A 1463 gene cattle-human comparative map with anchor points defined by human genome sequence coordinates

A second-generation 5000 rad radiation hybrid (RH) map of the cattle genome was constructed primarily using cattle ESTs that were targeted to gaps in the existing cattle-human comparative map, as well as to sparsely populated map intervals. A total of 870 targeted markers were added, bringing the number of markers mapped on the RH(5000) panel to 1913. Of these, 1463 have significant BLASTN hits (E < e(-5)) against the human genome sequence. A cattle-human comparative map was created using human genome sequence coordinates of the paired orthologs. One-hundred and ninety-five conserved segments (defined by two or more genes) were identified between the cattle and human genomes, of which 31 are newly discovered and 34 were extended singletons on the first-generation map. The new map represents an improvement of 20% genome-wide comparative coverage compared with the first-generation map. Analysis of gene content within human genome regions where there are gaps in the comparative map revealed gaps with both significantly greater and significantly lower gene content. The new, more detailed cattle-human comparative map provides an improved resource for the analysis of mammalian chromosome evolution, the identification of candidate genes for economically important traits, and for proper alignment of sequence contigs on cattle chromosomes.     

Variable MHC class I engagement by Ly49 natural killer cell receptors demonstrated by the crystal structure of Ly49C bound to H-2K(b)

The Ly49 family of natural killer (NK) receptors regulates NK cell function by sensing major histocompatibility complex (MHC) class I. Ly49 receptors show complex patterns of MHC class I cross-reactivity and, in certain cases, peptide selectivity. To investigate whether specificity differences result from topological differences in MHC class I engagement, we determined the structure of the peptide-selective receptor Ly49C in complex with H-2K(b). The Ly49C homodimer binds two MHC class I molecules in symmetrical way, a mode distinct from that of Ly49A, which binds MHC class I asymmetrically. Ly49C does not directly contact the MHC-bound peptide. In addition, MHC crosslinking by Ly49C was demonstrated in solution. We propose a dynamic model for Ly49-MHC class I interactions involving conformational changes in the receptor, whereby variations in Ly49 dimerization mediate different MHC-binding modes.     

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Relationship of epileptic seizures to sleep stage and sleep depth

STUDY OBJECTIVES: Interictal epileptiform discharges (IEDs) are facilitated by NREM stages 3 and 4 sleep and as sleep is deepening. To determine whether sleep influences seizures in a similar way to IEDs, we examined seizure rates in various stages of sleep in epilepsy patients undergoing overnight video-EEG-polysomnography (VPSG). DESIGN: Cross-sectional study. SETTING: Neurology Department. PATIENTS, MEASUREMENTS, AND INTERVENTIONS: We reviewed VPSGs from our Sleep and Epilepsy Laboratories to identify patients with recorded seizures during sleep. A total of 55 patients having 117 seizures were identified. RESULTS: Ninety-five percent of seizures occurred in NREM sleep (61% in stage 2, 20% in stage 1, 14% in stages 3 and 4 combined), and 5% in REM sleep. Adjusting for time spent in each stage of sleep, patients had 0.34 seizures per hour in stage 1, 0.38 seizures per hour in stage 2, 0.29 seizures/hr in stage 3 and 4 combined, and 0.09 seizures per hour in REM sleep. Seizures/hour was higher in NREM sleep (0.35 for NREM and 0.09 for REM; p=0.0001). For single seizures occurring in 1 night, seizure rate was significantly higher in NREM stages 1 and 2 as compared to NREM stages 3 and 4 sleep. A significant increase in log delta power, an automated measure of sleep depth, was observed in the 10 minutes prior to seizures. CONCLUSIONS: Both seizures and IEDs are facilitated by NREM sleep. While deeper stages of NREM sleep activate IEDs, lighter stages of NREM sleep promote seizures, at least for single seizures occurring in 1 night.     

Effect of sodium arsenite on peripheral lymphocytes in vitro: individual susceptibility among a population exposed to arsenic through the drinking water

Arsenic (As) contamination in ground water has affected more than 19 countries. Approximately 36 million people in the Bengal delta alone are exposed to this toxicant via drinking water (>50 microg/l) and are at potential health risk. Chronic ingestion of As via drinking water is associated with occurrence of skin lesions, cancer and other arsenic-induced diseases in West Bengal, India. An in vitro cytogenetic study was performed utilizing chromosomal aberrations (CA) in lymphocytes treated with sodium arsenite (0-5 microM) in six symptomatic (having arsenic-related skin lesions) individuals, six age- and sex-matched As-exposed asymptomatic (no arsenic-related skin lesions) individuals and six control individuals with similar socio-economic status residing in non-affected districts of West Bengal with no evidence of As exposure. The mean As content in nails and hair was 9.61 and 5.23 microg/g in symptomatic, 3.48 and 2.17 microg/g in asymptomatic and 0.42 and 0.33 microg/g in the control individuals, respectively. The main aim of our study was to determine whether genotoxic effects differed in the lymphocytes of the control (no exposure to arsenic), asymptomatic and symptomatic individuals after in vitro treatment with sodium arsenite. Although both the exposed groups had chronic exposure to As through the drinking water, individuals with skin lesions accumulated more As in their nails and hair and excreted less in urine (127.80 versus 164.15 microg/l). The results show that sodium arsenite induced a significantly higher percentage of aberrant cells in the lymphocytes of control individuals than in the lymphocytes of both the exposed groups. Within the two exposed groups As induced higher incidences of CA in the symptomatic than the asymptomatic individuals. These results suggest that asymptomatic individuals have relatively lower sensitivity and susceptibility to induction of genetic damage by As compared with the symptomatic individuals.     

Reduction of adhesion formation in rabbits by intraperitoneal administration of lazaroid formulations

Adhesion formation is a major source of postoperative morbidity and mortality. In this study, the ability of a variety of lazaroid formulations [the antioxidant 21-aminosteroid PNU74006F (tirilazad) and the non-steroidal 2-methylaminochroman derivative PNU83,836E] to reduce i.p. adhesion formation in three rabbit models was examined. In initial studies, PNU83836E was administered via Alzet miniosmotic pump to the site of injury. In the sidewall and double uterine horn models, PNU83,836E was administered via Alzet miniosmotic pump for the entire postoperative interval. In the sidewall model, there was a dose- dependent reduction in the area of the sidewall injury that was involved in adhesions. In the double uterine horn model, PNU83,836E was administered via Alzet miniosmotic pump to the area of injury for 1, 2, 3 or 7 days. Administration for as little as 24 h after surgery significantly reduced the extent of adhesion formation and the reduction was increased if it was administered for longer. Further studies were conducted in which various lazaroid formulations were administered as a bolus at the end of surgery. In both the sidewall and double uterine horn models, administration of either PNU83,386E (in citrate buffer) or PNU74006F (in cyclodextrin or lipid emulsion vehicles) at the end of surgery reduced adhesion formation. Administration of a bolus of PNU74006F 10 min prior to initiation of surgery with or without additional treatment at the end of surgery further increased its efficacy in the reduction of adhesion formation. Administration of a minimum of 1.5 mg before and after surgery (3 mg total) was required for maximal efficacy. These studies demonstrate that pre- and postoperative administration of either a steroidal (PNU74006F) or non-steroidal (PNU83,836E) lazaroid intraperitoneally reduced the formation and reformation of postoperative adhesions in three animal models.      

Diagnosis of retinoblastoma by fine-needle aspiration and aqueous cytology

This article reports the cytodiagnosis of three cases of retinoblastoma in children aged 1.5, 2.5, and 5 yr. Two of them were diagnosed by fine-needle aspiration cytology of the primary tumor and one by aqueous cytology. The tumor cells were usually round to oval, small and uniform, with scanty cytoplasm; they generally occurred in closely packed clusters of variable sizes. We discuss the differential diagnosis of retinoblastoma with other round-cell tumors of childhood involving the orbit.