SPACRCAN, a novel human interphotoreceptor matrix hyaluronan-binding proteoglycan synthesized by photoreceptors and pinealocytes.
AuthorsAcharya S, Foletta VC, Lee JW, Rayborn ME, Rodriguez IR, Young WS, Hollyfield JG
JournalJ Biol Chem
PubMed ID10702256
The interphotoreceptor matrix is a unique extracellular complex occupying the interface between photoreceptors and the retinal pigment epithelium in the fundus of the eye. Because of the putative supportive role in photoreceptor maintenance, it is likely that constituent molecules play key roles in photoreceptor function and may be targets for ... More
Lateral-flow and up-converting phosphor reporters to detect single-stranded nucleic acids in a sandwich-hybridization assay.
AuthorsCorstjens PL, Zuiderwijk M, Nilsson M, Feindt H, Sam Niedbala R, Tanke HJ
JournalAnal Biochem
PubMed ID12531205
'Up-converting Phosphor Technology (UPT) particles were used as reporters in lateral-flow (LF) assays to detect single-stranded nucleic acids. The 400-nm phosphor particles exhibit strong visible luminescence upon excitation with infrared (IR) light resulting in the total absence of background autofluorescence from other biological compounds. A sandwich-type hybridization assay was applied ... More
Application of digital image analysis and flow cytometry to enumerate marine viruses stained with SYBR gold.
AuthorsChen F, Lu JR, Binder BJ, Liu YC, Hodson RE
JournalAppl Environ Microbiol
PubMed ID11157214
'A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of ... More
The stability of tristetraprolin mRNA is regulated by mitogen-activated protein kinase p38 and by tristetraprolin itself.
AuthorsTchen CR, Brook M, Saklatvala J, Clark AR
JournalJ Biol Chem
PubMed ID15187092
'Tristetraprolin (TTP) is an mRNA-destabilizing protein that negatively regulates the expression of proinflammatory mediators such as tumor necrosis factor alpha, granulocyte/macrophage colony-stimulating factor, and cyclooxygenase 2. Here we investigate the regulation of TTP expression in the mouse macrophage cell line RAW264.7. We show that TTP mRNA is expressed in a ... More
A new approach to determine the genetic diversity of viable and active bacteria in aquatic ecosystems.
'BACKGROUND: Discrimination among viable, active, and inactive cells in aquatic ecosystems is of great importance to understand which species participate in microbial processes. In this study, a new approach combining flow cytometry (FCM), cell sorting, and molecular analyses was developed to compare the diversity of viable cells determined by different ... More
Naturally occurring mutations in human mitochondrial pre-tRNASer(UCN) can affect the transfer ribonuclease Z cleavage site, processing kinetics, and substrate secondary structure.
AuthorsYan H, Zareen N, Levinger L
JournalJ Biol Chem
PubMed ID16361254
'tRNAs are transcribed as precursors with a 5'' end leader and a 3'' end trailer. The 5'' end leader is processed by RNase P, and in most organisms in all three kingdoms, transfer ribonuclease (tRNase) Z can endonucleolytically remove the 3'' end trailer. Long ((L)) and short ((S)) forms of ... More
Does the high nucleic acid content of individual bacterial cells allow us to discriminate between active cells and inactive cells in aquatic systems?
AuthorsLebaron P, Servais P, Agogué H, Courties C, Joux F
JournalAppl Environ Microbiol
PubMed ID11282632
'The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated ... More
Protein-free parallel triple-stranded DNA complex formation.
'A 14 nt DNA sequence 5''-AGAATGTGGCAAAG-3'' from the zinc finger repeat of the human KRAB zinc finger protein gene ZNF91 bearing the intercalator 2-methoxy,6-chloro,9-amino acridine (Acr) attached to the sugar-phosphate backbone in various positions has been shown to form a specific triple helix (triplex) with a 16 bp hairpin (intramolecular) ... More
Phylogenetic assessment of length variation at a microsatellite locus.
AuthorsOrtí G, Pearse DE, Avise JC
JournalProc Natl Acad Sci U S A
PubMed ID9380705
'Sixty-six haplotypes at a locus containing a simple dinucleotide (CA)n microsatellite repeat were isolated by PCR-single-strand conformational polymorphism from populations of the horseshoe crab Limulus polyphemus. These haplotypes were sequenced to assess nucleotide variation directly. Thirty-four distinct sequences (alleles) were identified in a region 570 bp long that included the ... More
Flow cytometric analysis of bacteria- and virus-like particles in lake sediments.
AuthorsDuhamel S, Jacquet S
JournalJ Microbiol Methods
PubMed ID16081175
'Flow cytometry (FCM) was successfully used to analyze freshwater bacteria and viruses in lake sediments after relatively simple sample treatment and optimization of dilution/fixation/staining procedures. Biological particles from Lakes Geneva and Bourget were first separated from the sediments by using both Sodium Pyrophosphate (0.01 M final concentration) and Polyoxyethylene-Sorbitan Monooleate ... More
Separation of miRNA and its methylation products by capillary electrophoresis.
AuthorsLi N, Nguyen A, Diedrich J, Zhong W,
JournalJ Chromatogr A
PubMed ID18649888
'Methylation is a crucial step in plant microRNA biogenesis. To improve our understanding of the methylation process and its regulation, a rapid and convenient high-throughput method should be sought to help with the study of reaction kinetics and assist the search for chemical inhibitors of the methyltransferase, HEN1. In this ... More
Normalization of RNA hybridization signals by means of SYBR green II-stained 28S or 18S ribosomal RNA and a phosphor imager.
AuthorsSpiess AN, Ivell R
JournalBiotechniques
PubMed ID9894590
A rapid, sensitive, and nonradioactive method for assay of FHIT Ap3A hydrolase activity by fluorescence thin-layer chromatographic image analysis.
AuthorsJi L, Fang B, Roth JA
JournalAnal Biochem
PubMed ID10361018
A fluorometric assay for the quantification of RNA in solution with nanogram sensitivity.
AuthorsSchmidt DM, Ernst JD
JournalAnal Biochem
PubMed ID8600826
An evaluation of four staining methods for the detection of DNA in nondenaturing polyacrylamide gels.
AuthorsStothard JR, Frame IA, Miles MA
JournalAnal Biochem
PubMed ID9367513
Rapid and simple quantification of bacterial cells by using a microfluidic device.
AuthorsSakamoto C, Yamaguchi N, Nasu M,
JournalAppl Environ Microbiol
PubMed ID15691978
This study investigated a microfluidic chip-based system (on-chip flow cytometry) for quantification of bacteria both in culture and in environmental samples. Bacterial numbers determined by this technique were similar to those obtained by direct microscopic count. The time required for this on-chip flow cytometry was only 30 min per 6 ... More
Utility of green fluorescent nucleic acid dyes and aluminum oxide membrane filters for rapid epifluorescence enumeration of soil and sediment bacteria.
AuthorsWeinbauer MG, Beckmann C, Höfle MG
JournalAppl Environ Microbiol
PubMed ID9835595
High background fluorescence and unspecific staining hampered the epifluorescence enumeration of bacteria in 45% of the tested soil and sediment samples with 4',6-diamidino-2-phenylindole (DAPI) and polycarbonate membrane filters. These problems of the determination of total cell counts can be circumvented by using green fluorescent high-affinity nucleic acid dyes and aluminum ... More
Development of an adhesive sheet for direct counting of bacteria on solid surfaces.
AuthorsYamaguchi N, Ishidoshiro A, Yoshida Y, Saika T, Senda S, Nasu M
JournalJ Microbiol Methods
PubMed ID12689718
An adhesive sheet was developed for direct counting of microorganisms on solid surfaces. The sheet consists of a polyurethane film base and water insoluble adhesive. SYBR Green II (for total direct counting) or 6-carboxyfluorescein diacetate (6CFDA) (for fluorescent vital staining) was used for fluorescent microscopy of bacteria collected on the ... More
Analysis of DNA microarrays by non-destructive fluorescent staining using SYBR green II.
AuthorsBattaglia C, Salani G, Consolandi C, Bernardi LR, De Bellis G
JournalBiotechniques
PubMed ID10907080
A simple, non-destructive procedure is described to determine the quality of DNA arrays before they are used. It consists of a preliminary staining step of the DNA microarray by using SYBR green II, a fluorophore with specific affinity for ssDNA, followed by a laser scan analysis. The surface quality, integrity ... More
Fluorometric quantification of RNA and DNA in solutions containing both nucleic acids.
A fluorescence-based method for quantitative determination of RNA and DNA in probes containing both nucleic acids has been developed. The total concentration of nucleic acids is determined using SYBR Green II dye under conditions providing independent binding of the fluorophore with DNA and RNA. The concentration of DNA is specifically ... More
Spectral imaging detection and counting of microbial cells in marine sediment.
AuthorsSunamura M, Maruyama A, Tsuji T, Kurane R
JournalJ Microbiol Methods
PubMed ID12609724
Semiautomated detection and counting techniques for microbial cells in soil and marine sediment using microscopic-spectral-imaging analysis were developed. Microbial cells in microscopic fields were selectively detected from other fluorescent particles by their fluorescent spectrum, based on the spectral shift between the conjunction and nonconjunction of DNA fluorochrome (SYBR Green II) ... More
Immunofluorescent detection and quantitation of hepatitis A virus in sewage treatment effluent and on agri-food surfaces using scanning confocal microscopy.
AuthorsKukavica-Ibrulj I, Darveau A, Fliss I
JournalJ Virol Methods
PubMed ID12565149
An immunofluorescent (IF) assay was developed for specific detection and quantitation of hepatitis A virus (HAV) in sewage treatment effluent or attached to stainless steel, copper, high density polyethylene and polyvinyl chloride surfaces. Polyclonal antibodies were produced in rabbits and characterized for specificity to HAV. Purified anti-HAV antibodies were used ... More
Flow cytometric detection of viruses.
AuthorsBrussaard CP, Marie D, Bratbak G
JournalJ Virol Methods
PubMed ID10716350
Representatives from several different virus families (Baculoviridae, Herpesviridae, Myoviridae, Phycodnaviridae, Picornaviridae, Podoviridae, Retroviridae, and Siphoviridae) were stained using a variety of highly fluorescent nucleic acid specific dyes (SYBR Green I, SYBR Green II, OliGreen, PicoGreen) and examined using a standard flow cytometer equipped with a standard 15 mW argon-ion laser. ... More
Nucleic acid detection using non-radioactive labelling methods.
AuthorsMansfield ES, Worley JM, McKenzie SE, Surrey S, Rappaport E, Fortina P
JournalMol Cell Probes
PubMed ID7477006
Nucleic acid probe-based assays are now widely used in genetic research, human identification, forensics and in a broad spectrum of clinical assays in the fields of microbiology, haematology/oncology and virology. Labelled probes are used in a variety of assay formats including dot-blots, Southern blots (DNA target), Northern blots (RNA target), ... More
Analysis of the microbial functional diversity within water-stressed soil communities by flow cytometric analysis and CTC+ cell sorting.
AuthorsWhiteley AS, Griffiths RI, Bailey MJ
JournalJ Microbiol Methods
PubMed ID12782381
Total and active cell counts within soil samples were determined by culture-independent methods using flow cytometry and preparative Nycodenz gradient centrifugation. Whole cells were purified from soil cores and total extractable cell counts assessed by SYBR Green II fluorescence, while active cell counts were determined by 5-cyano-2,3-ditolyl tetrazolium chloride reduction ... More
Herp, a new ubiquitin-like membrane protein induced by endoplasmic reticulum stress.
AuthorsKokame K, Agarwala KL, Kato H, Miyata T
JournalJ Biol Chem
PubMed ID10922362
Hyperhomocysteinemia, a risk factor for vascular disease, injures endothelial cells through undefined mechanisms. We previously identified several homocysteine-responsive genes in cultured human vascular endothelial cells, including the endoplasmic reticulum (ER)-resident molecular chaperone GRP78/BiP. Here, we demonstrate that homocysteine induces the ER stress response and leads to the expression of a ... More
Microarray optimizations: increasing spot accuracy and automated identification of true microarray signals.
AuthorsTran PH, Peiffer DA, Shin Y, Meek LM, Brody JP, Cho KW
JournalNucleic Acids Res
PubMed ID12060692
In this paper, fluorescent microarray images and various analysis techniques are described to improve the microarray data acquisition processes. Signal intensities produced by rarely expressed genes are initially correctly detected, but they are often lost in corrections for background, log or ratio. Our analyses indicate that a simple correlation between ... More
In situ analysis of single-stranded and duplex siRNA integrity in living cells.
AuthorsRaemdonck K, Remaut K, Lucas B, Sanders NN, Demeester J, De Smedt SC
JournalBiochemistry
PubMed ID16939213
To attain the full therapeutic promise of short interfering RNA (siRNA), it is believed that improvements such as increased biostability are critical. Regrettably, thus far, insufficient in situ data are on hand regarding the intracellular stability of siRNAs. We report on the use of an advanced fluorescence-based method to probe ... More
Mutational analysis of the transforming growth factor beta receptor type II gene in human ovarian carcinoma.
AuthorsLynch MA, Nakashima R, Song H, DeGroff VL, Wang D, Enomoto T, Weghorst CM
JournalCancer Res
PubMed ID9766642
In the present study, we evaluated a series of sporadic ovarian carcinomas for mutations within the entire coding region of TbetaR-II. Using reverse transcription-PCR and "Cold" single-strand conformational polymorphism analysis, 6 of 24 samples (25%) were found to contain code-altering mutations in TbetaR-II: (a) four mutations resulting in amino acid ... More
Enumeration of water-borne bacteria using viability assays and flow cytometry: a comparison to culture-based techniques.
AuthorsHoefel D, Grooby WL, Monis PT, Andrews S, Saint CP
JournalJ Microbiol Methods
PubMed ID14607402
Maintaining optimal conditions in catchments or distribution systems relies heavily on water authorities having access to rapid and accurate water quality data, including an indication of bacteriological quality. In this study, the BacLight bacterial viability kit and carboxyfluorescein diacetate (CFDA) were coupled with flow cytometry (FCM) for rapid detection of ... More
Molecular and biochemical characterization of a novel oxysterol-binding protein (OSBP2) highly expressed in retina.
AuthorsMoreira EF, Jaworski C, Li A, Rodriguez IR
JournalJ Biol Chem
PubMed ID11278871
We are interested in understanding the possible function(s) of the oxysterol-binding proteins in mediating oxysterol cytotoxicity in the retina. In this study we describe the cloning, localization, and biological activity of a novel oxysterol-binding protein (OSBP2), and complete the molecular characterization of the previously known OSBP1. Both OSBP genes contain ... More
Sensitive determination of microbial growth by nucleic acid staining in aqueous suspension.
AuthorsMartens-Habbena W, Sass H
JournalAppl Environ Microbiol
PubMed ID16391029
The determination of cell numbers or biomass in laboratory cultures or environmental samples is usually based on turbidity measurements, viable counts, biochemical determinations (e.g., protein and lipid measurements), microscopic counting, or recently, flow cytometric analysis. In the present study, we developed a novel procedure for the sensitive quantification of microbial ... More
Detailed characterization of the posttranscriptional gene-silencing-related small RNA in a GUS gene-silenced tobacco.
AuthorsHutvágner G, Mlynárová L, Nap JP
JournalRNA
PubMed ID11073220
Posttranscriptional gene-silencing phenomena such as cosuppression and RNA interference are associated with the occurrence of small, about 21-23 nt short RNA species homologous to the silenced gene. We here show that the small RNA present in silenced transgenic plants can easily be detected in total RNA isolated according to standard ... More