A Modified Murine Embryonic Stem Cell Test for Evaluating the Teratogenic Effects of Drugs on Early Embryogenesis

Mammalian fetal development is easily disrupted by exogenous agents, making it essential to test new drug candidates for embryotoxicity and teratogenicity. To standardize the testing of drugs that might be used to treat pregnant women, the U.S. Food and Drug Administration (FDA) formulated special grade categories, labeled A, B, C, D and X, that define the level of risk associated with the use of a specific drug during pregnancy. Drugs in categories (Cat.) D and X are those with embryotoxic and/or teratogenic effects on humans and animals. However, which stages of pregnancy are affected by these agents and their molecular mechanisms are unknown. We describe here an embryonic stem cell test (EST) that classifies FDA pregnancy Cat.D and Cat.X drugs into 4 classes based on their differing effects on primitive streak formation. We show that ~84% of Cat.D and Cat.X drugs target this period of embryogenesis. Our results demonstrate that our modified EST can identify how a drug affects early embryogenesis, when it acts, and its molecular mechanism. Our test may thus be a useful addition to the drug safety testing armamentarium.     

Feeding behaviour of the ladybeetle,Pseudoscymnus kurohime

A laboratory study was made of the feeding behaviour of the ladybeetlePseudoscymnus kurohime (Miyatake) when attacking the sugar cane woolly aphidCeratovacuna lanigera Zehntner. The 1st-instar ladybeetle larva was smaller than the 1st instar aphid nymph. All larval stages of the ladybeetle sucked out the body fluids of aphids and left their emptied corpses. The 1st, 2nd, and 3rd instar ladybeetle larvae mostly attacked 1st instar aphids, whereas the 4th-instar ladybeetle larvae attacked all stages of aphids. Ladybeetle adults ate mostly 1st-instar aphids. Young larvae attacked aphids in several different ways: (1) They crawled under an aphid, seized it by its underside and lifted it up. (2) They attacked new born nymphs at birth or shortly afterwards. (3) They fed on an aphid that had been captured by an older larva. The larvae preferred to seize with their mandibles the head or thorax of an aphid, while adults seized their prey by the abdomen. When attacked by an adult, 82% of the aphids secreted droplets from their abdominal cornicles, whereas only 7.2–12% secreted droplets when attacked by larvae. The 4th instar larvae more voracious than the younger larvae.     

Interaction between substrate and oxygen ligand responsible for effective O-O bond cleavage in bovine cytochrome P450 steroid 21-hydroxylase proved by Raman spectroscopy

We investigated structural and functional properties of bovine cytochrome P450 steroid 21-hydroxylase (P450c21), which catalyzes hydroxylation at C-21 of progesterone and 17alpha-hydroxyprogesterone. The uncoupled H(2)O(2) formation was higher in the hydroxylation of progesterone (26% of NADPH consumed) than that of 17alpha-hydroxyprogesterone (15% of NADPH consumed), indicating that 17alpha-hydroxyprogesterone can better facilitate the O-O bond scission. In relation to this, it is noted that the O-O stretching mode (nu(O-O)) of the oxygen complex of P450c21 was sensitive to the substrate; the progesterone- or 17alpha-hydroxyprogesterone-bound enzyme gave single (at 1137 cm(-1)) or split nu(O-O) bands (at 1124 and 1138 cm(-1)), respectively, demonstrating the presence of two forms for the latter. In contrast to nu(O-O), no corresponding difference was observed for the Fe-O(2) stretching mode between two different substrate-bound forms. The Fe-S(Cys) stretching mode in the ferric state was also identical (349 cm(-1)) for each substrate-bound form, suggesting that modulation through the axial thiolate by the substrate is unlikely. Therefore, it is deduced that the hydroxyl group at C-17 of 17alpha-hydroxyprogesterone forms a hydrogen bond with the terminal oxygen atom of the FeOO complex in one form, yielding a lower nu(O-O) frequency with higher reactivity for O-O cleavage, whereas the other form in which the substrate does not provide a hydrogen bond to the oxygen ligand is essentially the same between the two kinds of substrates. In the hydrogen-bonded species, the substrate changes the geometry of the FeOO moiety, thereby performing the hydroxylation reaction more effectively in 17alpha-hydroxyprogesterone than in progesterone.     

GLP-1 receptor signaling protects pancreatic beta cells in intraportal islet transplant by inhibiting apoptosis

To clarify the cytoprotective effect of glucagon-like peptide-1 receptor (GLP-1R) signaling in conditions of glucose toxicity in vivo, we performed murine isogenic islet transplantation with and without exendin-4 treatment. When a suboptimal number of islets (150) were transplanted into streptozotocin-induced diabetic mice, exendin-4 treatment contributed to the restoration of normoglycemia. When 50 islets expressing enhanced green fluorescent protein (EGFP) were transplanted, exendin-4 treatment reversed loss of both the number and mass of islet grafts one and 3 days after transplantation. TUNEL staining revealed that exendin-4 treatment reduced the number of apoptotic beta cells during the early posttransplant phase, indicating that GLP-1R signaling exerts its cytoprotective effect on pancreatic beta cells by inhibiting their apoptosis. This beneficial effect might be used both to ameliorate type 2 diabetes and to improve engraftment rates in clinical islet transplantation.     

Production of transgenic rats via lentiviral transduction and xenogeneic transplantation of spermatogonial stem cells

Spermatogonial stem cells (SSCs) continue to proliferate in the testis to support spermatogenesis throughout life, which makes them ideal targets for germline modification. Although recent success in the production of transgenic and knockout animals using SSCs has opened up new experimental possibilities, several problems, including the low efficiency of germ cell transplantation and poor fertility rates, remain to be resolved. In the present study, we took advantage of the xenogeneic transplantation to resolve these problems. Rat SSCs were transduced in vitro with a lentiviral vector that expressed enhanced green fluorescent protein (EGFP), and then transplanted into the testes of immunodeficient mice. The transduced rat SSCs produced EGFP-expressing spermatogenic cells, and microinsemination using these cells was used to produce transgenic rats, which stably transmitted the transgene to the next generation. Thus, xenogeneic transplantation is a powerful strategy for transgenesis, and smaller xenogeneic surrogates can be used for male germline modification using SSCs.