Fluorescent Vital Stains for Complementary Labelling of Protoplasts from Trichoderma Spp

In this study several fluorescent vital stains were evaluated for their ability to provide complementary vital staining of protoplasts of Trichoderma spp. for selection of heterokaryons following protoplast fusion. Tetramethyl rhodamine isothiocyanate and fluorescein isothiocyanate were rejected because they stained only a small proportion of protoplasts. Fluorescein diacetate stained all protoplasts, but the chromophore leaked rapidly from stained cells. A mixture of FluoroBora T and acriflavine stained all cells, but intensity was low and fading upon illumination was rapid. Nile red stained lipid bodies in all cells, but the stain was lost upon protoplast fusion in polyethylene glycol. Rhodamine 6G, on the other hand, stained all cells, fluoresced green, and was stable through fusion and upon illumination. Hydroethidine also stained all protoplasts, and staining was relatively stable through fusion and upon illumination. Hydroethidine fluoresced red and stained nuclei more prominently than the cytoplasm. Rhodamine 6G and hydroethidine were tested on a number of strains to determine whether they were toxic to protoplasts. No toxicity to any strain was noted with rhodamine 6G. Hydroethidine, however, was toxic at the higher concentrations tested, especially when stained protoplasts were exposed to light. When protoplasts were stained with the minimum concentration giving ready visualization and were incubated in darkness, hydroethidine also was nontoxic. Hydroethidine and rhodamine 6G are useful complementary vital stains of Trichoderma protoplasts for visualization of frequency and type (dicell, multicell) of fusion.     

荧光活性染料DiO标记腹腔巨噬细胞的示踪与应用研究

目的 以细胞膜绿色荧光活性染料DiO （DiOC18（3））标记腹腔巨噬细胞（peritoneal macrophage）,探讨在巨噬细胞消失反应（macrophage disappearance reaction,MDR）中腹腔巨噬细胞的示踪研究。方法 DiO标记腹腔巨噬细胞,过继移植给C57BL/6小鼠;以脂多糖（lipopolysaccharide,LPS）诱导体内MDR。采用荧光显微镜和流式细胞术检测DiO标记的腹腔巨噬细胞数量及荧光强度;分离收集小鼠的各组织,进行冰冻切片,检测DiO标记的腹腔巨噬细胞分布情况。结果 荧光显微镜和流式细胞仪观察发现,腹腔注射LPS能显著降低腹腔中DiO标记的腹腔巨噬细胞数量及荧光强度。在MDR过程中消失的腹腔巨噬细胞,通过冰冻切片发现在肝脏、胸腺及脾脏中有分布。结论 DiO标记对腹腔巨噬细胞的存活无影响且能长效保持荧光,是一种安全、有效的示踪腹腔巨噬细胞分布的技术手段。     

Rapid Chemical Synthesis of Oligodeoxyribonucleotides by the Improved Phosphotriester Method

Abstract

An efficient phosphotriester methodology based on the use of condensing agents in the presence of several new nucleophilic catalysts has been developed.     

The Incorporation of 2,6-Diaminopurine Into Oligodeoxyribonucleotides by the Phosphoramidite Method

Abstract

A method has been developed to allow the synthesis of oligodeoxyribonucleotides containing 2,6-diaminopurine and avoiding depurination.     

定量PCR的荧光技术

荧光定量PCR是在普通PCR基础上,利用荧光技术对核酸进行绝对定量的一项新兴技术,其灵敏度高、特异性高、操作简便和定量准确,已被广泛应用于临床和科研中。为更好地发挥荧光定量PCR的优点,荧光技术领域的研发工作十分活跃。     

Synthesis of Oligodeoxyribonucleotides Containing 2,6-Diaminopurine

Abstract

The preparation of a new protected derivative of 2,6-diaminopurine 2′-deoxyriboside carrying two phenoxyacetyl groups is described. The new derivative is useful to prepare oligonucleotides containing 2,6-diaminopurine and it is deprotected at the same time as the standard protecting groups of the natural bases.     

Synthesis of Oligodeoxyribonucleotides Containing Oleylamine Moieties

A method for directional introduction of oleylamine residues to any position of oligodeoxyribonucleotides during their automated synthesis was developed. The presence of oleylamine residues in 3"- or 5"-terminal nucleotides was shown to have no effect on the thermodynamic stability of DNA duplexes formed by such oligonucleotides and the complementary sequences. The rate of hydrolysis of oligonucleotides containing oleylamine residues in the 3"-terminal units by the snake venom phosphodiesterase was shown to be markedly lower than that of natural oligonucleotides.     

Fluorescent and Ferritin Labelling of Cuticle Surface Carbohydrates of Caenorhabditis elegans and Panagrellus redivivus

Caenorhabditis elegans and Panagrellus redivivus were investigated for surface carbohydrates using fluorescent-labelled and ferritin-labelled lectins. Rhodamine-labelled Concanavalin A was specifically located in the cephalic region of both species. Rhodamine-labelled wheat germ agglutinin was located over the entire cuticle of P. redivivus but was absent on C. elegans. Rhodamine-labelled peanut agglutinin and Limax flavus agglutinin did not label nematodes of either species. Galactose and sialic acid were not detected on either species, whereas mannose-glucose residues were specifically localized in the head areas of both species. No detectable N-acetylglucosamine occurred on C. elegans, but it was evenly distributed over the cuticle surface of P. redivivus.     

Polymerization of Horseradish Peroxidase by a Laccase‐Catalyzed Tyrosine Coupling Reaction

The polymerization of proteins can create newly active and large bio‐macromolecular assemblies that exhibit unique functionalities depending on the properties of the building block proteins and the protein units in polymers. Herein, the first enzymatic polymerization of horseradish peroxidase (HRP) is reported. Recombinant HRPs fused with a tyrosine‐tag (Y‐tag) through a flexible linker at the N‐ and/or C‐termini are expressed in silkworm, Bombyx mori. Trametes sp. laccase (TL) is used to activate the tyrosine of Y‐tagged HRPs with molecular O₂ to form a tyrosyl‐free radical, which initiates the tyrosine coupling reaction between the HRP units. A covalent dityrosine linkage is also formed through a HRP‐catalyzed self‐crosslinking reaction in the presence of H₂O₂. The addition of H₂O₂ in the self‐polymerization of Y‐tagged HRPs results in lower activity of the HRP polymers, whereas TL provides site‐selectivity, mild reaction conditions and maintains the activity of the polymeric products. The cocrosslinking of Y‐tagged HRPs and HRP‐protein G (Y‐HRP‐pG) units catalyzed by TL shows a higher signal in enzyme‐linked immunosorbent assay (ELISA) than the genetically pG‐fused HRP, Y‐HRP‐pG, and its polymers. This new enzymatic polymerization of HRP promises to provide highly active and functionalized polymers for biomedical applications and diagnostics probes.     

New Labelling Technology for Molecular Probes Applied to the Ligation Detection Reaction–Universal Array System

The ligation detection reaction (LDR) associated with universal arrays (UA) uses a fluorescently labelled probe (DP) and a Zip Code-extended probe to detect single nucleotide polymorphisms in DNA target sequences. When used for genotyping, the LDR-UA technique uses two DPs, each specific to an allele and labelled with a different fluorophore. The fluorescent signals are processed to calculate the genotype. The uneven decay of fluorophores due to ageing and freezing/thawing cycles and the consequent unequal fluoresce level can lead to erroneous genotype calls. To circumvent this problem, an indirect labelling strategy was developed based on the substitution of the fluorophore with allele-specific 22 bp universal labelling sequences (ULS). Labelling is achieved with fluorescently labelled oligos complementary to the ULS (cULS). The strategy improved the uniformity in probe labelling, and generated results comparable to those using direct-labelled probes, as shown by genotyping 22 polymorphic sites in 70 samples with both strategies. This method can be easily implemented in the routine screening with LDR-UA or other techniques. Moreover, the approach results in a significant cost reduction over traditional direct labelling, and offers the possibility to interchange fluorophores and to increase the fluorescent signal by using multiple-labelled cULS.     

Possibilities of Triplex-Formation by 21-Mer Oligodeoxyribonucleotides as Analogs of Tetrahymena Pre-rRNA Fragments

Abstract

A potential DNA triple helix of 21-mer oligodeoxyribonucleotides was synthesized and characterized. The strands were chosen to study the interaction of internal guide and intervening sequences analogs as well as adjacent 3′and 5′exon parts around the splicing site of Tetrahymena pre-rRNA. Further in parallel works a series of different RNA and DNA strands was synthesized and combined yielding a suitable order of stability. Here we want to show an isolated examination of a DNA-strand triple helix with defined sequences containing a central mismatched base arrangement and T-A-T bases at the ends.     

Automated Synthesis of Cyclic Oligodeoxyribonucleotides via Phosphoramidite Method

Abstract

The functionalized polyethylene glycol/polystyrene copolymer support 4 was shown to be suitable for a completely automated synthesis of small- to medium-sized cyclic oligodeoxy-ribonucleotides. Syntheses of the linear precursors were achieved by the phosphoramidite method, whereas the cyclization reactions were based on the phosphotriester chemistry.     

Quantitative Parametrs of Cooperative Interactions of the Oligodeoxyribonucleotides on the Complementary Template

Abstract

The cooperative interactions of oligonucleotides on the complementary template were studied using the quantitative analysis of the template alkylation with the oligonucleotides bearing covalently attached 4-[N-(2-chloroethyl)-N-methylamino]benzyl group at 5′-end. The influence of the mismatched nucleotides and the stabilizing N-(2-hydroxyethyl)phenazinium group at the 5′- and 3′-ends of the oligonucleotides on the parameters of cooperativity was evaluated.     

Reaction Fields in the Environment of Fluorescent Probes: Polarity Profiles in Membranes

Fluorescent probes in biological systems are sensitive to environmental polarity by virtue of their response to the reaction field created by polarization of the dielectric medium. Classically, fluorophore solvatochromism is analyzed in terms of the Lippert equation and later variants, all of which rely upon the original reaction field of Onsager. A recent survey of the solvent dependence of EPR spin-label probes, which are responsive solely to the reaction field in the ground state without the complication of excited states, shows that the reaction field of Block and Walker performs best in describing the polarity dependence. In this model, the step-function transition to the bulk dielectric medium used by Onsager is replaced by a graded transition. Analysis of the Stokes shifts for representative fluorescent membrane probes, such as PRODAN, DANSYL, and anthroyl fatty acid, reveals that, of several different reaction fields (including that of Onsager), the Block-Walker model best describes the dependence on solvent dielectric constant and refractive index for the different probes simultaneously. This is after full allowance is made for all contributions involving polarizability of the fluorophore, a point that is frequently neglected or treated incorrectly in studies using biological fluorescent probes. By using the full range of polar and apolar solvents, it is then possible to establish a common reference for the polarity dependence of different fluorophores and to relate this also to the polarity dependence of biologically relevant spin-label EPR probes. An important application is calibration of the transmembrane polarity profile recorded by fluorescent probes in terms of the high-resolution profile obtained from site-specifically spin-labeled lipid chains.     

Diastereomer Separation of Azobenzene-Tethered Oligodeoxyribonucleotides and Determination of Their Absolute Configurations by Enzymatic Digestion

Two diastereomers were produced by the introduction of azobenzene-tethering prochiral linker (2,2-bis(hydroxymethyl)propionic acid) in the modified ODN, which had been used for the photoregulation of DNA functions. We found that this modified ODN with sequence 5′-…pNpXpN…-3′ (p = phosphate; N = nucleoside; X = azobenzene residue) could be digested to pX (the phosphate at the 5′ side of X was left) by an over excess of Phosphodiesterase I. By comparing the retention time of pX from the separated diastereomer with that of authentic R- or S-pX on chiral HPLC, absolute configuration could be easily determined.