Time course of isotonic shortening and the underlying contraction mechanism in airway smooth muscle

Although the structure of the contractile unit in smooth muscle is poorly understood, some of the mechanical properties of the muscle suggest that a sliding-filament mechanism, similar to that in striated muscle, is also operative in smooth muscle. To test the applicability of this mechanism to smooth muscle function, we have constructed a mathematical model based on a hypothetical structure of the smooth muscle contractile unit: a side-polar myosin filament sandwiched by actin filaments, each attached to the equivalent of a Z disk. Model prediction of isotonic shortening as a function of time was compared with data from experiments using ovine tracheal smooth muscle. After equilibration and establishment of in situ length, the muscle was stimulated with ACh (100 μM) until force reached a plateau. The muscle was then allowed to shorten isotonically against various loads. From the experimental records, length-force and force-velocity relationships were obtained. Integration of the hyperbolic force-velocity relationship and the linear length-force relationship yielded an exponential function that approximated the time course of isotonic shortening generated by the modeled sliding-filament mechanism. However, to obtain an accurate fit, it was necessary to incorporate a viscoelastic element in series with the sliding-filament mechanism. The results suggest that a large portion of the shortening is due to filament sliding associated with muscle activation and that a small portion is due to continued deformation associated with an element that shows viscoelastic or power-law creep after a step change in force.     

Association of cooking oil fumes exposure with lung cancer: involvement of inhibitor of apoptosis proteins in cell survival and proliferation in vitro

Cooking oil fumes (COF) have been shown to be associated with lung cancer incidence in Chinese women. Our recent report indicates that inhibitor of apoptosis protein 2 (IAP2) induced by COF may contribute to the survival and proliferation of A549 lung cancer cells. In this study, to further verify whether other antiapoptosis proteins including IAP1, X-linked IAP (XIAP), and survivin, were linked with lung cancer cell survival and proliferation, these IAPs expressions in A549 cells after treatment with COF and its two major components, benzo[a]pyrene (BaP) and 2,4-decadienal (2,4-DDE) were evaluated by Western blotting. Our data showed that IAP2 was significantly induced by COF, BaP, and 2,4-DDE, but XIAP was decreased by COF and 2,4-DDE, but not by BaP. Even though different effects of COF and 2,4-DDE on IAP2 and XIAP protein expressions were observed, the caspase-3 expression was diminished by COF and 2,4-DDE. In addition, induction of IAP2 and phosphorylated Akt proteins by COF and 2,4-DDE were simultaneously abolished by LY294002. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis showed that the proportion of A549 cells at the S-phase was increased significantly after treatment with COF or 2,4-DDE. The cell proliferation induced by COF is associated with the attenuation of p21(Cip/Waf1) expression. Therefore, increases of IAP1, IAP2, survivin, and cyclin D1 expressions and decreases of XIAP, caspase-3, and p21 expressions might partly contribute to the survival and proliferation of lung cancer cells after exposure to 2,4-DDE and COF. In conclusion, the lung cancer cell growth promoted by COF might support previous epidemiological reports indicating that exposure of COF was associated with lung cancer development among Chinese women.     

Physical integrity of smooth muscle myosin filaments is enhanced by phosphorylation of the regulatory myosin light chain.

BACKGROUND AND AIMS: Smooth muscle myosin monomers self-assemble in solution to form filaments. Phosphorylation of the 20-kD regulatory myosin light chain (MLC20) enhances filament formation. It is not known whether the phosphorylated and non-phosphorylated filaments possess the same structural integrity. METHODS: We purified myosin from bovine trachealis to form filaments, in ATP-containing zero-calcium solution during a slow dialysis that gradually reduced the ionic strength. Sufficient myosin light chain kinase and phosphatase, as well as calmodulin, were retained after the myosin purification and this enabled phosphorylation of MLC20 within 20-40s after addition of calcium to the filament suspension. The phosphorylated and non-phosphorylated filaments were then partially disassembled by ultrasonification. The extent of filament disintegration was visualized and quantified by atomic force microscopy. RESULTS: MLC20 phosphorylation reduced the diameter of the filaments and rendered the filaments more resistant to ultrasonic agitation. Electron microscopy revealed a similar reduction in filament diameter in intact smooth muscle when the cells were activated. CONCLUSION: Modification of the structural and physical properties of myosin filaments by MLC20 phosphorylation may be a key regulation step in smooth muscle where formation and dissolution of the filaments are required in the cells' adaptation to different cell length.     

IL-17A and Serum Amyloid A Are Elevated in a Cigarette Smoke Cessation Model Associated with the Persistence of Pigmented Macrophages,Neutrophils and Activated NK Cells

While global success in cessation advocacy has seen smoking rates fall in many developed countries, persistent lung inflammation in ex-smokers is an increasingly important clinical problem whose mechanistic basis remains poorly understood. In this study, candidate effector mechanisms were assessed in mice exposed to cigarette smoke (CS) for 4 months following cessation from long term CS exposure. BALF neutrophils, CD4⁺ and CD8⁺ T cells and lung innate NK cells remained significantly elevated following smoking cessation. Analysis of neutrophil mobilization markers showed a transition from acute mediators (MIP-2α, KC and G-CSF) to sustained drivers of neutrophil and macrophage recruitment and activation (IL-17A and Serum Amyoid A (SAA)). Follicle-like lymphoid aggregates formed with CS exposure and persisted with cessation, where they were in close anatomical proximity to pigmented macrophages, whose number actually increased 3-fold following CS cessation. This was associated with the elastolytic protease, MMP-12 (macrophage metallo-elastase) which remained significantly elevated post-cessation. Both GM-CSF and CSF-1 were significantly increased in the CS cessation group relative to the control group. In conclusion, we show that smoking cessation mediates a transition to accumulation of pigmented macrophages, which may contribute to the expanded macrophage population observed in COPD. These macrophages together with IL-17A, SAA and innate NK cells are identified here as candidate persistence determinants and, we suggest, may represent specific targets for therapies directed towards the amelioration of chronic airway inflammation.     

Helminth Colonization Is Associated with Increased Diversity of the Gut Microbiota

Soil-transmitted helminths colonize more than 1.5 billion people worldwide, yet little is known about how they interact with bacterial communities in the gut microbiota. Differences in the gut microbiota between individuals living in developed and developing countries may be partly due to the presence of helminths, since they predominantly infect individuals from developing countries, such as the indigenous communities in Malaysia we examine in this work. We compared the composition and diversity of bacterial communities from the fecal microbiota of 51 people from two villages in Malaysia, of which 36 (70.6%) were infected by helminths. The 16S rRNA V4 region was sequenced at an average of nineteen thousand sequences per samples. Helminth-colonized individuals had greater species richness and number of observed OTUs with enrichment of Paraprevotellaceae, especially with Trichuris infection. We developed a new approach of combining centered log-ratio (clr) transformation for OTU relative abundances with sparse Partial Least Squares Discriminant Analysis (sPLS-DA) to enable more robust predictions of OTU interrelationships. These results suggest that helminths may have an impact on the diversity, bacterial community structure and function of the gut microbiota.     

Cdk5 phosphorylation of doublecortin ser297 regulates its effect on neuronal migration

Mutations in the doublecortin (DCX) gene in human or targeted disruption of the cdk5 gene in mouse lead to similar cortical lamination defects in the developing brain. Here we show that Dcx is phosphorylated by Cdk5. Dcx phosphorylation is developmentally regulated and corresponds to the timing of expression of p35, the major activating subunit for Cdk5. Mass spectrometry and Western blot analysis indicate phosphorylation at Dcx residue Ser297. Phosphorylation of Dcx lowers its affinity to microtubules in vitro, reduces its effect on polymerization, and displaces it from microtubules in cultured neurons. Mutation of Ser297 blocks the effect of Dcx on migration in a fashion similar to pharmacological inhibition of Cdk5 activity. These results suggest that Dcx phosphorylation by Cdk5 regulates its actions on migration through an effect on microtubules.     

Biliverdin-promoted lateral root formation is mediated through heme oxygenase in rice

In this study, we examined the effect of biliverdin (BV), a product of heme oxygenase (HO) catalyzed reaction, on lateral root (LR) formation in rice. Treatment with BV induced LR formation and HO activity. As well, BV, could induce OsHO1 mRNA expression. Zn protoporphyrin IX (the specific inhibitor of HO) reduced LR number, HO activity and OsHO1 mRNA level induced by BV. Our data suggest that HO is required for BV-induced LR formation in rice.     

Human umbilical cord blood‐derived mesenchymal stem cells (hUCB‐MSC) inhibit the proliferation of K562 (human erythromyeloblastoid leukaemic cell line)

hUCB‐MSC (human umbilical cord blood‐derived mesenchymal stem cells) offer an attractive alternative to bone marrow‐derived MSC for cell‐based therapy by being less invasive a source of biological material. We have evaluated the effect of hUCB‐MSC on the proliferation of K562 (an erythromyeloblastoid cell line) and the cytokine secretion pattern of hUCB‐MSC. Co‐culturing of hUCB‐MSC and K562 resulted in inhibition of proliferation of K562 in a dose‐dependent manner. However, the anti‐proliferative effect was reduced in transwells, suggesting the importance of direct cell‐to‐cell contact. hUCB‐MSC inhibited proliferation of K562, arresting them in the G₀/G₁ phase. NO (nitric oxide) was not involved in the hUCB‐MSC‐mediated tumour suppression. The presence of IL‐6 (interleukin 6) and IL‐8 were obvious in the hUCB‐MSC conditioned media, but no significant increase was found in 29 other cytokines. Th1 cytokines, IFNα (interferon α), Th2 cytokine IL‐4 and Th17 cytokine, IL‐17 were not secreted by hUCB‐MSC. There was an increase in the number of hUCB‐MSC expressing the latent membrane‐bound form of TGFβ1 co‐cultured with K562. The anti‐proliferative effect of hUCB‐MSC was due to arrest of the growth of K562 in the G₀/G₁ phase. The mechanisms underlying increased IL‐6 and IL‐8 secretion and LAP (latency‐associated peptide; TGFβ1) by hUCB‐MSC remains unknown.