Two nuclear export signals of Cdc6 are differentially associated with CDK-mediated phosphorylation residues for cytoplasmic translocation

Cdc6 is cleaved at residues 442 and 290 by caspase-3 during apoptosis producing p49-tCdc6 and p32-tCdc6, respectively. While p32-tCdc6 is unable to translocate into the cytoplasm, p49-tCdc6 retains cytoplasmic translocation activity, but it has a lower efficiency than wild-type Cdc6. We hypothesized that a novel nuclear export signal (NES) sequence exists between amino acids 290 and 442. Cdc6 contains a novel NES in the region of amino acids 300–315 (NES2) that shares sequence similarity with NES1 at residues 462–476. In mutant versions of Cdc6, we replaced leucine with alanine in NES1 and NES2 and co-expressed the mutant constructs with cyclin A. We observed that the cytoplasmic translocation of these mutants was reduced in comparison to wild-type Cdc6. Moreover, the cytoplasmic translocation of a mutant in which all four leucine residues were mutated to alanine was significantly inhibited in comparison to the translocation of wild-type Cdc6. The Crm1 binding activities of Cdc6 NES mutants were consistent with the efficiency of its cytoplasmic translocation. Further studies have revealed that L468 and L470 of NES1 are required for cytoplasmic translocation of Cdc6 phosphorylated at S74, while L311 and L313 of NES2 accelerate the cytoplasmic translocation of Cdc6 phosphorylated at S54. These results suggest that the two NESs of Cdc6 work cooperatively and distinctly for the cytoplasmic translocation of Cdc6 phosphorylated at S74 and S54 by cyclin A/Cdk2.     

The dependence of residence time distribution on flow in co-axial cylinder devices

Numerical simulations are presented showing the effects of operating and geometrical parameters on the transition of laminar to Taylor vortex flow for induced rotational-axial flow in the gap of a pair of rotating cylinders. These simulations indicate that annular rotational flow becomes more stable in the presence of a small degree of axial flow and as gap width increases. The effect of rotational speed on the breakdown of laminar flow is more complex and for given radius ratio and axial flow rate depends on both the speed ratio and the direction of the rotation of the cylinders, counter-rotating flow generally producing a more stable flow than co-rotating. Limited experimental data are provided on the residence time distribution for flow of Newtonian liquids through the gap of two rotating cylinders. The data include results from experiments in which flow transition occurred from laminar to Taylor vortex flow. The findings from these experiments are successfully analyzed and assessed using the simulations studies.     

Endothelial intercellular cell adhesion molecule 1 contributes to cell aggregate formation in CHO cells cultured in serum-free media

Chinese hamster ovary (CHO) cells have been adapted to grow in serum-free media and in suspension culture to facilitate manufacturing needs. Some CHO cell lines, however, tend to form cell aggregates while being cultured in suspension. This can result in reduced viability and capacity for single cell cloning (SCC) via limiting dilution, and process steps to mitigate cell aggregate formation, for example, addition of anti-cell-aggregation agents. In this study, we have identified endothelial intercellular cell adhesion molecule 1 (ICAM-1) as a key protein promoting cell aggregate formation in a production competent CHO cell line, which is prone to cell aggregate formation. Knocking out (KO) the ICAM-1 gene significantly decreased cell aggregate formation in the culture media without anti-cell-aggregation reagent. This trait can simplify the process of transfection, selection, automated clone isolation, and so on. Evaluation in standard cell line development of ICAM-1 KO and wild-type CHO hosts did not reveal any noticeable impacts on titer or product quality. Furthermore, analysis of a derived nonaggregating cell line showed significant reductions in expression of cell adhesion proteins. Overall, our data suggest that deletion of ICAM-1 and perhaps other cell adhesion proteins can reduce cell aggregate formation and improve clonality assurance during SCC.     

Cover Image,Volume 116, Number 5, May 2019

Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) present an attractive alternative to primary EC sources for vascular grafting. However, there is a need to mature them towards either an arterial or venous subtype. A vital environmental factor involved in the arteriovenous specification of ECs during early embryonic development is fluid shear stress; therefore, there have been attempts to employ adult arterial shear stress conditions to mature hPSC-ECs. However, hPSC-ECs are naïve to fluid shear stress, and their shear responses are still not well understood. Here, we used a multiplex microfluidic platform to systematically investigate the dose-time shear responses on hPSC-EC morphology and arterial-venous phenotypes over a range of magnitudes coincidental with physiological levels of embryonic and adult vasculatures. The device comprised of six parallel cell culture chambers that were individually linked to flow-setting resistance channels, allowing us to simultaneously apply shear stress ranging from 0.4 to 15 dyne/cm ². We found that hPSC-ECs required up to 40 hr of shear exposure to elicit a stable phenotypic change. Cell alignment was visible at shear stress <1 dyne/cm ², which was independent of shear stress magnitude and duration of exposure. We discovered that the arterial markers NOTCH1 and EphrinB2 exhibited a dose-dependent increase in a similar manner beyond a threshold level of 3.8 dyne/cm ², whereas the venous markers COUP-TFII and EphB4 expression remained relatively constant across different magnitudes. These findings indicated that hPSC-ECs were sensitive to relatively low magnitudes of shear stress, and a critical level of ~4 dyne/cm ² was sufficient to preferentially enhance their maturation into an arterial phenotype for future vascular tissue engineering applications.     

Purification and characteristics of hydrophobic membrane protein(s) required for DCCD sensitivity of ATPase in mycobacterium phlei

The energy-transducing N,N′-dicyclohexylcarbodiimide-sensitive (DCCD-sensitive) ATPase complex consists of two parts, a soluble catalytic protein (F₁), and an intrinsic membrane protein (F₀). The bacterial coupling factor complex, BCF₀-BCF₁, has recently been purified from Mycobacterium phlei, and used to reconstitute oxidative phosphorylation in detergent-extracted membranes. The BCF₀ moiety has been purified by being recovered from the purified BCF₀-BCF₁ complex by affinity chromatography. BCF₀ is a lipoprotein or lipoprotein complex with an approximate molecular weight of 60,000. The preparation contained 0.15 mg of phospholipid per milligram protein. There appear to be three polypeptides, with approximate molecular weights of 24,000, 18,000, and 8,000 as determined by sodium dodecylsulfate a crylamide gel electrophoresis. Purified BCF₀ conferred DCCD sensitivity on a purified BCF₁ preparation. Reconstitution of oxidative phosphorylation was achieved after incubation of detergent-extracted membranes with purified BCF₀ and purified BCF₁.