Germinal vesicle material is essential for nucleus remodeling after nuclear transfer

Successful cloning by nuclear transfer has been reported with somatic or embryonic stem (ES) cell nucleus injection into enucleated mouse metaphase II oocytes. In this study, we enucleated mouse oocytes at the germinal vesicle (GV) or pro-metaphase I (pro-MI) stage and cultured the cytoplasm to the MII stage. Nuclei from cells of the R1 ES cell line were injected into both types of cytoplasm to evaluate developmental potential of resulting embryos compared to MII cytoplasmic injection. Immunocytochemical staining revealed that a spindle started to organize 30 min after nucleus injection into all three types of cytoplasm. A well-organized bipolar spindle resembling an MII spindle was present in both pro-MI and MII cytoplasm 1 h after injection with ES cells. However, in the mature GV cytoplasm, chromosomes were distributed throughout the cytoplasm and a much bigger spindle was formed. Pseudopronucleus formation was observed in pro-MI and MII cytoplasm after activation treatment. Although no pronucleus formation was found in GV cytoplasm, chromosomes segregated into two groups in response to activation. Only 8.1% of reconstructed embryos with pro-MI cytoplasm developed to the morula stage after culture in CZB medium. In contrast, 53.5% of embryos reconstructed with MII cytoplasm developed to the morula/blastocyst stage, and 5.3% of transferred embryos developed to term. These results indicate that GV material is essential for nucleus remodeling after nuclear transfer.     

A single cell density-sensing factor stimulates distinct signal transduction pathways through two different receptors

In Dictyostelium discoideum, cell density is monitored by levels of a secreted protein, conditioned medium factor (CMF). CMFR1 is a putative CMF receptor necessary for CMF-induced G protein-independent accumulation of the SP70 prespore protein but not for CMF-induced G protein-dependent inositol 1,4,5-trisphosphate production. Using recombinant fragments of CMF, we find that stimulation of the inositol 1,4,5-trisphosphate pathway requires amino acids 170-180, whereas SP70 accumulation does not, corroborating a two-receptor model. Cells lacking CMFR1 do not aggregate, due to the lack of expression of several important early developmentally regulated genes, including gp80. Although many aspects of early developmental cAMP-stimulated signal transduction are mediated by CMF, CMFR1 is not essential for cAMP-stimulated cAMP and cGMP production or Ca(2+) uptake, suggesting the involvement of a second CMF receptor. Exogenous application of antibodies against either the region between a first and second or a second and third possible transmembrane domain of CMFR1 induces SP70 accumulation. Antibody- and CMF-induced gene expression can be inhibited by recombinant CMFR1 corresponding to the region between the first and third potential transmembrane domains, indicating that this region is extracellular and probably contains the CMF binding site. These observations support a model where a one- or two-transmembrane CMFR1 regulates gene expression and a G protein-coupled CMF receptor mediates cAR1 signal transduction.     

ARFGAP1 promotes the formation of COPI vesicles,suggesting function as a component of the coat

The role of GTPase-activating protein (GAP) that deactivates ADP-ribosylation factor 1 (ARF1) during the formation of coat protein I (COPI) vesicles has been unclear. GAP is originally thought to antagonize vesicle formation by triggering uncoating, but later studies suggest that GAP promotes cargo sorting, a process that occurs during vesicle formation. Recent models have attempted to reconcile these seemingly contradictory roles by suggesting that cargo proteins suppress GAP activity during vesicle formation, but whether GAP truly antagonizes coat recruitment in this process has not been assessed directly. We have reconstituted the formation of COPI vesicles by incubating Golgi membrane with purified soluble components, and find that ARFGAP1 in the presence of GTP promotes vesicle formation and cargo sorting. Moreover, the presence of GTPgammaS not only blocks vesicle uncoating but also vesicle formation by preventing the proper recruitment of GAP to nascent vesicles. Elucidating how GAP functions in vesicle formation, we find that the level of GAP on the reconstituted vesicles is at least as abundant as COPI and that GAP binds directly to the dilysine motif of cargo proteins. Collectively, these findings suggest that ARFGAP1 promotes vesicle formation by functioning as a component of the COPI coat.     

P2P-R protein overexpression restricts mitotic progression at prometaphase and promotes mitotic apoptosis

Mitotic cells show a tenfold increase in immunoreactive P2P-R protein. During mitosis, the distribution of P2P-R protein also changes from a primary nucleolar localization in interphase cells to the periphery of chromosome in mitotic cells. These findings suggest that P2P-R might serve a functional role in mitosis. To test this possibility, human Saos2 cells were stably transfected with P2P-R DNA constructs and the biological effects of P2P-R overexpression were evaluated. Overexpression of near full-length P2P-R was found to have paradoxical effects on the relationship between proliferation and mitosis in the nine Saos2 cell clones that were studied. A significant repression in the population doubling rates was observed in all nine clones even though a significant increase in the frequency of easily detached cells with a mitotic morphology was apparent. Flow cytometric analysis confirmed that greater than two thirds of the cells with a mitotic morphology had a 4n DNA content. Confocal microscopy further established that 85% of the mitotic cell population had prometaphase characteristics suggesting that P2P-R overexpression restricts mitotic progression at prometaphase. Many cells with a mitotic morphology also showed signs of apoptosis with prominent cell surface blebs. Confocal microscopy confirmed that 25-40% of such mitotic cells were apoptotic with chromosomal abnormalities and cell surface blebbing. In association with mitotic apoptosis, P2P-R protein appears to dissociate from the periphery of chromosomes and localize in the cytoplasm and in cell surface blebs. The presence of P2P-R in cell surface blebs was confirmed by analysis of highly enriched populations of apoptotic cell surface blebs wherein Western blotting documented the presence of 250 kDa P2P-R. These results therefore suggest that P2P-R overexpression promotes both prometaphase arrest in mitosis and mitotic apoptosis.     

Evaluation of the natural refuge function for Helicoverpa arnigera (Lepidoptera: Noctuidae) within Bacillus thuringiensis transgenic cotton growing areas in north China

The density of Helicoverpa armigera (Hübner) populations on Bacillus thuringiensis Berliner (Bt) transgenic cotton, corn, peanut, and soybean; differences in its development on Bt cotton and common (nontransgenic) cotton; and the potential for mating among populations from Bt cotton fields and other crop fields were investigated in the suburbs of Xinxiang City (Henan Province) and Langfang City (Hebei Province) in the southern and northern parts of north China, respectively. Although development of H. armigera on Bt cotton was much slower than on common cotton, there was a still high probability of mating between populations from Bt cotton and other sources due to the scattered emergence pattern of H. armigera adults, and overlap of the second and third generations. In a cotton and corn growing region, early and late planted corn provided suitable refugia for the third and fourth generations of H. armigera, but not for the second generation. In a cotton and soybean/ peanut mix system, noncotton crops provided a natural refugia from the second- to fourth-generation H. armigera, but function of the refuge would closely depend on the proportion of Bt cotton. Consequently, it may be necessary to compensate the original mixed cropping patterns in different areas for delaying resistance development of H. armigera to Bt cotton.     

Molecular cloning and characterization of a novel human protein phosphatase,LMW-DSP3

Reversible phosphorylation is recognized to be a major mechanism for the control of intracellular events in eukaryotic cells. From a human fetal brain cDNA library, we isolated a cDNA clone encoding a novel dual specificity protein phosphatase, which showed 88% identity with previously reported mouse LMW-DSP3 at the amino acid level. The deduced protein had a single dual-specificity phosphatase catalytic domain, and lacked a cdc25 homology domain. LMW-DSP3 was expressed in the heart, lung, liver, and pancreas, and the expression level in the pancreas was highest. The LMW-DSP3 gene was located in human chromosome 2q32, and consisted of five exons spanning 21kb of human genomic DNA. LMW-DSP3 fused to GST showed phosphatase activity towards p-nitrophenyl phosphate which was optimal at pH 7.0 and 40 degrees C, and the activity was enhanced by Ca(2+) and Mn(2+). The phosphatase activity of LMW-DSP3 was inhibited by orthovanate. LMW-DSP3 showed phosphatase activity toward oligopeptides containing pSer/Thr and pTyr, indicating that LMW-DSP3 is a protein phosphatase with dual substrate specificity.     

Morphologies and phylogenetic classification of cellulolytic myxobacteria

The evolutionary distances of the 16S rDNA sequences in cellulolytic myxobacteria are less than 3%, which units all the strains into a single genus, Sorangium. The size of myxospores and the shape of sporangioles, rather than fruiting body colors or swarm morphologies are consistent with the changes of the 16S rDNA sequences. It is suggested that there are at least two species in the genus Sorangium: one includes strains with small myxospores and spherical sporangioles, and the color of the fruiting bodies is normally orange or brown, though sometimes yellow or black. The second species has large myxospores, polyhedral sporangioles with many inter-cystic substrates, and normally deep brown to black color.     

Specific regulation of lipocalin-type prostaglandin D synthase in mouse heart by estrogen receptor beta

Estrogens have important physiological roles in the cardiovascular system. We use DNA microarray technology to study the molecular mechanism of estrogen action in the heart and to identify novel estrogen-regulated genes. In this investigation we identify genes that are regulated by chronic estrogen treatment of mouse heart. We present our detailed characterization of one of these genes, lipocalin-type prostaglandin D synthase (L-PGDS). Northern and Western blot analysis revealed that L-PGDS was induced both by acute and chronic estrogen treatment. Northern blot analysis, using estrogen receptor (ER)-disrupted mice, suggests that L-PGDS is specifically induced by ERbeta in vivo. In further support of ERbeta-selective regulation, we identify a functional estrogen-responsive element in the L-PGDS promoter, the activity of which is up-regulated by ERbeta, but not by ERalpha. We demonstrate that a one-nucleotide change (A to C) in the L-PGDS estrogen-responsive element affects receptor selectivity.