Evidence by allelic association-dependent methods for a type 1 diabetes polygene (IDDM6) on chromosome 18q21

Type 1 diabetes is a common polygenic disease. Fine mapping of polygenes by affected sibpair linkage analysis is not practical and allelic association or linkage disequilibrium mapping will have to be employed to attempt to detect founder chromosomes. Given prior evidence of linkage of the Jk-D18S64 region of chromosome 18q12-q21 to type 1 diabetes, we evaluated the 12 informative microsatellite markers in the region for linkage with disease by the transmission disequilibrium test (TDT) in a UK data set of type 1 diabetic families (n = 195). Increased transmission of allele 4 of marker D18S487 to affected children was detected (P = 0.02). Support for this was extended in a total of 1067 families from four different countries by isolating, and evaluating by the TDT, two novel microsatellites within 70 kb of D18S487. Evidence for linkage and association was P = 5 x 10(-5) and 3 x 10(-4), respectively. There was no evidence for increased transmission of associated alleles to nonaffected siblings. Analysis of an additional 390 families by the TDT did not extend the evidence further, and reduced support in the total 1457 families to P = 0.001 for linkage and P = 0.003 for association. However, evidence for linkage by affected sibpair allele sharing was strong (P = 3.2 x 10(-5)) in the second data set. Heterogeneity in TDT results between data sets was, in part, accounted for by the presence of more than one common disease-associated haplotype (allelic heterogeneity) which confounds the analysis of individual alleles by the TDT. Guidelines for strategies for the mapping of polygenes are suggested with the emphasis on collections of large numbers of families from multiple populations that should be as genetically homogeneous as possible.     

Malignant progenitors from patients with acute myelogenous leukemia are sensitive to a diphtheria toxin-granulocyte-macrophage colony-stimulating factor fusion protein

We have previously demonstrated that human granulocyte-macrophage colony-stimulating factor (GM-CSF) fused to a truncated diphtheria toxin (DT388-GMCSF) kills acute myelogenous leukemia (AML) cell lines bearing the GM-CSF receptor. We now report that exposure of malignant cells from 50 different patients with AML for 48 hours in culture to DT388-GMCSF reduces by a median of 1.6 logs (range, 0 to 3.7 logs) the number of leukemic cells capable of forming colonies in semisolid media (leukemic colony-forming cells [CFU-L]) with a median IC50 of 3 x 10(-12) mol/L (range, 5 to >4,000 x 10(-12) mol/L). Furthermore, the cell kill is dependent on the presence of high-affinity GM-CSF receptors on leukemic blasts, because CFU-L from 27 of 28 AML samples expressing > or = 35 GM-CSF receptors per cell were inhibited by the toxin, whereas the colony growth from all 4 leukemic samples (2 AML, 1 acute lymphoblastic leukemia [ALL], and 1 prolymphocytic leukemia [PLL]) that had less than 35 receptors per cell was unaffected by the drug. Sensitivity of CFU-L to DT388-GMCSF was seen regardless of the clinical responsiveness of the patient's leukemia to standard chemotherapy agents. In contrast, clonogenic cells from normal bone marrow formed colonies at near control numbers after exposure to much higher toxin concentrations (4 x 10(-9) mol/L) than those required to kill CFU-L from most patients. Thus, leukemic progenitors isolated directly from the peripheral blood of most AML patients show the same sensitivity to DT388-GMCSF as previously demonstrated for AML cell lines. Under the same conditions of exposure, normal hematopoietic progenitors are relatively unaffected by DT388-GMCSF, suggesting its potential as a therapeutic agent in AML.     

An optimal implementation of broadcasting with selective reduction

A model of parallel computation called broadcasting with selective reduction (BSR) can be viewed as a concurrent-read concurrent-write (CRCW) parallel random access machine (PRAM) with one extension. An additional type of concurrent memory access is permitted in BSR, namely the BROADCAST instruction by means of which all N processors may gain access to all M memory locations simultaneously for the purpose of writing. At each memory location, a subset of the incoming broadcast data is selected and reduced to one value finally stored in that location. For several problems, BSR algorithms are known which require fewer steps than the corresponding best-known PRAM algorithms, using the same number of processors. A circuit is introduced to implement the BSR model, and it is shown that, in size and depth, the circuit presented is of the same order as an optimal circuit implementing the PRAM. Thus, if it is reasonable to assume that CRCW PRAM instructions execute in constant time, the assumption of a constant time BROADCAST instruction is no less reasonable     

Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure

Retrovirus-mediated gene transfer of the human beta-globin gene into hematopoietic stem cells is an attractive approach to the therapy of human beta-globin gene disorders. However, expression of the transduced beta-globin gene linked to its proximal cis-acting sequences (-0.8 to +0.3 kb from the cap site) is considerably below the level required for a significant therapeutic effect. The discovery of the beta-locus control region (beta-LCR), organized in four major DNase I hypersensitive sites far upstream of the human beta-like globin gene cluster, provided a potential means to achieve a high level of expression of a linked human beta-globin gene, but initial attempts to incorporate beta-LCR derivatives in retroviral vectors resulted in the production of low-titer viruses with multiple rearrangements of the transmitted proviral structures. We now describe how extensive mutagenesis of the transduced beta-globin gene, eliminating a 372 bp intronic segment and multiple reverse polyadenylation and splicing signals, increases viral titer significantly and restores stability of proviral transmission upon infection of cell lines and bone marrow-repopulating cells. These optimized vectors have enabled us to analyze the expression properties of various retrovirally transduced beta-LCR derivatives in dimethylsulfoxide-induced murine erythroleukemia cells and to achieve ratios of human beta-globin/murine beta maj-globin mRNA, on a per gene basis, as high as 80%.     

Computer simulation of sample size and experimental design in human psychogenetics.

Applied a method of computer simulation to the investigation of problems connected with the genetic analysis of continuously variable behavioral characters in human populations. The efficiency with which various components of genetic and environmental variation could be detected was related to sample size. Results indicate that a convincing partition of the genetic variance into its additive and nonadditive components required much larger samples than those frequently employed in human psychogenetics. (15 ref.) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Application of the FLIPSY pulse sequence for increased sensitivity in 1H NMR-based metabolic profiling studies

Metabolite profiling relies on optimal precision of the acquired data, which requires, among others, a high signal-to-noise ratio (S/N). In addition, increased S/N will increase the likelihood of identification of new biomarkers. Here we introduce, for the first time in metabolite profiling studies by 1H NMR, an approach to enhance the precision of multivariate regression models by use of the FLIPSY (flip angle adjustable one-dimensional NOESY) pulse sequence, augmented by a homospoil pulse after the presaturation period to provide superior baseline quality. Unlike NOESYPRESAT, the standard one-dimensional (1D) sequence generally used in metabonomic studies, FLIPSY incorporates a variable flip angle, allowing use of the Ernst angle for excitation and thus optimization of S/N ratios according to spin lattice relaxation times (T1) of individual resonances. T1 values of metabolites present in human urine were determined by inversion-recovery experiments and subsequently used in calculations of optimal experimental conditions. Comparison of human urine analysis by the FLIPSY and NOESYPRESAT demonstrated an increase of S/N ratio in the former case that amounts to approximately 7% when measured for the hippurate doublet at delta 7.84. An orthogonal projection to latent structures discriminant analysis (O-PLS-DA) model exhibited superior discrimination between controls and simulated phenylketonuria urines when using data generated by the FLIPSY as compared to NOESYPRESAT.     

Heteronuclear 19F-1H statistical total correlation spectroscopy as a tool in drug metabolism: study of flucloxacillin biotransformation

We present a novel application of the heteronuclear statistical total correlation spectroscopy (HET-STOCSY) approach utilizing statistical correlation between one-dimensional 19F/1H NMR spectroscopic data sets collected in parallel to study drug metabolism. Parallel one-dimensional (1D) 800 MHz 1H and 753 MHz 19F{1H} spectra (n = 21) were obtained on urine samples collected from volunteers (n = 6) at various intervals up to 24 h after oral dosing with 500 mg of flucloxacillin. A variety of statistical relationships between and within the spectroscopic datasets were explored without significant loss of the typically high 1D spectral resolution, generating 1H-1H STOCSY plots, and novel 19F-1H HET-STOCSY, 19F-19F STOCSY, and 19F-edited 1H-1H STOCSY (X-STOCSY) spectroscopic maps, with a resolution of approximately 0.8 Hz/pt for both nuclei. The efficient statistical editing provided by these methods readily allowed the collection of drug metabolic data and assisted structure elucidation. This approach is of general applicability for studying the metabolism of other fluorine-containing drugs, including important anticancer agents such as 5-fluorouracil and flutamide, and is extendable to any drug metabolism study where there is a spin-active X-nucleus (e.g., 13C, 15N, 31P) label present.