Comparative studies of embryotoxic action of ethylenethiourea in rat whole embryo and embryonic cell culture

The effects of ethylenethiourea (ETU) were investigated using rat (Wistar-imamichi) embryos cultured from days 11 to 13 of gestation or cultured rat embryonic cells extracted on day 11. Malformations in cultured embryos at the concentration of 30 micrograms/ml of ETU were found in the head and tail, which were severely affected, as well as the limb and face. All embryos exposed to 150 and 300 micrograms/ml of ETU had malformed heads, tails, limbs, and facial configurations. Protein contents of the cultured embryos were decreased dose-dependently at the concentrations ranging from 30 to 300 micrograms/ml. In the histological studies of the cultured embryos with ETU, thinner neuroepithelium in head was observed. In the embryonic cells extracted on day 11 of gestation, ETU dose-dependently inhibited the differentiation of midbrain (MB) cells into neurons and that of limb bud (LB) cells into chondrocytes at the concentrations ranging from 30 to 600 micrograms/ml of ETU. The concentrations of ETU that inhibited the production of differentiated foci by 50% (IC50) were 170 micrograms/ml in LB cells of day 11, and greater than 600 micrograms/ml in LB cells on day 12 of development. Therefore, differentiation of MB cells was more sensitive to ETU than the differentiation of LB cells. These results indicated that there was a reasonable correlation of ETU induced changes in cultured whole embryos and embryonic cells.     

Nonradioactive labeling with chemically modified cytosine tails by the polymerase chain reaction.

We developed a modified nonradioactive method for the detection of DNA. This method makes use of the polymerase chain reaction for preparation of probes; that is, a DNA fragment inserted in the polylinker region of an M13 or pUC vector is amplified with primers that have a modified cytosine tail at the 5' terminus (C-tailed primers). By this method, large amounts of labeled probes can be obtained easily. After hybridization, modified cytosine tails can be detected immunologically. DNA labeled by this method could be used in plaque hybridization. We could detect 0.05 pg of dot-blotted labeled DNA in 30 min with an enzyme-catalyzed chemiluminescence reaction.     

Effects of 22K or 20K human growth hormone on lipolysis, leptin production in adipocytes in the presence and absence of human growth hormone binding protein.

OBJECTIVE AND METHOD: We studied the effects of human growth hormone (hGH) on leptin production and lipolysis stimulation in the presence or absence of human growth hormone binding protein (hGHBP) using 3T3- L1-hGHR adipocytes which efficiently express human growth hormone receptor. RESULTS AND CONCLUSION: It was clarified that (1) hGH decreases leptin secretion after hGH-induced lipolysis stimulation, and (2) the reduction of leptin production and lipolysis stimulation by 22K hGH was attenuated with hGHBP, whereas that by 20K hGH, which is a naturally occurring isoform of 22K hGH, was not affected with hGHBP.     

Distribution of F-actin during mouse facial morphogenesis and its perturbation with cytochalasin D using whole embryo culture.

Histological and experimental studies were performed in mouse embryos to elucidate possible roles of actin filaments in the nasal epithelium during facial morphogenesis. C57BL/6 mouse embryos (8.5-11.5 days of gestation) were fixed and frozen sections were stained with rhodamine-phalloidin. Before formation of the nasal placode, there was no specific localization of F-actin. After the nasal placode was formed, intense staining of F-actin was observed at the apical side of the placode. Conversely, it was located at the basal side of the epithelium of developing nasal prominences. By using the whole embryo culture system, perturbation experiments were conducted with cytochalasin D (CD), which inhibits the polymerization of actin filaments. When day-10 embryos were exposed to CD at several concentrations for 24 hr, fusion of nasal prominences was inhibited in a dose-dependent manner. Treatment with a high dose of CD for 2 hr also prevented the same development irreversibly. In contrast, when day-9 embryos were exposed to CD at several concentrations for 24 hr, invagination of the nasal placode was not perturbed at all. The results suggest that apical F-actin plays an essential role in maintaining the close apposition state of the nasal prominences and in the following fusion. During the invagination stage, F-actin might be important in maintaining the epithelial structure, but is not crucial to the initiation of placode invagination.     

LIRF, a gene induced during hippocampal long-term potentiation as an immediate-early gene, encodes a novel RING finger protein.

We describe here an LTP-induced gene, LIRF, which encodes a novel protein with RING finger and B30.2 domains in its N- and C-terminal portions, respectively. Each domain is encoded by one exon, suggesting that the organization of the gene was generated by exon shuffling. The amino acid sequences of the mouse, rat, and human LIRF proteins are highly conserved and contain a putative PEST sequence. LIRF is an immediate-early gene in hippocampal granule cells, and its expression is upregulated immediately after the induction of long-lasting long-term potentiation at perforant pathway-dentate gyrus synapses and returns to the basal level within 150 min. A heterologously expressed LIRF protein fused to EGFP localizes specifically to the cytoplasm in COS-7 cells. These findings suggest a possible involvement of LIRF in a limited, early phase of synaptic plasticity.