The Role of the N-Terminus of the Myosin Essential Light Chain in Cardiac Muscle Contraction

To study the regulation of cardiac muscle contraction by the myosin essential light chain (ELC) and the physiological significance of its N-terminal extension, we generated transgenic (Tg) mice by partially replacing the endogenous mouse ventricular ELC with either the human ventricular ELC wild type (Tg-WT) or its 43-amino-acid N-terminal truncation mutant (Tg-Δ43) in the murine hearts. The mutant protein is similar in sequence to the short ELC variant present in skeletal muscle, and the ELC protein distribution in Tg-Δ43 ventricles resembles that of fast skeletal muscle. Cardiac muscle preparations from Tg-Δ43 mice demonstrate reduced force per cross-sectional area of muscle, which is likely caused by a reduced number of force-generating myosin cross-bridges and/or by decreased force per cross-bridge. As the mice grow older, the contractile force per cross-sectional area further decreases in Tg-Δ43 mice and the mutant hearts develop a phenotype of nonpathologic hypertrophy while still maintaining normal cardiac performance. The myocardium of older Tg-Δ43 mice also exhibits reduced myosin content. Our results suggest that the role of the N-terminal ELC extension is to maintain the integrity of myosin and to modulate force generation by decreasing myosin neck region compliance and promoting strong cross-bridge formation and/or by enhancing myosin attachment to actin.     

Adult Cardiac Progenitor Cell Aggregates Exhibit Survival Benefit Both In Vitro and In Vivo

Background

A major hurdle in the use of exogenous stems cells for therapeutic regeneration of injured myocardium remains the poor survival of implanted cells. To date, the delivery of stem cells into myocardium has largely focused on implantation of cell suspensions.

Methodology and Principal Findings

We hypothesize that delivering progenitor cells in an aggregate form would serve to mimic the endogenous state with proper cell-cell contact, and may aid the survival of implanted cells. Microwell methodologies allow for the culture of homogenous 3D cell aggregates, thereby allowing cell-cell contact. In this study, we find that the culture of cardiac progenitor cells in a 3D cell aggregate augments cell survival and protects against cellular toxins and stressors, including hydrogen peroxide and anoxia/reoxygenation induced cell death. Moreover, using a murine model of cardiac ischemia-reperfusion injury, we find that delivery of cardiac progenitor cells in the form of 3D aggregates improved in vivo survival of implanted cells.

Conclusion

Collectively, our data support the notion that growth in 3D cellular systems and maintenance of cell-cell contact improves exogenous cell survival following delivery into myocardium. These approaches may serve as a strategy to improve cardiovascular cell-based therapies.     

How Nature Modulates Inherent Fluctuations for Biological Self-Organization – The Case of Membrane Fusion

Biological systems in nano-scale, due to the weak electrostatic interactions and structural connectivity therein, are flexible so that they undergo conformational transition subject to thermal fluctuations and external noises. In the presence of barriers, nature utilizes the fluctuations to give rise to self-organization, typically accompanied by conformational transitions. In two opposing membranes with like-charges, the cooperative coupling between the undulation and charge fluctuations give rise to a dynamic instability to spontaneous growth of the in-phase membrane undulation, and thus a great reduction of the energy barrier to fusion. The multivalent counter-ions, the Ca²⁺ for example, enhance the necessary charge density fluctuation leading to surface charge inversion and overcondensation.     

Viral and cellular kinases are potential antiviral targets and have a central role in varicella zoster virus pathogenesis

Herpesviruses utilize viral and cellular kinases for replication, and these mediate essential functions that are important for viral pathogenesis. Elucidating the roles of kinases in herpesvirus infections may highlight virus-host interactions that are possible targets for kinase inhibitors with antiviral activity. Varicella zoster virus (VZV) encodes two kinases that phosphorylate viral proteins involved in regulation, assembly, and virulence. VZV infection also induces the activity of host cell cyclin-dependent kinases (cdk4 and cdk2) in nondividing cells, causing a disregulation of the cell cycle. Roscovitine and Purvalanol, kinase inhibitors that target cdks, prevent VZV replication at concentrations with few cytotoxic effects. Cdk inhibitors therefore have potential as antivirals that may extend to a broad range of viruses and have the added advantage that resistance does not arise easily.     

Superoxide production at phagosomal cup/phagosome through beta I protein kinase C during Fc gamma R-mediated phagocytosis in microglia

Protein kinase C (PKC) plays a prominent role in immune signaling. To elucidate the signal transduction in a respiratory burst and isoform-specific function of PKC during FcgammaR-mediated phagocytosis, we used live, digital fluorescence imaging of mouse microglial cells expressing GFP-tagged molecules. betaI PKC, epsilonPKC, and diacylglycerol kinase (DGK) beta dynamically and transiently accumulated around IgG-opsonized beads (BIgG). Moreover, the accumulation of p47(phox), an essential cytosolic component of NADPH oxidase and a substrate for betaI PKC, at the phagosomal cup/phagosome was apparent during BIgG ingestion. Superoxide (O(2)(-)) production was profoundly inhibited by G?6976, a cPKC inhibitor, and dramatically increased by the DGK inhibitor, R59949. Ultrastructural analysis revealed that BIgG induced O(2)(-) production at the phagosome but not at the intracellular granules. We conclude that activation/accumulation of betaI PKC is involved in O(2)(-) production, and that O(2)(-) production is primarily initiated at the phagosomal cup/phagosome. This study also suggests that DGKbeta plays a prominent role in regulation of O(2)(-) production during FcgammaR-mediated phagocytosis.     

DHP-Derivative and Low Oxygen Tension Effectively Induces Human Adipose Stromal Cell Reprogramming

Background and Methods

In this study, we utilized a combination of low oxygen tension and a novel anti-oxidant, 4-(3,4-dihydroxy-phenyl)-derivative (DHP-d) to directly induce adipose tissue stromal cells (ATSC) to de-differentiate into more primitive stem cells. De-differentiated ATSCs was overexpress stemness genes, Rex-1, Oct-4, Sox-2, and Nanog. Additionally, demethylation of the regulatory regions of Rex-1, stemnesses, and HIF1α and scavenging of reactive oxygen species were finally resulted in an improved stem cell behavior of de-differentiate ATSC (de-ATSC). Proliferation activity of ATSCs after dedifferentiation was induced by REX1, Oct4, and JAK/STAT3 directly or indirectly. De-ATSCs showed increased migration activity that mediated by P38/JUNK and ERK phosphorylation. Moreover, regenerative efficacy of de-ATSC engrafted spinal cord-injured rats and chemical-induced diabetes animals were significantly restored their functions.

Conclusions/Significance

Our stem cell remodeling system may provide a good model which would provide insight into the molecular mechanisms underlying ATSC proliferation and transdifferentiation. Also, these multipotent stem cells can be harvested may provide us with a valuable reservoir of primitive and autologous stem cells for use in a broad spectrum of regenerative cell-based disease therapy.