To investigate possible involvement of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 (extracellular signal-regulated kinases) in somatic cell mitosis, we have used indirect immunofluorescence with a highly specific phospho-MAP kinase antibody and found that a portion of the active MAP kinase is localized at kinetochores, asters, and the midbody during mitosis. Although the aster labeling was constant from the time of nuclear envelope breakdown, the kinetochore labeling first appeared at early prometaphase, started to fade during chromosome congression, and then disappeared at midanaphase. At telophase, active MAP kinase localized at the midbody. Based on colocalization and the presence of a MAP kinase consensus phosphorylation site, we identified the kinetochore motor protein CENP-E as a candidate mitotic substrate for MAP kinase. CENP-E was phosphorylated in vitro by MAP kinase on sites that are known to regulate its interactions with microtubules and was found to associate in vivo preferentially with the active MAP kinase during mitosis. Therefore, the presence of active MAP kinase at specific mitotic structures and its interaction with CENP-E suggest that MAP kinase could play a role in mitosis at least in part by altering the ability of CENP-E to mediate interactions between chromosomes and microtubules.

A large number of hDAF transgenic pigs to be used for xenotransplantation research were generated by using sperm-mediated gene transfer (SMGT). The efficiency of transgenesis obtained with SMGT was much greater than with any other method. In the experiments reported, up to 80% of pigs had the transgene integrated into the genome. Most of the pigs carrying the hDAF gene transcribed it in a stable manner (64%). The great majority of pigs that transcribed the gene expressed the protein (83%). The hDAF gene was transmitted to progeny. Expression was stable and found in caveolae as it is in human cells. The expressed gene was functional based on in vitro experiments performed on peripheral blood mononuclear cells. These results show that our SMGT approach to transgenesis provides an efficient procedure for studies involving large animal models.SMGT ͉ hyperacute rejection ͉ swine

The effect of cytochalasin B (Cyt-B; 3 and 6 micrograms/ml; for the last 28 h) on micronuclei (MN) was studied in 72-h purified lymphocyte cultures of three male donors. The frequency of MN was much higher in multinucleate cells (mean 100-204 MN per 1000 cells) than in binucleate cells (mean 8.2-21.0 MN per 1000 cells), tetranucleate cells containing more MN than trinucleate cells. The presence of whole chromosomes in the MN was studied in two separate experiments by immunofluorescence using antikinetochore (CREST) serum and by a centromeric alphoid DNA oligomer probe (in situ hybridization, ISH). In the tri- and tetra-nucleate cells produced by Cyt-B, MN were clearly more often kinetochore-positive (K+) (mean 82-86%) and centromere-positive (C+) (mean 73-83%) than in mononucleate cells of cultures containing no Cyt-B (mean 63% for CREST and 50% for ISH), indicating that most of the excess MN in the multinucleate cells were due to whole chromosomes. The binucleate lymphocytes had about as high prevalence of K+MN (mean 79-84%) as the tri- and tetra-nucleate cells, despite their low MN count. Also in the ISH analysis, the majority of MN in binucleate cells were positively stained (mean 58-62%). If it is assumed that the extra labelled MN are due to Cyt-B, the present findings suggest that Cyt-B could be responsible for approximately 45-57% (CREST data) or approximately 17-23% (ISH data) of MN in binucleate cells.(ABSTRACT TRUNCATED AT 250 WORDS)