Evolution of unbalanced gain of distal chromosome 2p in neuroblastoma

Neuroblastoma, one of the most common tumors of childhood, presents at diagnosis with a vast number of recurrent chromosomal imbalances that include hyperdiploidy for whole chromosomes, partial loss of 1p, 3p, 4p, 11q, 14q, partial gain of 1q, 7q, 17q and amplification of MYCN. These abnormalities are nonrandomly distributed in neuroblastoma as loss of 3p and 11q rarely occur in MYCN amplified neuroblastomas. Here, we report on a patient who had a non-MYCN amplified 3p-/11q- neuroblastoma at diagnosis who subsequently developed a high level of MYCN amplification in bone marrow metastases 41 months after induction of complete remission. The tumor at diagnosis had low level unbalanced gain of distal 2p. In order to assess the frequency of low level gain of distal 2p in neuroblastoma, we examined the comparative genomic hybridization results from 60 neuroblastomas. Among non-MYCN amplified neuroblastomas, 8/45 (18%) had low level gain of distal 2p. Low level gain for a segment of 2p (i.e. a region larger than the 2p23-->p24 undergoing amplification) was also detected in five of the 15 tumors that had high level MYCN amplification. The possibility that low level gain of distal 2p is a risk factor for high level MYCN amplification is discussed.     

Evidence of linkage of familial hypoalphalipoproteinemia to a novel locus on chromosome 11q23

Coronary heart disease (CHD) accounts for half of the 1 million deaths annually ascribed to cardiovascular disease and for almost all of the 1.5 million acute myocardial infarctions. Within families affected by early and apparently heritable CHD, dyslipidemias have a much higher prevalence than in the general population; 20%-30% of early familial CHD has been ascribed to primary hypoalphalipoproteinemia (low HDL-C). This study assesses the evidence for linkage of low HDL-C to chromosomal region 11q23 in 105 large Utah pedigrees ascertained with closely related clusters of early CHD and expanded on the basis of dyslipidemia. Linkage analysis was performed by use of 22 STRP markers in a 55-cM region of chromosome 11. Two-point analysis based on a general, dominant-phenotype model yielded LODs of 2.9 for full pedigrees and 3.5 for 167 four-generation split pedigrees. To define a localization region, model optimization was performed using the heterogeneity, multipoint LOD score (mpHLOD). This linkage defines a region on 11q23.3 that is approximately 10 cM distal to-and apparently distinct from-the ApoAI/CIII/AIV gene cluster and thus represents a putative novel localization for the low HDL-C phenotype.     

Two distinct binding affinities of poliovirus for its cellular receptor

To study the kinetics and equilibrium of poliovirus binding to the poliovirus receptor, we used surface plasmon resonance to examine the interaction of a soluble form of the receptor with poliovirus. Soluble receptor purified from mammalian cells is able to bind poliovirus, neutralize viral infectivity, and induce structural changes in the virus particle. Binding studies revealed that there are two binding sites for the receptor on the poliovirus type 1 capsid, with affinity constants at 20 degrees C of K(D)(1) = 0.67 microm and K(D)(2) = 0.11 microm. The relative abundance of the two binding sites varies with temperature. At 20 degrees C, the K(D)(2) site constitutes approximately 46% of the total binding sites on the sensor chip, and its relative abundance decreased with decreasing temperature such that at 5 degrees C, the relative abundance of the K(D)(2) site is only 12% of the total binding sites. Absolute levels of the K(D)(1) site remained relatively constant at all temperatures tested. The two binding sites may correspond to docking sites for domain 1 of the receptor on the viral capsid, as predicted by a model of the poliovirus-receptor complex. Alternatively, the binding sites may be a consequence of structural breathing, or could result from receptor-induced conformational changes in the virus.     

Characterization of two inducible phosphate transport systems in Rhizobium tropici

Rhizobium tropici forms nitrogen-fixing nodules on the roots of the common bean (Phaseolus vulgaris). Like other legume-Rhizobium symbioses, the bean-R. tropici association is sensitive to the availability of phosphate (P(i)). To better understand phosphorus movement between the bacteroid and the host plant, P(i) transport was characterized in R. tropici. We observed two P(i) transport systems, a high-affinity system and a low-affinity system. To facilitate the study of these transport systems, a Tn5B22 transposon mutant lacking expression of the high-affinity transport system was isolated and used to characterize the low-affinity transport system in the absence of the high-affinity system. The K(m) and V(max) values for the low-affinity system were estimated to be 34 +/- 3 microM P(i) and 118 +/- 8 nmol of P(i) x min(-1) x mg (dry weight) of cells(-1), respectively, and the K(m) and V(max) values for the high-affinity system were 0.45 +/- 0.01 microM P(i) and 86 +/- 5 nmol of P(i) x min(-1) x mg (dry weight) of cells(-1), respectively. Both systems were inducible by P(i) starvation and were also shock sensitive, which indicated that there was a periplasmic binding-protein component. Neither transport system appeared to be sensitive to the proton motive force dissipator carbonyl cyanide m-chlorophenylhydrazone, but P(i) transport through both systems was eliminated by the ATPase inhibitor N,N'-dicyclohexylcarbodiimide; the P(i) transport rate was correlated with the intracellular ATP concentration. Also, P(i) movement through both systems appeared to be unidirectional, as no efflux or exchange was observed with either the wild-type strain or the mutant. These properties suggest that both P(i) transport systems are ABC type systems. Analysis of the transposon insertion site revealed that the interrupted gene exhibited a high level of homology with kdpE, which in several bacteria encodes a cytoplasmic response regulator that governs responses to low potassium contents and/or changes in medium osmolarity.     

Electronic effects in transition metal porphyrins. 5. Thermodynamics of axial ligand addition and spectroscopic trends of a series of symmetrical and unsymmetrical derivatives of tetraphenylporphinatozinc(II)

The electronic spectra and equilibrium constants for addition of 3-picoline to a series of symmetrically and unsymmetrically phenyl-substituted ZnTPP derivatives have been measured. It is found that the α band energy varies slightly nonlinearly with the sum of the Hammett sigma constants of the substituents within the series (p-Cl)_x(p-NEt₂)_yTPPZn(II), while the smaller variation in the β band appears to be linear. The log of the intensity ratio of the α and β bands, log A_β/A^?_α, however, varies linearly with the band energies of both the α and β bands for both 4- and 5-coordinate complexes of unsymmetrical as well as symmetrical ZnTPP derivatives. Likewise, log K_eq for 3-picoline addition varies linearly with the sum of the Hammett sigma constants for all complexes investigated. Thus the electronic effects of unsymmetrically placed substituents are averaged by the metal Zn to yield a Lewis acid strength toward 3-picoline which is dependent only upon the sum of the electronic effects and not on the identity of the substituents or the symmetry of their distribution.     

Comparing apples and oranges--model-based assessment of different tsetse-transmitted trypanosomosis control strategies

The current control strategies for tsetse-transmitted trypanosomosis in cattle (trypanocidal drugs, tsetse control and trypanotolerant cattle) are briefly reviewed and their adoption rates in different geographic regions of sub-Saharan Africa are presented. The impact of these control strategies and the potential use of vaccines, should they be developed, on trypanosomosis transmission were compared using a mathematical model. The relative trypanosomosis prevalence compared with no control was estimated across a range of control coverages (from none to complete control coverage) by varying the change in specific model parameters influenced by individual control measures. Based on this comparison, the relative rankings of the effect of control strategies on reducing disease prevalence were: vector control, vaccination, and drug use, in that order. In this model, trypanotolerance was assumed to decrease disease prevalence, but not to influence transmission. Differences in the predicted impact of control measures on the transmission of human sleeping sickness are discussed. Finally, the role of transmission model outputs as inputs for economic models to guide investment decisions for trypanosomosis control is emphasised.     

A recombinant group 1 house dust mite allergen, rDer f 1, with biological activities similar to those of the native allergen

Serum IgE directed against Der f 1, a protease found in the feces of Dermatophagoides farinae, correlates well with allergic sensitization to house dust mite in humans and is a risk factor for developing asthma. Native Der f 1 (nDer f 1) is produced as a pre-pro form and processed to an approximately 25-kDa mature form. We have expressed recombinant forms of Der f 1 (rDer f 1) in Pichia pastoris using AOX1-promoter expression vectors. Fusion of either the pro-enzyme form or the mature form to the Saccharomyces cerevisiae alpha factor pre-pro sequence resulted in secretion of the mature form of the protein from P. pastoris. The secreted protein was heterogeneously glycosylated at a single N-glycosylation site and had an apparent molecular mass of 35-50 kDa. Both the alpha factor signal peptide and the pro-enzyme region were efficiently processed during secretion. A version of the pro-enzyme with a mutated consensus N-linked glycosylation site was secreted from P. pastoris as a mature, unglycosylated, approximately 25-kDa protein. The IgE binding activity of this unglycosylated rDer f 1 was similar to that of glycosylated forms produced by P. pastoris and to nDer f 1 obtained from mites. Thus, oligosaccharides are not required for secretion from P. pastoris or for IgE binding in vitro. Recombinant and native versions of Der f 1 displayed protease activity on casein zymogram gels. The availability of a highly purified recombinant Der f 1 will facilitate experimental and clinical studies of mite allergy.     

Comprehensive Molecular Profiling Identifies FOXM1 as a Key Transcription Factor for Meningioma Proliferation

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Identification and Validation of miRNAs as Endogenous Controls for RQ-PCR in Blood Specimens for Breast Cancer Studies

Introduction

A prerequisite to accurate interpretation of RQ-PCR data is robust data normalization. A commonly used method is to compare the cycle threshold (C_T) of target miRNAs with those of a stably expressed endogenous (EC) miRNA(s) from the same sample. Despite the large number of studies reporting miRNA expression patterns, comparatively few appropriate ECs have been reported thus far. The purpose of this study was to identify stably expressed miRNAs with which to normalize RQ-PCR data derived from human blood specimens.

Methods

MiRNA profiling of approximately 380 miRNAs was performed on RNA derived from blood specimens from 10 women with breast cancer and 10 matched controls. Analysis of mean expression values across the dataset (GME) identified stably expressed candidates. Additional candidates were selected from the literature and analyzed by the geNorm algorithm. Further validation of three candidate ECs by RQ-PCR was performed in a larger cohort (n = 40 cancer, n = 20 control) was performed, including analysis by geNorm and NormFinder algorithms.

Results

Microarray screening identified 10 candidate ECs with expression patterns closest to the global mean. Geometric averaging of candidate ECs from the literature using geNorm identified miR-425 as the most stably expressed miRNA. MiR-425 and miR-16 were the best combination, achieving the lowest V-value of 0.185. Further validation by RQ-PCR confirmed that miR-16 and miR-425 were the most stably expressed ECs overall. Their combined use to normalize expression data enabled the detection of altered target miRNA expression that reliably differentiated between cancers and controls in human blood specimens.

Conclusion

This study identified that the combined use of 2 miRNAs, (miR-16 and miR-425) to normalize RQ-PCR data generated more reliable results than using either miRNA alone, or use of U6. Further investigation into suitable ECs for use in miRNA RQ-PCR studies is warranted.