RESUMO
BACKGROUND: Biological laboratories and companies involved in antibody development need convenient and versatile methods to detect highly active antibodies. METHODS: To develop a mammalian cell-based ZZ display system for antibody quantification, the eukaryotic ZZ-displayed plasmid was constructed and transfected into CHO cells. After screening by flow cytometric sorting, the stable ZZ display cells were incubated with reference IgG and samples with unknown IgG content for 40 min at 4â, the relative fluorescence intensity of cells was analyzed and the concentration of IgG was calculated. RESULTS: By investigating the effects of different display-associated genetic elements, a eukaryotic ZZ-displaying plasmid with the highest display efficiency were constructed. After transfection and screening, almost 100% of the cells were able to display the ZZ peptide (designated CHO-ZZ cells). These stable CHO-ZZ cells were able to capture a variety of IgG, including human, rabbit, donkey and even mouse and goat. CHO-ZZ cells could be used to quantify human IgG in the range of approximately 12.5-1000 ng/mL, and to identify high-yielding engineered monoclonal cell lines. CONCLUSIONS: We have established a highly efficient CHO-ZZ display system in this study, which enables the quantification of IgG from various species under physiological conditions. This system offers the advantage of eliminating the need for antibody purification and will contribute to antibody development.
Assuntos
Imunoglobulina G , Cricetinae , Camundongos , Coelhos , Animais , Humanos , Cricetulus , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Citometria de Fluxo , PlasmídeosRESUMO
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.
Assuntos
Artrite , Receptor Tirosina Quinase Axl , Receptores Proteína Tirosina Quinases , Receptores de IgG , c-Mer Tirosina Quinase , Animais , Camundongos , Anticorpos , Receptor Tirosina Quinase Axl/metabolismo , c-Mer Tirosina Quinase/metabolismo , Proteínas de Transporte , Citocinas/metabolismo , Inflamação , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de IgG/metabolismo , TirosinaRESUMO
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.
Assuntos
Malus , Mariposas , Penicillium , Prunus persica , Compostos Orgânicos Voláteis , Animais , Feminino , Frutas/microbiologia , Malus/microbiologia , Mariposas/fisiologia , Compostos Orgânicos Voláteis/farmacologiaRESUMO
Secreted platelet protein disulfide isomerases, PDI, ERp57, ERp5, and ERp72, have important roles as positive regulators of platelet function and thrombosis. Thioredoxin-related transmembrane protein 1 (TMX1) was the first described transmembrane member of the protein disulfide isomerase family of enzymes. Using a specific antibody, the recombinant extracellular domain of TMX1 (rTMX1) protein, a knockout mouse model, and a thiol-labeling approach, we examined the role of TMX1 in platelet function and thrombosis. Expression of TMX1 on the platelet surface increased with thrombin stimulation. The anti-TMX1 antibody increased platelet aggregation induced by convulxin and thrombin, as well as potentiated platelet ATP release. In contrast, rTMX1 inhibited platelet aggregation and ATP release. TMX1-deficient platelets had increased aggregation, ATP release, αIIbß3 activation, and P-selectin expression, which were reversed by addition of rTMX1. TMX1-knockout mice had increased incorporation of platelets into a growing thrombus in an FeCl3-induced mesenteric arterial injury model, as well as shortened tail-bleeding times. rTMX1 oxidized thiols in the αIIbß3 integrin and TMX1-deficient platelets had increased thiols in the ß3 subunit of αIIbß3, consistent with oxidase activity of rTMX1 against αIIbß3. Thus, TMX1 is the first identified extracellular inhibitor of platelet function and the first disulfide isomerase that negatively regulates platelet function.
Assuntos
Plaquetas/patologia , Proteínas de Membrana/metabolismo , Ativação Plaquetária , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Tiorredoxinas/metabolismo , Trombose/patologia , Animais , Plaquetas/metabolismo , Hemostasia , Humanos , Camundongos , Camundongos Knockout , Trombose/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Coagulação Sanguínea , Inflamação/etiologia , Lesão Pulmonar/etiologia , Material Particulado/efeitos adversos , Calicreína Plasmática/imunologia , Animais , Deleção de Genes , Humanos , Inflamação/sangue , Inflamação/genética , Inflamação/imunologia , Lesão Pulmonar/sangue , Lesão Pulmonar/genética , Lesão Pulmonar/imunologia , Camundongos , Camundongos Knockout , Material Particulado/imunologia , Calicreína Plasmática/análise , Calicreína Plasmática/genéticaRESUMO
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.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Ciclina D1/metabolismo , Ginsenosídeos/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Oxaliplatina/farmacologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Ginsenosídeos/administração & dosagem , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Oxaliplatina/administração & dosagemRESUMO
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.
Assuntos
Artrite/metabolismo , Artrite/patologia , Autoanticorpos/metabolismo , Calicreína Plasmática/metabolismo , Animais , Artrite/genética , Artrite/imunologia , Bradicinina/metabolismo , Citocinas/metabolismo , Fator XII/genética , Fator XII/metabolismo , Feminino , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Knockout , Monócitos/metabolismo , Calicreína Plasmática/genética , Reação em Cadeia da PolimeraseRESUMO
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.
Assuntos
Ativação Plaquetária , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Trombose/metabolismo , c-Mer Tirosina Quinase/metabolismo , Animais , Humanos , Camundongos , Fosforilação , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Receptor Tirosina Quinase AxlRESUMO
The moss Physcomitrella patens is a model system for studying Plant developmental processes. To better understand the biochemical and physiological changes involved in developmental reprogramming, we conducted a quantitative proteomics analysis for protonemata, protoplasts made therefrom and protoplasts regenerated for 2 d. Using an iTRAQ peptide labeling strategy and liquid chromatography-tandem mass spectrometry (LC-MS/MS), >3,000 peptides and 1,000 proteins were quantified. Of these, 162 proteins were identified as having differential abundances during developmental reprogramming. These proteins were involved in various biological functions, such as defense, energy production, translation, metabolism, protein destination and storage, transcription, transport, cell growth/division, cell structure and signal transduction. Of these, the proteins involved in energy production and translation increased in abundance, while many of the metabolism and defense proteins decreased in abundance. In addition, most of the cell growth/division, protein stability and cell structure proteins were also down-regulated. This is the first report on the metabolic changes involved in developmental reprogramming in protoplasts. The significance of metabolic networks in developmental programming is beginning to emerge. Our study suggested that stress signals, energy metabolism and ribosomal proteins are pivotal components during developmental programming.
Assuntos
Bryopsida/metabolismo , Proteínas de Plantas/metabolismo , Proteômica/métodos , Protoplastos/metabolismo , Transdução de Sinais/fisiologiaRESUMO
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.
Assuntos
Macrófagos/imunologia , Fagocitose/fisiologia , Fosfatidilserinas/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Apoptose/imunologia , Células HEK293 , Humanos , Immunoblotting , Imunoprecipitação , Cininogênio de Alto Peso Molecular/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/fisiologia , Ressonância de Plasmônio de Superfície , Transfecção , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias Mamárias Animais/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Estilbenos/administração & dosagem , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Mamárias Animais/patologia , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Nitroimidazóis/administração & dosagem , Fosfatidilinositol 3-Quinases/genética , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Artrite Experimental/imunologia , Artrite Experimental/fisiopatologia , Bradicinina/fisiologia , Receptores da Bradicinina/fisiologia , Animais , Anticorpos Anti-Idiotípicos/efeitos adversos , Artrite Experimental/metabolismo , Colágeno/efeitos adversos , Colágeno/imunologia , Modelos Animais de Doenças , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Articulações/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores da Bradicinina/deficiência , Receptores da Bradicinina/genéticaRESUMO
Spermatogenesis requires precise posttranslational control in the endoplasmic reticulum (ER), but the mechanism remains largely unknown. The protein disulfide isomerase (PDI) family is a group of thiol oxidoreductases responsible for catalyzing the disulfide bond formation of nascent proteins. In this study, we generated 14 strains of KO mice lacking the PDI family enzymes and found that only PDI deficiency caused spermatogenesis defects. Both inducible whole-body PDI-KO (UBC-Cre/Pdifl/fl) mice and premeiotic PDI-KO (Stra8-Cre/Pdifl/fl) mice experienced a significant decrease in germ cells, testicular atrophy, oligospermia, and complete male infertility. Stra8-Cre/Pdifl/fl spermatocytes had significantly upregulated ER stress-related proteins (GRP78 and XBP1) and apoptosis-related proteins (Cleaved caspase-3 and BAX), together with cell apoptosis. PDI deletion led to delayed DNA double-strand break repair and improper crossover at the pachytene spermatocytes. Quantitative mass spectrometry indicated that PDI deficiency downregulated vital proteins in spermatogenesis such as HSPA4L, SHCBP1L, and DDX4, consistent with the proteins' physical association with PDI in normal testes tissue. Furthermore, PDI served as a thiol oxidase for disulfide bond formation of SHCBP1L. Thus, PDI plays an essential role in protein quality control for spermatogenesis in mice.
Assuntos
Chaperona BiP do Retículo Endoplasmático , Camundongos Knockout , Isomerases de Dissulfetos de Proteínas , Espermatogênese , Testículo , Animais , Masculino , Espermatogênese/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Camundongos , Testículo/metabolismo , Chaperona BiP do Retículo Endoplasmático/metabolismo , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Apoptose , Espermatócitos/metabolismo , Estresse do Retículo Endoplasmático , Oligospermia/genética , Oligospermia/metabolismo , Oligospermia/patologiaRESUMO
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.
Assuntos
Antineoplásicos , Apoptose , Sobrevivência Celular , Cisplatino , Neoplasias Pulmonares , Fotoquimioterapia , Fármacos Fotossensibilizantes , Humanos , Fotoquimioterapia/métodos , Cisplatino/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Sobrevivência Celular/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Antineoplásicos/farmacologia , Células A549 , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma de Pulmão/tratamento farmacológico , Hematoporfirinas/farmacologia , Derivado da Hematoporfirina/farmacologia , Linhagem Celular TumoralRESUMO
The essence of difference between hemostasis and thrombosis is that the clotting reaction is a highly fine-tuned process. Vascular protein disulfide isomerase (PDI) represents a critical mechanism regulating the functions of hemostatic proteins. Herein we show that histidine-rich glycoprotein (HRG) is a substrate of PDI. Reduction of HRG by PDI enhances the procoagulant and anticoagulant activities of HRG by neutralization of endothelial heparan sulfate (HS) and inhibition of factor XII (FXIIa) activity, respectively. Murine HRG deficiency (Hrg-/-) leads to delayed onset but enhanced formation of thrombus compared to WT. However, in the combined FXII deficiency (F12-/-) and HRG deficiency (by siRNA or Hrg-/-), there is further thrombosis reduction compared to F12-/- alone, confirming HRG's procoagulant activity independent of FXIIa. Mutation of target disulfides of PDI leads to a gain-of-function mutant of HRG that promotes its activities during coagulation. Thus, PDI-HRG pathway fine-tunes thrombosis by promoting its rapid initiation via neutralization of HS and preventing excessive propagation via inhibition of FXIIa.
Assuntos
Isomerases de Dissulfetos de Proteínas , Proteínas , Trombose , Animais , Camundongos , Dissulfetos , Fator XII/metabolismo , Heparitina Sulfato , Isomerases de Dissulfetos de Proteínas/genética , Proteínas/metabolismo , Trombose/genética , Trombose/metabolismoRESUMO
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.
Assuntos
Artrite Experimental/imunologia , Endotélio Vascular/imunologia , Células-Tronco Hematopoéticas/imunologia , Sistema Calicreína-Cinina/imunologia , Receptor B2 da Bradicinina/imunologia , Membrana Sinovial/imunologia , Doença Aguda , Animais , Artrite Experimental/patologia , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Bradicinina/imunologia , Movimento Celular/imunologia , Células Cultivadas , Endotélio Vascular/citologia , Feminino , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Calicreínas/sangue , Calicreínas/imunologia , Neovascularização Patológica/imunologia , Neovascularização Patológica/patologia , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos Lew , Ratos Transgênicos , Receptores CXCR4/genética , Receptores CXCR4/imunologia , Membrana Sinovial/irrigação sanguínea , Membrana Sinovial/patologiaRESUMO
Critical examinations of specimens, with literature reviews, have shown that Rubusdavidianus is conspecific with R.lambertianus. Therefore, we treat R.davidianus as a new synonym within Rubus. We propose a new name, Rubusloirensis Ti R. Huang nom. nov. to replace the later homonym of R.pycnanthus Genev. Additionally, lectotypification of three names, R.davidianus Kuntze, R.malifolius Focke and R.viburnifolius Franch., are designated here after examination of previous works.
RESUMO
Pulmonary fibrosis is a chronic interstitial lung disease that causes irreversible and progressive lung scarring and respiratory failure. Activation of fibroblasts plays a central role in the progression of pulmonary fibrosis. Here we show that platelet endothelial aggregation receptor 1 (PEAR1) in fibroblasts may serve as a target for pulmonary fibrosis therapy. Pear1 deficiency in aged mice spontaneously causes alveolar collagens accumulation. Mesenchyme-specific Pear1 deficiency aggravates bleomycin-induced pulmonary fibrosis, confirming that PEAR1 potentially modulates pulmonary fibrosis progression via regulation of mesenchymal cell function. Moreover, single cell and bulk tissue RNA-seq analysis of pulmonary fibroblast reveals the expansion of Activated-fibroblast cluster and enrichment of marker genes in extracellular matrix development in Pear1-/- fibrotic lungs. We further show that PEAR1 associates with Protein Phosphatase 1 to suppress fibrotic factors-induced intracellular signalling and fibroblast activation. Intratracheal aerosolization of monoclonal antibodies activating PEAR1 greatly ameliorates pulmonary fibrosis in both WT and Pear1-humanized mice, significantly improving their survival rate.