IDH1 and IDH2 mutations in pediatric acute leukemia.

To investigate the frequency of isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) mutations in pediatric acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL), we sequenced these genes in diagnostic samples from 515 patients (227 AMLs and 288 ALLs). Somatic IDH1/IDH2 mutations were rare in ALL (N=1), but were more common in AML, occurring in 3.5% (IDH1 N=3 and IDH2 N=5), with the frequency higher in AMLs with a normal karyotype (9.8%). The identified IDH1 mutations occurred in codon 132 resulting in replacement of arginine with either cysteine (N=3) or histidine (N=1). By contrast, mutations in IDH2 did not affect the homologous residue but instead altered codon 140, resulting in replacement of arginine with either glutamine (N=4) or tryptophan (N=1). Structural modeling of IDH2 suggested that codon 140 mutations disrupt the enzyme's ability to bind its substrate isocitrate. Accordingly, recombinant IDH2 R140Q/W were unable to carry out the decarboxylation of isocitrate to α-ketoglutarate (α-KG), but instead gained the neomorphic activity to reduce α-KG to R(-)-2-hydroxyglutarete (2-HG). Analysis of primary leukemic blasts confirmed high levels of 2-HG in AMLs with IDH1/IDH2 mutations. Interestingly, 3/5 AMLs with IDH2 mutations had FLT3-activating mutations, raising the possibility that these mutations cooperate in leukemogenesis.

The dynamics of Salmonella occurrence in commercial laying hen flocks throughout a laying period.

Contaminated eggs and egg products have been recognized for many years as an important source of Salmonella infections in humans in the European Union and in the United States. Longitudinal studies can help to increase our knowledge about the dynamics of the occurrence of Salmonella in the course of a laying period. The total of 41 laying hen flocks-18 in Belgium, six in Denmark and 17 in Germany-were followed during an entire laying period. Samples taken from the empty cleaned and disinfected poultry houses were all negative for Salmonella. After hens arrived on the farms, five pooled faecal samples, one pooled dust sample and 40 cloacal swabs (Belgium and Germany) or 40 swabs from fresh droppings (Denmark) were taken four times from 18 flocks, three times from 21 flocks and two times from two flocks in the course of the laying period. Ten flocks (two Belgian and eight German flocks) tested up to three times positive for Salmonella. Forty-three out of 50 positive samples contained Salmonella Enteritidis phage type 4 (29 isolates) or phage type 8 (14 isolates). The probability of subsequent Salmonella-positive findings increased significantly in Salmonella-positive flocks (P<0.05, odds ratio = 6.4). However, the probability of finding Salmonella did not depend on the time of sampling in the laying period or the season.

Identification of acquired copy number alterations and uniparental disomies in cytogenetically normal acute myeloid leukemia using high-resolution single-nucleotide polymorphism analysis.

Recent advances in genome-wide single-nucleotide polymorphism (SNP) analyses have revealed previously unrecognized microdeletions and uniparental disomy (UPD) in a broad spectrum of human cancers. As acute myeloid leukemia (AML) represents a genetically heterogeneous disease, this technology might prove helpful, especially for cytogenetically normal AML (CN-AML) cases. Thus, we performed high-resolution SNP analyses in 157 adult cases of CN-AML. Regions of acquired UPDs were identified in 12% of cases and in the most frequently affected chromosomes, 6p, 11p and 13q. Notably, acquired UPD was invariably associated with mutations in nucleophosmin 1 (NPM1) or CCAAT/enhancer binding protein-alpha (CEBPA) that impair hematopoietic differentiation (P=0.008), suggesting that UPDs may preferentially target genes that are essential for proliferation and survival of hematopoietic progenitors. Acquired copy number alterations (CNAs) were detected in 49% of cases with losses found in two or more cases affecting, for example, chromosome bands 3p13-p14.1 and 12p13. Furthermore, we identified two cases with a cryptic t(6;11) as well as several non-recurrent aberrations pointing to leukemia-relevant regions. With regard to clinical outcome, there seemed to be an association between UPD 11p and UPD 13q cases with overall survival. These data show the potential of high-resolution SNP analysis for identifying genomic regions of potential pathogenic and clinical relevance in AML.

Pediatric acute myeloid leukemia with NPM1 mutations is characterized by a gene expression profile with dysregulated HOX gene expression distinct from MLL-rearranged leukemias.

Somatic mutations in nucleophosmin (NPM1) occur in approximately 35% of adult acute myeloid leukemia (AML). To assess the frequency of NPM1 mutations in pediatric AML, we sequenced NPM1 in the diagnostic blasts from 93 pediatric AML patients. Six cases harbored NPM1 mutations, with each case lacking common cytogenetic abnormalities. To explore the phenotype of the AMLs with NPM1 mutations, gene expression profiles were obtained using Affymetrix U133A microarrays. NPM1 mutations were associated with increased expression of multiple homeobox genes including HOXA9, A10, B2, B6 and MEIS1. As dysregulated homeobox gene expression is also a feature of MLL-rearranged leukemia, the gene expression signatures of NPM1-mutated and MLL-rearranged leukemias were compared. Significant differences were identified between these leukemia subtypes including the expression of different HOX genes, with NPM1-mutated AML showing higher levels of expression of HOXB2, B3, B6 and D4. These results confirm recent reports of perturbed HOX expression in NPM1-mutated adult AML, and provide the first evidence that the NPM1-mutated signature is distinct from MLL-rearranged AML. These findings suggest that mutated NPM1 leads to dysregulated HOX expression via a different mechanism than MLL rearrangement.

[3H]serotonin binding sites in goldfish retinal membranes.

Serotonin (5HT) binding sites were studied in goldfish retinal membranes by radioligand experiments. The binding site of [3H]5HT was sensitive to pre-treatment of the membranes at 40 degrees or 60 degrees C. 5HT and 5-methoxy-N,N-dimethyltryptamine were the best inhibitors of [3H]5HT binding to retinal membranes. The 5HT2 agonist, 1-(-naphthyl)piperazine, was also a potent inhibitor, however, (+)-1-2,5-dimethoxy-4-iodophenyl-2-aminopropane was less efficient. The catecholaminergic agents haloperidol and clonidine did not display an important inhibition. Propranolol, also reported as 5HT1B antagonist, was a relatively potent blocker. Monoamine uptake blockers did not show potent inhibition. The GTP analog, GppNHp, inhibited the binding. The iterative analysis of saturation curves revealed two classes of binding sites, a high affinity component (B(max) 2.45 pmol/mg of protein, kd 6.86 nM), and a low affinity component (B(max) 53.46 pmol/mg of protein, Kd 232.07 nM). Analysis of the association and dissociation kinetics suggested a binding site (Kd 2 nM). The semilogarithmic plot of the dissociation kinetics gave curves concave to the upper side. The selectivity of the binding and the inhibition by GppNHp suggest the existence of 5HT1 receptors in goldfish retina. The low affinity interaction probably represents the transporter of 5HT or a subtype of receptor expressed in glial cells.

Purification and characterization of poly(A) polymerase from Saccharomyces cerevisiae.

Poly(A) polymerase was purified 22,000-fold to homogeneity from a whole cell extract of Saccharomyces cerevisiae with a yield of 22%. The enzyme is a monomeric polypeptide with a denatured molecular weight of 63,000. Incorporation of labeled ATP into acid-precipitable material by the purified enzyme proceeds faster with manganese than with magnesium ions. Various RNA homopolymers as well as Escherichia coli tRNA or rRNA can serve as primers. An RNA that terminates at the natural poly(A) site of the CYC1 gene is not more efficiently elongated than several nonspecific substrates, indicating the requirement for additional factors to provide specificity. Elongation of the primer is distributive. Covering of a poly(A) primer with poly(A)-binding protein reduces the enzyme's activity more than 10-fold.