Kinetic Analysis of Substrate Utilization by Native and TNAP-, NPP1-, or PHOSPHO1-Deficient Matrix Vesicles

During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix by promoting the formation of hydroxyapatite seed crystals in the sheltered interior of membrane-limited matrix vesicles (MVs). Here, we have studied phosphosubstrate catalysis by osteoblast-derived MVs at physiologic pH, analyzing the hydrolysis of ATP, ADP, and PPi by isolated wild-type (WT) as well as TNAP-, NPP1- and PHOSPHO1-deficient MVs. Comparison of the catalytic efficiencies identified ATP as the main substrate hydrolyzed by WT MVs. The lack of TNAP had the most pronounced effect on the hydrolysis of all physiologic substrates. The lack of PHOSPHO1 affected ATP hydrolysis via a secondary reduction in the levels of TNAP in PHOSPHO1-deficient MVs. The lack of NPP1 did not significantly affect the kinetic parameters of hydrolysis when compared with WT MVs for any of the substrates. We conclude that TNAP is the enzyme that hydrolyzes both ATP and PPi in the MV compartment. NPP1 does not have a major role in PPi generation from ATP at the level of MVs, in contrast to its accepted role on the surface of the osteoblasts and chondrocytes, but rather acts as a phosphatase in the absence of TNAP. © 2010 American Society for Bone and Mineral Research

Characterization of transcriptional networks in blood stem and progenitor cells using high-throughput single-cell gene expression analysis

Cellular decision-making is mediated by a complex interplay of external stimuli with the intracellular environment, in particular transcription factor regulatory networks. Here we have determined the expression of a network of 18 key haematopoietic transcription factors in 597 single primary blood stem and progenitor cells isolated from mouse bone marrow. We demonstrate that different stem/progenitor populations are characterized by distinctive transcription factor expression states, and through comprehensive bioinformatic analysis reveal positively and negatively correlated transcription factor pairings, including previously unrecognized relationships between Gata2, Gfi1 and Gfi1b. Validation using transcriptional and transgenic assays confirmed direct regulatory interactions consistent with a regulatory triad in immature blood stem cells, where Gata2 may function to modulate cross-inhibition between Gfi1 and Gfi1b. Single-cell expression profiling therefore identifies network states and allows reconstruction of network hierarchies involved in controlling stem cell fate choices, and provides a blueprint for studying both normal development and human disease

Percutaneous treatment of patients with heart diseases: selection, guidance and follow-up. A review

Aortic stenosis and mitral regurgitation, patent foramen ovale, interatrial septal defect, atrial fibrillation and perivalvular leak, are now amenable to percutaneous treatment. These percutaneous procedures require the use of Transthoracic (TTE), Transesophageal (TEE) and/or Intracardiac echocardiography (ICE). This paper provides an overview of the different percutaneous interventions, trying to provide a systematic and comprehensive approach for selection, guidance and follow-up of patients undergoing these procedures, illustrating the key role of 2D echocardiography

PARP16/ARTD15 is a novel endoplasmic-reticulum-associated mono-ADP-ribosyltransferase that interacts with, and modifies karyopherin-ß1

Background: Protein mono-ADP-ribosylation is a reversible post-translational modification that modulates the function of target proteins. The enzymes that catalyze this reaction in mammalian cells are either bacterial pathogenic toxins or endogenous cellular ADP-ribosyltransferases. The latter include members of three different families of proteins: the well characterized arginine-specific ecto-enzymes ARTCs, two sirtuins and, more recently, novel members of the poly(ADP-ribose) polymerase (PARP/ARTD) family that have been suggested to act as cellular mono-ADP-ribosyltransferases. Here, we report on the characterisation of human ARTD15, the only known ARTD family member with a putative C-terminal transmembrane domain. Methodology/Principal Findings: Immunofluorescence and electron microscopy were performed to characterise the sub-cellular localisation of ARTD15, which was found to be associated with membranes of the nuclear envelope and endoplasmic reticulum. The orientation of ARTD15 was determined using protease protection assay, and is shown to be a tail-anchored protein with a cytosolic catalytic domain. Importantly, by combining immunoprecipitation with mass spectrometry and using cell lysates from cells over-expressing FLAG-ARTD15, we have identified karyopherin-ß1, a component of the nuclear trafficking machinery, as a molecular partner of ARTD15. Finally, we demonstrate that ARTD15 is a mono-ADP-ribosyltransferase able to induce the ADP-ribosylation of karyopherin-ß1, thus defining the first substrate for this enzyme. Conclusions/Significance: Our data reveal that ARTD15 is a novel ADP-ribosyltransferase enzyme with a new intracellular location. Finally, the identification of karyopherin-ß1 as a target of ARTD15-mediated ADP-ribosylation, hints at a novel regulatory mechanism of karyopherin-ß1 functions

Heart function analysis in cardiac patients with focus on sex-specific aspects

Cardiac function is best described by investigating the pressure-volume relationships. This information permits description in terms of the ventricular volume regulation graph (VRG), estimation of systolic elastance, evaluation of lusitropic properties, and assessment of ventriculo-arterial coupling. Current techniques yield noninvasive determination of cardiac compartmental volumes, along with systolic/diastolic arterial pressure, while ventricular end-diastolic pressure can be inferred from an echocardiography-based surrogate measure. Ventricular volume is known to vary with age, as well as to be affected by intrinsic cardiac disease and abnormalities of the vascular system. Moreover, 35 years ago it has been shown in healthy adults that left ventricular volume is significantly smaller in women compared to men. This important observation has serious implications for several metrics which are routinely used in clinical practice, e.g., ejection fraction. The remarkable difference between ventricular size in men and women is also a powerful starting point for the study of aging and the investigation of interventions such as exercise. In this review we evaluate sex-specific characteristics of the VRG and the implications for various cardiac patient populations, during basal conditions and intervention such as exercise