Effects of neonatal treatment with phytoestrogens, genistein and daidzein, on sex difference in female rat brain function: estrous cycle and lordosis

It is well known that neonatal exposure to estrogen induces masculinization or defeminization of the brain. In this study, the effects of neonatal treatment with two kinds of soybean isoflavone aglycone, genistein (GS) and daidzein (DZ), on the estrous cycle and lordosis behavior were investigated. Female rats were injected subcutaneously with 1 mg GS, 1 mg DZ, 100 microg estradiol (E2), or oil daily for 5 days from birth. As a result, vaginal opening was advanced in GS- or E2-treated females. A vaginal smear check indicated that oil- or DZ-treated females showed a constant 4- or 5-day estrous cycle, whereas GS- or E2-treated rats showed a persistent or prolonged estrus. Ovariectomy was performed in all females at 60 days of age. The ovaries in the GS- or E2-treated groups were smaller than those in the oil- and DZ-treated groups and contained no corpora lutea. In the DZ group, although corpora lutea were seen, ovaries were smaller than that of control females. Behavioral tests were carried out after implantation of E2-tubes. All of the oil- or DZ-treated females showed lordosis with a high lordosis quotient (LQ). On the other hand, as male rats, LQs were extremely low in the E2-treated group, when compared to the oil-treated group. In the GS-treated group, the mean LQ was lower than that in the oil-treated group, but higher than those in the E2-treated female or male groups. These results suggest that genistein acts as an estrogen in the sexual differentiation of the brain and causes defeminization of the brain in regulating lordosis and the estrous cycle in rats. In addition, neonatal daidzein also has some influence on ovarian function.     

Protective role of hydroxysteroid sulfotransferase in lithocholic acid-induced liver toxicity

Supplement of 1% lithocholic acid (LCA) in the diet for 5-9 days resulted in elevated levels of the marker for liver damage aspartate aminotransferase and alkaline phosphatase activities in both farnesoid X receptor (FXR)-null and wild-type female mice. The levels were clearly higher in wild-type mice than in FXR-null mice, despite the diminished expression of a bile salt export pump in the latter. Consistent with liver toxicity marker activities, serum and liver levels of bile acids, particularly LCA and taurolithocholic acid, were clearly higher in wild-type mice than in FXR-null mice after 1% LCA supplement. Marked increases in hepatic sulfating activity for LCA (5.5-fold) and hydroxysteroid sulfotransferase (St) 2a (5.8-fold) were detected in liver of FXR-null mice. A 7.4-fold higher 3alpha-sulfated bile acid concentration was observed in bile of FXR-null mice fed an LCA diet compared with that of wild-type mice. Liver St2a content was inversely correlated with levels of alkaline phosphatase. In contrast, microsomal LCA 6beta-hydroxylation was not increased and was in fact lower in FXR-null mice compared in wild-type mice. Clear decreases in mRNA encoding sodium taurocholate cotransporting polypeptide, organic anion transporting polypeptide 1, and liver-specific organic anion transporter-1 function in bile acid import were detected in LCA-fed mice. These transporter levels are higher in FXR-null mice than wild-type mice after 1% LCA supplement. No obvious changes were detected in the Mrp2, Mrp3, and Mrp4 mRNAs. These results indicate hydroxysteroid sulfotransferase-mediated LCA sulfation as a major pathway for protection against LCA-induced liver damage. Furthermore, Northern blot analysis using FXR-null, pregnane X receptor-null, and FXR-pregnane X receptor double-null mice suggests a repressive role of these nuclear receptors on basal St2a expression.     

Specification and differentiation processes of secondary mesenchyme-derived cells in embryos of the sea urchin Hemicentrotus pulcherrimus

Four types of mesoderm cells (pigment cells, blastocoelar cells, coelomic pouch cells and circumesophageal muscle cells) are derived from secondary mesenchyme cells (SMC) in sea urchin embryos. To gain information on the specification and differentiation processes of SMC-derived cells, we studied the exact number and division cycles of each type of cell in Hemicentrotus pulcherrimus. Numbers of blastocoelar cells, coelomic pouch cells and circumesophageal muscle fibers were 18.0 +/- 2.0 (36 h post-fertilization (h.p.f.)), 23.0 +/- 2.5 (36 h.p.f.) and 9.5 +/- 1.3 (60 h.p.f.), respectively, whereas the number of pigment cells ranged from 40 to 60. From the diameters of blastocoelar cells and coelomic pouch cells, the numbers of division cycles were elucidated; these two types of cells had undertaken 11 rounds of cell division by the prism stage, somewhat earlier than pigment cells. To determine the relationship among the four types of cells, we tried to alter the number of pigment cells with chemical treatment and found that CH3COONa increased pigment cells without affecting embryo morphology. Interestingly, the number of blastocoelar cells became smaller in CH3COONa-treated embryos. In contrast, blastocoelar cells were markedly increased with NiCl2 treatment, whereas the number of pigment cells was markedly decreased. The number of coelomic pouch cells and circumesophageal muscle fibers was not affected with these treatments, indicating that coelomic pouch and muscle cells are specified independently of, or at much later stages, than pigment and blastocoelar cells.     

Adaptor protein Shc is an isoform-specific direct activator of the tyrosine kinase c-Src

The activity of c-Src protein-tyrosine kinase is up-regulated under a number of receptor signaling pathways. However, the activation mechanism of c-Src under physiological conditions has remained unclear. We show here that the Shc adaptor protein is a novel direct activator of c-Src in epidermal growth factor receptor signaling in A431 human epidermoid carcinoma cells. Among the three Shc isoforms, P66 and P52, but not P46, were found to interact with and activate c-Src in vitro and in vivo. Activation of c-Src accompanied autophosphorylation of c-Src in the activation segment, but the carboxyl-terminal dephosphorylation was not observed. We have identified the interaction sites between Shc and c-Src and constructed a point mutant of Shc that abolishes the c-Src activation. Using this mutant, we have confirmed that the Shc-mediated c-Src activation triggers Stat-p21/WAF1/Cip1 pathway that has been implicated in the cell cycle arrest and apoptosis of epidermal growth factor-stimulated A431 cells.     

Genotoxic activation of benzophenone and its two metabolites by human cytochrome P450s in SOS/umu assay

The genotoxic potential of benzophenone and its metabolically related compounds, benzhydrol and p-benzoylphenol, was investigated using human cytochrome P450 (P450) enzymes. Benzophenone and its two metabolites (0.1-1mM) showed a suppression of bacterial growth without any P450 system, but no induction of umu gene expression was observed in Salmonella typhimurium TA1535/pSK1002. Human liver microsomes induced the bacterial cytotoxicity of these compounds without any umu gene expression. On the other hand, with the addition of Escherichia coli membranes expressing recombinant human P450 2A6 and NADPH-cytochrome P450 reductase (NPR), benzophenone showed umu gene expression (64 umu units/min/nmol) P450 2A6). Moderate activation of benzophenone by P450 1A1/NPR membranes, 1A2/NPR membranes, or 1B1/NPR membranes was also observed. Activation of benzhydrol and p-benzoylphenol by the P450/NPR system was similar to that of benzophenone. These results suggest that benzophenone and its metabolically related benzhydrol and p-benzoylphenol can be bioactivated by P450 2A6 and P450 family 1 enzymes. Until now, benzophenone has been investigated mainly in terms of estrogenic activity and cytotoxicity, however, the genotoxic activation of benzophenone by human cytochrome P450s should be examined in terms of the risks to humans.     

Behavior of sister copies of mini-F plasmid after synchronized plasmid replication in Escherichia coli cells

To clarify whether sister copies of mini-F plasmid are immediately separated from each other after replication, we analyzed the behavior of sister mini-F copies after synchronized replication of mini-F. Sister copies of mini-F were separated immediately or shortly after replication, in contrast to sister oriC copies of the Escherichia coli chromosome.     

SCB1, a BURP-domain protein gene,from developing soybean seed coats

We describe a gene, SCB1 (Seed Coat BURP-domain protein 1), that is expressed specifically within the soybean (Glycine max [L.] Merrill) seed coat early in its development. Northern blot analysis and mRNA in situ hybridization revealed novel patterns of gene expression during seed development. SCB1 mRNA accumulated first within the developing thick-walled parenchyma cells of the inner integument and later in the thick- and thin-walled parenchyma cells of the outer integument. This occurred prior to the period of seed coat maturation and seed filling and before either of the layers started to degrade. SCB1 may therefore play a role in the differentiation of the seed coat parenchyma cells. In addition, the protein product appears to be located within cell walls. The SCB1 gene codes for a new member of a class of modular proteins that possess a carboxy-terminal BURP domain and a variety of different repeated sequences. The sequence of the genomic clone revealed the insertion of a Tgm transposable element in the upstream promoter region but it is not certain whether it contributes to the tissue-specific pattern of SCB1 expression.