Heptad repeats regulate protein phosphatase 2a recruitment to I-kappaB kinase gamma/NF-kappaB essential modulator and are targeted by human T-lymphotropic virus type 1 tax

The switching on-and-off of I-kappaB kinase (IKK) and NF-kappaB occurs rapidly after signaling. How activated IKK becomes down-regulated is not well understood. Here we show that following tumor necrosis factor-alpha stimulation, protein phosphatase 2A (PP2A) association with IKK is increased. A heptad repeat in IKKgamma, helix 2 (HLX2), mediates PP2A recruitment. Two other heptad repeats downstream of HLX2, termed coiled-coil region 2 (CCR2) and leucine zipper (LZ), bind HLX2 and negatively regulate HLX2 interaction with PP2A. HTLV-1 transactivator Tax also binds HLX2, and this interaction is enhanced by CCR2 but reduced by LZ. In the presence of Tax, PP2A-IKKgamma binding is greatly strengthened. Interestingly, peptides spanning CCR2 and/or LZ disrupt IKKgamma-Tax and IKKgamma-PP2A interactions and potently inhibit NF-kappaB activation by Tax and tumor necrosis factor-alpha. We propose that when IKK is resting, HLX2, CCR2, and LZ form a helical bundle in which HLX2 is sequestered. The HLX2-CCR2-LZ bundle becomes unfolded by signal-induced modifications of IKKgamma or after Tax binding. In this conformation, IKK becomes activated. IKKgamma then recruits PP2A via the exposed HLX2 domain for rapid down-regulation of IKK. Tax-PP2A interaction, however, renders PP2A inactive, thus maintaining Tax-PP2A-IKK in an active state. Finally, CCR2 and LZ possibly inhibit IKK activation by stabilizing the HLX2-CCR2-LZ bundle.     

Role of myosin light chain kinase in regulation of basal blood pressure and maintenance of salt-induced hypertension

Vascular tone, an important determinant of systemic vascular resistance and thus blood pressure, is affected by vascular smooth muscle (VSM) contraction. Key signaling pathways for VSM contraction converge on phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin. This phosphorylation is mediated by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) but Ca(2+)-independent kinases may also contribute, particularly in sustained contractions. Signaling through MLCK has been indirectly implicated in maintenance of basal blood pressure, whereas signaling through RhoA has been implicated in salt-induced hypertension. In this report, we analyzed mice with smooth muscle-specific knockout of MLCK. Mesenteric artery segments isolated from smooth muscle-specific MLCK knockout mice (MLCK(SMKO)) had a significantly reduced contractile response to KCl and vasoconstrictors. The kinase knockout also markedly reduced RLC phosphorylation and developed force. We suggest that MLCK and its phosphorylation of RLC are required for tonic VSM contraction. MLCK(SMKO) mice exhibit significantly lower basal blood pressure and weaker responses to vasopressors. The elevated blood pressure in salt-induced hypertension is reduced below normotensive levels after MLCK attenuation. These results suggest that MLCK is necessary for both physiological and pathological blood pressure. MLCK(SMKO) mice may be a useful model of vascular failure and hypotension.     

Viral tegument proteins restrict cGAS-DNA phase separation to mediate immune evasion

1. Download : Download high-res image (223KB)
2. Download : Download full-size image

     

Bioactive thin film of acidic fibroblast growth factor fabricated by layer-by-layer assembly

A new class of bioactive thin films using growth factors as building blocks has been fabricated via layer-by-layer assembly (LBL) technique. Acid fibroblast growth factor (aFGF) in the presence of heparin was used as negatively charged polyelectrolytes, while poly(ethyleneimine) (PEI) was chosen as a positively charged counterpart. The self-deposition process and surface morphology of the resultant multilayers were monitored and detected by UV-vis absorbance spectra, advanced contact angle measurements, and scanning force microscopy (SFM) observations. Cell culture was performed to assess the efficiency of the growth factors. The fibroblasts proliferated faster on the surface assembled with five bilayers of (aFGF/heparin)/PEI with apparent higher cytoviability than on those surfaces modified by one bilayer of (aFGF/heparin)/PEI, five bilayers of aFGF/PEI, or five bilayers of heparin/PEI, and tissue culture polystyrene. Enhanced secretion of collagen type I and interleukin 6 (IL-6) by the fibroblasts seeded on the five bilayers of (aFGF/heparin)/PEI was also verified by immunohistochemical examination. The bioactivity of the (aFGF/heparin)/PEI multilayers could be largely preserved when stored at -20 degrees C.     

Experimental Study of Low Dose Ultrashortwave Promoting Nerve Regeneration after Acellular Nerve Allografts Repairing the Sciatic Nerve Gap of Rats

Objectives To observe the effect of ultrashortwave (USW) therapy on nerve regeneration after acellular nerve allografts(ANA) repairing the sciatic nerve gap of rats and discuss its acting mechanisms. Methods Sixteen Wistar rats weighing 180–220 g were randomly divided into four groups with four rats in each group: normal control group; acellular group (ANA, treated by hypotonic-chemical detergent, was applied for bridging a 10 mm-long sciatic nerve defect); USW group (After 24 h of ANA repairing the sciatic nerve gap, low dose USW was administrated for 7 min, once a day, 20 times a course of treatment, three courses of treatment in all); and autografts group. 12 weeks after operation, a series of examinations was performed, including electrophysiological methods, the restoring rate of tibialis anterior muscle wet weight, histopathological observation (myelinated nerve number, myelin sheath thickness, and axon diameter), vascular endothelial growth factor (VEGF) mRNA expression of spinal cord, and muscle at injury site, and analyzed statistically. Results Compared to acellular nerve allografts alone, USW therapy can increase nerve conductive velocity, the restoring rate of tibialis anterior muscle wet weight, myelinated nerve number, axon diameter, VEGF mRNA expression of spinal cord, and muscle at injury site, the difference is significant. There were no differences between USW group and autografts group except myelin sheath thickness. Conclusions USW therapy can promote nerve axon regeneration and Schwann cells proliferation after ANA repairing the sciatic nerve gap of rats, the upregulation of VEGF mRNA expression of spinal cord and muscle may play an important role.     

Optogenetic manipulation of cyclic guanosine monophosphate to probe phosphodiesterase activities in megakaryocytes

Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types, including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatio-temporal regulation in megakaryocytes (MKs) is lacking. Optogenetics is a technique which allows spatio-temporal manipulation of molecular events in living cells or organisms. We took advantage of this method and expressed a photo-activated guanylyl cyclase, Blastocladiella emersonii Cyclase opsin (BeCyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. BeCyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide-triggered cGMP generation in BM MKs. In summary, we established for the first-time optogenetics in primary MKs and show that PDE5 is the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.