Coral Fluorescent Proteins as Antioxidants

Background

A wide array of fluorescent proteins (FP) is present in anthozoans, although their biochemical characteristics and function in host tissue remain to be determined. Upregulation of FP''s frequently occurs in injured or compromised coral tissue, suggesting a potential role of coral FPs in host stress responses.

Methodology/Principal Findings

The presence of FPs was determined and quantified for a subsample of seven healthy Caribbean coral species using spectral emission analysis of tissue extracts. FP concentration was correlated with the in vivo antioxidant potential of the tissue extracts by quantifying the hydrogen peroxide (H₂O₂) scavenging rates. FPs of the seven species varied in both type and abundance and demonstrated a positive correlation between H₂O₂ scavenging rate and FP concentration. To validate this data, the H₂O₂ scavenging rates of four pure scleractinian FPs, cyan (CFP), green (GFP), red (RFP) and chromoprotein (CP), and their mutant counterparts (without chromophores), were investigated. In vitro, each FP scavenged H₂O₂ with the most efficient being CP followed by equivalent activity of CFP and RFP. Scavenging was significantly higher in all mutant counterparts.

Conclusions/Significance

Both naturally occurring and pure coral FPs have significant H₂O₂ scavenging activity. The higher scavenging rate of RFP and the CP in vitro is consistent with observed increases of these specific FPs in areas of compromised coral tissue. However, the greater scavenging ability of the mutant counterparts suggests additional roles of scleractinian FPs, potentially pertaining to their color. This study documents H₂O₂ scavenging of scleractinian FPs, a novel biochemical characteristic, both in vivo across multiple species and in vitro with purified proteins. These data support a role for FPs in coral stress and immune responses and highlights the multi-functionality of these conspicuous proteins.     

Tumor Endothelial Cell-Specific Drug Delivery System Using Apelin-Conjugated Liposomes

Background

A drug delivery system specifically targeting endothelial cells (ECs) in tumors is required to prevent normal blood vessels from being damaged by angiogenesis inhibitors. The purpose of this study was to investigate whether apelin, a ligand for APJ expressed in ECs when angiogenesis is taking place, can be used for targeting drug delivery to ECs in tumors.

Methods and Results

Uptake of apelin via APJ stably expressed in NIH-3T3 cells was investigated using TAMRA (fluorescent probe)-conjugated apelin. Both long and short forms of apelin (apelin 36 and apelin 13) were taken up, the latter more effectively. To improve efficacy of apelin- liposome conjugates, we introduced cysteine, with its sulfhydryl group, to the C terminus of apelin 13, resulting in the generation of apelin 14. In turn, apelin 14 was conjugated to rhodamine-encapsulating liposomes and administered to tumor-bearing mice. In the tumor microenvironment, we confirmed that liposomes were incorporated into the cytoplasm of ECs. In contrast, apelin non-conjugated liposomes were rarely found in the cytoplasm of ECs. Moreover, non-specific uptake of apelin-conjugated liposomes was rarely detected in other normal organs.

Conclusions

ECs in normal organs express little APJ; however, upon hypoxic stimulation, such as in tumors, ECs start to express APJ. The present study suggests that apelin could represent a suitable tool to effectively deliver drugs specifically to ECs within tumors.     

烟曲霉的遗传操作系统

建立遗传操作系统是研究基因功能的一种重要手段,在研究烟曲霉Aspergillus fumigatus致病性、感染机制以及耐药性等方面发挥重要作用。文中介绍了烟曲霉中主要存在的遗传操作系统包括根癌农杆菌介导的转化、PEG介导的原生质体转化法,并重点介绍了非同源末端连接DNA修复技术、RNA沉默技术以及CRISP-Cas9转化技术。     

人工转录因子研究进展

转录因子是真核表达调控中非常重要的一类反式作用因子,通常由DNA结合结构域与效应结构域两部分组成,研究发现这两个结构域可以各自独立发生作用。基于转录因子的这种结构特点,可以人为地选择针对特定序列的DNA结合结构域与具有特定作用的效应结构域构建人工转录因子。目前人工转录因子的DNA结合结构域多为C₂H₂ 型锌指结构,每一个锌指单元由大约30个氨基酸组成,识别DNA双螺旋大沟中相连的3bp序列,并可通过氢键作用与相应的碱基结合;多个锌指可以串联成簇,从而识别并结合较长的DNA序列区域。常见的人工转录因子的效应结构域有激活结构域以及抑制结构域,不同的效应结构域赋予人工转录因子不同的功能。目前人工转录因子已经在基础研究、药物设计以及基因治疗等领域得到了广泛的应用。     

Transposon Assisted Gene Insertion Technology (TAGIT): A Tool for Generating Fluorescent Fusion Proteins

We constructed a transposon (transposon assisted gene insertion technology, or TAGIT) that allows the random insertion of gfp (or other genes) into chromosomal loci without disrupting operon structure or regulation. TAGIT is a modified Tn5 transposon that uses Kan^R to select for insertions on the chromosome or plasmid, β-galactosidase to identify in-frame gene fusions, and Cre recombinase to excise the kan and lacZ genes in vivo. The resulting gfp insertions maintain target gene reading frame (to the 5′ and 3′ of gfp) and are integrated at the native chromosomal locus, thereby maintaining native expression signals. Libraries can be screened to identify GFP insertions that maintain target protein function at native expression levels, allowing more trustworthy localization studies. We here use TAGIT to generate a library of GFP insertions in the Escherichia coli lactose repressor (LacI). We identified fully functional GFP insertions and partially functional insertions that bind DNA but fail to repress the lacZ operon. Several of these latter GFP insertions localize to lacO arrays integrated in the E. coli chromosome without producing the elongated cells frequently observed when functional LacI-GFP fusions are used in chromosome tagging experiments. TAGIT thereby faciliates the isolation of fully functional insertions of fluorescent proteins into target proteins expressed from the native chromosomal locus as well as potentially useful partially functional proteins.     

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle¹. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions^2-5.Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner^4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes¹², has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.     

Cell-Specific Targeting Strategies for Electroporation-Mediated Gene Delivery in Cells and Animals

The use of electroporation to facilitate gene transfer is an extremely powerful and useful method for both in vitro and in vivo applications. One of its great strengths is that it induces functional destabilization and permeabilization of cell membranes throughout a tissue leading to widespread gene transfer to multiple cells and cell types within the electric field. While this is a strength, it can also be a limitation in terms of cell-specific gene delivery. The ability to restrict gene delivery and expression to particular cell types is of paramount importance for many types of gene therapy, since ectopic expression of a transgene could lead to deleterious host inflammatory responses or dysregulation of normal cellular functions. At present, there are relatively few ways to obtain cell-specific targeting of nonviral vectors, molecular probes, small molecules, and imaging agents. We have developed a novel means of restricting gene delivery to desired cell types based on the ability to control the transport of plasmids into the nuclei of desired cell types. In this article, we discuss the mechanisms of this approach and several applications in living animals to demonstrate the benefits of the combination of electroporation and selective nuclear import of plasmids for cell-specific gene delivery.     

锌指核酸内切酶:基因操作的有力工具

针对动植物进行基因靶向操作的技术,是解析基因功能、研究疾病,以及农业经济生产中一个有用的工具。至今,基因靶向操作主要是通过在胚胎干细胞(embryonic stem cell,ES cell)中进行同源重组或者是体细胞核转移的方法进行,但同源重组方法由于需要ES细胞而被限制在个别物种,而核转移方法存在核去分化、效率低、成本高的缺陷。近几年,一种基于锌指核酸内切酶(zinc-finger nuclease,ZFN)基因靶向修饰的新技术被应用于包括植物、果蝇、爪蟾、斑马鱼和大鼠等不同物种的基因操作。通过胚胎注射ZFN的质粒或是mRNA可以有效地定靶并迅速地在内源基因上引起可遗传的突变。ZFN介导基因靶向敲除的可行性,使得那些无法获得ES细胞和克隆技术支持的物种的基因靶向修饰成为可能。     

A Two-Plasmid System for Independent Genetic Manipulation of Subunits of Homodimeric Proteins and Selective Isolation of Chimeric Dimers

We have designed and tested a modular two-plasmid expression system which allows coexpression of two different subunits of recombinant dimeric protein in Escherichia coli and selective purification of heterodimers. We have constructed a new expression vector, pBIOEx, with p15a replication origin which allows its stable coexistence with different ColE1 group plasmids. The expression cassette of this plasmid under control of the T7 promoter contains cloning site, followed by a short sequence coding for the C-terminal extension of the recombinant protein which is a target of the in vivo biotinylation by BirA protein. The expression unit is bicistronic, the second expressed protein being BirA. We have used this plasmid together with pET30a to clone kinesin heavy-chain fragment and coexpressed the two polypeptide chains differing by tags on their C-termini and we purified heterodimers made of two recombinant molecules. The heterodimeric protein had a normal biochemical activity. There was no discrimination against heterodimer formation at the dimerization step. The system is a powerful tool in studies of different aspects of interactions between subunits of the homodimeric proteins since it makes possible separate genetic manipulations on each subunit of the dimer.     

Targeted Gene Manipulation in Plants Using the CRISPR/Cas Technology

The CRISPR/Cas technology is emerging as a revolutionary genome editing tool in diverse organisms including plants,and has quickly evolved into a suite of versatile tools for sequence-specific gene manipulations beyond genome editing.Here,we review the most recent applications of the CRISPR/Cas toolkit in plants and also discuss key factors for improving CRISPR/Cas performance and strategies for reducing the off-target effects.Novel technical breakthroughs in mammalian research regarding the CRISPR/Cas toolkit will also be incorporated into this review in hope to stimulate prospective users from the plant research community to fully explore the potential of these technologies.     

Assessment of Transient Gene Expression in Plant Tissues Using the Green Fluorescent Protein as a Reference

Four different promoters (35S and enhanced 35S of the cauliflower mosaic virus, polyubiquitin of maize and actin1 of rice) were compared in a transient assay using maize leaves and particle bombardment. A gene encoding the jellyfish green fluorescent protein (GFP) driven by the 35S promoter was used as an internal standard to monitor the effectiveness of each bombardment. Normalisation of the transient expression assay using the GFP reference significantly reduced the variability between separate bombardments and allowed for a rapid and accurate evaluation of different promoters in microprojectile-bombarded leaves.     

MSJ-1, a New Member of the DNAJ Family of Proteins, Is a Male Germ Cell-Specific Gene Product

A cDNA encoding for a new member of the DnaJ protein family has been isolated by screening a mouse spermatogenic cell expression library. The full-length cDNA obtained by extension of the original clone with RT-PCR has been characterized with respect to its DNA sequence organization and expression. The predicted open reading frame encodes a protein of 242 amino acid residues whose sequence is similar to that of bacterial DnaJ proteins in the amino-terminal portion since it contains the highly conserved J domain which is present in all DnaJ-like proteins and is considered to have a critical role in DnaJ protein–protein interactions. In contrast, the middle and carboxyl-terminal regions of the protein are not similar to any other DnaJ proteins, with the exception of the human neuronal HSJ-1 with which displays a 48% identity in a 175-amino-acid overlap. Analysis of RNAs from a wide spectrum of mouse somatic tissues, including the brain, and from ovary and testis reveals that the gene is specifically expressed in testis only. Developmental Northern blot analysis of testis RNA from mice of different ages andin situhybridization on juvenile and adult testis sections demonstrate that the mRNA is first transcribed in spermatids. A similar pattern of expression is exhibited also in rat testis. Based upon all these observations, we have named this novel mouse gene, MSJ-1, for mouse sperm cell-specific DNAJ first homolog.     

分泌蛋白特异性基因陷阱的设计与验证

蛋白质分子向细胞外分泌的过程有赖于信号肽的介导.基因陷阱是功能性基因克隆的一种有效方法,经典的基因陷阱以geo作为报告基因,对所克隆的基因类型没有选择性.将绿脓杆菌外毒素、2A序列、IL-2受体穿膜区以及新霉素磷酸转移酶的基因依次融合构建新型报告基因——peo,该报告基因能够特异性地甄别具有信号肽编码序列的基因.为验证peo对信号肽编码序列的特异性甄别作用,以peo为报告基因,构建了3种质粒载体,分别模拟用基因陷阱载体进行筛选时可能出现的3种情况,通过转染HeLa细胞、G418筛选以及碱性磷酸酶检测,证明peo能够有效地区分信号肽和非信号肽编码序列,以peo为报告基因改造的基因陷阱载体可以用于分泌蛋白基因的筛选.     

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at www.impactaging.com. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.Download video file.^{(158M, mp4)}     

An Arsenic Fluorescent Compound as a Novel Probe to Study Arsenic-Binding Proteins

Arsenic-binding proteins are under continuous research. Their identification and the elucidation of arsenic/protein interaction mechanisms are important because the biological effects of these complexes may be related not only to arsenic but also to the arsenic/protein structure. Although many proteins bearing a CXXC motif have been found to bind arsenic in vivo, new tools are necessary to identify new arsenic targets and allow research on protein/arsenic complexes. In this work, we analyzed the performance of the fluorescent compound APAO-FITC (synthesized from p-aminophenylarsenoxide, APAO, and fluorescein isothiocyanate, FITC) in arsenic/protein binding assays using thioredoxin 1 (Trx) as an arsenic-binding protein model. The Trx-APAO-FITC complex was studied through different spectroscopic techniques involving UV?CVis, fluorescence, atomic absorption, infrared and circular dichroism. Our results show that APAO-FITC binds efficiently and specifically to the Trx binding site, labeling the protein fluorescently, without altering its structure and activity. In summary, we were able to study a protein/arsenic complex model, using APAO-FITC as a labeling probe. The use of APAO-FITC in the identification of different protein and cell targets, as well as in in vivo biodistribution studies, conformational studies of arsenic-binding proteins, and studies for the design of drug delivery systems for arsenic anti-cancer therapies, is highly promising.