Sequence-dependent effects of hematoporphyrin derivatives (HPD) photodynamic therapy and cisplatin on lung adenocarcinoma cells.

BACKGROUND: Hematoporphyrin derivatives (HPD)-Photodynamic therapy (PDT) in combination with cisplatin (DDP) is an effective anticancer strategy. However, whether the order of combination affects efficacy has not been studied. METHODS: The human lung adenocarcinoma (LUAD) A549 cells were used as the study subjects. After A549 cells were treated with a single medication (PDT/DDP) or a sequential combination (PDT + DDP / DDP + PDT), the cell viability was assayed using the cell counting kit-8 method. Hoechst staining, Annexin-V/propidium iodide (PI) double staining, western blotting, and a real-time quantitative polymerase chain reaction (RT-qPCR) were performed to examine the mechanisms behind the combined effects. RESULTS: A synergistic impact between HPD-PDT and DDP was found. The cell viability in the PDT+DDP group was significantly lower than in the DDP+PDT group. A significant apoptotic profile and a high apoptotic rate were seen in the PDT + DDP group. The western blot showed that the expression levels of Bcl2-associated x(Bax) and cleaved-poly ADP-ribose polymerase (PARP) increased, and those of B-cell lymphoma-2 (Bcl-2) and Caspase-9 decreased in the PDT + DDP group. At the same time, the RT-qPCR revealed the upregulation of Bax and PARP mRNA and the downregulation of Bcl-2 and Caspase-9 mRNA. CONCLUSION: The order of the combination therapy (PDT + DDP / DDP + PDT) was important. The HPD-PDT followed by DDP significantly inhibited LUAD cell viability, which may be related to the mitochondrial apoptotic pathway.

Receptor tyrosine kinases Tyro3, Axl, and Mertk differentially contribute to antibody-induced arthritis.

Tyro3, Axl, and Mertk (abbreviated TAMs) comprise a family of homologous type 1 receptor tyrosine kinases (RTKs) that have been implicated as inhibitory receptors that dampen inflammation, but their roles in the pathogenesis of rheumatoid arthritis remains understudied. Here, to investigate TAMs in an inflammatory arthritis model, antibody-induced arthritis in single TAM-deficient mice (Tyro3- KO, Axl-KO, Mertk-KO) was induced by K/BxN serum injection. Subsequently, joint inflammation and cytokine levels, as well as the expression of Fcγ Rs and complement receptors were assessed in WT and TAM-deficient mice. Compared with littermate control mice, Axl-/- and Mertk-/- mice developed more severe antibody-induced arthritis, while in contrast, Tyro3-/- mice showed diminished joint inflammation. Concomitantly, the levels of cytokines in joints of Axl-/- and Mertk-/- mice were also significantly increased, while cytokines in the Tyro3-/- joint tissues were decreased. At the molecular and cellular level, TAMs showed distinct expression patterns, whereby monocytes expressed Axl and Mertk, but no Tyro3, while neutrophils expressed Axl and Tyro3 but little Mertk. Moreover, expression of Fcγ receptors and C5aR showed different patterns with TAMs expression, whereby FcγRIV was higher in monocytes of Axl-/- and Mertk-/- mice compared to wild-type mice, while Tyro3-/- neutrophils showed lower expression levels of FcγRI, FcγRIII and FcγRIV. Finally, expression of C5aR was increased in Mertk-/- monocytes, and was decreased in Tyro3-/- neutrophils. These data indicate that Axl, Mertk and Tyro3 have distinct functions in antibody-induced arthritis, due in part to the differential regulation of cytokines production, as well as expression of FcγRs and C5aR. Video Abstract.

Penicillium fungi mediate behavioral responses of the yellow peach moth, Conogethes punctiferalis (Guenée) to apple fruits via altering the emissions of host plant VOCs.

Plant-associated microbes have been reported as important but overlooked drivers of plant-herbivorous insect interactions. Influence of plant-associated microbes on plant-insect interactions is diverse, including beneficial, detrimental, and neutral. Here, we determined the effects of three Penicillium fungi, including Penicillium citrinum, Penicillium sumatrense, and Penicillium digitatum, on the oviposition selection and behavior of the yellow peach moth (YPM), Conogethes punctiferalis (Guenée). Compared with fungi noninfected apples (NIA), mechanically damaged apples (MDA), and P. citrinum in potato dextrose agar medium (PC), the oviposition selection and four-arm olfactometer experiments both showed that mated YPM females preferred to P. citrinum-infected apples (PCA). For P. sumatrense or P. digitatum, we also found that mated YPM females preferred to P. sumatrense-infected apples (PSA) or P. digitatum-infected apples (PDA), respectively. Among three Penicillium fungi-infected apples, the selection rates including oviposition and olfactometer behavior of mated YPM females on PDA were both higher than those on PSA and PCA. Further analyses of host plant volatile organic compounds (VOCs) by GC-MS showed that the absolute contents of ethyl hexanoate and (Z, E)-α-farnesene in PCA, PSA, and PDA were all higher than those in NIA, and a total of 16 novel VOCs were detected in fungi-infected apples (PCA, PSA, and PDA), indicating that fungi infection changed the components and proportions of apple VOCs. Taken together, three Penicillium fungi play significant roles in mediating the host selection of YPMs via altering the emissions of VOCs. These findings will be beneficial for developing formulations for field trapping of YPMs in the future.

Icaritin-Induced FAM99A Affects GLUT1-Mediated Glycolysis via Regulating the JAK2/STAT3 Pathway in Hepatocellular Carcinoma.

Icaritin is a potential treatment option for hepatocellular carcinoma (HCC) based on the results of its phase 2 stage trial. Glucose transporter 1 (GLUT1), a critical gene in regulating glycolysis, has been recognized as a promising target in HCC treatment. Previous studies have reported that FAM99A, a new long noncoding (lncRNA), is associated with HCC metastasis. It has also been demonstrated that the JAK2/STAT3 pathway is related to HCC and is the target of icaritin treatment. However, whether FAM99A participates in icaritin treatment and regulates GLUT1-mediated glycolysis via the JAK2/STAT3 pathway in HCC cells remains to be explored. Our study aimed to clarify the mechanisms underlying glycolysis and understand the regulating effects of the FAM99A and JAK2/STAT3 pathway in HCC cells in icaritin treatment. Molecular mechanism studies were conducted to verify whether FAM99A could bind to the JAK2/STAT3 pathway and to identify the regulatory mechanisms in the HCC cells. It was revealed that icaritin inhibited proliferation, GLUT1 level, and the glycolysis of the HCC cells. FAM99A in HCC cells was upregulated after a high concentration treatment of icaritin. FAM99A inhibited GLUT1 by blocking the JAK2/STAT3 pathway. Mechanically, FAM99A interacted with EIF4B to inhibit gp130 and gp80 translation, which then interacted with miR-299-5p to upregulate SOCS3, causing the JAK2 pathway to inhibit STAT3 phosphorylation, so that JAK2/STAT3 was blocked in HCC cells. Overall, our study proved that icaritin-induced FAM99A can inhibit HCC cell viability and GLUT1-mediated glycolysis via blocking the JAK2/STAT3 pathway.

The disulfide bond Cys2724-Cys2774 in the C-terminal cystine knot domain of von Willebrand factor is critical for its dimerization and secretion.

BACKGROUND: Type 3 von Willebrand disease (VWD) exhibits severe hemorrhagic tendency with complicated pathogenesis. The C-terminal cystine knot (CTCK) domain plays an important role in the dimerization and secretion of von Willebrand factor (VWF). The CTCK domain has four intrachain disulfide bonds including Cys2724-Cys2774, Cys2739-Cys2788, Cys2750-Cys2804 and Cys2754-Cys2806, and the single cysteine mutation in Cys2739-Cys2788, Cys2750-Cys2804 and Cys2754-Cys2806 result in type 3 VWD, demonstrating the crucial role of these three disulfide bonds in VWF biosynthesis, however, the role of the remaining disulfide bond Cys2724-Cys2774 remains unclear. METHOD AND RESULTS: In this study, by the next-generation sequencing we found a missense mutation a c.8171G>A (C2724Y) in the CTCK domain of VWF allele in a patient family with type 3 VWD. In vitro, VWF C2724Y protein was expressed normally in HEK-293T cells but did not form a dimer or secrete into cell culture medium, suggesting that C2724 is critical for the VWF dimerization, and thus for VWF multimerization and secretion. CONCLUSIONS: Our findings provide the first genetic evidence for the important role of Cys2724-Cys2774 in VWF biosynthesis and secretion. Therefore, all of the four intrachain disulfide bonds in CTCK monomer contribute to VWF dimerization and secretion.

Plasma kallikrein contributes to ambient particulate matter-induced lung injury.

Particulate matter (PM) is a key component of air pollutants and is associated with mortality of cardiovascular and respiratory diseases. PM-induced tissue injury involves inflammation and coagulation. Plasma prekallikrein (pKal), along with coagulation factor XII (FXII) and high-molecular-weight kininogen (HK), form the plasma kallikrein-kinin system (KKS), a component of the innate immune response that generates proinflammatory products in response to injury. When the KKS proteins contact with activation surface such as negatively charged molecules, this system becomes activated. Activated kallikrein (Kal) activates FXII to initiate the intrinsic coagulation pathway, and cleaves HK to release bradykinin to enhance vascular permeability and systemic inflammation. In his study we determined the role of plasma pKal in the PM2.5-induced lung injury. Using TALEN technology, we generated a new mouse strain lacking the gene for pKal. In PM2.5-induced lung injury model, Klkb1-/- mice exhibited a decrease in total protein, cells numbers in bronchoalveolar lavage fluid (BALF) and histologic lung injury score. The TNF-α and IL-6 levels in BALF were significantly decreased in PM2.5-treated Klkb1-/- mice. Plasma thrombin-antithrombin (TAT) complex levels were significantly decreased in PM2.5-treated Klkb1-/- mice. PM2.5 induces pKal activation, HK cleavage and bradykinin production. PM2.5-induced HK cleavage in plasma was completely blocked by a Kal inhibitor, as well as in pKal-deficient plasma. PM2.5 markedly induced thrombin generation in human plasma and wild-type mouse plasma, which was inhibited by both blockade and deficiency of pKal. Taken together, plasma pKal is activated by PM2.5 and the activated Kal plays an important role in PM2.5-induced lung injury.

Ginsenoside Rg3 Combined with Oxaliplatin Inhibits the Proliferation and Promotes Apoptosis of Hepatocellular Carcinoma Cells via Downregulating PCNA and Cyclin D1.

The present study aims to investigate the effects of ginsenoside Rg3 combined with oxaliplatin on the proliferation and apoptosis of hepatocellular carcinoma cells and the related mechanism. In this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied to examine the proliferation rate of hepatocellular carcinoma cell SMMC-7721 with different treatment. Flow cytometry was performed to examine apoptosis rate of hepatocellular carcinoma cells with different treatment. Immunofluorescence and Western blot methods were used to evaluate the expressions of proliferating cell nuclear antigen (PCNA) and cyclin D1 in different groups. We found that ginsenoside Rg3, oxaliplatin or ginsenoside Rg3 + oxaliplatin significantly suppressed the proliferation and promoted the apoptosis of SMMC-7721. Meanwhile, ginsenoside Rg3, oxaliplatin or ginsenoside Rg3 + oxaliplatin also significantly inhibited the expressions of PCNA and cyclin D1. Moreover, compared with ginsenoside Rg3 group and oxaliplatin group, the effect of ginsenoside Rg3 + oxaliplatin was more remarkable. Taken together, cells treated with oxaliplatin+ ginsenoside enhanced the anti-tumor effect and may inhibit the proliferation and promoted apoptosis of hepatocellular carcinoma via regulating the expression of PCNA and cyclin D1.

Tyro3, Axl, and Mertk receptors differentially participate in platelet activation and thrombus formation.

BACKGROUND: Previously, several studies have shown that Tyro3, Axl, and Mertk (TAM) receptors participate in platelet activation and thrombosis. However, the role of individual receptors is not fully understood. METHODS: Using single receptor-deficient platelets from TAM knockout mice in the C57BL/6 J strain, we performed a knockout study using single TAM-deficient mice. We treated platelets isolated from TAM knockout mice with the Glycoprotein VI (GPVI) agonists convulxin, poly(PHG), and collagen-related triple-helical peptide (CRP), as well as thrombin for in-vitro experiments. We used a laser-induced cremaster arterial injury model for thrombosis experiments in vivo. RESULTS: Deficiency of the tyrosine kinase receptors, Axl or Tyro3, but not Mertk, inhibited aggregation, spreading, JON/A binding, and P-selectin expression of platelets in vitro. In vivo, platelet thrombus formation was significantly decreased in Axl-/- and Tyro3-/- mice, but not in Mertk-/- mice. Upon stimulation with glycoprotein VI (GPVI) agonists, tyrosine phosphorylation of signaling molecules, including spleen tyrosine kinase (Syk) and phospholipase C-γ2 (PLCγ2), was decreased in Axl-/- and Tyro3-/- platelets, but not in Mertk-/- platelets. While platelet aggregation induced by agonists did not differ in the presence or absence of the Gas6 neutralizing antibody, the platelet aggregation was inhibited by anti-Axl or anti-Tyro3 neutralizing antibodies antibody, but not the anti-Mertk antibody. Additionally, the recombinant extracellular domain of Axl or Tyro3, but not that of Mertk, also inhibited platelet aggregation. CONCLUSIONS: These data suggest that Axl and Tyro3, but not Mertk, have an important role in platelet activation and thrombus formation, and mechanistically may do so by a pathway that regulates inside to outside signaling and heterotypic interactions via the extracellular domains of TAMs.

A critical role for plasma kallikrein in the pathogenesis of autoantibody-induced arthritis.

The plasma kallikrein-kinin system (KKS) consists of serine proteases, prekallikrein (pKal) and factor XII (FXII), and a cofactor, high-MW kininogen (HK). Upon activation, activated pKal and FXII cleave HK to release bradykinin. Activation of this system has been noted in patients with rheumatoid arthritis, and its pathogenic role has been characterized in animal arthritic models. In this study, we generated 2 knockout mouse strains that lacked pKal and HK and determined the role of KKS in autoantibody-induced arthritis. In a K/BxN serum transfer-induced arthritis (STIA) model, mice that lacked HK, pKal, or bradykinin receptors displayed protective phenotypes in joint swelling, histologic changes in inflammation, and cytokine production; however, FXII-deficient mice developed normal arthritis. Inhibition of Kal ameliorated arthritis severity and incidence at early stage STIA and reduced the levels of major cytokines in joints. In addition to releasing bradykinin from HK, Kal directly activated monocytes to produce proinflammatory cytokines, up-regulated their C5aR and FcRIII expression, and released C5a. Immune complex increased pKal activity, which led to HK cleavage. The absence of HK is associated with a decrease in joint vasopermeability. Thus, we identify a critical role for Kal in autoantibody-induced arthritis with pleiotropic effects, which suggests that it is a new target for the inhibition of arthritis.-Yang, A., Zhou, J., Wang, B., Dai, J., Colman, R. W., Song, W., Wu, Y. A critical role for plasma kallikrein in the pathogenesis of autoantibody-induced arthritis.

Focal adhesion kinase overexpression and its impact on human osteosarcoma.

Focal adhesion kinase (FAK) has been implicated in tumorigenesis in various malignancies. We sought to examine the expression patterns of FAK and the activated form, phosphorylated FAK (pFAK), in human osteosarcoma and to investigate the correlation of FAK expression with clinicopathologic parameters and prognosis. In addition, the functional consequence of manipulating the FAK protein level was investigated in human osteosarcoma cell lines. Immunohistochemical staining was used to detect FAK and pFAK in pathologic archived materials from 113 patients with primary osteosarcoma. Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognoses. The role of FAK in the cytological behavior of MG63 and 143B human osteosarcoma cell lines was studied via FAK protein knock down with siRNA. Cell proliferation, migration, invasiveness and apoptosis were assessed using the CCK8, Transwell and Annexin V/PI staining methods. Both FAK and pFAK were overexpressed in osteosarcoma. There were significant differences in overall survival between the FAK-/pFAK- and FAK+/pFAK- groups (P = 0.016), the FAK+/pFAK- and FAK+/pFAK+ groups (P = 0.012) and the FAK-/pFAK- and FAK+/pFAK+ groups (P < 0.001). There were similar differences in metastasis-free survival between groups. The Cox proportional hazards analysis showed that the FAK expression profile was an independent indicator of both overall and metastasis-free survival. SiRNA-based knockdown of FAK not only dramatically reduced the migration and invasion of MG63 and 143B cells, but also had a distinct effect on osteosarcoma cell proliferation and apoptosis. These results collectively suggest that FAK overexpression and phosphorylation might predict more aggressive biologic behavior in osteosarcoma and may be an independent predictor of poor prognosis.

Combretastatin A4 phosphate treatment induces vasculogenic mimicry formation of W256 breast carcinoma tumor in vitro and in vivo.

The purpose of this study was to investigate the effect of combretastatin A4 phosphate (CA4P) on vasculogenic mimicry (VM) channel formation in vitro and in vivo after a single-dose treatment and the underlying mechanism involved in supporting VM. In vitro model of three-dimensional cultures was used to test the effect of CA4P on the tube formation of Walker 256 cells. Western blot analysis was conducted to assess the expression of hypoxia-inducible factor (HIF)-1α and VM-associated markers. W256 tumor-bearing rat model was established to demonstrate the effect of CA4P on VM formation and tumor hypoxia by double staining and a hypoxic marker pimonidazole. Anti-tumor efficacy of CA4P treatment was evaluated by tumor growth curve. Under hypoxic conditions for 48 h in vitro, W256 cells formed VM network associated with increased expression of VM markers. Pretreatment with CA4P did not influence the amount of VM in 3-D culture as well as the expression of these key molecules. In vivo, W256 tumors showed marked intratumoral hypoxia after CA4P treatment, accompanied by increased VM formation. CA4P exhibited only a delay in tumor growth within 2 days but rapid tumor regrowth afterward. VM density was positively related to tumor volume and tumor weight at day 8. CA4P causes hypoxia which induces VM formation in W256 tumors through HIF-1α/EphA2/PI3K/matrix metalloproteinase (MMP) signaling pathway, resulting in the consequent regrowth of the damaged tumor.

Raltitrexed induces mitochondrial­mediated apoptosis in SGC7901 human gastric cancer cells.

Raltitrexed is a specific inhibitor of thymidylate synthase (TS), which has been considered as a potential chemotherapeutic agent for the treatment of advanced gastric cancer. In the present study, the apoptosis mechanisms of raltitrexed in SGC7901 human gastric cancer cells were investigated. The cytotoxic activity of raltitrexed on SGC7901 cells was determined by cell counting kit-8 (CCK-8) assay. The CCK­8 assay indicated that raltitrexed inhibits SGC7901 cell growth in a dose- and time-dependent manner. The morphological changes were observed by fluorescent microscopy, and characteristic morphological changes, including nuclear shrinkage and apoptotic bodies, were observed following Hoechst 33258 staining. The effects on apoptosis, cell cycle, mitochondrial transmembrane potential and reactive oxygen species (ROS) were measured by flow cytometry. The analysis revealed that raltitrexed exerted a growth inhibitory effect by inducing time-dependent apoptosis and cell-cycle arrest at the G0/G1 phase. In addition, a compromised mitochondrial membrane potential and overproduction of ROS demonstrated the involvement of the mitochondrial signaling pathway. Raltitrexed­induced caspase­3­dependent apoptosis was identified using a caspase-3 activity assay and pretreatment with the caspase-3 inhibitor, Ac­DEVD­CHO (sequence, Ac-Asp-Glu-Val-Asp-CHO). The activity of caspase-3 was analyzed with a spectrometer. The protein expression levels of Bax, Bcl-2, cytochrome c, cleaved caspase-3 and TS were examined by western blot and the mRNA expression level of TS was detected by quantitative polymerase chain reaction. The analysis revealed that the protein levels of Bax, cytochrome c and cleaved caspase­3 were significantly increased by raltitrexed, while Bcl-2 expression levels were reduced. Furthermore, raltitrexed increased the expression of the TS protein and mRNA in a time­dependent manner. These results indicate that raltitrexed induces the apoptosis of SGC7901 cells through the caspase­3­dependent mitochondrial signaling pathway and upregulates the expression of the TS protein and mRNA.

High molecular weight kininogen binds phosphatidylserine and opsonizes urokinase plasminogen activator receptor-mediated efferocytosis.

Phagocytosis of apoptotic cells (efferocytosis) is essential for regulation of immune responses and tissue homeostasis and is mediated by phagocytic receptors. In this study, we found that urokinase plasminogen activator receptor (uPAR) plays an important role in internalization of apoptotic cells and also characterized the underlying mechanisms. In a flow cytometry-based phagocytic assay, uPAR-deficient macrophages displayed significant defect in internalization but not tethering of apoptotic cells. When uPAR-deficient mice were challenged with apoptotic cells, they exhibited pronounced splenomegaly resulting from accumulation of abundant apoptotic cells in spleen. Overexpression of uPAR in HEK-293 cells enhanced efferocytosis, which was inhibited by Annexin V and phosphatidylserine (PS) liposome, suggesting that uPAR-mediated efferocytosis is dependent on PS. In serum lacking high m.w. kininogen (HK), a uPAR ligand, uPAR-mediated efferocytosis was significantly attenuated, which was rescued by replenishment of HK. As detected by flow cytometry, HK selectively bound to apoptotic cells, but not viable cells. In purified systems, HK was specifically associated with PS liposome. HK binding to apoptotic cells induced its rapid cleavage to the two-chain form of HK (HKa) and bradykinin. Both the H chain and L chain of HKa were associated with PS liposome and apoptotic cells. HKa has higher binding affinity than HK to uPAR. Overexpression of Rac1/N17 cDNA inhibited uPAR-mediated efferocytosis. HK plus PS liposome stimulated a complex formation of CrkII with p130Cas and Dock-180 and Rac1 activation in uPAR-293 cells, but not in control HEK-293 cells. Thus, uPAR mediates efferocytosis through HK interaction with PS on apoptotic cells and activation of the Rac1 pathway.

A role for bradykinin in the development of anti-collagen antibody-induced arthritis.

OBJECTIVES: Clinical and experimental observations have suggested that bradykinin, a major activation product of the plasma kallikrein-kinin system, is involved in the pathogenesis of arthritis, but the pathogenic role of bradykinin receptors remains inconclusive. In this study we examined whether bradykinin receptors are important in the pathogenesis of anti-collagen antibody-induced arthritis (CAIA) using double receptor-deficient (B1RB2R(-/-)) mice. METHODS: CAIA was induced in B1RB2R(+/+) and B1RB2R(-/-) mice by injection of an anti-collagen antibody cocktail on day 0 and lipopolysaccharide on day 3. Severity of disease was evaluated by measurement of joint diameter and histological analysis. The expression of proinflammatory cytokines in joint tissue and peripheral mononuclear cells was determined by ELISA and real-time RT-PCR. RESULTS: The absent expression of B1R and B2R mRNA in B1RB2R(-/-) mice was confirmed by RT-PCR. Although B1RB2R(+/+) mice developed severe CAIA, the severity of the disease was significantly attenuated in B1RB2R(-/-) mice. In B1RB2R(+/+) mice bearing CAIA, both B1R and B2R mRNA levels were increased in joint tissue and peripheral mononuclear cells. Compared with B1RB2R(+/+) mice, the production of IL-1ß and IL-6 in joint tissue and their mRNA expression in peripheral mononuclear cells were remarkably reduced in B1RB2R(-/-) mice. CONCLUSION: These observations provide genetic evidence that bradykinin plays an important role in the pathogenesis of CAIA. B1R, whose expression is induced in inflamed joint tissue and peripheral inflammatory cells, is important in the development of CAIA.

Basic fibroblast growth factor is a key factor that induces bone marrow mesenchymal stem cells towards cells with Schwann cell phenotype.

Bone marrow mesenchymal stem cells (MSCs) can be differentiate towards a Schwann cells (SCs) lineage when exposed to pre-inducing reagents ß-mercaptoethanol (BME) and retinoic acid (RA), followed by inducing factors: forskolin (FSK), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), and heregulin (HRG). However, the underlying mechanisms remain unclear. Here, we investigated the individual effects of these inducing factors on the differentiation of MSCs towards SC phenotype in rats. We show that the omission of either HRG or PDGF from the induction medium is not sufficient to change the SC-like phenotype or the expression level of the SC marker, S100ß. However, the omission of bFGF from the induction medium effectively blocked neural induction of the MSCs. Moreover, only bFGF was found to inhibit MSC proliferation during differentiation. To clarify the mechanism responsible for the effect of bFGF, we also investigated the activation of the extracellular signal-regulated kinase (ERK) pathway in the induced cells. Our results suggest that morphological changes in MSCs induced by bFGF depend on the activation of ERK, and bFGF may be an indispensable factor that induces MSCs to differentiate into cells with SCs phenotype.

Role of plasma kallikrein-kinin system activation in synovial recruitment of endothelial progenitor cells in experimental arthritis.

OBJECTIVE: To examine whether activation of the plasma kallikrein-kinin system (KKS) mediates synovial recruitment of endothelial progenitor cells (EPCs) in arthritis. METHODS: EPCs were isolated from Lewis rat bone marrow, and expression of progenitor cell-lineage markers and functional properties were characterized. EPCs were injected intravenously into Lewis rats with arthritis, and their recruitment and formation of de novo blood vessels in inflamed synovium were evaluated. The role of plasma KKS was examined using a plasma kallikrein inhibitor (EPI-KAL2) and an antikallikrein antibody (13G11). A transendothelial migration assay was used to determine the role of bradykinin and its receptor in EPC mobilization. RESULTS: EPCs from Lewis rats exhibited a strong capacity to form tubes and vacuoles and expressed increased levels of bradykinin type 2 receptor (B2R) and progenitor cell markers CD34 and Sca-1. In Lewis rats with arthritis, EPCs were recruited into inflamed synovium at the acute phase of disease and formed de novo blood vessels. Inhibition of plasma kallikrein by EPI-KAL2 and 13G11 significantly suppressed synovial recruitment of EPCs and hyperproliferation of synovial cells. Bradykinin stimulated transendothelial migration of EPCs in a concentration-dependent manner. This was mediated by B2R, as demonstrated by the finding that knockdown of B2R with silencing RNA completely blocked bradykinin-stimulated transendothelial migration. Moreover, bradykinin selectively up-regulated expression of the homing receptor CXCR4 in EPCs. CONCLUSION: These observations demonstrate a novel role of plasma KKS activation in the synovial recruitment of EPCs in arthritis, acting via kallikrein activation and B2R-dependent mechanisms. B2R might be involved in the mobilization of EPCs via up-regulation of CXCR4.

mRNA profiles of cytokine receptors in unstimulated peripheral blood mononuclear cells from patients with chronic idiopathic urticaria.

This present study was aimed to investigate the roles of the receptors of Th1/Th2 cytokines and chemokines in the pathogenesis of chronic idiopathic urticaria (CIU). Thirty patients with CIU, 30 patients with dermographism and 30 healthy controls were randomly enrolled. Reverse transcription-PCR (RT-PCR) was used to analyze the mRNA of cytokine receptors in peripheral blood mononuclear cells (PBMCs). The mRNA levels of tumor necrosis factor receptor (TNFR), interferon-γ receptor (IFN-γR), and interleukin-10 receptor (IL-10R) were statistically increased in the CIU group (P < 0.05), while IL-2R, IL-4R, IL-6R, and IL-13R showed no significant differences between the CIU and other groups. The mRNA levels of CCR3 and CCR6 were statistically increased in the CIU group (P < 0.05). The toll-like receptor 2 (TLR2) mRNA level was significantly lower in the CIU group than the healthy control group (P < 0.05). These findings indicate that the regulation of mRNA of TNFR, IFN-γR, IL-10R, CCR3, CCR6 and TLR2 may be involved in the pathogenesis of CIU.

[Effects of exogenous betaine on physiological responses of peach tree under water stress].

With four-year-old potted Prunus persica L. cv. Qingfeng as test material, this paper studied the change pattern of its leaf betaine content under water stress, and its physiological responses under effects of foliage-spraying exogenous betaine. The results showed that under normal water supply, the betaine content in Qingfeng' s leaf was 75.9-80.5 microg x g(-1) FM, which was increased with increasing water stress, and up to 278.9 microg x g(-1) FM on the 16th day after cutting off the water supply. The leaf plasma membrane permeability was 8.06% - 8.61% under normal water supply, but increased to 28.62% under water stress. When 100 and 500 mg x L(-1) of betaine were applied exogenously, the plasma membrane permeability was 26.25% and 21.79% after 16 days, respectively. The hydrogen peroxide (H2O2) content increased from 27.2-32.5 micromol g(-1) FM to 76.4 micromol x g(-1) FM in the course of water stress, and decreased to 73.2 and 68.5 micromol x g(-1) FM after spraying 100 and 500 mg betaine x L(-1), respectively. During the period of intensified water stress, the peak value of ascorbate peroxidase (AsA-POD) activity was 0.435 mg x g(-1) FM, and up to 0.490 mg x g(-1) FM when treated with exogenous betaine. When the peach tree was subjected to water stress, the contents of free proline and soluble sugar accumulated dramatically, but produced on approximately decrease in 500 mg x L(-1) endogenous betaine application on the 16th day which was slightly less than that of control and 100 mg x L(-1) betaine application. There was a gradual decline in the content of soluble protein under water stress, and an increment of 20. 3% was observed when betaine was applied exogenously. These results strongly suggested that foliage-spraying exogenous betaine could increase the drought resistance of peach tree through decreasing its leaf plasma membrane permeability and H2O2, free proline and soluble sugar contents and increasing its leaf AsA-POD activity and soluble protein content.

Inhibitory effects of clofilium on membrane currents associated with Ca channels, NMDA receptor channels and Na+, K+-ATPase in cortical neurons.

The class III antiarrhythmic agent 4-chloro-N,N-diethyl-N-heptyl-benzene butanaminium (clofilium) is known as a K+ channel open-channel blocker and has either anti- or proapoptotic property due to undefined mechanisms. Based on the evidence that neuronal viability is largely, sometimes critically, affected by voltage- and ligand-gated Ca2+ channels and the Na+, K+-ATPase, we tested the hypothesis that clofilium might additionally act on Ca2+ permeable ion channels and the Na+, K+-ATPase. Membrane currents associated with activities of voltage-gated Ca2+ channels, N-methyl-D-aspartate (NMDA) receptor channels and Na+, K+-ATPase were recorded using whole-cell recordings in cultured murine cortical neurons. Clofilium (0.1-100 micromol/l) inhibited high voltage-activated Ca2+ currents in concentration- and use-dependent manners. Clofilium acted as a potent antagonist of NMDA receptor channels, preferably blocked the NMDA steady-state current at a low concentration (0.1 micromol/l). At concentrations of >100 micromol/l, clofilium blocked both peak and steady-state NMDA currents in a voltage-independent manner. Clofilium also inhibited the Na+, K+-ATPase current with an IC50 of 7.5 micromol/l. Our data suggest that the pharmacological action of clofilium is far more complex than recognized before; the multiple actions of clofilium on membrane conductance may explain its diverse effects on cellular events and cell viability.

Apoptotic insults impair Na+, K+-ATPase activity as a mechanism of neuronal death mediated by concurrent ATP deficiency and oxidant stress.

The Na+, K+-ATPase (Na+, K+-pump) plays critical roles in maintaining ion homeostasis. Blocking the Na+, K+-pump may lead to apoptosis. By contrast, whether an apoptotic insult may affect the Na+, K+-pump activity is largely undefined. In cultured cortical neurons, the Na+, K+-pump activity measured as a membrane current Ipump was time-dependently suppressed by apoptotic insults including serum deprivation, staurosporine, and C2-ceramide, concomitant with depletion of intracellular ATP and production of reactive oxygen species. Signifying a putative relationship among these events, Ipump was highly sensitive to changes in ATP and reactive oxygen species levels. Moreover, the apoptosis-associated Na+, K+-pump failure and serum deprivation-induced neuronal death were antagonized by pyruvate and succinate in ATP- and reactive-oxygen-species-dependent manners. We suggest that failure of the Na+, K+-pump as a result of a combination of energy deficiency and production of reactive oxygen species is a common event in the apoptotic cascade; preserving the pump activity provides a neuroprotective strategy in certain pathological conditions.