Diffusion-based transport of nascent ribosomes in the nucleus

Although the complex process of ribosome assembly in the nucleolus is beginning to be understood, little is known about how the ribosomal subunits move from the nucleolus to the nuclear membrane for transport to the cytoplasm. We show here that large ribosomal subunits move out from the nucleolus and into the nucleoplasm in all directions, with no evidence of concentrated movement along directed paths. Mobility was slowed compared with that expected in aqueous solution in a manner consistent with anomalous diffusion. Once nucleoplasmic, the subunits moved in the same random manner and also sometimes visited another nucleolus before leaving the nucleus.     

Glucose-dependent insulinotropic polypeptide receptor null mice exhibit compensatory changes in the enteroinsular axis

The incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are gut hormones that act via the enteroinsular axis to potentiate insulin secretion from the pancreas in a glucose-dependent manner. Both GLP-1 receptor and GIP receptor knockout mice (GLP-1R(-/-) and GIPR(-/-), respectively) have been generated to investigate the physiological importance of this axis. Although reduced GIP action is a component of type 2 diabetes, GIPR-deficient mice exhibit only moderately impaired glucose tolerance. The present study was directed at investigating possible compensatory mechanisms that take place within the enteroinsular axis in the absence of GIP action. Although serum total GLP-1 levels in GIPR knockout mice were unaltered, insulin responses to GLP-1 from pancreas perfusions and static islet incubations were significantly greater (40-60%) in GIPR(-/-) than in wild-type (GIPR(+/+)) mice. Furthermore, GLP-1-induced cAMP production was also elevated twofold in the islets of the knockout animals. Pancreatic insulin content and gene expression were reduced in GIPR(-/-) mice compared with GIPR(+/+) mice. Paradoxically, immunocytochemical studies showed a significant increase in beta-cell area in the GIPR-null mice but with less intense staining for insulin. In conclusion, GIPR(-/-) mice exhibit altered islet structure and topography and increased islet sensitivity to GLP-1 despite a decrease in pancreatic insulin content and gene expression.     

Premature structural changes at replication origins in a yeast minichromosome maintenance (MCM) mutant

The Cdc7p protein kinase in the budding yeast Saccharomyces cerevisiae is thought to help trigger DNA replication by modifying one or more of the factors that assemble at replication origins (ARSs). To investigate events catalyzed by Cdc7p, we compared the structure of replication origins in cells containing conditional mutations in Cdc7p and Cdc8p, a thymidylate kinase that is required for DNA synthesis. High resolution genomic footprinting indicated that the presumptive lagging strand template in ARS1 became highly sensitive to KMnO(4) modification after the CDC7 execution point. These results suggested that Cdc7p triggers DNA unwinding. The transition from late G(1) phase to the CDC7 execution point and from the CDC7 to the CDC8 execution points was accompanied by small but ARS-dependent changes in DNA topology. These results suggested that DNA unwinding before the CDC8 execution point either is highly localized or that the torsional stress associated with initial DNA unwinding is minimized by compensatory protein-DNA structural changes. The ARS DNA structural attributes evident in cells blocked at the CDC8 execution point were also evident in alpha-factor-blocked, G(1) phase cells containing the CDC7 bypass mutant mcm5/cdc46-bob1. This result strongly suggests that the structural changes during the transition from the CDC7 to CDC8 execution points depend on the Cdc7p protein kinase and involve alteration of the minichromosome maintenance protein complex.     

Adjuvant activities of immune response modifier R-848: comparison with CpG ODN

R-848 and imiquimod belong to a class of immune response modifiers that are potent inducers of cytokines, including IFN-alpha, TNF-alpha, IL-12, and IFN-gamma. Many of these cytokines can affect the acquired immune response. This study examines the effects of R-848 on aspects of acquired immunity, including immunoglobulin secretion, in vivo cytokine production, and Ag-specific T cell cytokine production. Results are compared with those of Th1 CpG ODN. R-848 and CpG ODN are effective at skewing immunity in the presence of Alum toward a Th1 Ab response (IgG2a) and away from a Th2 Ab response (IgE). R-848 and CpG ODN are also capable of initiating an immune response in the absence of additional adjuvant by specifically enhancing IgG2a levels. Both R-848 and imiquimod showed activity when given subcutaneously or orally, indicating that the compound mechanism was not through generation of a depot effect. Although CpG ODN behaves similarly to R-848, CpG ODN has a distinct cytokine profile, is more effective than R-848 when given with Alum in the priming dose, and is active only when given by the same route as the Ag. The mechanism of R-848's adjuvant activity is linked to cytokine production, where increases in IgG2a levels are associated with IFN-alpha, TNF-alpha, IL-12, and IFN-gamma induction, and decreases in IgE levels are associated with IFN-alpha and TNF-alpha. Imiquimod also enhances IgG2a production when given with Ag. The above results suggest that the imidazoquinolines R-848 and imiquimod may be attractive compounds for use as vaccine adjuvants and in inhibiting pathological responses mediated by Th2 cytokines.     

Ser2]- and [SerP2] incretin analogs: comparison of dipeptidyl peptidase IV resistance and biological activities in vitro and in vivo

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP; also known as gastric inhibitory polypeptide) are incretin hormones that reduce postprandial glycemic excursions via enhancing insulin release but are rapidly inactivated by enzymatic N-terminal truncation. As such, efforts have been made to improve their plasma stability by synthetic modification or by inhibition of the responsible protease, dipeptidyl peptidase (DP) IV. Here we report a parallel comparison of synthetic GIP and GLP-1 with their Ser2- and Ser(P)2-substituted analogs, examining receptor binding and activation, metabolic stability, and biological effects in vivo. Both incretins and their Ser2-substituted analogs showed similar EC50s (0.16-0.52 nm) and IC50s (4.3-8.1 nm) at their respective cloned receptors. Although both phosphoserine 2-modified (Ser(PO3H2); Ser(P)) peptides were able to stimulate maximal cAMP production and fully displace receptor-bound tracer, they showed significantly right-shifted concentration-response curves and binding affinities. Ser2-substituted analogs were moderately resistant to DP IV cleavage, whereas [Ser(P)2]GIP and [Ser(P)2] GLP-1 showed complete resistance to purified DP IV. It was shown that the Ser(P) forms were dephosphorylated in serum and thus in vivo act as precursor forms of Ser2-substituted analogs. When injected subcutaneously into conscious Wistar rats, all peptides reduced glycemic excursions (rank potency: [Ser(P)2]incretins > or = [Ser2] incretins > native hormones). Insulin determinations indicated that the reductions in postprandial glycemia were at least in part insulin-mediated. Thus it has been shown that despite having low in vitro bioactivity using receptor-transfected cells, in vivo potency of [Ser(P)2] incretins was comparable with or greater than that of native or [Ser2]peptides. Hence, Ser(P)2-modified incretins present as novel glucose-lowering agents.     

Control of misincorporation of serine for asparagine during antibody production using CHO cells

A recombinant monoclonal antibody produced by Chinese hamster ovary (CHO) cell fed‐batch culture was found to have amino acid sequence misincorporation upon analysis by intact mass and peptide mapping mass spectrometry. A detailed analysis revealed multiple sites for asparagine were being randomly substituted by serine, pointing to mistranslation as the likely source. Results from time‐course analysis of cell culture suggest that misincorporation was occurring midway through the fed‐batch process and was correlated to asparagine reduction to below detectable levels in the culture. Separate shake flask experiments were carried out that confirmed starvation of asparagine and not excess of serine in the medium as the root cause of the phenomenon. Reduction in serine concentration under asparagine starvation conditions helped reduce extent of misincorporation. Supplementation with glutamine also helped reduce extent of misincorporation. Maintenance of asparagine at low levels in 2 L bench‐scale culture via controlled supplementation of asparagine‐containing feed eliminated the occurrence of misincorporation. This strategy was implemented in a clinical manufacturing process and scaled up successfully to the 200 and 2,000 L bioreactor scales. Biotechnol. Bioeng. 2010;107: 116–123. © 2010 Wiley Periodicals, Inc.