Flagellin, the structural component of flagellar filament in various locomotive bacteria, is the ligand for Toll-like receptor 5 (TLR5) of host cells. TLR stimulation by various pathogen-associated ...molecular patterns leads to activation of innate and subsequent adaptive immune responses. Therefore, TLR ligands are considered attractive adjuvant candidates in vaccine development. In this study, we show the highly potent mucosal adjuvant activity of a Vibrio vulnificus major flagellin (FlaB). Using an intranasal immunization mouse model, we observed that coadministration of the flagellin with tetanus toxoid (TT) induced significantly enhanced TT-specific immunoglobulin A (IgA) responses in both mucosal and systemic compartments and IgG responses in the systemic compartment. The mice immunized with TT plus FlaB were completely protected from systemic challenge with a 200x minimum lethal dose of tetanus toxin. Radiolabeled FlaB administered into the nasal cavity readily reached the cervical lymph nodes and systemic circulation. FlaB bound directly to human TLR5 expressed on cultured epithelial cells and consequently induced NF-kappaB and interleukin-8 activation. Intranasally administered FlaB colocalized with CD11c as patches in putative dendritic cells and caused an increase in the number of TLR5-expressing cells in cervical lymph nodes. These results indicate that flagellin would serve as an efficacious mucosal adjuvant inducing protective immune responses through TLR5 activation.
B23/NPM is a major nucleolar phosphoprotein that has a critical role in cell proliferation and cell death. Here, we show that it forms a complex with Akt on growth factor (GF) stimulation in both the ...cytoplasm and the nucleus, for which Akt activation is indispensable. The C terminus of B23 (239-294 residues) potently binds pleckstrin homology (PH) domain of Akt. Akt binding to B23 protects it from proteolytic degradation by caspase-3, leading to the up-regulation of cell survival. Interestingly, unsumoylated B23 K263R, but not wild-type B23, strongly interacts with Akt in the nucleoplasm in the absence of GFs. Furthermore, we show that Akt2, but not other isoforms, specifically regulates B23 sumoylation and protein stability. Also, nuclear Akt regulates the cell cycle progression activity of B23. Therefore, our findings support that nuclear Akt binds and stabilizes B23 in the nucleoplasm, and regulates its activities in cell survival and cell cycle.
Background
Sevoflurane is commonly used in general anesthesia for premature neonates. The main mechanism of retinopathy of prematurity (ROP) is increased levels of vascular endothelial growth factor ...(VEGF). For the investigation of sevoflurane’s effect on angiogenesis, the angiogenesis and VEGF expression in the retina were measured after administering sevoflurane in an oxygen-induced retinopathy mice model.
Materials and methods
The mice were divided into the normoxic group (Nc and Ns group;
n
= 6) and the ROP group (C, Rc, and Rs group;
n
= 6). Rc group were exposed to 75% oxygen for 5 days beginning on postnatal day (P) 7, and then returned to room air. Age-matched mice in the C group were exposed to room air. To observe angiogenesis of the retina, the mice were sacrificed on P16. The Rs group was exposed to 2 vol% sevoflurane for 2 h on P12, P13, and P14 with 40% oxygen.
Results
The angiogenic area and the spreading distance of vessels on P4 were statistically decreased in the Ns group, compared to the Nc group. The avascular area on P16 was significantly increased and the expression of VEGF was suppressed in the Rs group compared to the Rc group.
Conclusions
Sevoflurane can inhibit retinal angiogenesis via suppressing VEGF expression in an OIR mice model with exposure to relative hypoxia. Nevertheless, it is still difficult to apply the results of this study immediately to humans because of the heterogeneity of responses to sevoflurane.
Angiogenesis plays a critical role in embryo development, tissue repair, tumor growth and wound healing. In the present study, we investigated the role of the serine/threonine kinase Akt in ...angiogenesis. Silencing of Akt1 in human umbilical vein endothelial cells significantly inhibited vascular endothelial growth factor (VEGF)‐induced capillary‐like tube formation. Mice lacking Akt1 exhibited impaired retinal angiogenesis with delayed endothelial cell (EC) proliferation. In addition, VEGF‐induced corneal angiogenesis and tumor development were significantly inhibited in mice lacking Akt1. Loss of Akt1 resulted in reduced angiogenic sprouting, as well as the proliferation of ECs and mural cells. Addition of culture supernatant of vascular smooth muscle cells (VSMCs) in which Akt1 was silenced suppressed tube formation, the stability of preformed tubes and the proliferation of ECs. In addition, attachment of VSMCs to ECs was significantly reduced in cells in which Akt1 was silenced. Mural cell coverage of retinal vasculature was reduced in mice lacking Akt1. Finally, mice lacking Akt1 showed severe retinal hemorrhage compared to the wild‐type. These results suggest that the regulation of EC function and mural cell coverage by Akt1 is important for blood vessel maturation during angiogenesis.
Loss of Akt1 resulted in delayed retinal, cornea and tumor angiogenesis concomitant with vascular leakage. In particular, mural cell coverage was significantly reduced in mice lacking Akt1. In addition, conditioned medium from vascular smooth muscle cells (VSMCs) in which Akt1 was silenced markedly suppressed endothelial cell proliferation, tube formation and preformed tube stability, suggesting that Akt1 has an important role in VSMCs for angiogenesis and vascular stability.
To establish the standard of procedure in preparing benign and cancerous prostate tissues and evaluate the quality of proteomics and phosphoproteomics during transurethral resection of the prostate ...(TUR-P) with different surgical conditions.
TUR-P tissue samples from three patients, two diagnosed with prostate cancer and one with benign prostatic hyperplasia, were each analyzed under three different conditions, based on differences in energy values, tissue locations, and surgical techniques. Global- and phosphorylated proteomic profiles of prostate tissues were analyzed by liquid chromatography-tandem mass spectrometry.
A total of 6,019 global proteins and 4,280 phosphorylated peptides were identified in the nine tissues. The quantitative distributions of proteins and phosphorylation in tissues from the same patient were not affected by changes in the surgical conditions, but indirect relative comparisons differed among patients. Phosphorylation levels, especially of proteins involved in the androgen receptor pathway, important in prostate cancer, were preserved in each patient.
Proteomic profiles of prostate tissue collected by TUR-P were not significantly affected by energy levels, tissue location, or surgical technique. In addition, since protein denaturation of samples through TUR-P is rarely confirmed in this study, we think that it will be an important guide for tissue samples in castration resistant prostate cancer patients, where it is difficult to obtain tissue. This result is the first report about proteomic and phosphoproteomic results with TUR-P samples in prostate cancer and will be theoretical basis in protein analysis research with prostate cancer tissues.
Schisandra chinensis (SC) is a known medical herb for the treatment of cardiovascular symptoms associated with menopausal symptoms in Korea. However, the pharmacological action mechanisms involved ...have not been well studied. This study was aimed to investigate the vascular effects of SC in rat thoracic aorta.
We isolated the hexane, chloroform, and methanol extracts from SC and evaluated their vasodilatory effects in the rat thoracic aorta.
Hexane extracts of SC (SCHE, 5
×
10
−5 to 10
−3
g/L) caused a concentration-dependent relaxation in both endothelium-intact and -denuded aortas. The relaxant effect of SCHE on the endothelium-intact aorta was more prominent than on the endothelium-denuded aorta. The former was significantly attenuated by L-NAME, a nitric oxide synthase inhibitor, and ODQ, a soluble guanyl cyclase inhibitor, but not by tetraethylammonium, a nonselective blocker of K
+ channels, and indomethacin, a cyclooxygenase inhibitor. Furthermore, SCHE caused nitrite production as well as eNOS activation in aortic segments, suggesting implication of NO signal pathway in SCHE-induced relaxation. In endothelium-denuded aorta, SCHE-induced vasorelaxation was also attenuated by calyculin A, an inhibitor of myosin light chain (MLC) phosphatase, but not by ML-9, a MLC kinase inhibitor, suggestive of implication of MLC phosphatase activation. Phenylephrine-enhanced MLC phosphorylation ratio was significantly attenuated by SCHE, which was recovered to the control level by pretreatment with calyculin A.
Taken collectively, these findings suggest that the vascular relaxation evoked by SCHE was mediated by not only endothelium dependent NO pathway but also direct effect on vascular smooth muscle cell via dephosphorylation of MLC.
Translationally controlled tumor protein (TCTP), a repressor for Na,K-ATPase has been implicated in the development of systemic hypertension, as proved by TCTP-over-expressing transgenic (TCTP-TG) ...mice. Aorta of TCTP-TG exhibited hypercontractile response compared to that of non-transgenic mice (NTG) suggesting dys-regulation of signaling pathways involved in the vascular contractility by TCTP. Because dys-regulation of RhoA/Rho kinase pathway is implicated in increased vascular contractility, we examined whether TCTP induces alterations in RhoA pathway in vascular smooth muscle cells (VSMCs). We found that TCTP over-expression by adenovirus infection up-regulated RhoA pathway including the expression of RhoA, and its downstream signalings, phosphorylation of myosin phosphatase target protein (MYPT-1), and myosin light chain (MLC). Conversely, lentiviral silencing of TCTP reduced the RhoA expression and Rho kinase signalings. Using immunohistochemical and Western blotting studies on aortas from TCTP-TG confirmed the elevated expression of RhoA and increase in p-MLC (phosphorylated MLC). In contrast, down-regulation of RhoA and p-MLC were found in aortas from heterozygous mice with deleted allele of TCTP (TCTP+/-). We conclude that up-regulation of TCTP induces RhoA-mediated pathway, and that TCTP-induced RhoA plays a role in the regulation in vasculature. Modulation of TCTP may offer a therapeutic target for hypertension and in vascular contractility dysfunction.
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