含Dsred类似生色团的红色荧光蛋白的发展及应用

自从绿色荧光蛋白(GFP)被发现以来,荧光蛋白在生物医学领域已经成为一种重要的荧光成像工具．随着红色荧光蛋白DsRed的出现,各种优化的DsRed突变体和远红荧光蛋白也不断涌现．其中荧光蛋白生色团的形成机制对改建更优的荧光蛋白变种影响很大,对于红色荧光蛋白而言,大多数的红色荧光蛋白的生色团类型为DsRed类似生色团,在此基础上又出现了Far-red DsRed类似生色团．目前,含DsRed类似生色团的荧光蛋白主要有单体红色荧光蛋白、光转换荧光蛋白、斯托克斯红移蛋白、荧光计时器等．这些优化的荧光蛋白作为分子探针可以实现对活细胞、细胞器或胞内分子的时空标记和追踪,已经在生物工程学、细胞生物学、基础医学领域得到广泛应用．本文综述了含DsRed类似生色团的荧光蛋白的研究进展及其应用,以及由此发展起来的远红荧光蛋白在活体显微成像技术中的应用,并展望了荧光探针技术研究的新方向．     

珊瑚和海葵来源红荧光蛋白的研究和应用

绿色荧光蛋白作为标记蛋白和报告蛋白在生物学研究中应用越来越广。但在荧光共振能量转移(fluorescenceresonanceenergytransfer,FRET)等技术中存在一些缺陷,需要更大波长范围的荧光蛋白。最近研究发现了多种来源于珊瑚和海葵的红荧光蛋白,这些长波长的荧光蛋白对绿色荧光蛋白是一种很好的代替和补充,可以实现细胞内多荧光标记,提供更理想的FRET荧光对。经随机突变和定点突变等方法改建获得的红荧光蛋白变种显示出更高的荧光强度,成熟时间也更短。目前应用较多的是来源于香菇珊瑚(Discosomasp.)的红荧光蛋白DsRed。     

磁场和声音可能有助于向大脑给药

澳大利亚科学家近日在该国东南部海岸发现大量彩色荧光珊瑚，其中一些泛着红色荧光的珊瑚相当罕见，对于人类进一步认识癌细胞、研发新的抗癌药物具有重要意义。     

桃叶珊瑚苷在初级糖尿病脑病中的神经保护作用

在初级糖尿病脑病中海马神经元细胞的凋亡与认知能力相关．在本研究中,研究桃叶珊瑚苷在初级糖尿病脑病大鼠中的神经保护机制．实验大鼠被随机分为3组：空白对照组、STZ诱导糖尿病组及STZ诱导糖尿病桃叶珊瑚苷治疗组．除空白对照组以外,所有大鼠接受60mg／kg链脲佐菌素腹腔注射诱导糖尿病．桃叶珊瑚苷治疗组的大鼠在第65天开始连续腹腔注射5mg／kg的桃叶珊瑚苷,共注射15天,利用Y迷宫检测动物行为学变化．在第87天进行脑组织的组织学检测,用光学显微镜进行海马CA1区存活神经元细胞计数,应用透射电子显微镜对CA1区神经元细胞的超微结构观察和凋亡细胞数量计数．为了弄清桃叶珊瑚苷的神经保护机制,应用Western Blot和免疫组化方法对Bcl-2和Bax两种凋亡蛋白的表达进行了分析．结果显示,桃叶珊瑚苷抑制了海马CA1区神经元细胞凋亡,并起到降低血糖浓度、增加体重、改善糖尿病大鼠的学习状况,最终使Bcl-2和Bax两种凋亡蛋白的表达比例得到了平衡．结果暗示桃叶珊瑚苷抑制神经元细胞的凋亡可能是通过调控Bcl-2和Bax两种凋亡蛋白的表达来完成．     

红色荧光蛋白的研究进展

从荧光蛋白的首次发现至今,其不断丰富的荧光光谱以及日益增多的光化学特性促使其在生物学研究中的地位不断提升。1999年出现的红色荧光蛋白以其卓越的优势在生物学研究中占领了重要地位。主要介绍了从红色荧光蛋白发现至今日趋重要的原因,以及它的显著优势。另外,重讨论了目前几种常见的红色荧光蛋白的特性及其在生物学研究中存在的优势和不足之处,为今后红色荧光蛋白的研究提供一个选择。     

珊瑚菜中香豆素的组织化学定位

利用冷冻切片技术和常规石蜡制片技术,分别通过荧光显微镜和光学显微镜对珊瑚菜的根、叶片、叶柄等部位中的香豆素进行了组织化学定位。研究表明:香豆素存在于珊瑚菜的分泌道中,在荧光显微镜下发蓝色荧光;分泌道广泛分布于植物体中,在根中,分布在次生韧皮部中;在叶片中,分布在叶脉的薄壁组织中;在叶柄中,分布在维管束周围以及厚角组织内侧的薄壁组织中。     

板栗疫病菌绿色荧光与红色荧光蛋白共定位载体的构建

低毒病毒-板栗疫病菌组合是研究病毒与宿主相互作用的一个优秀的模式系统.我们构建了含绿色荧光蛋白基因gfp的载体pCPXHY2GFP与含红色荧光蛋白基因rfp的载体pCPXG418RFP,并用于转化野生型菌株EP155,获得了以潮霉素为筛选标记、表达绿色荧光蛋白的转化株pCPXHY2GFP/EP155和以G418为筛选标记、表达红色荧光蛋白的转化株pCPXG418RFP/EP155.将载体pCPXG418RFP转化pCPXHY2GFP/EP155,获得的转化株能观察到绿色荧光蛋白与红色荧光蛋白共定位的现象.板栗疫病菌绿色荧光与红色荧光共定位载体pCPXHY2GFP与pCPXG418RFP的构建,为深入研究病毒与宿主相互作用的分子机制提供了强有力的研究材料.     

带红色荧光蛋白靶向线粒体载体的构建与表达

选取由核表达的线粒体蛋白细胞色素C氧化亚单位Ⅷ（COX8）的前导序列为靶序列,从人胚胎肺成纤维细胞中扩增出COX8的前导序列,插入到pcDNA3．1／myc—HisA中,并将pDsRED1-n1中的红色荧光蛋白序列RFP克隆至COX8的下游,形成融合蛋白。在脂质体的介导下,将重组载体转染至肿瘤细胞中,在荧光显微镜下观察其在细胞内的表达及分布情况。构建的靶向线粒体的载体以及以红色荧光蛋白为报告基因的靶向线粒体的载体,经酶切、DNA序列测定,结果表明构建正确。将pcDNAmito—RFP转染到HeLa细胞的线粒体中,16h即可见散在荧光,72h达高峰,第10d开始减弱。以上结果表明成功构建了以红色荧光蛋白为报告基因的线粒体靶向的特异表达载体,在靶序列的引导下将红色荧光蛋白输入到线粒体中,为进一步对线粒体疾病的基因治疗研究提供了重要工具。     

人PIRH2b与ARF4相互作用的初步研究

目的 利用酵母双杂交技术筛选PIRH2b的相互作用蛋白。方法 以PIRH2b为诱饵蛋白,利用酵母双杂交技术筛选人胎肝cDNA文库,用GST—pull down验证PIRH2b与ARF4在体外的相互作用,并用绿色荧光蛋白标记PIRH2b,红色荧光蛋白标记ARF4,观察两者在肝癌细胞株Hep3B中的亚细胞定位。结果利用酵母双杂交筛选到一个能与PIRH2b相互作用的蛋白ARF4,GST—pull down验证了两者在体外的相互作用,荧光标记共定位结果显示两个蛋白共定位于Hep3B细胞的核周区域。结论首次发现并证实了PIRH2b与ARF4的相互作用,PIRH2b对ARF4的功能可能有重要影响。     

一种快捷可靠评价链霉菌噬菌体ФC31整合酶功能的双荧光报告系统

在NIH3T3细胞中构建了一种链霉菌噬菌体ФC31整合酶报告系统．该报告载体同时编码红色荧光蛋白和绿色荧光蛋白,与编码ФC31整合酶的载体共转染可以反映ФC31整合酶的活性．细胞中从红色荧光到绿色荧光的变化和百分比的变化可经流式细胞仪检出．随着转染中ФC31整合酶表达载体的比例升高,表达绿色荧光的细胞比例上升．ФC31整合酶表达载体和报告系统载体比例在10∶1时,可达最高约90%的红绿荧光转变率．这表明该ФC31整合酶报告系统提供了一种在细胞中快捷可靠的评价ФC31整合酶功能的方法．     

Ex and in vivo characterization of the wavelength‐dependent 3‐photon action cross‐sections of red fluorescent proteins covering the 1700‐nm window

Multiphoton action cross‐sections are the prerequisite for excitation light selection. At the 1700‐nm window suitable for deep‐tissue imaging, wavelength‐dependent 3‐photon action cross‐sections ησ₃ for RFPs are unknown, preventing wavelength selection. Here we demonstrate: (1) ex vivo measurement of wavelength‐dependent ησ₃ for purified RFPs; (2) a multiphoton imaging guided measurement system for in vivo measurement; and (3) in vivo measurement of wavelength‐dependent ησ₃ in RFP labeled cells. These fundamental results will provide guidelines for excitation wavelength selection for 3‐photon fluorescence imaging of RFPs at the 1700‐nm window, and augment the existing database of multiphoton action cross‐sections for fluorophores.      

The profiles of red fluorescent proteins with antinucleopolyhedrovirus activity in races of the silkworm Bombyx mori

Partially purified red fluorescent proteins (RFPs) secured from the gut juice of 5th-instar multivoltine and bivoltine silkworm races were observed as several bands in electrophoretograms and chromatographic eluates. Interestingly, different races of silkworms had varying numbers of fluorescent protein bands: 11 in Pure Mysore (resistant), 11 in Nistari (resistant), 4 in CSR₂ (moderately susceptible) and 1 in NB₄D₂ (highly susceptible). Bioassay experiments indicated that the fluorescent bands had antinucleopolyhedrovirus (antiNPV) activity. The molar extinction coefficients and fluorescence quantum yields of all RFPs were estimated. The purified tetrapyrroles were characterized by UV–visible absorption and fluorescence spectral analyses. All tetrapyrrole moieties associated with RFPs were found to be different and characteristic of the fluorescent bands. The resulting qualitative and quantitative differences among the individual RFPs from various races of silkworm were related to the susceptibilities of the silkworms to the viral disease. Moreover, light was found to be essential for the synthesis of RFPs, and, therefore, the role of light in the synthesis of RFPs was evaluated. Thus, this work may elucidate the process of RFP synthesis in silkworm, which may be used as a biomarker to measure the degree of susceptibility of silkworm races to NPV. Therefore, the characteristic band pattern may be used as an indicator to define the relative resistance of a race towards the specific virus.     

PSEUDO-NITZSCHIA PUNGENS AND P. MULTISENES (BACILLARIOPHYCEAE): NUCLEAR RIBOSOMAL DNAS AND SPECIES DIFFERENCES1

The region of the nuclear ribosomal DNA (rDNA) operon containing the small subunit (SSU), internal transcribed spacer 1 (ITS1), and a portion of the 5.8s rDNA gene was sequenced in one isolate each of Pseudo-nitzschia multiseries (Hasle) Hasle and Pseudo-nitzschia pungens (Grunow in Cleve & Möller) Hasle. The SSUs of these two species were highly similar, differing only in 14 point mutations and one insertion/deletion in 1774 bp. The ITS1 sequences were more variable, with 57 point mutations and three insertion/deletions in 257 bp. There were no differences in 44 bp of the 5.8S sequences. Restriction fragment patterns (RFPs) for the restriction endonucleases HaeIII, Hha1, and Rsa1 for 13 isolates of P. multiseries from the Atlantic, Pacific, and Gulf coasts of the United States and 16 isolates of P. pungens from the three coasts of the United States, in addition to Japan and China, were compared. There were differences between the RFPs of P. multiseries and P. pungens that corresponded to sites mapped by the DNA sequences, but no infraspecific variation in RFPs was observed for either species. The differences in RFPs correlate with morphological, immunological, and other rDNA differences and support the recognition of these taxa as separate species.     

RFP tags for labeling secretory pathway proteins

Red fluorescent proteins (RFPs) are useful tools for live cell and multi-color imaging in biological studies. However, when labeling proteins in secretory pathway, many RFPs are prone to form artificial puncta, which may severely impede their further uses. Here we report a fast and easy method to evaluate RFPs fusion properties by attaching RFPs to an environment sensitive membrane protein Orai1. In addition, we revealed that intracellular artificial puncta are actually colocalized with lysosome, thus besides monomeric properties, pKa value of RFPs is also a key factor for forming intracellular artificial puncta. In summary, our current study provides a useful guide for choosing appropriate RFP for labeling secretory membrane proteins. Among RFPs tested, mOrange2 is highly recommended based on excellent monomeric property, appropriate pKa and high brightness.     

Recovery of Red Fluorescent Protein Chromophore Maturation Deficiency through Rational Design

Red fluorescent proteins (RFPs) derived from organisms in the class Anthozoa have found widespread application as imaging tools in biological research. For most imaging experiments, RFPs that mature quickly to the red chromophore and produce little or no green chromophore are most useful. In this study, we used rational design to convert a yellow fluorescent mPlum mutant to a red-emitting RFP without reverting any of the mutations causing the maturation deficiency and without altering the red chromophore’s covalent structure. We also created an optimized mPlum mutant (mPlum-E16P) that matures almost exclusively to the red chromophore. Analysis of the structure/function relationships in these proteins revealed two structural characteristics that are important for efficient red chromophore maturation in DsRed-derived RFPs. The first is the presence of a lysine residue at position 70 that is able to interact directly with the chromophore. The second is an absence of non-bonding interactions limiting the conformational flexibility at the peptide backbone that is oxidized during red chromophore formation. Satisfying or improving these structural features in other maturation-deficient RFPs may result in RFPs with faster and more complete maturation to the red chromophore.     

Restriction fragment polymorphisms as probes for plant diversity and their development as tools for applied plant breeding

Summary Maize and tomato cDNA clones have been hybridized in Southern blotting experiments to plant genomic DNA prepared from different lines to detect restriction fragment polymorphisms (RFPs). In maize we have found that a high degree of genetic variability is present, even among domestic inbred lines. Most randomly chosen maize cDNA clones can be used to detect elements of this variability. Similar levels of polymorphism are observed when genomic DNA is digested with any of a number of different restriction enzymes and probed with individual clones. When a clone is hybridized to genomic DNAs prepared from several different maize lines, a number of different alleles are often detected at a single locus. At the same time one clone can often detect more than one independently segregating locus by cross hybridization to related sequences at other loci. As expected these markers are inherited as simple codominant Mendelian alleles from one generation to the next and colinkage of these markers can be demonstrated in the progeny from a heterozygous parent. In similar studies with tomato, remarkably different results were found. Few RFPs were demonstrable among domestic Lycopersicon esculentum lines although a higher level of variability could be detected when comparing esculentum with its wild Lycopersicon relatives. These results are discussed in relation to the applied uses of RFPs in plant breeding as well as the inherent variability of different plant genomes.This work was supported in part by funds from Sandoz Ltd. (Basel, Switzerland) and its subsidiary company, Northrup King Co. (Minneapolis, Minn., U.S.A.) as well as by NSF SBIR grant #BSR-8360870.     

Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus pneumoniae and Efficient Transformation with DNA Assembled via the Gibson Assembly Method

During the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single-cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogen Streptococcus pneumoniae and other low-GC Gram-positive bacteria. In the present work, we constructed and compared four red fluorescent proteins (RFPs) in S. pneumoniae. Two orange-red variants, mOrange2 and TagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson assembly (isothermal assembly) products, a method that speeds up the strain construction process significantly. Our data show that mCherry is the fastest-maturing RFP in S. pneumoniae and is best suited for studying gene expression, while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multicolor labeling in S. pneumoniae and related organisms.     

Spectral correction for handheld optoacoustic imaging by means of near‐infrared optical tomography in reflection mode

In vivo imaging of tissue/vasculature oxygen saturation levels is of prime interest in many clinical applications. To this end, the feasibility of combining two distinct and complementary imaging modalities is investigated: optoacoustics (OA) and near‐infrared optical tomography (NIROT), both operating noninvasively in reflection mode. Experiments were conducted on two optically heterogeneous phantoms mimicking tissue before and after the occurrence of a perturbation. OA imaging was used to resolve submillimetric vessel‐like optical absorbers at depths up to 25 mm, but with a spectral distortion in the OA signals. NIROT measurements were utilized to image perturbations in the background and to estimate the light fluence inside the phantoms at the wavelength pair (760 nm, 830 nm). This enabled the spectral correction of the vessel‐like absorbers' OA signals: the error in the ratio of the absorption coefficient at 830 nm to that at 760 nm was reduced from 60%‐150% to 10%‐20%. The results suggest that oxygen saturation (SO ₂) levels in arteries can be determined with <10% error and furthermore, that relative changes in vessels' SO ₂ can be monitored with even better accuracy. The outcome relies on a proper identification of the OA signals emanating from the studied vessels.      

Imaging of the Blue,Green, and Red Fluorescence Emission of Plants: An Overview

An overview is given on the fluorescence imaging of plants. Emphasis is laid upon multispectral fluorescence imaging in the maxima of the fluorescence emission bands of leaves, i.e., in the blue (440 nm), green (520 nm), red (690 nm), and far-red (740 nm) spectral regions. Details on the origin of these four fluorescence bands are presented including emitting substances and emitting sites within a leaf tissue. Blue-green fluorescence derives from ferulic acids covalently bound to cell walls, and the red and far-red fluorescence comes from chlorophyll (Chl) a in the chloroplasts of green mesophyll cells. The fluorescence intensities are influenced (1) by changes in the concentration of the emitting substances, (2) by the internal optics of leaves determining the penetration of excitation radiation and partial re-absorption of the emitted fluorescence, and (3) by the energy distribution between photosynthesis, heat production, and emission of Chl fluorescence. The set-up of the Karlsruhe multispectral fluorescence imaging system (FIS) is described from excitation with UV-pulses to the detection with an intensified CCD-camera. The possibilities of image processing (e.g., formation of fluorescence ratio images) are presented, and the ways of extraction of physiological and stress information from the ratio images are outlined. Examples for the interpretation of fluorescence images are given by demonstrating the information available for the detection of different developmental stages of plant material, of strain and stress of plants, and of herbicide treatment. This novel technique can be applied for near-distance screening or remote sensing.     

Multispectral near infrared absorption imaging for histology of skin cancer

Multispectral imaging combines the spectral resolution of spectroscopy with the spatial resolution of imaging and is therefore very useful for biomedical applications. Currently, histological diagnostics use mainly stainings with standard dyes (eg, hematoxylin + eosin) to identify tumors. This method is not applicable in vivo and provides low amounts of chemical information. Biomolecules absorb near infrared light (NIR, 800‐1700 nm) at different wavelengths, which could be used to fingerprint tissue. Here, we built a NIR multispectral absorption imaging setup to study skin tissue samples. NIR light (900‐1500 nm) was used for homogenous wide‐field transmission illumination and detected by a cooled InGaAs camera. In this setup, images I(x, y, λ) from dermatological samples (melanoma, nodular basal‐cell carcinoma, squamous‐cell carcinoma) were acquired to distinguish healthy from diseased tissue regions. In summary, we show the potential of multispectral NIR imaging for cancer diagnostics.