Mutational analysis reveals a single binding interface between RhoA and its effector, PRK1

Protein kinase C-related kinases (PRKs) are serine/threonine kinases that are members of the protein kinase C superfamily and can be activated by binding to members of the Rho family of small G proteins via a Rho binding motif known as an HR1 domain. The PRKs contain three tandem HR1 domains at their N-termini. The structure of the HR1a domain from PRK1 in complex with RhoA [Maesaki, R., et al. (1999) Mol. Cell 4, 793-803] identified two potential contact interfaces between the G protein and the HR1a domain. In this work, we have used an alanine scanning mutagenesis approach to identify whether both contact sites are used when the two proteins interact in solution and also whether HR1b, the second HR1 domain from PRK1, plays a role in binding to RhoA. The mutagenesis identified just one contact site as being relevant for binding of RhoA and HR1a in solution, and the HR1b domain was found not to contribute to RhoA binding. The folded state and thermal stability of the HR1a and HR1b domains were also investigated. HR1b was found to be more thermally stable than HR1a, and it is hypothesized that the differences in the biophysical properties of these two domains govern their interaction with small G proteins.     

Selection of CHO host cell subclones with increased specific antibody production rates by repeated cycles of transient transfection and cell sorting

Optimization of host cell lines both for transient and stable protein production is typically hampered by the inherent heterogeneity of cells within a population. This heterogeneity is caused not only by “hard fact” gene mutations, but also by subtle differences in the cellular network of regulation, which may include epigenetic variations. Taking advantage of this heterogeneity, we sorted for naturally occurring variants of CHO‐K1 and CHO‐S host cells that possess an improved cellular machinery for transient antibody production. The long‐term goal of this study was both to identify host cells that yield recombinant cell lines with on average higher productivity, but also to study the molecular differences that characterize such cells, independent of the site of gene integration or gene amplification. To identify such cells we optimized the procedure for transient transfection by electroporation to a degree that gave uniform transfer of plasmid DNA into nearly 100% of the cells and resulted in reproducible average productivities, with a standard deviation of 16% between independent experiments. Using this optimized protocol, the 1% of cells with the highest specific productivity was sorted and subcloned with a cold capture secretion assay. Upon re‐transfection, the resulting subclones showed the same specific productivity as their respective parental cell line. To enrich for cells with potentially stable improved properties, the 1% highest producers were sorted three times, 2 days after transient transfection each, and the enriched population was again sorted into microtiter plates for subcloning. For each of the two parental cell lines tested, three subclones were obtained that had a threefold higher specific productivity after transient transfection. This property was stable for approximately 3 months, indicating that the changes in productivity were regulatory and not mutational. Biotechnol. Bioeng. 2011;108: 386–394. © 2010 Wiley Periodicals, Inc.     

Cellular inhibitor of apoptosis 1 (cIAP-1) degradation by caspase 8 during TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis

TNF-related apoptosis-inducing ligand (TRAIL) is a potential chemotherapeutic agent with high selectivity for malignant cells. Many tumors, however, are resistant to TRAIL cytotoxicity. Although cellular inhibitors of apoptosis 1 and 2 (cIAP-1 and -2) are often over-expressed in cancers, their role in mediating TRAIL resistance remains unclear. Here, we demonstrate that TRAIL-induced apoptosis of liver cancer cells is associated with degradation of cIAP-1 and X-linked IAP (XIAP), whereas cIAP-2 remains unchanged. Lower concentrations of TRAIL causing minimal or no apoptosis do not alter cIAP-1 or XIAP protein levels. Silencing of cIAP-1 expression, but not XIAP or cIAP-2, as well as co-treatment with a second mitochondrial activator of caspases (SMAC) mimetic (which results in rapid depletion of cIAP-1), sensitizes the cells to TRAIL. TRAIL-induced loss of cIAP-1 and XIAP requires caspase activity. In particular, caspase 8 knockdown stabilizes both cIAP-1 and XIAP, while caspase 9 knockdown prevents XIAP, but not cIAP-1 degradation. Cell-free experiments confirmed cIAP-1 is a substrate for caspase 8, with likely multiple cleavage sites. These results suggest that TRAIL-mediated apoptosis proceeds through caspase 8-dependent degradation of cIAP-1. Targeted depletion of cIAP-1 by SMAC mimetics in conjunction with TRAIL may be beneficial for the treatment of human hepatobiliary malignancies.     

A new, vapour-phase mechanism for stomatal responses to humidity and temperature

A new mechanism for stomatal responses to humidity and temperature is proposed. Unlike previously-proposed mechanisms, which rely on liquid water transport to create water potential gradients within the leaf, the new mechanism assumes that water transport to the guard cells is primarily through the vapour phase. Under steady-state conditions, guard cells are assumed to be in near-equilibrium with the water vapour in the air near the bottom of the stomatal pore. As the water potential of this air varies with changing air humidity and leaf temperature, the resultant changes in guard cell water potential produce stomatal movements. A simple, closed-form, mathematical model based on this idea is derived. The new model is parameterized for a previously published set of data and is shown to fit the data as well as or better than existing models. The model contains mathematical elements that are consistent with previously-proposed mechanistic models based on liquid flow as well as empirical models based on relative humidity. As such, it provides a mechanistic explanation for the realm of validity for each of these approaches.     

Calcium Supplementation Increases Blood Creatinine Concentration in a Randomized Controlled Trial

Background

Calcium supplements are widely used among older adults for osteoporosis prevention and treatment. However, their effect on creatinine levels and kidney function has not been well studied.

Methods

We investigated the effect of calcium supplementation on blood creatinine concentration in a randomized controlled trial of colorectal adenoma chemoprevention conducted between 2004–2013 at 11 clinical centers in the United States. Healthy participants (N = 1,675) aged 45–75 with a history of colorectal adenoma were assigned to daily supplementation with calcium (1200 mg, as carbonate), vitamin D₃ (1000 IU), both, or placebo for three or five years. Changes in blood creatinine and total calcium concentration were measured after one year of treatment and multiple linear regression was used to estimate effects on creatinine concentrations.

Results

After one year of treatment, blood creatinine was 0.013±0.006 mg/dL higher on average among participants randomized to calcium compared to placebo after adjustment for other determinants of creatinine (P = 0.03). However, the effect of calcium treatment appeared to be larger among participants who consumed the most alcohol (2–6 drinks/day) or whose estimated glomerular filtration rate (eGFR) was less than 60 ml/min/1.73 m² at baseline. The effect of calcium treatment on creatinine was only partially mediated by a concomitant increase in blood total calcium concentration and was independent of randomized vitamin D treatment. There did not appear to be further increases in creatinine after the first year of calcium treatment.

Conclusions

Among healthy adults participating in a randomized clinical trial, daily supplementation with 1200 mg of elemental calcium caused a small increase in blood creatinine. If confirmed, this finding may have implications for clinical and public health recommendations for calcium supplementation.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00153816","term_id":"NCT00153816"}}NCT00153816     

Stomatal responses to non-local changes in PFD: evidence for long-distance hydraulic interactions

Interactions among stomata within a single areole have recently been reported, and evidence suggests that hydraulic mechanisms may be responsible for these interactions. Such interactions may play a role in patchy stomatal behaviour by coordinating stomatal behaviour within areoles. However, models suggest that longer‐distance interactions may be required to produce the large‐scale discoordination that is characteristic of stomatal patchiness. This study was undertaken to characterize long‐distance interactions between ‘artificial patches’ of stomata under varying conditions of evaporative demand and soil water stress. Gas‐exchange was monitored in two adjacent regions (‘patches’) of a wheat leaf by two independent gas mixing and analysis systems. When photon flux density (PFD) was changed in only one of these patches, stomatal conductance responded in both patches in a manner consistent with hydraulic interactions propagated by changes in xylem water potential. These data are discussed in the context of mechanisms for patchy stomatal conductance and implications for the design and analysis of gas‐exchange experiments.