State-of-the art data normalization methods improve NMR-based metabolomic analysis

Extracting biomedical information from large metabolomic datasets by multivariate data analysis is of considerable complexity. Common challenges include among others screening for differentially produced metabolites, estimation of fold changes, and sample classification. Prior to these analysis steps, it is important to minimize contributions from unwanted biases and experimental variance. This is the goal of data preprocessing. In this work, different data normalization methods were compared systematically employing two different datasets generated by means of nuclear magnetic resonance (NMR) spectroscopy. To this end, two different types of normalization methods were used, one aiming to remove unwanted sample-to-sample variation while the other adjusts the variance of the different metabolites by variable scaling and variance stabilization methods. The impact of all methods tested on sample classification was evaluated on urinary NMR fingerprints obtained from healthy volunteers and patients suffering from autosomal polycystic kidney disease (ADPKD). Performance in terms of screening for differentially produced metabolites was investigated on a dataset following a Latin-square design, where varied amounts of 8 different metabolites were spiked into a human urine matrix while keeping the total spike-in amount constant. In addition, specific tests were conducted to systematically investigate the influence of the different preprocessing methods on the structure of the analyzed data. In conclusion, preprocessing methods originally developed for DNA microarray analysis, in particular, Quantile and Cubic-Spline Normalization, performed best in reducing bias, accurately detecting fold changes, and classifying samples.

     

The human PDGF receptor α-subunit gene maps to chromosome 4 in close proximity to c-kit

Summary The gene encoding the -subunit of the human platelet-derived growth factor receptor (PDGFRA) maps to band q11–q12 of chromosome 4 by in situ hybridization, which was confirmed by Southern analysis of a Chinese hamster × human cell hybrid that retains only human chromosome 4.     

Atrazine Resistance in a Velvetleaf (Abutilon theophrasti) Biotype Due to Enhanced Glutathione S-Transferase Activity

We previously reported that a velvetleaf (Abutilon theophrasti Medic) biotype found in Maryland was resistant to atrazine because of an enhanced capacity to detoxify the herbicide via glutathione conjugation (JW Gronwald, Andersen RN, Yee C [1989] Pestic Biochem Physiol 34: 149-163). The biochemical basis for the enhanced atrazine conjugation capacity in this biotype was examined. Glutathione levels and glutathione S-transferase activity were determined in extracts from the atrazine-resistant biotype and an atrazine-susceptible or “wild-type” velvetleaf biotype. In both biotypes, the highest concentration of glutathione (approximately 500 nanomoles per gram fresh weight) was found in leaf tissue. However, no significant differences were found in glutathione levels in roots, stems, or leaves of either biotype. In both biotypes, the highest concentration of glutathione S-transferase activity measured with 1-chloro-2,4-dinitrobenzene or atrazine as substrate was in leaf tissue. Glutathione S-transferase measured with 1-chloro-2,4-dinitrobenzene as substrate was 40 and 25% greater in leaf and stem tissue, respectively, of the susceptible biotype compared to the resistant biotype. In contrast, glutathione S-transferase activity measured with atrazine as substrate was 4.4- and 3.6-fold greater in leaf and stem tissue, respectively, of the resistant biotype. Kinetic analyses of glutathione S-transferase activity in leaf extracts from the resistant and susceptible biotypes were performed with the substrates glutathione, 1-chloro-2,4-dinitrobenzene, and atrazine. There was little or no change in apparent K_m values for glutathione, atrazine, or 1-chloro-2,4-dinitrobenzene. However, the V_max for glutathione and atrazine were approximately 3-fold higher in the resistant biotype than in the susceptible biotype. In contrast, the V_max for 1-chloro-2,4-dinitrobenzene was 30% lower in the resistant biotype. Leaf glutathione S-transferase isozymes that exhibit activity with atrazine and 1-chloro-2,4-dinitrobenzene were separated by fast protein liquid (anion-exchange) chromatography. The susceptible biotype had three peaks exhibiting activity with atrazine and the resistant biotype had two. The two peaks of glutathione S-transferase activity with atrazine from the resistant biotype coeluted with two of the peaks from the susceptible biotype, but peak height was three- to fourfold greater in the resistant biotype. In both biotypes, two of the peaks that exhibit glutathione S-transferase activity with atrazine also exhibited activity with 1-chloro-2,4-dinitrobenzene, with the peak height being greater in the susceptible biotype. The results indicate that atrazine resistance in the velvetleaf biotype from Maryland is due to enhanced glutathione S-transferase activity for atrazine in leaf and stem tissue which results in an enhanced capacity to detoxify the herbicide via glutathione conjugation.     

Differential regulation of expression of two platelet-derived growth factor receptor subunits by transforming growth factor-beta

The binding of three radiolabeled isoforms of platelet-derived growth factor (PDGF), 125I-PDGF-AA, 125I-PDGF-AB, and 125I-PDGF-BB, is differentially affected by exposure of quiescent 3T3 cells to transforming growth factor-beta (TGF-beta). By 24 h after exposure to TGF-beta, binding of 125I-PDGF-AA and 125I-PDGF-AB is almost completely lost, whereas binding of 125I-PDGF-BB is reduced by only 40%. The loss of PDGF-binding sites caused by TGF-beta is time- and concentration-dependent and reflects a change in the pattern of expression of receptor subunits; the number of alpha-subunits decreases, and the number of beta-subunits increases. The loss of binding sites for PDGF-AA is accompanied by a decreased mitogenic response to PDGF-AA but not to PDGF-AB or PDGF-BB. These results suggest that TGF-beta may differentially regulate the expression of PDGF-binding sites and the mitogenic responsiveness toward the three PDGF isoforms. TGF-beta did not stimulate synthesis of PDGF A-chain mRNA or PDGF-AA protein, and PDGF-AA receptors could not be restored by the presence of suramin, suggesting that the loss of binding sites may result from direct effects on receptor expression rather than autocrine down-regulation by PDGF-AA.     

Selection and characterization of sethoxydim- tolerant maize tissue cultures

`Black Mexican Sweet' (BMS) maize (Zea mays L.) tissue cultures were selected for tolerance to sethoxydim. Sethoxydim, a cyclohexanedione, and haloxyfop, an aryloxyphenoxypropionate, exert herbicidal activity on most monocots including maize by inhibiting acetyl-coenzyme A carboxylase (ACCase). Selected line B10S grew on medium containing 10 micromolar sethoxydim. Lines B50S and B100S were subsequent selections from B10S that grew on medium containing 50 and 100 micromolar sethoxydim, respectively. Growth rates of BMS, B10S, B50S, and B100S were similar in the absence of herbicide. Herbicide concentrations reducing growth by 50% were 0.6, 4.5, 35, and 26 micromolar sethoxydim and 0.06, 0.5, 5.4, and 1.8 micromolar haloxyfop for BMS, B10S, B50S, and B100S, respectively. Sethoxydim and haloxyfop concentrations that inhibited ACCase by 50% were similar for BMS, B10S, B50S, and B100S. However, ACCase activities were 6.01, 10.7, 16.1, and 11.4 nmol HCO₃⁻ incorporated per milligram of protein per minute in extracts of BMS, B10S, B50S, and B100S, respectively, suggesting that increased wild-type ACCase activity conferred herbicide tolerance. Incorporation of [¹⁴C]acetate into the nonpolar lipid fraction was higher for B50S than for BMS in the absence of sethoxydim providing further evidence for an increase in ACCase activity in the selected line. In the presence of 5 micromolar sethoxydim, [¹⁴C]acetate incorporation by B50S was similar to that for untreated BMS. The levels of a biotin-containing polypeptide (about 220,000 molecular weight), presumably the ACCase subunit, were increased in the tissue cultures that exhibited elevated ACCase activity indicating overproduction of the ACCase enzyme.     

Purification and Characterization of Acetyl-Coenzyme A Carboxylase from Diclofop-Resistant and -Susceptible Lolium multiflorum

Acetyl-coenzyme A carboxylase (ACCase) was purified >100-fold (specific activity 3.5 units mg-1) from leaf tissue of diclofopresistant and -susceptible biotypes of Lolium multiflorum. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified fractions from both biotypes contained a single 206-kD biotinylated polypeptide. The molecular mass of the native enzyme from both biotypes was approximately 520 kD. In some cases the native dimer from both biotypes dissociated during gel filtration to form a subunit of approximately 224 kD. The inclusion of 5% (w/v) polyethylene glycol 3350 (PEG) in the elution buffer prevented this dissociation. Steady-state substrate kinetics were analyzed in both the presence and absence of 5% PEG. For ACCase from both biotypes, addition of PEG increased the velocity 22% and decreased the apparent Km values for acetyl-coenzyme A (acetyl-CoA), but increased the Km values for bicarbonate and ATP. In the presence of PEG, the Km values for bicarbonate and ATP were approximately 35% higher for the enzyme from the susceptible biotype compared with the resistant enzyme. In the absence of PEG, no differences in apparent Km values were observed for the enzymes from the two biotypes. Inhibition constants (Ki app) were determined for CoA, malonyl-CoA, and diclofop. CoA was an S-hyperbolic (slope replots)-I-hyperbolic (intercept replots) noncompetitive inhibitor with respect to acetyl-CoA, with Ki app values of 711 and 795 [mu]M for enzymes from the resistant and susceptible biotypes, respectively. Malonyl-CoA competitively inhibited both enzymes (versus acetyl-CoA) with Ki app values of 140 and 104 [mu]M for ACCase from resistant and susceptible biotypes, respectively. Diclofop was a linear noncompetitive inhibitor of ACCase from the susceptible biotype and a nonlinear, or S-hyperbolic-I-hyperbolic, noncompetitive inhibitor of ACCase from the resistant biotype. For ACCase from the susceptible biotype the slope (Kis) and intercept (Kii) inhibition constants for diclofop versus acetyl-CoA were 0.08 and 0.44 [mu]M, respectively. ACCase from the resistant biotype had a Ki app value of 6.5 [mu]M. At a subsaturating acetyl-CoA concentration of 50 [mu]M, the Hill coefficients for diclofop binding were 0.61 and 1.2 for ACCase from the resistant and susceptible biotypes, respectively. The Hill coefficients for diclofop binding and the inhibitor replots suggest that the resistant form of ACCase exhibits negative cooperativity in binding diclofop. However, the possibility that the nonlinear inhibition of ACCase activity by diclofop in the enzyme fraction isolated from the resistant biotype is due to the presence of both resistant and susceptible forms of ACCase cannot be excluded.     

Characterization of Maize Acetyl-Coenzyme A Carboxylase

Maize (Zea mays L.) leaf acetyl-CoA carboxylase (ACCase) was purified about 500-fold by ammonium sulfate fractionation and gel filtration and blue Sepharose affinity and anion-exchange chromatography. Most ACCase activity (85%) recovered from the anion-exchange column was found in a highly purified fraction (specific activity 5.5 [mu]mol acid-stable product min-1 mg-1) that consisted primarily of a single 227-kD biotinylated polypeptide. The fraction represented 29% of the original activity and was designated ACCase I. A second partially purified ACCase activity (ACCase II) eluted earlier during anion-exchange chromatography, contained a single biotinylated polypeptide of 219 kD, was poorly recognized by antiserum raised against the ACCase I polypeptide, and was less inhibited by the herbicides haloxyfop or sethoxydim than was ACCase I. ACCase I and II both utilized propionyl-CoA as substrate about 50% as effectively as acetyl-CoA, and neither utilized methylcrotonyl-CoA. Immunoprecipitation with antiserum and protein blotting of crude extracts of leaf, embryo, and endosperm tissue and suspension cells indicated that most ACCase activity in these tissues was immunologically similar and consisted of ACCase I. Only leaves contained significant amounts of the ACCase II polypeptide; however, no ACCase II polypeptide was found in isolated mesophyll chloroplasts. The ACCase I and II polypeptides appear to be subunits of distinct ACCase isoforms.     

Expression of the Acc1 Gene-Encoded Acetyl-Coenzyme A Carboxylase in Developing Maize (Zea mays L.) Kernels

A mutation (Acc1-S2) in the structural gene for maize (Zea mays L.) acetyl-coenzyme A carboxylase (ACCase) that significantly reduces sethoxydim inhibition of leaf ACCase activity was used to investigate the gene-enzyme relationship regulating ACCase activity during oil deposition in developing kernels. Mutant embryo and endosperm ACCase activities were more than 600-fold less sensitive to sethoxydim inhibition than ACCase in wild-type kernel tissues. Moreover, in vitro cultured mutant kernels developed normally in the presence of sethoxydim concentrations that inhibited wild-type kernel development. The results indicate that the Acc1-encoded ACCase accounts for the majority of ACCase activity in developing maize kernels, suggesting that Acc1-encoded ACCase functions not only during membrane biogenesis in leaves but is also the predominant form of ACCase involved in storage lipid biosynthesis in maize embryos.     

Structure of the leech protein saratin and characterization of its binding to collagen

The leech protein Saratin from Hirudo medicinalis prevents thrombocyte aggregation by interfering with the first binding step of the thrombocytes to collagen by binding to collagen. We solved the three-dimensional structure of the leech protein Saratin in solution and identified its collagen binding site by NMR titration experiments. The NMR structure of Saratin consists of one α-helix and a five-stranded β-sheet arranged in the topology ββαβββ. The C-terminal region, of about 20 amino acids in length, adopts no regular structure. NMR titration experiments with collagen peptides show that the collagen interaction of Saratin takes place in a kind of notch that is formed by the end of the α-helix and the β-sheet. NMR data-driven docking experiments to collagen model peptides were used to elucidate the putative binding mode of Saratin and collagen. Mainly, parts of the first and the end of the fifth β-strand, the loop connecting the α-helix and the third β-strand, and a short part of the loop connecting the fourth and fifth β-strand participate in binding.     

Large BRCA1 and BRCA2 genomic rearrangements in Polish high-risk breast and ovarian cancer families

BRCA1 and BRCA2 are two major genes associated with familial breast and ovarian cancer susceptibility. In Poland standard BRCA gene test is usually limited to Polish founder BRCA1 mutations: 5382insC, C61G and 4153delA. To date, just a few single large genomic rearrangements (LGRs) of BRCA1 gene have been reported in Poland. Here we report the first comprehensive analysis of large mutations in BRCA1 and BRCA2 genes in this country. We screened LGRs in BRCA1 and BRCA2 genes by multiplex ligation-dependent probe amplification in 200 unrelated patients with strong family history of breast/ovarian cancers and negative for BRCA1 Polish founder mutations. We identified three different LGRs in BRCA1 gene: exons 13-19 deletion, exon 17 deletion and exon 22 deletion. No LGR was detected in BRCA2 genes. Overall, large rearrangements accounted for 3.7 % of all BRCA1 mutation positive families in our population and 1.5 % in high-risk families negative for Polish founder mutation.