Stable expression of mammalian beta 1,4-galactosyltransferase extends the N-glycosylation pathway in insect cells

An established lepidopteran insect cell line (Sf9) was cotransfected with expression plasmids encoding neomycin phosphotransferase and bovine beta 1,4-galactosyltransferase. Neomycin-resistant transformants were selected, assayed for beta 1,4-galactosyltransferase activity, and the transformant with the highest level of enzymatic activity was characterized. Southern blots indicated that this transformed Sf9 cell derivative contained multiple copies of the galactosyltransferase- encoding expression plasmid integrated at a single site in its genome. One-step growth curves showed that these cells supported normal levels of baculovirus replication. Baculovirus infection of the transformed cells stimulated beta 1,4-galactosyltransferase activity almost 5-fold by 12 h postinfection. This was followed by a gradual decline in activity, but the infected cells still had about as much activity as uninfected controls as late as 48 h after infection and they were able to produce a beta 1,4-galactosylated virion glycoprotein during infection. Infection of the transformed cells with a conventional recombinant baculovirus expression vector encoding human tissue plasminogen activator also resulted in the production of a galactosylated end-product. These results demonstrate that stable transformation can be used to add a functional mammalian glycosyltransferase to lepidopteran insect cells and extend their N- glycosylation pathway. Furthermore, stably-transformed insect cells can be used as modified hosts for conventional baculovirus expression vectors to produce foreign glycoproteins with "mammalianized" glycans which more closely resemble those produced by higher eucaryotes.      

The remodeling of glycoconjugates in mice

A role for glycoconjugates in mediating cellular interactions is well established. To further understand the formation, function and regulation of various glycoconjugates in vivo, gene targeting approaches have been applied to glycosyltransferase and glycosidase enzymes involved in different biosynthetic pathways. The growing number of gene targeted mice generated have brought confirmations of the importance of both core and terminal glycosylation enzymes in normal development and physiology. Of particular interest has been the degree of cell and tissue specificity of phenotypes generated by systemic null mutations as well as the number of enzymes belonging to multigene families having overlapping activities.     

A quantitative method for assessing stages of the rat estrous cycle

The impact of gender and/or hormone variations on a wide variety of neural functions makes the choice between studying males or females (or both) of a given species difficult. Although female rats are widely used experimentally, few studies control for the stage of estrus. More detailed information about how to distinguish the various stages of the estrous cycle is needed. For the present study, vaginal smears were obtained once a day and stained using an adaptation of the Papanicolaou (PAP) procedure. Images are provided of unstained “wet” samples and the corresponding PAP stained smears illustrating the cellular profile for each stage of the cycle as well as post-ovariectomy. The different cell populations across the cycle were quantified and ratios determined to show trends between the predominant and other cell types in each stage of the estrous cycle. Both stained and unstained images and cell quantification data provide valuable guidelines for distinguishing the stages of the estrous cycle.     

Statistical analysis of an RNA titration series evaluates microarray precision and sensitivity on a whole-array basis

Background

It is one of the ultimate goals for modern biological research to fully elucidate the intricate interplays and the regulations of the molecular determinants that propel and characterize the progression of versatile life phenomena, to name a few, cell cycling, developmental biology, aging, and the progressive and recurrent pathogenesis of complex diseases. The vast amount of large-scale and genome-wide time-resolved data is becoming increasing available, which provides the golden opportunity to unravel the challenging reverse-engineering problem of time-delayed gene regulatory networks.

Results

In particular, this methodological paper aims to reconstruct regulatory networks from temporal gene expression data by using delayed correlations between genes, i.e., pairwise overlaps of expression levels shifted in time relative each other. We have thus developed a novel model-free computational toolbox termed TdGRN (Time-delayed Gene Regulatory Network) to address the underlying regulations of genes that can span any unit(s) of time intervals. This bioinformatics toolbox has provided a unified approach to uncovering time trends of gene regulations through decision analysis of the newly designed time-delayed gene expression matrix. We have applied the proposed method to yeast cell cycling and human HeLa cell cycling and have discovered most of the underlying time-delayed regulations that are supported by multiple lines of experimental evidence and that are remarkably consistent with the current knowledge on phase characteristics for the cell cyclings.

Conclusion

We established a usable and powerful model-free approach to dissecting high-order dynamic trends of gene-gene interactions. We have carefully validated the proposed algorithm by applying it to two publicly available cell cycling datasets. In addition to uncovering the time trends of gene regulations for cell cycling, this unified approach can also be used to study the complex gene regulations related to the development, aging and progressive pathogenesis of a complex disease where potential dependences between different experiment units might occurs.     

Novel O-palmitolylated beta-E1 subunit of pyruvate dehydrogenase is phosphorylated during ischemia/reperfusion injury

Background  

During and following myocardial ischemia, glucose oxidation rates are low and fatty acids dominate as a source of oxidative metabolism. This metabolic phenotype is associated with contractile dysfunction during reperfusion. To determine the mechanism of this reliance on fatty acid oxidation as a source of ATP generation, a functional proteomics approach was utilized.     

A nuclear gene for higher level phylogenetics: phosphoenolpyruvate carboxykinase tracks mesozoic-age divergences within Lepidoptera (Insecta)

The sequence of phosphoenolpyruvate carboxykinase (PEPCK) has been previously identified as a promising candidate for reconstructing Mesozoic-age divergences (Friedlander, Regier, and Mitter 1992, 1994). To test this hypothesis more rigorously, 597 nucleotides of aligned PEPCK coding sequence (approximately 30% of the coding region) were generated from 18 species representing Mesozoic-age lineages of moths (Insecta: Lepidoptera) and outgroup taxa. Relationships among basal Lepidoptera are well established by morphological analysis, providing a strong test for the utility of a gene which has not previously been used in systematics. Parsimony and other phylogenetic analyses were conducted on nucleotides by codon positions (nt1, nt2, nt3) separately and in combination, and on amino acids, for comparison to the test phylogeny. The highest concordance was achieved with nt1 + nt2, for which one of two most-parsimonious trees was identical to the test phylogeny, and with all nucleotides when nt3 was down-weighted sevenfold or higher, for which a single most-parsimonious tree identical to the test phylogeny resulted. Substitutions in nt3 approached saturation in many, but not all, pairwise comparisons and their exclusion or severe downweighting greatly increased the degree of concordance with the test phylogeny. Neighbor-joining analysis confirms this finding. The utility of PEPCK for phylogenetics is demonstrated over a time span for which few other suitable genes are currently available.      

Statistical analysis of an RNA titration series evaluates microarray precision and sensitivity on a whole-array basis

Background  

Concerns are often raised about the accuracy of microarray technologies and the degree of cross-platform agreement, but there are yet no methods which can unambiguously evaluate precision and sensitivity for these technologies on a whole-array basis.     

EDC3 phosphorylation regulates growth and invasion through controlling P‐body formation and dynamics

Regulation of mRNA stability and translation plays a critical role in determining protein abundance within cells. Processing bodies (P‐bodies) are critical regulators of these processes. Here, we report that the Pim1 and 3 protein kinases bind to the P‐body protein enhancer of mRNA decapping 3 (EDC3) and phosphorylate EDC3 on serine (S)161, thereby modifying P‐body assembly. EDC3 phosphorylation is highly elevated in many tumor types, is reduced upon treatment of cells with kinase inhibitors, and blocks the localization of EDC3 to P‐bodies. Prostate cancer cells harboring an EDC3 S161A mutation show markedly decreased growth, migration, and invasion in tissue culture and in xenograft models. Consistent with these phenotypic changes, the expression of integrin β1 and α6 mRNA and protein is reduced in these mutated cells. These results demonstrate that EDC3 phosphorylation regulates multiple cancer‐relevant functions and suggest that modulation of P‐body activity may represent a new paradigm for cancer treatment.