Functional Effect of Pim1 Depends upon Intracellular Localization in Human Cardiac Progenitor Cells

Human cardiac progenitor cells (hCPC) improve heart function after autologous transfer in heart failure patients. Regenerative potential of hCPCs is severely limited with age, requiring genetic modification to enhance therapeutic potential. A legacy of work from our laboratory with Pim1 kinase reveals effects on proliferation, survival, metabolism, and rejuvenation of hCPCs in vitro and in vivo. We demonstrate that subcellular targeting of Pim1 bolsters the distinct cardioprotective effects of this kinase in hCPCs to increase proliferation and survival, and antagonize cellular senescence. Adult hCPCs isolated from patients undergoing left ventricular assist device implantation were engineered to overexpress Pim1 throughout the cell (PimWT) or targeted to either mitochondrial (Mito-Pim1) or nuclear (Nuc-Pim1) compartments. Nuc-Pim1 enhances stem cell youthfulness associated with decreased senescence-associated β-galactosidase activity, preserved telomere length, reduced expression of p16 and p53, and up-regulation of nucleostemin relative to PimWT hCPCs. Alternately, Mito-Pim1 enhances survival by increasing expression of Bcl-2 and Bcl-X_L and decreasing cell death after H₂O₂ treatment, thereby preserving mitochondrial integrity superior to PimWT. Mito-Pim1 increases the proliferation rate by up-regulation of cell cycle modulators Cyclin D, CDK4, and phospho-Rb. Optimal stem cell traits such as proliferation, survival, and increased youthful properties of aged hCPCs are enhanced after targeted Pim1 localization to mitochondrial or nuclear compartments. Targeted Pim1 overexpression in hCPCs allows for selection of the desired phenotypic properties to overcome patient variability and improve specific stem cell characteristics.     

Nuclear Calcium/Calmodulin-dependent Protein Kinase II Signaling Enhances Cardiac Progenitor Cell Survival and Cardiac Lineage Commitment

Ca²⁺/Calmodulin-dependent protein kinase II (CaMKII) signaling in the heart regulates cardiomyocyte contractility and growth in response to elevated intracellular Ca²⁺. The δB isoform of CaMKII is the predominant nuclear splice variant in the adult heart and regulates cardiomyocyte hypertrophic gene expression by signaling to the histone deacetylase HDAC4. However, the role of CaMKIIδ in cardiac progenitor cells (CPCs) has not been previously explored. During post-natal growth endogenous CPCs display primarily cytosolic CaMKIIδ, which localizes to the nuclear compartment of CPCs after myocardial infarction injury. CPCs undergoing early differentiation in vitro increase levels of CaMKIIδB in the nuclear compartment where the kinase may contribute to the regulation of CPC commitment. CPCs modified with lentiviral-based constructs to overexpress CaMKIIδB (CPCeδB) have reduced proliferative rate compared with CPCs expressing eGFP alone (CPCe). Additionally, stable expression of CaMKIIδB promotes distinct morphological changes such as increased cell surface area and length of cells compared with CPCe. CPCeδB are resistant to oxidative stress induced by hydrogen peroxide (H₂O₂) relative to CPCe, whereas knockdown of CaMKIIδB resulted in an up-regulation of cell death and cellular senescence markers compared with scrambled treated controls. Dexamethasone (Dex) treatment increased mRNA and protein expression of cardiomyogenic markers cardiac troponin T and α-smooth muscle actin in CPCeδB compared with CPCe, suggesting increased differentiation. Therefore, CaMKIIδB may serve as a novel modulatory protein to enhance CPC survival and commitment into the cardiac and smooth muscle lineages.     

X-ray structure of human acid-beta-glucosidase covalently bound to conduritol-B-epoxide. Implications for Gaucher disease

Gaucher disease is an inherited metabolic disorder caused by mutations in the lysosomal enzyme acid-beta-glucosidase (GlcCerase). We recently determined the x-ray structure of GlcCerase to 2.0 A resolution (Dvir, H., Harel, M., McCarthy, A. A., Toker, L., Silman, I., Futerman, A. H., and Sussman, J. L. (2003) EMBO Rep.4, 704-709) and have now solved the structure of Glc-Cerase conjugated with an irreversible inhibitor, conduritol-B-epoxide (CBE). The crystal structure reveals that binding of CBE to the active site does not induce a global conformational change in GlcCerase and confirms that Glu340 is the catalytic nucleophile. However, only one of two alternative conformations of a pair of flexible loops (residues 345-349 and 394-399) located at the entrance to the active site in native GlcCerase is observed in the GlcCerase-CBE structure, a conformation in which the active site is accessible to CBE. Analysis of the dynamics of these two alternative conformations suggests that the two loops act as a lid at the entrance to the active site. This possibility is supported by a cluster of mutations in loop 394-399 that cause Gaucher disease by reducing catalytic activity. Moreover, in silico mutational analysis demonstrates that all these mutations stabilize the conformation that limits access to the active site, thus providing a mechanistic explanation of how mutations in this loop result in Gaucher disease.     

Importance of cooperation and affiliation in the evolution of primate sociality

The idea that competition and aggression are central to an understanding of the origins of group‐living and sociality among human and nonhuman primates is the dominant theory in primatology today. Using this paradigm, researchers have focused their attention on competitive and aggressive behaviors, and have tended to overlook the importance of cooperative and affiliative behaviors. However, cooperative and affiliative behaviors are considerably more common than agonistic behaviors in all primate species. The current paradigm often fails to explain the context, function, and social tactics underlying affiliative and agonistic behavior. Here, we present data on a basic question of primate sociality: how much time do diurnal, group‐living primates spend in social behavior, and how much of this time is affiliative and agonistic? These data are derived from a survey of 81 studies, including 28 genera and 60 species. We find that group‐living prosimians, New World monkeys, Old World monkeys, and apes usually devote less than 10% of their activity budget to active social interactions. Further, rates of agonistic behaviors are extremely low, normally less than 1% of the activity budget. If the cost to the actors of affiliative behavior is low even if the rewards are low or extremely variable, we should expect affiliation and cooperation to be frequent. This is especially true under conditions in which individuals benefit from the collective environment of living in stable social groups. Am J Phys Anthropol 128:84–97, 2005. © 2005 Wiley‐Liss, Inc.     

The effect of a genetically reduced plasma membrane protonmotive force on vegetative growth of Arabidopsis

The plasma membrane proton gradient is an essential feature of plant cells. In Arabidopsis (Arabidopsis thaliana), this gradient is generated by the plasma membrane proton pump encoded by a family of 11 genes (abbreviated as AHA, for Arabidopsis H(+)-ATPase), of which AHA1 and AHA2 are the two most predominantly expressed in seedlings and adult plants. Although double knockdown mutant plants containing T-DNA insertions in both genes are embryonic lethal, under ideal laboratory growth conditions, single knockdown mutant plants with a 50% reduction in proton pump concentration complete their life cycle without any observable growth alteration. However, when grown under conditions that induce stress on the plasma membrane protonmotive force (PMF), such as high external potassium to reduce the electrical gradient or high external pH to reduce the proton chemical gradient, aha2 mutant plants show a growth retardation compared with wild-type plants. In this report, we describe the results of studies that examine in greater detail AHA2's specific role in maintaining the PMF during seedling growth. By comparing the wild type and aha2 mutants, we have measured the effects of a reduced PMF on root and hypocotyl growth, ATP-induced skewed root growth, and rapid cytoplasmic calcium spiking. In addition, genome-wide gene expression profiling revealed the up-regulation of potassium transporters in aha2 mutants, indicating, as predicted, a close link between the PMF and potassium uptake at the plasma membrane. Overall, this characterization of aha2 mutants provides an experimental and theoretical framework for investigating growth and signaling processes that are mediated by PMF-coupled energetics at the cell membrane.     

Backbone and side chain NMR assignments for the intrinsically disordered cytoplasmic domain of human neuroligin-3

Neuroligins act as heterophilic adhesion molecules at neuronal synapses. Their cytoplasmic domains interact with synaptic scaffolding proteins, and have been shown to be intrinsically disordered. Here we report the backbone and side chain ¹H, ¹³C and ¹⁵N resonance assignments for the cytoplasmic domain of human neuroligin 3.