Combining Affymetrix microarray results

Background  

As the use of microarray technology becomes more prevalent it is not unusual to find several laboratories employing the same microarray technology to identify genes related to the same condition in the same species. Although the experimental specifics are similar, typically a different list of statistically significant genes result from each data analysis.     

Estrogen metabolism in rat liver microsomal and isolated hepatocyte preparations--II. Inhibition studies

The abilities of various inhibitors and metabolism modifiers to alter the metabolism of estradiol and the irreversible binding of estradiol to proteins were examined in subcellular microsomal incubations and in intact hepatocyte preparations. In studies with rat liver microsomal preparations containing estradiol and an NADPH-generating system, the irreversible binding of radiolabeled steroid metabolite(s) to the microsomal proteins was 77.59 pmol/mg/min (SD 6.1; 7.6% of total steroid). 2-Bromoestradiol and 4-bromoestradiol, inhibitors of estrogen 2-hydroxylase, effectively decreased this irreversible binding of radiolabeled estradiol metabolite(s) to microsomal proteins to 17 pmol mg-1 min-1 (2.1% of total estradiol). These haloestrogens were also effective inhibitors in the intact hepatocyte cells, decreasing the amounts of organic metabolites, aqueous-soluble conjugates, and protein-bound materials. The HPLC radiochromatograms of the organic-extracted fractions from the 2 h hepatocyte incubations demonstrate that the catechol estrogen products, i.e. 2-hydroxyestrogens and 2-methoxyestrogens, were present in lower amounts in the incubations containing the bromoestrogens than in control incubations containing no inhibitor. Ascorbic acid and cysteine, general modifiers of oxidative pathways of metabolism, also affected estradiol metabolism in microsomal and hepatocyte preparations. Both these agents were able to decrease the irreversible binding of estradiol to proteins in the microsomal assays. Ascorbic acid decreased the general metabolism of estradiol in the hepatocyte incubations but did not decrease irreversible binding to proteins. The addition of cysteine to the hepatocyte incubation resulted in an increased metabolism of estradiol and the production of more aqueous-soluble radiolabeled metabolites than the control incubations; however, cysteine did not decrease the amounts of estradiol metabolite(s) irreversibly bound to proteins. Investigations of steroid metabolism in the isolated hepatocytes thus provide an effective in vitro technique for examining the overall oxidative, reductive, and conjugative pathways that are functional in the liver and enables one to investigate the abilities of inhibitors, regulators, and modifiers to affect the metabolic processes. Also, these hepatocyte studies demonstrate that the inhibitors of estrogen 2-hydroxylase, 2-bromoestradiol and 4-bromoestradiol, can enter and act in the intact cells. Consequently, these agents may be useful pharmacological probes for examining the functions of catechol estrogens in other tissues.     

Monoclonal antibody 91.9H raised against sulfated mucins is specific for the 3'-sulfated Lewisa tetrasaccharide sequence

The IgG1hybridoma antibody, 91.9H, was originally raised against sulfated mucins isolated from normal human colonic mucosa. Previous studies have shown that the 91.9H antigen is expressed on normal colonic epithelial cells and the sulfomucins that they produce, but not in the normal small intestine and stomach. Tissue-specific changes occur in 91.9H antigen expression in disease: the antigen diminishes in colonic carcinomas, whereas in regions of gastric mucosa showing intestinal metaplasia and in gastric carcinomas, the antigen is expressed as a "neo-antigen." This report is concerned with elucidation, by the neoglycolipid technology, of the determinant recognized by antibody 91.9H using sulfated and sialyl oligosaccharides of Lewisa(Lea) and Lextypes, and analogs that lack sulfate, sialic acid, or fucose. Binding experiments with the lipid-linked oligosaccharides immobilized on chromatograms or on microwells, and inhibition of binding experiments with free oligosaccharides based on di-, tri- and tetrasaccharide backbones, show that the 91.9H antigenic determinant is based on a trisaccharide backbone, and consists of the 3'-sulfated Leatetrasaccharide sequence, which is a potent ligand for the E- and L-selectins. The antibody gives a relatively low signal with the 3'-sulfated non-fucosylated backbone, and has no detectable cross- reaction with the 3'-sulfated Lexisomer, nor with sialyl-Leaand - Lexanalogues. Antibody 91.9H is a valuable addition, therefore, to the repertoire of reagents for mapping details of the distribution, and determining the relative importance of sulfated and sialyl oligosaccharides as ligands for the selectins, in normal and pathological epithelia and endothelia.      

Human responses to propionic acid. I. Quantification of within- and between-participant variation in perception by normosmics and anosmics

The objective of this study was to fully characterize normosmic perception of stimuli expected to cause widely varying degrees of olfactory and nasal trigeminal stimulation and to directly evaluate the possible role of olfactory nerve stimulation in nasal irritation sensitivity. During each of four identical test sessions, four anosmic and 31 normosmic participants were presented with a range of concentrations extending from peri-threshold for normosmics to supra- threshold for anosmics. For each session, odor (O) and nasal irritation (NI) sensitivities were summarized in terms of the concentrations required to produce four sensation levels ('iso-response' concentrations). Within-participant variation in these iso-response concentrations was < 10-fold for 95% of normosmics, for both O and NI. For O but not NI, these apparent fluctuations in sensitivity were largely accounted for by the uncertainty surrounding the iso-response concentrations calculated for each session. Anosmics exhibited minimal within- and between-participant variation in NI and required, for all but the highest perceptual level, a higher concentration than almost all normosmics. Between-participant variation, expressed in terms of 90% confidence interval widths, was approximately 0.5 log units for both O and NI for the highest perceptual level, but increased to approximately 0.8 and 1.8 log units, respectively, for the lowest (peri- threshold) level. Our findings suggest that: (i) most apparent variation over time in O sensitivity is actually a reflection of the uncertainty surrounding estimates of sensitivity obtained for each session; (ii) within- and between-participant variation in O sensitivity is far less than is commonly reported; and (iii) low to moderate levels of NI in normosmics are the result of relatively weak trigeminal stimulation combined with much greater olfactory activation.      

Gossypolone suppresses progesterone synthesis in bovine luteal cells

Gossypolone, a proposed major metabolite of gossypol, was synthesized and investigated for its effect on progesterone synthesis in cultured bovine luteal cells. Gossypolone inhibited human chorionic gonadotropin(hCG)-stimulated progesterone secretion, reduced substrate-enhanced conversions of 25-hydroxycholesterol to pregnenolone and of pregnenolone to progesterone in a dose-dependent fashion. These findings indicate that gossypolone inhibits not only 3β-hydroxysteroid dehydrogenase (3β-HSD) activity, as gossypol does, but also side-chain cleavage enzyme complex (cytochrome P450scc activity. However, the two compounds appear to have a similar potency in inhibiting progesterone secretion. Both gossypolone and gossypol (8.5 μM) induced morphological changes in cellular organelles.     

Aromatase inhibition by 7-substituted steroids in human choriocarcinoma cell culture.

Androstenedione analogs containing 7 alpha-substituents have proven to be potent inhibitors of aromatase both in vitro and in vivo. Several of these agents have exhibited higher affinity for the enzyme complex than the substrate. In order to examine further the interaction(s) of 7-substituted steroids with aromatase, 7-substituted 4,6-androstadiene-3,17-diones were synthesized and demonstrated competitive inhibition of aromatase activity in human placental microsomes. 7-Substituted 1,4,6-androstatriene-3,17-diones demonstrated mechanism-based inhibition of placental aromatase activity. These agents were evaluated for inhibition of aromatase activity in the JAr human choriocarcinoma line. The 7-substituted 4,6-androstadiene-3,17-diones produced dose dependent inhibition of aromatase activity in the cell cultures, with IC50 values ranging from 490 nM to 4.5 microM. However, these agents are less effective when compared to other steroidal inhibitors, such as 7 alpha-thiosubstituted androstenediones. These results on the 7-substituted 4,6-androstadiene-3,17-diones are consistent with the data from biochemical enzyme inhibition studies using human placental aromatase. On the other hand, 7-phenethyl-1,4,6-androstatriene-3,17-dione exhibits greater inhibitory activity, with an IC50 value of 80 nM. Other mechanism-based inhibitors, 7 alpha-(4'-amino)phenylthio-1,4-androstadiene-3,17-dione and 4-hydroxyandrostenedione, also exhibited potent inhibition of aromatase activity in JAr cells. In summary, the most effective B-ring modified steroidal aromatase inhibitors are those derivatives that can project the 7-aryl substituent into the 7 alpha-position.     

7-substituted 1,4,6-androstatriene-3,17-diones as enzyme-activated irreversible inhibitors of aromatase

7-Phenyl-1,4,6-androstatriene-3,17-dione (4), 7-benzyl-1,4,6-androstatriene-3,17-dione (5) and 7-phenethyl-1,4,6-androstatriene-3,17-dione (6) were synthesized and evaluated in vitro in human placental microsomes as enzyme-activated irreversible inhibitors of aromatase. The compounds were synthesized from appropriate 7-substituted 4,6-androstadiene-3,17-diones by reaction with DDQ under neutral conditions. All the compounds produced a first order inactivation of aromatase in the presence of NADPH but not in the absence of NADPH. Substrate 4-androstene-3,17-dione protected the enzyme from inactivation by the inhibitors. Furthermore, cysteine failed to protect aromatase from inactivation by compounds 5 and 6. In contrast, cysteine partially protected aromatase from inactivation by compound 4. Irreversibility studies illustrated the covalent nature of the inactivation by 4, 5 and 6. The above experimental evidence demonstrated that compounds 5 and 6 are effective enzyme-activated irreversible inhibitors of aromatase.     

Phylogenetic utility of elongation factor-1 alpha in noctuoidea (Insecta: Lepidoptera): the limits of synonymous substitution

To test its phylogenetic utility, nucleotide sequence variation in a 1,240-bp fragment of the elongation factor-1 alpha (EF-1 alpha) gene was examined in 49 moth species representing the major groups of the superfamily Noctuoidea. Both parsimony and distance analyses supported the monophyly of nearly all groups for which there are clear morphological synapomorphies. Clades of subfamily rank and lower, probably mid-Tertiary and younger, were strongly supported. The third codon position contains 88% of variable sites, and approaches saturation at approximately 20% sequence divergence, possibly due to among-site rate heterogeneity and composition bias; higher divergences occur only in association with shifts in composition. Surprisingly, the few nonsynonymous changes appear no more phylogenetically reliable than synonymous changes. Signal strength for basal divergences is weak and fails to improve with character weighting; thus, dense taxon sampling is probably needed for strong inference from EF-1 alpha regarding deeper splits in Noctuoidea (probably early Tertiary). EF-1 alpha synonymous changes show promise for phylogeny reconstruction within Noctuidae and other groups of Tertiary age.      

Effects of matrix components on aromatase activity in breast stromal cells in culture

Local estradiol production within breast tissue is maintained by the aromatase cytochrome P450_arom complex, which has been localized primarily to the stromal component of tumors but also has been detected in the breast epithelial cells. Paracrine interactions between stromal and epithelial components of the breast are critical to the sustained growth and progression of breast tumors. Maintenance of the differentiated state, including hormone and growth factor responsiveness, requires extracellular matrix proteins as substrata for cells. This research has focused on developing a cell culture system that more closely mimics in vivo interactions in order to dissect actual paracrine signaling between these two cell types. Human fibroblasts were isolated from breast tissue and were maintained in a cell culture system grown on plastic support or on a collagen I support matrix. The collagen I matrix model supports cell maintenance and subsequent differentiation on collagen rather than maximal proliferation, therefore allowing for a more accurate environment for the study of hormonal control and cellular communication. Initial experiments compared aromatase activity of patient fibroblasts grown on plastic versus collagen I using the tritiated water release method. Constitutive aromatase activity was found to be lower when cells were grown on a collagen gel for 4–7 days (7.7 fold lower) using DMEM/F12 containing 10% dextran coated charcoal stripped serum. However, fibroblasts grown on collagen I appeared to be significantly more responsive to stimulation by 100 nM dexamethasone (plastic: 6.0 fold induction, collagen: 33.2 fold induction) when pretreated for 12 h prior to measurement of aromatase activity. In an effort to examine paracrine interactions between the stromal and epithelial cells in breast tissue, experiments using conditioned media from fibroblast cultures were performed. Testosterone administration to fibroblasts results in the production of estradiol into the media in sufficient concentrations to elicit an increase in pS2 expression when the conditioned media is administered to MCF-7 cells. The addition of a potent aromatase inhibitor resulted in a complete suppression of fibroblast-derived estrogens and showed only a modest increase in pS2 expression. Culturing breast fibroblasts and epithelial cells on extracellular matrix allows for a more meaningful examination of the paracrine interactions between these cell types within the context of an appropriate extracellular environment. This study highlights the need for evaluation of gene expression in cell culture systems that accurately reflect the tissue microenvironment.