Role of PKA in the anti-Thy-1 antibody-induced neurite outgrowth of dorsal root ganglionic neurons

Thy-1 is highly expressed in the mammalian nervous system. Our previous study showed that addition of anti-Thy-1 antibody to cultured dorsal root ganglionic (DRG) neurons promotes neurite outgrowth. In this study, we identified a novel signaling pathway mediating this event. Treatment with function-blocking anti-Thy-1 antibodies enhanced neurite outgrowth of DRG neurons in terms of total neurite length, longest neurite length, and total neurite branching points. To elucidate the possible signal transduction pathway involved, activation of kinases was evaluated by Western blotting. Transient phosphorylation of protein kinase A (PKA) and mitogen-activated kinase kinase (MEK) was induced after 15 min of anti-Thy-1 antibody treatment. Pretreatment with a PKA inhibitor (PKI) or an MEK inhibitor, PD98059, significantly decreased the neurite outgrowth response triggered by anti-Thy-1 antibody, indicating the involvement of both kinases. In addition, anti-Thy-1 antibody treatment also induced transient phosphorylation of cyclic AMP-response element-binding protein (CREB) and this effect was also blocked by a PKI or PD98059. Furthermore, the fact that PKI abolished anti-Thy-1 antibody-induced MEK phosphorylation showed that PKA acts upstream of the MEK-CREB cascade. In summary, the PKA-MEK-CREB pathway is a new pathway involved in the neurite outgrowth-promoting effect of anti-Thy-1 antibody.     

Daidzein induces neuritogenesis in DRG neuronal cultures

Background

Phorbol myristate acetate (PMA) is a strong neutrophil activator and has been used to induce acute lung injury (ALI). Niacinamide (NAC) is a compound of B complex. It exerts protective effects on the ALI caused by various challenges. The purpose was to evaluate the protective effects of niacinamide (NAC) on the PMA-induced ALI and associated changes.

Methods

The rat's lungs were isolated in situ and perfused with constant flow. A total of 60 isolated lungs were randomized into 6 groups to received Vehicle (DMSO 100 ??g/g), PMA 4 ??g/g (lung weight), cotreated with NAC 0, 100, 200 and 400 mg/g (lung weight). There were 10 isolated lungs in each group. We measured the lung weight and parameters related to ALI. The pulmonary arterial pressure and capillary filtration coefficient (K_fc) were determined in isolated lungs. ATP (adenotriphosphate) and PARP [poly(adenosine diphophate-ribose) polymerase] contents in lung tissues were detected. Real-time PCR was employed to display the expression of inducible and endothelial NO synthases (iNOS and eNOS). The neutrophil-derived mediators in lung perfusate were determined.

Results

PMA caused increases in lung weight parameters. This agent produced pulmonary hypertension and increased microvascular permeability. It resulted in decrease in ATP and increase in PARP. The expression of iNOS and eNOS was upregulated following PMA. PMA increased the neutrophil-derived mediators. Pathological examination revealed lung edema and hemorrhage with inflammatory cell infiltration. Immunohistochemical stain disclosed the presence of iNOS-positive cells in macrophages and endothelial cells. These pathophysiological and biochemical changes were diminished by NAC treatment. The NAC effects were dose-dependent.

Conclusions

Our results suggest that neutrophil activation and release of neutrophil-derived mediators by PMA cause ALI and associated changes. NO production through the iNOS-producing cells plays a detrimental role in the PMA-induced lung injury. ATP is beneficial, while PARP plays a deteriorative effect on the PMA-induced ALI. NAC exerts protective effects on the inflammatory cascade leading to pulmonary injury. This B complex compound may be applied for clinical usage and therapeutic regimen.     

Free anterolateral thigh adipofascial perforator flap

The anterolateral thigh adipofascial flap is a vascularized flap prepared from the adipofascial layer of the anterolateral thigh region. It is a perforator flap based on septocutaneous or musculocutaneous perforators of the lateral circumflex femoral system. With methods similar to those used for the free anterolateral thigh flap, only the deep fascia of the anterolateral thigh and a 2-mm-thick to 3-mm-thick layer of subcutaneous fatty tissue above the fascia were harvested. In 11 cases, this flap (length, 5 to 11 cm; width, 4 to 8 cm) was used for successful reconstruction of extremity defects. Split-thickness skin grafts were used to immediately resurface the adipofascial flaps for eight patients, and delayed skin grafting was performed for the other three patients. The advantage of the anterolateral thigh adipofascial flap is its ability to provide vascularized, thin, pliable, gliding coverage. In addition, the donor-site defect can be closed directly. Other advantages of this flap, such as safe elevation, a long wide vascular pedicle, a large flap territory, and flow-through properties that allow simultaneous reconstruction of major-vessel and soft-tissue defects, are the same as for the conventional anterolateral thigh flap. The main disadvantage of this procedure is the need for a skin graft, with the possible complications of subsequent skin graft loss or hyperpigmentation.     

A Rickettsia WASP-like protein activates the Arp2/3 complex and mediates actin-based motility

Spotted fever group Rickettsia are obligate intracellular pathogens that exploit the host cell actin cytoskeleton to promote motility and cell-to-cell spread. Although other pathogens such as Listeria monocytogenes use an Arp2/3 complex-dependent nucleation mechanism to generate comet tails consisting of Y-branched filament arrays, Rickettsia polymerize tails consisting of unbranched filaments by a previously unknown mechanism. We identified genes in several Rickettsia species encoding proteins (termed RickA) with similarity to the WASP family of Arp2/3-complex activators. Rickettsia rickettsii RickA activated both the nucleation and Y-branching activities of the Arp2/3 complex like other WASP-family proteins, and was sufficient to direct the motility of microscopic beads in cell extracts. Actin tails generated by RickA-coated beads consisted of Y-branched filament networks. These data suggest that Rickettsia use an Arp2/3 complex-dependent actin-nucleation mechanism similar to that of other pathogens. We propose that additional Rickettsia or host factors reorganize the Y-branched networks into parallel arrays in a manner similar to a recently proposed model of filopodia formation.     

我国实验小鼠群中巨细胞病毒自然感染情况的调查

迄今为止,有关MCMV在实验小鼠群中的自然感染情况报道极少。我们在MCMV实验感染的基础上对国内常用的小鼠品系进行MCMV自然感染的调查。结果发现在334份不同品系小鼠颌下腺中无1份分离出MCMV,而特异性抗体存在于所检查的10个品系中,阳性率为10.9—87.5％。抗体阳性率与鼠龄有关,随鼠龄的增长而上升。对全国部分省市40家实验动物机构的556份小鼠血清的调查结果表明MCMV的抗体阳性率达61.0％,提示在我国实验小鼠群中MCMV的自物感染是相当普遍的。这种病毒分离阴性而抗体阳性的状态表明隐性感染和潜伏感染是MCMV自然感染的主要方式。     

Synthesis and biological activity of novel potent endothelin-converting enzyme-1 inhibitors

Through directed screening of metalloprotease inhibitors, CGS 30084 (1) has been identified as a potent endothelin-converting enzyme-1 (ECE-1) inhibitor in vitro (IC50 = 77 nM). Herein we report the syntheses and biological activities of analogues derived from this lead, based on modifications of the biphenyl moiety. Compound 10, the thioacetate methyl ester prodrug derivative of compound 6m, was found to be an orally active and potent inhibitor of ECE-1 activity in rats.     

A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation

Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and temporal distribution of cAMP at the subcellular level could be important for developing new strategies for the prevention or treatment of unfavorable responses associated with different disease states.