In situ hybridization detection of short viral amplicon sequences within cultured cells and body fluids after the in situ polymerase chain reaction

Using single primer pairs, intracellular gene sequences of cytomegalovirus (CMV-Towne's strain) and alpha-tubulin were amplified (in situ PCR) from cells in human body fluids and in suspensions. Visualization of CMV amplificants was carried out by in situ hybridization (ISH), using both a biotinylated double-stranded DNA probe and a radiolabelled oligonucleotide probe. Visualization of alpha-tubulin amplificants was achieved using both radiolabelled single-stranded cRNA and oligonucleotide probes. Liberated amplificants were also identified by bands of expected size by gel electrophoresis. The specificity of the PCR products was confirmed by Southern blot analysis. Intracellular amplification was identified both in unfixed cells and, optimally, after brief alcohol fixation, whilst maintaining relative isotonicity in all working solutions. For CMV, enhanced signal was observed in cells (cultured fibroblasts or urine sediment) undergoing in situ PCR using either biotinylated or radiolabelled probes compared with controls undergoing ISH alone. For alpha-tubulin, radiolabelled riboprobes and oligoprobes only produced signals within cells (human peripheral lymphocytes, ascitic fluid and bladder washings from routine cytological specimens) after in situ PCR, but not after ISH alone. Morphological evaluation was superior with biotinylated probes, and minimal back-diffusion effect was found compared with radiolabelled probes. Up to 80% of cells survived thermal cycling. In situ PCR detected short sequence (100 bp) foreign DNA and low copy number genomic DNA, and was superior to ISH alone. In contrast to radiolabelled probes, very small CMV amplificants could be detected without a significant 'back-diffusion' effect when using the large biotinylated probe in this model system.     

Senescence mutants of Saccharomyces cerevisiae with a defect in telomere replication identify three additional EST genes

The primary determinant for telomere replication is the enzyme telomerase, responsible for elongating the G-rich strand of the telomere. The only component of this enzyme that has been identified in Saccharomyces cerevisiae is the TLC1 gene, encoding the telomerase RNA subunit. However, a yeast strain defective for the EST1 gene exhibits the same phenotypes (progressively shorter telomeres and a senescence phenotype) as a strain deleted for TLC1, suggesting that EST1 encodes either a component of telomerase or some other factor essential for telomerase function. We designed a multitiered screen that led to the isolation of 22 mutants that display the same phenotypes as est1 and tlc1 mutant strains. These mutations mapped to four complementation groups: the previously identified EST1 gene and three additional genes, called EST2, EST3 and EST4. Cloning of the EST2 gene demonstrated that it encodes a large, extremely basic novel protein with no motifs that provide clues as to function. Epistasis analysis indicated that the four EST genes function in the same pathway for telomere replication as defined by the TLC1 gene, suggesting that the EST genes encode either components of telomerase or factors that positively regulate telomerase activity.     

Fine genetic and comparative mapping of the deafness mutation Ames waltzer on mouse chromosome 10

The fundamental properties of a flow cytometer that govern its capacity to detect and resolve dim fluorescent particles are 1) the efficiency with which it can convert a photon emitted by a fluorochrome into a photoelectron at the photocathode of the photomultiplier tube and 2) the amount of optical background noise that is collected along with the fluorescence emission from the particle. Either of these properties alone is insufficient to predict an instrument's performance. Thus, to characterize a flow cytometer, both of these properties need to be determined. To determine these properties, one must not only consider the optical characteristics of the instruments but also understand the impact of the signal processing on the data collected. After the collection efficiency and the optical background noise level are determined, it is possible to predict the instrument's performance. A specific flow cytometer performance level is not unique to only one pair of these properties. To achieve a specific level of instrument performance, whole families of pairs of these properties are possible.     

Corneal asphericity in eye bank eyes implanted with the intrastromal corneal ring

The endogenous circadian rhythm of melatonin in humans provides information regarding the resetting response of the human circadian timing system to changes in the light-dark (LD) cycle. Alterations in the LD cycle have both acute and chronic effects on the observed melatonin rhythm. Investigations to date have firmly established that the melatonin rhythm can be reentrained following an inversion of the LD cycle. Exposure to bright light and darkness given over a series of days can rapidly induce large-magnitude phase shifts of the melatonin rhythm. Even single pulses of bright light can shift the timing of the melatonin rhythm. Recent data have demonstrated that lower light intensities than originally believed are capable of resetting the melatonin rhythm and that stimulation of photopically sensitive photoreceptors (i.e., cones) is sufficient to reset the endogenous circadian melatonin rhythm. In addition to phase resetting, exposure to light of critical timing, strength, and duration can attenuate the amplitude of the endogenous circadian rhythm of melatonin. Measurement of melatonin throughout resetting trials provides a dynamic view of the resetting response of the human circadian pacemaker to light. Future studies of the melatonin rhythm in humans may further characterize the resetting response of the human circadian timing system to light.     

Effect of immunotherapy on sCD23 levels in patients allergic to Hymenoptera venom

BACKGROUND: sCD23 is the designation given to the low affinity IgE receptor. The soluble fragment of this receptor (sCD23) participates in the regulation of IgE synthesis. OBJECTIVE: The purpose of this study is to examine the effect of a venom immunotherapy regimen on sCD23 levels. METHODS: We measured sCD23 levels by ELISA in Hymenoptera venom-allergic patients (positive skin tests and a history of systemic reactions to Hymenoptera sting) in serial sera collected over a course of venom immunotherapy with a mean duration of 54 months. Mean pre-sCD23 and post-sCD23 levels were compared using a Student's two-tailed t test. RESULTS: sCD23 levels were found to be unchanged over the course of venom immunotherapy. CONCLUSIONS: This is the first longitudinal study that has been done. It suggests that while both immunotherapy and sCD23 are known to be involved in the regulations of IgE synthesis in the atopic patient, the immunomodulation seen in venom immunotherapy is not mediated through sCD23 in any simple regulatory manner.     

Enhancement of oxidative cell injury and antitumor effects of localized 44 degrees C hyperthermia upon combination with respiratory hyperoxia and xanthine oxidase

Polymerase chain reaction (PCR) assays were designed to amplify 56- and 99-base regions of the pmoA gene from Methylosinus trichosporium OB3b and Methylomicrobium albus BG8, two species of methanotrophic bacteria that are of interest for monitoring bioremediation activity. The PCR product sizes are in a mass range that is accessible to analysis by MALDI-TOF mass spectrometry. A rapid purification procedure using commercially available reversed-phase cartridges was applied prior to MALDI-TOF analysis. A small aliquot (1.5%, 1.5 microL) from a single 100-microL PCR reaction was sufficient for reliable detection. No cross-amplification products were observed when primers designed for one bacterial species were used with genomic DNA of the other species. The methodology described here has potential to allow less expensive and faster characterization of the ability of microbial populations to destroy pollutants in groundwater and soil at contaminated industrial sites.     

Gastric pneumatosis associated with preduodenal portal vein, duodenal atresia, and asplenia

An 8-day-old newborn presented with non-bilious vomiting, upper abdominal fullness, and failure to pass meconium. Plain radiographs revealed gastric pneumatosis (GP). At operation, he was found to have a non-obstructive preduodenal portal vein, preampullary duodenal atresia, asplenia, and malrotation. The baby was treated by duodeno-duodenostomy without mobilizing the portal vein and correction of the malrotation according to Ladd's procedure. He made an uneventful recovery and the GP resolved spontaneously. The malformative process was believed to have occurred at or soon after the 5th week of gestation, and the GP probably resulted from intramural air tracking through mucosal tears caused by high intragastric pressure.