Conservation Potential Trough In Vitro Regeneration of Two Threatened Medicinal Plants Ungernia sewertzowii and U. victoris.

Ungernia sewertzowii (US) and U. victoris (UV) are medicinal plants and sources of biologically active compounds for pharmaceutical needs. The leaves of US contain 0.29-0.81% sum of alkaloids with a predominance of lycorine, which is 0.04-0.46% in leaves and 0.15-0.38% in bulbs. Lycorine is used to treat acute and chronic bronchitis. The leaves of UV contain 0.27-0.71% sum of alkaloids with a predominance of galanthamine-0.13-1.15%. Galanthamine is used to treat mild-to-moderate dementia (Alzheimer's disease). The natural populations of US and UV are in danger as sources of income for local people. To resolve this problem, two protocols for microclonal propagation were developed to replace natural raw materials with in vitro regenerated plants. Callusogenesis of US and UV was induced on Murashige and Skoog (MS) nutrient media with 2.4D (0.5 mg/L) in combination with BAP (0.5 mg/L), Kin (0.5 mg/L), or Zea (0.5 mg/L). Direct (for US) and indirect (for US and UN) organogenesis were observed on MS with BAP (0.5 mg/L) or Kin (0.5 mg/L) in combination with IAA (0.5 mg/L) or NAA (0.5 mg/L). Direct organogenesis resulted in 3-5 bulbs of US on one explant; indirect organogenesis resulted in up to 100-150 bulbs of US and UV on one explant within 6 months, or five to six subcultures after transferring the callus to the nutrient medium. The tissue cultures of US and UV were characterized by very low data on antioxidant activity based on IC50 values for DPPH and ABTS radical scavenging activities, whereas in vitro regenerated plants (leaves and bulbs) had higher data. We concluded that in vitro regenerated plants are valuable sources of lycorine and galanthamine, which allow the protection of the natural populations of these two species from extinction.

Differential expression of uterine calcium transporter 1 and plasma membrane Ca2+ ATPase 1b during rat estrous cycle.

Calcium-related proteins include the calcium transporters 1 and 2 (CaT1 and CaT2), plasma membrane Ca2+-ATPase 1b (PMCA1b), and calbindin-D9k and -D28k. The expression of CaT1 and PMCA1b and their potential roles in the uterine tissue remain to be clarified. Thus, in the present study, the expression patterns of CaT1 and PMCA1b were examined to predict their roles in rat uterus during the estrous cycle. Both CaT1 and PMCA1b mRNAs were detected in rat uterus. Uterine CaT1 mRNA was highly expressed at diestrus compared with proestrus, whereas PMCA1b expression was not altered during the estrus cycle. To evaluate the sex steroids involved in uterine CaT1 mRNA regulation, 17beta-estradiol (E2) and/or progesterone (P4) were injected into immature rats. Treatment with P4 or E2 plus P4 resulted in an increase in CaT1 mRNA, but a synergetic effect of E2 plus P4 was not detected. Uterine CaT1 mRNA was induced by P4 in a time- and dose-dependent manner, with maximal transcript detected 12 h after the final P4 injection. Treatment with RU486, a progesterone receptor (PR) antagonist, completely blocked P4-induced CaT1 mRNA, indicating that P4 regulates CaT1 mRNA expression via a PR-mediated pathway. In addition, CaT1 mRNA was expressed in uterine endometrium and glandular endometrium at diestrus in P4-treated rats. Together, these results suggest that CaT1 is regulated by P4 at diestrus via a PR-dependent pathway.

Estrogen receptor alpha pathway is involved in the regulation of Calbindin-D9k in the uterus of immature rats.

It has been demonstrated in our previous studies that Calbindin-D9k (CaBP-9k) is a potent biomarker for screening estrogen-like chemicals in the rat model. Although treatments with 17beta-estradiol (E2) and endocrine disrupting compounds resulted in the up-regulation of uterine CaBP-9k, the mechanism of CaBP-9k induction by these compounds through two subtypes of estrogen receptors (ERalpha and ERbeta) is unclear. Thus, in the present study, immature rats were treated with propyl pyrazole triol (PPT, an ERalpha-selective ligand), diarylpropionitrile (DPN, an ERbeta-selective ligand), E2, or dimethyl sulfoxide (DMSO, a vehicle control) for three days in order to clarify which subtype of ER is involved in the uterine CaBP-9k induction. Following injection with these ER ligands, uterine CaBP-9k expression was analyzed by Northern blot and immunoblot assays. Uterine CaBP-9k expression is mainly mediated by PPT in a dose- and time-dependent manner in immature rats, whereas no significant alteration of the uterine CaBP-9k gene was observed after DPN treatment. In addition, an estrogenicity of PPT in inducing CaBP-9k expression was completely blocked by the anti-estrogen ICI 182,780, implying that uterine CaBP-9k is solely induced by ERalpha. A single treatment with PPT rapidly increased the protein levels of ERalpha and PR, an E2-mediated gene, in these tissues. Taken together, these results indicate that uterine CaBP-9k is induced by E2 and endocrine disrupting chemicals via the ERalpha pathway, but not ERbeta, in the uterus of immature rats.

Effect of genistein as a selective estrogen receptor beta agonist on the expression of Calbindin-D9k in the uterus of immature rats.

Genistein, a phytoestrogen possessing a high affinity for estrogen receptor beta (ERbeta), is of increasing interest because of its possible influence on the physiology of mammalian reproductive tracts. Although estrogen has been demonstrated to regulate Calbindin-D9k (CaBP-9k) in the rat uterus as with other calcium binding proteins, the role of ERbeta on the modulation of CaBP-9k remains to be elucidated. To elucidate the effect of genistein as a selective ERbeta agonist on uterine expression of CaBP-9k mRNA and protein, immature female rats were injected with genistein daily for three consecutive days in a dose-dependent (0.4, 4, and 40 mg/kg/day) and time-dependent (40 mg/kg/day; 3, 6, 12, 24, 48, and 72 h) manner. Then, the expression of CaBP-9k mRNA and protein was analyzed by Northern hybridization and Western blot, respectively, in the absence or presence of ICI 182,780 (ICI), an estrogen antagonist. In addition, the protein levels of ERalpha and ERbeta and mRNA level of progesterone receptor (PR) were further measured following genistein treatment to elucidate which of ERs is involved in CaBP-9k modulation. In a dose-dependent experiment, the highest dose of genistein (40 mg/kg/day) for 3 days significantly induced uterine CaBP-9k protein as 17beta-estradiol (E2) did. In addition, its maximal mRNA expression was observed at 3 and 6 h, and it returned to control level at 24 h in a time-dependent experiment. In parallel with its mRNA level, the protein level of CaBP-9k was significantly induced by genistein at 3 h and sustained up to 48 h. The pretreatment with ICI, followed by genistein or E2, completely blocked genistein- and E2-induced CaBP-9k protein in the uterus of immature rats. Interestingly, genistein was demonstrated to induce ERalpha protein, but not ERbeta and PR mRNA, an E2-responsive gene, in this tissue. These results imply that genistein, an ERbeta ligand, may regulate CaBP-9k gene through ERalpha pathway. Taken together, the present study demonstrated that genistein enhanced CaBP-9k gene via ERalpha in the uterus of immature rats, suggesting that ERalpha may be a key mediator in uterine CaBP-9k gene induction in immature rats.