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Molecular Probes (molecular + probe)

Distribution by Scientific Domains

Chemistry	36%
Life Sciences	26%
Medical Sciences	20%
Polymers and Materials Science	13%

Selected Abstracts

5-Ethynyl-2,-deoxyuridine labeling detects proliferating cells in the regenerating avian cochlea,

THE LARYNGOSCOPE, Issue 9 2009
Christina L. Kaiser PhD
Abstract Objectives/Hypothesis: The avian cochlea regenerates hair cells following aminoglycoside treatment through supporting cell proliferation. Immunocytochemical labeling of 5-bromo-2,-deoxyuridine (BrdU), a thymidine analog, is a popular nonradioactive marker for identifying cells in the DNA synthesis (S phase) of the cell cycle. However, it requires harsh treatments to denature double-stranded DNA for the antibody to bind BrdU. We explored a new method using 5-ethynyl-2,-deoxyuridine (EdU) as a thymidine analog and a nonantibody azide/alkyne reaction between EdU and the fluorescent probe. We propose that EdU is as effective as BrdU, but without the requirement for harsh denaturation or the use of antibodies for detection. Study Design: Two-week-old chicks received a single gentamicin injection followed by a single EdU injection 72 hours later. Cochleae were extracted 4,8 hours later, fixed, and processed for fluorescent detection of EdU. Methods: Cochleae were processed for detection of incorporated EdU using the Click-iT Imaging Kit (Invitrogen/Molecular Probes, Carlsbad, CA) and colabeled with Sox2, myosin VI, or myosin VIIa antibodies. Whole-mount cochlear preparations were examined with confocal microscopy. Results: Supporting cells incorporated EdU into their newly synthesized DNA during the 4,8 hours following the EdU injection and were readily detected with little background signal. The intensity and quantity of cells labeled were similar to or better than that seen for BrdU. Conclusions: The EdU method is as effective as BrdU, without requiring harsh denaturation or secondary antibodies to identify proliferating cells. Thus, the nonantibody EdU system allows more flexibility by enabling colabeling with multiple antibodies to other cellular proteins involved in regeneration. Laryngoscope, 2009 [source]

A Highly Sensitive Hybrid Colorimetric and Fluorometric Molecular Probe for Cyanide Sensing Based on a Subphthalocyanine Dye,

ADVANCED FUNCTIONAL MATERIALS, Issue 9 2006
E. Palomares
Abstract A highly sensitive, selective colorimetric and fluorometric molecular probe based on a subphthalocyanine dye has been developed for cyanide-anion determination in aqueous solution. It has also been shown that a carboxysubphthalocyanine derivative can be covalently anchored to transparent mesoporous nanocrystalline high-surface-area metal oxide films to detect low concentrations of cyanide anion in pure water with no interference from other anionic or cationic species. [source]

A Molecular Probe for the Detection of Homopurine Sequences

CHEMBIOCHEM, Issue 1 2007
Ivan Trkulja
The highly selective detection of homopurine target strands with a triplex-forming molecular probe is described. The binding of a clamp-type oligonucleotide containing two terminally attached pyrene molecules to the target sequence is easily monitored through excimer formation. The oligonucleotide probe allows the efficient discrimination of single nucleotide mismatches because of the high mismatch sensitivity of the triplex formation. [source]

A Water-Soluble, Octacationic Zinc Phthalocyanine as Molecular Probe for Nucleic Acid Secondary Structure

CHEMISTRY & BIODIVERSITY, Issue 2 2007
An-Ming Zhang
Abstract The interaction between CT-DNA and the zinc phthalocyanine ZnPc (1) was studied by UV/VIS and fluorescence titration, as well as by thermal denaturation. ZnPc was found to strongly bind to CT-DNA (Kapp=7.35×105, M,1) in a non-intercalative mode. The photosensitized cleavage of pBR322 DNA was found to efficiently proceed via singlet-oxygen (1O2) production. Further, ZnPc (1) caused site-specific scission of guanine (G) bases around the bulge of the hairpin oligonucleotides OD1,OD3, as clearly shown by gel-electrophoresis experiments. [source]

Switchable Fluorescent and Solvatochromic Molecular Probes Based on 4-Amino- N -methylphthalimide and a Photochromic Diarylethene

EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 15 2008
Sergey F. Yan
Abstract New fluorescent photochromic compounds (1 -H and 1 -Boc) have been synthesized and characterized in different solvents. The fluorescence emission can be switched "on" and "off" with visible light and UV, respectively, by means of the photochromic reaction. The emission wavelength and efficiency strongly depend on the polarity of the solvent. The compounds show a positive solvatochromic effect in the emission maxima, and their fluorescence quantum yield decreases as the solvent's polarity increases (from cyclohexane to dioxane). In solvents more polar than dioxane the emission is too weak and therefore undetectable, and thus 1 -H and 1 -Boc behave as "normal" photochromic compounds. The photochromic reaction is also sensitive to the environment. A decrease of more than an order of magnitude was found for the quantum yield of the colouring reaction (,OF,CF) for 1 -H in ethanol compared with cyclohexane, and an about threefold decrease in ,OF,CF was observed for the compound 1 -Boc in polar solvents (compared with apolar solvents). For both compounds the ring-opening reaction was found not to dependent on the solvent. The novel fluorescent molecular switches 1 -H and 1 -Boc are able to probe the polarity of their microenvironment. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008) [source]

Quantitative assessment of chondrocyte viability after laser mediated reshaping: A novel application of flow cytometry

LASERS IN SURGERY AND MEDICINE, Issue 1 2003
Alexandre Rasouli BS
Abstract Background and Objectives Lasers can be used to reshape cartilage by accelerating mechanical stress relaxation. In this study, fluorescent differential cell viability staining and flow cytometry were used to determine chondrocyte viability following laser heating. Study Design/Materials and Methods Porcine septal cartilages were irradiated with an Nd:YAG laser (,,= 1.32 ,m, 25 W/cm2) while surface temperature, stress relaxation, and diffuse reflectance were recorded. Each slab received one, two, or three laser exposures (respective exposure times of 6.7, 7.2, 10 seconds). Irradiated samples were then divided into two groups analyzed immediately and at 5 days following laser exposure. Chondrocytes were isolated following serial enzymatic digestion, and stained using SYTO®/DEAD RedÔ (Molecular Probes, Eugene, OR). A flow cytometer was then used to detect differential cell fluorescence; size; granularity; and the number of live cells, dead cells, and post-irradiation debris in each treatment population. Results Nearly 60% of chondrocytes from reshaped cartilage samples isolated shortly after one irradiation, were viable while non-irradiated controls were 100% viable. Specimens irradiated two or three times demonstrated increasing amounts of cellular debris along with a reduction in chondrocyte viability: 31 and 16% after two and three exposures, respectively. In those samples maintained in culture medium and assayed 5 days after irradiation, viability was reduced by 28,88%, with the least amount of deterioration in untreated and singly irradiated samples. Conclusions Functional fluorescent dyes combined with flow cytometric analysis successfully determines the effect of laser irradiation on the viability of reshaped cartilage. Lasers Surg. Med. 32:3,9,2003. © 2003 Wiley-Liss, Inc. [source]

Dual Chromophore-Nitroxides: Novel Molecular Probes, Photochemical and Photophysical Models and Magnetic Materials

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2007
Gertz I. Likhtenstein
Over the last decades scientists have faced growing requirements in novel methods of fast and sensitive analysis of antioxidant status of biological systems, spin redox probing and spin trapping, investigation of molecular dynamics, and of convenient models for studies of photophysical and photochemical processes. In approaching this problem, methods based upon the use of dual chromophore-nitroxide (CN) compounds have been suggested and developed. A CN consists of two molecular sub-functionality (a chromophore and a stable nitroxide radical) tethered together by spacers. In the dual compound the nitroxide is a strong intramolecular quencher of the fluorescence from the chromophore fragment. Reduction to hydroxylamine, oxidation of the nitroxide fragment or addition of an active radical yield the fluorescence increase and the parallel decay of the fragment electron spin resonance (ESR) signal. At certain conditions the dual molecules undergo photomagnetic switching and form excited state multi-spin systems. These unique properties of CN were intensively exploited as the basis for several methodologies, which include molecular probing, modeling intramolecular photochemical and photophysical processes, and construction of new magnetic materials. [source]

Synthesis and Application of Fluorescein- and Biotin-Labeled Molecular Probes for the Chemokine Receptor CXCR4

CHEMBIOCHEM, Issue 7 2008
Shinya Oishi Dr.
Abstract The design, synthesis, and bioevaluation of fluorescence- and biotin-labeled CXCR4 antagonists are described. The modification of D -Lys8 at an ,-amino group in the peptide antagonist Ac-TZ14011 derived from polyphemusin II had no significant influence on the potent binding of the peptide to the CXCR4 receptor. The application of the labeled peptides in flow cytometry and confocal microscopy studies demonstrated the selectivity of their binding to the CXCR4 receptor, but not to CXCR7, which was recently reported to be another receptor for stromal cell-derived factor 1 (SDF-1)/CXCL12. [source]

Synthesis of Postulated Molecular Probes: Stereoselective Free-Radical-Mediated C-Glycosylation in Tandem with Hydrogen Transfer.

CHEMINFORM, Issue 24 2005
Yvan Guindon
Abstract For Abstract see ChemInform Abstract in Full Text. [source]

Ecological niche partitioning in the picoplanktonic green alga Micromonas pusilla: evidence from environmental surveys using phylogenetic probes

ENVIRONMENTAL MICROBIOLOGY, Issue 9 2008
Elodie Foulon
Summary Very few studies have analysed the niches of pelagic protist in details. This is because for most protists, both an accurate species definition and methods for routine detection and quantification of cells are lacking. The morphospecies Micromonas pusilla, a marine unicellular green alga, is the most ubiquitous and cosmopolitan picoeukaryote described to date. This species comprises several independent genetic lineages or clades, which are not currently distinguishable based on comparison of their morphology or biogeographical distribution. Molecular probes were used to detect and quantify the genetic clades of M. pusilla in samples from temperate, polar and tropical environments in order to assess potential ecological niche partitioning. The three clades were detected in all biogeographical regions studied and were commonly found in sympatry. Cell abundances recorded for clades A and B were high, especially at coastal stations. Clade C, when detected, was always at low abundances and is suggested to be a low-light clade. Shifts in the contribution of clades to total M. pusilla abundance were observed along environmental gradients, both at local and basin-wide scales. This suggests that the phylogenetic clades occupy specific niches and confirms the existence of cryptic species within the morphospecies M. pusilla. Parameters which can precisely explain the distribution of these cryptic species remain to be elucidated. [source]

A Highly Sensitive Hybrid Colorimetric and Fluorometric Molecular Probe for Cyanide Sensing Based on a Subphthalocyanine Dye,

Facing the challenge of biosample imaging by FTIR with a synchrotron radiation source

JOURNAL OF SYNCHROTRON RADIATION, Issue 1 2010
Cyril Petibois
Fourier-transform infrared (FTIR) synchrotron radiation (SR) microspectroscopy is a powerful molecular probe of biological samples at cellular resolution (<10,µm). As the brilliance of SR is 100,1000 times higher than that of a conventional Globar source, FTIR microscopes are now available in almost all advanced SR facilities around the world. However, in spite of this superior performance, the expected advances in IR SR microscopy have not yet been realised, particularly with regard to bio-analytical studies of single cells and soft tissues. In recent decades solid-state array detectors have revolutionized the fields of molecular spectroscopy and chemical imaging, and now new IR focal plane array detectors implemented at ultra-bright SR facilities will extend the performance and overcome the existing limitations, possibly allowing IR SR instrumentation to achieve the highest sensitivity and resolution of molecular imaging. The impact of IR imaging on large tissue area and the complexity of the analysis are discussed. In view of the high brilliance of SR sources, a comparison of published microscope images is given. Finally, it is briefly outlined how an optimized combination of IR instrumentation and SR optical systems could reach the expected advantages of a SR-based FTIR imaging system. [source]

Encapsulation and Controlled Release of a Hydrophobic Drug Using a Novel Nanoparticle-Forming Hyperbranched Polyester,

MACROMOLECULAR BIOSCIENCE, Issue 7 2005
Jianhua Zou
Abstract Summary: An amphiphilic, hyperbranched polymer suitable for use in controlled drug delivery is reported. This polymer was obtained by modification of the hyperbranched aliphatic polyester BoltornÔ H20 (H20) with succinic anhydride and then glycidyl methacrylate, and formed nanoparticles in aqueous solution. The critical association concentration was 7.4,×,10,3 g,·,L,1, as determined by fluorescence spectroscopy using pyrene as a molecular probe. A static/dynamic laser light scattering (LLS) study revealed that the average particle size was 39.4 nm with a low particle size distribution (PDI = 0.04), and that each particle was composed of about 350 amphiphilic molecules. Daidzein, a hydrophobic traditional Chinese medicine, was encapsulated during particle formation and the release properties were determined. The optimal feeding concentration of daidzein to hyperbranched polyester was 4.9,×,10,5 g,·,mL,1 to 5.0,×,10,3 g,·,mL,1 with a loading efficiency of 76.1%. In the presence of the enzyme Lipase PS, the drug loaded nanoparticles degraded in a random one-by-one manner and released the drug over a few days. This system is therefore a novel controlled drug release system based on nanoparticles formed of hyperbranched polyester. Encapsulation of daidzein by hyperbranched polyester particles. [source]

Pax-3 and Pax-7 Label Muscle Progenitor Cells During Myotomal Myogenesis in Coregonus lavaretus (Teleostei: Coregonidae)

ANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 6 2009
A. Kacperczyk
Summary In Coregonus lavaretus, prior the mesoderm segmentation, in cells adjacent to the notochord called adaxial cells MyoD and slow myosin heavy chain (MyHC-slow) proteins were observed. After somite formation, adaxial cells migrate towards the lateral part of the myotome and form a layer of red muscles. Deeper cells differentiate into white muscle fibres. In situ hybridization using Pax-3 molecular probe revealed, that after somitogenesis, Pax-3 is expressed in a layer of cells superficial to the myotome resembling the "external cells" (found in many teleosts species) or dermomyotome described in Amniota. During later developmental stages Pax-3 gene is expressed in cells in intermyotomal space and then in myoblasts between myotubes. In these cells Pax-7 protein was also observed. Pax-3/7 positive cells which have migrated into the myotomes differentiate into satellite cells/secondary myoblasts and participate in hypertrophic and hyperplastic growth of muscles. [source]

A Molecular Probe for the Detection of Homopurine Sequences

Synthesis, and DNA-Binding and DNA-Photocleavage Properties of Multiply Charged Porphyrins

CHEMISTRY & BIODIVERSITY, Issue 3 2007
Kai Wang
Abstract Four new cationic porphyrins, compounds 1,4, with five to seven positive charges, were synthesized, characterized, and investigated for their binding properties towards calf-thymus DNA (CT-DNA). UV/VIS and fluorescence-titration data indicated strong binding, the apparent binding constants (Kapp; (1.3,10)×10,6M) increasing with increasing number of charges, as determined by competitive fluorescence titration using ethidium bromide (EB) as molecular probe. These results were qualitatively confirmed by the observed photocleavage efficiency of the porphyrins towards plasmid pBR322 DNA. [source]

Improved synthesis of DOTA tetraamide ligands for lanthanide(III) ions: A tool for increasing the repertoire of potential PARACEST contrast agents for MRI and/or fluorescent sensors

CONTRAST MEDIA & MOLECULAR IMAGING, Issue 3 2010
Luis M. De León-Rodríguez
Abstract The synthesis of new DOTA tetraamide (DOTAMR4) compounds is of great interest given their application in the formation of Ln(III) complexes as potential PARACEST contrast agents in MRI or fluorescent molecular probes. In this context amino acid and peptide DOTAMR4 derivatives are particularly attractive since the amino-acid and/or peptide moiety can show responsive properties dependent on a given stimuli which might translate to changes in water exchange rates of the corresponding Ln(III) complex. Current synthesis of DOTAMR4 derivatives is typically carried out by reacting haloacetamide intermediates with cyclen. However, this method fails to generate the tetra-substituted products when bulky substituents are present in the haloacetamide and in some cases this intermediate cannot be prepared by conventional acylation procedures limiting the number of DOTAMR4 compounds available for study. As a solution to these limitations, an improved methodology for the synthesis of DOTAMR4 by coupling DOTA to an appropriate amine containing reagent (i.e. protected amino-acids with the , -amino group free) is presented in this work. Several DOTAMR4 derivatives which are difficult or impossible to prepare with the traditional methodologies were easily obtained starting with DOTA. A new protocol was derived using this methodology for the solution-phase synthesis of DOTA peptide derivatives. With this methodology, many other DOTAMR4 peptide and non-peptide derivatives have been prepared in our laboratories with several of these new compounds showing interesting properties for molecular imaging. Copyright © 2010 John Wiley & Sons, Ltd. [source]

Fluorescence lifetime imaging of activatable target specific molecular probes

CONTRAST MEDIA & MOLECULAR IMAGING, Issue 1 2010
Raphael Alford
Abstract In vivo optical imaging using fluorescently labeled self-quenched monoclonal antibodies, activated through binding and internalization within target cells, results in excellent target-to-background ratios. We hypothesized that these molecular probes could be utilized to accurately report on cellular internalization with fluorescence lifetime imaging (FLI). Two imaging probes were synthesized, consisting of the antibody trastuzumab (targeting HER2/neu) conjugated to Alexa Fluor750 in ratios of either 1:8 or 1:1. Fluorescence intensity and lifetime of each conjugate were initially determined at endosomal pHs. Since the 1:8 conjugate is self-quenched, the fluorescence lifetime of each probe was also determined after exposure to the known dequencher SDS. In vitro imaging experiments were performed using 3T3/HER2+ and BALB/3T3 (HER2,) cell lines. Changes in fluorescence lifetime correlated with temperature- and time-dependent cellular internalization. In vivo imaging studies in mice with dual flank tumors [3T3/HER2+ and BALB/3T3 (HER2,)] detected a minimal difference in FLI. In conclusion, fluorescence lifetime imaging monitors the internalization of target-specific activatable antibody,fluorophore conjugates in vitro. Challenges remain in adapting this methodology to in vivo imaging. Copyright © 2010 John Wiley & Sons, Ltd. [source]

Xenopus, the next generation: X. Tropicalis genetics and genomics

DEVELOPMENTAL DYNAMICS, Issue 4 2002
Nicolas Hirsch
Abstract A small, fast-breeding, diploid relative of the frog Xenopus laevis, Xenopus tropicalis, has recently been adopted for research in developmental genetics and functional genomics. X. tropicalis shares advantages of X. laevis as a classic embryologic system, but its simpler genome and shorter generation time make it more convenient for multigenerational genetic, genomic, and transgenic approaches. Its embryos closely resemble those of X. laevis, except for their smaller size, and assays and molecular probes developed in X. laevis can be readily adapted for use in X. tropicalis. Genomic manipulation techniques such as gynogenesis facilitate genetic screens, because they permit the identification of recessive phenotypes after only one generation. Stable transgenic lines can be used both as in vivo reporters to streamline a variety of embryologic and molecular assays, or to experimentally manipulate gene expression through the use of binary constructs such as the GAL4/UAS system. Several mutations have been identified in wild-caught animals and during the course of generating inbred lines. A variety of strategies are discussed for conducting and managing genetic screens, obtaining mutations in specific sequences, achieving homologous recombination, and in developing and taking advantage of the genomic resources for Xenopus tropicalis. © 2002 Wiley-Liss, Inc. [source]

Detection of nematode antagonistic bacteria by fluorogenic molecular probes,

EPPO BULLETIN, Issue 3-4 2000
A. Ciancio
Last-generation DNA probes include molecules yielding a fluorogenic emission through an intramolecular change occurring after hybridization to a complementary sequence. They display a high sequence specificity and may detect even single-base mutations in polymerase chain reaction (PCR) amplification products. We applied Scorpion primers for the detection of an unculturable nematode-parasitic bacterium, Pasteuria sp., with potential as a biological control agent. A 16S rDNA oligonucleotide sequence unique to Pasteuria spp. was used to detect the parasite in juveniles of Heterodera goettingiana or in soil. The parasitized nematodes came from a population with a Pasteuria prevalence of 40,80% and were individually checked for parasitism. Probes with 6-carboxy-fluorescein (FAM) at the 5'terminus, used for PCR with nematodes or soil, successfully detected the parasite from both samples. The amplification of the expected 139bp fragment was shown both by fluorescence observed under UV excitation in the eppendorfs and by gel electrophoresis of the corresponding amplicons. The potential of this detection method for the study of unculturable bacteria is discussed. [source]

The transient nature of maximum maleic anhydride grafting of polypropylene: A mechanistic approach based on a consecutive reaction model.

JOURNAL OF APPLIED POLYMER SCIENCE, Issue 1 2007

Abstract This article compares the batch solution and molten state chemical modification of an atactic polypropylene to yield a grafted polypropylene. Short reaction times appear to be sufficient and indeed necessary for the highest graft yields to be obtained if degradative processes occurring in both reaction media are to be avoided. The consecutive reactions for the optimized grafting reaction pathway were proposed for the solution process in an earlier article. The present work attempts to correlate this pathway with that of the molten state process. Grafted succinic anhydride groups react with two resorcine molecules to yield grafted succinyl-fluorescein groups. This work considers the resorcine units as true molecular probes, to be able to stabilize and activate the complexes formed between the succinic anhydride groups and the propylene sequence. This work shows the unsteady and later dynamic character of the process. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 345,351, 2007 [source]

Molecular imaging: The application of small animal positron emission tomography

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue S39 2002
Douglas J. Rowland
Abstract The extraordinary advances in genomic technologies over the last decade have led to the establishment of new animal models of disease. The use of molecular imaging techniques to examine these models, preferably with non-destructive imaging procedures, such as those offered by positron emission tomography (PET), are especially valuable for the timely advancement of research. With the use of small animal PET imaging it is possible to follow individual subjects of a sample population over an extended time period by using highly specific molecular probes and radiopharmaceuticals. In this Prospect small animal PET imaging will be described, specifically focusing on the current technologies, its applications in molecular imaging and the logistics of performing small animal PET. J. Cell. Biochem. Suppl. 39: 110,115, 2002. © 2002 Wiley-Liss, Inc. [source]

Investigation of dyed human hair fibres using apertureless near-field scanning optical microscopy

JOURNAL OF MICROSCOPY, Issue 2 2006
F. FORMANEK
Summary We present the first studies of dyed human hair fibres performed with an apertureless scanning near-field optical microscope. Samples consisted of 5-µm-thick cross-sections, the hair fibres being bleached and then dyed before being cut. Hair dyed with two molecular probes diffusing deep inside the fibre or mainly spreading at its periphery were investigated at a wavelength of 655 nm. An optical resolution of about 50 nm was achieved, well below the diffraction limit; the images exhibited different optical contrasts in the cuticle region, depending on the nature of the dye. Our results suggest that the dye that remains confined at the hair periphery is mainly located at its surface and in the endocuticle. [source]

Raman spectroscopy in comparative investigations of mechanisms of binding of three molecular probes , fluorescein, eosin, and erythrosin , to human serum albumin

LASER PHYSICS LETTERS, Issue 11 2008
I.M. Vlasova
Abstract The comparative analysis of binding of three molecular fluorescent probes (fluorescein, eosin, and erythrosin), belonging to one homologous family, to human serum albumin (HSA) is made by Raman spectroscopy method. The binding of all three probes to binding Center I of HSA is registered. The character of binding of initial probe of the given homologous family , fluorescein , to protein differs from character of binding of its halogen-derivatives (eosin and erythrosin) to protein. The differences in binding of these three probes to HSA are determined by value of electronegativity of atoms of lateral radicals in structural formulas of probes and, therefore, by value of pK of their ionized groups. (© 2008 by Astro Ltd., Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA) [source]

Presumptive pre-sertoli cells express genes involved in cell proliferation and cell signalling during a critical window in early testis differentiation

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 12 2007
Aron T. Cory
In mammals, the pre-Sertoli cell of the male genital ridge is the first cell type to display sex specific differentiation and differential gene expression. The genetic cascade driving the differentiationof pre-Sertoli cells and ultimately testis formationis beginning to be unravelled, but many questions remain. A better understanding of the transcriptome of pre-Sertoli cells immediately after sex determination is essential in order to further understand this differentiationprocess. A mouse model expressing Red Fluorescent Protein (RFP) under the control of a hybrid mouse/pig SRY promoter (HybSRYp-RFP) was used to purify cells from embryonic day 12.0 (e12.0) male genital ridges. To compare the transcriptomes of HybSRYp-RFP cell populations versus age matched whole female genital ridges, RNA was extracted and used to generate molecular probes that were hybridized onto Affymetrix Mouse Genome 430 2.0 micro-arrays. The expression of genes considered markers for pre-Sertoli cells, including Sox9, Mis, Dhh and Fgf9 were identified within the HybSRYp-RFP expressing cell population, while markers for germ cells (Oct4, SSEA-1) and endothelial cells (Ntrk3) were not identified. In contrast, markers for ovarian somatic cell expression, including Fst and Bmp2, were identified as overexpressed within the ovarian cell population. In a general fashion, genes identified as 2.5-fold over expressed in HybSRYp-RFP expressing cells coded notably for cell signalling and extra cellular proteins. The expression of Sox10, Stc2, Fgf18, Fgf13 and Wnt6 were further characterized via whole mount in situ hybridization (WISH) on male and female genital ridges between e11.5 and e14.5. Sox10, Fgf18, Fgf13 and Stc2 gene expression was detected within the male genital ridges while Wnt6 was found diffusely within both the male and female genital ridges. These data represent the earliest comprehensive microarray expression analysis of purified presumptive pre-Sertoli cells available to date. Mol. Reprod. Dev. 74: 1491,1504, 2007. © 2007 Wiley-Liss, Inc. [source]

Identification of a Second rRNA Gene Unit in the Perkinsus andrewsi Genome

THE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2 2004
WOLF T. PECHER
ABSTRACT. Perkinsus species are parasitic protozoa of mollusks, currently classified within the Perkinsozoa, a recently established phylum that is basal to the Apicomplexa and Dinozoa. Ribosomal RNA (rRNA) genes and their intergenic spacers have been used to support the taxonomy of Perkinsus species, the description of new species, and to develop molecular probes for their detection and identification. We previously described ultrastructure, behavior in culture, and partial sequence of the rRNA locus of a Perkinsus species isolated from the baltic clam Macoma balthica. The rRNA genes and intergenic spacers of this Perkinsus isolate differed from those described in the currently accepted species to a degree that led to its designation as a new species, Perkinsus andrewsi. In this study, we identify an additional rRNA gene unit (rRNA-B) in the P. andrewsi holotype, and report the complete sequences of both rRNA gene units. Except for the 5. 8S, all regions of the rRNA-B gene unit exhibited sequence differences from that initially described (rRNA-A). Each rRNA gene unit is arranged in a "head-to-tail" tandem repeat. This is the first report demonstrating two distinct rRNA units in a Perkinsus species. [source]

ADLOC: An Aptamer-Displacement Assay Based on Luminescent Oxygen Channeling

CHEMISTRY - A EUROPEAN JOURNAL, Issue 36 2010
Dipl.-Chem.
Abstract Functional nucleic acids, such as aptamers and allosteric ribozymes, can sense their ligands specifically, thereby undergoing structural alterations that can be converted into a detectable signal. The direct coupling of molecular recognition to signal generation enables the production of versatile reporters that can be applied as molecular probes for various purposes, including high-throughput screening. Here we describe an unprecedented type of a nucleic acid-based sensor system and show that it is amenable to high-throughput screening (HTS) applications. The approach detects the displacement of an aptamer from its bound protein partner by means of luminescent oxygen channeling. In a proof-of-principle study we demonstrate that the format is feasible for efficient identification of small drug-like molecules that bind to a protein target, in this case to the Sec7 domain of cytohesin. We extended the approach to a new cytohesin-specific single chain DNA aptamer, C10.41, which exhibits a similar binding behavior to cytohesins but has the advantage of being more stable and easier to synthesize and to modify than the RNA-aptamer M69. The results obtained with both aptamers indicate the general suitability of the aptamer-displacement assay based on luminescent oxygen channelling (ADLOC) for HTS. We also analyzed the potential for false positive hits and identified from a library of 18,000 drug-like small molecules two compounds as strong singlet-oxygen quenchers. With full automation and the use of commercially available plate readers, we estimate that the ADLOC-based assay described here could be used to screen at least 100,000 compounds per day. [source]

BINOL-3,3,-Triflone N,N -Dimethyl Phosphoramidites: Through-Space 19F,31P Spin,Spin Coupling with a Remarkable Dependency on Temperature and Solvent Internal Pressure

CHEMISTRY - A EUROPEAN JOURNAL, Issue 26 2008
Matthias Kruck
Abstract A combined computational and experimental study of the effects of solvent, temperature and stereochemistry on the magnitude of the through-space spin,spin coupling between 31P and 19F nuclei which are six-bonds apart is described. The reaction of 3-trifluoromethylsulfonyl-2,,2-dihydroxy-1,1,-binaphthalene (3-SO2CF3 -BINOL) with hexamethylphosphorous triamide (P(NMe2)3) generates a pair of N,N -dimethylphosphoramidites which are diastereomeric due to their differing relative configurations at the stereogenic phosphorous centre and the axially chiral (atropisomeric) BINOL unit. Through-space NMR coupling of the 31P and 19F nuclei of the phosphoramidite and sulfone is detected in one diastereomer only. In the analogous N,N -dimethylphosphoramidite generated from 3,3,-(SO2CF3)2 -BINOL only one of the diastereotopic trifluoromethylsulfone moieties couples with the 31P of the phosphoramidite. In both cases, the magnitude of the coupling is strongly modulated (up to 400,%) by solvent and temperature. A detailed DFT analysis of the response of the coupling to the orientation of the CF3 moiety with respect to the P-lone pair facilitates a confident assignment of the stereochemical identity of the pair of diastereomers. The analysis shows that the intriguing effects of environment on the magnitude of the coupling can be rationalised by a complex interplay of solvent internal pressure, molecular volume and thermal access to a wider conformational space. These phenomena suggest the possibility for the design of sensitive molecular probes for local environment that can be addressed via through-space NMR coupling. [source]

Assembly of a Series of Malarial Glycosylphosphatidylinositol Anchor Oligosaccharides

CHEMISTRY - A EUROPEAN JOURNAL, Issue 8 2005
Yong-Uk Kwon Dr.
Abstract We report an efficient and convergent synthesis of a series of oligosaccharides comprised of the malaria GPI glycan (2,a), a promising anti-malaria vaccine candidate currently in preclinical trials and several related oligosaccharide sequences (3,8) that are possible biosynthetic precursors of the malarial GPI. A flexible synthetic strategy is disclosed that relies on a late-stage coupling between oligomannosides of varying length and pseudo-disaccharide glycosyl acceptor 11 to readily access various malarial GPI structures. Phosphorylation was accomplished by mild and efficient H-phosphonate chemistry before the final deprotection was carried out by using sodium in ammonia. The direct connection of a thiol group via a phosphate diester linkage to the inositol moiety provides a handle for easy conjugation of the GPI glycan to carrier proteins, immobilization on carbohydrate microarrays and photo-affinity labels identification. These synthetic oligosaccharides will serve as molecular probes. [source]