No abstract

Older adults suffer from weakened and delayed bone healing due to age-related alterations in bone cells and in the immune system. Given the interaction between the immune system and skeletal cells, therapies that address deficiencies in both the skeletal and the immune system are required to effectively treat bone injuries of older patients. The sequence of macrophage activation observed in healthy tissue repair involves a transition from a pro-inflammatory state followed by a pro-reparative state. In older patients, inflammation is slower to resolve and impedes healing. The goal of this study was to design a novel drug delivery system for temporal guidance of the polarization of macrophages using bone grafting materials. A biomimetic calcium phosphate coating (bCaP) physically and temporally separated the pro-inflammatory stimulus interferon-gamma (IFNγ) from the pro-reparative stimulus simvastatin (SIMV). Effective doses were identified using a human monocyte line (THP-1) and testing culminated with bone marrow macrophages obtained from old mice. Sequential M1-to-M2 activation was achieved with both cell types. These results suggest that this novel immunomodulatory drug delivery system holds potential for controlling macrophage activation in bones of older patients.

Although the influenza virus only infects the respiratory system, myalgias are commonly experienced during infection. In addition to a greater risk of hospitalization and death, older adults are more likely to develop disability following influenza infection; however, this relationship is understudied. We hypothesized that upon challenge with influenza, aging would be associated with functional impairments, as well as upregulation of skeletal muscle inflammatory and atrophy genes. Infected young and aged mice demonstrated decreased mobility and altered gait kinetics. These declines were more prominent in hind limbs and in aged mice. Skeletal muscle expression of genes involved in inflammation, as well as muscle atrophy and proteolysis, increased during influenza infection with an elevated and prolonged peak in aged mice. Infection also decreased expression of positive regulators of muscle mass and myogenesis components to a greater degree in aged mice. Gene expression correlated to influenza-induced body mass loss, although evidence did not support direct muscle infection. Overall, influenza leads to mobility impairments with induction of inflammatory and muscle degradation genes and downregulation of positive regulators of muscle. These effects are augmented and prolonged with aging, providing a molecular link between influenza infection, decreased resilience and increased risk of disability in the elderly.

No abstract

Myristoylated alanine-rich C kinase substrate (MARCKS) is an unfolded protein that contains well characterized actin-binding sites within the phosphorylation site domain (PSD), yet paradoxically, we now find that intact MARCKS does not bind to actin. Intact MARCKS also does not bind as well to calmodulin as does the PSD alone. Myristoylation at the N terminus alters how calmodulin binds to MARCKS, implying that, despite its unfolded state, the distant N terminus influences binding events at the PSD. We show that the free PSD binds with site specificity to MARCKS, suggesting that long-range intramolecular interactions within MARCKS are also possible. Because of the unusual primary sequence of MARCKS with an overall isoelectric point of 4.2 yet a very basic PSD (overall charge of ؉13), we speculated that ionic interactions between oppositely charged domains of MARCKS were responsible for long-range interactions within MARCKS that sterically influence binding events at the PSD and that explain the observed differences between properties of the PSD and MARCKS. Consistent with this hypothesis, chemical modifications of MARCKS that neutralize negatively charged residues outside of the PSD allow the PSD to bind to actin and increase the affinity of MARCKS for calmodulin. Similarly, both myristoylation of MARCKS and cleavage of MARCKS by calpain are shown to increase the availability of the PSD so as to activate its actin-binding activity. Because abundant evidence supports the conclusion that MARCKS is an important protein in regulating actin dynamics, our data imply that post-translational modifications of MARCKS are necessary and sufficient to regulate actin-binding activity. Myristoylated alanine-rich C kinase substrate (MARCKS)1 is a well characterized, charge-polarized, natively unfolded molecule (1-3) with a centrally located active site known as the phosphorylation site domain (PSD). Consistent with the paradigm for natively unfolded proteins, MARCKS is thought to interact with several ligands so as to integrate information from various signal transduction pathways to produce an output signal that regulates cell motile and contractile function. Numerous studies of the MARCKS protein have utilized a peptide with a sequence that corresponds to the PSD peptide as a substitute for studying interactions between the intact protein and its multiple ligands (3, 4). Although this approach intuitively appears to be logical, given the unfolded state of the native protein, the substitution of the PSD peptide for the intact protein has never been rigorously justified. In fact, there are several reported experiments that imply that the PSD peptide behaves differently from intact MARCKS. The nonphosphorylated PSD peptide is known to have extended structure, to nucleate polymerization, and to cross-link F-actin filaments (5-7), presumably because of two binding sites with a site-specific K d of ϳ0.5 M for F-actin (8). Although the PSD of MARCKS and its homolog MARCKS-related protein have both been shown to bind to actin with si...