Differential impact of mitochondrial positioning on mitochondrial Ca(2+) uptake and Ca(2+) spark suppression in skeletal muscle

Muscle contraction requires ATP and Ca(2+) and, thus, is under direct control of mitochondria and the sarcoplasmic reticulum. During postnatal skeletal muscle maturation, the mitochondrial network exhibits a shift from a longitudinal ("longitudinal mitochondria") to a mostly transversal orientation as a result of a progressive increase in mitochondrial association with Ca(2+) release units (CRUs) or triads ("triadic mitochondria"). To determine the physiological implications of this shift in mitochondrial disposition, we used confocal microscopy to monitor activity-dependent changes in myoplasmic (fluo 4) and mitochondrial (rhod 2) Ca(2+) in single flexor digitorum brevis (FDB) fibers from 1- to 4-mo-old mice. A robust and sustained Ca(2+) accumulation in triadic mitochondria was triggered by repetitive tetanic stimulation (500 ms, 100 Hz, every 2.5 s) in FDB fibers from 4-mo-old mice. Specifically, mitochondrial rhod 2 fluorescence increased 272 ± 39% after a single tetanus and 412 ± 45% after five tetani and decayed slowly over 10 min following the final tetanus. Similar results were observed in fibers expressing mitochondrial pericam, a mitochondrial-targeted ratiometric Ca(2+) indicator. Interestingly, sustained mitochondrial Ca(2+) uptake following repetitive tetanic stimulation was similar for triadic and longitudinal mitochondria in FDB fibers from 1-mo-old mice, and both mitochondrial populations were found by electron microscopy to be continuous and structurally tethered to the sarcoplasmic reticulum. Conversely, the frequency of osmotic shock-induced Ca(2+) sparks per CRU density decreased threefold (from 3.6 ± 0.2 to 1.2 ± 0.1 events·CRU(-1)·min(-1)·100 μm(-2)) during postnatal development in direct linear correspondence (r(2) = 0.95) to an increase in mitochondrion-CRU pairing. Together, these results indicate that mitochondrion-CRU association promotes Ca(2+) spark suppression but does not significantly impact mitochondrial Ca(2+) uptake.     

Multivalent display of proteins on viral nanoparticles using molecular recognition and chemical ligation strategies

Multivalent display of heterologous proteins on viral nanoparticles forms a basis for numerous applications in nanotechnology, including vaccine development, targeted therapeutic delivery, and tissue-specific bioimaging. In many instances, precise placement of proteins is required for optimal functioning of the supramolecular assemblies, but orientation- and site-specific coupling of proteins to viral scaffolds remains a significant technical challenge. We have developed two strategies that allow for controlled attachment of a variety of proteins on viral particles using covalent and noncovalent principles. In one strategy, an interaction between domain 4 of anthrax protective antigen and its receptor was used to display multiple copies of a target protein on virus-like particles. In the other, expressed protein ligation and aniline-catalyzed oximation was used to display covalently a model protein. The latter strategy, in particular, yielded nanoparticles that induced potent immune responses to the coupled protein, suggesting potential applications in vaccine development.     

Synthesis and application of cNGR-containing imaging agents for detection of angiogenesis

Angiogenesis is a multi-step process regulated by pro- and anti-angiogenic factors. Inhibition of angiogenesis is a potential anti cancer treatment strategy that is now investigated clinically. In addition, advances in the understanding of the angiogenic process have led to the development of new angiogenesis therapies for ischemic heart disease.Currently, researchers search for objective measures that indicate pharmacological responses to pro- and anti-angiogenic drugs and therefore, there is a great interest in techniques to visualize angiogenesis noninvasively. As CD13 is selectively expressed in angiogenic blood vessels, it can serve as a target for molecular imaging tracers to noninvasively visualize angiogenic processes in animal models and patients. Here, an overview on the currently used CD13 targeted molecular imaging probes for noninvasive visualization of angiogenesis is given.     

Differentiated mouse epithelial cell line with hepatocyte characteristics

An epithelial cell line, 3105, with an unusual growth pattern has been derived from the liver of an (NZB x NZW)F1 mouse. When confluent, it forms a monolayer of closely packed cells interspersed with holes that do not fill in during cultivation. By electron microscopy, the line has tight and intermediate junctions as well as desmosomes typical of epithelial cells. It produces several enzymes normally present in liver including hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, glucose-6-phosphatase, and alkaline phosphatase; has cytochromes P-450 and b5; and spontaneously release xenotropic but not ecotropic endogenous mouse type C viruses. Inoculation of the cell line into athymic nude mice gives rise to benign cysts in 2-3 months. This mouse epithelial line with hepatocyte characteristics should be helpful to investigators as a cell model of normal liver cell differentiation.     

Adiponectin mediates antiproliferative and apoptotic responses in human MCF7 breast cancer cells

It is well established that obesity is a risk factor for breast cancer and that blood levels of adiponectin, a hormone mainly secreted by white adipocytes, are inversely correlated with the body fat mass. As adiponectin elicits anti-proliferative effects in some cell types, we tested the hypothesis that adiponectin could influence human breast cancer MCF-7 cell growth. Here we show that MCF-7 cells express adiponectin receptors and respond to human recombinant adiponectin by reducing their growth, AMPkinase activation, and p42/p44 MAPkinase inactivation. Further, we demonstrate that the anti-proliferative effect of adiponectin involves activation of cell apoptosis and inhibition of cell cycle. These findings suggest that adiponectin could act in vivo as a paracrine/endocrine growth inhibitor towards mammary epithelial cells. Moreover, adipose adiponectin production being strongly reduced in obesity, this study may help to explain why obesity is a risk factor of developing breast cancers.     

Design and structure-activity relationship of heterocyclic analogs of 4-amino-3-benzimidazol-2-ylhydroquinolin-2-ones as inhibitors of receptor tyrosine kinases

Herein are described a series of novel heterocyclic analogs of the 4-amino-3-benzimidazol-2-ylhydroquinolin-2-one scaffold. These compounds are potent inhibitors of receptor tyrosine kinases and exhibit favorable pharmacokinetic profiles. The synthesis and SAR of these compounds are described.     

Temperature and RyR1 Regulate the Activation Rate of Store-Operated Ca(2+) Entry Current in Myotubes

Store-operated calcium entry (SOCE) is an important Ca²⁺ entry pathway in skeletal muscle. However, direct electrophysiological recording and full characterization of the underlying SOCE current in skeletal muscle cells (I_SkCRAC) has not been reported. Here, we characterized the biophysical properties, pharmacological profile, and molecular identity of I_SkCRAC in skeletal myotubes, as well as the regulation of its rate of activation by temperature and the type I ryanodine receptor (RyR1). I_SkCRAC exhibited many hallmarks of Ca²⁺ release activated Ca²⁺ currents (I_CRAC): store dependence, strong inward rectification, positive reversal potential, limited cesium permeability, and sensitivity to SOCE channel blockers. I_SkCRAC was reduced by siRNA knockdown of stromal interaction molecule 1 and expression of dominant negative Orai1. Average I_SkCRAC current density at −80mV was 1.00 ± 0.05 pA/pF. In the presence of 20 mM intracellular EGTA, I_SkCRAC activation occurred over tens of seconds during repetitive depolarization at 0.5Hz and was inhibited by treatment with 100 μM ryanodine. The rate of SOCE activation was reduced threefold in myotubes from RyR1-null mice and increased 4.6-fold at physiological temperatures (35–37°C). These results show that I_SkCRAC exhibits similar biophysical, pharmacological, and molecular properties as I_CRAC in nonexcitable cells and its rate of activation during repetitive depolarization is strongly regulated by temperature and RyR1 activity.