Localization of the locus causing Spider Lamb Syndrome to the distal end of ovine Chromosome 6

Spider Lamb Syndrome (SLS) is a semi-lethal congenital disorder, causing severe skeletal abnormalities in sheep. The syndrome has now been disseminated into several sheep breeds in the United States, Canada, and Australia. The mode of inheritance for SLS is autosomal recessive, making the identification and culling of carrier animals difficult due to their normal phenotype. Two large pedigrees segregating for the SLS mutation were established, and a genome scan with genetic markers from previously published genome maps of cattle and sheep was used to map the locus causing SLS. Genetic linkage between SLS and several microsatellite markers, OarJMP8, McM214, OarJMP12, and BL1038, was detected, thereby mapping the SLS locus to the telomeric end of ovine Chromosome (Chr) 6. Alignment of ovine Chr 6 with its evolutionary ortholog, human Chr 4, revealed a positional candidate gene, fibroblast growth factor receptor 3 (FGFR3). Received: 10 June 1998 / Accepted: 23 September 1998     

Normal Human Telomeres Are Not Late Replicating

Telomeres in yeast are late replicating. Genes placed next to telomeres in yeast can be repressed (telomere positional effects), leading to the hypothesis that telomeres may be heterochromatic and may control the expression of subtelomeric genes. In addition, yeast telomeres are processed to have a transient long overhang at the end of S phase. The applicability of the yeast data to human biology was examined by determining the timing of telomere replication and processing in normal human diploid fibroblasts. Telomeres were purified from synchronized cells that had been labeled with 5-bromodeoxyuridine (BrdU) at hourly intervals, and the fraction of labeled telomeres was analyzed by retrieval with anti-BrdU antibodies. We determined that normal human telomeres replicate throughout S phase rather than being very late replicating. Furthermore, the overall timing of replication was unaffected by telomere length in young versus old cells or cells whose telomeres had been elongated following transfection with the catalytic subunit of telomerase. Finally, the asymmetry in the length of the G-rich overhang in daughter telomeres produced by leading versus lagging strand synthesis was shown to be established within 1 h of telomere replication, indicating there is no significant delay between synthesis and the processing events that contribute to the establishment of asymmetric overhangs. Therefore, the timings of replication and processing of human telomeres are very different from those of yeast.     

The transition from day-to-night activity is a risk factor for the development of CNS oxygen toxicity in the diurnal fat sand rat (Psammomys obesus)

Performance and safety are impaired in employees engaged in shift work. Combat divers who use closed-circuit oxygen diving apparatus undergo part of their training during the night hours. The greatest risk involved in diving with such apparatus is the development of central nervous system oxygen toxicity (CNS-OT). We investigated whether the switch from day-to-night activity may be a risk factor for the development of CNS-OT using a diurnal animal model, the fat sand rat (Psammomys obesus). Animals were kept on a 12:12 light–dark schedule (6 a.m. to 6 p.m. at 500?lx). The study included two groups: (1) Control group: animals were kept awake and active during the day, between 09:00 and 15:00. (2) Experimental group: animals were kept awake and active during the night, between 21:00 and 03:00, when they were exposed to dim light in order to simulate the conditions prevalent during combat diver training. This continued for a period of 3?weeks, 5?days a week. On completion of this phase, 6-sulphatoxymelatonin (6-SMT) levels in urine were determined over a period of 24?h. Animals were then exposed to hyperbaric oxygen (HBO). To investigate the effect of acute melatonin administration, melatonin (50?mg/kg) or its vehicle was administered to the animals in both groups 20?min prior to HBO exposure. After the exposure, the activity of superoxide dismutase, catalase and glutathione peroxidase was measured, as were the levels of neuronal nitric oxide synthase (nNOS) and overall nitrotyrosylation in the cortex and hippocampus. Latency to CNS-OT was significantly reduced after the transition from day-to-night activity. This was associated with alterations in the level of melatonin metabolites secreted in the urine. Acute melatonin administration had no effect on latency to CNS-OT in either of the groups. Nevertheless, the activity of superoxide dismutase and catalase, as well as nitrotyrosine and nNOS levels, were altered in the hippocampus following melatonin administration. On the basis of these results, we suggest that a switch from diurnal to nocturnal activity may represent an additional risk factor for the development of CNS-OT. Utilizing a diurnal animal model may contribute to our understanding of the heightened risk of developing CNS-OT when diving with closed-circuit oxygen apparatus at night.     

DIABLA: a new screening method for the discovery of protein targets

Dynamic isoelectric/anisotropy binding ligand assay (DIABLA) is a new method to identify proteins in a complex sample that bind to a molecule of interest. This is accomplished by first using capillary isoelectric focusing (cIEF) to separate the proteins in a capillary based on their isoelectric point. This separation is performed while the compound being tested is present in the separation buffer. When the proteins are focused, the entire capillary is scanned to identify regions of nonzero anisotropy, which are locations where the test compound is interacting with a focused protein band. DIABLA was demonstrated by observing the binding of fluorescein-tagged progesterone to an MCF-7 breast cancer cell lysate. The proteins were tagged with rhodamine to permit their observation and then focused in the presence of the tagged progesterone. Anisotropy measurements show that progesterone binds to six different proteins bands in the sample.     

New Electropositive Filter for Concentrating Enteroviruses and Noroviruses from Large Volumes of Water

The U.S. Environmental Protection Agency''s information collection rule requires the use of 1MDS electropositive filters for concentrating enteric viruses from water, but unfortunately, these filters are not cost-effective for routine viral monitoring. In this study, an inexpensive electropositive cartridge filter, the NanoCeram filter, was evaluated for its ability to concentrate enteroviruses and noroviruses from large volumes of water. Seeded viruses were concentrated using the adsorption-elution procedure. The mean percent retention of seeded polioviruses by NanoCeram filters was 84%. To optimize the elution procedure, six protocols, each comprising two successive elutions with various lengths of filter immersion, were evaluated. The highest virus recovery (77%) was obtained by immersing the filters in beef extract for 1 minute during the first elution and for 15 min during the second elution. The recovery efficiencies of poliovirus, coxsackievirus B5, and echovirus 7 from 100-liter samples of seeded tap water were 54%, 27%, and 32%, respectively. There was no significant difference in virus recovery from tap water with a pH range of 6 to 9.5 and a water flow rate range of 5.5 liters/min to 20 liters/min. Finally, poliovirus and Norwalk virus recoveries by NanoCeram filters were compared to those by 1MDS filters, using tap water and Ohio River water. Poliovirus and Norwalk virus recoveries by NanoCeram filters from tap and river water were similar to or higher than those by the 1MDS filters. These data suggest that NanoCeram filters can be used as an inexpensive alternative to 1MDS filters for routine viral monitoring of water.Viruses that primarily infect and replicate in the gastrointestinal tract are known as enteric viruses. More than 140 different enteric viruses are known to infect humans. These include the enteroviruses, rotaviruses, hepatitis A virus, noroviruses, adenoviruses, and reoviruses, among others. Enteric viruses are capable of causing a wide range of illnesses, including gastroenteritis, paralysis, aseptic meningitis, herpangina, respiratory illness, fevers, myocarditis, etc. Given the potential public health impact of the enteric viruses, enteroviruses (echovirus and coxsackievirus), adenoviruses, and caliciviruses are on the U.S. Environmental Protection Agency''s contaminant candidate list 2 for regulatory consideration for drinking water (11). Within the Caliciviridae family, noroviruses are the primary viruses of concern for drinking water.Contaminated drinking water is considered to be a potential transmission route, and an infectious dose in humans may consist of only a small number of virus particles. Enteric viruses are introduced in aquatic environments through natural or human activities, such as leaking sewage and septic systems, urban runoff, landfills, injection of treated wastewater into aquifers, wastewater discharge, sewage outfall, etc. These viruses have been found in surface water, groundwater, and drinking water (1, 6, 13, 22, 26). Between 1971 and 2004, 789 drinking water outbreaks and 575,207 cases of illness were reported in the United States, and 8% of the reported outbreaks were due to enteric viruses (2, 5, 28, 29, 30, 46).The levels of enteric viruses in natural waters are often low, and as such, typical virus sampling involves a primary concentration of viruses from large volumes of water (hundreds to thousands of liters). Unlike other waterborne pathogens (such as bacteria and parasites), viruses are smaller, and thus, size exclusion filtration is often not practical, especially for turbid waters. In addition, viruses are negatively charged in natural environments and can be adsorbed onto a number of different matrices by electrostatic and hydrophobic interactions (16). Consequently, different types of matrices have been used to isolate enteric viruses from water. These include negatively and positively charged membranes or cartridge filters (10, 17, 32, 34, 35, 39), gauze pad (31), and glass powder or glass wool (14, 27). Of all of these methods, electronegative and electropositive filters are most commonly used. In the case of electronegative filters, the acidification of the water and addition of multivalent cations are required for optimal virus adsorption. Because of this need to condition the water to attain acceptable recoveries, it is difficult to use electronegative filters for field sampling. In contrast, electropositive filters do not require conditioning of the water. Among all the filters, 1MDS electropositive filters (Cuno, Meriden, CT) are the most commonly used filter for fresh and drinking water sampling; however, they are not cost-effective for routine viral monitoring of water and require pH adjustment for waters with pH values exceeding 8.0 (12).Viruses adsorbed on the filter are usually eluted and recovered using 1 to 1.6 liters of eluting solution (6, 12). Many different procedures are described in the literature to elute viruses from filters. These procedures include the use of different eluting solutions, such as 0.3%, 1.5% or 3% beef extract, urea-arginine phosphate buffer, glycine buffer, etc. (10, 12, 24, 37). There are also different elution processes, such as single elution, recirculation of eluents, or successive elution of filters (6, 8, 15, 43). Sobsey and Hickey (40) used only one elution with 0.3% beef extract in 50 mM glycine. Sobsey et al. (43) suggested that 1 liter of 1.5% beef extract be recirculated through the filters for 5 min. Dahling and Wright (8) reported that the highest virus recoveries were obtained by three elutions, each using 1.6 liters of 3% beef extract. Dahling (6) reported that the highest virus recoveries were obtained with two separate beef extract elutions, one being an overnight filter immersion in beef extract.Although methods for concentration of many enteric viruses have been developed, limited studies have been conducted for concentrating noroviruses from water. Huang et al. (21) described a norovirus concentration method using porcine calicivirus (Pan-1) as a surrogate. Pan-1 was sensitive to the high pH (9.5) of the eluting solution, which is commonly used. Myrmel et al. (33) described a method of norovirus concentration using feline calicivirus as a surrogate organism. The method used electronegative filters, and the recovery of virus was 5 to 10%. Many other studies reported detection of human noroviruses in environmental waters (18, 19, 25); however, none of these studies evaluated the recovery efficiencies of human noroviruses from large volumes of water.The objective of this study was to evaluate the NanoCeram (Argonide, Sanford, FL) cartridge filter for the concentration of enteroviruses and noroviruses from large volumes of water. NanoCeram filters have an active component of nano alumina (AlOOH) fibers, which give them a naturally occurring electropositive charge.