Improvement of biological and pharmacokinetic features of human interleukin-11 by site-directed mutagenesis

Recombinant human interleukin-11 (rhIL-11) has been shown to increase platelet counts in animals and humans and is the only drug approved for its use in chemotherapy-induced thrombocytopenia (CIT). However, due to its serious side effects, its clinical use has been limited. The current work presents significantly improved efficacy of rhIL-11 via knowledge based re-designing process. The interleukin-11 mutein (mIL-11) was found to endure chemical and proteolytic stresses, while retaining the biological activity of rhIL-11. The improved efficacy of mIL-11 was evident after subcutaneous administration of mIL-11 and rhIL-11 in the rodent and primate models. More than three-fold increase in maximum plasma concentration (C_max) and area-under-the curve (AUC) was observed. Furthermore, three-fold higher increase in the platelet counts was obtained after seven consecutive daily subcutaneous mIL-11 injections than that with rhIL-11. The mIL-11 demonstrated not only improved stability but also enhanced efficacy over the currently used rhIL-11 regimen, thereby suggesting less toxicity.     

Exercise training increases membrane bound form of tumor necrosis factor-alpha receptors with decreases in the secretion of soluble forms of receptors in rat adipocytes

We examined the effects of exercise training (treadmill running over 9 weeks) on the ability of isolated adipocytes to secrete tumor necrosis factor-alpha (TNF-alpha) and type 1 soluble TNF receptor (sTNFR1) in vitro in Wistar rats. We also examined the effects of exercise training on the expression of membrane bound forms of type 1 TNF receptor (mTNFR1) in adipocyte crude membranes of the same rat subjects. Exercise training significantly increased the secretions of TNF-alpha from isolated adipocytes. Treatment with a cyclooxygenase inhibitor, either indomethacin (100 microM) or eicosatetraynoic acid (100 microM), significantly blocked the release of TNF-alpha from adipocytes in both exercise-trained rat group and sedentary control rat group, suggesting that some cyclooxygenase metabolite(s) acts as a ligand in TNF-alpha synthesis. Decreased amounts of TNF-alpha were found to be significantly greater in both exercise-trained rat group than in sedentary control rat group after incubation with inhibitors. Thus, the inhibitory effect of both indomethacin and eicosatetraynoic acid was significantly greater in adipocytes from exercise-trained rats. Both plasma sTNFR1 levels and adipocytes-derived sTNFR1 were found to be significantly less in the exercise-trained rat group. Western blot analysis revealed that exercise training remarkably increased the expressions of mTNFR1 in adipocyte crude membrane. Thus, exercise training enhanced the ability of isolated adipocytes to secrete TNF-alpha with reduced secretion of sTNFR1, and provoked the greater expressions of mTNFR1 in adipocyte crude membrane. These alterations may induce enhanced the autocrine effects of TNF-alpha within adipocytes in exercise-trained rats.     

Synergistic induction of hepatocyte growth factor in human skin fibroblasts by the inflammatory cytokines interleukin-1 and interferon-gamma

Hepatocyte growth factor (HGF) is one of the vital factors for wound healing. HGF expression markedly increases in wounded skin and is mainly localized in dermal fibroblasts. HGF expression level in human dermal fibroblasts in vitro, however, is low and thus may be stimulated by some factors in the process of wound healing. Candidates of the factors are inflammatory cytokines released by polymorphonuclear and mononuclear cells infiltrating the wounded area, but HGF production in human dermal fibroblasts is only slightly induced by interleukin (IL)-1, tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma. We here report that a combination of IL-1beta and IFN-gamma or a combination of TNF-alpha and IFN-gamma very markedly induced HGF production. The synergistic effect of the former was more marked than that of the latter. Synergistic effects of IL-1beta and IFN-gamma were observed at more than 10 pg/ml and 10 IU/ml, respectively, and were detectable as early as 12 h after addition. Neither IFN-alpha nor IFN-beta was able to replace IFN-gamma. HGF mRNA expression was also synergistically upregulated by IL-1beta and IFN-gamma. IL-1beta plus IFN-gamma-induced synergistic production of HGF was potently inhibited by treatment of cells with the extracellular signal-regulated kinase (ERK) kinase inhibitor PD98059 and the p38 inhibitor SB203580 but not by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. Taken together, our results indicate that a combination of IL-1beta and IFN-gamma synergistically induced HGF production in human dermal fibroblasts and suggest that activation of ERK and p38 but not of JNK is involved in the synergistic effect.     

Inhibition of hepatocyte growth factor induction in human dermal fibroblasts by interleukin-1 and its prevention by interferon-gamma

Hepatocyte growth factor (HGF) is one of the vital factors for liver regeneration. HGF production is induced by the activation of protein kinase A and protein kinase C-mediated pathways, interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and epidermal growth factor (EGF) in mesenchymal cells. We here report that IL-1 and TNF-alpha, hitherto regarded as HGF inducers, potently inhibited HGF production stimulated by other HGF inducers. IL-1alpha, IL-1beta, and TNF-alpha alone had minimal stimulating effects on HGF production in human dermal fibroblasts, but they strongly inhibited production of HGF induced by cholera toxin, 8-bromo-cAMP, EGF, and phorbol 12-myristate 13-acetate (PMA). Moreover, although the high level of HGF production in MRC-5 cells was enhanced by PMA and less markedly by IL-1beta, HGF production in MRC-5 cells treated with PMA plus IL-1beta was less than that in the cells treated with PMA alone. In the presence of interferon (IFN)-gamma, however, cholera toxin- and 8-bromo-cAMP-induced HGF production was not inhibited by IL-1beta. Pretreatment of cells with IL-1beta suppressed the phosphorylation of cAMP-responsive element-binding protein induced by cholera toxin but not that induced by 8-bromo-cAMP. Taken together, our results indicate that IL-1 inhibited HGF production stimulated by various inducers, including protein kinase A-activating agents, and that IFN-gamma overcame this inhibition of induction of HGF production.     

大肠埃希菌脂多糖对高脂饮食动物血脂及炎性反应的影响

目的研究大肠埃希菌脂多糖对高脂饮食兔血脂和炎性反应的影响。方法给含0．5％胆固醇的饲料,3周后,分别在第4、8、12周采用耳动脉内、颈部、腹股沟处肌肉注射大肠埃希菌脂多糖（LPS）,并设立正常组和单纯高脂组。16周后观察兔的一般状态,取血清检查血脂六项、C-反应蛋白和TNF—α,取耳动脉、颈动脉、主动脉弓、胸主动脉、腹主动脉、髂动脉、肝脏,放置4％多聚甲醛中过夜,常规行HE染色,检查血管病变和相关脏器病变情况。结果单纯高脂组血清中胆固醇和LDL-C较正常组增加,复合模型组动物血清中胆固醇和LDL-C均明显高于单纯高脂组,单纯高脂组TNF-α较正常组高,复合模型组TNF-α比单纯高脂组高。病理显示主动脉弓变化明显,复合模型组内膜斑块弥漫,而单纯高脂组内膜只出现单个小斑块,单纯高脂组和复合模型组心脏病变区别不大,均见轻度水肿和小脂肪滴;单纯模型组肝脏细胞轻度水肿,而复合模型组肝脏脂肪滴明显。结论大肠埃希菌脂多糖加重了内膜斑块的形成,加剧了血脂代谢的紊乱和炎性反应。     

Role of extracellular signal-regulated kinases 1 and 2 and p38 mitogen-activated protein kinase pathways in regulating replication of Penicillium marneffei in human macrophages

Penicillium marneffei (P. marneffei) is a human pathogen which persists in macrophages and threatens the immunocompromised patients. To elucidate the mechanisms involved, we investigated the role of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (p38) pathways in cytokine expression, phagosome–lysosome fusion and replication of P. marneffei in P. marneffei-infected human macrophages. Analysis of both ERK1/2 and p38 showed rapid phosphorylation in response to P. marneffei. Using specific inhibitors of p38 (SB203580) and MAP kinase kinase-1 (PD98059), we found that ERK1/2 and p38 were essential for P. marneffei-induced tumor necrosis factor-α production, whereas p38, but not that of ERK, was essential for IL-10 production. Furthermore, the presence of PD98059 always decreased phagosomal acidification and maturation and increased intracellular multiplication of P. marneffei, whereas the use of SB203580 always increased phagosomal acidification and maturation and decreased intracellular replication. These data suggest that a proper balance of between ERK1/2 and p38 may play an important role in controlling the replication of P. marneffei. Our findings further indicate a novel therapeutic avenue for treating P. marneffei by stimulating ERK1/2 or activating ERK1/2-dependent mechanisms.     

Proteomic analysis effects of ginsenoside Rg1 on human umbilical vein endothelial cells stimulated by tumor necrosis factor-alpha

Ginsenoside Rg1 (derived from ginseng root) has been found to have many vasoprotective activities. The present study was undertaken to examine effect of ginsenoside Rg1 on the secretion of nitric oxide (NO) in human umbilical vein endothelial cells (HUVECs) stimulated with or without tumor necrosis factor-alpha (TNF-alpha). We showed here that ginsenoside Rg1 can increase the basal and TNF-alpha-attenuated NO production in a dose-dependent manner. As little is known regarding the vascular molecular mechanism of ginsenoside Rg1 on HUVECs and proteomic technique has more advantages in molecular identification, we attempted to use proteomic analysis to explain vascular molecular mechanism of ginsenoside Rg1 on HUVECs. Proteomic analytical result showed that 21 protein spots were changed in TNF-alpha stimulated HUVECs, including 9 up-regulated spots, 11 down-regulated spots, and 1 spot detected in TNF-alpha stimulated group only. The expression level of proteins such as MEKK3, phosphoglycerate mutase was increased, and nitric-oxide synthase, mineralocorticoid receptor were decreased in TNF-alpha stimulated HUVECs, while ginsenoside Rg1 could prevent this change or reverse to some degree. This study suggested that NO production increased via ginsenoside Rg1 played an important role in the protective effect on TNF-alpha stimulated HUVECs and was helpful to deeply understand the active mechanism of ginsenoside Rg1 to HUVECs at the molecular level.     

Structure-activity relationship study of human interleukin-3. Identification of residues required for biological activity by site-directed mutagenesis

Recombinant human interleukin-3 (rhuIL-3) variants were generated by site-directed mutagenesis and expression in Escherichia coli. Amino acid deletions and substitutions were made in the previously identified epitopes of two huIL-3-specific neutralizing monoclonal antibodies (mAbs). The rhuIL-3 variants were analyzed for their ability to bind to the IL-3 receptor and to induce the proliferation of the human IL-3-dependent cell line M-O7. Several deletion mutants spanning the epitopes of these neutralizing mAbs indicated the importance of residues Pro33 and Leu34 for biological activity. Further, substitution of Pro33 with Asn (Asn33) showed an enhanced proliferative activity (4-fold) and a moderate increase in receptor binding (2-fold) compared to wild-type (wt) rhuIL-3. The most remarkable change, however, was seen with variant Gly33, which showed a 14-fold increase in promoting the growth of M-O7 cells without a significant modification in its receptor binding capacity. In contrast, substitution of Leu34 with Gly (Gly34) yielded an IL-3 variant that had a 25-fold decreased receptor binding capacity and proliferative activity, while Glu34 had properties similar to wild-type rhuIL-3. Analysis of the binding of these variants to different rhuIL-3-specific monoclonal antibodies suggested that no major modification had occurred in their conformations. These results indicate that both residues, Pro33 and Leu34, play a critical role in modulating the activity of rhuIL-3.     

FAPP2 promotes tumor cell growth in human colon cancer through activation of Wnt signaling

Human phosphatidylinositol-4-phosphate adaptor protein-2 (FAPP2) is well-known to function as a cytoplasmic lipid transfer protein during vesicle maturation. However, the expression and role of FAPP2 in tumor remain elusive. In this study, data from immunohistochemical assays displayed that FAPP2 was remarkably upregulated (57.8%) in 90 cases of colon cancer samples in contrast to their corresponding adjacent tissues. Disruption of FAPP2 by CRISPR/Cas9 technique in colon cancer cells led to an attenuated effect on cell growth analyzed by CCK8 and colony formation assays. Meanwhile, the tumorigenicity of FAPP2 downregulated cells also decreased in nude mice model. Accordantly, CCK8 assays also indicated that FAPP2 overexpression could promote colon cancer cell growth. In addition, dual luciferase reporter assays and western blot analyses revealed that Wnt/β-catenin signaling was involved in the FAPP2-regulated tumor cell growth. These findings suggest that FAPP2 could act as an oncogene in the regulation of tumor growth and may provide a new therapeutic target for human colon cancer.     

BATF2对Wnt信号通路影响的研究

摘要wnt信号通路在各种生物体内高度进化保守,与癌症的发生发展密切相关。BATF2是一个新近发现的基因,研究表明其具有抑癌基因的作用。目前,BATF2与wnt信号通路的关系尚不清楚,该文用荧光素酶报告基因检测、Real－timePCR和Western blot发现BATF2能影响wnt信号通路。过表达BArF2可明显下调TCF4／p－catenin的转录活性和Wnt信号通路下游基因的表达,可以下调细胞核中的β-caenin。推测BATF2可能通过下调细胞核中的p－catenin来实现对wnt信号通路的下调。上述结果为抑制Wnt信号通路用于肿瘤治疗提供了一定的依据。     

TACE/ADAM-17 maturation and activation of sheddase activity require proprotein convertase activity

Proprotein convertases (PCs) have been proposed to play a role in tumor necrosis factor-alpha converting enzyme (TACE) processing/activation. Using the furin-deficient LoVo cells, as well as the furin-proficient synoviocytes and HT1080 cells expressing the furin inhibitor alpha(1)-PDX, we demonstrate that furin activity alone is not sufficient for effective maturation and activation of the TACE enzyme. Data from in vitro and in vivo cleavage assays indicate that PACE-4, PC5/PC6, PC1 and PC2 can directly cleave the TACE protein and/or peptide. PC inhibition in macrophages reduced the release of soluble TNF-alpha from transmembrane pro-TNF-alpha. We therefore conclude that furin, in addition to other candidate PCs, is involved in TACE maturation and activation.     

Characterization of rat and human Kupffer cells after cryopreservation

Kupffer cells (KC) are the resident macrophages of the liver and represent about 80% of the total fixed macrophage population. They are involved in disease states such as endotoxin shock, alcoholic liver diseases and other toxic-induced liver injury. They release physiologically active substances such as eicosanoids and inflammatory cytokines (IL-1, IL-6, TNFalpha), and produce free radical species. Thus, KC are attractive targets for anti-inflammatory therapies and potential candidates responsible for differences in inflammation in liver disease seen between different individuals. However, to perform parallel in vitro experiments with KC from different donors a suitable method for conservation of KC would be necessary. Therefore, the present study evaluated, whether rat and human KC can be frozen, stored and recovered without losing their functional integrity. Rat and human KC were isolated and either cultured under standard conditions (fresh KC) or cryopreserved in special freezing medium (cryopreserved KC). At least 24 h later, cryopreserved KC were thawed, brought into suspension and seeded in the same density as fresh cells for subsequent experiments. Viability of cultured KC was analyzed by trypan blue exclusion. LPS (or PBS as control) stimulation was performed at different time points and cytokine release was analyzed with IL-6 and TNFalpha ELISAs, respectively. Phagocytic capacity was investigated by using a specific phagocytosis assay and FACS analysis. The recovery rate after thawing was around 57% for rat and around 65% for human cryopreserved KC. The results indicate, that KC can successfully be cryopreserved with an adequate recovery rate of viable cells. The properties of fresh and frozen KC can also be compared after thawing. Freshly isolated and cryopreserved cultured KC showed near-normal morphology and did not differ in the cultivation profiles over a period of 72 h. One to three days after seeding, frozen rat or human KC also retained inducible functions such as the production of TNFalpha or IL-6 after LPS challenge. Finally, regardless if they were cryopreserved or not, no differences in the phagocytic activities of the cells were obtained. Taken together, it is concluded that cryopreservation of KC does not change the physiological characteristics of the cells in vitro. Therefore, the method used here for cryopreservation of especially human KC allows the accumulation of KC from several donors for parallel in vitro experiments.     

Promotion of osteogenesis through beta-catenin signaling by desferrioxamine

Desferrioxamine, an iron chelator with “hypoxia-mimetic” activity, promotes bone mineralization when used in aluminum-overloaded dialysis patients. However, the effect of desferrioxamine on osteoblastic differentiation from pluripotent mesenchymal stem cells (MSCs) has not been reported. In this study, pluripotent human MSCs and murine mesenchymal C3H10T1/2 cells were simultaneously treated with desferrioxamine and bone morphogenetic protein-2 (BMP2). In BMP2-treated MSCs, desferrioxamine levels of 15 μΜ were found to increase alkaline phosphatase (ALP) activity and calcium deposition, which were the markers of osteoblastic differentiation. These effects of desferrioxamine were accompanied by promoted phosphorylation of glycogen synthase kinase 3β (GSK-3β) and increased β-catenin protein content, a direct GSK-3β substrate. Knockdown of β-catenin by RNA interference eliminates this positive effect of desferrioxamine on ALP activity. Taken together, these data demonstrate that desferrioxamine plays a direct role in the differentiation of mesenchymal stem cells by activating β-catenin signaling cascades.     

Canonical Wnt signaling negatively regulates branching morphogenesis of the lung and lacrimal gland

Key gene families such as FGFs and BMPs are important mediators of branching morphogenesis. To understand whether Wnt genes, and in particular, the canonical Wnt signaling pathway also function in the branching process, we have used a combination of experimental and genetic gain and loss of function approaches to perturb the levels of canonical Wnt signaling in two arborized structures, the lung and the lacrimal gland. Here, we show that the addition of Wnt3a conditioned medium or LiCl strongly represses growth and proliferation of the lung and lacrimal gland, a result that was confirmed in vivo using a dominant stable mutation of beta-catenin conditionally expressed in the lacrimal gland epithelium. In agreement with these data, knockdown of Wnt signaling with beta-catenin morpholinos results in a greater number of branches and increased cell proliferation. In addition, we show that canonical Wnt signaling is able to modulate the levels of Fgf10 and suppress BMP-induced proliferation in the lacrimal gland. Thus, canonical Wnt signaling negatively regulates branching morphogenesis providing a balance to FGFs and BMPs which positively regulate this process. This multilayered control of growth and proliferation ensures that branched structures attain the morphology required to function efficiently.     

Improvement of alkali stability and thermostability of Paenibacillus campinasensis Family-11 xylanase by directed evolution and site-directed mutagenesis

The extreme process condition of high temperature and high alkali limits the applications of most of natural xylanases in pulp and paper industry. Recently, various methods of protein engineering have been used to improve the thermal and alkalic tolerance of xylanases. In this work, directed evolution and site-directed mutagenesis were performed to obtain a mutant xylanase improved both on alkali stability and thermostability from the native Paenibacillus campinasensis Family-11 xylanase (XynG1-1). Mutant XynG1-1B43 (V90R/P172H) with two units increased in the optimum pH (pH 7.0–pH 9.0) and significant improvement on alkali stability was selected from the second round of epPCR library. And the further thermoduric mutant XynG1-1B43cc16 (V90R/P172H/T84C-T182C/D16Y) with 10 °C increased in the optimum temperature (60–70 °C) was then obtained by introducing a disulfide bridge (T84C-T182C) and a single amino acid substitution (D16Y) to XynG1-1B43 using site-directed mutagenesis. XynG1-1B43cc16 also showed higher thermostability and catalytic efficiency (k _cat /K _m ) than that of wild-type (XynG1-1) and XynG1-1B43. The attractive improved properties make XynG1-1B43cc16 more suitable for bioleaching of cotton stalk pulp under the extreme process condition of high temperature (70 °C) and high alkali (pH 9.0).     

Wnt/Lrp/beta-catenin signaling suppresses adipogenesis by inhibiting mutual activation of PPARgamma and C/EBPalpha

Wnt/beta-catenin signaling has been implicated in repressing adipogenesis. Several lines of evidence show that the possible mechanism is blockade of PPARgamma induction. However, the precise mechanisms remain to be elucidated. In this study, we demonstrated that Wnt3a conditioned medium suppresses C/EBPbeta/delta-induced adipogenesis of 3T3-L1 cells by inhibiting PPARgamma induction. In addition, the mutual activation of PPARgamma and C/EBPalpha was also repressed in the presence of Wnt3a. To further investigate the role of the canonical Wnt pathway in adipogenesis, we used mouse embryonic fibroblasts (MEFs) isolated from Lrp6-deficient embryos. Contrary to wild-type MEFs, Lrp6-deficient MEFs showed spontaneous adipogenesis and escaped the suppressive effect of exogenous Wnt3a. These findings suggest a critical role of Wnt/Lrp6/beta-catenin signaling in adipogenesis and cell fate decision of mesenchymal stem cells.     

Normal hematopoiesis and hematologic malignancies: Role of canonical Wnt signaling pathway and stromal microenvironment

Wnts are a family of evolutionary-conserved secreted signaling molecules critically involved in a variety of developmental processes and in cell fate determination. A growing body of evidence suggests that Wnt signaling plays a crucial role in the influence of bone marrow stromal microenvironment on the balance between hematopoietic stem cell self-renewal and differentiation. Emerging clinical and experimental evidence also indicates Wnt signaling involvement in the disruption of the latter balance in hematologic malignancies, where the stromal microenvironment favors the homing of cancer cells to the bone marrow, as well as leukemia stem cell development and chemoresistance. In the present review, we summarize and discuss the role of the canonical Wnt signaling pathway in normal hematopoiesis and hematologic malignancies, with regard to recent findings on the stromal microenvironment involvement in these process and diseases.     

Up-regulation of protease-activated receptor-2 by bFGF in cultured human synovial fibroblasts

Protease-activated receptors (PARs) have been implicated in the development of acute and chronic inflammatory responses. We have examined the expression of mRNA for PARs and their regulation by growth factors and cytokines in synovial fibroblasts derived from patients with rheumatoid arthritis (RA). Messenger RNA for PAR-1, -2 and -3 was detected in these cells, but not that for PAR-4. Expression of mRNA for PAR-2 was up-regulated by bFGF in a concentration-dependent manner, whereas expression of mRNA for PAR-1 and PAR-3 was not affected. Levels of mRNA encoding PAR-1, PAR-2 and PAR-3 did not increase in response to IL-1beta and TNF-alpha. Expression of mRNA for PAR-2 was maximal 12 h after addition of bFGF, and maximal levels of immunoreactive PAR-2 were reached after 24 h. Furthermore, PAR-2 agonist peptide (SLIGKV-NH(2)), but not the inactive reverse peptide (VKGILS-NH(2)), induced transitory cytosolic Ca(2+) mobilization in cells, and its response was increased by pretreatment with bFGF. An important role could be played by bFGF in the regulation of functional PAR-2 expression in cultured RA synovial fibroblasts.