Construction of alpha-alpha domains structure in recombinant monkey metallothionein-1

The differences in metal-thiolate coordination and reactivity of mammalian metallothionein (MT) domains are closely related to their distinct, highly conservative cysteine number and position. Monkey metallothionein-1, containing a beta-domain with Cd(3)S(9) cluster and an alpha-domain with Cd(4)S(11) cluster, was used to evaluate the role of cysteine residues in the formation of MT's metal-thiolate clusters. The possible influence of cysteine residues on the binding and stability of MT domains has been examined with the metallothionein mutants: N4C, T27C and N4C/T27C, which possess ten or eleven cysteine residues in the re-constructed beta-domain, respectively. Assisted by study of UV, CD and electrospray ionization mass spectroscopy (ESI-MS) and their reactivity with DTNB (5,5'-dithiobis (2-nitrobenzoic acid)), we found that besides the original alpha-domain, some kinds of new domain containing 4-cadmium-thiolate clusters were formed in the N4C and N4C/T27C mutants of mkMT1. These new domains displayed metal binding and kinetic reactivity with DTNB similar to the alpha-domain. However, the thermal stability of the mutants was less stable than that of WT mkMT1. This might result from the disturbance of the inter-domains hydrogen bonds and of the non-cysteine amino acid residue arrangement.     

Complex physiological and molecular processes underlying root gravitropism

Gravitropism allows plant organs to guide their growth in relation to the gravity vector. For most roots, this response to gravity allows downward growth into soil where water and nutrients are available for plant growth and development. The primary site for gravity sensing in roots includes the root cap and appears to involve the sedimentation of amyloplasts within the columella cells. This process triggers a signal transduction pathway that promotes both an acidification of the wall around the columella cells, an alkalinization of the columella cytoplasm, and the development of a lateral polarity across the root cap that allows for the establishment of a lateral auxin gradient. This gradient is then transmitted to the elongation zones where it triggers a differential cellular elongation on opposite flanks of the central elongation zone, responsible for part of the gravitropic curvature. Recent findings also suggest the involvement of a secondary site/mechanism of gravity sensing for gravitropism in roots, and the possibility that the early phases of graviresponse, which involve differential elongation on opposite flanks of the distal elongation zone, might be independent of this auxin gradient. This review discusses our current understanding of the molecular and physiological mechanisms underlying these various phases of the gravitropic response in roots.     

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.