Control of the pericentrosomal H2O2 level by peroxiredoxin I is critical for mitotic progression

Proteins associated with the centrosome play key roles in mitotic progression in mammalian cells. The activity of Cdk1-opposing phosphatases at the centrosome must be inhibited during early mitosis to prevent premature dephosphorylation of Cdh1—an activator of the ubiquitin ligase anaphase-promoting complex/cyclosome—and the consequent premature degradation of mitotic activators. In this paper, we show that reversible oxidative inactivation of centrosome-bound protein phosphatases such as Cdc14B by H₂O₂ is likely responsible for this inhibition. The intracellular concentration of H₂O₂ increases as the cell cycle progresses. Whereas the centrosome is shielded from H₂O₂ through its association with the H₂O₂-eliminating enzyme peroxiredoxin I (PrxI) during interphase, the centrosome-associated PrxI is selectively inactivated through phosphorylation by Cdk1 during early mitosis, thereby exposing the centrosome to H₂O₂ and facilitating inactivation of centrosome-bound phosphatases. Dephosphorylation of PrxI by okadaic acid–sensitive phosphatases during late mitosis again shields the centrosome from H₂O₂ and thereby allows the reactivation of Cdk1-opposing phosphatases at the organelle.     

Lack of Significant Elevation of Myeloid-Derived Suppressor Cells in Peripheral Blood of Chronically Hepatitis C Virus-Infected Individuals

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with immunosuppressive function. Compared to the level in healthy controls (HC), no elevation of MDSC in chronic hepatitis C (cHEP-C) patients was found, and there was no difference in MDSC based on genotype or viral load (P > 0.25). Moreover, MDSC of cHEP-C patients inhibited CD8 T cell function as efficiently as MDSC of HC did. Since we detected neither quantitative nor qualitative differences in MDSC of cHEP-C patients relative to those of HC, we postulate that MDSC in peripheral blood are most likely not significant regarding immune dysfunction in cHEP-C.     

Cox model with interval‐censored covariate in cohort studies

In cohort studies the outcome is often time to a particular event, and subjects are followed at regular intervals. Periodic visits may also monitor a secondary irreversible event influencing the event of primary interest, and a significant proportion of subjects develop the secondary event over the period of follow‐up. The status of the secondary event serves as a time‐varying covariate, but is recorded only at the times of the scheduled visits, generating incomplete time‐varying covariates. While information on a typical time‐varying covariate is missing for entire follow‐up period except the visiting times, the status of the secondary event are unavailable only between visits where the status has changed, thus interval‐censored. One may view interval‐censored covariate of the secondary event status as missing time‐varying covariates, yet missingness is partial since partial information is provided throughout the follow‐up period. Current practice of using the latest observed status produces biased estimators, and the existing missing covariate techniques cannot accommodate the special feature of missingness due to interval censoring. To handle interval‐censored covariates in the Cox proportional hazards model, we propose an available‐data estimator, a doubly robust‐type estimator as well as the maximum likelihood estimator via EM algorithm and present their asymptotic properties. We also present practical approaches that are valid. We demonstrate the proposed methods using our motivating example from the Northern Manhattan Study.     

Mutations affecting the catalytic activity of Bacillus cereus 5/B/6 beta-lactamase II

Random in vitro mutagenesis of a cloned Bacillus cereus 5/B/6 beta-lactamase II gene was used to select defective genes unable to confer ampicillin or cephalosporin C resistance to Escherichia coli. DNA sequencing of mutant genes identified histidine at position 28 as important to beta-lactamase II function. In addition, the isolation of six identical frameshift mutants established that the carboxyl-terminal end of beta-lactamase II is critical for enzyme function. Random mutagenesis also revealed that His88 (implicated previously as one of 4 residues acting as a zinc ligand) is crucial to enzymatic activity and that a glycine to glutamic acid substitution at position 148 produced a defective beta-lactamase. Oligonucleotide mutagenesis directed at Glu37 and Glu212 suggests that these residues are inconsequential to enzyme function but that histidine at position 28 may be involved in substrate binding or recognition.     

Regulation of c-Myc Expression by Ahnak Promotes Induced Pluripotent Stem Cell Generation

We have previously reported that Ahnak-mediated TGFβ signaling leads to down-regulation of c-Myc expression. Here, we show that inhibition of Ahnak can promote generation of induced pluripotent stem cells (iPSC) via up-regulation of endogenous c-Myc. Consistent with the c-Myc inhibitory role of Ahnak, mouse embryonic fibroblasts from Ahnak-deficient mouse (Ahnak^−/− MEF) show an increased level of c-Myc expression compared with wild type MEF. Generation of iPSC with just three of the four Yamanaka factors, Oct4, Sox2, and Klf4 (hereafter 3F), was significantly enhanced in Ahnak^−/− MEF. Similar results were obtained when Ahnak-specific shRNA was applied to wild type MEF. Of note, expressionof Ahnak was significantly induced during the formation of embryoid bodies from embryonic stem cells, suggesting that Ahnak-mediated c-Myc inhibition is involved in embryoid body formation and the initial differentiation of pluripotent stem cells. The iPSC from 3F-infected Ahnak^−/− MEF cells (Ahnak^−/−-iPSC-3F) showed expression of all stem cell markers examined and the capability to form three primary germ layers. Moreover, injection of Ahnak^−/−-iPSC-3F into athymic nude mice led to development of teratoma containing tissues from all three primary germ layers, indicating that iPSC from Ahnak^−/− MEF are bona fide pluripotent stem cells. Taken together, these data provide evidence for a new role for Ahnak in cell fate determination during development and suggest that manipulation of Ahnak and the associated signaling pathway may provide a means to regulate iPSC generation.