊蜂的营养需求及明确其对采集的食物是否存在酿制过程。【方法】利用白砂糖溶液(糖浓度50%)饲喂弗里熊蜂Bombus friseanus蜂群,收集并检测其贮藏在蜜罐中1~7 d的糖液,作为处理组样品;同时将上述白砂糖溶液置于灭菌离心管中,排除熊蜂取食,作为对照组样品,测定贮藏期间处理组和对照组糖液的pH值、糖浓度、糖组分及α-淀粉酶和转化酶活性。【结果】弗里熊蜂B. friseanus贮藏在蜜中1~7d的糖液pH值平均为3.74±0.13,显著低于对照组(6.55±0.15);糖浓度与贮藏时间显著正相关,贮藏6d后糖浓度显著高于贮藏1~3 d时的;糖液组分更加丰富,除蔗糖外利用HPLC还检出果糖、葡萄糖、麦芽糖和海藻糖,贮藏4~5 d的糖液中己糖含量极显著高于贮藏1~3 d和6~7 d时的,己糖和麦芽糖含量分别与蔗糖含量极显著负相关,总糖中果糖和麦芽糖含量与贮藏时间极显著负相关,葡萄糖、蔗糖和海藻糖的含量分别与贮藏时间极显著正相关。贮藏6 d的糖液中α-淀粉酶活性显著高于其他时间,其他贮藏时间样品间α-淀粉酶活性差异不显著,而所有样本的转化酶活性在21.17~38.05 U/g FW之间,随贮藏时间的延长差异不显著。【结论】弗里熊蜂B. friseanus采集人工饲喂的糖溶液贮藏在蜜罐中,经其加工后发生了物理和生物化学变化,揭示熊蜂存在酿蜜能力。本研究的结果为熊蜂生物学及繁育研究提供了参考。     

Real time quantitative analysis of lipid storage and lipolysis pathways by confocal spectral imaging of intracellular micropolarity

Organisms store fatty acids in triacylglycerols in the form of lipid droplets, or hydrolyze triacylglycerols in response to energetic demands via activation of lipolytic or storage pathways. These pathways are complex sets of sequential reactions that are finely regulated in different cell types. Here we present a high spatial and temporal resolution-based method for the quantification of the turnover of fatty acids into triglycerides in live cells without introducing sample preparation artifacts.We performed confocal spectral imaging of intracellular micropolarity in cultured insulin secreting beta cells to detect micropolarity variations as they occur in time and at different pixels of microscope images. Acquired data are then analyzed in the framework of the spectral phasors technique.The method furnishes a metabolic parameter, which quantitatively assesses fatty acids - triacylglycerols turnover and the activation of lipolysis and storage pathways. Moreover, it provides a polarity profile, which represents the contribution of hyperpolar, polar and non-polar classes of lipids. These three different classes can be visualized on the image at a submicrometer resolution, revealing the spatial localization of lipids in cells under physiological and pathological settings.This new method allows for a fine-tuned, real-time visualization of the turnover of fatty acids into triglycerides in live cells with submicrometric resolution. It also detects imbalances between lipid storage and usage, which may lead to metabolic disorders within living cells and organisms.     

Processing of ovine cardiac valve allografts: 1. Effects of preservation method on structure and mechanical properties.

It is essential to have some method of preservation of allograft valves during the time between procurement and implantation. Cryopreservation is the most commonly-used storage method today but it has the major disadvantage of high cost, and because its aim is to preserve living cells only relatively gentle antimicrobial treatments are used. This study addresses two interrelated questions: Is it necessary to maintain living donor cells in the tissue graft?Can more effective measures be used to reduce the risk of transmission of diseases, especially viral diseases, via human tissue grafts. In this paper, were port an investigation of four preservation methods that could be combined with more effective disinfection: cryopreservation with dimethyl sulphoxide, storage at ～4 °C in a high concentration of glycerol as used for the preservation of skin, snap-freezing by immersion in liquid nitrogen and vitrification. Snap freezing was mechanically damaging and vitrification proved to be impracticable but two methods, cryopreservation and storage in 85%glycerol, were judged worthy of further study. Cryopreservation was shown to maintain cellular viability and excellent microscopic structure with unchangedmechanical properties. The glycerol-preserved valves did not contain any living cells but the connective tissue matrix and mechanical properties were well preserved. The importance of living cells in allograft valves is uncertain. If living cells are unimportant then either method could be combined with more effective disinfection methods: in that case the simplicity and economy of the glycerol method would be advantageous. These questions are addressed in the two later papers in this series.     

Viability and Estimation of Shelf-Life of Bacterial Populations

Mathematical concepts associated with the exponential and probit models are developed, and the similarity of the two methods is discussed. Because of its greater flexibility in design, the probit method was used to estimate the shelf-life for four bacterial populations, wet and dry spores of Bacillus anthracis and wet and dry cells of Pasteurella tularensis. On the basis of data gained by storing these organisms at high temperature, the probit method was used to predict the time at which 50% viability would occur for cells stored at 3 C. A plane passing through a three-space showing change in percentage viability of bacteria was formulated by a multiple regression method. With this functional technique, the percentage viability, expressed as a probit, was linearily related to a logarithm of storage time and storage temperature. The use of this method to study the effect of controlled variables on the viability of cells is demonstrated by comparing the effect of viability associated with three additives used prior to drying. The results of the test gave shelf-life estimates which were too low for all additives; however, the order of stability was ranked properly as confirmed by long-term tests.     

New assays for detection and localization of endogenous lipid peroxidation products in living boar sperm after BTS dilution or after freeze-thawing

Reactive oxygen species have been implicated in sperm aberrations causing multiple pathologies including sub- and infertility. Freeze/thawing of sperm samples is routinely performed in the cattle breeding industries for semen storage prior to artificial insemination but unusual in porcine breeding industries as semen dilution and storage at 17 degrees C is sufficient for artificial insemination within 2-3 days. However, longer semen storage requires cryopreservation of boar semen. Freeze/thawing procedures induce sperm damage and induce reactive oxygen species in mammalian sperm and boar sperm seems to be more vulnerable for this than bull sperm. We developed a new method to detect reactive oxygen species induced damage at the level of the sperm plasma membrane in bull sperm. Lipid peroxidation in freshly stored and frozen/thawed sperm cells was assessed by mass spectrometric analysis of the main endogenous lipid classes, phosphatidylcholine and cholesterol and by fluorescence techniques using the lipid peroxidation reporter probe C11-BODIPY(581/591). Peroxidation as reported by the fluorescent probe, clearly corresponded with the presence of hydroxy- and hydroperoxyphosphatidylcholine in the sperm membranes, which are early stage products of lipid peroxidation. This allowed us, for the first time, to correlate endogenous lipid peroxidation with localization of this process in the living sperm cells. Cytoplasmatic droplets in incompletely matured sperm cells were intensely peroxidized. Furthermore, lipid peroxidation was particularly strong in the mid-piece and tail of frozen/thawed spermatozoa and significantly less intense in the sperm head. Induction of peroxidation in fresh sperm cells with the lipid soluble reactive oxygen species tert-butylhydroperoxide gave an even more pronounced effect, demonstrating antioxidant activity in the head of fresh sperm cells. Furthermore, we were able to show using the flow cytometer that spontaneous peroxidation was not a result of cell death, as only a pronounced subpopulation of living cells showed peroxidation after freeze-thawing. Although the method was established on bovine sperm, we discuss the importance of these assays for detecting lipid peroxidation in boar sperm cells.     

The effect of prolonged ischaemia and cryopreservation on the cell viability of human aortic and femoral artery allografts

The viability of the human arterial allograft cells depends on the time and method of vessel procurement and storage. In this study, an evaluation of the effect of the duration of 4 degrees C ischaemia and cryopreservation on human aortic and femoral artery allograft viability was performed. After the isolation of arterial wall cells, the identification of cultured cells was performed using mRNA analysis for estimation of smooth-muscle markers of differentiation: desmin and heavy-caldesmon. The viability of cells from the medial layer of the aortic wall ranged from 74 to 90% (61-79% for femoral arteries). Cold ischaemia time (from harvesting until the beginning of the preparation) is a statistically significant factor influencing smooth muscle cell viability. Smooth muscle cells represented the majority of live cell population.     

Influence of fermentation time, cryoprotectant and neutralization of cell concentrate on freeze-drying survival, storage stability, and acid and bile exposure of Bifidobacterium animalis ssp. lactis cells produced without milk-based ingredients

AIMS: To investigate the stability of Bifidobacterium animalis ssp. lactis VTT E-012010 (=Bb-12) during freeze-drying, storage and acid and bile exposure. The effect of harvesting time and composition and pH of the cryoprotectant on the survival was evaluated. The procedure was performed by using a milk-free culture medium and cryoprotectants to produce cells for nonmilk-based applications. METHODS AND RESULTS: Bifidobacterial cells were grown in fermenters in general edible medium for 15 or 22 h. The cell mass was freeze-dried either as non-neutralized or neutralized using sucrose, betaine or reconstituted skim milk (control) as cryoprotectants. For stability studies freeze-dried powders were stored at 37, 5 and -20 degrees C for 2-6 months. In addition, acid and bile tolerance of the powders was tested. Sucrose-formulated B. animalis ssp. lactis preparations had an excellent stability during storage at refrigerated and frozen temperatures for 5-6 months. They also had a good survival during storage at 37 degrees C for 2 months as well as during exposure to pH 3 and 1% bile acids. No difference was observed between 15 and 22 h grown cells or between non-neutralized and neutralized cells. Betaine proved to be a poor cryoprotectant compared with sucrose. CONCLUSIONS: Fermentation time and neutralization of cell concentrate before freeze-drying had no impact on the storage stability and bile and acid tolerance of freeze-dried bifidobacterial cells. The nonmilk-based production protocol using sucrose as a cryoprotectant yielded powdery preparations with excellent stability in adverse conditions (storage at elevated temperatures and during acid and bile exposure). SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that it is feasible to develop nonmilk-based production technologies for probiotic cultures. This provides new possibilities for the development of nondairy-based probiotic products.     

Evidence of temporal postdischarge decontamination of bacteria by gliding electric discharges: application to Hafnia alvei

This study aimed to characterize the bacterium-destroying properties of a gliding arc plasma device during electric discharges and also under temporal postdischarge conditions (i.e., when the discharge was switched off). This phenomenon was reported for the first time in the literature in the case of the plasma destruction of microorganisms. When cells of a model bacterium, Hafnia alvei, were exposed to electric discharges, followed or not followed by temporal postdischarges, the survival curves exhibited a shoulder and then log-linear decay. These destruction kinetics were modeled using GinaFiT, a freeware tool to assess microbial survival curves, and adjustment parameters were determined. The efficiency of postdischarge treatments was clearly affected by the discharge time (t*); both the shoulder length and the inactivation rate k(max) were linearly modified as a function of t*. Nevertheless, all conditions tested (t* ranging from 2 to 5 min) made it possible to achieve an abatement of at least 7 decimal logarithm units. Postdischarge treatment was also efficient against bacteria not subjected to direct discharge, and the disinfecting properties of "plasma-activated water" were dependent on the treatment time for the solution. Water treated with plasma for 2 min achieved a 3.7-decimal-logarithm-unit reduction in 20 min after application to cells, and abatement greater than 7 decimal logarithm units resulted from the same contact time with water activated with plasma for 10 min. These disinfecting properties were maintained during storage of activated water for 30 min. After that, they declined as the storage time increased.     

Process parameters for the high-scale production of alginate-encapsulated stem cells for storage and distribution throughout the cell therapy supply chain

With the ever-increasing clinical application of cell-based therapies, it is considered critical to develop systems that facilitate the storage and distribution of cell therapy products (CTPs) between sites of manufacture and the clinic. For such systems to be realized, it is essential that downstream bioprocessing strategies be established that are scalable, reproducible and do not influence the viability or function of the living biologic. To this end, we examined alginate-encapsulation as a method to heighten the preservation of human adipose-derived stem cells (hASCs) during hypothermic storage, and establish a scalable process for high-volume production. A drop-wise method for scalable alginate bead generation, using calcium as the cross-linker, was modified to enable the yield of up to 3500 gelled beads per minute. The effect of alginate concentration on the viscosity of non-gelled sodium alginate and the mechanical properties and internal structure of calcium-crosslinked alginate in response to different alginate and calcium concentrations were investigated. Mechanical strength was chiefly dependent on alginate concentration and 1.2% alginate cross-linked with 100 mM calcium chloride could withstand stress in the order of 35 kPa. Upon selection of appropriate parameters, we demonstrated the suitability of using this method for immobilizing human stem cells. Encapsulated hASCs demonstrated no loss in cell viability, and had a uniform distribution after high-volume production. Following storage, released cells were able to attach and recover a normal morphology upon return to culture conditions. Thus we present a scalable method for stem cell encapsulation and storage for application within the cell therapy supply chain.