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Biomedical subjects

A Sage

Publications and source records attributed to A Sage.

16 recordsLinked to original sources
PubMed2001

Combination of ATP-bioluminescence and PNA probes allows rapid total counts and identification of specific microorganisms in mixed populations.

We have combined ATP-dependent bioluminescence with a novel chemiluminescent in situ hybridization (CISH) method using peroxidase-labeled peptide nucleic acid (PNA) probes targeting species-specific rRNA sequences to provide total counts and subsequent identification of specific microorganisms. Both methods are applied to the same membrane filter following a short incubation time and both methods provide results in the form of spots of light that are captured by the MicroStar detection system. Each spot of light represents individual micro-colonies detected by either ATP bioluminescence or PNA CISH. This new concept is particularly intended for in process and quality control of non-sterile products to rapidly provide total counts as well as presence/absence of specific indicators and/or pathogens in non-sterile, filterable samples.

Adenosine Triphosphate↗
PubMed2001

Spectroscopic characterisation and identification of ecdysteroids using high-performance liquid chromatography combined with on-line UV--diode array, FT-infrared and 1H-nuclear magnetic resonance spectroscopy and time of flight mass spectrometry.

A prototype multiply hyphenated reversed-phase HPLC system has been applied to the analysis of a mixture of pure ecdysteroids and an ecdysteroid-containing plant extract. Characterisation was achieved via a combination of diode array UV, 1H NMR, FT-IR spectroscopy and time of flight (TOF) mass spectrometry. This combination of spectrometers allowed the collection of UV, 1H NMR, IR and mass spectra for a mixture of pure standards enabling almost complete structural characterisation to be performed. The technique was then applied to a partially purified plant extract in which 20-hydroxyecdysone and polypodine B were identified despite incomplete chromatographic resolution and the presence of co-chromatographing interferents. The experimental difficulties in the use of such a systems for these analytes are described.

Chromatography, High Pressure Liquid↗
PubMed2001

Rapid detection, identification, and enumeration of Escherichia coli cells in municipal water by chemiluminescent in situ hybridization.

A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturable Escherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35 degrees C, individual microcolonies of E. coli were detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targeting P. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other than E. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.

Base Sequence↗
PubMed2001

Identification of Dekkera bruxellensis (Brettanomyces) from wine by fluorescence in situ hybridization using peptide nucleic acid probes.

A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity.

Base Sequence↗
PubMed2000

Reversed-phase high-performance liquid chromatography combined with on-line UV diode array, FT infrared, and 1H nuclear magnetic resonance spectroscopy and time-of-flight mass spectrometry: application to a mixture of nonsteroidal antiinflammatory drugs.

A prototype multiply hyphenated system has been applied to the analysis of a mixture of nonsteroidal antiinflammatory drugs separated by reversed-phase HPLC. Characterization of the model NSAIDs was achieved via a combination of diode array UV, 1H NMR, FT-IR spectroscopy, and time-of-flight mass spectrometry. This combination of spectrometers allowed the collection of UV, 1H NMR, IR, and mass spectra together with atomic composition data enabling almost complete structural characterization to be performed.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2000

Rapid detection, identification, and enumeration of Pseudomonas aeruginosa in bottled water using peptide nucleic acid probes.

A new chemiluminescent in situ hybridization (CISH) method that provides simultaneous detection, identification, and enumeration of Pseudomonas aeruginosa in bottled water within 1 working day has been developed. Individual micro-colonies of P. aeruginosa were detected directly on membrane filters following 5 h of growth by use of soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeted to a species-specific sequence in P. aeruginosa rRNA. Within each micro-colony, reaction of the peroxidase with a chemiluminescent substrate generated light that was subsequently captured by film or with a digital camera system. Each spot of light represented one micro-colony of P. aeruginosa. Sensitivity and specificity for the identification of P. aeruginosa were 100% as determined by testing 28 P. aeruginosa strains and 17 other bacterial species that included closely related Pseudomonas species. Furthermore, the number of micro-colonies of P. aeruginosa represented by light spots correlated with counts of visible colonies following sustained growth. We conclude that PNA CISH speeds up traditional membrane filtration techniques and adds the specificity of PNA probe technology to generate fast and definitive results.

Bacteriological Techniques↗
PubMed2000

Flow injection spectroscopic analysis of model drugs using on-line UV-diode array, FT-infrared and 1H-nuclear magnetic resonance spectroscopy and time-of-flight mass spectrometry.

A prototype flow injection analysis (FIA) system for the characterisation of compounds via a combination of diode array UV, 1H NMR, FT-IR spectroscopy and time-of-flight (TOF) mass spectrometry has been investigated using a number of pharmaceuticals and related compounds as model compounds. This combination of spectrometers allowed the on-flow collection of UV, 1H NMR, IR and mass spectra together with atomic composition data, enabling almost complete structural characterisation to be performed. Practical detection limits with the current system were in the region of 50 micrograms, however, the use of state of the art spectrometers would result in a significant reduction in the amount of material required.

Flow Injection Analysis↗
PubMed1994

Two genes for carbohydrate catabolism are divergently transcribed from a region of DNA containing the hexC locus in Pseudomonas aeruginosa PAO1.

The hexC locus of Pseudomonas aeruginosa PAO1 was localized to a 247-bp segment of chromosomal DNA on the multicopy broad-host-range vector pRO1614. The presence of this plasmid (pPZ196) in strain PAO1 produced the so-called "hexC effect," a two- to ninefold increase in the activities of four carbohydrate catabolism enzymes, glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase. The extent of the hexC effect was restricted, since three independently regulated metabolic enzymes were not affected by the presence of the hexC plasmid. Furthermore, the hexC-containing plasmid did not suppress catabolite repression control. Nucleotide sequence analysis of the segment of DNA encompassing hexC revealed a 128-bp region rich in adenosine-plus-thymine (AT) content separating two divergent open reading frames (ORFs). Transcriptional start sites for these two genes were mapped to the intergenic region, demonstrating that this sequence contained overlapping divergent promoters. The intergenic region contained potential regulatory sequences such as dyad symmetry motifs, polydeoxyadenosine tracts, and a sequence matching the integration host factor recognition site in Escherichia coli. One of the ORFs encoded a 610-amino-acid protein with 55 to 60% identity to 6-phosphogluconate dehydratase from E. coli and Zymomonas mobilis. The second ORF coded for a protein of 335 amino acids that displayed 45 to 60% identity to the NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAP) family of enzymes. The NAD-dependent GAP gene on the P. aeruginosa chromosome was previously unmapped. GAP was found to exhibit the hexC-dependent increase in its basal activity, establishing it as a fifth catabolic enzyme in the multioperonic hex regulon.

Amino Acid Sequence↗
PubMedDate not supplied

Ultrasonographic evaluation of tarsocrural joint cartilage in normal adult horses.

Ultrasonographic examination of the tarsus was performed on four clinically and radiographically normal limbs of adult horses. Particular attention was paid to the articular cartilage surfaces of the trochlear ridges of the talus and the distal intermediate ridge of the tibia. Two separate measurements of articular cartilage thickness were acquired from a longitudinal view at each site. Anatomy was confirmed with post mortem dissection. Ultrasonography was found to be a practical method for imaging the articular cartilage over the trochlear ridges of the talus and distal intermediate ridge of the tibia. The cartilage appeared as a hypoechoic band overlying the hyperechoic subchondral bone. The mean cartilage thickness over the lateral and medial trochlear ridges of the talus and the distal intermediate ridge of the tibia were 0.57 mm, 0.58 mm and 0.7 mm respectively. These measurements may have value for comparison to thickened cartilage and lesions of osteochondrosis and abnormally thinned cartilage of osteoarthritis. Ultrasound examination was not helpful in evaluating the proximal and distal intertarsal and tarsometatarsal joints, the close proximity of the articular surfaces obscured visualization of the articular cartilage.

Animals↗