Fetal origins of the TEL-AML1 fusion gene in identical twins with leukemia

The TEL (ETV6)−AML1 (CBFA2) gene fusion is the most common reciprocal chromosomal rearrangement in childhood cancer occurring in ≈25% of the most predominant subtype of leukemia— common acute lymphoblastic leukemia. The TEL-AML1 genomic sequence has been characterized in a pair of monozygotic twins diagnosed at ages 3 years, 6 months and 4 years, 10 months with common acute lymphoblastic leukemia. The twin leukemic DNA shared the same unique (or clonotypic) but nonconstitutive TEL-AML1 fusion sequence. The most plausible explanation for this finding is a single cell origin of the TEL-AML fusion in one fetus in utero, probably as a leukemia-initiating mutation, followed by intraplacental metastasis of clonal progeny to the other twin. Clonal identity is further supported by the finding that the leukemic cells in the two twins shared an identical rearranged IGH allele. These data have implications for the etiology and natural history of childhood leukemia.

Human ATP-binding cassette transporter 1 (ABC1): Genomic organization and identification of the genetic defect in the original Tangier disease kindred

Tangier disease is characterized by low serum high density lipoproteins and a biochemical defect in the cellular efflux of lipids to high density lipoproteins. ABC1, a member of the ATP-binding cassette family, recently has been identified as the defective gene in Tangier disease. We report here the organization of the human ABC1 gene and the identification of a mutation in the ABC1 gene from the original Tangier disease kindred. The organization of the human ABC1 gene is similar to that of the mouse ABC1 gene and other related ABC genes. The ABC1 gene contains 49 exons that range in size from 33 to 249 bp and is over 70 kb in length. Sequence analysis of the ABC1 gene revealed that the proband for Tangier disease was homozygous for a deletion of nucleotides 3283 and 3284 (TC) in exon 22. The deletion results in a frameshift mutation and a premature stop codon starting at nucleotide 3375. The product is predicted to encode a nonfunctional protein of 1,084 aa, which is approximately half the size of the full-length ABC1 protein. The loss of a Mnl1 restriction site, which results from the deletion, was used to establish the genotype of the rest of the kindred. In summary, we report on the genomic organization of the human ABC1 gene and identify a frameshift mutation in the ABC1 gene of the index case of Tangier disease. These results will be useful in the future characterization of the structure and function of the ABC1 gene and the analysis of additional ABC1 mutations in patients with Tangier disease.

Gilbert syndrome and glucose-6-phosphate dehydrogenase deficiency: A dose-dependent genetic interaction crucial to neonatal hyperbilirubinemia

Severe jaundice leading to kernicterus or death in the newborn is the most devastating consequence of glucose-6-phosphate dehydrogenase (EC 1.1.1.49; G-6-PD) deficiency. We asked whether the TA repeat promoter polymorphism in the gene for uridinediphosphoglucuronate glucuronosyltransferase 1 (EC 2.4.1.17; UDPGT1), associated with benign jaundice in adults (Gilbert syndrome), increases the incidence of neonatal hyperbilirubinemia in G-6-PD deficiency. DNA from term neonates was analyzed for UDPGT1 polymorphism (normal homozygotes, heterozygotes, variant homozygotes), and for G-6-PD Mediterranean deficiency. The variant UDPGT1 promoter allele frequency was similar in G-6-PD-deficient and normal neonates. Thirty (22.9%) G-6-PD deficient neonates developed serum total bilirubin ≥ 257 μmol/liter, vs. 22 (9.2%) normals (P = 0.0005). Of those with the normal homozygous UDPGT1 genotype, the incidence of hyperbilirubinemia was similar in G-6-PD-deficients and controls (9.7% and 9.9%). In contrast, in the G-6-PD-deficient neonates, those with the heterozygous or homozygous variant UDPGT1 genotype had a higher incidence of hyperbilirubinemia than corresponding controls (heterozygotes: 31.6% vs. 6.7%, P < 0.0001; variant homozygotes: 50% vs. 14.7%, P = 0.02). Among G-6-PD-deficient infants the incidence of hyperbilirubinemia was greater in those with the heterozygous (31.6%, P = 0.006) or variant homozygous (50%, P = 0.003) UDPGT1 genotype than in normal homozygotes. In contrast, among those normal for G-6-PD, the UDPGT1 polymorphism had no significant effect (heterozygotes: 6.7%; variant homozygotes: 14.7%). Thus, neither G-6-PD deficiency nor the variant UDPGT1 promoter, alone, increased the incidence of hyperbilirubinemia, but both in combination did. This gene interaction may serve as a paradigm of the interaction of benign genetic polymorphisms in the causation of disease.

Mitogen-activated protein kinase kinase 7 is an activator of the c-Jun NH2-terminal kinase

The c-Jun NH2-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases is activated by phosphorylation on Thr and Tyr. Here we report the molecular cloning of a new member of the mammalian MAP kinase kinase group (MKK7) that functions as an activator of JNK. In vitro protein kinase assays demonstrate that MKK7 phosphorylates and activates JNK, but not the p38 or extracellular signal-regulated kinase groups of MAP kinase. Expression of MKK7 in cultured cells causes activation of the JNK signal transduction pathway. MKK7 is therefore established to be a novel component of the JNK signal transduction pathway.

Selective Perturbation of Apical Membrane Traffic by Expression of Influenza M2, an Acid-activated Ion Channel, in Polarized Madin–Darby Canine Kidney Cells

The function of acidification along the endocytic pathway is not well understood, in part because the perturbants used to modify compartmental pH have global effects and in some cases alter cytoplasmic pH. We have used a new approach to study the effect of pH perturbation on postendocytic traffic in polarized Madin–Darby canine kidney (MDCK) cells. Influenza M2 is a small membrane protein that functions as an acid-activated ion channel and can elevate the pH of the trans-Golgi network and endosomes. We used recombinant adenoviruses to express the M2 protein of influenza virus in polarized MDCK cells stably transfected with the polymeric immunoglobulin (Ig) receptor. Using indirect immunofluorescence and immunoelectron microscopy, M2 was found to be concentrated at the apical plasma membrane and in subapical vesicles; intracellular M2 colocalized partly with internalized IgA in apical recycling endosomes as well as with the trans-Golgi network marker TGN-38. Expression of M2 slowed the rate of IgA transcytosis across polarized MDCK monolayers. The delay in transport occurred after IgA reached the apical recycling endosome, consistent with the localization of intracellular M2. Apical recycling of IgA was also slowed in the presence of M2, whereas basolateral recycling of transferrin and degradation of IgA were unaffected. By contrast, ammonium chloride affected both apical IgA and basolateral transferrin release. Together, our data suggest that M2 expression selectively perturbs acidification in compartments involved in apical delivery without disrupting other postendocytic transport steps.

A genome-wide search for chromosomal loci linked to mental health wellness in relatives at high risk for bipolar affective disorder among the Old Order Amish

Bipolar affective disorder (BPAD; manic-depressive illness) is characterized by episodes of mania and/or hypomania interspersed with periods of depression. Compelling evidence supports a significant genetic component in the susceptibility to develop BPAD. To date, however, linkage studies have attempted only to identify chromosomal loci that cause or increase the risk of developing BPAD. To determine whether there could be protective alleles that prevent or reduce the risk of developing BPAD, similar to what is observed in other genetic disorders, we used mental health wellness (absence of any psychiatric disorder) as the phenotype in our genome-wide linkage scan of several large multigeneration Old Order Amish pedigrees exhibiting an extremely high incidence of BPAD. We have found strong evidence for a locus on chromosome 4p at D4S2949 (maximum genehunter-plus nonparametric linkage score = 4.05, P = 5.22 × 10−4; sibpal Pempirical value <3 × 10−5) and suggestive evidence for a locus on chromosome 4q at D4S397 (maximum genehunter-plus nonparametric linkage score = 3.29, P = 2.57 × 10−3; sibpal Pempirical value <1 × 10−3) that are linked to mental health wellness. These findings are consistent with the hypothesis that certain alleles could prevent or modify the clinical manifestations of BPAD and perhaps other related affective disorders.

Randomised controlled trial of laparoscopic versus open mesh repair for inguinal hernia: outcome and cost

Objective: To compare tension-free open mesh hernioplasty under local anaesthetic with transabdominal preperitoneal laparoscopic hernia repair under general anaesthetic.

Comparative analysis of the gene-dense ACHE/TFR2 region on human chromosome 7q22 with the orthologous region on mouse chromosome 5

Chromosome 7q22 has been the focus of many cytogenetic and molecular studies aimed at delineating regions commonly deleted in myeloid leukemias and myelodysplastic syndromes. We have compared a gene-dense, GC-rich sub-region of 7q22 with the orthologous region on mouse chromosome 5. A physical map of 640 kb of genomic DNA from mouse chromosome 5 was derived from a series of overlapping bacterial artificial chromosomes. A 296 kb segment from the physical map, spanning Ache to Tfr2, was compared with 267 kb of human sequence. We identified a conserved linkage of 12 genes including an open reading frame flanked by Ache and Asr2, a novel cation-chloride cotransporter interacting protein Cip1, Ephb4, Zan and Perq1. While some of these genes have been previously described, in each case we present new data derived from our comparative sequence analysis. Adjacent unfinished sequence data from the mouse contains an orthologous block of 10 additional genes including three novel cDNA sequences that we subsequently mapped to human 7q22. Methods for displaying comparative genomic information, including unfinished sequence data, are becoming increasingly important. We supplement our printed comparative analysis with a new, Web-based program called Laj (local alignments with java). Laj provides interactive access to archived pairwise sequence alignments via the WWW. It displays synchronized views of a dot-plot, a percent identity plot, a nucleotide-level local alignment and a variety of relevant annotations. Our mouse–human comparison can be viewed at http://web.uvic.ca/~bioweb/laj.html. Laj is available at http://bio.cse.psu.edu/, along with online documentation and additional examples of annotated genomic regions.

Protein Information Resource: a community resource for expert annotation of protein data

The Protein Information Resource, in collaboration with the Munich Information Center for Protein Sequences (MIPS) and the Japan International Protein Information Database (JIPID), produces the most comprehensive and expertly annotated protein sequence database in the public domain, the PIR-International Protein Sequence Database. To provide timely and high quality annotation and promote database interoperability, the PIR-International employs rule-based and classification-driven procedures based on controlled vocabulary and standard nomenclature and includes status tags to distinguish experimentally determined from predicted protein features. The database contains about 200 000 non-redundant protein sequences, which are classified into families and superfamilies and their domains and motifs identified. Entries are extensively cross-referenced to other sequence, classification, genome, structure and activity databases. The PIR web site features search engines that use sequence similarity and database annotation to facilitate the analysis and functional identification of proteins. The PIR-Inter­national databases and search tools are accessible on the PIR web site at http://pir.georgetown.edu/ and at the MIPS web site at http://www.mips.biochem.mpg.de. The PIR-International Protein Sequence Database and other files are also available by FTP.

iProClass: an integrated, comprehensive and annotated protein classification database

The iProClass database is an integrated resource that provides comprehensive family relationships and structural and functional features of proteins, with rich links to various databases. It is extended from ProClass, a protein family database that integrates PIR superfamilies and PROSITE motifs. The iProClass currently consists of more than 200 000 non-redundant PIR and SWISS-PROT proteins organized with more than 28 000 superfamilies, 2600 domains, 1300 motifs, 280 post-translational modification sites and links to more than 30 databases of protein families, structures, functions, genes, genomes, literature and taxonomy. Protein and family summary reports provide rich annotations, including membership information with length, taxonomy and keyword statistics, full family relationships, comprehensive enzyme and PDB cross-references and graphical feature display. The database facilitates classification-driven annotation for protein sequence databases and complete genomes, and supports structural and functional genomic research. The iProClass is implemented in Oracle 8i object-relational system and available for sequence search and report retrieval at http://pir.georgetow n.edu/iproclass/.

FM1-43 dye ultrastructural localization in and release from frog motor nerve terminals.

Previous work has shown that the fluorescent styryl dye FM1-43 stains nerve terminals in an activity-dependent fashion. This dye appears to label the membranes of recycled synaptic vesicles by being trapped during endocytosis. Stained terminals can subsequently be destained by repeating nerve stimulation in the absence of dye; the destaining evidently reflects escape of dye into the bathing medium from membranes of exocytosing synaptic vesicles. In the present study we tested two key aspects of this interpretation of FM1-43 behavior, namely: (i) that the dye is localized in synaptic vesicles, and (ii) that it is actually released into the bathing medium during destaining. To accomplish this, we first photolyzed the internalized dye in the presence of diaminobenzidine. This created an electron-dense reaction product that could be visualized in the electron microscope. Reaction product was confined to synaptic vesicles, as predicted. Second, using spectrofluorometry, we quantified the release of dye liberated into the medium from tubocurarine-treated nerve-muscle preparations. Nerve stimulation increased the amount of FM1-43 released, and we estimate that normally a stained synaptic vesicle contains a few hundred molecules of the dye. The key to the successful detection of released FM1-43 was to add the micelle-forming detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), which increased FM1-43 quantum yield by more than two orders of magnitude.