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Alkaline phosphatase inhibitors as labels of DNA probes.

A new approach to nucleic acid labeling was developed by preparing bifunctional reagents containing, in addition to the DNA-linking group, a competitive inhibitor of the chromogenic enzyme alkaline phosphatase. The nucleic acids labeled in such a way were able to bind themselves to the enzyme, whose activity was restored in the presence of a chromogenic substrate. Five phosphonic-acid-containing reagents were synthesized and coupled to linearized pBR322 plasmid DNA by different condensation methods. Eight probes thus obtained were assayed in a modified dot-blot detection procedure obtaining the best nucleic acid detection sensitivity of 25 pg. Finally, five of the above probes were tested in hybridization experiments, reaching sensitivity of 50 pg.

Alkaline Phosphatase↗

Histochemical techniques for locating Escherichia coli beta-galactosidase activity in transgenic organisms.

Escherichia coli beta-galactosidase is a commonly used reporter molecule for analyzing gene expression. Recently, beta-galactosidase fusions have been applied to a variety of eukaryotic systems. The techniques for constructing and introducing beta-galactosidase fusion constructs as well as soluble assays for total enzyme function have been described in detail elsewhere. This article describes histochemical techniques for analyzing organisms that contain a functional beta-galactosidase fusion construct. The object is to determine semiquantitatively which cells are expressing the beta-galactosidase fusion protein, as well as the subcellular localization of the protein. Due to its prevalence in the author's laboratory, Caenorhabditis elegans is used as a canonical organism for the detailed methods described.

Animals↗

Enhanced sensitivity and precision in an enzyme-linked immunosorbent assay with fluorogenic substrates compared with commonly used chromogenic substrates.

Quantitative enzyme-linked immunosorbent assay (ELISA) is a widely used tool for analyzing biopharmaceutical and vaccine products. The superior sensitivity of the ELISA format is conferred by signal amplification through the enzymatic oxidation or hydrolysis of substrates to products with enhanced color or fluorescence. The extinction coefficient for a colored product or the quantum yield of a fluorescent product, coupled with the efficiency of the immobilized enzyme, is the determining factor for the sensitivity and precision of a given ELISA. The enhancement of precision and sensitivity using fluorogenic substrates was demonstrated in a direct-binding ELISA in a low-analyte concentration range compared with commonly used chromogenic substrates. The enhancement in precision was demonstrated quantitatively with lower coefficients of variation in measurements of signal intensities, approximately a five- to six-fold enhancement in signal-to-noise ratio at a given analyte concentration with fluorogenic substrates. Similarly, the amplitude of the enhancement in sensitivity, as reflected by relative limits of detection or quantitation, is approximately two- to five-fold when compared with commonly used chromogenic substrates. Additional advantages of a fluorescence-based ELISA format include the continuous monitoring of initial rates of enzymatic reactions, the measurement of fluorescence changes in the presence of particulate materials, the absence of a quench step, and a larger quantifiable analyte range.

Chromogenic Compounds↗

Immunofluorescent labeling of cancer cells with quantum dots synthesized in aqueous solution.

Thioglycolic-acid-stabilized CdTe quantum dots, synthesized directly in aqueous solution, are successfully conjugated with biotin and polyethylene glycol. Using these conjugates, we report the development of this kind of water-soluble quantum dot for immunofluorescent labeling of cancer cells. The results show that these conjugates have very low nonspecific binding and good stability against photobleaching, enabling them to be applied in many biological fields, such as cellular labeling, intracellular tracking, and other imaging applications.

Cell Line, Tumor↗

The activity and inhibition of the food vacuole plasmepsin from the rodent malaria parasite Plasmodium chabaudi.

The rodent malaria parasite Plasmodium chabaudi encodes one food vacuole plasmepsin-the aspartic proteinases important in haemoglobin degradation. A recombinant form of this enzyme was found to cleave a variety of peptide substrates and was susceptible to a selection of naturally occurring and synthetic inhibitors, displaying an inhibition profile distinct from that of aspartic proteinases from other malaria parasites. In addition, inhibitors of HIV proteinase that kill P. chabaudi in vivo were also inhibitors of this new plasmepsin. P. chabaudi is a widely used model for human malaria species and, therefore, the characterisation of this plasmepsin is an important contribution towards understanding its biology.

Amino Acid Sequence↗

Fetal membranes act as a barrier for adenoviruses: gene transfer into exocoelomic cavity of rat fetuses does not affect cells in the fetus.

OBJECTIVES: In utero gene therapy has a potential to correct genetic disorders before the first clinical symptoms appear. Our aim was to examine whether the exocoelomic cavity between amniotic and chorionic membranes offers a minimally invasive route for gene transfer to the fetus during early pregnancy. STUDY DESIGN: We injected lacZ-adenovirus (4 x 10(9) pfu) during open surgery into the exocoelomic cavity of rat fetuses (n=50) and analyzed the fetuses and rat dams for transgene expression with X-gal staining and polymerase chain reaction. RESULTS: Giant cells around Reichert's membrane, the outermost extraembryonic membrane in rodents, were transduced; but no transduction was observed in the cells of the fetuses or rat dams. CONCLUSION: In rodents, the exocoelomic cavity does not offer a route for gene transfer into the fetus. It was concluded that fetal membranes act as a barrier that prevents adenoviral particles from passing between embryonic cavities.

Adenoviridae↗

Bayesian fluorescence in situ hybridisation signal classification.

Previous research has indicated the significance of accurate classification of fluorescence in situ hybridisation (FISH) signals for the detection of genetic abnormalities. Based on well-discriminating features and a trainable neural network (NN) classifier, a previous system enabled highly-accurate classification of valid signals and artefacts of two fluorophores. However, since this system employed several features that are considered independent, the naive Bayesian classifier (NBC) is suggested here as an alternative to the NN. The NBC independence assumption permits the decomposition of the high-dimensional likelihood of the model for the data into a product of one-dimensional probability densities. The naive independence assumption together with the Bayesian methodology allow the NBC to predict a posteriori probabilities of class membership using estimated class-conditional densities in a close and simple form. Since the probability densities are the only parameters of the NBC, the misclassification rate of the model is determined exclusively by the quality of density estimation. Densities are evaluated by three methods: single Gaussian estimation (SGE; parametric method), Gaussian mixture model assuming spherical covariance matrices (GMM; semi-parametric method) and kernel density estimation (KDE; non-parametric method). For low-dimensional densities, the GMM generally outperforms the KDE that tends to overfit the training set at the cost of reduced generalisation capability. But, it is the GMM that loses some accuracy when modelling higher-dimensional densities due to the violation of the assumption of spherical covariance matrices when dependent features are added to the set. Compared with these two methods, the SGE and NN provide inferior and superior performance, respectively. However, the NBC avoids the intensive training and optimisation required for the NN, demanding extensive resources and experimentation. Therefore, when supporting these two classifiers, the system enables a trade-off between the NN performance and NBC simplicity of implementation.

Algorithms↗

Advances in fluorescence imaging with quantum dot bio-probes.

After much effort in surface chemistry development and optimization by several groups, fluorescent semiconductor nanocrystals probes, also known as quantum dots or qdots, are now entering the realm of biological applications with much to offer to biologists. The road to success has been paved with hurdles but from these efforts has stemmed a multitude of original surface chemistries that scientists in the biological fields can draw from for their specific biological applications. The ability to easily modulate the chemical nature of qdot surfaces by employing one or more of the recently developed qdot coatings, together with their exceptional photophysics have been key elements for qdots to acquire a status of revolutionary fluorescent bio-probes. Indeed, the unique properties of qdots not only give biologists the opportunity to explore advanced imaging techniques such as single molecule or lifetime imaging but also to revisit traditional fluorescence imaging methodologies and extract yet unobserved or inaccessible information in vitro or in vivo.

Chromogenic Compounds↗

Practical application of a chromogenic FXIIa assay.

Autohydrolysis of blood factor XII (FXII+FXIIa-->2FXIIa) is found to be a facile reaction in neat-buffer buffer solutions of FXII but an insignificant reaction in the presence of plasma proteins. Autohydrolysis causes a chromogenic assay for FXIIa in buffer solution to strongly deviate from the traditional plasma-coagulation assay. Autohydrolysis can be accommodated by performing chromogenic detection of FXIIa as a rate assay in swamping concentrations of FXII. Rate-assay results performed in this way are shown to be in analytical agreement with the plasma-coagulation assay. Autohydrolysis can be used as a means of amplifying FXIIa produced by contacting neat-buffer solutions of FXII with biomaterials, suggesting a route to highly sensitive measurement of biomaterial hemocompatibility.

Biological Assay↗

Application of chromogenic reagents in surface plasmon resonance (SPR).

In this paper, a new simple approach for sensitivity optimization in surface plasmon resonance (SPR) chemosensors based on colorimetric ligands is presented. A new design of SPR sensor with tunable analytical wavelength (lambda(SPR)) was constructed for this purpose, to perform studies on the ligand absorbance spectra related sensitivity enhancement. Unlike commercial SPR sensors which operate at one lambda(SPR), the new device can be used for sensitivity analysis at selected lambda(SPR) in the range 550-750 nm, offering the possibility to identify the highest sensitivity lambda(SPR) in regard to the spectral changes of the selected ligand. Measurements can be easily done in ligand bulk solutions without immobilization steps. Sensitivity enhancement analysis and optimization of lambda(SPR) on chromogenic reagents with hypsochromic shift in their absorption spectra are demonstrated in this contribution. Optimal selection of analytical wavelength, set at the absorbance peak of chromogenic reagent Eriochrome Black T (EBT) was observed to result in up to two times increased SPR sensitivity to Cd(2+) compared to wavelengths selected in other parts of the ligand absorbance spectra, with a limit of detection (LOD) 0.2 ppm. The sensitivity enhancement at optimal lambda(SPR) was observed to be related to increased refractive index (n), drop in extinction coefficient (alpha) and simultaneous hypsochromic shift of the EBT absorbance spectra causing the lambda(SPR) to match the absorbance peak shoulder.

Chromogenic Compounds↗

Resorbable poly(D,L)lactide plates and screws for osteosynthesis of condylar neck fractures in sheep.

We made osteotomies in the condylar neck in 12 adult sheep to simulate fractures, and joined the two ends with 2 poly(D,L)lactide (PDLLA) plates and 8 PDLLA screws 2mm in diameter. The animals were killed after 2, 6, and 12 months and bony healing was assessed macroscopically and histologically. The plates and screws remained intact and there was no displacement of the bony ends. The degrading plates, which were still visible in the specimens after 6 months, had been replaced by bone. At 12 months the PDLLA had been resorbed with no foreign body reaction and no resorption of underlying bone. The articular discs showed no signs of degeneration.

Absorbable Implants↗

Design and synthesis of chromogenic thiopeptolide substrates as MetAPs active site probes.

Twenty one chromogenic thiopeptolide substrates were designed and synthesized as the active site probes and analyzed with each S1 site of mutant residues and enzymes of wild-type MetAP1s. The preliminary enzymatic experiments indicate that cysteine 70 or 202, at either Escherichia coli or human MetAP1, played a crucial role in the methionine hydrolysis.

Amino Acid Sequence↗

A chromism-based assay (CHROBA) technique for in situ detection of protein kinase activity.

A unique chromism-based assay technique (CHROBA) using photochromic spiropyran-containing peptides has been firstly established for detection of protein kinase A-catalyzed phosphorylation. The alternative method has advantages that avoid isolation and/or immobilization of kinase substrates to remove excess reagents including nonreactive isotope-labeled ATP or fluorescently-labeled anti-phosphoamino acid antibodies from the reaction mixture. Such a novel protocol based on thermocoloration of the spiropyran moiety in the peptide can offer not only an efficient screening method of potent kinase substrates but also a versatile analytical tool for monitoring other post-translational modification activities.

Benzopyrans↗

A fluorogenic and chromogenic probe that detects the esterase activity of trace copper(II).

Bis(picolinoyl)fluorescein, a new fluorogenic and chromogenic probe for monitoring the esterase activity of metal ions, has been synthesized. The probe is highly selective for Cu2+ and is applicable both to quantification of the free ion and detection of esterolytic activity of Cu bound to organic ligands or biomolecules, with a detection limit of 100 nM by fluorimetry.

Chromogenic Compounds↗

Magnifying chromoendoscopy for the detection of premalignant gastrointestinal lesions.

The prognosis for patients with malignancies of the gastrointestinal tract is strictly dependent on the early detection of premalignant and malignant lesions. At present, endoscopy can be performed with new, powerful high-resolution or magnifying endoscopes. Comparable to the rapid development in chip technology, the optic features of the newly designed endoscopes offer resolutions that allow new mucosal surface details to be seen. In conjunction with chromoendoscopy, the newly discovered tool of video endoscopy is much easier to use and more impressive than previously used fibreoptic endoscopy. This review summarises the value of magnifying endoscopy in the upper and lower gastrointestinal tract and focuses on gastroesophageal reflux disease and early gastric and colorectal cancer.

Chromogenic Compounds↗

HistoGreen: a new alternative to 3,3'-diaminobenzidine-tetrahydrochloride-dihydrate (DAB) as a peroxidase substrate in immunohistochemistry?

Abide its toxicity, 3,3'-diaminobenzidine-tetrahydrochloride-dihydrate (DAB) was the most potent marker for immunochemistry at the light and electron microscopic level in the last decades. Recently, a sensitive substrate for immunohistochemical staining methods and in-situ hybridization, HistoGreen, was developed for the use with peroxidase. In peroxidase reactions, HistoGreen delivers a green staining product which is suitable for permanent embedding without water. In contrast to DAB, HistoGreen is not toxic. To evaluate its usefulness, we performed comparative immunohistochemistry on angiotensin II (AT1)-receptors with DAB- and HistoGreen-staining on paraffin embedded slices of the rat brain at the light microscopic level. This also included counterstaining with Mayer's Hemalum and Nuclear Fast Red, respectively. We could demonstrate that HistoGreen delivers a coarsely grained label which is fast detectable in light microscopy. HistoGreen equals DAB in the exact localization of the immunoreaction to a large degree but its reaction product is considerably less stable in alcohol and water than DAB. In combination with Nuclear Fast Red, HistoGreen provides excellent imaging properties for the visualization and documentation of immunoreactive structures paired with an adequate demonstration of cellular details. Its tendency towards rapid over-staining as well as its low stability will restrict the use of HistoGreen in some areas of immunohistochemical research, yet the new chromogen represents an interesting alternative to DAB at the light microscopic level.

3,3'-Diaminobenzidine↗

Isolation and characterization of a trypsin fraction from the pyloric ceca of chinook salmon (Oncorhynchus tshawytscha).

A trypsin fraction was isolated from the pyloric ceca of New Zealand farmed chinook salmon (Oncorhynchus tshawytscha) by ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The chinook salmon enzyme hydrolyzed the trypsin-specific synthetic substrate benzoyl-DL-arginine-p-nitroanilide (DL-BAPNA), and was inhibited by the general serine protease inhibitor phenyl methyl sulfonyl fluoride (PMSF), and also by the specific trypsin inhibitors - soybean trypsin inhibitor (SBTI) and benzamidine. The enzyme was active over a broad pH range (from 7.5 to at least pH 10.0) at 25 degrees C and was stable from pH 4.0 to pH 10.0 when incubated at 20 degrees C, with a maximum at pH 8.0. The optimum temperature for the hydrolysis of DL-BAPNA by the chinook salmon enzyme was 60 degrees C, however, the enzyme was unstable at temperatures above 40 degrees C. The molecular mass of the chinook salmon trypsin was estimated as 28 kDa by SDS-PAGE.

Animals↗