Structural and phylogenetic comparison of three pepsinogens from Pacific bluefin tuna: molecular evolution of fish pepsinogens.

The amino acid sequences of three pepsinogens (PG1, PG2 and PG3) of Pacific bluefin tuna (Thunnus orientalis) were deduced by cloning and nucleotide sequencing of the corresponding cDNAs. The amino acid sequences of the pre-forms of PG1, PG2 and PG3 were composed of a signal peptide (16 residues each), a propeptide (41, 37 and 35 residues, respectively) and a pepsin moiety (321, 323 and 332 residues, respectively). Amino acid sequence comparison and phylogenetic analysis indicated that PG1 and PG2 belong to the pepsinogen A family and PG3 to the pepsinogen C family. Homology modeling of the three-dimensional structure suggested that the remarkably high specific activity of PG2 toward hemoglobin, which had been found previously, was partly due to a characteristic deletion of several residues in the S1'-loop region that widens the space of the active site cleft region so as to accommodate protein and larger polypeptide substrates more efficiently. Including the tuna and all other fish pepsinogen sequences available to date, the molecular phylogenetic comparison was made with reference to evolution of fish pepsinogens. It was suggested that functional divergences of pepsinogens (pepsins) occurring in fishes as well as in mammals, correlated with differences in various aspects of fish physiology.

Evaluation of estrogenic activity of phthalate esters by gene expression profiling using a focused microarray (EstrArray).

Phthalates are used industrially as plasticizers and are known to contaminate natural environments, mostly as di-ester or mono-ester complexes. Because they are structurally similar to natural estrogens, they could act as endocrine disruptors. Here, we used a DNA microarray containing estrogen responsive genes (EstrArray) to examine gene expression profiles in MCF-7 cells treated with 10 microM butylbenzyl phthalate (BBP), dibutyl phthalate (DBP), diethyl phthalate (DEP), and diisopropyl phthalate (DIP) along with the natural estrogen 17beta-estradiol ([E(2)], 10 nM). The profiles for phthalate esters and E(2) were examined by correlation analysis using correlation coefficients (r-values) and cluster analysis. We found that BBP showed the highest correlation with E(2) (r = 0.85), and DEP and DIP showed moderate r-values (r = 0.52 and r = 0.49, respectively). Dibutyl phthalate exhibited the lowest (but still significant) correlation with E(2) (r = 0.36). Furthermore, among the pairs of chemicals, DEP-DIP and DIP-DBP showed very high correlations (r = 0.90 and r = 0.80, respectively), and the other pairs showed moderate relationships, which reflected how structurally close they are to each other. The analysis of six functional groups of genes (enzymes, signaling, proliferation, transcription, transport, and others) indicated that the genes belonging to the enzyme, transcription, and other functional groups showed common responses to phthalate esters and E(2). Although the effect of BBP was similar to that of E(2), the other phthalate esters showed different types of effects. These results indicate that the structure of estrogenic chemicals is strongly related to their estrogenic activity and can be evaluated by appropriate grouping of the responsive genes by focused microarray analysis.

Activation of rapid signaling pathways and the subsequent transcriptional regulation for the proliferation of breast cancer MCF-7 cells by the treatment with an extract of Glycyrrhiza glabra root.

Glycyrrhiza glabra root is one of the common traditional Chinese medicines and used as flavoring and sweetening agents for tobaccos, chewing gums, candies, toothpaste and beverages. While glycyrrhizin is one of the main components in the extract of G. glabra root and has been characterized, the other components have not been well characterized. The mechanism of growth activation of breast cancer MCF-7 cells, including the activation of Erk1/2 and Akt, and the transcriptional regulation of estrogen-responsive genes, was examined by means of sulforhodamine B, luciferase reporter gene, real-time RT-PCR and Western blotting assays after the induction of the cells with the extract of G. glabra root. The extract has similar activity to that induced by 17beta-estradiol (E(2)), although glycyrrhizin did not show such an activity. Moreover, the estrogen receptor alpha-dependent neurite outgrowth induced by the extract was similar to that by E(2), whereas glycyrrhizin had no effect. Furthermore, the expression profile examined by cDNA microarray assay using a set of 120 estrogen-responsive genes, which were related to proliferation, transcription, transport, enzymes and signaling, showed a statistically significant correlation (R=0.47, P<0.0001) between the profiles for E(2) and the extract. However, the expression profile for glycyrrhizin was different from that of the extract and E(2). The results indicate that rapid signaling pathways, including Erk1/2 and Akt, and the subsequent transcriptional regulation are involved in the proliferation of MCF-7 cells induced by the extract of G. glabra root. Furthermore, the extract had estrogenic activity and a distinguishable profile of gene expression, suggesting the presence of potentially useful components other than glycyrrhizin in G. glabra root for hormone and anti-cancer therapies.

Comparative profiling of the gene expression for estrogen responsiveness in cultured human cell lines.

It is important to know the difference as well as the similarity in estrogen responsiveness among cell lines for understanding the effects of estrogenic chemicals. Here, using 120 estrogen responsive genes, we examined comparative expression profiles between the profile in breast cancer MCF-7 cells treated with 17beta-estradiol and the profiles in other cell lines derived from breast (T-47D and HBC-4 cells), endometrium (Ishikawa cells) and kidney (RXF-631L cells) treated with estrogenic chemicals. First, comparative profiling between MCF-7 and T-47D cells showed similar (correlation coefficient or R value=0.49-0.87) profiles for all chemicals examined: 17beta-estradiol, estrone, estriol, diethylstilbestrol, bisphenol A, nonylphenol and genistein. The analysis using other cell lines indicated that significant correlations to the profile in MCF-7 cells treated with 17beta-estradiol were observed for the profiles in Ishikawa cells treated with 17beta-estradiol, diethylstilbestrol and bisphenol A, and HBC-4 cells treated with 17beta-estradiol. The profiles for diethylstilbestrol and bisphenol A in HBC-4 cells and all three chemicals in RXF-631L cells did not show significant correlation with those in MCF-7 cells. Hierarchical cluster analysis revealed that there are cell-specific responses to estrogenic chemicals (T-47D and HBC-4 cells for example). Correlation analysis using six (proliferation, transcription, transport, enzymes, signaling and others) functionally-categorized gene groups indicated that the genes related to enzymes showed greater correlations for all chemicals tested in T-47D cells and some chemicals in Ishikawa and HBC-4 cells while those related to transcription contributed to variations.

Purification and characterization of pepsinogens from the gastric mucosa of African coelacanth, Latimeria chalumnae, and properties of the major pepsins.

Two major pepsinogens, PG1 and PG2, and one minor pepsinogen, PG3, were purified from the gastric mucosa of African coelacanth, Latimeria chalumnae (Actinistia). PG1 and PG2 were much less acidic than PG3. Their molecular masses were estimated by SDS-PAGE to be 37.0, 37.0 and 39.3 kD, respectively. When incubated at pH 2.0, PG1 and PG2 were converted autocatalytically to the mature pepsins through an intermediate form, whereas PG3 was converted to an intermediate form, but not to the mature pepsin autocatalytically. The N-terminal sequencing indicated that the 42 residue sequences of the propeptides of PG1 and PG2 were essentially identical with each other, but different from that of PG3. A phylogenetic tree based on the N-terminal propeptide sequences indicates that PG1 and PG2 belong to the pepsinogen A group, and PG3 to the pepsinogen C group. From the phylogenetic comparison, coelacanth PG1 and PG2 appear to be evolutionally closer to tetrapod pepsinogens A than ray-finned fish pepsinogens A, consistent with the traditional systematics. Pepsins 1 and 2 were essentially identical with each other and rather similar to mammalian pepsins A in the pH optimum toward hemoglobin (pH 2-2.5), the cleavage specificity toward oxidized insulin B chain and strong inhibition by pepstatin, except that they possessed a significant level of activity in the higher pH range unlike mammalian pepsins A.

Expression profiling of estrogen-responsive genes in breast cancer cells treated with alkylphenols, chlorinated phenols, parabens, or bis- and benzoylphenols for evaluation of estrogenic activity.

We examined expression profiles of estrogen-responsive genes after treatment with alkylphenols (p-cresol (pC), 4-n-ethylphenol (4EP), 4-n-heptylphenol (4HP), 4-t-octylphenol (4OP) and nonylphenol (NP)), chlorinated phenols (4-chlorophenol (4CP), 4-chloro-3,5-dimethylphenol (CDP), 2,4-dichlorophenol (DCP) and pentachlorophenol (PCP)), parabens (methylparaben (MPB), ethylparaben (EPB) propylparaben (PPB) and butylparaben (BuPB)), or bis- and benzoylphenols (bisphenols A and B and p-hydroxybenzophenone (pHBP)) by means of a DNA microarray assay first to evaluate the estrogenic activity of these chemicals and then to understand the structural basis for the activity. By selecting a set of 120 genes showing greater statistical reliability for estrogen, a more reliable assay for each of the chemicals was achieved and, for the chemicals for which data were available, the results were consistent with those of previously reported estrogen receptor-binding and yeast two-hybrid assays except for chlorinated and few other phenols. Evaluation of the chemicals based on gene function indicated that the genes related to proliferation, transcription and transport were mostly up-regulated while significant numbers of genes related to enzymes and signaling were down-regulated. The genes related to transcription showed the highest degree of variation among the six functional categories (enzymes, signaling, proliferation, transcription, transport and others) for the chemicals with relatively high levels of estrogenic activity. These results indicate that the variations in chemicals and their biological effects can be monitored by the appropriate grouping of estrogen-responsive genes.

Expression profiling of the estrogen responsive genes in response to phytoestrogens using a customized DNA microarray.

Here, we examined phytoestrogens, isoflavones (genistein, daidzein, glycitein, biochanin A and ipriflavone), flavones (chrysin, luteolin and apigenin), flavonols (kaempferol and quercetin), and a coumestan, a flavanone and a chalcone (coumestrol, naringenin and phloretin, respectively) by means of a DNA microarray assay. A total of 172 estrogen responsive genes were monitored with a customized DNA microarray and their expression profiles for the above phytoestrogens were compared with that for 17beta-estradiol (E2) using correlation coefficients, or R values, after a correlation analysis by linear regression. While R values indicate the similarity of the response by the genes, we also examined the genes by cluster analysis and by their specificity to phytoestrogens (specific to genistein, daidzein or glycitein) or gene functions. Several genes were selected from p53-related genes (CDKN1A, TP53I11 and CDC14), Akt2-related genes (PRKCD, BRCA1, TRIB3 and APPL), mitogen-activated protein kinase-related genes (RSK and SH3BP5), Ras superfamily genes (RAP1GA1, RHOC and ARHGDIA) and AP-1 family and related genes (RIP140, FOS, ATF3, JUN and FRA2). We further examined the extracts from two local crops of soy beans (Kuro-daizu or Mochi-daizu) by comparing the gene expression profiles with those of E2 or phytoestrogens as a first step in utilizing the expression profiles for various applications.

Using a customized DNA microarray for expression profiling of the estrogen-responsive genes to evaluate estrogen activity among natural estrogens and industrial chemicals.

We developed a DNA microarray to evaluate the estrogen activity of natural estrogens and industrial chemicals. Using MCF-7 cells, we conducted a comprehensive analysis of estrogen-responsive genes among approximately 20,000 human genes. On the basis of reproducible and reliable responses of the genes to estrogen, we selected 172 genes to be used for developing a customized DNA microarray. Using this DNA microarray, we examined estrogen activity among natural estrogens (17beta-estradiol, estriol, estrone, genistein), industrial chemicals (diethylstilbestrol, bisphenol A, nonylphenol, methoxychlor), and dioxin. We obtained results identical to those for other bioassays that are used for detecting estrogen activity. On the basis of statistical correlations analysis, these bioassays have shown more sensitivity for dioxin and methoxychlor.

Positional cloning of rice semidwarfing gene, sd-1: rice "green revolution gene" encodes a mutant enzyme involved in gibberellin synthesis.

A rice semidwarfing gene, sd-1, known as the "green revolution gene," was isolated by positional cloning and revealed to encode gibberellin 20-oxidase, the key enzyme in the gibberellin biosynthesis pathway. Analysis of 3477 segregants using several PCR-based marker technologies, including cleaved amplified polymorphic sequence, derived-CAPS, and single nucleotide polymorphisms revealed 1 ORF in a 6-kb candidate interval. Normal-type rice cultivars have an identical sequence in this region, consisting of 3 exons (558, 318, and 291 bp) and 2 introns (105 and 1471 bp). Dee-Geo-Woo-Gen-type sd-1 mutants have a 383-bp deletion from the genome (278-bp deletion from the expressed sequence), from the middle of exon 1 to upstream of exon 2, including a 105-bp intron, resulting in a frame-shift that produces a termination codon after the deletion site. The radiation-induced sd-1 mutant Calrose 76 has a 1-bp substitution in exon 2, causing an amino acid substitution (Leu [CTC] to Phe [TTC]). Expression analysis suggests the existence of at least one more locus of gibberellin 20-oxidase which may prevent severe dwarfism from developing in sd-1 mutants.