Proliferating human cells hypomorphic for origin recognition complex 2 and pre-replicative complex formation have a defect in p53 activation and Cdk2 kinase activation

The Origin Recognition Complex (ORC) is a critical component of replication initiation. We have previously reported generation of an Orc2 hypomorph cell line (Delta/-) that expresses very low levels of Orc2 but is viable. We have shown here that Chk2 is phosphorylated, suggesting that DNA damage checkpoint pathways are activated. p53 was inactivated during the derivation of the Orc2 hypomorphic cell lines, accounting for their survival despite active Chk2. These cells also show a defect in the G1 to S-phase transition. Cdk2 kinase activation in G1 is decreased due to decreased Cyclin E levels, preventing progression into S-phase. Molecular combing of bromodeoxyuridine-labeled DNA revealed that once the Orc2 hypomorphic cells enter S-phase, fork density and fork progression are approximately comparable with wild type cells. Therefore, the low level of Orc2 hinders normal cell cycle progression by delaying the activation of G1 cyclin-dependent kinases. The results suggest that hypomorphic mutations in initiation factor genes may be particularly deleterious in cancers with mutant p53 or increased activity of Cyclin E/Cdk2.     

Alphavbeta3 integrin and cofilin modulate K1735 melanoma cell invasion

Cytoskeletal reorganization is partially mediated through cofilin, an actin assembly regulatory protein. Cofilin activity is modulated by reversible phosphorylation at Ser3. In this study, using K1735 murine melanoma cells, we examined the relationship between beta3-integrin expression, phosphorylation of cofilin, and metalloproteinase production. The levels of phosphorylated cofilin were 10-fold higher in cells expressing alphavbeta3 than in alphavbeta3-negative cells when plated on vitronectin for 30 min. However, by 60 min, phosphorylation of cofilin was greater in the beta3-negative cells. Expression of the wild type (WT) or non-phosphorylatable cofilin (A3 mutant) increased melanoma cell migration on vitronectin and invasion through a reconstituted basement membrane. Expression of a pseudophosphorylated, poorly active cofilin (E3 mutant) reduced cell motility. Expression of active cofilin accelerated the phosphorylation of FAK at Y397 and at Y576, strongly implicating cofilin as a mediator of cell signaling. The expression of MT1-MMP and MMP2 was also increased by expression of wild type or A3 cofilin. A 50% reduction of both enzymes was observed by the expression of the E3 cofilin. Overexpression of non-phosphorylatable cofilin was sufficient to induce the expression of MT1-MMP and MMP2 in the beta3-negative M2Tbeta3 cells. Interestingly, the invasion of M2Tbeta3 cells could be sustained by overexpression of cofilin A3. These results suggest that the integrin alphavbeta3 and cofilin together regulate K1735 melanoma cell invasion.     

Phytochromes from Agrobacterium tumefaciens: difference spectroscopy with extracts of wild type and knockout mutants

Phytochromes are photoreceptors that occur in plants, fungi and bacteria, among others in the phytopathogen Agrobacterium tumefaciens. We constructed single and double knockout mutants of the two A. tumefaciens phytochromes Agp1 and Agp2. In liquid culture, the double mutant revealed a reduced growth rate, whereas the growth rates of the single mutants did not differ significantly from that of the wild type. Using these mutants, we analyzed the spectral properties of native A. tumefaciens phytochromes. A wild-type A. tumefaciens cell contains about 10 molecules of Agp1 and about 19 molecules of Agp2. Dark conversion of native Agp1 and Agp2 proceeds from Pfr to Pr and from Pr to Pfr, respectively, as has already been reported for the recombinant proteins. The spectral properties of recombinant and native Agp2 were significantly different. Mixing experiments with extracts from the double mutant and recombinant Agp2 imply that the spectral properties of Agp2 are modulated by components of the extract.     

CD72 down-modulates BCR-induced signal transduction and diminishes survival in primary mature B lymphocytes

CD72, a 45-kDa type II transmembrane glycoprotein carrying an ITIM motif, is believed to be an inhibitory coreceptor of the BCR. Mature B cells lacking CD72 show enhanced Ca(2+) mobilization and are hyperproliferative in response to BCR ligation. However, the signal transduction pathways downstream of BCR signaling that transmit the inhibitory effect of CD72 in mature B cells remain unknown. To address this question, we used hen egg lysozyme-specific BCR transgenic mice to elucidate the differential cell signaling between wild-type and CD72-deficient B cells in response to hen egg lysozyme Ag stimulation. Our results demonstrate that CD72 predominantly down-regulates the major signal transduction pathways downstream of the BCR, including NF-AT, NF-kappaB, ERK, JNK, p38-MAPK, and PI3K/Akt in mature B cells. CD72 ligation with anti-CD72 Ab (K10.6), which mimics the binding of CD100 (a natural ligand for CD72) to release the inhibitory function of CD72, augments cell proliferation, Ca(2+) flux, IkappaBalpha activation, and ERK MAPK activity upon Ag stimulation in wild-type B cells. In addition, we show direct evidence that CD72 promotes cell cycle arrest and apoptosis after Ag stimulation in mature B cells. Taken together, our findings conclude that CD72 plays a dominant role as a negative regulator of BCR signaling in primary mature B lymphocytes.     

Grb10 mediates insulin-stimulated degradation of the insulin receptor: a mechanism of negative regulation

Growth factor receptor-bound protein 10 (Grb10) is an adapter protein that interacts with a number of tyrosine-phosphorylated growth factor receptors, including the insulin receptor (IR). To investigate the role of Grb10 in insulin signaling, we generated cell lines in which the expression levels of Grb10 are either overexpressed by stable transfection or suppressed by RNA interference. We found that suppressing endogenous Grb10 expression led to increased IR protein levels, whereas overexpression of Grb10 led to reduced IR protein levels. Altering Grb10 expression levels had no effect on the mRNA levels of IR, suggesting that the modulation occurs at the protein level. Reduced IR levels were also observed in cells with prolonged insulin treatment, and this reduction was inhibited in Grb10-deficient cells. The insulin-induced IR reduction was greatly reversed by MG-132, a proteasomal inhibitor, but not by chloroquine, a lysosomal inhibitor. IR underwent insulin-stimulated ubiquitination in cells, and this ubiquitination was inhibited in the Grb10-suppressed cell line. Together, our results suggest that, in addition to inhibiting IR kinase activity by directly binding to the IR, Grb10 also negatively regulates insulin signaling by mediating insulin-stimulated degradation of the receptor.     

Genetic diversity in European pigs utilizing amplified fragment length polymorphism markers

The use of DNA markers to evaluate genetic diversity is an important component of the management of animal genetic resources. The Food and Agriculture Organisation of the United Nations (FAO) has published a list of recommended microsatellite markers for such studies; however, other markers are potential alternatives. This paper describes results obtained with a set of amplified fragment length polymorphism (AFLP) markers as part of a genetic diversity study of European pig breeds that also utilized microsatellite markers. Data from 148 AFLP markers genotyped across samples from 58 European and one Chinese breed were analysed. The results were compared with previous analyses of data from 50 microsatellite markers genotyped on the same animals. The AFLP markers had an average within-breed heterozygosity of 0.124 but there was wide variation, with individual markers being monomorphic in 3-98% of the populations. The biallelic and dominant nature of AFLP markers creates a challenge for their use in genetic diversity studies as each individual marker contains limited information and AFLPs only provide indirect estimates of the allelic frequencies that are needed to estimate genetic distances. Nonetheless, AFLP marker-based characterization of genetic distances was consistent with expectations based on breed and regional distributions and produced a similar pattern to that obtained with microsatellites. Thus, data from AFLP markers can be combined with microsatellite data for measuring genetic diversity.     

Pervasive purifying selection characterizes the evolution of I2 homologs

We sampled 384 sequences related to the Solanum pimpinellifolium (=Lycopersicon pimpinellifolium) disease resistance (R) gene 12 from six species, potato, S. demissum, tomato, eggplant, pepper, and tobacco. These species represent increasing phylogenetic distance from potato to tobacco, within the family Solanaceae. Using sequence data from the nucleotide binding site (NBS) region of this gene, we tested models of gene family evolution and inferred patterns of selection acting on the NBS gene region and I2 gene family. We find that the I2 family has diversified within the family Solanaceae for at least 14 million years and evolves through a slow birth-and-death process requiring approximately 12 million years to homogenize gene copies within a species. Analyses of selection resolved a general pattern of strong purifying selection acting on individual codon positions within the NBS and on NBS lineages through time. Surprisingly, we find nine codon positions strongly affected by positive selection and six pairs of codon positions demonstrating correlated amino acid substitutions. Evolutionary analyses serve as bioinformatic tools with which to sort through the vast R gene diversity in plants and find candidates for new resistance specificities or to identify specific amino acid positions important for biochemical function. The slow birth-and-death evolution of I2 genes suggests that some NBS-leucine rich repeat-mediated resistances may not be overcome rapidly by virulence evolution and that the natural diversity of R genes is a potentially valuable source for durable resistance.     

Protein-ligand docking: current status and future challenges

Understanding the ruling principles whereby protein receptors recognize, interact, and associate with molecular substrates and inhibitors is of paramount importance in drug discovery efforts. Protein-ligand docking aims to predict and rank the structure(s) arising from the association between a given ligand and a target protein of known 3D structure. Despite the breathtaking advances in the field over the last decades and the widespread application of docking methods, several downsides still exist. In particular, protein flexibility-a critical aspect for a thorough understanding of the principles that guide ligand binding in proteins-is a major hurdle in current protein-ligand docking efforts that needs to be more efficiently accounted for. In this review the key concepts of protein-ligand docking methods are outlined, with major emphasis being given to the general strengths and weaknesses that presently characterize this methodology. Despite the size of the field, the principal types of search algorithms and scoring functions are reviewed and the most popular docking tools are briefly depicted. Recent advances that aim to address some of the traditional limitations associated with molecular docking are also described. A selection of hand-picked examples is used to illustrate these features.