Phosphorylation of caldesmon by myosin light chain kinase increases its binding affinity for phosphorylated myosin filaments

Phosphorylation of myosin by myosin light chain kinase (MLCK) is essential for smooth muscle contraction. In this study we show that caldesmon (CaD) is also phosphorylated in vitro by MLCK. The phosphorylation is calcium- and calmodulin (CaM)-dependent and requires a MLCK concentration close to that found in vivo. On average, approximately 2 mol Pi per mol of CaD are incorporated at Thr-626 and Thr-693, with additional partial phosphorylation at Ser-658 and Ser-702. The phosphorylation rate for CaD is 20- to 50-fold slower than that for filamentous myosin; faster relative rates were obtained with CaD added to purified actomyosin or myosin preparations containing endogenous MLCK/CaM complex. Addition of CaM also augmented CaD phosphorylation. We further demonstrate that [32P] labeled CaD binds much more readily to phosphorylated filamentous myosin than to unphosphorylated myosin. For actomyosin, CaD binding affinity doubles after myosin phosphorylation, without a significant change in binding stoichiometry (approx. one CaD per myosin molecule). Unphosphorylated CaD is ineffective in competing with the phosphorylated protein for the binding site(s) on myosin filaments. The ATPase activity of reconstituted actomyosin is inhibited by unphosphorylated CaD, and this inhibition was removed by CaD phosphorylation. Our results suggest that CaD phosphorylation plays a role in modifying actomyosin interaction in vivo, particularly during prolonged muscle activation

Elastogenic Inductability of Smooth Muscle Cells from a Rat Model of Late Stage Abdominal Aortic Aneurysms

Although abdominal aortic aneurysms (AAA) can be potentially stabilized by inhibiting inflammatory cell recruitment and their release of proteolytic enzymes, active AAA regression is not possible without regeneration of new elastic matrix structures. Unfortunately, postneonatal vascular smooth muscle cells (SMCs), healthy, and likely more so, diseased cells, poorly synthesize or remodel elastic fibers, impeding any effort directed at regenerative AAA treatment. Previously, we determined the eleastogenic benefits of oligomers (HA-o; 4–6 mers) of the glycosaminoglycan, hyaluronan (HA) and transforming growth factor-β1 (TGF-β1) to healthy SMCs. Since AAAs are often diagnosed only late in development when matrix disruption is severe, we now determine if elastogenic upregulation of SMCs from late-stage AAAs (>100% diameter increase) is possible. AAAs were induced by perfusion of rat infrarenal aortae with porcine pancreatic elastase. Elastic matrix degradation, vessel expansion (∼120%), inflammatory cell infiltration, and enhanced activity of matrix-metalloproteases (MMPs) 2 and 9 resulted, paralleling human AAAs. Aneurysmal SMCs (EaRASMCs) maintained a diseased phenotype in 2D cell culture and exhibited patterns of gene expression different from healthy rat aortic SMCs (RASMCs). Relative to passage-matched healthy RASMCs, unstimulated EaRASMCs produced far less tropoelastin and matrix elastin. Exogenous TGF-β and HA-o (termed “factors”) significantly decreased EaRASMC proliferation and enhanced tropoelastin synthesis, though only at the highest provided dose combination (20 mg/mL of HA-o, 10 ng/mL of TGF-β); despite such enhancement, tropoelastin amounts were only ∼40% of amounts synthesized by healthy RASMC cultures. Differently, elastic matrix synthesis was enhanced beyond amounts synthesized by healthy RASMCs (112%), even at lower doses of factors (2 mg/mL of HA-o and 5 ng/mL of TGF-β). The factors also enhanced elastic fiber deposition over untreated EaRASMC cultures and restored several genes whose expression was altered in EaRASMC cultures back to levels expressed by healthy RASMCs. However, the activity of MMPs 2 and 9 generated by EaRASMC cultures was unaffected by the factors/factor dose. The study confirms that SMCs from advanced AAAs can be elastogenically induced, although much higher doses of elastogenic factors are required for induction relative to healthy SMCs. Also, the factors do not appear to inhibit MMP activity, vital to preserve existing elastic matrix structures that serve as nucleation sites for new elastic fiber deposition. Thus, to enhance net accumulation of newly regenerated elastic matrix, toward possibly regressing AAAs, codelivery of MMP inhibitors may be necessitated

Seasonal influenza vaccination predicts pandemic H1N1 vaccination uptake among healthcare workers in three countries

The aim of this study was to identify the common barriers and facilitators for acceptance of pandemic influenza vaccination across different countries. This study utilized a standardized, anonymous, self-completed questionnaire-based survey recording the demographics and professional practice, previous experience and perceived risk and severity of influenza, infection control practices, information of H1N1 vaccination, acceptance of the H1N1 vaccination and reasons of their choices and opinions on mandatory vaccination. Hospital-based doctors, nurses and allied healthcare workers in Hong Kong (HK), Singapore (SG) and Leicester, United Kingdom (UK) were recruited. A total of 6318 (HK: 5743, SG: 300, UK: 275) questionnaires were distributed, with response rates of 27.1% (HK), 94.7% (SG) and 94.5% (UK). The uptake rates for monovalent 2009 pandemic H1N1 vaccine were 13.5% (HK), 36.2% (SG) and 41.3% (UK). The single common factor associated with vaccine acceptance across all sites was having seasonal influenza vaccination in 2009. In UK and HK, overestimation of side effect reduced vaccination acceptance; and fear of side effect was a significant barrier in all sites. In HK, healthcare workers with more patient contact were more reluctant to accept vaccination. Drivers for vaccination in UK and HK were concern about catching the infection and following advice from health authority. Only a small proportion of respondents agreed with mandatory pandemic influenza vaccination (HK: 25% and UK: 42%), except in Singapore where 75.3% were in agreement. Few respondents (&lt;5%) chose scientific publications as their primary source of information, but this group was more likely to receive vaccination. The acceptance of pandemic vaccine among healthcare workers was poor (13-41% of respondents). Breaking barriers to accept seasonal influenza vaccination should be part of the influenza pandemic preparedness plan. Mandatory vaccination even during pandemic is likely to arouse substantial discontent.<br/