Genomic analysis of the nuclear receptor family: new insights into structure, regulation, and evolution from the rat genome

Completion of the Rattus norvegicus genome sequence enabled a global inventory and analysis of the nuclear receptors (NRs) in three mammalian species. Forty-nine NR members were found in mouse, 48 in human. Forty-seven were found in the rat, with gaps at the locations expected for the other two. Pairwise comparisons of their distribution in rat, mouse, and human identified 11 syntenic NR gene blocks, including three small clusters of two or three closely related genes, each spanning 40 kb to 1700 kb. The exon structure of the ligand-binding domain suggests that exon shuffling has played a role in the evolution of this family. An invariant splice junction in all members of the NR family except LXRbeta suggests a functional role for the intron. The ligand-binding domains of PXR and CAR are among the most divergent in the family. Their higher nucleotide substitution rates may be related to the central role played by these two NRs in the metabolism of the foreign compounds and may have resulted from limited positive selection.     

Hydrogen peroxide-induced alterations in prostaglandin secretion in the rat colon in vitro

Although the specific cause(s) of inflammatory bowel diseases (IBD) has not been identified, one theory suggests ischemia as the early event that occurs in IBD and reperfusion causes sustained release of oxyradicals, leading to inflammation and ulceration. In this study, we have confirmed that H₂O₂ in the concentration seen during ischemia/reperfusion is primarily responsible for cellular membrane damage in the rat colonic fragments in vitro. Hydrogen peroxide caused a time and dose-dependent increase in 6-keto-PGF₁ and TXB₂ release. Hydrogen peroxide-stimulated 6-keto-PGF₁ release was blocked (50%) by phospholipase A₂ (PLA₂) inhibitors quinacrine and dimethyleicosodienoic acid at 5 min. Hydrogen peroxide-stimulated 6-keto-PGF₁ release was completely blocked by idomethacin, significantly blocked (69%) by nordihydroguiaretic acid, and completely blocked by catalase. superoxide dismutase and uric acid failed to inhibit H₂O₂-stimulated 6-keto-PGF₁ release. Endogenous catalase inhibitors 3-aminotriazole and sodium azide further enhanced the release of 6-keto-PGF₁ stimulated by H₂O₂ by 29% and 73%, respectively. Xanthine-xanthine oxidase also increased 6-keto-PGF₁ release from the fragments by 110%. This release was not inhibited by superoxide dismutase and uric acid, but was completely inhibited by catalase. These studies suggest a direct effect of H₂O₂ on colonic fragments leading to submicroscopic cellular membrane damage and excess prostanoid production utilizing a PLA₂/cyclooxygenase and catalase-sensitive pathway without the formation of toxic hydroxyl ions. The quick release of 6-keto-PGF₁ also suggests an early manifestation of H₂O₂-induced damage in rat colonic fragments.     

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.      

Mouse homologues of the human AZF candidate gene RBM are expressed in spermatogonia and spermatids, and map to a Y chromosome deletion interval associated with a high incidence of sperm abnormalities

An RNA-binding motif (RBM) gene family has been identified on the human Y chromosome that maps to the same deletion interval as the 'azoospermia factor' (AZF). We have identified the homologous gene family (Rbm) on the mouse Y with a view to investigating the proposal that this gene family plays a role in spermatogenesis. At least 25 and probably >50 copies of Rbm are present on the mouse Y chromosome short arm located between Sry and the centromere. As in the human, a role in spermatogenesis is indicated by a germ cell-specific pattern of expression in the testis, but there are distinct differences in the pattern of expression between the two species. Mice carrying the deletion Yd1, that maps to the proximal Y short arm, are female due to a position effect resulting in non-expression of Sry ; sex-reversing such mice with an Sry transgene produces males with a high incidence of abnormal sperm, making this the third deletion interval on the mouse Y that affects some aspect of spermatogenesis. Most of the copies of Rbm map to this deletion interval, and the Yd1males have markedly reduced Rbm expression, suggesting that RBM deficiency may be responsible for, or contribute to, the abnormal sperm development. In man, deletion of the functional copies of RBM is associated with meiotic arrest rather than sperm anomalies; however, the different effects of deletion are consistent with the differences in expression between the two species.      

Relationship between surface antigens of two variants of influenza A (H3N2) virus, as revealed by hemagglutination inhibition, kinetic neutralization, and neuraminidase inhibition.

Rabbit antisera were raised against plaque-purified influenza virus strains of A/Victoria/75 and A/Texas/77 isolated from Seattle influenza patients. The antigenic specificity of hemagglutinins was compared by hemagglutination inhibition (HI) and kinetic neutralization tests. Anti-A/Victoria/75 had equally high HI titers and neutralization rate constants (kappa values) for A/Victoria/75 and A/Texas/77. In contrast, anti-A/Texas/77 had a high HI titer and kappa value to A/Texas/77 and a low HI titer and kappa value to A/Victoria/75. Similar results were obtained with antisera to recombinants with hemagglutinin specific for A/Victoria/3/75 or A/Texas/1/77 and with irrelevant neuraminidase. Seven wild-type isolates, three each of A/Texas and A/Victoria, and one strain characterized as a bridging strain were tested by HI and kinetic neutralization. Characterization as A/Texas or A/Victoria was confirmed by the results. No significant difference in neuraminidase specific for A/Victoria/75 or A/Texas/77 was hown when recombinants with an irrelevant hemagglutinin were compared by the neuraminidase inhibition test. These results suggest that A/Victoria/75 strains are "senior" to A/Texas/77 strains. The epidemiological implications of this observation are discussed.     

Pulmonary carcinoid tumours: a comparative regional study.

Twenty-two pulmonary carcinoid tumours (18 central, 4 peripheral) were seen in this department over an 11-year period. The majority of the tumours displayed a mixed pattern on microscopic examination, and cell-nest formation was a prominent feature in sixteen. The findings are at variance with the results of a similar series reported from Japan and suggest that there is regional variation in the tumour pattern of pulmonary carcinoids. Various aspects of the histopathology are discussed, and a causal relationship between ossification of bronchial cartilage in these tumours and locally produced calcitonin is postulated.     

L1 knockout mice show dilated ventricles, vermis hypoplasia and impaired exploration patterns

L1 is a neural cell adhesion molecule mainly involved in axon guidance and neuronal migration during brain development. Mutations in the human L1 gene give rise to a complex clinical picture, with mental retardation, neurologic abnormalities and a variable degree of hydrocephalus. Recently, a transgenic mouse model with a targeted null mutation in the L1 gene was generated. These knockout (KO) mice show hypoplasia of the corticospinal tract. Here we have performed further studies of these KO mice including magnetic resonance imaging of the brain, neuropathological analysis and behavioral testing. The ventricular system was shown to be abnormal with dilatation of the lateral ventricles and the 4th ventricle, and an altered shape of the Sylvius aqueduct. Additionally, the cerebellar vermis of the KO mice is hypoplastic. Their exploratory behavior is characterized by stereotype peripheral circling reminiscent of that of rodents with induced cerebellar lesions.      

Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung

Lung carcinoids occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). There are no well defined genetic abnormalities known to occur in these tumors. We studied 11 sporadic lung carcinoids for loss of heterozygosity (LOH) at the locus of the MEN1 gene on chromosome 11q13, and for mutations of the MEN1 gene using dideoxy fingerprinting. Additionally, a lung carcinoid from a MEN1 patient was studied. In four of 11 (36%) sporadic tumors, both copies of the MEN1 gene were inactivated. All four tumors showed the presence of a MEN1 gene mutation and loss of the other allele. Observed mutations included a 1 bp insertion, a 1 bp deletion, a 13 bp deletion and a single nucleotide substitution affecting a donor splice site. Each mutation predicts truncation or potentially complete loss of menin. The remaining seven tumors showed neither the presence of a MEN1 gene mutation nor 11q13 LOH. The tumor from the MEN1 patient showed LOH at chromosome 11q13 and a complex germline MEN1 gene mutation. The data implicate the MEN1 gene in the pathogenesis of sporadic lung carcinoids, representing the first defined genetic alteration in these tumors.