Identification of differentially expressed genes involved in the regression and development of the chicken Müllerian duct

Müllerian ducts of male chickens undergo regression around day 12 of incubation, but the underlining mechanisms remain unclear. The purpose of this study was to identify factors that contribute to regression of the Müllerian duct in the chicken. We first employed annealing control primer-based RT-PCR to screen candidate genes differentially expressed in the Müllerian ducts between male and female. Four differentially expressed genes (MSX2, GAL10, VCP and PLCH1) were partially sequenced. The expression of mRNA of the latter genes and MSX1 in the male and female Müllerian ducts were compared at 7.5, 8 and 9 days of incubation using semi-quantitative RT-PCR. The results indicated that both MSX1 and MSX2 mRNA was highly expressed in the male Müllerian duct at day 9 of incubation, whereas, PLCH1 mRNA was lower in the male duct at day 9 of incubation compared to that of the female duct. Although VCP mRNA was expressed in both left and right female Müllerian ducts, no expression was detected in the male duct. Whole mount in situ hybridyzation analysis showed that the expression of MSX1 and MSX2 mRNA were localized specifically in the mesenchymal cells of the male Müllerian duct at day 9 of incubation. In contrast, VCP mRNA expression was observed in both mesenchymal and epithelial cells of the female Müllerian duct but not detected in the male duct. These results suggest that both up-regulation of MSX1 and MSX2 mRNA expression is involved in the regression of the Müllerian duct in male chicken embryo, whereas VCP expression is involved in development of the female duct.     

Critical roles of the CuB site in efficient proton pumping as revealed by crystal structures of mammalian cytochrome c oxidase catalytic intermediates

Mammalian cytochrome c oxidase (CcO) reduces O₂ to water in a bimetallic site including Fe_a3 and Cu_B giving intermediate molecules, termed A-, P-, F-, O-, E-, and R-forms. From the P-form on, each reaction step is driven by single-electron donations from cytochrome c coupled with the pumping of a single proton through the H-pathway, a proton-conducting pathway composed of a hydrogen-bond network and a water channel. The proton-gradient formed is utilized for ATP production by F-ATPase. For elucidation of the proton pumping mechanism, crystal structural determination of these intermediate forms is necessary. Here we report X-ray crystallographic analysis at ∼1.8 Å resolution of fully reduced CcO crystals treated with O₂ for three different time periods. Our disentanglement of intermediate forms from crystals that were composed of multiple forms determined that these three crystallographic data sets contained ∼45% of the O-form structure, ∼45% of the E-form structure, and ∼20% of an oxymyoglobin-type structure consistent with the A-form, respectively. The O- and E-forms exhibit an unusually long Cu_B²⁺-OH⁻ distance and Cu_B¹⁺-H₂O structure keeping Fe_a3³⁺-OH⁻ state, respectively, suggesting that the O- and E-forms have high electron affinities that cause the O→E and E→R transitions to be essentially irreversible and thus enable tightly coupled proton pumping. The water channel of the H-pathway is closed in the O- and E-forms and partially open in the R-form. These structures, together with those of the recently reported P- and F-forms, indicate that closure of the H-pathway water channel avoids back-leaking of protons for facilitating the effective proton pumping.     

Characteristics and inhibition by flavonoids of 20alpha-hydroxysteroid dehydrogenase activity in mouse tissues

Progesterone was stereoselectively reduced to a metabolite 20alpha-hydroxy-4-pregnen-3-one in the cytosolic fraction from the liver of male mice, indicating that the reduction of progesterone is catalyzed by 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD). The cytosolic 20alpha-HSD activity was observed not only in the liver, but also in the kidney and lung. In liver cytosol, both NADPH and NADH were effective as cofactors for 20alpha-HSD activity, although NADPH was better than NADH for the enzyme activity. On the other hand, 20alpha-HSD activity in kidney cytosol required only NADPH as a cofactor. No significant sex-related difference of 20alpha-HSD activity was observed in liver and kidney cytosols. Flavonoids have been reported to inhibit the biosynthesis and metabolism of steroids. However, little is known about inhibitory effects of flavonoids on 20alpha-HSD activity. Thus, the effects of 16 flavonoids on 20alpha-HSD activity were examined, using liver cytosol of male mice. Among flavonoids tested, fisetin, apigenin, naringenin, luteolin, quercetin and kaempferol exhibited high inhibitory potencies for the 20alpha-HSD activity. We propose the possibility that these flavonoids augment progesterone signaling by inhibiting potently 20alpha-HSD activity in non-reproductive tissues.     

Vacuolar citrate/H+ symporter of citrus juice cells

We have isolated a cDNA, designated Citrus sinensis citrate transporter 1 CsCit1 encoding a novel vacuolar citrate/symporter. Immunoblots using antibodies raised against CsCit1 showed that the protein is localized to the juice sac cell vacuoles. The highest expression of CsCit1 and the amount of protein in the juice sac cell vacuoles coincided with the developmental stage at which the vacuolar citrate content began declining with the concomitant increase in vacuolar pH. Vacuoles from Sacharomyces cereviseae expressing CsCit1 displayed a citrate-dependent H⁺ efflux, and our results clearly demonstrate that CsCit1 is able to mediate the electroneutral co-transport of H⁺ and citrate ions, since the citrate-dependent H⁺ fluxes are not affected by changing the electrical potential difference across the tonoplast. The roles of CsCit1 in mediating citrate efflux from the vacuole and on citric acid homoestasis in Citrus juice sac cells are discussed. T. Shimada and R. Nakano contributed equally to this work.     

Mammary tumorigenesis in ApcMin/+ mice is enhanced by X irradiation with a characteristic age dependence

The Apc(Min/+) (Min) mouse is genetically predisposed to both intestinal and mammary tumorigenesis. We investigated age-related changes in the susceptibility of mice (before, during and after puberty) to radiation-induced mammary tumorigenesis using this model. Female Min and wild-type mice having the C57BL/6J background were irradiated with 2 Gy of X rays at 2, 5, 7 and 10 weeks and killed humanely at 18 weeks of age. Min mice irradiated at 7-10 weeks of age (after puberty) developed mammary tumors with squamous metaplasia, whereas their wild-type littermates did not. Interestingly, irradiation of Min mice at 2-5 weeks (before and during puberty, respectively) did not induce mammary tumors but rather cystic nodules with metaplasia. The mammary tumors exhibited increased nuclear beta-catenin protein and loss of the wild-type Apc allele. Our results show that susceptibility to radiation-induced mammary tumorigenesis increases after puberty in Min mice, suggesting that the tumorigenic effect of ionizing radiation targets the lobular-alveolar progenitor cells, which increase in number with age and are controlled by beta-catenin signaling.     

Vasodilative effect of perillaldehyde on isolated rat aorta

The vasodilative effect of perillaldehyde, one of the major oil components in Perilla frutescens BRITTON, was studied using isolated rat aorta. Perillaldehyde at final concentrations of 0.01 to 1 mM showed dose-dependent relaxation of the aorta contracted by treatment with prostaglandin F2alpha or norepinephrine. Neither the presence of NG-nitro-L-arginine methyl ester nor removal of the aortic endothelium affected the vasodilatation, suggesting that perillaldehyde exerts a direct effect on vascular smooth muscle cells. The vasodilative effect of perillaldehyde was not inhibited by pretreatment with a beta-adrenergic receptor blocker (propranolol), an inhibitor of phosphodiesterase (theophylline), a delayed rectifier K+ channel blocker (tetraethylammonium chloride), or an ATP-sensitive K+ channel blocker (glibenclamide). However, perillaldehyde showed contrasting effects on vasodilatation of the aorta contracted by an influx of extracellular Ca2+ - perillaldehyde caused little vasodilatation on the aorta contracted by the Ca2+ ionophore A23187, while it inhibited the vasoconstriction induced by treatment with high-concentration K+, which dominantly opened the voltage-dependent Ca2+ channel. These results suggest that the vasodilative effect of perillaldehyde is derived from blocking the Ca2+ channels.     

Bombyx small RNAs: genomic defense system against transposons in the silkworm, Bombyx mori

Selfish genetic elements called transposons can insert themselves at new locations in host genomes to modify gene structure and alter gene expression. Expansion of transposons can occur when novel transposition events are transmitted to subsequent generations after germline hopping. Therefore, organisms seem likely to have evolved defense mechanisms to silence transposons in the germline. Recently, small RNAs interacting with Piwi proteins (piwi-interacting RNAs: piRNAs) have been demonstrated to be involved in genomic defense mechanism against transposons. Here, we show that piRNA-like small RNAs are present abundantly in the Bombyx ovary. We cloned 38,493 kinds of Bombyx small RNA from the ovary and performed functional characterization. Bombyx small RNAs showed a unimodal length distribution with a peak at 28nt and a strong bias for U at the 5' end. We found that 12,869 kinds of Bombyx small RNAs were associated with transposons or repetitive sequences. We classified them as repeat-associated small interfering RNAs (rasiRNAs), a subclass of piRNAs. Notably, antisense rasiRNAs have a strong bias toward U at 5' ends; in contrast, sense rasiRNAs have a strong bias toward A at nucleotide position 10, indicating that the piRNA amplification loop proposed in Drosophila is evolutionarily conserved in Bombyx. These results suggest that Bombyx small RNAs regulate transposon activity.