Mouse N-acetyltransferase type 2 , the homologue of human N-acetyltransferase type 1 There is increasing evidence that human arylamine N-acetyltransferase type 1 -LRB- NAT1 , EC 2.3.1.5 -RRB- , although first identified as a homologue of a drug-metabolising enzyme , appears to be a marker in human oestrogen receptor positive breast cancer . Mouse Nat2 is the mouse equivalent of human NAT1 . The development of mouse models of breast cancer is important , and it is essential to explore the biological role of mouse Nat2 . We have therefore produced mouse Nat2 as a recombinant protein and have investigated its substrate specificity profile in comparison with human NAT1 . In addition , we have tested the effects of inhibitors on mouse Nat2 , including compounds which are endogenous and exogenous steroids . We show that tamoxifen , genistein and diethylstilbestrol inhibit mouse Nat2 . The steroid analogue , bisphenol A , also inhibits mouse Nat2 enzymic activity and is shown by NMR spectroscopy , through shifts in proton peaks , to bind close to the active site . A three-dimensional structure for human NAT1 has recently been released , and we have used this crystal structure to generate a model of the mouse Nat2 structure . We propose that a conformational change in the structure is required in order for ligands to bind to the active site of the protein . 1 Introduction Arylamine N-acetyltransferases -LRB- NATs , EC 2.3.1.5 -RRB- have traditionally been identified as drug-metabolising enzymes responsible for the metabolism of arylamines , arylhydroxylamines and arylhydrazines . NATs were first identified in man and a range of eukaryotes and have had an important part to play in the early years of identification of pharmacogenetic variation in response to drug treatment -LSB- 1 -RSB- . The hydrazine drug isoniazid is polymorphically N-acetylated in humans to its therapeutically inactive form by the human isoenzyme now known as human NAT2 . There are two human NAT isoenzymes : human NAT2 , which metabolises isoniazid , and human NAT1 , which does not metabolise isoniazid or other arylhydrazines -LSB- 2 -RSB- , but does catalyse the acetylation of a distinct but overlapping series of arylamines -LSB- 2 -RSB- . The pattern of expression of the two human NAT genes also differs . As a result of genome-wide microarray -LSB- 3 -- 12 -RSB- and proteomic studies -LSB- 13 -RSB- , it is clear that human NAT1 is highly expressed in oestrogen receptor positive breast cancer -LSB- 14 -RSB- . There is also evidence that the level of expression of human NAT1 affects the growth of cultured breast cancer cells -LSB- 13 -RSB- . The role of the human NAT1 enzyme in breast cancer has not been extensively explored , although it has previously been demonstrated that the anti-oestrogen compound tamoxifen is an inhibitor of human NAT1 -LSB- 15 -- 17 -RSB- . It has also been demonstrated that both human NAT1 and the oestrogen receptor are down-regulated in tissues in which p53 is mutated -LSB- 5 -RSB- . It appears that human NAT1 is a marker for oestrogen receptor positive breast cancer , although information is still accumulating on the relationship of this marker to others , including the oestrogen receptor itself -LSB- 14 -RSB- . However , if the human NAT1 enzyme is to be considered as a target for breast cancer therapy , it is essential that there is a suitable in vivo animal model for testing -LSB- 18,19 -RSB- . Mice represent the most convenient animal models of disease . At present , the status of mouse Nat2 , the equivalent of human NAT1 , in a model of breast cancer has not been explored . We have previously demonstrated that mouse Nat2 is expressed in the epithelial cells lining the mammary ducts -LSB- 20 -RSB- . This is the same location as the human NAT1 enzyme in normal breast tissue -LSB- 13,21 -RSB- . In order to provide a firm foundation for establishing such a model , we have generated pure recombinant mouse Nat2 and investigated its activity with a wide range of substrates and the effects of a range of potential inhibitors , including endogenous and exogenous steroids . 2 Materials and methods 2.1 Chemicals All chemicals were purchased from Sigma -- Aldrich and all molecular biology reagents were purchased from Promega , unless otherwise stated . 2.2 Cloning and expression studies of mouse Nat2 The mouse Nat2 open-reading frame -LSB- 22 -RSB- was sub-cloned into pET28b -LRB- + -RRB- -LRB- Novagen -RRB- using the compatible restriction enzyme sites , NdeI and EcoRI . This allowed the production of recombinant mouse NAT protein with an N-terminal His-tag , for ease of downstream purification . The pET28b -LRB- + -RRB- plasmid containing mouse Nat2 was transformed into Escherichia coli strain JM109 and further transformed into Rosetta -LRB- DE3 -RRB- pLysS -LRB- Novagen -RRB- after confirming the correct insert sequence -LRB- DNA Sequencing Facility , Biochemistry , University of Oxford -RRB- . The positive transformant was grown in LB to mid-log phase at 37 °C with shaking -LRB- 180 rpm -RRB- and stored at − 80 °C with 10 % -LRB- v/v -RRB- glycerol . Thawed glycerol stock -LRB- 100 μL -RRB- was used to inoculate fresh LB media -LRB- 100 mL -RRB- supplemented with kanamycin -LRB- 30 μg/mL -RRB- and chloramphenicol -LRB- 34 μg/mL -RRB- and the culture was incubated at 37 °C for 16 h with shaking -LRB- 180 rpm -RRB- . The starter culture was then diluted 50-fold into fresh LB media -LRB- 2 L -RRB- supplemented with kanamycin -LRB- 30 μg/mL -RRB- , which was then incubated at 27 °C with shaking -LRB- 180 rpm -RRB- . When the absorbance at 600 nm reached 0.7 , the expression of mouse Nat2 was induced by addition of isopropyl-β-d-thiogalactopyranoside -LRB- IPTG -RRB- to a final concentration of 0.1 mM . The culture was grown for a further 16 h and the cells were harvested by centrifugation -LRB- 6000 × g , 4 °C , 20 min -RRB- . The cell pellet was resuspended in 20 mL lysis buffer -LRB- 300 mM NaCl , 20 mM Tris -- HCl -LRB- pH 8.0 -RRB- containing 1 × EDTA-free Complete Protease Inhibitor -LRB- Roche -RRB- -RRB- and then stored at − 80 °C . For expression of 15N-labelled mouse Nat2 , the starter culture was diluted 50-fold in 15N-labelled Standard-E . coli-OD2 N medium -LRB- Silantes -RRB- supplemented with kanamycin -LRB- 30 μg/mL -RRB- and grown at 37 °C until the absorbance at 600 nm reached approximately 0.6 . The cells were further grown at 27 °C until the absorbance reached 0.7 , when the expression was induced with 0.1 mM IPTG . After induction for 17 h , the cells were harvested and stored as described for unlabelled mouse Nat2 . The resuspended frozen cells were thawed at 37 °C and sonicated on ice -LRB- 25 cycles of 30 s on , 45 s off at 10 μm -RRB- . The soluble lysate was isolated from the cell debris by centrifugation -LRB- 12,000 × g , 4 °C , 20 min -RRB- and incubated -LRB- 4 °C , 5 min -RRB- with 5 mL of Ni-NTA resin -LRB- Qiagen -RRB- . The resin was sequentially washed with lysis buffer containing increasing imidazole concentrations in a step gradient at 4 °C -LRB- 2 washes each of 10 mL of 0 mM , 10 mM , 20 mM , 50 mM and 100 mM imidazole -RRB- . Fractions containing hexa-histidine tagged Nat2 -LRB- His-Nat2 -RRB- , were pooled and thrombin -LRB- 5 U/mg NAT -RRB- was added to remove the His-tag . After 16 h incubation at 4 °C , the protein sample was dialysed against 20 mM Tris -- HCl -LRB- pH 8.0 -RRB- , 1 mM dithiothreitol -LRB- DTT -RRB- , 1 mM EDTA buffer , and concentrated to 1 mg/mL by centrifugation in a 15 mL concentrator -LRB- Amicon -RRB- after addition of 5 % -LRB- v/v -RRB- glycerol . Protein samples in 100 μL aliquots were frozen in liquid nitrogen and stored at − 80 °C . Recombinant hamster Nat2 was expressed and purified as previously described -LSB- 23 -RSB- . 15N-labelled hamster Nat2 was grown and expressed as described for 15N-labelled mouse Nat2 and purified by the same method as for the unlabelled hamster Nat2 . The expression levels and activities of soluble Nat2 were monitored by visualizing the levels of recombinant protein expression on 12 % acrylamide Tris -- Glycine SDS-PAGE as well as by measuring the rate of acetylation of 4-aminobenzoic acid -LRB- PABA -RRB- as a substrate -LRB- see method below -RRB- -LSB- 24 -RSB- . 2.3 Enzymic assays 2.3.1 Acetylation of arylamines The rates of arylamine acetylation by NATs were determined colorimetrically as previously described -LSB- 25 -RSB- , but with minor modifications . Each assay contained the enzyme , arylamine substrate -LRB- see each figure legends for concentrations -RRB- and AcCoA -LRB- 400 μM -RRB- in a total volume of 100 μL in assay buffer -LRB- 20 mM Tris -- HCl -LRB- pH 8.0 -RRB- , 1 mM DTT -RRB- . Initially , the enzyme and substrate mixtures were pre-incubated at 25 °C for 5 min and AcCoA was added to start the reaction . The reaction was quenched using 20 % -LRB- w/v -RRB- trichloroacetic acid -LRB- TCA -RRB- at different time intervals . The stopped reaction was centrifuged -LRB- 16,000 × g rpm , 10 min -RRB- to pellet the precipitated proteins . The stopped reaction mixture -LRB- 200 μL -RRB- was added to 800 μL of 5 % -LRB- w/v -RRB- 4 - -LRB- N,N-dimethylamino -RRB- benzaldehyde -LRB- DMAB -RRB- in 9:1 acetonitrile : water to develop the colour . The absorption at 450 nm was measured with a Hitachi U-2001 UV -- vis spectrophotometer , with the amount of residual substrate in the reaction was determined by comparison with a standard curve . 2.3.2 Hydrolysis of AcCoA -LRB- free CoA production -RRB- The rate of production of the free thiol Coenzyme A by NAT , in the presence of a range of known NAT acetyl-acceptor substrates , was determined by using Ellman 's reagent , 5,5 ′ - dithio-bis -LRB- 2-nitrobenzoic acid -RRB- -LRB- DTNB -RRB- as previously described -LSB- 2,26 -RSB- . The substrate -LRB- 500 μM -RRB- and purified recombinant NAT were pre-incubated -LRB- 25 °C , 5 min -RRB- in 96-well flat-bottomed polystyrene plates -LRB- Costar ® , Corning Inc. -RRB- in 20 mM Tris -- HCl -LRB- pH 8.0 -RRB- . AcCoA -LRB- 400 μM -RRB- was added to start the reaction in a final volume of 100 μL . The reaction was quenched with the addition of 25 μL of guanidine -- HCl solution -LRB- 6.4 M guanidine -- HCl , 0.1 M Tris -- HCl -LRB- pH 7.3 -RRB- -RRB- containing 5 mM DTNB . The absorbance at 405 nm was measured on a plate-reader -LRB- Sunrise , Tecan -RRB- . Assay buffer was used to replace substrate , AcCoA or NAT for control reactions . The amount of CoA produced in the assay was determined in comparison with a CoA standard curve . Substrate abbreviations used for the substrate selectivity assays are as follows : aniline -LRB- ANL -RRB- , 4-aminobenzoic acid -LRB- PABA -RRB- , 4-aminosalicylic acid -LRB- 4AS -RRB- , 5-aminosalicylic acid -LRB- 5AS -RRB- , 4-chloroaniline -LRB- CLA -RRB- , 4-bromoaniline -LRB- BRA -RRB- , 4-iodoaniline -LRB- IOA -RRB- , 4-methoxyanline -LRB- ANS -RRB- , 4-ethoxyaniline -LRB- EOA -RRB- , 4-butoxyanline -LRB- BOA -RRB- , 4-hexyloxyaniline -LRB- HOA -RRB- , 4-phenoxyaniline -LRB- POA -RRB- , 4-aminoveratrole -LRB- 4AV -RRB- , 2-aminofluorene -LRB- 2AF -RRB- , 4-aminobenzoyl-l-glutamate -LRB- pABGlu -RRB- , sulfamethazine -LRB- SMZ -RRB- , procainamide -LRB- PRO -RRB- , 4-aminopyridine -LRB- APY -RRB- , isoniazid -LRB- INH -RRB- , hydralazine -LRB- HDZ -RRB- and phenylhydrazine -LRB- PHZ -RRB- . The stock concentration of substrate was 100 mM dissolved in DMSO , such that the final concentration of DMSO was less than 5 % in the assays . DMSO only controls were carried out . 2.4 NMR spectroscopy The 2D 1H-15N HSQC spectrum of mouse Nat2 was collected using a 750 MHz NMR spectrometer -LRB- Department of Biochemistry , University of Oxford -RRB- . The spectrum was collected at 20 °C for a sample containing 30 mg/mL of uniformly 15N-labelled mouse Nat2 in 95 % H2O/5 % D2O 10 mM Tris buffer at pH 7.0 . Spectral widths of 12,500 and 2500 Hz were used in F2 and F1 , respectively . The dataset contained 1024 and 128 complex points in t2 and t1 , respectively . 1D 1H NMR spectra for hamster and mouse Nat2 were collected at 20 °C using a jump-return sequence . This sequence enabled the observation of HN peaks which exchange to a significant extent with H2O . Spectra were collected using a 600 MHz spectrometer using a sweep width of 12,500 Hz . Unlabelled hamster and mouse Nat2 were used at a concentration of 10 mg/mL in the buffer described above . The titration of mouse Nat2 with bisphenol A was carried out by stepwise addition of small volumes -LRB- 5 -- 20 μL -RRB- of a concentrated solution of bisphenol A -LRB- 100 mM in deuterated DMSO -RRB- to the mouse Nat2 solution in the NMR tube . The highest concentration of DMSO in the sample used was 2.8 % . DMSO alone had no effect on the NMR spectrum of mouse Nat2 using concentrations up to 5 % . 3 Results 3.1 Cloning and expression of mouse Nat2 The mouse Nat2 was cloned into E. coli expression vector pET28b -LRB- + -RRB- and expressed in E. coli Rosetta -LRB- DE3 -RRB- pLysS with an N-terminal hexa-histidine tag . The recombinant mouse His-Nat2 was purified using immobilised metal affinity chromatography , using a Ni-NTA column -LRB- Novagen -RRB- , and eluted -LRB- Fig. 1a -RRB- . Pure Nat2 predominantly eluted in the 50 mM and 100 mM imidazole washes . The hexa-histidine tag was readily removed by thrombin digestion -LRB- Fig. 1b -RRB- . The purified recombinant Nat2 was active , readily catalysing the acetylation of PABA , a well-studied arylamine substrate of mouse Nat2 -LSB- 27 -RSB- -LRB- Table 1 -RRB- . The enzyme was relatively stable , and the enzymic activity was maintained -LRB- > 85 % -RRB- after incubation at temperatures ranging between 4 °C and 25 °C over a 72 h period . 3.2 Substrate specificity of mouse Nat2 To further characterise the purified recombinant mouse Nat2 , a panel of known NAT substrates -LRB- 20 compounds -RRB- was tested to determine the substrate selectivity of recombinant mouse Nat2 . In order to allow direct comparison with substrate selectivity profile for human NAT1 and NAT2 , the assay for mouse Nat2 was performed under the same conditions as previously described -LSB- 2 -RSB- . Mouse Nat2 shows high specific activities with a broad range of arylamines , including PABA , 5AS , 4AS and 2AF -LRB- Fig. 2 and Supplementary Table 1 -RRB- . In contrast , mouse Nat2 shows low specific activities against arylhydrazine substrates such as HDZ , INH , and PHZ , and against arylamine drugs procainamide and sulfamethazine -LRB- Fig. 2 -RRB- . This overall trend is in agreement with the substrate selectivity observed in previous studies with impure recombinant mouse Nat2 using a subset of these compounds -LSB- 22,27,28 -RSB- , although it is much less than the activity observed towards these substrates with human NAT2 -LRB- Fig. 2 -RRB- . There was low , yet measurable activity using arylhydrazine substrates with purified mouse Nat2 , which has not been observed in previous studies using recombinant mouse Nat2 expressed in a transient expression system -LSB- 28 -RSB- . Pure recombinant mouse Nat2 also readily acetylates 2-aminofluorene , the substrate which has been used to identify mouse strains carrying an active form of mouse Nat2 -- the fast acetylating C57Bl/6 strain , rather than the inactive form as is found in the slow acetylating A/J strain which is unstable and likely to be subject to rapid degradation -LSB- 29 -RSB- . The specific activity pattern of human NAT1 -LSB- 2 -RSB- overlaps with the mouse Nat2 profile -LRB- Fig. 2 -RRB- , reconfirming that mouse Nat2 is very similar to human NAT1 in substrate specificity . Mouse Nat2 shows differences from the activity profile of human NAT1 , however . It appears that mouse Nat2 generally has higher inherent specific activity than human NAT1 and also acetylates arylhydrazines , when the acetylation of these compounds by human NAT1 is not detected under the same conditions -LSB- 2 -RSB- . 3.3 Mouse Nat2 inhibition by steroidogenic compounds It had been demonstrated , with extracts of tissues and cells now known to express the human NAT1 gene , that tamoxifen is an inhibitor of human NAT1 -LSB- 15 -- 17 -RSB- . In view of the relationship between the expression of human NAT1 and oestrogen receptor positivity in breast cancer -LSB- 14 -RSB- , we wished to investigate the effects of steroidogenic compounds and xenobiotic oestrogenic compounds , including tamoxifen , on the activity of mouse Nat2 . From the inhibition study using a broad range of steroidogenic compounds , it has been demonstrated that mouse Nat2 is inhibited selectively by oestrogenic compounds , with 17-hydroxy-β-estradiol identified as the most potent inhibitor -LRB- Table 2 and Supplementary Fig. 1 -RRB- . This was substantiated by the inhibition of mouse Nat2 activity by xenobiotic oestrogens , such as tamoxifen , and the IC50 values for these inhibitors are shown in Table 3 . The phytoestrogens genistein , alpha-zearalenol and the synthetic oestrogen diethylstilbestrol show similar potency of mouse Nat2 inhibition to 17-hydroxy-β-estradiol , tamoxifen and 4-hydroxy tamoxifen . Bisphenol A was also shown to be an inhibitor of mouse Nat2 , albeit with lower potency than some of the other compounds tested . One key practical advantage of using bisphenol A as an inhibitor is the improved solubility of this compound relative to the other phytoestrogens and steroids . 3.4 Structure of mouse Nat2 Until recently , there has been no structural information on eukaryotic NAT enzymes . However , recently the three-dimensional structure of a human NAT1 mutant -LRB- Phe/Ser substitution at position 125 -RRB- was determined by X-ray crystallography -LRB- PDB accession code : 2IJA -LSB- 30 -RSB- -RRB- . A non-mutated human NAT1 crystal structure has also been deposited in which the active-site cysteine residue was modified to S - -LRB- 2-anilino-2-oxoethyl -RRB- - cysteine -LRB- PDB accession code : 2PQT -RRB- . It is considered likely that both the mutation of residue 125 and the modification of the active-site cysteine residue stabilise the structure of the human enzyme , because thousands of attempts to generate a crystal from the native human NAT1 protein have been unsuccessful -LRB- A. Kawamura , unpublished results -RRB- . The structure of human NAT1 is illustrated in comparison with the NAT from M. smegmatis -LRB- Fig. 3 -RRB- . The eukaryotic proteins contain a loop between the second and third domains . This inter-domain loop is not present in their prokaryotic counterparts -LSB- 31 -RSB- , as highlighted in Fig. 3 . It is interesting to note that in the structure of the human NAT1 enzyme , this inter-domain loop is folded back over the active site . Likewise , the C-terminus is shown to be folded over the active-site cleft . These observations indicate that a conformational change in the protein may be required to allow the substrates to access the active-site cysteine residue . However , a structure of human NAT2 with coenzyme A bound has also been deposited -LRB- PDB accession code : 2PFR -RRB- , and the position of the coenzyme A is such that the C-terminus and inter-domain loops regions are not significantly changed . This is in contrast to the recently reported structure of Mycobacterium marinum NAT in complex with coenzyme A -LSB- 32 -RSB- . In this bacterial NAT , the C-terminus is shorter than in the eukaryotic structures , and there is no inter-domain loop , and the ligand is found in the space where these regions are located in the eukaryotic structure . We have generated a structural model of the mouse Nat2 protein , whose amino acid sequence is over 80 % identical to human NAT1 , by using the program Modeller 8v2 -LSB- 33 -RSB- . Fig. 4 shows the structural model of mouse Nat2 and a comparison of the mouse Nat2 and human NAT1 protein structures . The two structures share 1191 equivalent atoms , over which the root mean squared deviation is 0.75 Å . In mouse Nat2 , as in human NAT1 , the inter-domain loop and the C-terminus occlude the active site . Despite the evidence for coenzyme A binding to human NAT2 without major conformational rearrangement of the C-terminus and inter-domain loop regions , it has not been possible to perform ligand docking into the mouse Nat2 or human NAT1 structures without disturbing these regions of the protein structure . Therefore , it remains possible that the inter-domain loop regions and C-termini of the eukaryotic NAT proteins are conformationally flexible , in order to accommodate substrate or inhibitor binding . 3.5 NMR studies NMR studies are ideal for investigating protein interactions with substrates and inhibitors in solution . Therefore , we have generated mouse Nat2 uniformly labelled with 15N to allow 2D NMR investigations of the protein . The 2D 1H-15N HSQC spectrum of mouse Nat2 is shown in Fig. 5 ; this spectrum contains a peak for each backbone amide -LRB- 1HN-15N -RRB- and additional peaks from the side chains of Asn , Gln and Trp . The HSQC spectrum is well resolved , as expected for a compact globular structure , and some cross peaks are relatively sharp for a 30 kDa protein , this may indicate some flexibility between the domains of mouse Nat2 or the presence of mobile loops . The HSQC spectrum of mouse Nat2 has been compared with the corresponding spectrum from hamster Nat2 for which a full assignment is available -LSB- 34 -RSB- . The pattern of peaks in the HSQC spectra is extremely similar for the two homologous proteins ; this allows some peaks observed in the mouse Nat2 spectrum to be assigned on the basis of the hamster Nat2 spectrum . The downfield region of the 1H 1D spectra , collected with a jump-return sequence , of mouse and hamster Nat2 also show a similar pattern of peaks -LRB- Fig. 6A and B -RRB- . The four peaks are compared in Table 4 , together with their assignments . Two of the peaks -LRB- 2 and 3 -RRB- have been assigned previously for hamster Nat2 and can be assigned in the mouse spectrum by homology ; these correspond to strongly hydrogen bonded tryptophan indole groups : the tryptophan associated with the P-loop -LRB- Trp132 , see Fig. 7 -RRB- and Trp67 , which is located adjacent to the active-site Cys68 , and appears to have a structural role . The strong hydrogen bonds are likely to be responsible for the large downfield shifts of these tryptophan peaks compared to random coil values -LRB- ∼ 9.5 ppm -RRB- . The two remaining peaks -LRB- 1 and 4 -RRB- are assigned to the imidazole 1HN of histidine on the basis of 15N decoupling experiments ; these peaks have not been assigned previously for hamster Nat2 . The side chain 1HN groups of histidine are usually not observed by NMR , particularly at pH 7 , due to their rapid exchange with solvent protons . The observation of these peaks indicates that the histidine residues are probably buried within the protein and that the side chain 1HN are involved in hydrogen bonds . Analysis of the homology model of mouse Nat2 suggests that at least one of these peaks might arise from the active site histidine , His107 . The homology model of mouse Nat 2 indicates that the side chain of His107 is involved in two hydrogen bonds in the active site and is the least accessible to solvent . In addition , recent enzymological studies with hamster Nat2 have confirmed that the active-site cysteine and histidine residues exist as a thiolate-imidazolium ion pair -LSB- 35 -RSB- , in which the histidine has two tightly bound and H-bonded protons . 3.6 Interaction of mouse Nat2 with exogenous steroid inhibitors In order to probe the molecular interactions of mouse Nat2 and the exogenous steroid inhibitors , we have investigated the effect of bisphenol A on the 1D 1H NMR spectrum of mouse Nat2 -LRB- Figs. 8 and 9 -RRB- . The observation of changes in chemical shift for particular amino acid residues which result from the addition of a ligand are usually interpreted as an indication that these residues are located in close proximity to the ligand-binding site . When bisphenol A was titrated into a solution of mouse Nat2 , the two downfield histidine peaks -LRB- 1 and 4 , Table 4 -RRB- were observed to shift by more than 0.05 ppm -LRB- Fig. 9 -RRB- ; peak 1 shifts downfield by ∼ 0.12 ppm and peak 4 shifts upfield by ∼ 0.08 ppm in the presence of 8 equivalents of bisphenol A. Additional small shifts were also observed for the side chain HN of Trp67 and Trp132 -LRB- Fig. 9 -RRB- . The observation of progressive changes in chemical shift upon addition of bisphenol A indicates fast exchange on the NMR timescale ; this would be expected given the relatively weak affinity of mouse Nat2 for this ligand -LRB- IC50 = 290 ± 10 μM -RRB- . We have postulated above that at least one of the histidine peaks arises from the active-site histidine , His107 ; therefore , these results show that binding of bisphenol A is likely to take place in close proximity to the active site of mouse Nat2 . 4 Discussion The possibility that human NAT1 may serve as a marker for sub-dividing populations of different breast cancers is intriguing . It may also be that human NAT1 will find a role as a target for breast cancer therapies . In order to exploit these findings fully , it will be essential to have an animal model . There have been , for a long time , suggestions that human NAT1 has an endogenous role . The mouse equivalent of human NAT1 is mouse Nat2 . Genetic deletion of mouse Nat2 has not created an overt phenotype ; however , some of these studies in mice indicate that Nat2 deletion gives rise to skewed sex ratios both on mixed -LSB- 36 -RSB- and pure -LSB- 37 -RSB- genetic backgrounds , indicating a gender-dependent phenotype , discernable at the population level , that is consistent with a role in endocrine function . There is now also clear evidence that mouse Nat2 has a role in vivo in folate catabolism -LSB- 38 -RSB- . These studies , together with the data on human NAT1 and breast cancer , are intriguing and are likely to provide clues as to the endogenous role of NAT1 . The data presented increases the knowledge of mouse Nat2 in comparison with human NAT1 and reinforce the notion that mouse Nat2 is a good homologue of human NAT1 . Mouse Nat2 has a similar substrate specific activity profile to human NAT1 . The inhibition studies show that mouse Nat2 is inhibited by endogenous steroids and , like human NAT1 , is inhibited by tamoxifen . Bisphenol A , which is used for making polycarbonates -LSB- 39 -RSB- , has been reported to activate the oestrogen receptor -LSB- 40 -RSB- . Like tamoxifen , it is also an inhibitor of mouse Nat2 . We have used bisphenol A to look at the binding of an oestrogenic stimulatory compound to mouse Nat2 and show by NMR that bisphenol A interacts with mouse Nat2 close to the active site region . These studies could not be done directly with tamoxifen because of the high concentrations of protein -LRB- and hence ligand -RRB- required for NMR experiments under which conditions tamoxifen is insoluble . Both endogenous and xenobiotic oestrogens have been shown to modify breast cancer risk , and tamoxifen is widely used for breast cancer therapy -LRB- reviewed in -LSB- 41 -RSB- -RRB- . The effect of different synthetic or natural oestrogens on cell proliferation has been analysed directly , in breast cancer cell lines , and in gene expression studies -LSB- 41 -- 44 -RSB- . Although the dose -- response curves for different oestrogenic compounds vary with the assay system , bisphenol A is generally less active than tamoxifen , genistein or diethylstilbestrol , which reflects the order of potency of mouse Nat2 inhibition . Circulating plasma concentrations of the phytoestogen genistein have been found to be as high as 18 μM after a soy-based meal -LSB- 45 -RSB- , and intratumoral levels of tamoxifen can reach micromolar concentrations -LSB- 46 -RSB- . Whether the effects on inhibition of Nat2 activity in vitro may be physiologically relevant for breast cancer prevention or therapy will require further analysis . These studies pave the way for investigation of the role of mouse Nat2 , the human NAT1 homologue , in studies of oestrogen receptor positive breast cancer . This is particularly relevant in view of the observation that mouse Nat2 is located histologically in the mammary gland epithelial cells -LSB- 20 -RSB- . The structural studies also provide a rational basis for identification of mouse Nat2 inhibitors . We have recently screened a 5000-strong compound library using human NAT1 , human NAT2 , mouse Nat1 and mouse Nat2 ; from these studies , several compounds been have identified which are potent and specific inhibitors of human NAT1 and its mouse homologue , Nat2 . An accumulation of information on the eukaryotic NAT proteins at a structural level will allow further development of chemical tools for investigation of the role of this marker in breast cancer in humans , and its potential in animal models .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O

selective estrogen receptor modulator/serm


Control and possible applications of a novel carrot-spoilage basidiomycete , Fibulorhizoctonia psychrophila A novel cold-tolerant fungus , Fibulorhizoctonia psychrophila , was isolated from a refrigerated carrot storage facility and identified as an anamorph of Athelia , often classified in Rhizoctonia s.l. Growth of this fungus was observed between 0 and 20 °C with an optimum at 9 -- 12 °C , while incubation of mycelium grown at 15 -- 32 °C resulted in absence of growth even after the fungus was transferred back to 15 °C . Growth was inhibited in the presence of the antifungals sorbic acid or natamycin , in particular when the fungus was incubated at 18 °C . F. psychrophila produces polysaccharide degrading enzymes that , when compared to enzymes from the ascomycete fungus Aspergillus niger , retain a larger proportion of their activity at lower temperatures . This indicates that F. psychrophila could be used as a source for novel industrial enzymes that are active at 4 -- 15 °C . Introduction Fungi occupy every natural environment on earth as well as man-made indoor environments . In fact , some fungi have ecological requirements and amplitudes that are ideally suited to some artificial -LRB- human made -RRB- environments . Some of these fungi create large problems with respect to human health -LRB- e.g. Aspergillus , Candida -RRB- or to food quality -LRB- e.g. Penicillium -RRB- . Among standard methodologies to prevent food spoilage are the use of preservatives and refrigeration of food . The latter method , however , is not effective when cold-tolerant spoilage fungi are present . Cold-tolerant fungi are commonly found in nature and comprise species of different branches of the fungal kingdom , such as Geomyces , Leucosporidium -LRB- Panikov and Sivoza 2007 -RRB- , Cystofilobasidium and Mrakia -LRB- Nagakawa et al. 2004 -RRB- . Cell walls form the majority of the plant biomass and consist mainly of polysaccharides . As fungi are not able to import polymeric compounds into their cells , they rely on extracellular enzyme systems for the degradation of polysaccharides into monosaccharides and short oligosaccharides . Hydrolytic enzymes acting on polysaccharides are commonly produced by fungi . These enzymes have many applications in the food and feed , paper and pulp , textile , and pharmaceutical industries and have therefore been the subject of many studies -LRB- de Vries and Visser 2001 ; de Vries 2003 -RRB- . Most of these studies and applications involve enzymes of saprophytic ascomycete fungi -LRB- mainly species from Aspergillus and Trichoderma -RRB- that have optimal growth temperatures between 30 and 37 °C . Consequently , most of these enzymes have optimum activities between 30 and 50 °C and their activity rapidly decrease at lower temperatures -LRB- de Vries and Visser 2001 -RRB- . Efficient use of these enzymes in industrial applications is therefore often only possible by incubation above 30 °C . This not only increases production costs due to the energy required to reach this temperature , but can also cause spoilage problems , especially with respect to food and feed applications . One of the most commonly known cold-tolerant spoilage fungi is the basidiomycete Rhizoctonia carotae -LRB- Adams and Kropp 1996 ; Jensen 1969 ; Jones and Aldwinckle 1990 ; Punja 1987 -RRB- , which is found in storage facilities of several vegetables such as carrot . Although the optimum growth-temperature of this fungus is between 15 and 20 °C -LRB- Punja 1987 -RRB- , this fungus causes crater rot characterized by sunken lesions and abundant mycelial growth on carrots stored at temperatures between 1 and 4 °C . The anamorph genus Fibulorhizoctonia -LRB- Adams and Kropp 1996 -RRB- -LRB- as Fibularhizoctonia -RRB- was proposed to accommodate Rhizoctonia carotae and R. centrifuga Lév -LRB- Rader 1948 -RRB- . The teleomorphs of these species belong to Athelia Pers. , while those of the Rhizoctonia solani-complex belong to Thanatephorus Donk , currently placed in a different order -LRB- Hibbett et al. 2007 -RRB- . The teleomorph of F. carotae was identified as Athelia arachnoidea -LRB- Berk . -RRB- Jülich -LRB- Adams and Kropp 1996 -RRB- . However , there is some controversion considering the scope of this species , as the name has been used for litter decomposing specimens and lichen parasites . Arvidsson -LRB- 1976 -RRB- concluded that the use of the name A. arachnoidea should be confined to the lichen parasite , while the material of Adams and Kropp -LRB- 1996 -RRB- contained only litter decomposers . Other basidiomycete species reported from cold storage are : Corticium centrifugum -LRB- Bielenin 1986 ; Stalpers and Loerakker 1984 ; Weresub and Illman 1980 -RRB- , from stored apples and pears , causing fisheye rot . Although the name C. centrifugum is connected with Athelia , the species concerned differs by having constant clamps at the septa , narrower hyphae and no sclerotia , and the production of a basidiome in culture , which is now known as Butlerelfia eustacei -LRB- Weresub and Illman 1980 -RRB- . This species is known from Europe and North America . In this paper we describe a new carrot-spoilage fungus , Fibulorhizoctonia psychrophila , which is a predominant species detected in refrigerated storage facilities for carrots in The Netherlands . We have analysed the cold-tolerance of this fungus and its sensitivity to commonly used fungicides . In addition , we have analysed F. psychrophila for production of plant polysaccharide degrading enzymes to assess its potential as a source of cold-active enzymes . Materials and methods Strains and growth conditions F. psychrophila CBS 109695 -LRB- IMI 395943 -RRB- was isolated as a mycelial sample by H.A.B. Wösten from a wooden crate containing Daucus carota in a refrigerated storage facility -LRB- 4 °C -RRB- in Bant , The Netherlands in 2002 . Athelia arachnoidea -LRB- Berkeley -RRB- Jülich CBS 418.72 was isolated from fallen leaf-litter of Populus sp. in The Netherlands . The type strain of Fibulorhizoctonia carotae -LRB- Rader -RRB- G.C. Adams & Kropp CBS 464.48 and Aspergillus niger N402 were described previously -LRB- Adams and Kropp 1996 ; Bos et al. 1988 -RRB- . F. psychrophila CBS 109695 and A. niger N402 were routinely propagated on malt extract agar and minimal medium -LRB- de Vries et al. 2004 -RRB- , respectively . For growth of F. psychrophila on carrot , potato and onion , these vegetables were ground using a coffee grinder . The vegetable pulp was used at a concentration of 10 % in 1.5 % agar plates in water . Liquid cultures of F. psychrophila and A. niger were performed in Schizophyllum commune minimal medium -LRB- Dons et al. 1979 -RRB- and Aspergillus minimal medium -LRB- de Vries et al. 2004 -RRB- , respectively , using 1 % of a crude arabinoxylan preparation obtained from wheat after extraction of starch and proteins as the substrate . Plate cultures to determine the influence of several fungicides on growth of F. psychrophila were performed on malt extract agar plates . Comparison of the optimal growth temperature for F. psychrophila , Athelia arachnoidea and F. carotae was performed on malt extract agar and cherry decoction agar at temperatures of 0 -- 27 °C with intervals of 3 °C . Powdered wood was obtained by grinding wood shavings of a storage crate in a coffee grinder until a fine powder was obtained . Of this powder , 1 g was added to 1.5 g agar and 100 ml water and autoclaved to prepare solid media . The crude arabinoxylan preparation was a gift from Latenstein -LRB- Nijmegen , The Netherlands -RRB- and is in fact a waste stream of a protein and starch extraction process from wheat . Molecular biology methods Genomic DNA of CBS 109695 was extracted using the FastDNA kit -LRB- Bio 101 Systems , Q-Biogene -RRB- . The 5.8 S gene and flanking ITS1 and ITS2 were amplified using the primers ITS1 and ITS4 -LRB- White et al. 1990 -RRB- and the sequence was deposited at genbank -LRB- Acc . Nr . EF492880 -RRB- . The sequence was compared with ITS sequences from F. carotae -LRB- U85789 -RRB- and A. arachnoidea -LRB- U85791 -RRB- . Enzyme assays F. psychrophila and Aspergillus niger were grown at 15 and 30 °C , respectively in liquid medium containing a crude arabinoxylan preparation . Culture filtrate was harvested over a nylon filter after 2 days of growth and analysed for enzyme activities at 4 , 15 and 30 °C . Enzyme activity was determined using p-nitrophenyl-β-d-galactopyranoside , p-nitrophenyl-β-d-glucopyranoside , p-nitrophenyl-α-d-galactopyranoside , and p-nitrophenyl-β-d-xylopyranoside -LRB- Sigma -RRB- as a substrate . A mixture was made consisting of 10 μl culture filtrate , 50 μl 50 mM sodium acetate -LRB- pH 5.0 -RRB- and 30 μl sterile MiliQ water . The reactions were performed in triplicate and were started by the addition of 10 μl of 0.1 % stock of the p-nitrophenyl-linked substrate and incubated for 2 h at 25 °C , unless stated otherwise . The reactions were stopped by the addition of 100 μl 0.25 M Na2CO3 and measured at 405 nm in a micro plate reader -LRB- model 550 , Bio-RAD -RRB- . The amount of free p-nitrophenol was calculated using a calibration curve . Results and discussion Taxonomy Fibulorhizoctonia psychrophila Stalpers & de Vries spec . nov. -LRB- Fig. 1 -RRB- . Fig. 1Colony morphology of Fibulorhizoctonia carotae -LRB- 464.84 -RRB- , Athelia arachnoidea -LRB- 418.72 -RRB- and Fibulorhizoctonia psychrophila -LRB- 109659 -RRB- after 2 weeks . -LRB- a -RRB- 0 °C ; -LRB- b -RRB- 6 °C ; -LRB- c -RRB- 18 °C Mycelium ad 9 °C cotoneum , albidum , ad 18 °C cremeum , avellaneum vel brunneum , velutinum vel crustosum . Liquor exsudatus brunneus . Sclerotia presentia vel absentia , irregularia , in statu maturitate subfusca a liquore exsiccata . Hyphae hyalinae vel subfuscae , -LRB- 2.5 -- -RRB- 3 -- 6 μm . Septa fibulatae vel afibulatae . Cellae sclerotiorum doliformes , tenui-tunicatae vel crasse-tunicatae , hyalinae vel fuscae . Fungus psychrophilus , ad temperaturas − 3 ° ad 21 °C crescens . Typus : CBS 109695 Colonies at − 3 to 9 °C are cottony , rather high and reaching the lid of the Petri dish , white . Margin raised , rather dense . At increasing temperatures the mycelium grows less high and becomes nearly velvety at 18 °C and nearly crustose at 21 °C . At 12 ° the centre of the colony is cream-coloured , at 15 °C becoming Light Cinnamon Drab to Avellaneous -LRB- Munsell 7.5 YR6/2 , 10YR6/2 , 10 YR6/3 -RRB- . At these temperatures exudate droplets are not present . At 18 °C the colony is low , nearly velvety , Ochraceous Tawny -LRB- 7.5 YR5/8 -RRB- caused by exudate drops and at 21 °C there is hardly any growth ; the mycelium is crustose and Cinnamon Brown to Prouts Brown -LRB- 5YR4/3 , 5YR4/4 -RRB- , and finally Mummy Brown . Sclerotia are generally not produced within 6 weeks , but have occurred in a tube ; they start as white semiglobose pustules , generally up to 1 mm diameter , aggregated , often fusing , and producing a brown exudate , which dries in , leaving a dark brown sclerotium , up to 5 mm diam , finally Mummy Brown . Marginal hyphae at − 3 to 3 °C are irregular , rather long-celled , with swellings and granular contents , thin-walled , -LRB- 2.5 -- -RRB- 3 -- 6 μm wide , swellings up to 12 μm wide . Clamps present , not abundant . Branching at various angles , sometimes a constriction is present at the base of the side branch . At 6 °C and above the hyphae are regular , thin - to firm-walled , 2.5 -- 6 -LRB- -- 7.5 -RRB- μm wide , contents hyaline to pale brownish , with abundant clamps , especially at the wider hyphae , but the narrower hyphae -LRB- 2.5 -- 3.4 -- 4 μm -RRB- generally have clampless septa . Cell wall hyaline , but irregular brownish encrustations present at some hyphae , probably dried exudate . Aerial mycelium regular , thin - to firm-walled , -LRB- 2.5 -- -RRB- 3.2 -- 6.5 μm wide , with hyaline to brownish contents . Most septa with clamps ; clamps regular , but sometimes of the medallion type . Sclerotia rarely produced , uniform , not forming a distinct cortex , consisting of thin - to thick-walled hyphae , usually consisting of slightly elongated swollen cells with granular contents , 4 -- 10 μm wide , not unlike the -LRB- thin-walled -RRB- advancing hyphae at lower temperatures.Cardinal temperatures : minimum below − 3 °C , optimum range between 9 and 12 °C , maximum 20 °C . Holotype : CBS 109695 -LRB- IMI 395943 -RRB- , also preserved dried in herb . CBS.Mycobank number : MB501325.The sterile , sclerotium producing mycelia are traditionally classified in the artificial genus Rhizoctonia , which is currently restricted to basidiomycetous fungi and comprises anamorphs of various , not closely related genera such as Tulasnella , the Ceratobasidium -- Thanatephorus complex and Athelia -LRB- Stalpers and Anderson 1996 -RRB- . These groups have been elevated to the genus level as Epulorhiza , Rhizoctonia s.str . and Fibulorhizoctonia . Although teleomorphs have not been observed in F. carotae and F. psychrophila , morphological , physiological and molecular characters indicate that F. psychrophila , F. carotae and Athelia arachnoidea are closely related and belong to Athelia , a corticioid genus , currently classified in the Atheliales -LRB- Rader 1948 -RRB- . F. carotae has been described from cold stored carrots . It differs from F. psychrophila in growing between 18 and 27 °C -LRB- Fig. 2 -RRB- , abundant production of sclerotia , and colour of the mat above 15 °C . A. arachnoidea has been described from both lichens and leaf litter . It differs from F. psychrophila in its growth at higher temperatures -LRB- Fig. 2 -RRB- , less abundant aerial mycelium , and pale colour at higher temperatures . ITS sequencing of F. psychrophila revealed 95 % identity to the ITS sequence of A. arachnoidea and A. carotae -LRB- data not shown -RRB- , indicating that F. psychrophila is very closely related to these two species . Fig. 2Growth comparison at different temperatures of Fibulorhizoctonia psychrophila , Fibulorhizoctonia carotae and Athelia arachnoidea . The means of duplicate values are represented Both morphological and molecular characters indicate without doubt , that F. psychrophila , F. carotae and Athelia arachnoidea are closely related . Temperature-tolerance and growth of F. psychrophila F. psychrophila was inoculated on malt extract agar plates and incubated between 0 and 27 °C . Growth was monitored by measuring the colony diameter . Optimal growth occurred at 9 -- 12 °C -LRB- Fig. 2 -RRB- and growth was absent above 20 °C . When plates incubated for 3 days at 20 and 25 °C were placed back at 15 °C the fungus re-initiated growth , but incubation for 3 days at 32 °C prevented growth of F. psychrophila after the plates were transferred to 15 °C -LRB- data not shown -RRB- . Optimal growth of F. carotae and A. arachnoidea was at between 18 and 21 °C -LRB- Fig. 2 -RRB- . The strong decrease in growth of F. psychrophila when the incubation temperature is raised above 15 °C , suggests an even stronger adaptation of this fungus to cold biotopes . Growth of F. psychrophila was tested on three vegetable crops that are commonly stored under refrigerated conditions -LRB- carrot , potato and onion -RRB- in comparison to agar plates without carbon source . Water agar alone already permits growth of F. psychrophila , but growth is significantly improved in the presence of carrot -LRB- data not shown -RRB- . The presence of potato only resulted in a small increase in growth compared to water agar , but onion reduced growth of F. psychrophila . These data demonstrate a preference of F. psychrophila for carrot as a substrate . The reduced growth on onion is likely caused by the high levels of phenolic compounds in onions . Inhibition of growth of F. psychrophila using antifungals Sorbic acid and natamycin are compounds commonly used for the inhibition of growth of spoilage fungi and the effectiveness of these compounds was tested on F. psychrophila on malt - and water agar plates with powdered wood . F. psychrophila is believed to survive on the crates used for the storage of carrots and therefore causes repeated spoilage when these crates are re-used . The presence of 250 mM sorbic acid resulted in a significant reduction in growth on malt extract agar , but not on powdered wood at 4 °C -LRB- Fig. 3a -RRB- . However , higher levels of sorbic acid -LRB- 625 and 1,250 mM -RRB- prevented growth of F. psychrophila on both substrates . In light of the high concentrations of sorbic acid required to inhibit growth , the expected effectiveness of this compound in preventing spoilage by F. psychrophila is limited . Fig. 3Influence of antifungals on the growth of Fibulorhizoctonia psychrophila grown on malt extract agar -LRB- MA -RRB- or powdered wood from storages crates -LRB- HW -RRB- . -LRB- a -RRB- sorbic acid , 18 °C ; -LRB- b -RRB- natamycin , 4 °C ; -LRB- c -RRB- natamycin , 18 °C Natamycin was more effective in preventing growth of F. psychrophila , resulting in a significant decrease in growth at 2 g/l at 4 °C . Increasing levels of natamycin reduced growth of the fungus at 4 °C similarly on both malt extract agar and powdered wood , although a significant difference was observed at 50 and 100 g/l -LRB- Fig. 3b -RRB- . However , at 100 g/l natamycin , some residual growth could still be observed for powdered wood . A stronger inhibition with natamycin was observed when the incubations occurred at 18 °C instead of 4 °C , resulting in nearly complete inhibition of growth on both media at 0.5 g/l natamycin -LRB- Fig. 3c -RRB- . The results described here indicate that spraying wooden crates with natamycin and incubating them at 18 °C or higher likely reduces spoilage F. psychrophila . F. psychrophila produces cold-active hydrolytic enzymes during growth on polysaccharides Both carrots and powdered wood consist largely of polysaccharides . As these are the main carbon sources for F. psychrophila during growth in the storage facilities , it can be expected that the fungus produces polysaccharide degrading enzymes that are active at low temperatures . To study whether these enzymes have in fact a higher relative activity at low temperatures than those currently used in industrial applications , we compared enzyme activities from F. psychrophila to those from Aspergillus niger . A. niger is one of the most commonly used fungi for the production of industrial enzyme preparations and produces a wide range of polysaccharide degrading enzymes -LRB- de Vries and Visser 2001 -RRB- . However , these enzymes are mainly active at higher temperatures . For A. niger , enzyme activities dropped to 30 -- 50 % when incubation at 15 °C was compared to 30 °C -LRB- Fig. 4a -RRB- . At 4 °C , only 10 -- 20 % of the 30 °C - activity was observed . For F. psychrophila only a 5 -- 35 % drop was detected when comparing 30 °C to 15 °C and 30 -- 60 % of the activity at 30 °C was still observed at 4 °C -LRB- Fig. 4b -RRB- . The strongest difference was observed for α-galactosidase and β-glucosidase , where more than 90 % of the activity was still detected at 15 °C for F. psychrophila , while for A. niger only 35 -- 45 % of the activity was detected at this temperature . Fig. 4Relative hydrolase activities in culture filtrate of Fibulorhizoctonia psychrophila and Aspergillus at different temperatures . -LRB- a -RRB- A. niger culture filtrate ; -LRB- b -RRB- F. psychrophila culture filtrate . AGL = α-galactosidase , LAC = β-galactosidase , BGL = β-glucosidase , BXL = β-xylosidase . Activity at 30 ° is set at 100 % F. psychrophila and other cold-tolerant fungi are potential sources for enzymes with high activity at low temperatures . This idea is supported by studies with cold-tolerant yeast and fungi , which were shown to produce cold-active pectinases -LRB- Nagakawa et al. 2004 , 2005a , b -RRB- , although in these studies a direct comparison with industrially used enzymes was not made . Cold-active enzymes can be important for many applications , for example in detergents or for the removal of lactose from milk for lactose-intolerant people .

O O O O O O O O O O B I O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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carrot-spoilage
cold-active enzymes
cold-tolerant fungus


Integrative analysis of the mouse embryonic transcriptome Monitoring global gene expression provides insight into how genes and regulatory signals work together to guide embryo development . The fields of developmental biology and teratology are now confronted with the need for automated access to a reference library of gene-expression signatures that benchmark programmed -LRB- genetic -RRB- and adaptive -LRB- environmental -RRB- regulation of the embryonic transcriptome . Such a library must be constructed from highly-distributed microarray data . Birth Defects Systems Manager -LRB- BDSM -RRB- , an open access knowledge management system , provides custom software to mine public microarray data focused on developmental health and disease . The present study describes tools for seamless data integration in the BDSM library -LRB- MetaSample , MetaChip , CIAeasy -RRB- using the QueryBDSM module . A field test of the prototype was run using published microarray data series derived from a variety of laboratories , experiments , microarray platforms , organ systems , and developmental stages . The datasets focused on several developing systems in the mouse embryo , including preimplantation stages , heart and nerve development , testis and ovary development , and craniofacial development . Using BDSM data integration tools , a gene-expression signature for 346 genes was resolved that accurately classified samples by organ system and developmental sequence . The module builds a potential for the BDSM approach to decipher a large number developmental processes through comparative bioinformatics analysis of embryological systems at-risk for specific defects , using multiple scenarios to define the range of probabilities leading from molecular phenotype to clinical phenotype . We conclude that an integrative analysis of global gene-expression of the developing embryo can form the foundation for constructing a reference library of signaling pathways and networks for normal and abnormal regulation of the embryonic transcriptome . These tools are available free of charge from the web-site http://systemsanalysis.louisville.edu requiring only a short registration process . Background Animal development is fashioned by conserved signaling pathways that orchestrate morphogenesis , pattern formation , and cell differentiation - complex processes operating jointly in different parts of an embryo and in stages associated with sequential gene activation . Monitoring local and temporal changes in gene expression can provide insight into how genes and regulatory signals work together to guide development . -LSB- 1 -RSB- This knowledge is important for understanding the pathogenesis of birth defects and to the central problems of defining precursor target cell susceptibility and the causal mechanisms of abnormal development triggered by diverse environmental and genetic perturbations to maternal-fetal unit . -LSB- 2 -RSB- Profiling gene expression on a global scale has become an important source of information for biological knowledge discovery . Despite well-known challenges confronting technology development , the analysis of global gene expression data can reveal themes in the biologically robust response patterns in gene activity . -LSB- 3 -RSB- Gene Expression Omnibus -LRB- GEO -RRB- is one of the main national repositories for high-information content transcript data from microarray analysis and serial analysis of gene expression -LRB- SAGE -RRB- . -LSB- 4 -RSB- GEO has grown from 18,235 records in June 2004 to 115,415 records in December 2006 , reflecting an average growth of over 100 new entries per day . A subset of this information describes the embryo proper and can be mined for major biological themes in developmental health and disease . Using keyword searches and trend analysis to mine PubMed and Medline literature databases , the public availability of embryo-based microarrays currently numbers 500-600 -LRB- 564 -RRB- , mostly studies on mouse embryos and differentiating human cell lines . The increasing volume of gene expression data on local and temporal states confronts the developmental biologist with the need for reference libraries and information management systems to handle optimal-scale gene-expression signatures and facilitate biological knowledge discovery . For example , Lamb et al. , -LSB- 5 -RSB- created a prototype reference collection of geneexpression signatures from cultured human cells exposed to bioactive molecules , which serves as a platform for pattern-matching software to establish a ` connectivity map ' between drugs , genes , and diseases . Another example is the integrative analysis of multi-study tumor profiles . -LSB- 6,7 -RSB- The emerging database model for tumor classification based on molecular abundance profiles has implied a 67-gene core ` common transcriptional program ' in multiple cancers . -LSB- 8 -RSB- In developmentteratogenesis , a preliminary meta-analysis across microarray studies in the mouse embryo returned a gene-expression signature of 512 developmentally regulated genes of which 16 % -LRB- ~ 82-genes -RRB- changed during exposure to teratogenic agents . -LSB- 2 -RSB- Given the promises and pitfalls of computational methods for solving gene expression problems , automated access to a reference collection of gene-expression signatures to benchmark the programmed -LRB- genetic -RRB- and adaptive -LRB- environmental -RRB- regulation of the embryonic transcriptome is scientifically needed . ` Birth Defects Systems Manager ' -LRB- BDSM -RRB- is a knowledge management system that provides custom software to mine public microarray data for interesting patterns across developmental stages , organ systems , and disease phenotypes . -LSB- 2,9 -RSB- This open resource enables : consolidation of communal data and metadata relevant to developmental health and disease ; interactions with current builds of national databases and data repositories ; efficient algorithms for cross-species annotation of symbolic gene annotations using the NCBI sequence homologybased annotations for corresponding homologues and orthologues ; specific queries across experiments to facilitate secondary analysis ; and data formats interoperable with analysis software for phenetic clustering , chromosomal mapping , gene ontology classification , pathway evaluation , and network identification . Since a comprehensive reference collection of gene-expression signatures for developing structures must be constructed from highly-distributed data , the present study was designed to empower BDSM with tools for seamless data integration : MetaSample , MetaChip , and CIAeasy . These tools are accessible at http://systemsanalysis.louisville.edu requiring only a short registration process to BDSM . A field test of the prototype run with published microarray data illustrates proof of concept for integrative analysis of the mouse embryonic transcriptome . Methodology Dataset collections Search of PubMed using keywords ` embryo ' and ` microarray ' returned 495 records of which 193 actually used the technology to study developing animal systems . GEO data sets -LRB- GDS -RRB- narrowed the list to 47 nonredundant microarray datasets , and including the keyword ` teratogen ' added a few more datasets , for a total of 564 public microarrays on the embryo . Raw and/or processed microarray sample-data files and associated metadata were parsed onto the server using LoadBDSM . -LSB- 9 -RSB- BDSM currently holds 25 developmental series containing 537 samples that are derived from the public domain and 3 series containing 43 samples which are private . These data represent 15 developing organ systems , 6 chemical exposures , and 5 drug interventions across 42 development stages . Tracking provenance For the present study , we restricted the analysis to 160 arrays in the BDSM library based on wellannotated experiments published on normal mouse embryogenesis using an Affymetrix technology platform . These conditions are identified by GEO Series Accession number -LRB- GSE -RRB- and/or literature citation as follows : preimplantation mouse embryo -LRB- GSE1749 -RRB- -LSB- 10 -RSB- ; heart -LRB- GSE1479 -RRB- GD10 - GD18 -LSB- 11 -RSB- ; nerve -LRB- GSE972 -RRB- GD9 .5 - birth -LSB- 12 -RSB- ; ovary -LRB- GSE1359 -RRB- and testis -LRB- GSE1358 -RRB- between GD11 .5 - birth -LSB- 13 -RSB- ; orofacial region -LRB- GSE1624 -RRB- -LSB- 14 -RSB- and secondary palate -LSB- 15 -RSB- between GD13-15 . The platforms for these series included : MG-U74Av2 -LRB- 12488 probes -RRB- , MG-U74Bv2 -LRB- 12478 probes -RRB- , MG430Av2 -LRB- 45104 probes -RRB- , MOE430Av2 -LRB- 22690 probes -RRB- , and MOE430Bv2 -LRB- 22576 probes -RRB- . Internal annotation of Affymetrix probe identifiers was performed to standardize gene labels across samples and improve cross-platform interoperability , as discussed previously . -LSB- 2 -RSB- Data integration Individual microarray sample-data files from the aforementioned developmental series were integrated using QueryBDSM , a module that for merging pre-processed , normalized samples from the BDSM library . Three metaanalysis tools written in PHP were designed to compare and analyze expression data across multiple chips and platforms : MetaSample , MetaChip , and CIAeasy . The workflow schema is diagrammed in Figure 1 . Individual sample-data files are selected from the BDSM library and added to a queue for integration . The formatted input files are tab-delimited expression ratios of probes -LRB- rows -RRB- x samples -LRB- columns -RRB- . QueryBDSM determines the number of distinct microarray platforms in the sample queue and merges the data as follows : if all samples come from the same microarray platform -LRB- number = 1 -RRB- , then QueryBDSM automatically runs MetaSample to create a merged-table having ` columns ' of normalized expression data -LRB- samples -RRB- in ` rows ' derived from the platform , with unique probe identifiers -LRB- ProbeID -RRB- expanded to include GenBank accession -LRB- GeneID -RRB- , and symbolic gene name -LRB- Gene Symbol -RRB- . If multiple platforms are represented by samples in the queue -LRB- number > 1 -RRB- , then QueryBDSM automatically runs MetaChip . MetaChip merges data when probe identifiers are different but represent the same annotation , such as across microarray platforms or phylogenetic species . The probe identifiers from each platform are converted to UniGene ID and then merged accordingly , with associated expression data , for those genes common across the datasets . The probes are annotated based on reverse-engineering the sequence homology-based annotations from GenBank . -LSB- 2 -RSB- For this purpose the system uses data flat files downloaded monthly from the HomoloGene and UniGene databases of NCBI . -LSB- 16 -RSB- In contrast to MetaSample and MetaChip , automated tools for merging samples , CIAeasy must be specified explicitly to compare datasets for the same samples on different platforms . CIAeasy was created from the ADE-4 for R statistical computing software package -LSB- 17 -RSB- and is adapted from ` coinertia analysis ' -LRB- CIA -RRB- for microarrays . -LSB- 18 -RSB- With CIAeasy users can perform CIA from the BDSM web site without detailed knowledge of R language programming . Since samples are aligned on a common space , CIA extracts information about the joint trends in expression patterns of genes independent of probe or sequence annotation . -LSB- 18 -RSB- CIAeasy automatically computes successive orthogonal axes with correspondence analysis and returns the percentage of total variance explained by each eigenvector to find the strongest trends in the co-structured datasets . Data analysis One of the problems confronting meta-analysis is the normalcy and spread of expression data . In order to derive expression ratios from the Affymetrix data , we computed a reference denominator , averaged for each gene at the earliest stage in a developmental series . These ratios were transformed to logarithm base 2 -LRB- log2 -RRB- to produce a continuous spectrum of values without biasing between up - and down-regulated genes and making the spread more normal . Each microarray set was centered to median of 0.00 and standardized by scaling to an average standard deviation of 0.5 . -LSB- 2 -RSB- The merged data were imported to GeneSpring v 7.2 using the UniGene cluster ID as the unique gene identifier . The data were clustered using Pearson correlation for the gene tree and two-sided Spearman Confidence for the developmental conditions . Functional annotation used the NIH/NIAID Database for Annotation , Visualization and Integrated Discovery -LRB- DAVID -RRB- . -LSB- 19 -RSB- The highest-ranking biological themes were stratified by Gene Ontology -LRB- GO -RRB- terms . Discussion Implementation of QueryBDSM For proof of concept we examined samples from GEO data source GSE1391 , a series describing global gene-expression profiles of the mouse embryo during preimplantation stages . -LSB- 10 -RSB- Samples included in this series represent development of the oocyte through fertilization -LRB- 1-cell embryo -RRB- , activation of the zygotic genome -LRB- 2-cell embryo -RRB- and first differentiation -LRB- 8-cell embryo -RRB- leading to divergent embryonal -LRB- inner cell mass -RRB- and trophectodermal -LRB- placental -RRB- lineages of the blastocyst . Biological replicates -LRB- 3-4 -RRB- arrayed at each stage used three different Affymetrix platforms : MOE430Av2 -LRB- 22690 probes -RRB- , MOE430Bv2 -LRB- 22576 probes -RRB- , and MG-U74Av2 -LRB- 12488 probes -RRB- . Gene-expression profiles were normalized to the ` oocyte ' in each platform as the earliest stage in the series . Derived data are log2-scale expression values computed from the ratio of signals to the oocyte reference . Using QueryBDSM , we merged datasets from the different samples to create three distinct datasets for the MOE430Av2 , MOE430Bv2 , and MG-U74Av2 platforms . Statistical -LRB- ANOVA -RRB- analysis , run at high stringency with Benjamini-Hochberg correction applied , returned 4417 probes -LRB- alpha = 0.0001 -RRB- , 1614 probes -LRB- alpha = 0.0001 -RRB- , and 2400 probes -LRB- alpha = 0.001 -RRB- that were differentially regulated . Aside from 34 probes that overlapped between the first two datasets , different genes were detected across these diverse platforms . Hierarchical clustering revealed two basic trajectories of gene-expression in all three platforms -LRB- Figure 2 -RRB- . One expression cluster contained genes that increased at the 2-cell stage to the blastocyst stage , and the other cluster contained genes that decreased over these stages -LRB- not shown -RRB- . These probes were mapped to the 307 reference pathways in the KEGG : Kyoto Encyclopedia of Genes and Genomes -LRB- http://www.genome.ad.jp/kegg/ -RRB- library to identify metabolic themes . The top significant KEGG pathways showed concordance between platforms MOE430Av2 and MGU74Av2 -LRB- Table 1 -RRB- , whereas MOE430Bv2 detected different pathways . Some pathways had marginal P-values with individual analysis that became significant in datasets joined by MetaChip -LRB- e.g. , Adherens Junction , Tight Junction pathways -RRB- . This illustrates the strength of the meta-analysis approach . We next used QueryBDSM to merge samples from platforms MOE430Av2 and MG-U74Av2 to illustrate the MetaChip and CIAeasy tools . These chips contained 13022 and 9562 nonredundant UniGene cluster identifiers , respectively , of which 7278 were common between the two platforms when passed through MetaChip . Statistical -LRB- ANOVA -RRB- analysis with the same parameters as before returned 3324 genes that were differentially regulated in this data subset . The top significant KEGG pathways for the combined 430A-U74A MetaChip are concordant with the individual analysis -LRB- Table 1 -RRB- . CIAeasy was used to compare the joint trend between 4417 , 1614 , and 2400 probes from MetaSample , resulting in a high-level of similarity between these three platforms -LRB- Figure 2 -RRB- . Of the 6812 nonredundant probes between MOE430Av2 and MG-U74Av2 -LRB- 4417 + 2400 -RRB- , only five probes were common . Annotating the probes which were not in common gave 4551 unique DAVID identifiers . Again , meta-analysis picked up most of the significant KEGG pathways identified in either of the singular analyses performed earlier and a few additional metabolic pathways -LRB- Table 1 -RRB- . Comparative bioinformatics analysis across developing systems An obvious limitation in combining data from several different platforms is that as more platforms are included , fewer representative genes are found to be common amongst all platforms . This problem increases when considering less comprehensive arrays , older arrays with outdated probe annotations , or arrays across animal species . Staging the entry of data from the most versatile arrays first can lessen the problem of losing information when data are combined across platforms ; however , in some data mining efforts the discriminating power gained by increasing samples and conditions might outweigh the loss of information . For example , multi-platform datasets have been found to discriminate tumor classification by expression profile with as few as 25 genes . -LSB- 6 -RSB- For this reason it may be possible to benchmark developmental stages using a limited number of genes across many diverse platforms . To illustrate this point we used BDSM-derived data to compare expression profiles across six unrelated studies and developing systems . We constructed a virtual meta-chip for probes common to all five technology platforms represented in these studies , yielding 346 genes . Unsupervised clustering and Pearson correlation of the gene-expression profiles correctly ordered the samples first by organ system and then by developmental sequence within each system -LRB- Figure 3 -RRB- . Within this hierarchy commonalities and differences across organ systems were evident for the patterns of expression in subsets of genes . Unfortunately , the number of genes returned from 346 by statistical -LRB- ANOVA -RRB- analysis of each individual system , or by K-means clustering of the entire matrix , was too small for insightful functional annotation . QueryBDSM is a simple and efficient solution that can be used to construct a self-evolving reference collection of geneexpression profiles from highly-distributed data on mouse development . Although robust mapping of biological themes and pathways that are expressed at particular developmental stages is straightforward when the same technology platform is considered -LSB- 2 -RSB- fuzzy-clustering methods will be needed when multiple platforms are considered . The MetaSample and MetaChip components of QueryBDSM are also available as standalone tools under the MetaBDSM module . In this way , the user can upload data outside the BDSM library . The user supplies details of the technology platform , organism , and information about the file format . Once all the required fields are entered and submitted , the files are checked for unique headers . Columns can be dropped by selecting the appropriate checkboxes . Clicking the Continue button combines the datasets with expression data and unique identifiers only for genes common between the datasets . These tools have been tested on Internet Explorer 6.0 or greater and Mozilla-based browsers , such as Netscape 6.0 and Firefox . Other tools are available to assemble data when the platform is same , such as Microarray data assembler . -LSB- 20 -RSB- This Excel-based program inherits Excel 's limitation from file sizes and number of samples -LRB- 256 columns and 65,000 data points -RRB- whereas the MetaSample and MetaChip tools do not have this limitation . These tools create temporary tables in the Oracle database and join them using the functionality of Oracle before putting it into a text file , reducing restrictions on the number of samples and size of files . Although users can theoretically combine 100 files at a single time , it is not recommended to load more then 25 files at a time . Conclusion The representation of experimental samples as developmentally contiguous groups is expected to yield a novel mosaic view of gene-expression signatures and genetic dependencies . Although sufficient data exists for data-mining efforts to begin , the ultimate goal of an unabridged reference collection must be viewed as a long-term effort . Regarding the embryo , a search of OVID MedLine using keywords ` embryo ' OR ` teratogen ' -LRB- 136,146 records -RRB- AND ` microarray ' OR ` SAGE ' -LRB- 16,906 records -RRB- returned 343 total records . At the current rate of 564 microarrays per 343 publication records -LRB- factor = 1.64 -RRB- , the trajectory of embryo-based microarray publications projects GEO to hold in excess of 1,476 microarrays relevant for embryogenesis or teratogenesis by the year 2010 . As studies unravel gene-expression signatures , the key principles in teratology -- namely , chemical effects on biological mechanisms , dose-response relationships , factors underlying genetic susceptibility , stage-dependent responses , and maternal influences , can be framed in a systems biology context to address an ` experience database ' for ranking pathways and networks by strength of association with anatomical landmarks and developmental abnormalities . -LSB- 2 -RSB- The BDSM resource would parallel efforts toward molecular diagnostics in cancer biology -LRB- http://www.oncomine.org/ -RRB- , which includes data sets profiling human tumor samples . -LSB- 21 -RSB- Since interpreting geneexpression signatures in birth defects will be predicated on posterior -LRB- prior -RRB- knowledge about developmental health and disease , an important payoff from this bioinformatics effort is to recognize and characterize how these biological states emerge from adaptation or adverse regulation of the embryonic transcriptome .

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transcriptome
mouse
expression
embryo
integrative analysis
birth defects