枸杞体细胞胚发生中蛋白质和DNA代谢动态的立体计量

本文以宁夏构杞无菌苗叶片为材料,离体培养,并诱导体细胞胚发生。根据细胞形态计量学原理,应用数字图像处理软件计量由光学底片经A/D转换成的数字图像中的蛋白质和DNA大分子,对于构杞体细胞胚发生过程中蛋白质和DNA分子的代谢动态进行了量化处理,并对量化结果分析了蛋白质和DNA代谢动态与体细胞胚发生、发育的关系。     

枸杞体细胞胚发生中DNA代谢动态的立体计量

本文以宁夏枸杞无菌苗叶片为材料,离体培养,并诱导体细胞胚胎发生。根据细胞形态计量学原理,应用数学图像处理软件计量由光学底片经Ａ／Ｄ转换成的数学图像中的ＤＮＡ大分子,对枸杞体细胞胚发生过程中ＤＮＡ分子的代谢动态进行量化分析。结果表明：在整个体细胞胚发和过程中ＤＮＡ代谢呈现动态变化。非胚性细胞与胚性细胞期的量化值分别为１．８２％和１．９１％;在二细胞胚、四细胞胚、多细胞胚时期ＤＮＡ缓慢增长,随着胚性愈     

枸杞体细胞胚发生中DNA代谢动态的立体计量

本文以宁夏枸杞无菌苗叶片为材料,离体培养,并诱导体细胞胚胎发生。根据细胞形态计量学原理,应用数字图像处理软件计量由光学底片经A/D转换成的数字图像中的DNA大分子,对枸杞体细胞胚发生过程中DNA分子的代谢动态进行量化分析。结果表明：在整个体细胞胚发生过程中DNA代谢呈现动态变化。非胚性细胞与胚性细胞期的量化值分别为1.82%和1.91%;在二细胞胚、四细胞胚、多细胞胚时期DNA缓慢增长,随着胚性愈伤组织的发育,DNA的含量在梨形胚时期达到高峰;成熟胚的DNA含量虽有所下降,但仍维持较高水平。因此DNA的合成动态变化与体胚生长发育和细胞增殖密切相关。     

宁夏枸杞体细胞胚发生过程中对金属离子的吸收速率和游离氨基酸含量的变化

采用多重示踪技术研究宁夏枸杞 (Lyciumbar barumL .)体细胞胚发生中对多种金属离子的吸收动态及其与游离氨基酸含量变化之间的关系。结果表明 :(1)在枸杞体细胞胚发生中对一些金属离子具有选择吸收特性 ,而且在体细胞胚发育不同时期对同一种金属离子的吸收量也不同 ;(2 )在枸杞体细胞胚发生早期对多数金属离子吸收量迅速增加 ,而后下降。到球形胚期吸收量达到第二个峰值 ,而且金属离子被吸收后提高了体细胞胚发生的频率 ;(3)枸杞体细胞胚发生中游离氨基酸总量从胚性细胞启动期开始下降 ,到胚性细胞形成期到达谷底 ,然后开始上升 ,到多细胞原胚期达到峰值 ,多数游离氨基酸含量变化与金属离子被吸收的量相交叉 ;(4 )外加RbCl和SrCl2 对枸杞体细胞胚发生具有促进作用 ,而且加大了几种游离氨基酸含量变化的幅度。文章讨论了它们之间的关系及其可能的作用机理     

体细胞胚发生的生化基础

在胚性细胞分化和分裂过程中ATP酶活性和分布的动态变化表明,这些胚性细胞进行着旺盛的主动物质吸收和活跃的新陈代谢过程。在多种植物的体细胞胚发生中过氧化物酶的活性与同工酶的种类都高于对照,而且在大麦中发现过氧化物酶、酯酶和酸性磷酸酶同工酶的结合应用可以作为体细胞胚发生的标志酶。胚性愈伤组织中可溶性蛋白质含量与组分远高于或多于非胚性愈伤组织。大多数材料中都存在45kD-55kD的胚胎发生特异性蛋白质组分。而且在体细胞胚发生中蛋白质和核酸代谢动态呈规律性变化,首先是RNA合成速率增加,继而是蛋白质的迅速合成,并在胚性细胞分化和发育过程中一直保持相对较高水平,其中mRNA种类丰富,不同发育时期mRNA种类不同,因此转译形成多种蛋白质。DNA的代谢相对较稳定,但在胚性细胞系中DNA合成量仍高于非胚性细胞系。加入蛋白质或核酸合成抑制剂,不仅抑制了蛋白质和核酸的合成,同时也抑制了体细胞胚的发生与发育,而且抑制剂加和时间愈早,影响愈严重。由此表明,蛋白质与核酸的合成为体细胞胚的分化和发育奠定了分子基础。     

枸杞体细胞胚发生过程中Ag~ 对痕量金属离子吸收的影响

以药用植物宁夏枸杞愈伤组织为材料,离体培养诱导体细胞胚发生。采用多重示踪剂和γ射线能谱分析法研究不同浓度AgNO_3处理的枸杞体细胞胚发生过程中对多种痕量金属元素离子的吸收。结果表明:(1)当AgNO_3的浓度小于50mg/L时,随着AgNO_3浓度的增加,多种痕量金属离子的吸收率也随之增加,而超过此浓度后,对多种痕量金属离子的吸收影响不同。Ag~ 对痕量金属离子的吸收有协同,拮抗或竞争的作用。(2)适当浓度的AgNO_3对细胞分化及体细胞胚发生有促进作用。当AgNO_3的浓度小于50mg/L时,随着AgNO_3浓度的增加,体细胞胚的发生频率随之增加。Ag~ 对枸杞体细胞胚发生表现促进作用,当AgNO_3的浓度为50mg/L时,可大大提高愈伤组织中体细胞胚发生,是对照(不加AgNO_3)组的3倍左右。而超过此浓度后,Ag~ 对枸杞体细胞胚发生表现毒害作用,体细胞胚的发生受到明显抑制。     

激素对枸杞体细胞胚发生及可溶性蛋白质含量和组分的影响

以宁夏枸杞无菌苗叶片为材料,对体细胞胚发生过程中激素作用、可溶性蛋白质变化及体细胞胚发生频率进行了研究。发现不同激素处理、可溶性蛋白质含量、组分和体细胞胚发生频率均有一定的差异,三者之间存在着相关性。结果如下：（１）ＭＳ＿１、ＭＳ＿２和ＭＳ＿３三种培养基上继代的愈伤组织难以诱导形成体细胞胚,为非胚性愈伤组织;而从ＭＳ＿１、ＭＳ＿２和ＭＳ＿３分别转到ＭＳ＿０和ＭＳ＿４培养基上的愈伤组织体细胞胚发生频率高,属于胚性愈伤组织。其中以ＭＳ＿３转至ＭＳ＿０后的体细胞胚发生频率最高。（２）可溶性蛋白质ＳＤＳ－ＰＡＧＥ分析表明：非胚性愈伤组织有特异性蛋白质６７ｋＤ,胚性愈伤组织有特异性蛋白质３５ｋＤ和４４ｋＤ;蛋白质３３ｋＤ和６７ｋＤ受２,４－Ｄ调控,蛋白质４９ｋＤ、５７ｋＤ受６ＢＡ调控,而蛋白质３７ｋＤ、５１ｋＤ受２,４－Ｄ和６ＢＡ协同调控。     

平贝母体细胞胚胎发生中的核酸与蛋白质合成研究

在获得比较理想的平贝母体细胞胚胎发生体系的基础上,我们应用放射性同位素液体闪烁计数技术测定了平贝母体细胞胚胎发生过程中球形胚、心形胚、鱼雷胚、子叶胚和成熟胚等时期的DNA、RNA和蛋白质合成动态,实验表明,从球形胚到子叶胚,核酸与蛋白质的合成速度递增。在子叶胚前期RNA合成达到高峰,在子叶胚期蛋白质合成达到高峰,在子叶胚后期DNA合成达到高峰,核酸与蛋白质合成速度的变化与胚体细胞增殖及器官分化相关联。     

植物体细胞胚发生的细胞生物学研究进展

体细胞胚发生是植物界的一个普遍现象,本文从组织细胞学、超微结构、电镜细胞化学、分子生物学等方面对体细胞胚发生发育过程中的形态建成和细胞结构,Ca2 和ATPase的动态变化,淀粉和蛋白质代谢,核糖体、线粒体等对极性变化的影响,畸形胚的发生和体胚同步化的诱导调控,基因调控等方面进行了综述,为进一步深入了解体细胞胚的发生发育规律及基因调控机制以及植物高效再生体系的建立提供参考。     

枸杞体细胞胚发生过程中内源多胺代谢动态的研究

完全脱分化的枸杞继代愈伤组织在转入分化培养后第1天就开始启动分化,接着体细胞转变为胚性细胞,继而分裂形成多细胞原胚,球形胚和成熟胚等。与此同时,Put含量迅速上升形成第1个峰值,随后有所下降,但到多细胞原胚期Put含量又上升,并达到最高峰,为对照的6倍,Spd只在胚性细胞分化早期出现,Spm仅在体细胞胚发育晚期存在,外源Put不仅可提高体细胞胚发生频率,而且使3种内源多胺含量均有所提高,加入多胺生物合成抑制剂DFMA后,多胺水平下降,体细胞胚发生几乎完全被抑制。补充外源Put后,多胺的生物合成得到部分恢复,DFMA对体细胞胚发生的抑制效应也被部分解除。结果表明,维持一定量的多胺水平是枸杞体细胞胚发生的必要因素。     

Quantitative stereologic analysis of metabolize dynamic of protein and DNA during somatic embryogensis in Lycium barbarum L.]

The protein and DNA content in somatic embryos of Lycium barbarum L were analysed using quantitative stereologicai procedure formed from Cell Morphometry. The photos were transformed into digital images by the Sharp JX-610 scanner. Then the quantity of protein and DNA was count applying digital images processing software. By the result we discuss the relationship between changes in protein and DNA metabolize dynamic and somatic embryogensis.     

Surface characterization of proteins using multi-fractal property of heat-denatured aggregates

Multi-fractal property of heat-denatured protein aggregates (HDPA) is characteristic of its individual form. The visual similarity between digitally generated microscopic images of HDPA with that of surface-image of its individual X-ray structures in protein databank (PDB) displayed using Visual Molecular Dynamics (VMD) viewer is the basis of the study. We deigned experiments to view the fractal nature of proteins at different aggregate scales. Intensity based multi-fractal dimensions (ILMFD) extracted from various planes of digital microscopic images of protein aggregates were used to characterize HDPA into different classes. Moreover, the ILMFD parameters extracted from aggregates show similar classification pattern to digital images of protein surface displayed by VMD viewer using PDB entry. We discuss the use of irregular patterns of heat-denatured aggregate proteins to understand various surface properties in native proteins.     

Digital images for interobserver variability comparison in cervicovaginal cytology

OBJECTIVE: To assess whether digital images can be useful in evaluating interobserver variability in cervical-vaginal cytology. STUDY DESIGN: In phase 1 of the study, to measure interobserver variability, a set of 160 digital images was submitted to 192 cytologists with experience ranging from 2 to > 30 years. The set was preceded by two days of immersion in lessons and practical exercises with digital images. In phase 2, to compare different procedures of interobserver variability, two different sets of slides and one set of digital images were used. RESULTS: In phase 1, kappa and weighted kappa w values computed against both the consensus and the target diagnosis showed good agreement, with few exceptions. In phase 2, the consensus and target diagnoses obtained on the slide sets and digital set were compared. Mean k and kw values obtained with the digital images in phase 2 were significantly lower as compared to those in phase 1. CONCLUSION: A set of digital images can be a useful tool for evaluating and improving interobserver reproducibility. A two-day course on digital images could be an ideal modality for introducing this new technology.     

大蒜体细胞胚胎发生过程中抗氧化酶活性变化及某些生理特征

以大蒜的发芽叶基(鳞茎)为外植体诱导体细胞胚胎发生,研究大蒜体胚发生过程中SOD、POD和CAT 3种抗氧化酶的活性以及可溶性糖和可溶性蛋白质含量变化.结果表明:在大蒜体胚发生过程中,SOD、POD和CAT活性变化与胚性愈伤组织的诱导及体胚的发育密切相关,POD对体胚的诱导起主导作用,而SOD和CAT在体胚的发育和成熟中起主导作用.可溶性糖和可溶性蛋白质累积与大蒜体细胞胚胎发生密切相关.     

Enolases: storage compounds in seeds? Evidence from a proteomic comparison of zygotic and somatic embryos of <Emphasis Type="Italic">Cyclamen persicum</Emphasis> Mill.

Somatic embryogenesis is well established for the economic relevant ornamental crop Cyclamen and thus could supplement the elaborate propagation via seeds. However, the use of somatic embryogenesis for commercial large scale propagation is still limited due to physiological disorders and asynchronous development within emerged embryos. To overcome these problems, profound knowledge of the physiological processes in Cyclamen embryogenesis is essential. Thus, the proteomes of somatic and zygotic embryos were characterised in a comparative approach. Protein separation via two dimensional IEF-SDS PAGE led to a resolution of more than 1,000 protein spots/gel. Overall, 246 proteins were of differential abundance in the two tissues compared. Mass spectrometry analysis of the 300 most abundant protein spots resulted in the identification of 247 proteins, which represent 90 distinct protein species. Fifty-five percent of the 247 proteins belong to only three physiological categories: glycolysis, protein folding and stress response. The latter physiological process was especially predominant in the somatic embryos. Remarkably, the glycolytic enzyme enolase was the protein most frequently detected and thus is supposed to play an important role in Cyclamen embryogenesis. Data are presented that indicate involvement of “small enolases” as storage proteins in Cyclamen. A digital reference map was established via a novel software tool for the web-based presentation of proteome data linked to KEGG and ExPasy protein-databases and both were made publicly available online.     

Microvessel density counting in breast cancer. Slides vs. digital images

OBJECTIVE: To develop a program to assist the pathologist in the acquisition and evaluation of digital images to determine microvessel density (MVD) in tissues. STUDY DESIGN: Ten cases of breast cancer with a high degree of neovascularization were selected. A standard immunohistochemical method was used to highlight the microvessels (monoclonal anti-factor VIII, avidinbiotin-peroxidase complex method). Two pathologists (one senior [S] and one junior [J]) evaluated four areas of highest neovascularization ("hot spots") in the tumors. Microscopically MVD was determined in four chosen areas (400:1). From the center of each area two digital images were acquired at a magnification of 200:1. All counts made by microscopic observation were compared with those made on the digital images. To compare MVD counting at different resolution, two sets of images at different sampling densities (320 x 240 and 1,600 x 1,200) were assessed by the two pathologists. RESULTS: We obtained a good correlation (r = .98 for S and .96 for J) between the MVD counts obtained at the microscope (192.8 MV/mm2 [mean of S] and 181.8 MV/mm2 [mean of J]) and the MVD counts from digital images (153.2 MV/mm2 [mean of S] and 171.0 MV/mm2 [mean of J]) at high resolution. The counts were lower for digital images at lower sampling density (125.0 MV/mm2 [mean of S] and 78.2 MV/mm2 [mean of J]). With low-resolution digital images only S maintained a good correlation (r = .96 for S and .34 for J) with the microscopic evaluation of MVD. Interobserver analysis showed a good correlation (r = .82 for the microscope and r = .78 for the digital images) of MVD evaluated either at the microscope or in high-resolution digital images. CONCLUSION: We demonstrated the functionality and usefulness of our program in performing MVD evaluation. Considering the capabilities of the program to store all images and microvessel marks and the reliability of MVD evaluation based on digital images, we consider this program the first step toward fully automated MVD assessment.     

Endogenous protein kinase-C activity and phosphorylated proteins in messenger ribonucleoprotein complexes of developing embryos of alfalfa.

Developing somatic and zygotic embryos of alfalfa (Medicago sativa L.) exhibited endogenous protein kinase activity and protein acceptors of phosphate groups using both cell-free translational extracts and oligo(dT)-cellulose-column-purified mRNPs. The cell-free-translation extracts from pre-cotyledonary-stage somatic embryos had approximately 50- and 100-fold more protein kinase activity than cotyledonary-stage somatic and zygotic embryos. Several polypeptides were phosphorylated; some of them were unique to the early stage and some to the late-stage developing embryos. A 65 kDa protein was phosphorylated heavily in pre-cotyledonary-stage somatic embryos. This phosphorylated protein was comprised of three main components, two of which were phosphorylated heavily. Heat-shock treated-embryos lost their exitant kinase activity and at the same time another form of protein kinase activity was activated which phosphorylated a novel 28 kDa protein. Endogenous protein kinase activity was also observed within the mRNPs of polysomal and non-polysomal fractions of developing embryos, and this phosphorylated only 65, 43 and 30 kDa proteins within these fractions. A 30 kDa protein from the pre-cotyledonary-stage somatic embryos showed a higher affinity for accepting phosphate groups than the proteins from cotyledonary-stage somatic or zygotic embryos. The activity of protein kinase was largely c-AMP-independent, but was dependent on Ca2+, phospholipid and phorbol ester. The enzyme belongs to the protein kinase-C family; the 65 kDa protein cross-reacts with antibodies made against protein kinase-C (alpha- and beta-isoforms) and it may be an autophosphorylated protein.     

Measuring beyond the microscope field of view using digital images in squamous cell carcinoma of the lip

OBJECTIVE: To study the impact of using digital images for measuring the size of the tumors, assisting with the prognostic evaluation of carcinomas of the oral mucosa. STUDY DESIGN: The depth of invasion of 12 cases of squamous cell carcinoma of the lower lip was assessed through a microscope and through digital images. All measurements of depth of invasion were assessed in a direction orthogonal to the lip surface. First, assessment of depth of invasion was done at the microscope, using an eyepiece reticule with an engraved scale. Second, depth of invasion was assessed by digital images, using a program module developed to assist pathologists with linear measuring. When the depth of the tumor was larger than the field of view at the proper magnification, several images were taken to include the whole area of invasion. The images were finally mounted in a single image and the depth of invasion measured. RESULTS: The results show positive and negative differences between assessments when assessing depths of < 2 mm. At greater depths (> or = 2 mm), the difference was always negative, showing that for deep invasion, measurements of longer distances were always performed on digital images. CONCLUSION: Measurements with digital images beyond the field of view at proper magnification could sig nificantly alter the diagnostic and prognostic assessment made using the microscope.     

Storage protein accumulation patterns in somatic embryos of cotton (Gossypium hirsutum L.)

Summary The storage protein content of somatic embryos of Gossypium hirsutum L. cv. Coker 201 was determined using extinction level, antigen/antibody association detection methods. Mature storage protein was first detected in early globular-stage somatic embryos at a total concentration of 0.36% of the embryo protein mass. Tulip-stage and mature somatic embryos were comprised of 3.0% and 1.3% mature storage protein, respectively. Maximum storage protein synthesis was found to occur during early globular- and early heart-stages. During this period of development, significant levels of protein precursors were found also to accumulate. The pattern of storage protein synthesis, processing and accumulation paralleled the pattern that has been reported for the zygotic system, although somatic embryos accumulate storage protein at much earlier stages and to a lesser degree. The possibility of using complex biochemical pathways to monitor embryogenic systems in vitro is discussed.