Targeted disruption of PDE3B, but not PDE3A, protects murine heart from ischemia/reperfusion injury.

Although inhibition of cyclic nucleotide phosphodiesterase type 3 (PDE3) has been reported to protect rodent heart against ischemia/reperfusion (I/R) injury, neither the specific PDE3 isoform involved nor the underlying mechanisms have been identified. Targeted disruption of PDE3 subfamily B (PDE3B), but not of PDE3 subfamily A (PDE3A), protected mouse heart from I/R injury in vivo and in vitro, with reduced infarct size and improved cardiac function. The cardioprotective effect in PDE3B(-/-) heart was reversed by blocking cAMP-dependent PKA and by paxilline, an inhibitor of mitochondrial calcium-activated K channels, the opening of which is potentiated by cAMP/PKA signaling.

Secretion of fatty acid binding protein aP2 from adipocytes through a nonclassical pathway in response to adipocyte lipase activity.

Adipocyte fatty acid binding protein 4, aP2, contributes to the pathogenesis of several common diseases including type 2 diabetes, atherosclerosis, fatty liver disease, asthma, and cancer. Although the biological functions of aP2 have classically been attributed to its intracellular action, recent studies demonstrated that aP2 acts as an adipokine to regulate systemic metabolism. However, the mechanism and regulation of aP2 secretion remain unknown.

In vivo microscopy reveals extensive embedding of capillaries within the sarcolemma of skeletal muscle fibers.

Objective: To provide insight into mitochondrial function in vivo, we evaluated the 3D spatial relationship between capillaries, mitochondria, and muscle fibers in live mice.

Methods: 3D volumes of in vivo murine TA muscles were imaged by MPM. Muscle fiber type, mitochondrial distribution, number of capillaries, and capillary-to-fiber contact were assessed.

Imaging flow cytometry for automated detection of hypoxia-induced erythrocyte shape change in sickle cell disease.

In preclinical and early phase pharmacologic trials in sickle cell disease, the percentage of sickled erythrocytes after deoxygenation, an ex vivo functional sickling assay, has been used as a measure of a patient's disease outcome. We developed a new sickle imaging flow cytometry assay (SIFCA) and investigated its application. To perform the SIFCA, peripheral blood was diluted, deoxygenated (2% oxygen) for 2 hr, fixed, and analyzed using imaging flow cytometry.

Autophagy regulates endothelial cell processing, maturation and secretion of von Willebrand factor.

Endothelial secretion of von Willebrand factor (VWF) from intracellular organelles known as Weibel-Palade bodies (WPBs) is required for platelet adhesion to the injured vessel wall. Here we demonstrate that WPBs are often found near or within autophagosomes and that endothelial autophagosomes contain abundant VWF protein. Pharmacological inhibitors of autophagy or knockdown of the essential autophagy genes Atg5 or Atg7 inhibits the in vitro secretion of VWF.

Imaging the post-fusion release and capture of a vesicle membrane protein.

The molecular mechanism responsible for capturing, sorting and retrieving vesicle membrane proteins following triggered exocytosis is not understood. Here we image the post-fusion release and then capture of a vesicle membrane protein, the vesicular acetylcholine transporter, from single vesicles in living neuroendocrine cells. We combine these measurements with super-resolution interferometric photo-activation localization microscopy and electron microscopy, and modelling to map the nanometer-scale topography and architecture of the structures responsible for the transporter's capture following exocytosis.

Deletion of mitochondrial associated ubiquitin fold modifier protein Ufm1 in Leishmania donovani results in loss of β-oxidation of fatty acids and blocks cell division in the amastigote stage.

Recently, we described the existence of the ubiquitin fold modifier 1 (Ufm1) and its conjugation pathway in Leishmania donovani. We demonstrated the conjugation of Ufm1 to proteins such as mitochondrial trifunctional protein (MTP) that catalyses β-oxidation of fatty acids in L. donovani.

High prevalence of respiratory ciliary dysfunction in congenital heart disease patients with heterotaxy.

Background: Patients with congenital heart disease (CHD) and heterotaxy show high postsurgical morbidity/mortality, with some developing respiratory complications. Although this finding is often attributed to the CHD, airway clearance and left-right patterning both require motile cilia function. Thus, airway ciliary dysfunction (CD) similar to that of primary ciliary dyskinesia (PCD) may contribute to increased respiratory complications in heterotaxy patients.

The role of agrin in synaptic development, plasticity and signaling in the central nervous system.

Development of the neuromuscular junction (NMJ) requires secretion of specific isoforms of the proteoglycan agrin by motor neurons. Secreted agrin is widely expressed in the basal lamina of various tissues, whereas a transmembrane form is highly expressed in the brain. Expression in the brain is greatest during the period of synaptogenesis, but remains high in regions of the adult brain that show extensive synaptic plasticity.

Actin cross-linking proteins cortexillin I and II are required for cAMP signaling during Dictyostelium chemotaxis and development.

Starvation induces Dictyostelium amoebae to secrete cAMP, toward which other amoebae stream, forming multicellular mounds that differentiate and develop into fruiting bodies containing spores. We find that the double deletion of cortexillin (ctx) I and II alters the actin cytoskeleton and substantially inhibits all molecular responses to extracellular cAMP. Synthesis of cAMP receptor and adenylyl cyclase A (ACA) is inhibited, and activation of ACA, RasC, and RasG, phosphorylation of extracellular signal regulated kinase 2, activation of TORC2, and stimulation of actin polymerization and myosin assembly are greatly reduced.

A cytosolic phospholipase A2-initiated lipid mediator pathway induces autophagy in macrophages.

Autophagy delivers cytoplasmic constituents to autophagosomes and is involved in innate and adaptive immunity. Cytosolic phospholipase (cPLA(2))-initiated proinflammatory lipid mediator pathways play a critical role in host defense and inflammation. The crosstalk between the two pathways remains unclear.

Mouse models of MYH9-related disease: mutations in nonmuscle myosin II-A.

We have generated 3 mouse lines, each with a different mutation in the nonmuscle myosin II-A gene, Myh9 (R702C, D1424N, and E1841K). Each line develops MYH9-related disease similar to that found in human patients. R702C mutant human cDNA fused with green fluorescent protein was introduced into the first coding exon of Myh9, and D1424N and E1841K mutations were introduced directly into the corresponding exons.

Disruption of caveolae blocks ischemic preconditioning-mediated S-nitrosylation of mitochondrial proteins.

Aims: Nitric oxide (NO) and protein S-nitrosylation (SNO) play important roles in ischemic preconditioning (IPC)-induced cardioprotection. Mitochondria are key regulators of preconditioning, and most proteins showing an increase in SNO with IPC are mitochondrial. The aim of this study was to address how IPC transduces NO/SNO signaling to mitochondria in the heart.

Expression of Y53A-actin in Dictyostelium disrupts the cytoskeleton and inhibits intracellular and intercellular chemotactic signaling.

We showed previously that phosphorylation of Tyr(53), or its mutation to Ala, inhibits actin polymerization in vitro with formation of aggregates of short filaments, and that expression of Y53A-actin in Dictyostelium blocks differentiation and development at the mound stage (Liu, X., Shu, S., Hong, M.

Induction of filopodia-like protrusions by transmembrane agrin: role of agrin glycosaminoglycan chains and Rho-family GTPases.

Filopodia sense the extracellular environment and direct movement in many cell types, including neurons. Recent reports suggest that the transmembrane form of the widely expressed proteoglycan agrin (TM-agrin) regulates formation and stability of neuronal filopodia. In order to elucidate the mechanism by which TM-agrin regulates filopodia, we investigated the role of agrin's glycosaminoglycan (GAG) chains in the induction of filopodia formation by TM-agrin over-expression in hippocampal neurons, and in the induction of filopodia-like processes in COS7 cells.

Mitochondrial dysfunction and oxidative stress mediate the physiological impairment induced by the disruption of autophagy.

Impaired or deficient autophagy is believed to cause or contribute to aging, as well as a number of age-related pathologies. The exact mechanism through which alterations in autophagy induce these various pathologies is not well understood. Here we describe the creation of two in vivo mouse models that allow for the characterization of the alteration in mitochondrial function and the contribution of the corresponding oxidative stress following deletion of Atg7.

Oxidative stress causes reversible changes in mitochondrial permeability and structure.

Mitochondria are a primary source as well a principal target of reactive oxygen species within cells. Using immunofluorescence microscopy, we have found that a number of mitochondrial matrix proteins are normally undetectable in formaldehyde-fixed cells permeabilized with the cholesterol-binding detergent saponin. However, exogenous or endogenous oxidative stress applied prior to fixation altered the permeability of mitochondria, rendering these matrix proteins accessible to antibodies.

Proteomic profiling of human plasma exosomes identifies PPARgamma as an exosome-associated protein.

Exosomes are nanovesicles that are released from cells as a mechanism of cell-free intercellular communication. Only a limited number of proteins have been identified from the plasma exosome proteome. Here, we developed a multi-step fractionation scheme incorporating gel exclusion chromatography, rate zonal centrifugation through continuous sucrose gradients, and high-speed centrifugation to purify exosomes from human plasma.

Autophagic dysfunction in mucolipidosis type IV patients.

Mutations in Mucolipin 1 (MCOLN1) have been linked to mucolipidosis type IV (MLIV), a lysosomal storage disease characterized by several neurological and ophthalmological abnormalities. It has been proposed that MCOLN1 might regulate transport of membrane components in the late endosomal-lysosomal pathway; however, the mechanisms by which defects of MCOLN1 function result in mental and psychomotor retardation remain largely unknown. In this study, we show constitutive activation of autophagy in fibroblasts obtained from MLIV patients.

Krp1 (Sarcosin) promotes lateral fusion of myofibril assembly intermediates in cultured mouse cardiomyocytes.

Krp1, also called sarcosin, is a cardiac and skeletal muscle kelch repeat protein hypothesized to promote the assembly of myofibrils, the contractile organelles of striated muscles, through interaction with N-RAP and actin. To elucidate its role, endogenous Krp1 was studied in primary embryonic mouse cardiomyocytes. While immunofluorescence showed punctate Krp1 distribution throughout the cell, detergent extraction revealed a significant pool of Krp1 associated with cytoskeletal elements.

Heterotaxy and complex structural heart defects in a mutant mouse model of primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder associated with ciliary defects and situs inversus totalis, the complete mirror image reversal of internal organ situs (positioning). A variable incidence of heterotaxy, or irregular organ situs, also has been reported in PCD patients, but it is not known whether this is elicited by the PCD-causing genetic lesion. We studied a mouse model of PCD with a recessive mutation in Dnahc5, a dynein gene commonly mutated in PCD.

Transmembrane agrin regulates filopodia in rat hippocampal neurons in culture.

Filopodia mediate axon guidance, neurite branching and synapse formation, but the membrane molecules that regulate neuronal filopodia in response to extracellular cues are largely unknown. The transmembrane isoform of the proteoglycan agrin, expressed predominantly in the CNS, may regulate neurite outgrowth, synapse formation and excitatory signaling. Here we demonstrate that agrin positively regulates neuronal filopodia.

Calcineurin localization in skeletal muscle offers insights into potential new targets.

The Ca(2+)/calmodulin-activated protein phosphatase, calcineurin, is believed to regulate the development and function of skeletal and cardiac muscle. Striated muscle contains many calcineurin substrates, a few of which have been colocalized or found in molecular complexes with calcineurin. We examined the subcellular distribution of calcineurin in developing rat skeletal muscle cells and adult mouse skeletal muscle fibers by immunofluorescence microscopy.